U.S. patent application number 14/434931 was filed with the patent office on 2015-09-10 for treatment of autophagy-based disorders and related pharmaceutical compositions, diagnostic and screening assays and kits.
The applicant listed for this patent is Eliseo F. CASTILLO, Vojo P. DERETIC, STC.UNM. Invention is credited to Eliseo F. Castillo, Vojo P. Deretic.
Application Number | 20150250808 14/434931 |
Document ID | / |
Family ID | 50488682 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150250808 |
Kind Code |
A1 |
Deretic; Vojo P. ; et
al. |
September 10, 2015 |
TREATMENT OF AUTOPHAGY-BASED DISORDERS AND RELATED PHARMACEUTICAL
COMPOSITIONS, DIAGNOSTIC AND SCREENING ASSAYS AND KITS
Abstract
In one embodiment, the invention provides a method of treating a
subject suffering from a Mycobacterium infection by administering
to the subject a therapeutically-effective amount of a degradative
autophagy agonist or a secretory autophagy antagonist. In another
embodiment, the invention provides a method of treating a subject
suffering from one or more diseases selected from the group
consisting of a Mycobacterium infection, an inflammatory disorder,
an immune disorder, a cancer and a neurodegenerative disorder by
administering to the subject a therapeutically-effective amount of
a TBK-1 antagonist (e.g. BX795 or amlexanox). Related
pharmaceutical compositions, diagnostic and screening assays and
kits are also provided.
Inventors: |
Deretic; Vojo P.; (Placitas,
NM) ; Castillo; Eliseo F.; (Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DERETIC; Vojo P.
CASTILLO; Eliseo F.
STC.UNM |
Placitas
Albuquerque
Albuquerque |
NM
NM
NM |
US
US
US |
|
|
Family ID: |
50488682 |
Appl. No.: |
14/434931 |
Filed: |
October 15, 2013 |
PCT Filed: |
October 15, 2013 |
PCT NO: |
PCT/US2013/064946 |
371 Date: |
April 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61713919 |
Oct 15, 2012 |
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61713843 |
Oct 15, 2012 |
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Current U.S.
Class: |
424/158.1 ;
424/172.1; 424/278.1; 435/18; 435/29; 435/6.11; 435/6.12; 435/7.1;
435/7.92; 506/15; 506/9 |
Current CPC
Class: |
A61K 31/4535 20130101;
G01N 33/6842 20130101; A61K 31/57 20130101; A61K 38/177 20130101;
A61P 11/12 20180101; G01N 2500/02 20130101; A61K 31/472 20130101;
A61K 31/506 20130101; G01N 2333/90212 20130101; A61K 31/4453
20130101; A61K 31/4184 20130101; G01N 2500/20 20130101; A61K
31/5415 20130101; A61K 38/1793 20130101; A61P 31/04 20180101; G01N
33/5695 20130101; G01N 2333/4719 20130101; A61K 31/00 20130101;
A61K 31/713 20130101; A61K 45/06 20130101; C07K 16/245 20130101;
A61K 31/65 20130101; A61K 31/277 20130101; A61K 31/495 20130101;
G01N 2333/4703 20130101; G01N 33/6893 20130101; A61P 11/10
20180101; A61K 31/381 20130101; G01N 2333/4724 20130101; A61K
9/0073 20130101; A61K 31/38 20130101; A61K 31/436 20130101; A61K
31/4365 20130101; A61P 31/06 20180101; A61K 31/473 20130101; C07K
2317/76 20130101; G01N 2333/54 20130101; A61K 38/00 20130101; G01N
33/5055 20130101; A61K 31/475 20130101; G01N 2333/545 20130101;
C07K 16/28 20130101; A61K 31/704 20130101 |
International
Class: |
A61K 31/704 20060101
A61K031/704; A61K 31/4184 20060101 A61K031/4184; A61K 31/473
20060101 A61K031/473; A61K 31/495 20060101 A61K031/495; A61K
31/4365 20060101 A61K031/4365; A61K 31/472 20060101 A61K031/472;
A61K 31/4535 20060101 A61K031/4535; A61K 31/65 20060101 A61K031/65;
A61K 31/4453 20060101 A61K031/4453; A61K 31/5415 20060101
A61K031/5415; A61K 31/277 20060101 A61K031/277; A61K 31/38 20060101
A61K031/38; A61K 31/475 20060101 A61K031/475; A61K 31/57 20060101
A61K031/57; A61K 38/17 20060101 A61K038/17; C07K 16/28 20060101
C07K016/28; A61K 31/381 20060101 A61K031/381; A61K 31/436 20060101
A61K031/436; A61K 31/506 20060101 A61K031/506; C07K 16/24 20060101
C07K016/24; G01N 33/68 20060101 G01N033/68; G01N 33/569 20060101
G01N033/569; A61K 45/06 20060101 A61K045/06 |
Goverment Interests
RELATED APPLICATIONS AND GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers R01 AI069345 and R01 AI042999 awarded by the National
Institutes of Health (NIH). The government has certain rights in
the invention.
Claims
1. A method of inhibiting, reducing the likelihood or treating a
tuberculosis infection in a patient or subj ect comprising
administering to a patient in need an effective amount of an
autophagy modulator.
2. The method according to claim 1 wherein said moldulator is an
autophagy agonist.
3. The method according to claim 1 wherein said modulator is
flubendazole, hexachlorophene, propidium iodide, bepridil,
clomiphene citrate (Z,E), GBR 12909, propafenone, metixene,
dipivefrin, fluvoxamine, dicyclomine, dimethisoquin, ticlopidine,
memantine, bromhexine, norcyclobenzaprine, diperodon,
nortriptyline, a mixture thereof or a pharmaceutically acceptable
salt thereof.
4. The method according to claim 1 wherein said modulator is
benzethonium, niclosamide, monensin, bromperidol, levobunolol,
dehydroisoandosterone 3-acetate, sertraline, tamoxifen, reserpine,
hexachlorophene, dipyridamole, harmaline, prazosin, lidoflazine,
thiethylperazine, dextromethorphan, desipramine, mebendazole,
canrenone, chlorprothixene, maprotiline, homochlorcyclizine,
loperamide, nicardipine, dexfenfluramine, nilvadipine, dosulepin,
biperiden, denatonium, etomidate, toremifene, tomoxetine,
clorgyline, zotepine, beta-escin, tridihexethyl, ceftazidime,
methoxy-6-harmalan, melengestrol, albendazole, rimantadine,
chlorpromazine, pergolide, cloperastine, prednicarbate,
haloperidol, clotrimazole, nitrofural, iopanoic acid, naftopidil,
Methimazole, Trimeprazine, Ethoxyquin, Clocortolone, Doxycycline,
Pirlindole mesylate, Doxazosin, Deptropine, Nocodazole,
Scopolamine, Oxybenzone, Halcinonide, Oxybutynin, Miconazole,
Clomipramine, Cyproheptadine, Doxepin, Dyclonine, Salbutamol,
Flavoxate, Amoxapine, Fenofibrate, Pimethixene, or a
pharmaceutically acceptable salt or mixture thereof.
5. (canceled)
6. (canceled)
7. A pharmaceutical composition in aerosol delivery form for
delivery to patient or subject in need of treatment for a
tuberculosis infection comprising an effective amount flubendazole,
hexachlorophene, propidium iodide, bepridil, clomiphene citrate
(Z,E), GBR 12909, propafenone, metixene, dipivefrin, fluvoxamine,
dicyclomine, dimethisoquin, ticlopidine, memantine, bromhexine,
norcyclobenzaprine, diperodon, nortriptyline, benzethonium,
niclosamide, monensin, bromperidol, levobunolol,
dehydroisoandosterone 3-acetate, sertraline, tamoxifen, reserpine,
hexachlorophene, dipyridamole, harmaline, prazosin, lidoflazine,
thiethylperazine, dextromethorphan, desipramine, mebendazole,
canrenone, chlorprothixene, maprotiline, homochlorcyclizine,
loperamide, nicardipine, dexfenfluramine, nilvadipine, dosulepin,
biperiden, denatonium, etomidate, toremifene, tomoxetine,
clorgyline, zotepine, beta-escin, tridihexethyl, ceftazidime,
methoxy-6-harmalan, melengestrol, albendazole, rimantadine,
chlorpromazine, pergolide, cloperastine, prednicarbate,
haloperidol, clotrimazole, nitrofural, iopanoic acid, naftopidil,
Methimazole, Trimeprazine, Ethoxyquin, Clocortolone, Doxycycline,
Pirlindole mesylate, Doxazosin, Deptropine, Nocodazole,
Scopolamine, Oxybenzone, Halcinonide, Oxybutynin, Miconazole,
Clomipramine, Cyproheptadine, Doxepin, Dyclonine, Salbutamol,
Flavoxate, Amoxapine, Fenofibrate, Pimethixene, or a
pharmaceutically acceptable salt or mixture thereof.
8. A pharmaceutical composition according to claim 7 wherein said
compound or compounds are further formulated in combination with an
additional anti-tuberculosis agent.
9. The pharmaceutical composition according to claim 7 wherein said
additional anti-tuberculosis agent is aminosalicylic acid,
isoniazid, ethionamide, myambutol, rifampin, rifabutin,
rifapentine, carpeomycin, cycloserine, or a pharmaceutically
acceptable salt or a mixture thereof.
10. (canceled)
11. (canceled)
12. A method of treating a subject suffering from a Mycobacterium
infection, the method comprising administering to the subject a
therapeutically-effective amount of a secretory autophagy
antagonist.
13. The method of claim 12, wherein the secretory autophagy
antagonist is selected from the group consisting of an IL-1RA
antagonist, an IL-1.beta. antagonist, an IL-18 (IL-1F4) antagonist,
a TSG101 antagonist, a HMGB1 antagonist, a Rab GTPase antagonist
and a GRASP55 or GRASP65 antagonist.
14. The method of claim 12, wherein the IL-1RA antagonist and
IL-1.beta. antagonist are Anakinra; the TSG101 antagonist is a
TSG101 siRNA; the HMGB1 antagonist is selected from the group
consisting of anti-HMGB1 antibody, ethyl pyruvate, a high mobility
group box (HMGB) A box or a biologically active fragment thereof,
an antibody to HMGB or an antigen-binding fragment thereof, an HMGB
small molecule antagonist, an antibody to TLR2 or an
antigen-binding fragment thereof, a soluble TLR2 polypeptide, an
antibody to RAGE or an antigen-binding fragment thereof, a soluble
RAGE polypeptide and a RAGE small molecule antagonist; the Rab
GTPase antagonist is
2-(benzoylcarbarnothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-
-3-carboxylic acid and the GRASP55 or GRASP65 antagonist is a
GRASP55 or GRASP65 siRNA.
15. The method of claim 13, wherein the HMGB 1 antagonist is
glycyrrhizin.
16. A method of treating a subject suffering from one or more
diseases selected from the group consisting of a Mycobacterium
infection, an inflammatory disorder, an immune disorder, a cancer
and a neurodegenerative disorder, the method comprising
administering a therapeutically-effective amount of a TBK-1
antagonist to the subject.
17. The method of claim 16, wherein the TBK-1 antagonist is BX795
or amlexanox.
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. A method of determining whether a subject suffers from, or is
likely to develop, one or more autophagy-related disorders selected
from the group consisting of an inflammatory disorder, an immune
disorder, a cancer and a neurodegenerative disorder, the method
comprising measuring expression levels in a cell sample obtained
from the subject of one or more biomarkers selected from the group
consisting of vimentin, galectin-1, galectin-3, ASC, ferritin and
thioredoxin and comparing measured expression levels of the one or
more biomarkers to expression levels of corresponding biomarkers in
a control cell sample, wherein elevated expression levels of the
one or more biomarkers when compared to control levels indicates
that the subject suffers from, or is likely to develop, one or more
of the autophagy-related disorders.
26. A method of determining whether a subject suffers from, or is
likely to develop, a Mycobacterium infection, the method comprising
measuring expression levels in a cell sample obtained from the
subject of one or more biomarkers selected from the group
consisting of Atg8 (LC3A, B and C, and GABARAP, GABARAPL1 and L2),
Rab8a, Atg9, FIP200, VMP1, WIPIs 91, DFCP-1 69, [L-1RA, IL-1.beta.,
IL-18 (IL-1F4), TSG101, HMGB1, a Rab GTPase, GRASP55 and GRASP65
and comparing measured expression levels of the one or more
biomarkers to expression levels of corresponding biomarkers in a
control cell sample, wherein elevated expression levels of the one
or more biomarkers when compared to control levels indicates that
the subject suffers from one or more of the autophagy-related
disorders.
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. A pharmaceutical composition comprising: (a) an amount of a
TBK-1 antagonist which is effective in treating one or more
diseases selected from the group consisting of a Mycobacterium
infection, an inflammatory disorder, an immune disorder, a cancer
and a neurodegenerative disorder; and optionally (b) a
pharmaceutically-acceptable excipient.
32. The pharmaceutical composition of claim 31, wherein the TBK-1
antagonist is BX795 or amlexanox.
33. (canceled)
34. A kit comprising: (a) at least one reagent which is selected
from the group consisting of: (i) reagents that detect a
transcription product of the gene coding for one or more biomarkers
selected from the group consisting of IL-1RA, IL-1.beta., IL-18
(IL-1F4A), TSG101, HMGB1, Rab GTPase, GRASP55 , GRASP65, Atg8
(LC3A, B and C, and GABARAP, GABARAPL1 and L2), Rab8a, Atg9,
FIP200, VMP1, WIPIs 91, DFCP-1 69, vimentin, galectin-1,
galectin-3, ASC, ferritin and thioredoxin; (ii) reagents that
detect a translation product of the gene coding for the one or more
biomarkers, and/or reagents that detect a fragment or derivative or
variant of said transcription or translation product; (b)
instructions for diagnosing, or prognosticating a Mycobacterium
infection, or determining the propensity or predisposition of a
subject to develop such a disease or of monitoring the effect of a
treatment by determining a level, or an activity, or both said
level and said activity, and/or expression of said transcription
product and/or said translation product and/or of fragments,
derivatives or variants of the foregoing, in a sample obtained from
said subject; and (1) comparing said level and/or said activity
and/or expression of said transcription product and/or said
translation product and/or fragments, derivatives or variants
thereof to a reference value representing a known Mycobacterium
infection status (patient) and/or to a reference value representing
a known health status (control) and/or to a reference value; and
(2) analyzing whether said level and/or said activity and/or
expression is varied compared to a reference value representing a
known health status, and/or is similar or equal to a reference
value representing a known disease status or a reference value; and
(3) diagnosing or prognosticating a Mycobacterium infection, or
determining the propensity or predisposition of said subject to
develop such a disease, wherein a varied or altered level,
expression or activity, or both said level and said activity, of
said transcription product and/or said translation product and/or
said fragments, derivatives or variants thereof compared to a
reference value representing a known health status (control) and/or
wherein a level, or activity, or both said level and said activity,
of said transcription product and/or said translation product
and/or said fragments, derivatives or variants thereof is similar
or equal to a reference value and/or to a reference value
representing a known Mycobacterium infection stage, indicates a
diagnosis or prognosis of a disease, or an increased propensity or
predisposition of developing such a disease, a high risk of
developing signs and symptoms of a disease.
35. (canceled)
36. A method of treating or reducing the likelihood of a disease
state or condition selected from the group consisting of sepsis, an
inflammatory disease state or disorder or cancer in a patient in
need comprising administering to said patient a composition
selected from the group consisting of a HMGB1 modulator, an
IL-1.beta. modulator, an IL-18 modulator, an IL-33 modulator, a
galectin modulator or mixtures thereof.
37. The method according to claim 36 wherein said HMGB1 modulator
is an inhibitor selected from the group consisting of an anti-HMGB
1 antibody or an antigen binding fragment thereof, ethyl pyruvate,
a HMGB 1 peptide, an HMGB small molecule antagonist, an anti-TLR2
antibody or an antigen-binding fragment thereof, a soluble TLR2
polypeptide, an antibody to RAGE or an antigen-binding fragment
thereof, a soluble RAGE polypeptide, a RAGE small molecule
antagonist and mixtures thereof.
38. The method according to claim 37 wherein said HMGB small
molecule antagonist is glycyrrhizin.
39. The method according to claim 36 wherein said IL-1.beta.
modulator is an anti-IL-1.beta. antibody or anakinra.
40. The method according to claim 36 wherein said IL-18 modulator
is an anti-IL-18 antibody or IL-18 binding protein (IL-18BP).
41. The method according to claim 36 wherein said IL-33 modulator
is an anti-IL-33 antibody or ST2.
42. The method according to claim 36 wherein said galectin
modulator is a galectin inhibitor selected from the group
consisting of GM-CT-01, GR-MD-02, GCS-100, taloside, pectin or a
mixture thereof.
43. The method according to claim 36 whererin said inflammatory
disease state or disorder is a lung disease, diabetes type I and
type II, severe insulin resistance, hyperinsulinemia, dyslipidemia,
elevated low-density lipoprotein (LDL), depressed high-density
lipoprotein (HDL), elevated triglycerides, Mendenhall's Syndrome,
Werner Syndrome, leprechaunism, lipoatrophic diabetes, acute and
chronic renal insufficiency, end-stage chronic renal failure,
glomerulonephritis, interstitial nephritis, pyelonephritis,
glomerulosclerosis, Kimmelstiel-Wilson disease in diabetic
patients, kidney failure after kidney transplantation, obesity,
GH-deficiency, GH resistance, Turner's syndrome, Laron's syndrome,
short stature, increased fat mass-to-lean ratios,
immunodeficiencies including decreased CD4.sup.+ T cell counts and
decreased immune tolerance or chemotherapy-induced tissue damage,
bone marrow transplantation, diseases or insufficiencies of cardiac
structure or function such as heart dysfunctions and congestive
heart failure, neuronal, neurological, or neuromuscular disorders,
e.g., diseases of the central nervous system including Alzheimer's
disease, or Parkinson's disease or multiple sclerosis, and diseases
of the peripheral nervous system and musculature including
peripheral neuropathy, muscular dystrophy, or myotonic dystrophy,
and catabolic states, including those associated with wasting
caused by any condition, including, e.g., mental health condition
(e.g., anorexia nervosa), trauma or wounding or infection such as
with a bacterium or human virus such as HIV, wounds, skin
disorders, gut structure and function that need restoration.
44. The method according to claim 36 wherein said inflammatory
disease state or disorder is an infectious disease, a disorder of
bone or cartilage growth in children, arthritis, osteoporosis,
heart dysfunction, kidney disorder, a neurological disorder, a bone
disorder, a whole body growth disorder or an immunological
disorder.
45. The method according to claim 44 wherein said neurological
disorder is Alzheimer's Dementia (AD), amyotrophic lateral
sclerosis, depression, epilepsy, Huntington's Disease, multiple
sclerosis, neurological complications of AIDS, spinal cord injury,
glaucoma and Parkinson's disease.
46. An assay for determining whether a compound of unknown activity
is an inhibitor or inducer of autophagy secretion comprising two
populations of cells, the first population of cells expressing a
sequestome-like receptor (SLR) and a galectin-GFP fusion protein
and emitting a green fluorescent signal in the absence of a test
compound (a first cell population control signal) wherein said SLR
and said galectin-GFP fusion protein interact to secrete said
galectin-GFP fusion protein from said first population of cells,
said second population of cells expressing a red fluorescent
protein and galectin receptors on the surface of the cells which
are capable of binding galectin-GFP secreted from said first
population of cells
Description
[0001] The present claims the benefit of priority of U.S.
Provisional Application Ser. Nos. U.S.61/713,919, filed Oct. 15,
2012 and entitled "Autophagy Protects Against Tuberculosis", and
filed U.S. 61/713,843, entitled "Mechanism of Secretive Autophagy"
filed Oct. 15, 2012, each of which applications is incorporated by
reference in its entirety herein.
FIELD OF THE INVENTION
[0003] In one embodiment, the invention provides a method of
treating a subject suffering from a Mycobacterium infection by
administering to the subject a therapeutically-effective amount of
a degradative autophagy agonist or a secretory autophagy
antagonist.
[0004] In another embodiment, the invention provides a method of
treating a subject suffering from one or more diseases selected
from the group consisting of a Mycobacterium infection, an
inflammatory disorder, an immune disorder, a cancer and a
neurodegenerative disorder by administering to the subject a
therapeutically-effective amount of a TBK-1 antagonist (e.g. BX795
or amlexanox).
[0005] Related pharmaceutical compositions, diagnostic and
screening assays and kits are also provided.
BACKGROUND OF THE INVENTION
[0006] Autophagy is a fundamental cell biological process (1) with
impact on aging, development, cancer, neurodegeneration,
myodegeneration, and metabolic disorders (2), idiopathic
inflammatory diseases and infection and immunity (3). Much of the
physiological effects of autophagy are the result of degradative
activities of autophagy (1), although biogenesis and secretory
roles (4-6) of autophagy are beginning to be recognized (7). The
execution of autophagy depends on factors collectively termed Atg
such as Atg5 (1) and Beclin 1 (Atg6) (8) whereas regulation of
autophagy responds to various inputs via mTOR, including presence
of microbes (9), TAB2/3-TAK1-IKK signaling axis (10), and processes
downstream of pattern recognition receptors and immune cytokine
activation (3, 11-13).
[0007] In the context of its immunological functions, autophagy
acts in four principal ways (14): (i) Autophagy cooperates with
conventional PRRs, such as TLRs, RLRs, and NLRs, and it plays the
role of both a regulator (11, 12, 15, 16) and an effector of PRR
signaling (17-19). (ii) Autophagy affects presentation of cytosolic
antigens in the context of MHC II molecules (20) in T cell
development, differentiation, polarization and homeostasis (21,
22). (iii) Most recently, autophagy has been shown to contribute to
both the negative (6, 7, 23-25) and positive regulation (6, 7) of
unconventional secretion of the leaderless cytosolic proteins known
as alarmins such as IL-1.beta. and HMGB1. (iv) Autophagy can
capture and eliminate intracellular microbes including M.
tuberculosis (17, 26-29) as one of the first two bacterial species
(26, 30) to be recognized as targets for autophagic removal. This
has been recently shown to depend on a recognition and capture by
adaptors that represent a specialized subset of pattern recognition
receptors (PRR) termed sequestosome-like receptors (SLRs) (31).
[0008] M. tuberculosis is one of the first microbes recognized as
being subject to elimination by immunological autophagy in ex vivo
systems in murine and human macrophages (17, 22, 26, 27, 29).
Although it has been shown that macrophages from
Atg5.sup.fl/flLysM-Cre.sup.+ mice defective for autophagy in
myeloid lineage fail to control M. tuberculosis H37Rv (32) the in
vivo role of autophagy in control of M. tuberculosis has not been
reported. Given the compelling reasons for testing whether
autophagy matters in control of M. tuberculosis in vivo, here we
used a mouse model of tuberculosis and employed transgenic mice
deficient in Atg5 in the myeloid lineage including macrophages, a
cell type parasitized by M. tuberculosis (33). We demonstrate that
autophagy controls tuberculosis infection in vivo and uncover a
parallel role of autophagy in preventing excessive inflammatory
reactions in the host.
[0009] The notion of autophagy as a purely degradative pathway was
recently challenged by the emergence of reports of the secretory
function of autophagy by three independent groups on the secretion
of Acb1 in yeast (25A,26A,32A) and IL-1.beta. secretion in
mammalian cells (17A,27A). These new developments assign to
autophagy a non-degradative function manifested as unconventional
protein secretion (FIG. 3A). Furthermore, it has become apparent
that autophagy even more broadly intersects with protein
trafficking to include effects on the constitutive biosynthetic
pathway (23A), regulated exocytosis (19A), and alternative sorting
of integral membrane proteins to the plasma membrane (28A).
SUMMARY OF THE INVENTION
[0010] We have characterized a conditional gene knockout mouse
model (Atg5.sup.fl/flLysM-Cre.sup.+) with a well documented Atg5
defect in macrophages and infected these mice aerogeneously with
the virulent M. tuberculosis strain H37Rv. An increase in bacterial
burden in the lungs and increased lung pathology was observed in
Atg5.sup.fl/flLysM-Cre.sup.+ compared to
Atg5.sup.fl/flLysM-Cre.sup.+ littermates (FIG. 1P, panel A). With
higher doses of M. tuberculosisAtg5.sup.flflLysM-Cre.sup.+ mice
succumbed sooner to infection. These findings demonstrate for the
first time that autophagy is important for control of Mycobacterium
infections such as M. tuberculosis in vivo.
[0011] As explained hereinafter, we have also identified and
proposed a variety of secretory autophagy-related biomarkers and
cellular processes that enable us to utilize the machinery and
mechanisms of autophagy-dependent unconventional protein secretion
in mammalian cells in novel therapeutic, diagnostic and screening
methods.
[0012] In one embodiment, our invention provides a method of
treating a subject suffering from a Mycobacterium infection by
administering to the subject a therapeutically-effective amount of
a degradative autophagy agonist.
[0013] In another embodiment, the invention provides a method of
treating a subject suffering from a Mycobacterium infection by
administering to the subject a therapeutically-effective amount of
a secretory autophagy antagonist (e.g. an IL-1 receptor (IL-1RA)
antagonist, an IL-1.beta. antagonist, an IL-18 (IL-1F4) antagonist,
an IL-33 antagonist, a galectin antagonist (antagonist of any one
or more of the human galectins galectin-1, galectin-2, galectin-3,
galectin-4, galectin-7, galectin-8, galectin-9, galectin-10,
galectin-12 and galectin-13), a TSG101 antagonist, a HMGB1
antagonist, a Rab GTPase antagonist or a GRASP55 or GRASP65
antagonist). In certain preferred embodiments, the IL-1RA
antagonist and IL-1.beta. antagonist are anti-IL-1RA antibodies,
IL-1.beta. antibodies or Anakinra; the TSG101 antagonist is a
TSG101 siRNA; the HMGB1 antagonist is selected from the group
consisting of anti-HMGB 1 antibody, ethyl pyruvate, a high mobility
group box (HMGB 1) peptide or a biologically active fragment
thereof, an antibody to HMGB or an antigen-binding fragment
thereof, an HMGB small molecule antagonist, an antibody to TLR2 or
an antigen-binding fragment thereof, a soluble TLR2 polypeptide, an
antibody to RAGE or an antigen-binding fragment thereof, a soluble
RAGE polypeptide and a RAGE small molecule antagonist; the Rab
GTPase antagonist is
2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran--
3-carboxylic acid and the GRASP55 or GRASP65 antagonist is a
GRASP55 or GRASP65 siRNA. In another preferred embodiment of this
method of treatment, the HMGB1 antagonist is glycyrrhizin. In
certain embodiments, the galectin antagonist/inhibitor is a
galactomannan based carbohydrate such as GM-CT-01, GR-MD-02
(Galectin Therapeutics, Inc., as described in U.S. Pat.
No.8,236,780 which is incorporated by reference herein), GCS-100
(CAS No. 531508-98-2) (a pectin have multiple side-branches
containing the sugar .beta.-galactose), taloside (a C-2 epimer of
galactose) or a pectin (apple, rhubarb, okra, onion), among others.
One or more of the above-described antagonists also may be used in
the treatment of a number of disease states and/or conditions such
as sepsis, inflammatory disease states and disorders and cancer as
described herein.
[0014] In another embodiment, the invention provides a method of
treating a subject suffering from one or more diseases selected
from the group consisting of a Mycobacterium infection, an
inflammatory disorder, an immune disorder, a cancer and a
neurodegenerative disorder, the method comprising administering a
therapeutically-effective amount of a TBK-1 antagonist to the
subject. In a preferred embodiment, the TBK-1 antagonist is BX795
or amlexanox.
[0015] In another embodiment, the present invention is directed to
the treatment or prophylaxis (reducing the likelihood) of a disease
state or condition modulated by secretory autophagy including
sepsis, an inflammatory disease state (as otherwise described
herein) and cancer in a patient in need comprising administering an
effective amount of agent which modulates (inhibits and/or
promotes/induces) secretory autophagy to said patient in need. The
disease state or condition may be modulated by any one or more of
HMGB.sub.1 (sepsis, inflammatory disease states and disorders and
cancer, often by inhibition of HMGB.sub.1), IL-1.beta. (sepsis,
cancer and inflammatory disease states and disorders, often by
inhibition of IL-1.beta.), IL-18 (sepsis, inflammatory disease
states and disorders and cancer, often by inhibition of IL-18 using
IL-18 binding protein IL-18BP, antibodies against IL-18 including
humanized antibodies or a mutein or fused protein thereof as
inhibitors), IL-33 (sepsis, inflammatory disease and cancer often
by inhibition the release of IL-33, or in the case of cancer, both
inhibition and inducing release of IL-33 by ST2 Inhibitor (ST2
protein) or IL-33 inhibitor or antibodies which bind to IL-33), or
one or more galectin inhibitor/antagonists described above. It is
noted that in the case of sepsis and inflammatory disease states
and disorders and conditions, the inhibition (including the
secretion of the modulator), rather than the induction or promotion
of the secretion of one or more of the above modulators is often
therapeutic. In the case of cancer therapy, the inhibition of one
or more of the above-modulators, including its secretion from
cells, may provide an anticancer benefit, and in certain instances,
the induction or promotion of secretion of one or more of the
above-modulators may prove therapeutically beneficial for the
treatment of cancer.
[0016] In another embodiment, the invention provides a method of
identifying a protein that is a substrate for secretory autophagy
(i.e. an autophagic secretome), the method comprising: [0017] (a)
providing a sample of Atg5-proficient cells and isogenic
Atg5-deficient cells; and [0018] (b) subjecting the sample to
subtractive analysis by inducing autophagy in the sample and
identifying protein entities released into supernatants from
Atg5-proficient cells but not from isogenic Atg5-deficient cells;
[0019] wherein protein entities released into supernatants from
Atg5-proficient cells are identified as substrates for secretory
autophagy.
[0020] In another embodiment, the invention provides a method of
identifying a protein that is a substrate for secretory autophagy
(i.e. an autophagic secretome), the method comprising: [0021] (a)
providing a sample of Atg5-proficient cells and isogenic
Atg5-deficient cells; and [0022] (b) subjecting the sample to
subtractive analysis by: (1) inducing autophagy in primary
macrophages of the sample for a period of time selected to avoid
nonspecific leakage of cytosolic proteins from cells; and (2)
identifying protein entities released into supernatants from
Atg5-proficient cells but not from isogenic Atg5-deficient cells;
[0023] wherein protein entities released into supernatants from
Atg5-proficient cells are identified as substrates for secretory
autophagy.
[0024] In another embodiment, the invention provides a method of
determining whether a composition is a secretory autophagy
antagonist, the method comprising contacting a eukaryotic cell
sample with the composition, measuring cellular expression levels
of one or more biomarkers selected from the group consisting of
vimentin, galectin-1, galectin-3 (or other galectins such as
galectin-2, -4, -7, -8, -9, -10, -12 and -13), ASC (an inflammasome
component), ferritin and thioredoxin, and comparing measured
cellular expression levels of the one or more biomarkers with
expression levels of corresponding biomarkers in a control
eukaryotic cell sample, wherein reduced expression levels of the
one or more biomarkers when compared to control expression levels
indicates that the composition is a secretory autophagy antagonist.
In a preferred embodiment of this screening method, the cells are
human primary peripheral blood monocyte-derived macrophages.
[0025] In another embodiment, the invention provides a method of
determining whether a composition is a secretory autophagy
antagonist, the method comprising contacting a eukaryotic cell
sample with the composition, measuring cellular expression levels
of one or more biomarkers selected from the group consisting of
Atg8 (LC3A, B and C, and GABARAP, GABARAPL1 and L2), Rab8a, Atg9,
FIP200, VMP1, WIPIs 91 and DFCP-1 69, and comparing measured
cellular expression levels of the one or more biomarkers with
corresponding expression levels of the biomarkers in a control
eukaryotic cell sample, wherein reduced expression levels of the
one or more biomarkers when compared to control expression levels
indicates that the composition is a secretory autophagy antagonist.
In a preferred embodiment of this screening method, the cells are
human primary peripheral blood monocyte-derived macrophages.
[0026] In another embodiment, the invention provides a method of
determining whether a composition is a secretory autophagy
antagonist, the method comprising contacting a eukaryotic cell
sample with the composition, measuring cellular expression levels
of at least one biomarkers selected from the group consisting of
IL-1RA, IL-1.beta., IL-18 (IL-1F4), IL-33, a galectin (galectin-1,
-2, -3, -4, -7, -8, -9, -10, -12 and -13), TSG101, HMGB1, a Rab
GTPase, GRASP55 and GRASP65 and comparing cellular expression
levels of the at least one biomarkers with expression levels of
corresponding biomarkers in a control eukaryotic cell sample,
wherein reduced measured expression levels of the at least one
biomarkers when compared to control expression levels indicates
that the composition is a secretory autophagy antagonist.
[0027] In still another embodiment, the invention provides a method
of determining whether a subject suffers from, or is likely to
develop, one or more autophagy-related disorders selected from the
group consisting of an inflammatory disorder, an immune disorder, a
cancer and a neurodegenerative disorder (as described herein), the
method comprising measuring expression levels in a cell sample
obtained from the subject of one or more biomarkers selected from
the group consisting of vimentin, galectin-1, galectin-3 (other
galectins including galectin-2, -4, -7, -8, -9, -10, -12 and -13),
ASC, ferritin and thioredoxin and comparing measured expression
levels of the one or more biomarkers to expression levels of
corresponding biomarkers in a control cell sample, wherein elevated
expression levels of the one or more biomarkers when compared to
control levels indicates that the subject suffers from, or is
likely to develop, one or more of the autophagy-related
disorders.
[0028] In still another embodiment, the invention provides a method
of determining whether a subject suffers from, or is likely to
develop, a Mycobacterium infection, the method comprising measuring
expression levels in a cell sample obtained from the subject of one
or more biomarkers selected from the group consisting of Atg8
(LC3A, B and C, and GABARAP, GABARAPL1 and L2), Rab8a, Atg9,
FIP200, VMP1, WIPIs 91, DFCP-1 69, IL-1RA, IL-1.beta., IL-18
(IL-1F4), IL-33, a galectin (galectin-1, -2, -3, -4, -7, -8, -9,
-10, -12 and -13), TSG101, HMGB1, a Rab GTPase, GRASP55 and GRASP65
and comparing measured expression levels of the one or more
biomarkers to expression levels of corresponding biomarkers in a
control cell sample, wherein elevated expression levels of the one
or more biomarkers when compared to control levels indicates that
the subject suffers from, or is likely to develop, one or more of
the autophagy-related disorders.
[0029] In still another embodiment, the invention provides a
pharmaceutical composition comprising an amount of a TBK-1
antagonist which is effective in treating a Mycobacterium infection
and, optionally, a pharmaceutically-acceptable excipient.
[0030] In one particular embodiment, a particular assay is provided
(see FIGS. 13A and 13B) comprising a population of two distinct
cells, the first population of cells expressing a sequestome-like
receptor (SLR) and a galectin-GFP (green fluorescent protein)
fusion protein (which provides a green fluorescent signal in the
first population of cells), the galectin-GFP fusion protein binding
to said SLR and being secreted from said first population of cells,
especially in the presence of an autophagy secretion inducer. The
second population of cells express red fluorescent protein and
galectin receptors on their surfaces which concentrate and take up
(by endocytosis) galectin bound on the surface of the cells. The
two populations of cells, when mixed together in the absence of an
inhibitor or promoter of autophagy secretion will provide a mixture
of green cells and red cells (and either no red fluorescent cells
or relatively few red fluorescent cells which also express green
fluorescent protein from galectin-GFP being taken up under control
conditions) from the two populations of cells (control). In the
presence of a compound which is an inducer of autophagy secretion,
the fluorescence emitted from the two populations of cells reflect
the induction of autophagy secretion (as evidenced by a decrease in
green fluorescence emanating from the first population of cells and
an increase in green fluorescence emanating from the second
population of cells). In the presence of an inhibitor of autophagy,
any green fluorescence which is emitted from the first population
of cells may be increased and any green fluorescence emitted from
the second population of cells which occurs naturally (control)
between the cells will be reduced, evidencing the compound as a
potential inhibitor of autophagy. In certain embodiments, the
release of galectin-GFP from the the first population of cells
under control conditions (i.e. in the absence of an inhibitor or
inducer of autophagy secretion) will be sufficiently high so that
the exposure of the cells to an inhibitor of autophagy secretion
will increase the green fluorescence emitted from the first
population of cells and reduce the green fluorescence emitted by
the second population of cells compared to control, and exposure to
an inducer of autophagy secretion will decrease the green
fluorescence emitted from the first population of cells and
increase the green fluorescence emitted from the second population
of cells. Through use of the above-described assays system, an
inducer or inhibitor of autophagy (galectin) may be readily
provided. The same system may be readily adapted for other
autophagy modulators described herein. Methods of using the
above-described assay(s) to identify a compound of unknown activity
as an inhibitor or inducer of autophagy secretion represent
additional embodiments of the present invention. The method can be
readily adapted for use in flow cytometry. A kit based upon the
above-described assay comprises a first cell population which
expresses sequestome-like receptor (SLR) and galectin-GFP fusion
protein and a second cell population which expresses red
fluorescent protein and galectin surface receptors which
concentrate and take up galectin into the cell. While any
eukaryotic cell may be readily engineered to provide a first
population of cells and a second population of cells, preferred
cells are human engineered cells including engineered HeLa
cells.
[0031] It is noted that any galectin-GFP fusion protein in the
above-described assays and methods, including galectin-1,
galectin-2, galectin-3, galectin-4, galectin-7, galectin-8,
galectin-9, galectin-10, galectin-12 and galectin-13, but often
galectin-1 and galectin-3 are the galectins which are most commonly
used in the assays and methods described hereinabove.
[0032] As described in detail herein, we have utilized degradative
and secretory autophagy processes to provide a wide variety of
therapeutic, diagnostic and screening methods. Our disoveries
relating to common and disparate aspects of degradative and
secretory autophagy enable diagnoses and treatments of a wide
variety of disorders.
[0033] These and other aspects of the invention are described
further in the detailed Description of the Invention.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1. Autophagy protects from excessive inflammation in a
mouse model of tuberculosis infection. (A) Bacterial burden (colony
forming units; cfu) in organs of Atg5.sup.fl/flLysM-Cre.sup.+ and
Atg5.sup.fl/flLysM-Cre.sup.- mice infected aerogenously with low
dose M. tuberculosis H37Rv. The data shown are representative of
>3 independent low dose experiments. (B) Weight loss in
Atg5.sup.fl/flLysM-Cre.sup.+ and Atg5.sup.fl/flLysM-Cre.sup.- mice
infected with low doseM. tuberculosis H37Rv. (C) Gross lung
pathology (low dose). (D) Lung histological sections (low dose, day
36). Panels: i-iv, H&E stain (arrows, necrotic lesions); v and
vi, acid-fast staining (arrows, bacilli; insets enlarged area).
Numbers: % of total area occupied by acid fast bacilli (AFB). (E)
Survival of Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+mice infected
with M. tuberculosis H37Rv (high dose). (F) Weight loss in
Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ mice infected with M.
tuberculosis H37Rv (high dose). Low infectious dose:
3.times.10e.sup.2 (.+-.30%) cfu of initial bacterial deposition per
lung following exposure to the infectious inoculum. High dose:
10e.sup.4 cfu per lung. Mouse survival statistics: Kaplan-Meier
survival analysis with the Log-Rank method. Other data, mean.+-.SE,
*p<0.05, **p<0.01, .dagger.>0.05 (ANOVA; n.gtoreq.3).
).
[0035] FIG. 2. Increased inflammatory cytokines in
Atg5.sup.fl/flLysM-Cre.sup.+ mice. Multiplex cytokine detection by
Luminex in the lungs of M. tuberculosis H37Rv infected
Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ mice (low dose). See SI
Appendix, FIG. S2 for additional cytokines. BDL, below detection
limit. Data, mean.+-.SE, *p<0.05, **p<0.01, .dagger.>0.05
(t test; n.gtoreq.3). Data (D) mean.+-.range from a single cohort
of infected mice (see SI Appendix, FIG. S2A for pooled IL-17
data).
[0036] FIG. 3. Activated phenotype of CD4 T cells from uninfected
Atg5.sup.fl/flLysM-Cre.sup.+ mice and their propensity to undergo
polarization into IL-17 producing cells. (A) CD44 expression on
lung T cells. Graph displays the percent of CD44.sup.high CD4 and
CD8 T cells in the lung of uninfected Atg5.sup.fl/flLysM-Cre.sup.-
or Cre.sup.+ mice. The uninfected mice were 10-12 weeks of age.
(B-E) Intracellular levels of IL-17A (top panel) and IFN.gamma.
(bottom panel) in CD4 T cells isolated from lungs of uninfected
Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ mice and stimulated with
phorbol 12-myristate 13-acetate and ionomycin ex vivo in the
presence of brefeldin A and monensin. Data: mean.+-.SE; *p<0.05,
**p<0.01 (t test; n.gtoreq.3).
[0037] FIG. 4. In vivo and ex vivo immune response to defined M.
tuberculosis antigens of Atg5.sup.fl/flLysM-Cre.sup.+ mice and
IL-17 production by their splenocytes upon ex vivo restimulation.
(A) DTH reaction (footpad induration) in BCG-infected
Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ mice footpad-injected
with the synthetic PPD at day 21 postinfection. Data, percent
change (footpad thickness) upon challenge with the synthetic PPD
relative to the contralateral PBS-challenged footpad. (B-E)
Cytokine production by splenocytes from
Atg5.sup.fl/flLysM-Cre.sup.31 and Cre.sup.+ mice (day 23
post-peritoneal injection of BCG) re-stimulated for 3 days ex vivo
with the synthetic PPD. All mice were 10-12 weeks of age at the
onset of the experiment. Data: mean.+-.SE; *p<0.05, **p<0.01,
p>0.05 (t test; n.gtoreq.3).
[0038] FIG. 5. Excess cytokine secretion is a cell-autonomous
property of autophagy-deficient macrophages and IL-1.alpha.
hypersecretion by Atg5.sup.fl/flLysM-Cre.sup.+ macrophages depends
on reactive oxygen intermediates and calpain. (A-C) In vitro
cytokine (IL-1.alpha., CXCL1, and IL-12p70) release (ELISA) from
LPS- and IFN-.gamma.-stimulated Atg5.sup.fl/flLysM-Cre.sup.- and
Cre.sup.+ bone marrow-derived macrophages (BMM). (D) CXCL1 released
from LPS- and IFN-.gamma.-stimulated Atg5.sup.fl/flLysM-Cre.sup.+
BMM in the absence of presence of IL-1 RA (0.5 .mu.g/mL). (E)
Fraction of 7-AAD.sup.+ BMM after LPS and IFN-.gamma. stimulation
in vitro. (F,G) IL-1.alpha. and CXCL1 levels (ELISA) in lung
homogenates of uninfected Atg5.sup.fl/flLysM-Cre.sup.- and
Cre.sup.+ mice. (H,I) IL-1.alpha. (ELISA) released from LPS- and
IFN-.gamma. stimulated Atg5.sup.fl/flLysM-Cre.sup.- BMM in the
presence of 50 .mu.g/ml rapamycin (Rap), 10 mM 3-MA, or 100 nM
Bafilomycin A1 (Baf A1) after 12 h of stimulation. (J) IL-1.alpha.
secretion during inflammasome activation. Atg5.sup.fl/flLysM-Cre
.sup.- and Cre.sup.+ BMM were pretreated overnight with LPS (100
ng/ml) then stimulated for 1 h in the absence or presence of the
inflammasome agonist silica (250 .mu.g/ml) in EBSS. (K) IL-1.alpha.
secretion in the presence of caspase 1 inhibitor YVAD.
Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ BMM were pretreated
overnight with LPS (100 ng/ml) then stimulated for 1 h in the
absence or presence of YVAD (50 .mu.M) during inflammasome
activation with silica as in J. (L) Effects of caspase 1 siRNA
knockdown (immunoblot, left) on IL-1.alpha. release (graph, right)
from Atg5.sup.fl/flLysM-Cre.sup.+ BMM. Graph (center), IL-1.alpha.
release from LPS-stimulated siRNA-treated
Atg5.sup.fl/flLysM-Cre.sup.+ BMM in full media or EBSS (Starv).
Graph (right), same as middle graph in full medium only. Casp 1,
caspase 1 siRNA: Scr, scrambled, control siRNA. (M) IL-1.alpha.
release from LPS- and IFN-.gamma.-stimulated
Atg5.sup.fl/flLysM-Cre.sup.+ BMM knocked down with siRNA for
inflammasome components ASC and NLRP3. (N,O) ROS inhibition and
IL-1 secretion: IL-1.alpha. (N) and IL-1.beta. (O) released (ELISA)
from LPS- and IFN-.gamma.-stimulated Atg5.sup.fl/flLysM-Cre.sup.-
and Cre.sup.+ BMM in the absence or presence of ROS antagonist APDC
(50 .mu.M) after 12 h of incubation. (P) Calpain and IFN-.gamma.
hypersecretion phenotype. IL-1.beta. (ELISA) released from LPS- and
IFN-y stimulated Atg5.sup.fl/flLysM-Cre.sup.- and Cre.sup.+ BMM in
the absence or presence of calpain inhibitor ALLN (100 .mu.M) after
12 h of stimulation. Data, mean.+-.SE, *p<0.05, **p<0.01,
.dagger.p>0.05 (t test; n.gtoreq.3).
[0039] FIG. S1. Atg5.sup.fl/flLysM-Cre.sup.+ lung tissue revealed
extensive necrotic centers (FIG. 1D, subpanels i-iv) with increase
in percent of involved lung area and total lung weight (SI
Appendix; FIG. S1A,B) and differential increase in
polymorphonucelar (PMN) leukocytes (Ly6G.sup.+) (SI Appendix; FIG.
S1C-E).
[0040] FIG. S2. Cytokines in the lungs of Atg5fl/fl LysM-Cre+ and
Cre-mice infected with M. tuberculosis H37Rv. (A-H) Multiplex
cytokine measurement of IL-17, IFN-.gamma., TNF-.alpha., IL-4,
IL-6, MIP-1.beta., GM-CSF, and IL-1.beta. as detected by Luminex in
lung homogenates of Atg5fl/fl LysM-Cre and Cre+mice infected with
low M. tuberculosis dose at weeks 3, 4 and 5 postinfection. IL-17
in panel A represents the combined data from 3 independent cohorts
of infections for weeks 3, 4, 5 and 6. Data: mean.+-.SE,
*p<0.05,**p<0.01 (t test; n.gtoreq.3).
[0041] FIG. S3. Cytokine and cellular analysis of uninfected
Atg5fl/fl LysM-Cre+ lungs.(A-E) IL-1.alpha., CXCL1, IL-12p70,
IL-113, and IL-17 levels (ELISA) in lung homogenates of uninfected
Atg5fl/fl LysM-Cre- and Atg5fl/fl LysM-Cre+ mice. (F, G) Flow
cytometric quantification of macrophages per organ tissues in
uninfected Atg5fl/fl LysM-Cre- and Cre+mice. (H) Activation state
of macrophages measured by surface markers CD1d, MHC II, DEC205 and
CD86 in the lungs of uninfected Atg5fl/fl LysM-Cre- (left plots)
and Cre+mice (right plots). (I,J) Flow cytometric quantification of
neutrophils in the lungs and bone marrow of uninfected Atg5fl/fl
LysM-Cre- and Cre+mice. Data, mean.+-.SE, n.gtoreq.3, *p<0.05,
.dagger.>0.05 (t test).
[0042] FIG. S4. T cell activation state and IL-1.alpha. role in T
cell IL-17 polarization. (A,B) CD44 and CD25 expression on CD4 T
cells from lungs of uninfected Atg5fl/fl LysM-Cre and Cre+mice.
(C-F) Intracellular IL-17A production (day 4; release blocked with
monensin) by naive CD4 T cells polarized in the presence of
cytokine cocktails: 5 ng/ml TGF-13 and 20 ng/ml IL-6, plus 20 ng/ml
IL-1a or 20 ng/ml IL-1.beta.. Dot plot (panel C), levels of IL-17A
in unstimulated cells (starting material). Histograms (D,E), IL-17A
in naive CD4 T cells polarized in the presence of TGF-.beta., IL-6
and IL-1.alpha. (panel D) or TGF-.beta., IL-6 and IL-1.beta. (panel
E). (F) Percent of IL-17A+CD4 T cells under respective polarizing
conditions. Data: mean.+-.SE; t p>0.05 (t test; n.gtoreq.3).
[0043] FIG. S5. Controls for pharmacological induction of
IL-1.alpha. hypersecretion phenotype in autophagy-competent
macrophages. These experiments were carried out as a controls for
effects of pharmacological induction of autophagy on IL-1.alpha.
secretion shown in FIG. 5F. IL-1.beta. secretion was examined in
autophagy-competent Atg5fl/fl LysM-CreBMM+ and IL-1.alpha.
secretion was measured in autophagy-deficient Atg5fl/fl LysM-Cre+
BMM. (A) IL-1.beta. (ELISA) released from Atg5fl/fl LysM-CreBMM
(identical activation as in FIG. 5F) treated with (A) 50 mg/ml
rapamycin (Rap) and 100 nM Bafilomycin A1 (Baf A1) after 12 h of
stimulation. (B) IL-1.alpha. (ELISA) released from Atg5fl/fl
LysM-Cre+BMM (identical activation as in FIG. 5F) in the presence
of (A) 50 mg/ml rapamycin (Rap) and 10 mM 3-MA after 12 h of
stimulation. Data: mean.+-.SE; *, p<0.05;.dagger. (or no symbol)
p>0.05 (t test; n.gtoreq.3).
[0044] FIG. S6. Analysis of p62, autophagosomes, and caspase 1 as
potentially contributing factors to the IL-1 hypersecretion
phenotype in Atg5fl/fl LysM-Cre+ macrophages. (A) Immunoblot
assessment of p62/sequestosome 1 knockdown in BMM. (B) IL-1.alpha.
release from Atg5fl/fl LysM-Cre+ BMM (stimulated with LPS and
IFN-.gamma.) subjected to p62/sequestosome 1 knockdown (p62 siRNA)
relative to siRNA control (scr, scramble). (C) IL-1.alpha. released
from LPS and IFN-y stimulated BMM from p62-/- knockout mice treated
with Scr (scrambled control) or Atg5 siRNA. (D) Transcriptional
analysis (QTRT PCR) of IL-1.alpha. gene expression. Total RNA was
isolated from Atg5fl/fl LysM-Creand Cre+BMM using RNeasy kit
(Qiagen) and cDNA was generated using QuantiTect Reverse
Transcription kit (Qiagen). (E) Confocal microcopy analysis of
IL-1.alpha. colocalization relative to LC3 in GFP-LC3 expressing
BMM induced for autophagy by starvation (EBSS) in the presence of
bafilomycin A1 for90 minutes. Scale bar, 5 .mu.M; Pearson's
colocalization coefficient for IL-1.alpha. vs. LC3. (F) Caspase 1
activation in the absence of autophagy. Atg5fl/fl LysM-Cre-or
Cre+BMM cells were starved (EBSS) with or without bafilomycin
(BafA1) for 2 or 4 h and subjected to immunoblot analysis;
Procasp-1, procaspase 1; Casp-1 p20, processed (activated) caspase
1. (G) Quantification of p20 bands from blots as in A, relative to
actin. (H) Atg5fl/fl LysM-Cre- or Cre+BMM were stimulated with LPS
overnight and caspase-1 activity was assessed by flow cytometry
using the FLICA caspase-1 reagent. Data, mean.+-.SE; *p<0.05,
**p<0.01 and .dagger. p>0.05 (t test; n.gtoreq.3).
[0045] FIG. S7. Analysis of mitochondrial content, mitochondrial
polarization state, calpain levels, and calpain localization
relative to autophagic organelles in Atg5fl/fl LysM-Cre+
macrophages. (A-C) Flow cytometry analysis of cellular
mitochondrial content in Atg5fl/fl LysM-Cre- and Cre+ BMM stained
with MitoTracker Green. A and B, histograms; C average mean
fluorescence intensity (MFI) of MitoTracker Green per cell. (D,E)
Polarization state of mitochondria in Atg5fl/fl LysM-Cre- and Cre+
BMM. (D), Overlay histogram, MitoTracker Red CMXRos in Atg5fl/fl
LysM-Cre- and Cre+ BMM. (E), Data and statistical analysis of
cumulative results represented in D. (F) Immunoblot analysis of
calpain I in unstimulated Atg5fl/fl LysM-Cre- or Cre+BMM and
quantification of calpain relative to actin. (G) Calpain does not
colocalize with the autophagosomal marker LC3. Confocal microscopy
image of endogenous calpain I (Capn1) and immunofluorescently
(anti-GFP) labeled GFP-LC3 in BMM from GFP-LC3 knock-in mice. Scale
bar, 10 .mu.m. Graph, Pearson's colocalization coefficient (n=3)
between calpain I and LC3 (note the negative value). Data,
mean.+-.SE, *p<0.05, .dagger.p>0.05 (t test; n.gtoreq.3).
[0046] FIG. 1A. Degradative (canonical) vs. secretory autophagy.
Left: Canonical autophagy digests cytoplasmic proteins following
fusion with lysosomes. Right: Secretory autophagy is a form of
unconventional protein secretion.
[0047] FIG. 2A. Three models of the autophagy pathway, as explained
further hereinafter.
[0048] FIG. 3A. Role of autophagy in conventional and
unconventional secretion, as explained further hereinafter (from
Trends in Cell Biology.sup.1).
[0049] FIG. 4A. The well-developed paradigm of conventional protein
secretion through endoplasmic reticulum (ER), Golgi and post-Golgi
trafficking (right arrow) versus autophagy-dependent unconventional
secretion of cytosolic proteins (secretory autophagy) (left arrow)
, as explained further hereinafter.
[0050] FIG. 5A. IL-1.crclbar. and LC3 colocalize in
macrophages.
[0051] FIG. 6A. GRASP55 affects IL-1.beta. secretion under
autophagy inducing conditions (starvation). Nig, nigericin
(inflam-masome agonist).
[0052] FIG. 7A. GRASP affects canonical autophagy.
[0053] FIG. 8A. The model and proposed points of divergence between
degradative and secretory autophagy.
[0054] FIG. 9A. Relocalization of GRASP55 to WIPI2 (mAtg18)
profiles upon induction of autophagy or autosecretion.
[0055] FIG. 10A. Duolink (PLA) method for detecting direct
interactions in situ. See text for explanation (presence of red
dots indicates direct protein-protein interactions, in this case
between Tab8b and TBK-1 but not between TBK-1 and NDP52 as they
have an adaptor in between.
[0056] FIG. 11A. Role of mammalian Atg8 (LC3s and GABARAP5) in
autosecretion.
[0057] FIG. 12A. Loss of autophagic adaptor p62 reduces IL-1.beta.
autosecretion.
[0058] FIG. 12B. Table 1. A selection of proteins identified as
released from macrophages induced for secretory autophagy
[0059] FIGS. 13A and B. These figures show an assay which is used
for determining whether a compound of unknown activity is a
potential inhibitor or inducer of authophagy secretion. FIG. 13A
shows the two populations of cells, the first of which expresses
sequestome-like receptor in the cystosol which binds to galectin
and a galectin-GFP fusion protein and secretes the galectin when
the cell is exposed to an inducer of autophagy secretion. As shown
in FIG. 13B, the second population of cells, which expresses red
fluorescent protein, has surface galectin receptors which bind the
galectin-GFP secreted from the first population of cells and
concentrate and take up the galectin-GFP into the cell. In the
presence of an autophagy inducer, the red fluorescent cells also
emit green fluorescence which can be identified and quantitated. In
addition, in the presence of an inducer, the green fluorescence in
the first population of cells will often be reduced. In the
presence of an autophagy inhibitor, the red fluorescent cells
remain unaffected or increase (depending on the autophagy secretion
which occurs for the control) and the fluorescence in the first
population of green fluorescent cells will remain unaffected or
increase (also depending on the autophagy secretion which occurs
for the control. By comparing the fluorescence emitted by the cells
compared to the control, a determination can be made as to the
activity of a compound with unknown activity as an inducer or
inhibitor of autophagy secretion or a compound with no
activity.
DETAILED DESCRIPTION OF THE INVENTION
[0060] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," include
plural referents unless expressly and unequivocally limited to one
referent. Thus, for example, reference to "a compound" includes two
or more different compound. As used herein, the term "include" and
its grammatical variants are intended to be non-limiting, such that
recitation of items in a list is not to the exclusion of other like
items that can be substituted or other items that can be added to
the listed items.
[0061] The term "compound" or "agent", as used herein, unless
otherwise indicated, refers to any specific chemical compound
disclosed herein and includes tautomers, regioisomers, geometric
isomers as applicable , and also where applicable, optical isomers
(e.g. enantiomers) thereof, as well as pharmaceutically acceptable
salts thereof. Within its use in context, the term compound
generally refers to a single compound, but also may include other
compounds such as stereoisomers, regioisomers and/or optical
isomers (including racemic mixtures) as well as specific
enantiomers or enantiomerically enriched mixtures of disclosed
compounds as well as diastereomers and epimers, where applicable in
context. The term also refers, in context to prodrug forms of
compounds which have been modified to facilitate the administration
and delivery of compounds to a site of activity.
[0062] The term "patient" or "subject" is used throughout the
specification within context to describe an animal, generally a
mammal, including a domesticated mammal including a farm animal
(dog, cat, horse, cow, pig, sheep, goat, etc.) and preferably a
human, to whom treatment, including prophylactic treatment
(prophylaxis), with the methods and compositions according to the
present invention is provided. For treatment of those conditions or
disease states which are specific for a specific animal such as a
human patient, the term patient refers to that specific animal,
often a human.
[0063] The terms "effective" or "pharmaceutically effective" are
used herein, unless otherwise indicated, to describe an amount of a
compound or composition which, in context, is used to produce or
affect an intended result, usually the modulation of secretive or
degradative autophagy within the context of a particular treatment
or alternatively, the effect of a bioactive agent which is
coadministered with the secretive or degradative autophagy
modulator in the treatment of disease.
[0064] The terms "treat", "treating", and "treatment", etc., as
used herein, refer to any action providing a benefit to a patient
at risk for or afflicted by a secretive or degradative autophagy
mediated disease state or condition as otherwise described herein.
The benefit may be in curing the disease state or condition,
inhibition its progression, or ameliorating, lessening or
suppressing one or more symptom of a secretive autophagy mediated
disease state or condition. Treatment, as used herein, encompasses
both prophylactic and therapeutic treatment. Prophylactic, when
used, refers to "reducing the likelihood" of a disease state,
condition or symptom associated with same occurring.
[0065] The term "co-administration" or "combination therapy" is
used to describe a therapy in which at least two active compounds
in effective amounts are used to treat a M. tuberculosis infection
or other secretive or degradative autophagy-related disorder as
otherwise described herein, either at the same time or within
dosing or administration schedules defined further herein or
ascertainable by those of ordinary skill in the art. Although the
term co-administration preferably includes the administration of at
least two active compounds to the patient at the same time, it is
not necessary that the compounds be administered to the patient at
the same time, although effective amounts of the individual
compounds will be present in the patient at the same time. In
addition, in certain embodiments, co-administration will refer to
the fact that two or more compounds are administered at
significantly different times, but the effects of the two compounds
are present at the same time. Thus, the term co-administration
includes an administration in which one active agent (especially a
traditional anti-tuberculosis agent such as aminosalicylic acid,
isoniazid, ethionamide, myambutol, rifampin, rifabutin,
rifapentine, carpeomycin, cycloserine, or a pharmaceutically
acceptable salt) is administered at approximately the same time
(contemporaneously), or from about one to several minutes to about
24 hours or more than the other bioactive agent coadministered with
the autophagy modulator as described herein.
[0066] Autophagy modulators include, but are not limited to,
autophagy agonists (such as flubendazole, hexachlorophene,
propidium iodide, bepridil, clomiphene citrate (Z,E), GBR 12909,
propafenone, metixene, dipivefrin, fluvoxamine, dicyclomine,
dimethisoquin, ticlopidine, memantine, bromhexine,
norcyclobenzaprine, diperodon, nortriptyline or a mixture thereof
or their pharmaceutically acceptable salts) to the patient or
subject at risk for or suffering from a tuberculosis infection.
Additional agents which may be used in the present invention to
inhibit, prevent and/or treat tuberculosis include one or more of
benzethonium, niclosamide, monensin, bromperidol, levobunolol,
dehydroisoandosterone 3-acetate, sertraline, tamoxifen, reserpine,
hexachlorophene, dipyridamole, harmaline, prazosin, lidoflazine,
thiethylperazine, dextromethorphan, desipramine, mebendazole,
canrenone, chlorprothixene, maprotiline, homochlorcyclizine,
loperamide, nicardipine, dexfenfluramine, nilvadipine, dosulepin,
biperiden, denatonium, etomidate, toremifene, tomoxetine,
clorgyline, zotepine, beta-escin, tridihexethyl, ceftazidime,
methoxy-6-harmalan, melengestrol, albendazole, rimantadine,
chlorpromazine, pergolide, cloperastine, prednicarbate,
haloperidol, clotrimazole, nitrofural, iopanoic acid, naftopidil,
Methimazole, Trimeprazine, Ethoxyquin, Clocortolone, Doxycycline,
Pirlindole mesylate, Doxazosin, Deptropine, Nocodazole,
Scopolamine, Oxybenzone, Halcinonide, Oxybutynin, Miconazole,
Clomipramine, Cyproheptadine, Doxepin, Dyclonine, Salbutamol,
Flavoxate, Amoxapine, Fenofibrate, Pimethixene and mixtures
thereof.
[0067] Additional autophagy modulators are those which inhibit or
induce the secretion of autophagy modulators, including HMGB1,
IL-1.beta., IL-18, IL-33 or a galectin (e.g. galectin-1,
galectin-2, galectin-3, galectin-4, galectin-7, galectin-8,
galectin-9, galectin 10, galectin-12 and galectin-13). Inhibitors
or inducers of the secrecy of these autophagy modulators include
(for HMGB1) anti-HMGB1 antibody, ethyl pyruvate, a high mobility
group box (HMGB1) peptide or a biologically active fragment
thereof, an antibody to HMGB or an antigen-binding fragment
thereof, an HMGB small molecule antagonist, an antibody to TLR2 or
an antigen-binding fragment thereof, a soluble TLR2 polypeptide, an
antibody to RAGE or an antigen-binding fragment thereof, a soluble
RAGE polypeptide and a RAGE small molecule antagonist. In a
preferred embodiment, the HMGB1 antagonist is glycyrrhizin. In the
case of IL-1.beta., the inhibitor is an anti-IL-1.beta. humanized
monoclonal antibody or Anakinra. In the case of IL-18, the
antagonist is IL-18 binding protein (IL-18BP), an antibody against
IL-18 including a humanized antibody or a mutein or fused protein.
In the case of IL-33, the antagonist is ST2, an anti-ST2 antibody
or an antibody, including a humanized antibody which binds to
IL-33. In certain embodiments, in the case of galectin (especially
galectin-1 and galectin-3), the galectin antagonist/inhibitor is a
galactomannan based carbohydrate such as GM-CT-01, GR-MD-02,
GCS-100 (CAS No. 531508-98-2) (a pectin have multiple side-branches
containing the sugar .beta.-galactose), taloside (a C-2 epimer of
galactose) or a pectin (apple, rhubarb, okra, onion), among
others.
[0068] Any one or more of the inhibitors or inducers of the
secretion of these autophagy modulators find use in the treatment
of sepsis, inflammatory disease states and disorders and conditions
as otherwise described herein and cancer.
[0069] The term "Mycobacterium", is used to describe a genus of
Actinobacteria, given its own family, the Mycobacteriaceae. The
genus includes pathogens known to cause serious diseases in
mammals, including tuberculosis and leprosy. The Latin prefix
"myco" means both fungus and wax; its use here relates to the
"waxy" compounds in the cell wall. Mycobacteria are aerobic and
non-motile bacteria (except for the species Mycobacterium marinum
which has been shown to be motile within macrophages) that are
characteristically acid-alcohol fast. Mycobacteria do not contain
endospores or capsules, and are usually considered Gram-positive.
While mycobacteria do not seem to fit the Gram-positive category
from an empirical standpoint (i.e. they do not retain the crystal
violet stain), they are classified as an acid-fast Gram-positive
bacterium due to their lack of an outer cell membrane. All
Mycobacterium species share a characteristic cell wall, thicker
than in many other bacteria, which is hydrophobic, waxy, and rich
in mycolic acids/mycolates. The cell wall makes a substantial
contribution to the hardiness of this genus.
[0070] Many Mycobacterium species adapt readily to growth on very
simple substrates, using ammonia or amino acids as nitrogen sources
and glycerol as a carbon source in the presence of mineral salts.
Optimum growth temperatures vary widely according to the species
and range from 25.degree. C. to over50.degree. C.
[0071] Some species can be very difficult to culture (i.e. they are
fastidious), sometimes taking over two years to develop in culture.
Further, some species also have extremely long reproductive cycles:
M. leprae (leprosy), may take more than 20 days to proceed through
one division cycle (for comparison, some E. coli strains take only
20 minutes), making laboratory culture a slow process.
[0072] A natural division occurs between slowly--and
rapidly--growing species. Mycobacteria that form colonies clearly
visible to the naked eye within 7 days on subculture are termed
rapid growers, while those requiring longer periods are termed slow
growers. Mycobacteria are slightly curved or straight rods between
0.2-0.6 .mu.m wide by 1.0-10 .mu.m long.
[0073] A "Mycobacterium infection" includes, but is not limited to,
tuberculosis and atypical mycobacterial infections cause by a
Mycobacterium species other than M. tuberculosis. Atypical
mycobacterial infections include, but are not limited to,
abscesses, septic arthritis, and osteomyelitis (bone infection).
They can also infect the lungs, lymph nodes, gastrointestinal
tract, skin, and soft tissues. Atypical mycobacterial infections
can be caused by Mycobacterium avium-intracellulare, which
frequently affects AIDS patients and causes lung disease.
Mycobacterium marinum cause skin infections and is also responsible
for swimming pool granuloma. Mycobacterium ulcerans cause skin
infections. Mycobacterium kansasii causes lung disease.
[0074] A particularly important Mycobacterium species to the
present invention is M. tuberculosis. The term "Tuberculosis" or
"TB" is used to describe the infection caused by the infective
agent "Mycobacterium tuberculosis" or "M. tuberculosis", a tubercle
bacillus bacteria. Tuberculosis is a potentially fatal contagious
disease that can affect almost any part of the body but is most
frequently an infection of the lungs. It is caused by a bacterial
microorganism, the tubercle bacillus or Mycobacterium
tuberculosis.
[0075] Tuberculosis is primarily an infection of the lungs, but any
organ system is susceptible, so its manifestations may be varied.
Effective therapy and methods of control and prevention of
tuberculosis have been developed, but the disease remains a major
cause of mortality and morbidity throughout the world. The
treatment of tuberculosis has been complicated by the emergence of
drug-resistant organisms, including multiple-drug-resistant
tuberculosis, especially in those with HIV infection.
[0076] Mycobacterium tuberculosis, the causative agent of
tuberculosis, is transmitted by airborne droplet nuclei produced
when an individual with active disease coughs, speaks, or sneezes.
When inhaled, the droplet nuclei reach the alveoli of the lung. In
susceptible individuals the organisms may then multiply and spread
through lymphatics to the lymph nodes, and through the bloodstream
to other sites such as the lung apices, bone marrow, kidneys, and
meninges.
[0077] The development of acquired immunity in 2 to 10 weeks
results in a halt to bacterial multiplication. Lesions heal and the
individual remains asymptomatic. Such an individual is said to have
tuberculous infection without disease, and will show a positive
tuberculin test. The risk of developing active disease with
clinical symptoms and positive cultures for the tubercle bacillus
diminishes with time and may never occur, but is a lifelong risk.
Approximately 5% of individuals with tuberculous infection progress
to active disease. Progression occurs mainly in the first 2 years
after infection; household contacts and the newly infected are thus
at risk.
[0078] Many of the symptoms oftuberculosis, whether pulmonary
disease or extrapulmonary disease, are nonspecific. Fatigue or
tiredness, weight loss, fever, and loss of appetite may be present
for months. A fever of unknown origin may be the sole indication of
tuberculosis, or an individual may have an acute influenza-like
illness. Erythema nodosum, a skin lesion, is occasionally
associated with the disease.
[0079] The lung is the most common location for a focus of
infection to flare into active disease with the acceleration of the
growth of organisms. Infections in the lung are the primary focus
of the present invention. There may be complaints of cough, which
can produce sputum containing mucus, pus- and, rarely, blood.
Listening to the lungs may disclose rales or crackles and signs of
pleural effusion (the escape of fluid into the lungs) or
consolidation if present. In many, especially those with small
infiltration, the physical examination of the chest reveals no
abnormalities.
[0080] Miliary tuberculosis is a variant that results from the
blood-borne dissemination of a great number of organisms resulting
in the simultaneous seeding of many organ systems. The meninges,
liver, bone marrow, spleen, and genitourinary system are usually
involved. The term miliary refers to the lung lesions being the
size of millet seeds (about 0.08 in. or 2 mm) These lung lesions
are present bilaterally. Symptoms are variable.
[0081] Extrapulmonary tuberculosis is much less common than
pulmonary disease. However, in individuals with AIDS,
extrapulmonary tuberculosis predominates, particularly with lymph
node involvement, with some pulmonary impact. For example, fluid in
the lungs and lung lesions are other common manifestations of
tuberculosis in AIDS. The lung is the portal of entry, and an
extrapulmonary focus, seeded at the time of infection, breaks down
with disease occurring.
[0082] Development of renal tuberculosis can result in symptoms of
burning on urination, and blood and white cells in the urine; or
the individual may be asymptomatic. The symptoms of tuberculous
meningitis are nonspecific, with acute or chronic fever, headache,
irritability, and malaise.
[0083] A tuberculous pleural effusion can occur without obvious
lung involvement. Fever and chest pain upon breathing are common
symptoms. Bone and joint involvement results in pain and fever at
the joint site. The most common complaint is a chronic arthritis
usually localized to one joint. Osteomyelitis is also usually
present. Pericardial inflammation with fluid accumulation or
constriction of the heart chambers secondary to pericardial
scarring are two other forms of extrapulmonary disease.
[0084] At present, the principal methods of diagnosis for
pulmonarytuberculosis are the tuberculin skin test (an
intracutaneous injection of purified protein derivative tuberculin
is performed, and the injection site examined for reactivity),
sputum smear and culture, and the chest x-ray. Culture and biopsy
are important in making the diagnosis in extrapulmonary
disease.
[0085] A combination of two or more drugs is often used in the
initial traditional therapy of tuberculous disease. Drug
combinations are used to lessen the chance of drug-resistant
organisms surviving. The preferred treatment regimen for both
pulmonary and extrapulmonary tuberculosis is a 6-month regimen of
the antibiotics isoniazid, rifampin, and pyrazinamide given for 2
months, followed by isoniazid and rifampin for 4 months. Because of
the problem of drug-resistant cases, ethambutol can be included in
the initial regimen until the results of drug susceptibility
studies are known. Once treatment is started, improvement occurs in
almost all individuals. Any treatment failure or individual relapse
is usually due to drug-resistant organisms.
[0086] An "inflammatory disorder" "inflammatory disease state" or
"inflammatory condition" includes, but is not limited to, lung
diseases, hyperglycemic disorders including diabetes and disorders
resulting from insulin resistance, such as Type I and Type II
diabetes, as well as severe insulin resistance, hyperinsulinemia,
and dyslipidemia (e.g. hyperlipidemia (e.g., as expressed by obese
subjects), elevated low-density lipoprotein (LDL), depressed
high-density lipoprotein (HDL), and elevated triglycerides) and
insulin-resistant diabetes, such as Mendenhall's Syndrome, Werner
Syndrome, leprechaunism, and lipoatrophic diabetes, renal
disorders, such as acute and chronic renal insufficiency, end-stage
chronic renal failure, glomerulonephritis, interstitial nephritis,
pyelonephritis, glomerulosclerosis, e.g., Kimmelstiel-Wilson in
diabetic patients and kidney failure after kidney transplantation,
obesity, GH-deficiency, GH resistance, Turner's syndrome, Laron's
syndrome, short stature, increased fat mass-to-lean ratios,
immunodeficiencies including decreased CD4.sup.+ T cell counts and
decreased immune tolerance or chemotherapy-induced tissue damage,
bone marrow transplantation, diseases or insufficiencies of cardiac
structure or function such as heart dysfunctions and congestive
heart failure, neuronal, neurological, or neuromuscular disorders,
e.g., diseases of the central nervous system including Alzheimer's
disease, or Parkinson's disease or multiple sclerosis, and diseases
of the peripheral nervous system and musculature including
peripheral neuropathy, muscular dystrophy, or myotonic dystrophy,
and catabolic states, including those associated with wasting
caused by any condition, including, e.g., mental health condition
(e.g., anorexia nervosa), trauma or wounding or infection such as
with a bacterium or human virus such as HIV, wounds, skin
disorders, .sub.gut structure and function that need restoration,
and so forth.
[0087] "Inflammatory disorder" also includes a cancer and an
"infectious disease" as defined herein, as well as disorders of
bone or cartilage growth in children, including short stature, and
in children and adults disorders of cartilage and bone in children
and adults, including arthritis and osteoporosis. An
"inflammation-associated metabolic disorder" includes a combination
of two or more of the above disorders (e.g., osteoporosis that is a
sequela of a catabolic state). Specific disorders of particular
interest targeted for treatment herein are diabetes and obesity,
heart dysfunctions, kidney disorders, neurological disorders, bone
disorders, whole body growth disorders, and immunological
disorders.
[0088] In one embodiment, an "inflammatory disorder" includes
central obesity, dyslipidemia including particularly
hypertriglyceridemia, low HDL cholesterol, small dense LDL
particles and postpranial lipemia; glucose intolerance such as
impaired fasting glucose; insulin resistance and hypertension, and
diabetes. The term "diabetes" is used to describe diabetes mellitus
type I or type II. The present invention relates to a method for
improving renal function and symptoms, conditions and disease
states which occur secondary to impaired renal function in patients
or subjects with diabetes as otherwise described herein. It is
noted that in diabetes mellitus type I and II, renal function is
impaired from collagen deposits, and not from cysts in the other
disease states treated by the present invention.
[0089] A "neurodegenerative disorder" or "neuroinflammation"
includes, but is not limited to inflammatory disorders such as
Alzheimer's Dementia (AD), amyotrophic lateral sclerosis,
depression, epilepsy, Huntington's Disease, multiple sclerosis, the
neurological complications of AIDS, spinal cord injury, glaucoma
and Parkinson's disease.
[0090] The term "cancer" is used throughout the specification to
refer to the pathological process that results in the formation and
growth of a cancerous or malignant neoplasm, i.e., abnormal tissue
that grows by cellular proliferation, often more rapidly than
normal and continues to grow after the stimuli that initiated the
new growth cease. Cancers generally show partial or complete lack
of structural organization and functional coordination with the
normal tissue and most invade surrounding tissues, metastasize to
several sites, and are likely to recur after attempted removal and
to cause the death of the patient unless adequately treated. As
used herein, the term cancer is used to describe all cancerous
disease states applicable to treatment according to the present
invention and embraces or encompasses the pathological process
associated with all virtually all epithelial cancers, including
carcinomas, malignant hematogenous, ascitic and solid tumors.
Examples of cancers which may be treated using methods according to
the present invention include, without limitation, carcinomas
(e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular
carcinomas, and renal cell carcinomas), particularly those of the
bladder, bowel, breast, cervix, colon, esophagus, head, kidney,
liver, lung, neck, ovary, pancreas, prostate, and stomach;
leukemias; benign and malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; benign and malignant
melanomas; myeloproliferative diseases; sarcomas, particularly
Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,
myosarcomas, peripheral neuroepithelioma, and synovial sarcoma;
tumors of the central nervous system (e.g., gliomas, astrocytomas,
oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas,
ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell
tumors, meningiomas, meningeal sarcomas, neurofibromas, and
Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,
prostate cancer, cervical cancer, uterine cancer, lung cancer,
ovarian cancer, testicular cancer, thyroid cancer, astrocytoma,
esophageal cancer, pancreatic cancer, stomach cancer, liver cancer,
colon cancer, and melanoma); mixed types of neoplasias,
particularly carcinosarcoma and Hodgkin's disease; and tumors of
mixed origin, such as Wilms' tumor and teratocarcinomas. See, for
example, The Merck Manual of Diagnosis and Therapy, 17.sup.th ed.
(Whitehouse Station, N.J.: Merck Research Laboratories,
1999)973-74, 976, 986, 988, 991).
[0091] In addition to the treatment of ectopic cancers as described
above, the present invention also may be used preferably to treat
eutopic cancers such as choriocarcinoma, testicular
choriocarcinoma, non-seminomatous germ cell testicular cancer,
placental cancer (trophoblastic tumor)and embryonal cancer, among
others.
[0092] An "immune disorder" includes, but is not limited to, lupus,
multiple sclerosis, rheumatoid arthritis, psoriasis, Type I
diabetes, complications from organ transplants, xeno
transplantation, diabetes, cancer, asthma, atopic dermatitis,
autoimmune thyroid disorders, ulcerative colitis, Crohn's disease,
Alzheimer's disease and leukemia.
[0093] A "biomarker" is any gene or protein whose level of
expression in a biological sample is altered compared to that of a
pre-determined level. The pre-determined level can be a level found
in a biological sample from a normal or healthy subject. Biomarkers
include genes and proteins, and variants and fragments thereof.
Such biomarkers include DNA comprising the entire or partial
sequence of the nucleic acid sequence encoding the biomarker, or
the complement of such a sequence. The biomarker nucleic acids also
include RNA comprising the entire or partial sequence of any of the
nucleic acid sequences of interest. A biomarker protein is a
protein encoded by or corresponding to a DNA biomarker of the
invention. A biomarker protein comprises the entire or partial
amino acid sequence of any of the biomarker proteins or
polypeptides. Biomarkers can be detected, e.g. by nucleic acid
hybridization, antibody binding, activity assays, polymerase chain
reaction (PCR), S1 nuclease assay and gene chip.
[0094] A "control" as used herein may be a positive or negative
control as known in the art and can refer to a control cell,
tissue, sample, or subject. The control may, for example, be
examined at precisely or nearly the same time the test cell,
tissue, sample, or subject is examined. The control may also, for
example, be examined at a time distant from the time at which the
test cell, tissue, sample, or subject is examined, and the results
of the examination of the control may be recorded so that the
recorded results may be compared with results obtained by
examination of a test cell, tissue, sample, or subject. For
instance, as can be appreciated by a skilled artisan, a control may
comprise data from one or more control subjects that is stored in a
reference database. The control may be a subject who is similar to
the test subject (for instance, may be of the same gender, same
race, same general age and/or same general health) but who is known
to not have a fibrotic disease. As can be appreciated by a skilled
artisan, the methods of the invention can also be modified to
compare a test subject to a control subject who is similar to the
test subject (for instance, may be of the same gender, same race,
same general age and/or same general health) but who is known to
express symptoms of a disease. In this embodiment, a diagnosis of a
disease or staging of a disease can be made by determining whether
protein or gene expression levels as described herein are
statistically similar between the test and control subjects.
[0095] The terms "level" and/or "activity" as used herein further
refer to gene and protein expression levels or gene or protein
activity. For example, gene expression can be defined as the
utilization of the information contained in a gene by transcription
and translation leading to the production of a gene product.
[0096] In certain non-limiting embodiments, an increase or a
decrease in a subject or test sample of the level of measured
biomarkers (e.g. proteins or gene expression) as compared to a
comparable level of measured proteins or gene expression in a
control subject or sample can be an increase or decrease in the
magnitude of approximately .+-.5,000-10,000%, or approximately
.+-.2,500-5,000%, or approximately .+-.1,000-2,500%, or
approximately .+-.500-1,000%, or approximately .+-.250-500%, or
approximately .+-.100-250%, or approximately .+-.50-100%, or
approximately .+-.25-50%, or approximately .+-.10-25%, or
approximately .+-.10-20%, or approximately .+-.10-15%, or
approximately .+-.5-10%, or approximately .+-.1-5%, or
approximately .+-.0.5-1%, or approximately .+-.0.1-0.5%, or
approximately .+-.0.01-0.1%, or approximately .+-.0.001-0.01%, or
approximately .+-.0.0001-0.001%.
[0097] The values obtained from controls are reference values
representing a known health status and the values obtained from
test samples or subjects are reference values representing a known
disease status. The term "control", as used herein, can mean a
sample of preferably the same source (e.g. blood, serum, tissue
etc.) which is obtained from at least one healthy subject to be
compared to the sample to be analyzed. In order to receive
comparable results the control as well as the sample should be
obtained, handled and treated in the same way. In certain examples,
the number of healthy individuals used to obtain a control value
may be at least one, preferably at least two, more preferably at
least five, most preferably at least ten, in particular at least
twenty. However, the values may also be obtained from at least one
hundred, one thousand or ten thousand individuals.
[0098] A level and/or an activity and/or expression of a
translation product of a gene and/or of a fragment, or derivative,
or variant of said translation product, and/or the level or
activity of said translation product, and/or of a fragment, or
derivative, or variant thereof, can be detected using an
immunoassay, an activity assay, and/or a binding assay. These
assays can measure the amount of binding between said protein
molecule and an anti-protein antibody by the use of enzymatic,
chromodynamic, radioactive, magnetic, or luminescent labels which
are attached to either the anti-protein antibody or a secondary
antibody which binds the anti-protein antibody. In addition, other
high affinity ligands may be used. Immunoassays which can be used
include e.g. ELISAs, Western blots and other techniques known to
those of ordinary skill in the art (see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999 and Edwards R,
Immunodiagnostics: A Practical Approach, Oxford University Press,
Oxford; England, 1999). All these detection techniques may also be
employed in the format of microarrays, protein-arrays, antibody
microarrays, tissue microarrays, electronic biochip or protein-chip
based technologies (see Schena M., Microarray Biochip Technology,
Eaton Publishing, Natick, Mass., 2000).
[0099] Certain diagnostic and screening methods of the present
invention utilize an antibody, preferably, a monocolonal antibody,
capable of specifically binding to a protein as described herein or
active fragments thereof. The method of utilizing an antibody to
measure the levels of protein allows for non-invasive diagnosis of
the pathological states of kidney diseases. In a preferred
embodiment of the present invention, the antibody is human or is
humanized. The preferred antibodies may be used, for example, in
standard radioimmunoassays or enzyme-linked immunosorbent assays or
other assays which utilize antibodies for measurement of levels of
protein in sample. In a particular embodiment, the antibodies of
the present invention are used to detect and to measure the levels
of protein present in a renal cell or urine sample.
[0100] Humanized antibodies are antibodies, or antibody fragments,
that have the same binding specificity as a parent antibody, (i.e.,
typically of mouse origin) and increased human characteristics.
Humanized antibodies may be obtained, for example, by chain
shuffling or by using phage display technology. For example, a
polypeptide comprising a heavy or light chain variable domain of a
non-human antibody specific for a disease related protein is
combined with a repertoire of human complementary (light or heavy)
chain variable domains. Hybrid pairings specific for the antigen of
interest are selected. Human chains from the selected pairings may
then be combined with a repertoire of human complementary variable
domains (heavy or light) and humanized antibody polypeptide dimers
can be selected for binding specificity for an antigen. Techniques
described for generation of humanized antibodies that can be used
in the method of the present invention are disclosed in, for
example, U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761; and
5,693,762. Furthermore, techniques described for the production of
human antibodies in transgenic mice are described in, for example,
U.S. Pat. Nos. 5,545,806 and 5,569,825.
[0101] In order to identify small molecules and other agents useful
in the present methods for treating or preventing a renal disorder
by modulating the activity and expression of a disease -related
protein and biologically active fragments thereof can be used for
screening therapeutic compounds in any of a variety of screening
techniques. Fragments employed in such screening tests may be free
in solution, affixed to a solid support, borne on a cell surface,
or located intracellularly. The blocking or reduction of biological
activity or the formation of binding complexes between the
disease-related protein and the agent being tested can be measured
by methods available in the art.
[0102] Other techniques for drug screening which provide for a high
throughput screening of compounds having suitable binding affinity
to a protein, or to another target polypeptide useful in
modulating, regulating, or inhibiting the expression and/or
activity of a disease, are known in the art. For example,
microarrays carrying test compounds can be prepared, used, and
analyzed using methods available in the art. See, e.g., Shalon, D.
et al., 1995, International Publication No. WO95/35505,
Baldeschweiler et al., 1995, International Publication No.
WO95/251116; Brennan et al., 1995, U.S. Pat. No. 5,474,796; Heller
et al., 1997, U.S. Pat. No. 5,605,662.
[0103] Identifying small molecules that modulate protein activity
can also be conducted by various other screening techniques, which
can also serve to identify antibodies and other compounds that
interact with proteins identified herein and can be used as drugs
and therapeutics in the present methods. See, e.g., Enna et al.,
eds., 1998, Current Protocols in Pharmacology, John Wiley &
Sons, Inc., New York N.Y. Assays will typically provide for
detectable signals associated with the binding of the compound to a
protein or cellular target. Binding can be detected by, for
example, fluorophores, enzyme conjugates, and other detectable
labels well known in the art. The results may be qualitative or
quantitative.
[0104] For screening the compounds for specific binding, various
immunoassays may be employed for detecting, for example, human or
primate antibodies bound to the cells. Thus, one may use labeled
anti-hlg, e.g., anti-hIgM, hIgG or combinations thereof to detect
specifically bound human antibody. Various labels can be used such
as radioisotopes, enzymes, fluorescers, chemiluminescers,
particles, etc. There are numerous commercially available kits
providing labeled anti-hlg, which may be employed in accordance
with the manufacturer's protocol.
[0105] In one embodiment, a kit can comprise: (a) at least one
reagent which is selected from the group consisting of (i) reagents
that detect a transcription product of the gene coding for a
protein marker as described herein (ii) reagents that detect a
translation product of the gene coding for proteins, and/or
reagents that detect a fragment or derivative or variant of said
transcription or translation product; (b) optionally, one or more
types of cells, including engineered cells in which cellular assays
are to be conducted; (c) instructions for diagnosing, or
prognosticating a disease, or determining the propensity or
predisposition of a subject to develop such a disease or of
monitoring the effect of a treatment by determining a level, or an
activity, or both said level and said activity, and/or expression
of said transcription product and/or said translation product
and/or of fragments, derivatives or variants of the foregoing, in a
sample obtained from said subject; and comparing said level and/or
said activity and/or expression of said transcription product
and/or said translation product and/or fragments, derivatives or
variants thereof to a reference value representing a known disease
status (patient) and/or to a reference value representing a known
health status (control) and/or to a reference value; and analyzing
whether said level and/or said activity and/or expression is varied
compared to a reference value representing a known health status,
and/or is similar or equal to a reference value representing a
known disease status or a reference value; and diagnosing or
prognosticating a disease, or determining the propensity or
predisposition of said subject to develop such a disease, wherein a
varied or altered level, expression or activity, or both said level
and said activity, of said transcription product and/or said
translation product and/or said fragments, derivatives or variants
thereof compared to a reference value representing a known health
status (control) and/or wherein a level, or activity, or both said
level and said activity, of said transcription product and/or said
translation product and/or said fragments, derivatives or variants
thereof is similar or equal to a reference value and/or to a
reference value representing a known disease stage, indicates a
diagnosis or prognosis of a disease, or an increased propensity or
predisposition of developing such a disease, a high risk of
developing signs and symptoms of a disease.
[0106] Reagents that selectively detect a transcription product
and/or a translation product of the gene coding for proteins can be
sequences of various length, fragments of sequences, antibodies,
aptamers, siRNA, microRNA, and ribozymes. Such reagents may be used
also to detect fragments, derivatives or variants thereof.
[0107] Compounds used in the methods of treatment of the present
invention may be used in pharmaceutical compositions having
biological/pharmacological activity for the treatment of, for
example, Mycobacterial infections, including a number of other
conditions and/or disease states which may appear or occur
secondary to the bacterial infection. These compositions comprise
an effective amount of any one or more of the compounds disclosed
hereinabove, optionally in combination with a pharmaceutically
acceptable additive, carrier or excipient. Compounds used in the
methods of treatment of the present invention may also be used as
intermediates in the synthesis of compounds exhibiting biological
activity as well as standards for determining the biological
activity of the present compounds as well as other biologically
active compounds.
[0108] The compounds used in the methods of treatment of the
present invention may be formulated in a conventional manner using
one or more pharmaceutically acceptable carriers. Pharmaceutically
acceptable carriers that may be used in these pharmaceutical
compositions include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as prolamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0109] The compounds used in the methods of treatment of the
present invention may be administered orally, parenterally, by
inhalation spray, topically, rectally, nasally, buccally, vaginally
or via an implanted reservoir. The term "parenteral" as used herein
includes subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally, or intravenously. Preferred routes of
administration include oral administration and pulmonary
administration (by inhaler/inhalation spreay).
[0110] Sterile injectable forms of the compounds used in the
methods of treatment of the invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example
as a solution in 1, 3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as Ph. Helv or
similar alcohol.
[0111] The pharmaceutical compositions used in the methods of
treatment of this invention may be orally administered in any
orally acceptable dosage form including, but not limited to,
capsules, tablets, aqueous suspensions or solutions. In the case of
tablets for oral use, carriers which are commonly used include
lactose and corn starch. Lubricating agents, such as magnesium
stearate, are also typically added. For oral administration in a
capsule form, useful diluents include lactose and dried corn
starch. When aqueous suspensions are required for oral use, the
active ingredient is combined with emulsifying and suspending
agents. If desired, certain sweetening, flavoring or coloring
agents may also be added.
[0112] Alternatively, the pharmaceutical compositions used in the
methods of treatment of this invention may be administered in the
form of suppositories for rectal administration. These can be
prepared by mixing the agent with a suitable non-irritating
excipient which is solid at room temperature but liquid at rectal
temperature and therefore will melt in the rectum to release the
drug. Such materials include cocoa butter, beeswax and polyethylene
glycols.
[0113] The pharmaceutical compounds used in the methods of
treatment of this invention may also be administered topically,
especially when the target of treatment includes areas or organs
readily accessible by topical application. Suitable topical
formulations are readily prepared for each of these areas or
organs.
[0114] Topical application also can be effected in a rectal
suppository formulation (see above) or in a suitable enema
formulation. Topically-transdermal patches may also be used.
[0115] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0116] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with or without a preservative such
as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
pharmaceutical compositions may be formulated in an ointment such
as petrolatum.
[0117] The pharmaceutical compositions used in the methods of
treatment of this invention may also be administered by nasal
aerosol or by inhalation. Such compositions are prepared according
to techniques well-known in the art of pharmaceutical formulation
and may be prepared as solutions in saline, employing benzyl
alcohol or other suitable preservatives, absorption promoters to
enhance bioavailability, fluorocarbons, and/or other conventional
solubilizing or dispersing agents.
[0118] The amount of compounds used in the methods of treatment of
the instant invention that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated, the particular mode of administration.
Preferably, the compositions should be formulated so that a
therapeutically effective dosage of between about 1 and 25 mg/kg,
about 5 to about 15 mg/kg of patient/day of the novel compound can
be administered to a patient receiving these compositions.
Preferably, pharmaceutical compositions in dosage form according to
the present invention comprise a therapeuticially effective amount
of at least 25 mg of isotopically labeled compound, at least 50 mg
of isotopically labeled compound, at least 60 mg of isotopically
labeled compound, at least 75 mg of isotopically labeled compound,
at least 100 mg of isotopically labeled, at least 150 mg of
isotopically labeled compound, at least 200 mg of isotopically
labeled compound, at least 250 mg of isotopically labeled compound,
at least 300 mg of isotopically labeled compound, about 350 mg of
isotopically labeled compound, about 400 mg of isotopically labeled
compound, about 500 mg of isotopically labeled compound, about 750
mg of isotopically labeled compound, about 1 g (1000mg) of
isotopically labeled compound, alone or in combination with a
therapeutically effective amount of at least one additional
anti-tuberculosis agent. Exemplary additional anti-tuberculosis
agents which may be used in pharmaceutical compositions include one
or more of aminosalicyclic acid/aminosalicylate sodium, capreomycin
sulfate, clofazimine, cycloserine, ethambutol hydrochloride
(myambutol), kanamycin sulfate, pyrazinamide, rifabutin, rifampin,
rifapentine, streptomycin sulfate, gatifloxacin and mixtures
thereof, all in therapeutically effective amounts.
[0119] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease or condition being treated.
[0120] Administration of the active compound may range from
continuous (intravenous drip) to several oral or inhalation
(intratracheal) administrations per day (for example, B.I.D. or
Q.I.D.) and may include oral, pulmonary, topical, parenteral,
intramuscular, intravenous, sub-cutaneous, transdermal (which may
include a penetration enhancement agent), buccal and suppository
administration, among other routes of administration. Enteric
coated oral tablets may also be used to enhance bioavailability of
the compounds from an oral route of administration. The most
effective dosage form will depend upon the pharmacokinetics of the
particular agent chosen as well as the severity of disease in the
patient. Oral dosage forms are particularly preferred, because of
ease of administration and prospective favorable patient
compliance.
[0121] To prepare the pharmaceutical compositions according to the
present invention, a therapeutically effective amount of one or
more of the compounds according to the present invention is
preferably intimately admixed with a pharmaceutically acceptable
carrier according to conventional pharmaceutical compounding
techniques to produce a dose. A carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, e.g., oral or parenteral. In preparing
pharmaceutical compositions in oral dosage form, any of the usual
pharmaceutical media may be used. Thus, for liquid oral
preparations such as suspensions, elixirs and solutions, suitable
carriers and additives including water, glycols, oils, alcohols,
flavoring agents, preservatives, colouring agents and the like may
be used. For solid oral preparations such as powders, tablets,
capsules, and for solid preparations such as suppositories,
suitable carriers and additives including starches, sugar carriers,
such as dextrose, mannitol, lactose and related carriers, diluents,
granulating agents, lubricants, binders, disintegrating agents
and'the like may be used. If desired, the tablets or capsules may
be enteric-coated or sustained release by standard techniques. The
use of these dosage forms may significantly the bioavailability of
the compounds in the patient.
[0122] For parenteral formulations, the carrier will usually
comprise sterile water or aqueous sodium chloride solution, though
other ingredients, including those which aid dispersion, also may
be included. Of course, where sterile water is to be used and
maintained as sterile, the compositions and carriers must also be
sterilized. Injectable suspensions may also be prepared, in which
case appropriate liquid carriers, suspending agents and the like
may be employed.
[0123] Liposomal suspensions (including liposomes targeted to viral
antigens) may also be prepared by conventional methods to produce
pharmaceutically acceptable carriers. This may be appropriate for
the delivery of free nucleosides, acyl/alkyl nucleosides or
phosphate ester pro-drug forms of the nucleoside compounds
according to the present invention.
[0124] The present invention also relates to the use of
pharmaceutical compositions in an oral dosage form comprising
therapeutically effective amounts of isotopically labeled compound
according to the present invention, optionally in combination with
a pharmaceutically acceptable carrier, additive or excipient.
Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, sachets,
capsules or tablets. Thickeners, diluents, flavorings, dispersing
aids, emulsifiers or binders may be desirable.
[0125] In preferred aspects of the invention, especially for
treatment of M. tuberculosis infections, the compound is
administered to the lungs of the subject via pulmonary
administration, including intratracheal administration. The
pharmaceutical composition of the invention for pulmonary
administration is usually used as an inhalant. The composition can
be formed into dry powder inhalants, inhalant suspensions, inhalant
solutions, encapsulated inhalants and like known forms of
inhalants. Such forms of inhalants can be prepared by filling the
pharmaceutical composition of the invention into an appropriate
inhaler such as a metered-dose inhaler, dry powder inhaler,
atomizer bottle, nebulizer etc. before use. Of the above forms of
inhalants, powder inhalants may be preferable.
[0126] When the pharmaceutical composition used in the methods of
treatment of the invention is used in the form of a powder, the
mean particle diameter of the powder is not especially limited but,
in view of the residence of the particles in the lungs, is
preferably that the particles fall within the range of about 0.1 to
20 .mu.m, and particularly about 1 to 5 .mu.m. Although the
particle size distribution of the powder pharmaceutical composition
of the invention is not particularly limited, it is preferable that
particles having a size of about 25 .mu.m or more account for not
more than about 5% of the particles, and preferably, 1% or less to
maximize delivery into the lungs of the subject.
[0127] The pharmaceutical composition in the form of a powder can
be produced by, for example, using the drying-micronization method,
the spray drying method and standard pharmaceutical methodology
well known in the art.
[0128] By way of example without limitation, according to the
drying-pulverization method, the pharmaceutical composition in the
form of a powder can be prepared by drying an aqueous solution (or
aqueous dispersion) containing the compound or mixtures with other
active agents thereof and excipients which provide for immediate
release in pulmonary tissue and microparticulating the dried
product. Stated more specifically, after dissolving (or dispersing)
a pharmaceutically acceptable carrier, additive or excipient in an
aqueous medium, compounds according to the present invention in
effective amounts are added and dissolved (or dispersed) by
stirring using a homogenizer, etc. to give an aqueous solution (or
aqueous dispersion). The aqueous medium may be water alone or a
mixture of water and a lower alcohol. Examples of usable lower
alcohols include methanol, ethanol, 1-propanol, 2-propanol and like
water-miscible alcohols. Ethanol is particularly preferable. After
the obtained aqueous solution (or aqueous dispersion) is dried by
blower, lyophilization, etc., the resulting product is pulverized
or microparticulated into fine particles using jet mills, ball
mills or like devices to give a powder having the above mean
particle diameter. If necessary, additives as mentioned above may
be added in any of the above steps.
[0129] According to the spray-drying method, the pharmaceutical
composition in the form of a powder of the invention can be
prepared, for example, by spray-drying an aqueous solution (or
aqueous dispersion) containing isoniazid, urea or mixtures thereof
and excipients, addtives or carriers for microparticulation. The
aqueous solution (or aqueous dispersion) can be prepared following
the procedure of the above drying-micronization method. The
spray-drying process can be performed using a known method, thereby
giving a powdery pharmaceutical composition in the form of globular
particles with the above-mentioned mean particle diameter.
[0130] The inhalant suspensions, inhalant solutions, encapsulated
inhalants, etc. can also be prepared using the pharmaceutical
composition in the form of a powder produced by the
drying-micronization method, the spray-drying method and the like,
or by using a carrier, additive or excipient and isoniazid, urea or
mixtures thereof that can be administered via the lungs, according
to known preparation methods.
[0131] Furthermore, the inhalant comprising the pharmaceutical
composition of the invention is preferably used as an aerosol. The
aerosol can be prepared, for example, by filling the pharmaceutical
composition of the invention and a propellant into an aerosol
container. If necessary, dispersants, solvents and the like may be
added. The aerosols may be prepared as 2-phase systems, 3-phase
systems and diaphragm systems (double containers). The aerosol can
be used in any form of a powder, suspension, solution or the
like.
[0132] Examples of usable propellants include liquefied gas
propellants, compressed gases and the like. Usable liquefied gas
propellants include, for example, fluorinated hydrocarbons (e.g.,
CFC substitutes such as HCFC-22, HCFC-123, HFC-134a, HFC-227 and
the like), liquefied petroleum, dimethyl ether and the like. Usable
compressed gases include, for example, soluble gases (e.g., carbon
dioxide, nitric oxide), insoluble gases (e.g., nitrogen) and the
like.
[0133] The dispersant and solvent may be suitably selected from the
additives mentioned above. The aerosol can be prepared, for
example, by a known 2-step method comprising the step of preparing
the composition of the invention and the step of filling and
sealing the composition and propellant into the aerosol
container.
[0134] As a preferred embodiment of the aerosol according to the
invention, the following aerosol can be mentioned: Examples of the
compounds to be used include isotopically labeled compound alone or
in mixtures with other compounds according to the present invention
or with other anti-Mycobacterial agents. As propellants,
fluorinated hydrocarbons such as HFC-134a, HFC-227 and like CFC
substitutes are preferable. Examples of usable solvents include
water, ethanol, 2-propanol and the like. Water and ethanol are
particularly preferable. In particular, a weight ratio of water to
ethanol in the range of about 0:1 to 10:1 may be used.
[0135] The aerosol of the invention contains excipient in an amount
ranging from about 0.01 to about 10.sup.4 wt. % (preferably about
0.1 to 10.sup.3 wt. %), propellant in an amount of about 10.sup.2
to 10.sup.7 wt. % (preferably about 10.sup.3 to 10.sup.6 wt. %),
solvent in an amount of about 0 to 10.sup.6 wt. % (preferably about
10 to 10.sup.5 wt. %), and dispersant in an amount of 0 to 10
.sup.3 wt. % (preferably about 0.01 to 10.sup.2 wt. %), relative to
the weight of compound according to the present invention which is
included in the final composition.
[0136] The pharmaceutical compositions of the invention are safe
and effective for use in the therapeutic methods according to the
present invention. Although the dosage of the compounds used in the
methods of treatment of the invention may vary depending on the
type of active substance administered (isoniazid, ethionamide,
propionamide and optional additional anti-tuberculosis agents) as
well as the nature (size, weight, etc.) of the subject to be
diagnosed, the composition is administered in an amount effective
for allowing the pharmacologically active substance to be cleaved
to cleavage products to be measured. For example, the composition
is preferably administered such that the active ingredient
(isotopically labeled compound) can be given to a human adult in a
dose of at least about 25 mg, at least about 50 mg, at least about
60 mg, at least about 75 mg., at least about 100 mg, at least about
150 mg, at least about 200 mg, at least about 250 mg, at least
about 300 mg, at least about 350 mg, at least about 400 mg, at
least about 500 mg, at least about 750 mg, at least about 1000 mg,
and given in a single dose, including sustained or controlled
release dosages once daily.
[0137] The form of the pharmaceutical composition of the invention
such as a powder, solution, suspension etc. may be suitably
selected according to the type of substance to be administered.
[0138] As an administration route, direct inhalation via the mouth
using an inhaler is usually administered into the airways and in
particular, directly to pulmonary tissue, the active substance
contained therein produces immediate effects. Furthermore, the
composition is formulated as an immediate release product so that
cleavage and analysis can begin soon after administration.
Autophagy Overview
[0139] During the canonical presentation of autophagy (FIG. 1A),
cells digest their cytoplasmic components as an endogenous source
of nutrients and energy at times of starvation or as a mechanism
for clearance of disused organelles and toxic intracellular
aggregates (2A,11A). The canonical autophagy pathway, also referred
to as macroautophagy, has been worked out (FIG. 2A) and includes a
set of autophagy-specific factors (termed Atgs) responsive to
upstream signaling by TOR, AMPK and other inputs (2A,44A,45A). The
Atg factors are responsible for the execution of autophagy and the
formation of the specialized double membrane organelles, termed
autophagosomes. The Atg factors include, among others, the
Atg5-Atg12/Atg16 complex. This complex acts as an equivalent to E3
ligases and regulates C-terminal lipidation of Atg8 (or its
mammalian equivalent LC3) with phosphatidylethanolamine (PE)
essential for autophagosomal membrane growth (2A). Atg8-PE may have
a role in membrane tethering and fusion (46A,47A), albeit this has
been disputed and instead SNAREs, the typical regulators of
membrane fusion have been invoked (48A). The membrane sources for
the formation of autophagosomes primarily originate from transient
domains of the ER, termed omegasomes (FIG. 2A), with potential (but
heavily debated) contributions of other compartments such as the
plasma membrane, mitochondria and Golgi. During degradative stages,
autophagosomes fuse with lysosomes to form autolysosomes where the
captured cargo is degraded.
[0140] FIG. 2A presents three autophagy models ("ER": endoplasmic
reticulum; "PM": plasma membrane; "MT": mitochondria). These models
are described below. [0141] Model 1. The central membranous
structure, omegasome (Q-some) is derived from the ER (ER cradle
model), and is believed to be an early precursor of autophagic
isolation membranes (IM) or phagophores. Phagophore crescents close
to form double membrane autophagosomes that fuse with lysosomal
intermediates to form the degradative organelles, autolysosomes.
[0142] Model 2. Mitochondria may contribute membrane or
phosphatidylinositol (PE) of relevance for LC3 (A, B and C; and
other Atg8 paralogs, GABARAP and GATE-16) C-terminal lipidation
into the LC3-II, autophagic membrane-associated form. Mitochondria
may also be a source of reactive oxygen species that inactivate
ATG4, an LC3delipidating enzyme. Mitochondria are also one of the
major target substrates for autophagic elimination. [0143] Model 3.
Plasma membrane Atg16L1-positive (initially LC3-negative) vesicles
may contribute to autophagic membrane growth. Factors in the left
upper corner represent upstream signaling systems (AMPK, mammalian
TOR -mTOR, Ral) controlling induction of autophagy in response to
nutritional and cellular energetics signals. Beclin 1 (BEC-1) and
class III phosphatidylinositol 3-kinase hVPS34 cooperate in control
of phosphatidylinositol 3-phosphate (PI3P) structures that start
with omegasome, identifiable by the marker DFCP-1 for which a
functional role in autophagy is yet to be established. NBR1 and p62
(also known as sequestosome 1) are autophagic adaptor proteins that
capture cargo and interact with LC3; p62 is also present very early
at the sites leading to omegasome formation, and is furthermore
found in complexes with mTOR that sense amino acid starvation (not
shown). Ambra and Atg14L are additional factors interacting with
Beclin 1 complexes that are responsible for the early
autophagosomal pathway. The lipid kinase hVPS34 interacts with
UVRAG and additional factors (not shown) to control autophagosomal
maturation into autolysosomes. Several tethering systems along the
different stages of the early secretory pathway and the Golgi
apparatus (TRAPP, COG, GRASP) influence the formation, expansion
(contributed by the only Atg integral membrane protein Atg9) and
maturation of autophagosomal organelles.
[0144] As stated above, autophagy has been assumed to represent
primarily a catabolic, lysosomal degradative pathway. The notion of
autophagy as a purely degradative pathway was recently challenged
by the emergence of reports of the secretory function of autophagy
by three independent groups on the secretion of Acb1 in yeast
(25A,26A,32A) and IL-1.beta. secretion in mammalian cells
(17A,27A). These new developments assign to autophagy a
non-degradative function manifested as unconventional protein
secretion (FIG. 3A). Furthermore, it has become apparent that
autophagy even more broadly intersects with protein trafficking to
include effects on the constitutive biosynthetic pathway (23A),
regulated exocytosis (19A), and alternative sorting of integral
membrane proteins to the plasma membrane (28A). In this proposal,
we will delineate the machinery and mechanisms of
autophagy-dependent unconventional protein secretion in mammalian
cells.
[0145] FIG. 3A illustrates the role of autophagy in conventional
and unconventional secretion. (From Trends in Cell Biology; 1A) 1.
Regulated secretion: secretory lysosomes, granules and other
organelles, partially derived from or affected by the post-Golgi
vesicles. ATG symbolizes that Atg factors affect regulated
secretion, delivering various biologically active cargo as
indicated. Others include non-proteinaceous cargo (e.g. ATP
secreted from drug-treated cancer cells), provided that they are
competent to undergo autophagy, with inflammatory consequences and
clearance of transplanted tumors. 2. Autophagy affects constitutive
secretion (e.g. IL-6, IL-8) via a compartment intermixed with
autophagic organelles, called TASCC (TOR-autophagy spatial coupling
compartment). 3. A subset of unconventional secretion processes
depend on autophagy (autophagy-based unconventional secretion;
secretory autophagy) for secretion of proinflammatory factors
IL-1.beta. and HMGB 1 in mammalian cells and Acb1 in yeast. GRASP
(note that GRASP is normally localized to the Golgi and that it
affects early stages of autophagy) is required for autophagy-based
unconventional secretion (secretory autophagy). CUPS, a yeast
structure implicated in autophagy-based unconventional secretion,
may be equivalent to omegasomes in mammalian cells. In addition,
autophagy plays a role in unconventional trafficking of the ER-form
of CFTR (cystic fibrosis transmembrane conductance regulator) to
the apical aspect of the plasma membrane, bypassing the Golgi and
rescuing function of mutant CFTR responsible for cystic fibrosis.
GRASP plays a role in autophagy-dependent unconventional traffickin
of CFTR and in unconventional trafficking of a-integrin to the
basolateral plasma membrane in Drosophila (a role for autophagy has
not been established as yet for .alpha.-integrin trafficking).
[0146] Our present view of protein secretion from eukaryotic cells
is dominated by an established, classical paradigm of conventional
secretion (FIG. 4A). FIG. 4A shows the well-developed paradigm of
conventional protein secretion through endoplasmic reticulum (ER),
Golgi and post-Golgi trafficking (right arrow) versus
autophagy-dependent unconventional secretion of cytosolic proteins
(secretory autophagy). The proteins destined for conventional
secretion enter ER via signal peptides, whereas cytosolic proteins
destined for secretory autophagy are sequestered into
autophagosomes to be exported from the cell.
The Secretory Role of Autophagy
[0147] Induction of autophagy promotes inflammasome-dependent
IL-1.beta. secretion. Whereas it has been found that basal
autophagy reduces extracellular release of the major
proinflammatory cytokine IL-1.beta. (50A,51A), we detected the
opposite when autophagy was induced in primary murine bone
marrow-derived macrophages (BMM). Stimulation of autophagy by
starvation strongly enhanced IL-1.beta. secretion in response to
the conventional NLRP3 (NALP3) inflammasome agonist nigericin. This
effect was also seen in Western blots of caspase 1 and mature
IL-1.beta. of culture supernatants from cells grown in the absence
of serum, as conventionally done when assessing IL-1.beta.
secretion by immunoblotting (52A). A reduced secretion in BMMs from
Atg5Fl/Fl LyzM-Cre+ mice, compared to BMMs from their Cre-
littermates, was accompanied and contrasted by the higher level of
cell-associated pro-IL-1.beta. in Cre- vs Cre+ BMMs. The effects of
induced autophagy on secretion of inflammasome substrates described
above were not limited to IL-1.beta., since secretion of another
inflammasome-dependent cytokine from the IL-1 family, IL-18
(IL-1F4), was enhanced when autophagy was induced. The increased
secretion of IL-1.beta. was not due to increased cell death or
non-specific membrane permeability as LDH release showed a kinetic
lag behind release of IL-1.beta. whether the inflammasome agonist
used was nigericin or silica.
[0148] IL-1.beta. and autophagic protein LC3 colocalize in the
cytoplasm. We considered a model in which autophagy, as a process
that can translocate cytosolic proteins and other targets (en masse
or specific components) from the cytosol to the inside of vesicular
compartments, brought IL-1.beta. into the lumen of autophagic
vacuoles followed by exocytosis. When we examined IL-1.beta. and
the key marker of autophagosomes, LC3, by immunofluorescence
confocal microscopy, LC3 and IL-1.beta. colocalized and displayed
major similarities in the overall intracellular organellar
distribution (FIG. 5A). The overlap between IL-1.beta. and LC3
remained detectable when cells were treated with nigericin. These
observations indicate that autophagic organelles and IL-.beta.
intersect.
[0149] Rab8a and exocyst components, regulator of polarized sorting
to plasma membrane, co-localize with IL-1.beta. and LC3 and control
IL-1.beta. secretion. We also addressed the features of the
compartment where LC3 and IL-1.beta. colocalized. We observed an
overlap between the LC3+ IL-1.beta.+ profiles and Rab8a (FIG. 5A).
Rab8a is a regulator of polarized membrane trafficking,
constitutive biosynthetic trafficking, and plasma membrane fusion
of insulin-responsive 53 and other vesicular carriers (54A-57A).
Rab8a also co-localized with LC3 and IL-1.beta. in cells exposed to
nigericin. Rab8a was required for enhanced IL-1.beta. secretion
caused by starvation-induced autophagy and inflammasome activation
with nigericin, since siRNA knockdown of Rab8a diminished
IL-1.beta. secretion from BMMs under these conditions. Rab8a
knockdown did not change pro-IL-1.beta. mRNA levels. Overexpression
of the Rab8 a dominant negative mutant (S22N) inhibited IL-1.beta.
secretion from RAW264.7 cells, employed in that experiment based on
their high efficiency of transfection (verified by flow cytometry
of GFP-Rab8a for equal yields). Additionally, LC3+IL-1.beta.+
profiles were positive for subunits of the exocyst complex. The
exocyst has been shown to cooperate with Rab8a in polarized plasma
membrane delivery of vesicular carriers (57A,58A). The presence of
exocyst components on IL-1.beta.+ autophagic organelles was also in
keeping with a recent report implicating exocyst in autophagy 59.
In summary, these experiments indicate that systems involved in
vectorial vesicular transport to the plasma membrane participate in
autophagy-based unconventional secretion and that Rab8a is required
for efficient autophagy-dependent secretion of IL-1.beta..
[0150] GRASP55 controls secretion of IL-1.beta. Two studies in
yeast (24A,26A) have reported that autophagic machinery is required
for unconventional secretion of the protein Acb1, and that this
pathway depends on the yeast equivalent of mammalian
Golgi-associated GRASPs (Golgi reassembly stacking proteins)
(30A,60A). Mammalian cells encode two GRASP paralogs, GRASP55
(GORASP2) and GRASP65 (GORASP1). We first tested whether any of the
mammalian GRASPs were required for IL-1.beta. secretion. We could
not obtain a good knockdown of GRASP65 (GORASP1) and thus could not
evaluate its involvement. However, a knockdown of GRASP55
diminished IL-1.beta. secretion (FIG. 6A). A similar downregulation
of IL-18 secretion was observed with GRASP55 knockdown. We next
tested whether GRASP55 showed any detectable response to
inflammasome stimulation. GRASP55 in resting cells is mostly
localized aligned within the perinuclear Golgi. However, a fraction
of it dispersed upon treatment of cells with the inflammasome
agonist nigericin and was found juxtaposed and partially
overlapping with LC3 profiles. Thus, GRASP55 responds to
inflammasome stimulation and is important for secretion of
IL-1.beta. and IL-18.
[0151] GRASP55 controls autophagy initiation. In addition to being
required for IL-1.beta. secretion, GRASP55 showed functional
effects on LC3 and autophagy, tested by employing two core assays
(61A): LC3-II lipidation and the RFP-GFP-LC3 tandem probe. When
GRASP55 was knocked down, autophagy initiation was negatively
affected, as LC3-II levels were lower in both untreated and
bafilomycin A1-treated cells. A partial down regulation of GRASP65
(to the extent that it could be achieved in BMMs) suggested a minor
synergistic effect with GRASP55 on LC3-II levels upon induction of
autophagy. Knocking down GRASP55 reduced the total number of
autophagic puncta, and selectively reduced the formation of
autophagosomes but not their maturation (FIG. 7A). This was
apparent from the data obtained with the RFP-GFP-LC3 probe
following published methods (62A), which showed reduced GFP+RFP+
LC3 profiles (early autophagosome) and equal number of GFP-RFP+LC3
profiles (mature autophagic organelles) in cells knocked down for
GRASP55 (FIG. 7A). Thus, mammalian GRASP55, a paralog of GRASP from
lower organisms that has thus far been the sole definitive
molecular factor associated with unconventional secretion (29A),
displays important and previously unappreciated positive regulatory
effects on autophagy induction. These findings strengthen the
connections between autophagy and GRASPs in general, and
specifically demonstrate the role of mammalian GRASP55 both in
autophagy initiation and in the secretion of leaderless
inflammasome substrates such as IL-1.beta. and IL-18.
[0152] Autophagy-based unconventional secretion is not limited to
proteolytically processed inflammasome substrates. We tested
whether induction of autophagy affected other proteins not
connected to proteolytic processing in the inflammasome, such as
HMGB1 (high mobility group box 1 protein). HMGB1 is a major
pro-inflammatory alarmin or DAMP (damage-associated molecular
pattern) normally present in the nucleus (34A). This
chromatin-associated nuclear protein (with additional intracellular
and extracellular signaling roles), upon exposure to inputs
including those that induce autophagy (63A,64A), undergoes a
complex set of biochemical and localization changes. In the
process, it first translocates from the nucleus into the cytoplasm
and then is released from the cytoplasm to act in tissue remodeling
signaling (when acting alone) or as an inflammatory mediator (when
combined with bacterial agonists or other alarmins such as
IL-1.beta.). When tested, starvation and nigericin co-treatment
caused HMGB1 extracellular release in an Atg5-dependent manner. An
HMGB 1 band was detected by immunoblots in BMM culture supernatants
upon stimulation of cells with nigericin, whereas HMGB 1 was
largely diminished when BMMs from Atg5Fl/Fl Cre-LysM mice were
tested. HMGB 1, along with additional unconventional substrates,
depends on inflammasome for secretion although the protein itself
is not subjected to proteolytic processing by caspase 1 (65A-68A).
These experiments show that autophagy-based unconventional
secretion affects release of HMGB1 in a manner similar to
IL-1.beta., broaden the spectrum of autophagy-based unconventional
secretion substrates, and establish this type of unconventional
secretion as a more general process in extracellular delivery of
cytosolic proteins.
Common versus Specialized Model for Secretory and Degradative
Autophagy
[0153] Our recently published work (17A,27A), presented in the
preliminary results section, indicates that GRASP55 (one of the two
mammalian GRASPs) is also necessary for canonical, degradative
autophagy (27A). Thus, the early secretory and degradative
autophagosomes may originate from the common ancestral membrane
domains. When and how these domains become sub-specialized is not
known. We will test in aim 1 whether such specialization may be
driven by different isoforms of early autophagy factors (see FIG.
8A, ER-associated structures entitled "Precursor specialization"
and "Precursor").
[0154] Secretory and degradative autophagy could diverge later in
the pathway. Thus, we seek to determine whether autophagic adapters
differ or are modified to select the correct cargo into secretory
autophagosomes vs. degradative autophagosomes. Furthermore, since
there are six different Atg8 paralogs (three LC3s and three
GABARAPs) in mammalian cells, there could be a specialization of a
subset of them for secretory autophagy.
[0155] We have identified a number of proteins (IL-1.beta., IL-18,
HMGB1) that start as cytosolic proteins and end up being secreted
through autophagy-dependent unconventional secretion (27A). These
proteins exert their known biological functions extracellularly
(IL-1.beta., IL-18) or both intracellularly and extracellularly
(HMGB 1).
[0156] The invention is described further in the following
non-limiting examples.
EXAMPLE 1
The In Vivo Role for Autophagy in Protecting Against
Tuberculosis
Overview
[0157] We used a characterized conditional gene knockout mouse
model (Atg5.sup.fl/flLysM-Cre.sup.+) with a well documented Atg5
defect in macrophages and infected these mice aerogeneously with
the virulent M. tuberculosis strain H37Rv. An increase in bacterial
burden in the lungs and increased lung pathology were observed in
Atg5.sup.fl/flLysM-Cre+ compared to Atg5.sup.fl/flLysM-Cre.sup.+
littermates (FIG. 1P, panel A). With higher doses of M.
tuberculosis Atg5.sup.fl/flLysM-Cre.sup.+ mice succumbed sooner to
infection. These findings demonstrate for the first time that
autophagy is important for control of M. tuberculosis in vivo.
[0158] It was noticed that the bacterial burden differences between
Atg5-proficient and Atg5-deficient mice were quite narrow; they
were in the range of one log increase in M. tuberculosis colony
forming units in the lungs of the autophagy-defective mice. This
prompted us to look beyond the previously in vitro established role
of autophagy in direct elimination of mycobacteria within
macrophages (2). Mice deficient for autophagy in myeloid lineage
showed elements of endogenous inflammation in the lung. We
furthermore found that lungs of infected autophagy-deficient
animals displayed higher levels of IL-17 and IL-1.alpha. (FIG. 1P,
panel A). We also observed that IL-1.alpha. was elevated even in
the uninfected lungs and that IL-1.alpha. was secreted at higher
levels from cultured Atg5-deficient macrophages (FIG. 1P, panel B).
Importantly, IL-1.alpha. promoted (similarly to the previously
known property of IL-1.alpha. (3)) a Th17 response in vitro.
Furthermore, mixed immune cells isolated from
Atg5.sup.fl/flLysM-Cre.sup.+ had the propensity to polarize T cells
into IL-17--producing phenotype after re-stimulation with specific
M. tuberculosis antigens. This for the first time implicates
autophagy as a negative regulator of Th17 inflammation, and
suggests that autophagy suppresses Th17 response and neutrophilia,
the potentiators of pathogenesis in tuberculosis (4).
[0159] We could not address in vivo the pathology-inducing role of
IL-1.alpha. inferred from the above experiments, because
IL-1.alpha. also confers a key protective role against M.
tuberculosis bacteria as recently shown in the IL-1.alpha. knockout
mice (5). We nevertheless determined the cell-autonomous mechanism
of its elevated secretion by Atg5.sup.fl/flLysM-Cre.sup.+
macrophages. The drivers of IL-1.alpha. hypersecretion differed
from the previously reported mechanisms of increased IL-1.alpha.
production by autophagy-deficient macrophages (6-8). They were
independent of inflammasome constituents and caspase 1, and instead
involved a calpain-dependent pathway (FIG. 1P, panel C). The
process is initiated by accumulation of unkempt mitochondria in
autophagy-deficient macrophages (autophagy normally removes
depolarized mitochondria), resulting in reactive oxygen
intermediates that lead to increased IL-1.alpha. secretion in a
calpain-dependent fashion.
[0160] Along with the previous in vitro studies addressing the
antimycobacterial effector mechanisms of autophagy (17, 22, 26-29,
50, 51) this establishes that autophagy is a bona fide barrier
again tuberculosis. Autophagy protects against tissue necrosis and
lung pathology, the hallmarks of active tuberculosis. This effect
is not a simple consequence of increased bacillary loads but is
compounded by the cell-autonomous action of autophagy in
macrophage-driven inflammatory processes. Autophagy-deficient
macrophages release excessive amounts of inflammatory mediators,
such as IL-1.alpha., even in the absence of infection. A model
emerges whereby these mediators, when in excess, pivot inflammation
with features of Th17 response, neutrophilic infiltration, tissue
necrosis and organ damage, the main features of active tuberculosis
and contagious state of the host.
[0161] The mechanisms of cell-autonomous elimination of M.
tuberculosis by autophagy have been extensively studied in vitro
and include direct microbial digestion in autophagolysosomes (26),
delivery of neoantimicrobial peptides generated in autolysosomes to
compartments harboring intracellular mycobacteria (27, 32, 50) and
an interplay of autophagy with conventional antimicrobial peptides
(28). Our previous work (32) has highlighted the role of the
sequestosome-like receptors (SLR) p62 in these processes,
complementing the examples of other SLRs engaging an array of
intracellular bacteria (31, 52-55) and viruses (56). In contrast to
a preponderance of studies in vitro, autophagic control of microbes
is not fully documented in vivo (34, 56). Altered intestinal tissue
and Paneth cell function has been noted in response to microbial
flora and viral co-infection in an Atg16L1 hypomorph mouse model of
Crohn's disease, a chronic inflammatory condition (57). In the
animal model of protection against lethal Sindbis virus infection,
the dominant contribution of autophagy was in preventing tissue
damage independently of viral loads (56). This dovetails with the
aspect of our study that shows autophagic protection against
excessive inflammation and necrosis in the murine model of
tuberculosis. We interpret our data and reports by others (23, 24,
57, 58) as evidence that partial seeds of endogenous inflammation
and predisposition to hyper-reactivity exist in autophagy deficient
uninfected animals. This is in keeping with the cell autonomous
IL-1.alpha. hypersecretion shown here, and eventually leads to
increased pathology in infected animals. Although leukocytes from
uninfected Atg5.sup.fl/flLysM-Cre.sup.+ animals show a propensity
to polarize into IL-17 cells when tested ex vivo, IL-17 has been
detected in vivo only in infected animals. Thus, the elevated IL-17
response represents a product of interactions between M.
tuberculosis and a host defective for autophagy in myeloid
cells.
[0162] The findings that a loss of autophagy in macrophages results
in increased release of IL-1.alpha. and fosters an environment
where T cells produce IL-17 link for the first time autophagy with
elements of the Th17 response. The associated elevated presence of
neutrophils in the lungs of Atg5.sup.fl/flLysM-Cre.sup.+ mice
infected with M. tuberculosis may be linked to increased pathology.
IL-17 and neutrophils play a complex role in tuberculosis (59) and
confer both positive (60-62) and negative elements of protection
(63-65). The latter aspect of the role of neutrophils in
tuberculosis has been recently highlighted in patient cohort
studies (66) further compounded by correlates between type I IFN
(not addressed in our study) and different participating cells (66,
67). The pathogenic effects of neutrophils are notably manifested
during repeat exposure to mycobacterial antigens (65), and at times
when a lingering Th17 response does not give way to Th1 control
(64) or is not suppressed by regulatory mechanisms (63). Our
findings indicate that autophagy, when functional, curbs
neutrophilic response, possibly at the time when it needs to be
diminished (63-65).
[0163] All reports thus far (6, 23-25, 58, 68) agree that autophagy
plays a negative role in inflammasome activation through a variety
of triggers or additional mechanisms. Autophagy suppresses basal
level of inflammasome activation by continually removing (23, 24)
endogenous sources of inflammasome agonists such as ROS and
mitochondrial DNA (23, 24). Our findings with the ROS-calpain axis
in IL-1.alpha. activation, and findings by others regarding the
ROS-RLR signaling (16) expand these pro-inflammatory phenomena to
non-inflammasome pathways downstream of the accumulation of
dysfunctional mitochondria and ROS in autophagy-deficient cells.
Other changes with inflammatory consequences have been noted in
mice with Atg5-deficient macrophages (57, 69).
[0164] Tuberculosis has been and remains one of the main global
public health hazards further augmented by the HIV co-pandemic
(70). The classical presentation of disease is often masked by the
untreated HIV co-infection (70), but in principle the majority of
humans have a well-developed capacity to contain the infection so
that the majority of the world's population infected with the
tubercle bacillus is asymptomatic and only approximately 10% of
individuals develop active disease. This tip of the iceberg is
nevertheless key to continuing thetuberculosis contagion in human
populations, since active disease is necessary for the transmission
of tuberculosis. We propose that autophagy plays a dual role: it
both protects against the microbe and guards against host-inflicted
tissue destruction and active disease. In this model autophagy
curbs tuberculosis transmission by helping maintain the majority of
the infected population asymptomatic. Strategies aimed at
pharmacological manipulation of autophagy may diminish tuberculosis
spread, which may prove vital in containing the emergence of the
increasingly drug-resistant tuberculosis strains.
[0165] In conclusion, this work demonstrates for the first time the
in vivo, role for autophagy in protection against tuberculosis and
reveals that autophagy acts beyond its known role as a cellular
antimycobacterial effector mechanism. Autophagy prevents excessive
inflammation with features of a Th17 response and neutrophilic
infiltration, tissue necrosis, and organ damage, the main features
of active uberculosis and contagious state of the host.
Results
[0166] Autophagy protects mice from M. tuberculosis. The in vivo
role of autophagy was investigated by selective genetic deletion of
Atg5 in myeloid cells (which include macrophages and granulocytes),
with macrophages being of principal interest as the cells both
successfully parasitized by intracellular M. tuberculosis (33) and
targeted by protective immune responses. We used the previously
reported conditional gene knockout mouse model
Atg5.sup.fl/flLysM-Cre.sup.+ with Atg5 deletion in myeloid lineage
(34). The Atg5.sup.+ mice (Atg5.sup.fl/flLysM-Cre) and their
Atg5.sup.fl/flLysM-Cre.sup.+ littermates, previously characterized
for lack of Atg5 and autophagy in macrophages (6), were subjected
to aerogenic infection with low dose (10e.sup.2-10e.sup.3 cfu)
virulent M. tuberculosis H37Rv. An increase in bacterial burden
(FIG. 1A) and weight loss (FIG. 1 B) were observed in
Atg5.sup.fl/flLysM-Cre.sup.+ compared to
Atg5.sup.fl/flLysM-Cre.sup.- littermates. The lung pathology in
Atg5.sup.fl/flLysM-Cre.sup.+ was remarkable for gross tubercle
lesions in contrast to smaller infected foci in the lungs of Atg5+
animals (FIG. 1 C). Atg5.sup.fl/flLysM-Cre.sup.+ lung tissue
revealed extensive necrotic centers (FIG. 1D, subpanels i-iv) with
increase in percent of involved lung area and total lung weight (SI
Appendix; FIG. S1A,B) and differential increase in
polymorphonucelar (PMN) leukocytes (Ly6G.sup.+) (SI Appendix; FIG.
S1C-E). Acid fast bacilli per unit area were twofold higher in
Atg5.sup.fl/flLysM-Cre.sup.+ compared to Atg5.sup.fl
flLysM-Cre.sup.- lung sections (FIG. 1 D, subpanels v and vi). In
keeping with the well-known general resistance of mice
totuberculosis, neither group of mice succumbed to the infection in
short term (36 days). When a 10-fold higher infection dose
(10e.sup.4 cfu) was employed, this resulted in animal mortality
with accelerated deaths (along with continuing weight loss) among
Atg5.sup.fl/flLysM-Cre.sup.+ mice relative to their
Atg5.sup.fl/flLysM-Cre.sup.- littermates, starting three weeks post
infection (FIG. 1E,F). The above data indicate that
Atg5.sup.fl/flLysM-Cre.sup.+ mice are more susceptible to M.
tuberculosis infection over a range of infectious doses and at the
same time suggest that the differences in lung pathology exceed the
observed differences in bacterial burden.
[0167] Atg5-deficiency in myeloid lineage results in excessive
inflammatory response during infection and reflects in part the
elevated basal markers of inflammation. The cytokine profile in the
lungs of M. tuberculosis infected animals was remarkable for
significant increase in IL-1.alpha., IL-12, and CXCL1 in the
Atg5.sup.fl/flLysM-Cre.sup.+ lungs relative to Cre.sup.-
littermates (FIG. 2A-C). Additionally, IL-17 was elevated in
infected mice with disabled autophagy in myeloid cells (FIG. 2D and
SI Appendix, FIG. S2A). No differences were observed in the lungs
of infected Atg5.sup.fl/flLysM-Cre.sup.+ vs. Cre.sup.- mice for
IFN.gamma. and TNF.alpha., the well-established anti-tuberculosis
cytokines (35), IL-4, a cytokine known to inhibit autophagy ex vivo
(22), IL-6, and MIP-1.beta. (SI Appendix ; FIG. S2B-F). Some
increase in infected Atg5.sup.fl/flLysM-Cre.sup.+ vs. Cre.sup.-
animals were detected for GM-CSF and IL-1.beta. but the absolute
levels of the latter were much lower compared to IL-1.alpha. (SI
Appendix; FIG. S2G,H).
[0168] The uninfected Atg5.sup.fl/flLysM-Cre.sup.+ and Cre.sup.-
animals did not display signs of or differences in mortality,
morbidity, overt disease or discomfort per standards of veterinary
care. However, even in the lungs of uninfected mice, IL-1.alpha.
was detectable at low basal levels and was higher in
Atg5.sup.fl/flLysM-Cre.sup.+ than in Atg5.sup.fl/flLysM-Cre.sup.-
littermates (SI Appendix; FIG. S3A). Increased basal levels of
CXCL1 were observed in Atg5.sup.fl/flLysM-Cre.sup.+ vs Cre.sup.-
lungs (SI Appendix; FIG. S3B) whereas IL-12p70 levels were equal in
both uninfected animal groups (SI Appendix; FIG. S3C). IL-1.beta.
and IL-17 were below detection limits in the lungs of both
uninfected Atg5.sup.fl/flLysM-Cre.sup.+ or Cre.sup.fl/fl mice (SI
Appendix; FIG. S3D,E). Thus, some aspects of cytokine profiles
detected during infection (notably IL-1.alpha. and CXCL1) were
present at low levels in uninfected animals.
[0169] In uninfected Atg5.sup.fl/flLysM-Cre.sup.+ and Cre.sup.-
mice, similar numbers of cells expressing macrophage markers
(F4/80.sup.+CD11b.sup.+; lineage-negative CD3.sup.-CD19.sup.-) were
detected in the lungs and bone marrow (FIG. S3F,G). However, unlike
in autophagy-competent littermates, a fraction of lung macrophages
obtained from uninfected Atg5.sup.fl/flLysM-Cre.sup.+ mice
displayed activated phenotype (FIG. S3H). Depending on the marker,
3-20% of Atg5.sup.fl/flLysM-Cre.sup.+ macrophages had increased
expression of MHC class II, DEC205, and CD86. An indication of
increased CD 11b.sup.+F4/80.sup.- cell numbers was observed in
uninfected Atg5.sup.fl/flLysM-Cre.sup.+ lungs (FIG. S3F; top left
quadrant). Further examination revealed that these cells were
Ly6G.sup.+ (1a8 clone) polymorphonuclear granulocytes (neutrophils;
PMN) (SI Appendix; FIG. S3I). This increase in PMN total number was
only observed in the lungs, as bone marrow PMN numbers were
comparable for both groups of mice (SI Appendix; FIG. S3J). The
innate immune cell analyses in uninfected animals, along with the
cytokine data indicate that the lungs of
Atg5.sup.fl/flLysM-Cre.sup.+ mice have elevated markers of immune
activation under basal conditions relative to
Atg5.sup.fl/flLysM-Cre littermates. Thus, autophagy in myeloid
cells of the lung, a peripheral organ where continual immune
surveillance is necessary, maintains a homeostatic balance of
immune cells and their activations states, a process that was
perturbed in Atg5.sup.fl/flLysM-Cre.sup.+ mice even prior to M.
tuberculosis exposure.
[0170] Functional autophagic machinery in myeloid lineage affects
CD4 T cell activation and IL-17 response in uninfected animals. The
PMN infiltrates, cytokines and the elevated IL-17 in the infected
animals suggest elements of a Th17 response (36, 37). In the
absence of infection, a fraction of lung CD4 and CD8 T cells with
activated/memory phenotype (CD44.sup.high; FIG. 3A) was
significantly increased in uninfected Atg5.sup.fl/flLysM-Cre.sup.+
relative to Atg5.sup.fl/fl LysM-Cre.sup.- uninfected littermates.
We next stimulated total lung leukocytes from the lungs of
uninfected mice with phorbol-12-myristate-13-acetate and ionomycin
in the presence of protein secretion inhibitors and assessed
intracellular levels of IL-17A and IFN.gamma. expressed by CD4 T
cells. CD4 T cells from uninfected Atg5.sup.fl/flLysM-Cre.sup.+
lungs but not those from uninfected Atg5.sup.fl/flLysM-Cre.sup.-
lungs produced IL-17A (FIG. 3B,C). There was no marked difference
between the same cells from two sources in their ability to mount
IFN.gamma. response (FIG. 3D,E). These findings show the propensity
of CD4 T cells from uninfected Atg5.sup.fl/flLysM-Cre.sup.+ mice to
produce IL-17A upon stimulation, perhaps due to the increase IL-1a
in the lung, reflecting the in vivo state of T cells induced by the
lack of autophagy in myeloid cells.
[0171] Defective autophagy in myeloid lineage of
Atg5.sup.fl/flLysM-Cre.sup.+ mice promotes IL-17 response to
defined M. tuberculosis antigens by T cells. The above
proinflammatory properties were next investigated using M.
tuberculosis immunologically active components. We employed a
cocktail of 5 well defined M. tuberculosis protein antigens (DnaK,
GroEL, Rv009, Rv0569, Rv0685) collectively referred to as synthetic
PPD (38) in reference to the purified protein derivative (PPD) used
clinically as tuberculin skin test for evidence of recent
tuberculosis infection or BCG vaccination. The synthetic PPD
reproduces the anatomical and molecular properties of the
tuberculin skin test while eliminating false positive inflammatory
reactions (seen in uninfected hosts) caused by the contaminating
lipoglycans and carbohydrates resident in conventional PPD (38).
Atg5.sup.fl/flLysM-Cre.sup.+ and Cre.sup.- mice were injected
peritoneally with live M. bovis BCG and evaluated for the quality
of their immune responses three weeks later. Mice were injected
with the synthetic PPD or PBS in the hind footpad and delayed type
hypersensitivity (DTH) induration was measured at 0, 2, 24 and 48 h
postinoculation (FIG. 4A). No differences were observed at 24 and
48 h time point between the autophagy-competent and mutant mice.
However, when splenocytes from the BCG-inoculated animals were
re-stimulated ex vivo with synthetic PPD, IL-17A was detected at
significantly higher levels with Atg5.sup.fl/flLysM-Cre.sup.+
splenocytes (FIG. 4B), whereas no differences were observed for
typical Th1 and Th2 cytokine signatures (FIG. 4C-E) indicating
polarization to IL-17 producing phenotype in
Atg5.sup.fl/flLysM-Cre.sup.+ mice.
[0172] Atg5-deficiency causes cell-autonomous increase in
IL-1.alpha. secretion by macrophages. The increased level of IL-17
in the lungs of infected Atg5.sup.fl/flLysM-Cre.sup.+ animals is a
product of T cell polarization downstream of the changes in myeloid
cells. The Atg5.sup.fl/flLysM-Cre.sup.+ macrophages are known to
possess increased inflammasome activation (6, 7, 23-25) downstream
of ROS (16) and mitochondrial DNA (24) released from unkempt
mitochondria in the absence of autophagy. A key proinflammatory
cytokine activated via inflammasome, IL-1.beta., can lead to Th17
differentiation via IL-1 receptor signaling (37). However, in the
lungs of Atg5.sup.fl/flLysM-Cre.sup.+ animals infected with M.
tuberculosis, IL-1.beta. was present only in minor quantities and
undetectable in uninfected lungs (SI Appendix; FIGS. S2H and S3D).
Nevertheless, IL-1.alpha., which also signals via IL-1 receptor,
was a dominant cytokine elevated in both infected and uninfected
Atg5.sup.fl/flLysM-Cre.sup.+ lungs (FIGS. 2A and S2A). When we
tested whether IL-1.alpha. can substitute for IL-1.beta. (in
combination with TGF-.beta. and IL-6) in driving Th17
differentiation ex vivo, IL-1.alpha.showed a capacity to promote
Th17 polarization (SI Appendix; FIG. S4C-F).
[0173] In vitro activated Atg5.sup.fl/flLysM-Cre.sup.+ bone marrow
macrophages (BMM) recapitulated the in vivo pattern of elevated
secretion of IL-1.alpha. (along with CXCL1 and IL-12p70) relative
to Atg5.sup.fl/flLysM-Cre.sup.- BMM (FIG. 5A-C). The CXCL1
phenotype was likely secondary to IL-1 increase, since IL-1
receptor antagonist (IL-1RA) lowered CXCL1 levels (FIG. 5D).
Differential release of IL-1.alpha., which is a cytosolic protein,
was not due to changes in cell death or increased membrane
permeability since in vitro activated BMM from
Atg5.sup.fl/flLysM-Cre.sup.+ and Atg5.sup.fl/flLysM-Cre.sup.- mice
showed no difference in staining with 7-AAD (FIG. 5E). The above
experiments, and additional data showing elevated secretion of
IL-1.alpha. in the lungs of uninfected Atg5.sup.fl/flLysM-Cre.sup.+
animals, whereas IL-12p70 and IL-1.beta. were below detection
levels in these mice (SI Appendix; FIG. S3D,E), singled out
IL-1.alpha. as a potential pivot of proinflammatory pathology
observed with Atg5.sup.fl/flLysM-Cre.sup.+ mice in the tuberculosis
model. We could however not test this in vivo, since IL-1.alpha.
also plays a critical protective role against bacterial burden as
recently shown in IL-1.alpha. knockout mice (39).
[0174] Cellular mechanism for increased secretion of IL-1.alpha. by
autophagy-deficient macrophages is inflammasome independent. We
wanted to understand the cellular mechanism of the IL-1.alpha.
hypersecretion phenotype in Atg5.sup.fl/flLysM-Cre.sup.+
macrophages. Autophagy was confirmed as a negative regulator of
IL-1.alpha. release by pharmacologically manipulating autophagy in
Atg5.sup.fl/flLysM-Cre.sup.- BMM. When autophagy was induced with
rapamycin in autophagy-competent macrophages, this reduced the
amount of IL-1.alpha. being secreted (FIG. 5F), paralleling the
effects on IL-1.beta. (SI Appendix; FIG. S5A), a cytokine whose
secretion has been previously reported to be affected by autophagy
by us (6) and others (23, 24). Conversely, when
Atg5.sup.fl/flLysM-Cre.sup.- BMM were treated with 3-methyladenine
(3MA), an inhibitor of autophagosome formation, the levels of
IL-1.alpha. were significantly increased (FIG. 5E). As a control,
autophagy-deficient Atg5.sup.fl/flLysM-Cre.sup.+ BMM showed no
response in IL-1.alpha. secretion to these pharmacological agents
(SI Appendix; FIG. S5B). An effect similar to 3MA was observed upon
treatment of Atg5.sup.fl/flLysM-Cre.sup.- BMM with bafilomycin A1
(Baf A1), an inhibitor of autophagic flux (FIG. 5G).
[0175] We next considered multiple levels at which absence of
autophagy could result in elevated IL-1.alpha. secretion. The
autophagic adaptor protein p62, which is consumed during autophagy
(40) and is the founding member of the SLR family of PRR (31), also
prominently functions in innate immunity signaling (41). It
accumulates in the absence of autophagy and has been shown to
perturb NF-.kappa.B responses and cytokine secretion (41, 42). As
IL-1.alpha. expression is controlled by NF-.kappa.B (43), we tested
whether p62-mediated NF-.kappa.B activation could be the cause of
elevated IL-1.alpha. expression. However, knocking down p62 via
siRNA in Atg5.sup.fl/flLysM-Cre.sup.+ BMM (FIG. S6A) did not
abrogate the elevated IL-1.alpha. secretion by these cells (FIG.
S6B). Atg5 was knocked down in BMM from p62.sup.-/- knockout mice
and this still caused more (albeit less pronounced due to residual
Atg5 levels) IL-1.alpha. secretion than in the scrambled siRNA
control (FIG. S6C). Finally, no increase in IL-1.alpha. mRNA levels
was detected in Atg5.sup.fl/flLysM-Cre.sup.+ BMM relative to
Atg5.sup.fl/flLysM-Cre.sup.- BMM (FIG. S6D). Thus, neither does the
p62-NF-.kappa.B axis contribute to the IL-1.alpha. phenotype in
Atg5-deficient cells nor is IL-1.alpha. expression
transcriptionally activated in Atg5.sup.fl/flLysM-Cre.sup.+
macrophages. We next considered whether IL-1.alpha. was a direct
target for removal by autophagic organelles. Endogenous LC3 and
IL-1.alpha. did not colocalize (SI Appendix; FIG. S6E, left image
panels) and displayed negative Pearson's colocalization coefficient
even upon treatment with Baf A1 (SI Appendix; FIG. S6E, graph)
while showing complete separation of respective profiles (SI
Appendix; FIG. S6E, right image). Thus, IL-1.alpha. is unlikely to
be a direct substrate for autophagic elimination.
[0176] Pathways leading to IL-1.alpha. secretion have been reported
to utilize inflammasome components (44, 45) although unlike
IL-1.beta., intracellular IL-1.alpha. is not an enzymatic substrate
for caspase 1. Atg5.sup.fl/flLysM-Cre.sup.+ BMM showed elevated
levels of p20 caspase 1 (its activated form) in comparison to
Atg5.sup.fl/flLysM-Cre.sup.- BMM (SI Appendix; FIG. S6F,G). FLICA
(fluorochrome-labeled inhibitor of caspase) assay confirmed
increased enzymatically active caspase 1 in
Atg5.sup.fl/flLysM-Cre.sup.+ BMM compared to
Atg5.sup.fl/flLysM-Cre.sup.- BMM (FIG. S6H). In keeping with the
potential role for inflammasome and caspase 1 in IL-1.alpha.
release (44, 45), adding silica to macrophages increased their
IL-1.alpha. output (FIG. 5H). However, both the basal and
inflammasome agonist (silica)-induced levels of IL-1.alpha.
released from macrophages were increased in
Atg5.sup.fl/flLysM-Cre.sup.+ BMM. Furthermore, when we tested
whether this release was caspase 1 dependent, neither the enzymatic
inhibitor of caspase 1 YVAD (FIG. 5I) nor caspase 1 knockdown (FIG.
5J-L) decreased relative IL-1.alpha. output. We next tested whether
the elevated IL-1.alpha. secretion by Atg5.sup.fl/flLysM-Cre.sup.+
BMM was dependent on other inflammasome components. Knocking down
the key inflammasome constituents ASC and NLRP3 did not diminish
IL-1 .alpha. output of Atg5.sup.fl/flLysM-Cre.sup.+ cells (FIG.
5M). These observations, while surprising, are in agreement with
the recent report of inflammasome/caspase 1-independent pathway for
IL-1.alpha. secretion (46), and show that although the inflammasome
is activated in Atg5.sup.f1/flLysM-Cre.sup.+ BMM it is not
responsible for the increase in IL-1.alpha.output.
[0177] Increased IL-1.alpha. in Atg5.sup.fl/flLysM-Cre.sup.+
macrophages defines a ROS-calpain pro-inflammatory pathway. We next
searched for potential sources of IL-1.alpha. activation upstream
of the inflammasome. Reactive oxygen species (ROS) released by
accummulated dysfuctional mitochondria in autophagy-deficient cells
have been implicated in inflammatory signaling both during
RIG-I-like receptors (RLR) response to viral products (16) and
inflammasome activation in IL-1.beta. production (23).
Atg5.sup.fl/flLysM-Cre.sup.+ BMM had elevated mitochondrial content
(increased MitoTracker Green staining; SI Appendix, FIG. S7A-C)
accompanied by reduced mitochondrial polarization (decrease in
MitoTracker Red staining; SI Appendix, FIG. S7D,E). We tested
whether ROS associated with the mitochondrial defect in other
inflammatory signaling (16, 23) played a role in elevated
IL-1.beta. release from Atg5.sup.fl/flLysM-Cre.sup.+ macrophages.
ROS inhibitor (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC)
abrogated excessive IL-1.alpha. (FIG. 50). In keeping with the
previous reports (23), APDC also inhibited excessive IL-1.beta.
release from Atg5.sup.fl/flLysM-Cre.sup.+ BMM (FIG. 5P). Thus, ROS
are mediators leading to both IL-1.alpha. and IL-1.beta.
hypersecretion by autophagy-deficient cells but the machinery
downstream of ROS differs for the two cytokines since IL-1.beta.
depends on the inflammasome (23) whereas IL-1.alpha., as shown
here, does not.
[0178] ROS can lead to calpain activation (47, 48) although this
pathway has not been previously implicated in inflammation. We used
ALLN, a calpain inhibitor, to test whether calpain was involved in
the IL-1 a hypersecretion phenotype of Atg5.sup.fl/flLysM-Cre.sup.+
cells. ALLN treatment of Atg5.sup.fl/flLysM-Cre.sup.+ completely
inhibited the excess IL-1.alpha. production normalizing its output
to the levels seen with Atg5.sup.fl/flLysM-Cre.sup.- cells (FIG.
5Q). An siRNA knockdown of the common calpain regulatory (small)
subunit Capns1, which forms heterodimers with and is required for
function of the conventional murine calpains Capn1 and Capn2 (49),
abrogated IL-1.alpha. hypersecretion (FIG. 5R). We also considered
the possibility that calpain may be a target for degradation by
autophagy; however, calpain levels were not different between
Atg5.sup.fl/flLysM-Cre.sup.+ vs. Cre.sup.- cells (SI Appendix; FIG.
S6F) and calpain did not colocalize with autophagic organelles
(FIG. S6G). We conclude that the IL-1.alpha. increase associated
with the Atg5 defect in macrophages is due to elevated ROS and
depends not on absolute levels of calpain but on its activation
downstream of ROS, thus defining a new pro-inflammatory pathway
downstream of autophagy.
Materials and Methods
[0179] Mice and infection. The transgenic
Atg5.sup.fl/flLysM-Cre.sup.+ (myeloid specific Atg5 deletion) and
Atg5.sup.fl/flLysM-Cre.sup.- mice have been previously
characterized (34) and the autophagy defect in BMM extensively
documented (6). LC3-GFP knock-in transgenic mice (71) and
p62.sup.-/- knockout mice (72) have been previously described. Mice
were maintained under specific pathogen-free conditions. F1 progeny
from Atg5.sup.fl/fl LysM-Cre.times.Atg5.sup.fl/fl crosses were
genotyped for presence (LysM-Cre.sup.+) or absence (LysM-Cre) of
the LysM-Cre allele by Transnetyx Inc. (Cordova, Tenn.). Infection
studies were carried out using murine respiratory infection model
(73) and virulent M. tuberculosis H37Rv with modifications (74, 75)
described in SI Appendix. The standard low dose resulted in the
initial bacterial deposition ranging in independent experiments
between 10e.sup.2 to 10e.sup.3 cfu of M. tuberculosis per lung. The
high dose had the deposition range of 10e.sup.4 cfu per lung.
[0180] Cells, flow cytometry and immunodetection. All cells were
pretreated with Stain FcX (anti-CD16/32) (Biolegend) before being
stained for: CD14 (Sa14-2), F4/80 (BM8), IFN.gamma. (XMG1.2),
IL-17A (TC11-18H10.1), CD11b (M1/70), DEC205 (NLDL-145), CD8
(53-6.7), CD86 (GL-1), Ly6G (1A8), CD25 (PC61), MHC II (M5
/114.15.2) (Biolegend). CD19 (eBio1D3), TCR.beta. (H57-597), CD3e
(145-2C11), CD44 (IM7), CD4 (GK1.5), CD1d (1B1), DEC205 (205yekta),
CD4 (RM4-5), CD45 (30-F11), CD3 (17A2), F4/80 (BM8), CD11b (M1/70),
B220 (RA3-6B2), CD8.alpha. (53-6.7), IL-12p35 (4D10p35),
IL-1.alpha. (ALF-161), MCH II (M5/114.15.2), CD25 (PC61.5)
(eBioscience), Ly6G (1a8)(BD Biosciences). Caspase 1 activity was
measured by flow cytometry using the FLICA caspase 1 reagent
(FAM-YVAD-FMK) (Immunochemistry Technologies). Cells were incubated
with 7-AAD for viability assessment. Secreted cytokines
(IL-1.alpha., IL-1.beta. CXCL 1, CXCL2 and IL-12p70) were measured
by ELISA (R&D Systems). For cytokine secretion, murine BMM,
prepared as described (32), were stimulated with 5 ng/ml
mIFN-.gamma. and 1 .mu.g/m1LPS, with autophagy agonist and
antagonists: rapamycin (Invivogen), 3-MA, and bafilomycin A1;
chemical inhibitors: brefeldin A (Biolegend), YVAD and ALLN
(Sigma); or IL-1RA (R&D Systems) all added 30 minutes prior to
LPS and IFN.gamma. stimulation. For autophagy-dependent
unconventional secretion of cytosolic cytokines as described (6),
BMM were stimulated for 1 h with 250 .mu.g/ml silica (MIN-U-SIL-15,
US Silica) with starvation (EBSS) to induce autophagy.
[0181] DTH and cell-mediated immunity. Mice were infected
intraperitoneally with 5.times.10.sup.6 BCG for 21 days, and then
injected with the synthetic PPD (a five antigens cocktail: Dnak,
GroEL, Rv009, Rv0569, and Rv0685) at 1.0.mu.g/ml in PBS, or PBS
control, 50 .mu.l in separate footpads. DTH was assessed as
described (38) by comparing swelling to a baseline value
immediately after injection. Splenocytes (5.0.times.10.sup.5
cells/well) were restimulated with the synthetic PPD adjusted for
2.0 .mu.g/ml(Dnak and GroEL), and 4.0 .mu.g/ml (Rv009, Rv0569 and
Rv0685) and culture supernatants assayed for IFN.gamma.,
TNF-.alpha., IL-4 and IL-17 secretion by ELISA (R&D
Systems).
[0182] T cell assays. Single cell suspensions from whole lungs
isolated from naive Atg5.sup.fl/flLysM-Cre.sup.+ and Cre.sup.- mice
were cultured in RPMI 10% FBS and Cell Stimulation Cocktail
(phorbol 12-myristate 13-acetate and ionomycin plus brefeldin A and
monensin; eBioscience) for 4 h and analyzed by flow cytometry. For
in vitro polarization, naive CD4.sup.+ T cell from spleens were
sorted CD44.sup.lowCD4.sup.+ TCR.sup..beta.+ cells in a MoFlo high
speed cell sorter (Beckman Coulter), sorted cells (5.times.10.sup.5
cells/well), incubated with plate-bound anti-CD3 antibody (Hu et
al., 2011) and stimulated with 20 ng/ml IL-6, 5 ng/ml TGF-.beta.,
20 ng/ml IL-1 or 20 ng/ml IL-1.beta. (R&D Systems) in the
presence of anti-CD28 (37.51), anti-IFN-.gamma. (R4.6A2), anti-IL-4
(11B11), anti-IL-2 (JE56-1A12) (eBioscience). After 4days, cells
were stimulated with 1.times. Cell Stimulation Cocktail in the
presence of protein transport inhibitors for 5 hours at 37.degree.
C. and analyzed by flow cytometry.
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EXAMPLE 2
GRASPs and Secretory Autophagy
[0258] In the model shown in FIG. 8A, the secretory and degradative
autophagosome precursor domains on the ER are interrelated. They
originate from the secretory aspect of ER oriented towards the
Golgi known as the ER-Golgi-intermediate compartment,
vesicular-tubular clusters, transitional ER (tER), or ER exit sites
(79A,80A). Our preliminary data indicate that GRASP55 relocalizes
to sites on the ER marked by WIPI2 (one of the four mammalian Atg18
paralogs) (FIG. 9A).
[0259] We hypothesize that mammalian GRASPs play a role in
organizing/tethering these precursors at or in the vicinity of the
tER sites into larger structures (omegasomes), which in turn lead
(69A) to the formation of autophagic membranes for secretion and
degradation end-purposes. This idea is based on: (i) the known
translocation of GRASP from the Golgi during induction of autophagy
observed in our published work (27A); (ii) the known mechanisms of
how GRASP tethers membrane domains into larger structures through
intermolecular homotypic oligomerization of GRASP 81,82; and (iii)
the control of GRASP oligomerization by protein kinases (e.g.
during mitosis) (81A,82A).
[0260] It is determined whether autophagy regulators such as Ulk1
and Ulk2 control redistribution of GRASP to the vicinity of tER
sites. Furthermore, GRASP interacting partners may change, and this
may permit coalescence of precursors into omegasomes. Whether the
resulting omegasomes specialize in degradative or secretory
autophagy may depend on the subsets of (at present seemingly
redundant) early autophagic factors, e.g. Ulk1 vs Ulk2 and four
different variants of mammalian Atg18s represented by WIPI-1, -2,
-3 and -4. Our preliminary data with GRASP55 relocalizition to
sites on the ER marked by WIPI2 (FIG. 9A) indicate that this
happens both during starvation-induced autophagy and
starvation-induced secretory autophagy (for the latter, the cells
are also treated with nigericin to activate inflammasome and
trigger extracellular release of IL-1(3 (27A)).
[0261] There are two types of factors that we expect to control
GRASP55 localization during degradative and secretory autophagy:
(i) kinase(s) that may affect GRASP55 homo-oligomerization state
and thus tethering of adjacent membranes (as in the Golgi ribbon);
and
[0262] (ii) compartment-specific interacting partners binding to
GRASPs' PDZ domains thus tethering them to the appropriate
membranes that eventually coalesce. GRASPs have the ability to link
membranes through their homotypic interactions between PDZ domains
and internal PDZ-binding motifs ("internal ligand") (see FIG. 8A,
top). This is morphologically best recognized in the formation,
maintenance and disruption of Golgi ribbons, where GRASPs already
play a role in linking of Golgi cistrenae (81A,82A) (FIG. 8A). The
ability of GRASPs to undergo homo-oligomerization depends on the
phosphorylation state of their Ser-Pro-rich C-terminal domains and
can be disrupted e.g. during mitosis (causing Golgi dispersal)
following complex phosphorylation patterns by several kinases
including ERK, CDK1 and PLK1 (81A,82A). In our model, Ulk1 or Ulk2
are the main kinase candidates for modulating GRASP55 translocation
following induction of degradative or secretory autophagy in our
system.
EXAMPLE 3
[0263] Common versus Specialized Precursors for Secretory and
Degradative Autophagy
[0264] Experiments and interpretations. Ulk1 and Ulk2 as well as
WIPI1, 2, 3 and 4 are knocked down and outputs of degradative and
secretory autophagy are measured. The physiological outputs of
secretory autophagy measured are IL-1.beta. and HMGB 1 in culture
supernatants (27A). For degradative autophagy, proteolysis of
stable proteins is measured as we have previously described (83A),
mitochondrial cellular content and quality by MitoTracker Green and
Red, microbial killing using our published procedures (84A), and
p62 degradation by immunoblotting and high content microscopy
quantification (85A). Differences in effects in the two categories
are normalized to each other, anticipating reciprocal
relationships. Our cells of choice are the primary murine bone
marrow-derived macrophages (BMM), since we know fully the
parameters of both processes from our published studies on
secretory autophagy (27A) and degradative functions of autophagy
(83A,85A,86A). Ulk1 and Ulk2 are targeted (seemingly redundant Atg1
orthologs in mammals) and WIPI1,2,3 and 4 (four Atg18 paralogs in
mammals) with the idea that perhaps they may specialize. Two
mammalian GRASPs, GRASP55 and GRASP65 are compared (FIG. 7A) for
potentially differential roles in degradative and secretory
autophagy.
Alternatives.
[0265] Conceptual. Differential effects on degradative vs secretory
autophagy upon knockdown of a given factor will be taken as
specialization. Of course, absence of differential effects will not
rule out specialization or divergence at a downstream point or
points, which will be addressed at different steps below.
[0266] Technical. We could not efficiently knock out GRASP65 in
macrophages, and have observed only a minor synergistic effect with
GRASP55 (but not GRASP65 alone) on IL-1.beta. secretion (27A). An
attempt to overcome this problem is made by using shRNA approaches.
As an alternative to siRNA and shRNA knockdowns, TALEN
(Transcription Activator-Like Effector Nucleases) knockout cell
lines are generated (87).
EXAMPLE 4
GRASP Translocation: Interacting Partners and Kinases.
[0267] Experiments and interpretations. myc-Ulk1 and myc-Ulk2 wild
type are employed, along with their equivalents to substrate
trapping KR mutants 88. These constructs are used to test whether
GRASP55 associates with Ulk1 or Ulk2. Duolink or PLA assay are used
(proximity ligation I in situ assay) to establish any transient
interactions between suspect kinases (e.g. Ulk1) and GRASP. The
Duolink (PLA) method (see FIG. 10A) has been invented specifically
for capture and detection of such transient protein-protein
interactions in whole cells (89A). As already preliminarily stated
above, an example of Duolink/PLA from our recently published paper
(85A) on Rab8b-TBK-1 interactions is given in FIG. 10A. In the
Duolink/PLA approach, stable and transient protein-protein
interactions are detected as fluorescent dots 89A. A PLA
fluorescent dot is a binary visual output (signifying positive
interaction) of a spatially-restricted rolling circle DNA
amplification event, which can occur only in locations where
successful circularization or ligation took place between the
connector oligonucleotides and primers covalently linked to
antibodies. The signal is generated only when the secondary
antibodies recognize in-coincidence binding of primary antibodies
to two proteins interacting within a cell at a range approximating
fluorescence resonance energy transfer distances (89A). For
experiments reported here, fixed BMM were incubated with primary
antibodies followed by Duolink/PLA probes. After ligation, rolling
circle amplification, and hybridization with fluorescent
nucleotides and counterstaining of nuclei with DAPI, red
fluorescent dots are imaged and quantified.
[0268] The Ulk-dependent phosphorylation of GRASP is tested using
protein chemistry methods as described in detail in our recent
publication (85A) of TBK-1-dependent p62 phosphorylation. We
postulate that Ulk1 or Ulk2 phosphorylate GRASP55 following
induction of degradative or secretory autophagy to allow its
translocation to the Sec23+ structures that are conventionally
considered to be ER exit sites but in the case of degradative or
secretory autophagy are specialized sites in the ER leading to the
formation of omegasome/CUPS (FIG. 8A). This is paralleled by
functional assays measuring IL-1.beta. and HMGB 1 secretion from
cells knocked down for Ulks. Complementary to these experiments, we
have generated mutants of GRASP55, fashioned after GRASP55 S441A
mutant (human S441 and mouse S443). These presumptive
phosphorylation sites (in our model for Ulk1/2) are key for
activation of GRASP in stimulating unconventional membrane protein
trafficking (CFTR) (28A).293T cells are transfected with GRASP55
wild type and S443A mutant and assayed to determine the effect on
HMGB 1 secretion as per our published methods (27A).
[0269] In a search for interacting partners that guide GRASP55
translocation to WIPI2-positve structures (autophagosomal
precursors; see FIG. 8), co-IPs with GRASP55 are conducted
following induction of degradative or secretory autophagy, probing
for autophagy initiation complex members in immunoprecipitates.
Among our candidate interacting partners are: Sec23 (based on
Ohsumi and colleagues, this part of COPII but not Sec13/31 plays a
key role in autophagy 90A); Atg9 (the only autophagy specific
integral membrane protein that is early on recruited to the
autophagosomal initiation sites in mammalian cells 2); FIP200 and
VMP 1 (believed to be among the first proteins at the sites where
Ulk1 and the rest of the autophagic machinery coalesces 2). WIPIs
91 and DFCP-1 69 are included. Positive co-IPs are confirmed using
an independent method (Duolink/PLA) designed to detect endogenous
protein-protein interactions in whole cells (89A).
[0270] Alternatives: Alternatively to Ulk1 and 2, JNKs and PKR are
tested. (i) JNKs, specifically JNK-1 (92A), is already known to
result in disinhibition of Beclin-1 activation of autophagy (92A).
Of interest for our model is that JNKs are know to act downstream
of IRE1 following ER stress and unfolded protein response (UPR)
(93A). UPR has been already linked to induction of autophagy 77.
The role of JNKs is tested using JNK1 and 2 knockout MEFs, IRE1 and
JNK expression constructs, and JNK inhibitor SP600125 with the idea
that the IRE1-JNK axis leads to phosphorylation and changes in
GRASP55 localization/activities and to IL-1.beta. or HMGB 1
secretion following ER stress (e.g. with thapsigargin, tunicamycin,
DTT, 2-deoxyglucose). (ii) PKR has recently been linked (94A) to
extracellular release of HMGB1, one of the autophagy-dependent
unconventionally secreted cargos (27A). The PKR substrates
identified thus far are components of inflammasome (e.g. NLRP3)
(94A). HMGB 1 release has been linked to inflammasome activation 65
upstream of unconventional secretion (27A). This is fully
compatible with our findings that inflammasome and
autophagy-dependent unconventional secretion cooperate in mammalian
cells, as an evolutionary adaptation in higher organisms (17A,27A).
Consequently, PKR phosphorylates and changes GRASP localization are
tested and is determined whether these effects may be responsible
for the previously reported links between PKR and autophagy
(95A).
EXAMPLE 5
Omegasome/CUPS Formation.
[0271] Experiments and interpretations. Morphometrically (imaging)
and biochemically (subcellular fractionation) quantify GRASP55
translocation to the WIPI-positive profiles (for initiation of
degradative or secretory autophagy) in kinase knockdown or
inhibition assays. Several methods are used to monitor
GRASP55-dependent coalescence, including measuring the area of WIPI
(FIG. 9A) and DFCP-1 profiles (DFCP1 is the distinguishing marker
for omegasome (69A)) using Cellomics HSC algorithms that can
quantify in a fully automated and unbiased mode (from acquisition
to processing) large number of profiles, routinely set as in flow
cytometry to gather data until a threshold (e.g. 5,000 analyzable
events) per sample has been reached (85A). GRASP effect upon
phosphorylation (e.g. by Ulk1) is tested; instead of normally
tethering Golgi cisternae, now tethers multiple ER exit sites
coalescing them into omegasomes, the large precursors to
degradative and secretory autophagosomes (FIG. 8A).
Alternatives:
[0272] Conceptual. (i) Indirect GRASP-induced effects model. As an
alternative to the model in which GRASP55 translocates and plays an
active role in tethering and coalescing ER exit site-associated
omegasome/CUPS precursors (FIG. 7A), a passive/indirect role for
GRASP55 is considered: GRASP phosphorylation may disrupt Golgi
structures to the extent that it indirectly causes backing up of
membrane flow at ER exit sites thus increasing their chance to
coalesce. The distinguishing feature between the passive and active
models is where GRASP55 localizes at the time of induction of
degradative or secretory autophagy: if GRASP55 remains associated
with the dispersed Golgi or its ministacks following starvation or
ER stress, then the passive model is more likely. If it is
translocated in the vicinity of well-defined ER exit sites or
ER-associated omegasomes/CUPS, then the active model is likely.
Biochemically, we should also be able to detect a switch to new
binding partners if the active GRASP-relocalization model is
correct, and retention of binding partners and loss of homotypic
interactions if the passive model is correct. (ii) Role for Atg9
and Atg9 isoforms (Atg9L1 and Atg9L2)? Atg9 is the only integral
membrane autophagy protein 2. Atg9 has been shown to affect
unconventional secretion of Acb1 in yeast (24A). Yeast GRASP (Grh1)
and Atg9 overlap upon induction of autophagy that leads to Acb1
unconventional secretion (25A). According to Y. Ohsumi's latest
work (75A), autophagosomal structures are formed or expanded in
yeast by fusion of small Atg9 vesicles derived from the Golgi. This
can be linked back to the observations by S. Tooze and colleagues
in mammalian cells whereby Atg9 was found to redistribute from TGN
to nascent autophagosomes upon induction of autophagy (73A). It is
determined whether knockdown of mammalian Atg9L1 affects secretory
autophagy of IL-1.beta.. (iii) Roles for COG and TRAPP A
potentially related question is how do other protein complexes in
the secretory pathway (between the ER and the Golgi and within the
Golgi) including factors affecting tethering and vectorial
transport such as TRAPP 97,98 and the COG complexes (74A) fit into
the above model. Both TRAPP and COG (97A,98A) affect canonical
autophagy (74A). It is determined whether TRAPP and COG complexes
affect secretory autophagy. It is determined whether TRAPP or COG
components interact with GRASP as it moves to form autophagosomal
precursors.
[0273] Technical. Additional morphological (EM), fluorescence (e.g.
FRET, fluorescence intensity), and biochemical/subcellular
fractionation of membranous organelles based assays will be
employed as needed.
EXAMPLE 6
Specialization and Selectivity Factors for Secretory Autophagy
[0274] Rationale. The determinants of selectivity for secretory and
degadative autophagy are defined. There are multiple mammalian Atg8
paralogs and multiple autophagic adaptors. Both of these groups
represent candidates for selectivity and specialization. There are
six mammalian Atg8s (LC3A, B and C, and GABARAP, GABARAPL1 and L2)
whose potential functional differences are not well-understood
(47A,99A) and may reflect selectivity and specialization for
secretory vsersus degradative autophagy. It is determined whether
adaptors that capture autophagic cargo destined for secretion
differ from the adaptors that capture cargo earmarked for
autophagic degradation. There is now considerable information
available regarding the adaptors for degradative autophagy. For
example, p62, NDP52, and optienurin, have been shown to find
ubiquitinated cargo tagged for elimination, be it a whole pathogen
13,100,101, a protein aggregate, mitochondrion, and another
cytoplasmic target (86A,102A,103A). NBR1 102 has yet to be assigned
a clearly identified function. Another source of selectivity can be
post-translational modifications of adaptors, which are known to
modify interactions either between the adaptors and the cargo
(85A,104A) or between the adaptors and Atg8/LC3 factors (100A).
[0275] The potential existence of new, previously uncharacterized
adaptors specializing in secretory autophagy is considered.
Finally, a potential for specialization is determined for exocyst
components and Rab8 isoforms (Rab8a vs Rab8b). Based on our
published data with two Rab8 isoforms, Rab8a 27 vs. Rab8b 85, and
based on work by others (59A) and reported observations by us (27A)
regarding exocyst components, it appears that these systems
participate in one but not the other type of autophagy, with an
appreciable degree of selectivity for the degradative or secretory
form. The small Rab GTPases act as pivotal regulators of membrane
trafficking 105A). We have found in a series of studies that Rab8
isoforms (there are two: Rab8a and Rab8b) affect both secretory 27
and degradative autophagy (85A). The published data suggest
specialization, with Rab8b being more important for degradative
autophagy 85 whereas Rab8a affects secretory autophagy (27A). Rab8b
binds to TBK-1 directly as shown by Duolink (FIG. 10A). Optineurin
binds to both Rab8a and Rab8b106 as well as to TBK-1 107 but
apparently is not needed for Rab8b-TBK-1 association (85A). These
relationships are examined as potential sources of specialization
and selectivity for secretory vs. degradative autophagy.
EXAMPLE 7
Role and Specialization of LC3/GABARAP Paralogs.
[0276] Experiments and interpretations. It is determined whether
there is a specific LC3 and a specific GABARAP specializing in
secretory autophagy. Our preliminary data support this notion,
based on differential effects of mammalian Atg8 knockdowns on
IL-1.beta. secretion (FIG. 11A). Biochemical (protein-protein
interactions) and subcellular fractionation analyses is conducted
to confirm the theory. It is also determined whether secretory
autophagy-specific mammalian Atg8 (e.g. LC3A) interacts with
secretory autophagy-specific adaptors or post-translationally
modified adaptors (as depicted in FIG. 8A). Furthermore, LC3B may
differentially co-fractionate with membranes containing cargo
destined for degradation (e.g. ubiquitinated proteins,
mitochondria, bacteria, polyQ-huntingtin, p62) vs LC3A's
cofractionation with cargo destined for unconventional secretion.
We often think of mammalian Atg8s as a uniform group of nearly
identical factors that are commonly referred to as simply "LC3".
The human and mouse genomes encode three LC3 variants (LC3A,
LC3B--the most popular variant, and LC3C) and three GABARAP
variants (GABARAP, GABARAPL1 and GABARAPL2). In fact, different
members of the mammalian Atg8 family are more divergent from each
other (LC3s differ by close to 50% and GABARAPs are below 40%
identical) than ubiquitin and NEDD, and yet nobody equates NEDD
with ubiquitin. Thus, different LC3s and GABARAPs are not a uniform
group of redundant proteins and they are unlikely to have identical
function. The divergence in functions among different LC3s and
GABARAPs have been studied (47A, 99A).
[0277] Alternatives: Recent work by Z. Elazar and colleagues (47A,
99A) has indicated that LC3 as a group specialize in elongation of
autophagosomes whereas GABARAPs may be more important for
autophagosome closure. It is determined whether a combination of an
LC3 and a GABARAP (based on our preliminary results in FIG. 11A,
possibly LC3A and GABARAP) exists to form a secretory autophagic
organelle.
EXAMPLE 8
Specialization of Cargo Adaptors.
[0278] Experiments and interpretations. Specific autophagic
adaptors are examined to determine whether the cargo (and
autophagic organelle) is destined for secretory or degradative
functions. NBR1, p62, NDP52, and optineurin are knocked-down, or
knockout cells are used (e.g. BMMs from the p62.sup.-/- mice
received from M. Komatsu). IL-1.beta. secretion is measured. Of
note, NBR1 (102A), despite being the closest homolog of p62 has yet
to be assigned a clearly defined function. Thus, it is knocked down
and tested to determine whether it may specialize in autosecretion
(measuring IL-1.beta. and HMGB1). Although p62/sequestosome 1 would
seem as the least likely candidate (since all the reports to date
have been focused on p62 in autophagic degradation), our
preliminary data using macrophages from p62-/- knockout mice
suggest that it may be engaged in secretion of IL-1.beta. (FIG.
12A). It is determined whether this effect is due to p62 alone or
p62 interactions with NBR1, by overexpressing mutant form of p62
(PB1 domain dual point mutation K7A,D69A precludes oligomerzation
with NBR1).
[0279] Whether post-translational modifications of autophagic
adaptors determine or modulate destination of their cargo for
degradative or secretory autophagy is also determined We have
reported the presence of multiple phospho-peptides in p62 85. A
determination of whether these patterns change during secretory
autophagy versus degradative autophagy is made by comparing p62
phospho-petidome in immunoprecipitates form starvation alone versus
starvation+nigericin treated cells. As shown by us and others
(85A,104A), phosphorylation of at least one of the Ser residues
(pSer-403) modulate p62's ability to bind to ubiquitinated
substrates. Our data show that TBK-1 (85A) can phosphorylate
Ser-403 in p62, whereas the Nukina laboratory reported that this
site in the p62 UBA domain is phosphorylated by CK2 (104A). Both
studies show that this is a requirement for ubiquitinated
cargo-recognition and clearance. Whether some of that clearance is
not only by degradation but may involve secretion (as we and others
have detected p62 in cell culture supernatants) is determined.
Furthermore, our published data (85A) indicate that, in cells
lacking TBK-1, p62 undergoes additional large post-translational
modifications and does not enter into degradative pathways (85A).
the nature (by mass spectrometry) of these post-translational
modifications (detectable only in TBK-1-negative cells) is
determined using the methods and materials colected for our just
published work 85. We will also knockdown and pharmacologically
inhibit TBK-1. Our preliminary studies with TBK-1 inhibitor BX795
show that IL-1.beta. secretion is reduced. It is determined whether
TBK-1 knockout MEFs 85 secrete less HMGB 1 (MEFs are not suitable
for IL-1.beta. secretion studies, so extracellular HMGB 1 will be
measured instead).
[0280] Alternatives: (i) Optineurin is also an autophagic adaptor
100, and since it binds to TBK-1 it is of interest. TBK-1
phosphorylates optineurin and increases its affinity for LC3B and
enhances its capacity for cargo delivery to autolysosomes (100A).
It is determined whether optineruin directs its cargo to secretion
in TBK-1 null (or inhibited) cells. (iii) Previously
uncharacterized secretory autophagy adaptors may exist.
Vps23/TSG101 is examined. CUPS structures in yeast have been shown
to contain Vps23 25, the equivalent of mammalian TSG101. The
significance of Vps23's presence in CUPS is not known and it is
curious that other ESCRT proteins are not found in CUPS 25. Vps23,
as a part of the ESCRT-I complex, is involved in multivesicular
endosomal sorting but also plays other cellular roles 108. Although
Vps23 is required for Acb1 unconventional secretion in yeast 26,
deletion of Vps23 did not affect Grhl+CUPS formation, suggesting
that Vps23 may be a part of the downstream sorting effector
functions of CUPS. A possible adaptor role for Vps23/TSG101 in
secretory autophagy is that VPS23/TSG101 in its sorting function
binds to ubiquitinated cargo or short peptide motifs. Thus, we may
include TSG101 on our list of potential adaptor candidates for
secretory autophagy.
EXAMPLE 9
[0281] Roles and Specialization of Rab8a vs. Rab8b and TBK-1 in
Secretory versus Degradative Autophagy.
[0282] Experiments and interpretations. A model in which Rab8a does
not bind directly to TBK-1 is tested, as opposed to direct
Rab8b-TBK-1 interaction (85A). Using co-immunoprecipitations and
Duolink assays, it is determined whether or not Rab8a associate
with TBK-1, and whether this association is direct (Duolink will
provide the resolution sufficient enough to rule out an
intermediate adaptor). This will be combined with optineurin
knockdowns. Optineurin, being a possible adaptor, might allow Rab8a
and TBK-1 to be present in a protein complex but not directly
interact, thus showing association by imunoprecipitation but its
absence by Duolink. Since interaction with TBK-1 is key for
entering the degradative autophagy pathway (85A), an absence of
Rab8a interaction with TBK-1 or a different type of association
between the two may explain how Rab8a directs autophagic organelles
for secretion instead of for degradation in autolysosomes.
Alternatives:
[0283] Conceptual. The exocyst complex (consisting of eight
subunits including Sec5, Sec6, and Exo84) has been recently
implicated in autophagy (59A). The exocyst is however best known
for its function in trafficking of post-Golgi carriers and their
tethering to the plasma membrane in preparation for exocytosis 109.
Exocyst is also known to cooperate with Rab8 109. Of further
interest is that the exocyst platform plays a role in TBK-1
activation, first observed in the context of innate type I
interferon response to pathogen products, with the Sec5 component
being in the complex 110. The exocyst has not yet been linked with
autophagy-dependent unconventional secretion, albeit this would
make sense given the known functions of the exocyst in secretion.
Nevertheless, according to a report by M. White's group, different
exocyst components can either promote (Exo84) or stall (Sec5)
autophagy (59A). Our published data indicate that IL-1.beta. in
LC3+ profiles en route for unconventional secretion colocalize with
Sec6, one of the exocyst components (27A). However, we could not
efficiently knock down any of the exocyst parts in primary murine
macrophages, the cell type where our entire study (27A) was
executed, and thus could not establish a functional role in
secretory autophagy. Human THP-1 cells, where knockdowns of exocyst
components are routinely achieved, are examined (M. White, personal
communication). It is determined whether Rab8, exocyst, and TBK-1
differentially regulate degradative autophagy versus secretory
autophagy.
[0284] Technical. Rab8a and Rab8b constitutively active and
dominant negative mutants in our collection and will be used to
probe the proposed effects on degradative versus secretory
autophagy.
EXAMPLE 10
Defining the Autophagic Secretome of a Mammalian Cell
[0285] Rationale. Autophagy has a highly relevant ability to act as
a topological inverter--taking a substrate that is in the cytoplasm
and placing it into the lumen of the autophagosome, from where it
can be either degraded upon fusion with lysosomes or secreted
following fusion with the plasma membrane. The second possibility
is examined and physical methods (mass spectrometry) are used to
identify the protein entities that are substrates for secretory
autophagy (autophagic secretome).
Identifying Autophagy-Dependent Unconventional Secretion Proteome
(Autophagic Secretome).
[0286] Experiments and interpretations. Mass spectrometry is used
to physically identify secretory cargo released from cells upon
stimulation of autophagy. A similar approach has been applied for
inflammasome-dependent release of proteins (65A). The difference
vis-a-vis the previously published inflammasome study (65A) will be
that we will use induction of autophagy and identify protein
entities released into supernatants from Atg5-proficient cells but
not from isogenic Atg5-deficient cells (subtractive analysis).
Primary macrophages (BMMs) will be induced for autophagy for the
period of time we have optimized to avoid nonspecific leakage of
cytosolic proteins from cells (27A). The concentrated supernatants
from Atg5fl/fl LysM-Cre+ vs Cre- BMMs will be digested with
trypsin. This is followed by labeling with isobaric chemical tags
using Tandem Mass Tag reagent (Thermo Scientific) specific for the
free N-ter Lys residues: TMT2-126 for Cre+ and TMT2-172 for Cre-.
Peptides are identified and quantified by tandem mass spectrometry
(MS/MS). In the first MS, the equivalent peptides are
indistinguishable from each other; in the tandem MS mode, each tag
generates a unique reporter ion. We have already carried out
preliminary studies and show in Table 1 (FIG. 12B) a number of
Atg5-dependent substrates released by secretory autophagy from bone
marrow-derived macrophages. A total of 153 proteins were found as
secreted in an Atg5-dependent manner as their presence was
decreased in the supernatants from Atg5 knock-out BMMs (negative
score in Table 1). Among species identified, in addition to new
candidates there are known unconventionally secreted
proteins--vimentin, galectin-1, galectin-3, ASC (an inflammasome
component), ferritin and thioredoxin. These experiments are
repeated with BMMs from Atg7fl/fl LysM-Cre+ mice to define the
autophagic secretome.
[0287] Additional analyses are carried out with other cell types
from mice with different Cre drivers (myeloid, lymphoid,
epithelial, fibroblast, neural, general/tamoxifen) crossed with
Atg5fl/fl and Atg7fl/fl mouse strains. Starvation and one cell type
physiologically relevant stimulus for induction of autophagy is
used, e.g.: ER stress, IFN-.gamma., microbial products, and
endogenous damage-associated molecular patterns (alarmins). This
permits testing of whether different cell types have different
autophagic secretomes and agonist-dependent composition response
patterns.
[0288] Changes in the secretory autophagy outputs should be
observed, depending on different cell types and autophagy agonists
used. In all experiments, release of cytosolic proteins
independently of secretory autophagy is recorded by monitoring
lactate dehydrogenase release and cell death, as in our published
work (27A).
[0289] Alternatives: (i) The murine proteins identified as
substrates for autophagic secretion using BMMs and for which there
are available antibodies are confirmed by immunoblots in autophagic
secretions from BMMs. (ii) Secretion of confirmed proteins is
tested with human primary peripheral blood monocyte-derived
macrophages. (ii) Given that the mass spectroscopy approach has
disadvantages due to the potential to miss a minor species of
potentially high biological significance, a complementary
biological activity-based tracking and purification approach to
complement the physical identification approach may be considered.
Addition of supernatants from autophagy-induced cells to the
reporter cells is used. Some of the biological activities
considered are: a) Programmed cell death (different types of cell
death such as apoptosis, necrosis, necroptosis, pyroptosis). Of
relevance here is that autophagy still has an incompletely defined
tumor suppressor role, whereas IL-1.beta. release is associated
with pyroptotic cell death and thus other secretary autophagy
substrates may have additional cell death modulating activities. b)
Immunological functions (e.g. Th1 and Th17 polarization signals; or
inflammasome agonists and neurodegenerative plaque generators such
as A1342 of significance for Alzheimer's disease). c)
Microbiological outputs such as direct killing of extracellular
bacteria; we have previously shown that autophagy can generate
intracellular neo-antimicrobial peptides from innocuous cytosolic
components such as ribosomal proteins (86A).
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