U.S. patent application number 16/389454 was filed with the patent office on 2019-11-07 for klebsiella pneumoniae strain that induces inflammation in the liver.
This patent application is currently assigned to KEIO UNIVERSITY. The applicant listed for this patent is KEIO UNIVERSITY. Invention is credited to Ryo Aoki, Takanori Kanai, Kentaro Miyamoto, Nobuhiro Nakamoto, Nobuo Sasaki, Toshiro Sato.
Application Number | 20190338374 16/389454 |
Document ID | / |
Family ID | 68384676 |
Filed Date | 2019-11-07 |
![](/patent/app/20190338374/US20190338374A1-20191107-D00001.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00002.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00003.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00004.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00005.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00006.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00007.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00008.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00009.png)
![](/patent/app/20190338374/US20190338374A1-20191107-D00010.png)
United States Patent
Application |
20190338374 |
Kind Code |
A1 |
Nakamoto; Nobuhiro ; et
al. |
November 7, 2019 |
KLEBSIELLA PNEUMONIAE STRAIN THAT INDUCES INFLAMMATION IN THE
LIVER
Abstract
To identify a microorganism causing the development of primary
sclerosing cholangitis associated with ulcerative colitis. A
Klebsiella pneumoniae strain inducing inflammation in the
liver.
Inventors: |
Nakamoto; Nobuhiro; (Tokyo,
JP) ; Sasaki; Nobuo; (Tokyo, JP) ; Aoki;
Ryo; (Tokyo, JP) ; Miyamoto; Kentaro; (Tokyo,
JP) ; Sato; Toshiro; (Tokyo, JP) ; Kanai;
Takanori; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KEIO UNIVERSITY |
Tokyo |
|
JP |
|
|
Assignee: |
KEIO UNIVERSITY
Tokyo
JP
|
Family ID: |
68384676 |
Appl. No.: |
16/389454 |
Filed: |
April 19, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01K 2207/25 20130101;
C12R 1/46 20130101; C12R 1/22 20130101; A01K 2267/0387 20130101;
A01K 2207/20 20130101; A01K 2207/12 20130101; A01K 2227/105
20130101; A01K 67/027 20130101; A01K 2267/035 20130101; C12R 1/37
20130101 |
International
Class: |
C12R 1/22 20060101
C12R001/22; C12R 1/37 20060101 C12R001/37; C12R 1/46 20060101
C12R001/46; A01K 67/027 20060101 A01K067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2018 |
JP |
2018-082192 |
Claims
1. A Klebsiella pneumoniae strain inducing inflammation in the
liver.
2. The Klebsiella pneumoniae strain according to claim 1, Inducing
a Th17 cell in the liver.
3. The Klebsiella pneumoniae strain according to claim 1, having an
ability to form a pore on the large intestinal epithelium.
4. The Klebsiella pneumoniae strain according to claim 1, having a
type 6 secretion system.
5. The Klebsiella pneumoniae strain according to claim 1, derived
from a patient suffering from both primary sclerosing cholangitis
and ulcerative colitis.
6. The Klebsiella pneumoniae strain according to claim 1,
comprising a DNA consisting of a nucleotide sequence registered in
the National Center for Biotechnology Information (NCBI) under
Assembly Name: ASM385182v1.
7. The Klebsiella pneumoniae strain according to claim 1, whose
deposit number is NITE BP-02879.
8. The Klebsiella pneumoniae strain according to claim 1,
comprising a DNA consisting of a nucleotide sequence registered in
the National Center for Biotechnology Information (NCBI) under
Assembly Name: ASM386511v1.
9. A Proteus mirabilis strain whose accession number is NITE
BP-02923.
10. An Enterococcus gallinarum strain whose accession number is
NITE BP-02922.
11. Use of a Klebsiella pneumoniae strain according to claim 1 for
prediction or diagnosis of the development of primary sclerosing
cholangitis.
12. Use of a Klebsiella pneumoniae strain according to claim 1, a
Proteus mirabilis strain, and an Enterococcus gallinarum strain for
prediction or diagnosis of the development of primary sclerosing
cholangitis.
13. The use for prediction or diagnosis of the development of
primary sclerosing cholangitis according to claim 12, wherein the
Proteus mirabilis strain is one whose accession number is NITE
BP-02923; and the Enterococcus gallinarum strain is one whose
accession number is NITE BP-02922.
14. A method for producing a mouse model suffering from both
primary sclerosing cholangitis and ulcerative colitis, the method
comprising: administering a bacterial solution containing a
Klebsiella pneumoniae strain according to claim 1 to a mouse; and
administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention relates to a Klebsiella pneumoniae
strain inducing inflammation in the liver.
Description of the Related Art
[0002] Primary sclerosing cholangitis (PSC) is an idiopathic
chronic cholestatic chronic liver disease and causes end-stage
hepatic cirrhosis through the progression of biliary stricture and
collapse of the biliary tree. To date, no definitive treatment
other than liver transplantation for this symptom has been
established, and a further understanding of the pathophysiology is
needed for treating this incurable disease.
[0003] Since portal bacteremia and high-level endotoxin in
cholangiocytes have been observed in patients with primary
sclerosing cholangitis, it is considered that bacterial
translocation, in which intestinal bacterial flora passes through
the damaged intestinal barrier, plays an important role in liver
inflammation. However, regardless of the presence of many evidences
of bacterial translocation in patients with primary sclerosing
cholangitis, the mechanisms that trigger collapse of intestinal
barrier and bacterial translocation are still unknown.
[0004] It is known that more than half of the patients with primary
sclerosing cholangitis suffer from ulcerative colitis, and it is
suggested that the bacterial translocation induced by chronic
colitis has a risk of causing hepatitis. In contrast, only a very
small number of patients with ulcerative colitis have primary
sclerosing cholangitis. Furthermore, the ulcerative colitis
patients having primary sclerosing cholangitis exhibit
discontinuous segmental colitis (right sided colitis). This is
remarkably contrast to continuous rectal lesions in typical
ulcerative colitis not complicated with primary sclerosing
cholangitis. Accordingly, only having colitis as the underlying
disease is insufficient for the onset of primary sclerosing
cholangitis complicated with ulcerative colitis, and it is
considered that other disease factors are involved in the
onset.
[0005] In recent years, microbial analysis has reported that the
intestinal bacterial flora of ulcerative colitis patients having
primary sclerosing cholangitis is different from those of healthy
subjects and typical ulcerative colitis patients not having primary
sclerosing cholangitis (Non-Patent Literatures 1 to 3). Considering
the difference in clinical symptoms of ulcerative colitis
complicated with primary sclerosing cholangitis and typical
ulcerative colitis not complicated with primary sclerosing
cholangitis, it is suggested that abnormality in the intestinal
bacterial flora (dysbiosis) may play an important role in the
development of primary sclerosing cholangitis associated with
ulcerative colitis.
CITATION LIST
Non-Patent Literature
[0006] [Non-Patent Literature 1] [0007] Sabino, J., et al., Primary
sclerosing cholangitis is characterised by intestinal dysbiosis
independent from IBD, Gut 65, 1681-1689, (2016) [0008] [Non-Patent
Literature 2] [0009] Kummen, M., et al., The gut microbial profile
in patients with primary sclerosing cholangitis is distinct from
patients with ulcerative colitis without biliary disease and
healthy controls, Gut 66, 611-619, (2017) [0010] [Non-Patent
Literature 3] [0011] Iwasawa, K., et al., Characterisation of the
faecal microbiota in Japanese patients with paediatric-onset
primary sclerosing cholangitis, Gut 66, 1344-1346, (2017)
[0012] However, there is not yet clear evidence showing that
dysbiosis causes the development of primary sclerosing cholangitis
associated with ulcerative colitis, and it is also unclear whether
a certain microbial species contributes to the development of
primary sclerosing cholangitis associated with ulcerative colitis.
Accordingly, there is a strong demand for identification of a
microorganism that causes the development of primary sclerosing
cholangitis associated with ulcerative colitis.
SUMMARY OF THE INVENTION
[0013] The present inventors have intensively studied in view of
the above problems and, as a result, have identified a Klebsiella
pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus
gallinarum strain that cause the development of primary sclerosing
cholangitis associated with ulcerative colitis, and the present
invention has been accomplished. That is, the present invention,
for example, relates to the following [1] to [14]:
[0014] [1] A Klebsiella pneumoniae strain inducing inflammation in
the liver;
[0015] [2] The Klebsiella pneumoniae strain according to [1],
inducing a Th17 cell in the liver;
[0016] [3] The Klebsiella pneumoniae strain according to [1] or
[2], having an ability to form a pore on the large intestinal
epithelium;
[0017] [4] The Klebsiella pneumoniae strain according to any one of
[1] to [3], having a type 6 secretion system;
[0018] [5] The Klebsiella pneumoniae strain according to any one of
[1] to [4], derived from a patient suffering from both primary
sclerosing cholangitis and ulcerative colitis;
[0019] [6] The Klebsiella pneumoniae strain according to any one of
[1] to [5], comprising a DNA consisting of the nucleotide sequence
registered in the National Center for Biotechnology Information
(NCBI) under Assembly Name: ASM385182v1;
[0020] [7] The Klebsiella pneumoniae strain according to any one of
[1] to [6], whose deposit number is NITE BP-02879;
[0021] [8] The Klebsiella pneumoniae strain according to any one of
[1] to [5], comprising a DNA consisting of the nucleotide sequence
registered in the National Center for Biotechnology Information
(NCBI) under Assembly Name: ASM386511v1;
[0022] [9] A Proteus mirabilis strain whose accession number is
NITE ABP-02923;
[0023] [10] An Enterococcus gallinarum strain whose accession
number is NITE ABP-02922;
[0024] [11] Use of the Klebsiella pneumoniae strain according to
any one of [1] to [8] for prediction or diagnosis of the
development of primary sclerosing cholangitis;
[0025] [12] Use of the Klebsiella pneumoniae strain according to
any one of [1] to [8], a Proteus mirabilis strain, and an
Enterococcus gallinarum strain for prediction or diagnosis of the
development of primary sclerosing cholangitis;
[0026] [13] The use for prediction or diagnosis of the development
of primary sclerosing cholangitis according to [12], wherein the
Proteus mirabilis strain is one whose accession number is NITE
ABP-02923; and the Enterococcus gallinarum strain is one whose
accession number is NITE ABP-02922; and
[0027] [14] A method for producing a mouse model suffering from
both primary sclerosing cholangitis and ulcerative colitis, the
method comprising:
[0028] administering a bacterial solution containing the Klebsiella
pneumoniae strain according to any one of [1] to [8] to a mouse;
and
[0029] administering 3,5-dicarbetoxy-1,4-dihydrocollidine to a
mouse.
[0030] According to the present invention, a Klebsiella pneumoniae
strain, a Proteus mirabilis strain, and an Enterococcus gallinarum
strain can be used for prediction of the development of primary
sclerosing cholangitis. In addition, a mouse model suffering from
both primary sclerosing cholangitis and ulcerative colitis can be
used for establishing a method for treating primary sclerosing
cholangitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1a is a graph showing the results of unweighted UniFrac
analysis of bacterial flora present in fecal samples of healthy
subjects (Hc 1 to 4), patients suffering from both primary
sclerosing cholangitis and ulcerative colitis (Psc/Uc 1 to 5), and
patients suffering from ulcerative colitis only (Uc 1 to 4) and in
fecal samples of humanized gnotobiotic mice administered the fecal
samples of these patients. FIG. 1b is a graph showing the results
of principal coordinates analysis based on the UniFrac analysis of
the human fecal samples and the humanized gnotobiotic mouse fecal
samples;
[0032] FIGS. 2a to 2c are graphs showing the results of measurement
of gene expression levels of serum amyloid protein A (Saa 1 to 3)
and IL-1.beta. (Il1b) in the livers (2A), colons (2B), and spleens
(2C) of GF mice (GF), humanized gnotobiotic mice administered a
fecal sample of a healthy subject (HC), and humanized gnotobiotic
mice administered a fecal sample of a patient suffering from both
primary sclerosing cholangitis and ulcerative colitis (PSCUC). * :
P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 (The
same applies to the following figures);
[0033] FIG. 3a includes graphs showing the results of measurement
of serum alkaline phosphatase (ALP, left) and serum total bilirubin
(TB, right) of GF, HC, and PSCUC. FIG. 3b includes photomicrographs
of samples of specimens prepared by Sirius red staining (above) and
hematoxylin-eosin staining and Masson-trichrome staining (below) of
liver tissue sections of GF, HC, and PSCUC. The scale bars indicate
100 .mu.m. FIG. 3c includes graphs showing the results of flow
cytometric analysis of the cells in the livers of GF, HC, and
PSCUC;
[0034] FIG. 4a includes graphs showing the results of counting the
cell number of the Klebsiella pneumoniae (KP), Proteus mirabilis
(PM), and Enterococcus gallinarum (EG) contained in fecal samples
of Hc, Psc/Uc, and Uc. FIG. 4b includes graphs showing correlations
between the proportion of Th17 cells or Th1 cells in the liver of
PSCUC and the number of Klebsiella pneumoniae cells in the fecal
samples thereof;
[0035] FIG. 5a includes graphs showing the results of flow
cytometric analysis of the cells in the livers, colons, and
mesenteric lymph nodes of humanized gnotobiotic mice administered
KP only (KP), PM and EG (PM+EG), and three strains of KP, PM, and
EG (3-mix), respectively, in fecal samples derived from Psc/Uc, GF
mice (GF), and SPF mice (SPF). FIG. 5b includes fluorescence
micrographs of the ilea of the SPF mouse and the humanized
gnotobiotic mice (KP gnoto, PM+EG gnoto, and 3-mix gnoto). The
scale bars indicate 50 .mu.m;
[0036] FIG. 6a includes scanning electron micrographs of large
intestinal epithelia co-cultured, in a monolayer organoid
co-culture system of the large intestine, with an enterohemorrhagic
Escherichia coli strain O-157:H7, a KP strain (KP JCM1662) not
having an ability to form a pore on the large intestinal
epithelium, and KP strains (KP-P1 and KP-P5) isolated from
mesenteric lymph nodes of mice administered fecal sample derived
from Psc/Uc. The scale bars indicate 20 .mu.m. FIG. 6b is a graph
showing the number of pores formed per unit area (mm.sup.2) of each
of the large intestinal epithelia. FIG. 6c includes fluorescence
micrographs of the co-cultured large intestinal epithelia
triple-stained with an anti-cleaved caspase-3 antibody, phalloidin,
and Hoechst 33324. The scale bars indicate 100 .mu.m;
[0037] FIG. 7 is a chart showing the results of comparative
analysis after whole genome sequencing of KP strains (ATCC 700603,
JCM1664, JCM1662, and JCM1663) not having an ability to form a pore
on the large intestinal epithelium and KP strains (JCM20694,
JCM20034, JCM20348, ATCC BAA2552, ATCC 700721, KP-P1, KP-P5, ATCC
BAA1705, and JCM20507) having an ability to form a pore on the
large intestinal epithelium was performed;
[0038] FIG. 8a is a schematic diagram illustrating an experimental
scheme. FIG. 8b includes fluorescence micrographs of the livers of
a humanized gnotobiotic mouse (3-mix gnoto) prepared by
administering KP-P1, PM, and EG to a GF mouse and of a humanized
gnotobiotic mouse (m3-mix gnoto) prepared by administering JCM1662,
PM, and EG to a mouse. The scale bars indicate 20 .mu.m. FIG. 8c is
a graph showing the measurement results of serum lipopolysaccharide
(LPS) of each mouse. FIG. 8d is a graph showing the results of flow
cytometric analysis of the cells in the liver of each mouse;
[0039] FIG. 9a is a schematic diagram illustrating an experimental
scheme. FIG. 9b includes graphs showing the results of flow
cytometric analysis of the cells in the livers of humanized
gnotobiotic mice (3-mix gnoto) treated with
3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) and ROR.gamma.t inverse
agonist (RORIA) or water as a control. FIG. 9c includes graphs
showing the measurement results of serum ALP (left) and serum TB
(right) of the humanized gnotobiotic mice; and
[0040] FIG. 10 includes micrographs of hematoxylin-eosin and
Masson-trichrome stained liver sections of humanized gnotobiotic
mice (3-mix gnoto) treated with DDC and RORIA. The scale bars
indicate 100 .mu.m.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] Embodiments of the present invention will now be described
with reference to the drawings, but the scope of the present
invention is not limited to the disclosed embodiments.
[0042] The present invention will now be described in detail.
<Klebsiella pneumoniae Strain>
[0043] An aspect of the present invention relates to a Klebsiella
pneumoniae strain inducing inflammation in the liver. The
inflammation of livers includes not only inflammation of livers but
also inflammation of intra- and extra-hepatic bile ducts, and
examples thereof include hepatitis and cholangitis. Examples of
hepatitis include viral hepatitis, drug-induced hepatitis,
alcoholic hepatitis, non-alcoholic steatohepatitis, and autoimmune
hepatitis. Examples of cholangitis include sclerosing cholangitis
(such as primary sclerosing cholangitis, IgG4-related sclerosing
cholangitis, and secondary sclerosing cholangitis), primary biliary
cholangitis (primary biliary cirrhosis), ascending cholangitis,
secondary sclerosing cholangitis, and recurrent pyogenic
cholangitis.
[0044] The Klebsiella pneumoniae strain of the present invention is
preferably a strain inducing Th17 cells in the liver. The induction
of Th17 cells can be verified by, for example, treating mononuclear
cells of the liver with a fluorescence labeled anti-CD4 antibody
and an anti-IL-17 antibody and verifying a significant increase in
IL-17-producing CD4 positive helper T (Th17) cells by flow
cytometric analysis.
[0045] The Klebsiella pneumoniae strain of the present invention
preferably has the ability to form pores which is capable of
forming pores on the large intestinal epithelia. The pores may have
any shape and size allowing enteric bacteria to leak through the
pores. For example, the diameter may be 0.1 to 20 .mu.m, more
preferably 0.5 to 15 .mu.m, in observation of large intestinal
epithelium with a scanning electron microscope. The leakage of
enteric bacteria through the pores can be verified by, for example,
hybridizing the DNA of the enteric bacteria with a fluorescent
probe, staining the large intestinal epithelial cells with a
fluorescent dye that emits fluorescence having a color different
from that of the fluorescence of the probe, and observing the
section of the large intestinal epithelium with a fluorescence
microscope. Although the details of the mechanism of forming pores
are unclear, it is inferred that the Klebsiella pneumoniae strain
of the present invention comes into direct contact with large
intestinal epithelium to induce apoptosis, resulting in the
formation of pores.
[0046] The Klebsiella pneumoniae strain of the present invention
preferably has a type 6 secretion system (T6SS). The type 6
secretion system can be verified by, for example, the presence of a
gene in comparative analysis of whole genome sequencing of the
strain.
[0047] The Klebsiella pneumoniae strain of the present invention is
preferably derived from a patient suffering from both primary
sclerosing cholangitis and ulcerative colitis described below. The
strain can be isolated from a fecal sample of the patient by a
known method using a generic growth medium. Examples of the medium
include a brain heart infusion (BHI) medium, a MacConkey agar
medium, and a VRBG medium.
[0048] The Klebsiella pneumoniae strain of the present invention
preferably has a DNA consisting of the nucleotide sequence
registered in the National Center for Biotechnology Information
(NCBI) under Assembly Name: ASM385182v1, and the Klebsiella
pneumoniae strain is more preferably one whose deposit number is
NITE BP-02879.
[0049] The Klebsiella pneumoniae strain of the present invention
preferably has a DNA consisting of the nucleotide sequence
registered in the National Center for Biotechnology Information
(NCBI) under Assembly Name: ASM386511v1.
<Primary Sclerosing Cholangitis>
[0050] Primary sclerosing cholangitis can be diagnosed in
accordance with known clinical guidelines. For example, as the
diagnosis items, when "bile duct image" (A) and "increase in
alkaline phosphatase level" (B) are defined as major items;
"complication of inflammatory bowel disease" (a) and "liver tissue
image (fibrous cholangitis/onion skin lesion)" (b) are defined as
minor items; and the bile duct image (A) is classified into
"recognition of findings of bile duct image characteristic to
primary sclerosing cholangitis" (A1) and "no recognition of
findings of bile duct image characteristic to primary sclerosing
cholangitis" (A2), only definite diagnosis and probable diagnosis
in the following Table 1 can be treated as primary sclerosing
cholangitis.
TABLE-US-00001 TABLE 1 Major item Minor item (A1) (A2) (B) (a) (b)
Diagnosis .smallcircle. -- .smallcircle. -- -- Definite diagnosis
.smallcircle. -- -- .smallcircle. -- Definite diagnosis
.smallcircle. -- -- -- .smallcircle. Definite diagnosis --
.smallcircle. .smallcircle. .smallcircle. .smallcircle. Definite
diagnosis .smallcircle. -- -- -- -- Probable diagnosis --
.smallcircle. .smallcircle. .smallcircle. -- Probable diagnosis --
.smallcircle. .smallcircle. -- .smallcircle. Probable diagnosis --
.smallcircle. -- .smallcircle. .smallcircle. Probable diagnosis --
.smallcircle. -- .smallcircle. -- Possible diagnosis --
.smallcircle. -- -- .smallcircle. Possible diagnosis
[0051] The alkaline phosphatase level can be measured by a known
method. For example, it can be measured in accordance with the
common standard method proposed by the Japan Society of Clinical
Chemistry (JSCC). When the measured value is, for example, 2 to 3
times as high as the reference value, the value can be determined
as an abnormal value.
[0052] The bile duct image can be judged to be (A1) or (A2)
mentioned above based on, for example, findings of diffuse wall
irregularities and strictures associated with inflammation of
intra- and extra-hepatic bile ducts characteristic to primary
sclerosing cholangitis by performing an endoscopic retrograde
cholangiography (ERC) or magnetic resonance
cholangiopancreatography (MRCP) test.
[0053] The above-mentioned diagnosis of primary sclerosing
cholangitis needs to exclude malignant tumors such as
cholangiocarcinoma, IgG4-related sclerosing cholangitis, and
secondary sclerosing cholangitis. The IgG4-related sclerosing
cholangitis can be diagnosed based on, for example, combination of
four criteria ((1) finding of characteristic bile duct image, (2)
increase in serum IgG4 level, (3) complication of IgG4-related
disease of an organ other than biliary tract, and (4)
characteristic histopathological findings) in Clinical diagnostic
criteria of IgG4-related sclerosing cholangitis, 2012 (Journal of
Hepato-Biliary-Pancreatic Sciences, vol. 19, pp. 536-542).
[0054] Examples of the secondary sclerosing cholangitis include
congenital diseases, such as Caroli's disease and cystic fibrosis;
chronic obstructive diseases, such as choledocholith, biliary
stricture, Mirizzi syndrome, anastomotic stenosis after liver
transplantation, and tumors; infectious diseases, such as bacterial
cholangitis, recurrent pyogenic cholangitis, parasitic infection,
and cytomegalovirus infection; poisoning diseases, such as
erroneous intrabiliary injection of alcohol, formaldehyde, or
hypertonic saline; immune abnormalities, such as eosinophilic
cholangitis and those with AIDS; ischemic diseases, such as those
related to vascular injury, post-traumatic sclerosing cholangitis,
hepatic artery embolism after liver transplantation, rejection
after liver transplantation, or coronary arterial anticancer drug
infusion; and invasive lesions, such as systemic vasculitis,
amyloidosis, sarcoidosis, systemic mastocytosis, eosinophilia,
Hodgkin's disease, and xanthogranulomatous cholangitis.
<Ulcerative Colitis>
[0055] Ulcerative colitis can be diagnosed in accordance with known
clinical guidelines. For example, ulcerative colitis can be clearly
diagnosed when the following item (B-1) or (B-2) is satisfied, in
addition to the items (A) and (C), and the diseases (D) are
excluded.
[0056] (A) Clinical symptom: Persistent or recurrent mucous or
bloody stool or medical history thereof is observed.
[0057] (B-1) Endoscopic examination: (1) The mucous membrane is
diffusely affected, the visible vascular pattern disappears, and
coarse or fine granular form is observed. Furthermore, friability
and hemorrhage-prone properties (contact bleeding) cause attachment
of mucous and bloody purulent secretion; or (2) multiple erosion,
ulcer, or pseudopolyposis is observed. (3) In general, lesions are
observed continuously from the rectum.
[0058] (B-2) Enema X-ray examination: (1) Diffuse change like a
coarse or fine granular form of the surface of the mucosal
membrane, (2) multiple erosion or ulcer, and (3) pseudopolyposis
are observed. In addition, disappearance of Haustra coli (lead pipe
appearance) or narrowing or shortening of the intestine is
observed.
[0059] (C) Biopsy histologic examination: In the active phase,
diffuse inflammatory cell infiltration, cryptic tumor, and a severe
decrease in goblet cells are observed in the entire mucosal layer.
Since all of them are nonspecific findings, comprehensive judgment
is performed. In the remission phase, abnormal arrangement of
glands (meander, bifurcation) and atrophy remain. These changes are
generally observed continuously from the rectum to the oral
side.
[0060] (D) Infectious enteritis, such as bacterial and Clostridium
difficile enteritis, amebic colitis, Salmonella enteritis,
Campylobacter enteritis, colonic tuberculosis, and Chlamydia
enteritis; Crohn's disease; radiation irradiated colitis;
drug-induced colitis; lymphoid follicular hyperplasia; ischemic
colitis; intestinal Behcet, and so on.
[0061] In addition to the above definite diagnosis cases, when the
items (B-1) or (B-2) and (C) are satisfied multiple times,
ulcerative colitis can be clearly diagnosed when findings grossly
and histologically characteristic to ulcerative colitis are
observed by excision surgery or autopsy.
<Use for Prediction of Development of Primary Sclerosing
Cholangitis>
[0062] Another aspect of the present invention relates to use of
the Klebsiella pneumoniae strain only or use of the Klebsiella
pneumoniae strain, a Proteus mirabilis strain, and an Enterococcus
gallinarum strain for predicting the development of primary
sclerosing cholangitis.
[0063] As described above, for example, a Klebsiella pneumoniae
strain derived from a patient suffering from both primary
sclerosing cholangitis and ulcerative colitis is characterized by
(1) having a type 6 secretion system, (2) having an ability to form
a pore on large intestinal epithelia, and (3) inducing Th17 cells
in the liver. This suggests that in a patient suffering from
ulcerative colitis, the Klebsiella pneumoniae strain having a type
6 secretion system forms pores on the large intestinal epithelium
to leak enteric bacteria, such as a Proteus mirabilis strain and an
Enterococcus gallinarum strain, and induce Th17 cells in the liver,
resulting in complication of primary sclerosing cholangitis.
[0064] Accordingly, if genome sequencing has revealed that the
Klebsiella pneumoniae strain in a fecal sample of a patient
suffering from ulcerative colitis has a type 6 secretion system,
the development of primary sclerosing cholangitis can be predicted.
In addition, if formation of pores is recognized on the large
intestinal epithelium when a biopsy tissue sample of the large
intestine is subsequently observed with a scanning electron
microscope, the prediction accuracy is increased. Furthermore, if
an increase in Th17 cells in the liver is recognized in, for
example, flow cytometry of a biopsy sample of the liver, the
prediction accuracy is further increased.
[0065] In addition, a fecal sample of a patient is cultured, and
the presence or absence of a Proteus mirabilis strain or an
Enterococcus gallinarum strain, in addition to the Klebsiella
pneumoniae strain, can be used as a predictor. The Proteus
mirabilis strain is preferably a Proteus mirabilis strain whose
accession number is NITE ABP-02923 and the Enterococcus gallinarum
strain is preferably an Enterococcus gallinarum strain whose
accession number is NITE ABP-02922.
<Method for Producing Mouse Model Suffering from Both Primary
Sclerosing Cholangitis and Ulcerative Colitis>
[0066] Another aspect of the present invention relates to a method
for producing a mouse model suffering from both primary sclerosing
cholangitis and ulcerative colitis. Specifically, the method for
producing a mouse model suffering from both primary sclerosing
cholangitis and ulcerative colitis includes a step of administering
a bacterial solution containing the Klebsiella pneumoniae strain
described in the paragraph <Klebsiella pneumoniae strain>
described above to a mouse and a step of administering
3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse. Although the step
of administering a bacterial solution containing the Klebsiella
pneumoniae strain to a mouse and the step of administering
3,5-dicarbetoxy-1,4-dihydrocollidine to a mouse may be performed in
any order, it is preferred to perform the step of administering a
bacterial solution containing the Klebsiella pneumoniae strain to a
mouse and then administering 3,5-dicarbetoxy-1,4-dihydrocollidine
to the mouse.
[0067] Although the original mouse to be used for producing the
mouse model may be any mouse that can suffer from both primary
sclerosing cholangitis and ulcerative colitis, a germ-free mouse is
preferred. Examples of the germ-free mouse include mice produced
from strains such as BALB/c, C57BL/6, and ICR.
[0068] In order to produce the mouse model, for example, a
suspension is prepared by suspending fecal samples collected from
primary sclerosing cholangitis and ulcerative colitis patients in,
for example, a phosphate-buffered saline (PBS) solution, and the
suspension is orally administered to original mice to produce
humanized gnotobiotic mice. Subsequently, the humanized gnotobiotic
mice 19 to 23 days after the administration can be given free
access to a diet containing 0.01% to 0.1%
3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 12 to 16 days to
produce a mouse model suffering from both primary sclerosing
cholangitis and ulcerative colitis.
[0069] Another aspect of the present invention relates to the
Proteus mirabilis strain whose accession number is NITE
ABP-02923.
[0070] Another aspect of the present invention relates to the
Enterococcus gallinarum strain whose accession number is NITE
ABP-02922.
[0071] Although the embodiments of the present invention have been
described in detail, they are merely examples, and the present
invention is not limited thereto. The scope of the present
invention should be interpreted by the claims and includes all
modifications within the meaning and scope equivalent to the
claims.
EXAMPLES
[0072] The present invention will now be further specifically
described based on Examples, but is not limited to these
Examples.
[Example 1] Comparison of Intestinal Bacterial Floras of Human and
Humanized Gnotobiotic Mouse
(1) Collection of Fecal Sample
[0073] Subjects were 14 patients suffering from both primary
sclerosing cholangitis and ulcerative colitis (hereinafter referred
to as "Psc/Uc" in some cases), 8 patients suffering from ulcerative
colitis only (hereinafter referred to as "Uc" in some cases), and
10 healthy subjects (hereinafter referred to as "Hc" in some
cases). Primary sclerosing cholangitis was diagnosed based on
clinical guidelines and findings by cholangiography and liver
biopsy. Ulcerative colitis was diagnosed by combination of
endoscopic examination, histopathologic examination, and
radiographic and serological examinations. Fecal samples were
collected using feces collection tubes and were suspended in a
solution containing 40% glycerol and an equivalent amount (w/v) of
PBS, and the resulting suspensions were rapidly frozen and were
stored at -80.degree. C. until use.
(2) Production of Humanized Gnotobiotic Mouse
[0074] The frozen stock of each fecal sample of the above (1) was
thawed and suspended in 6 volumes of PBS, and the suspension was
passed through a 70-.mu.m cell strainer to prepare a suspension for
oral administration. 200 .mu.L of the suspension for oral
administration was orally administered to 6 to 8-week old male
germ-free mice (GF mice, available from Sankyo Laboratories) using
a stainless steel oral gavage needle to produce humanized
gnotobiotic mice. Fecal samples were collected from the humanized
gnotobiotic mice 3 to 4 weeks after the administration.
(3) Collection of DNA in Fecal Sample
[0075] DNAs of the bacteria in the human fecal samples in the above
(1) and the humanized gnotobiotic mice fecal samples in the above
(2) were isolated by an enzymatic dissolution method using lysozyme
(manufactured by Sigma-Aldrich Co. LLC) and achromopeptidase
(manufactured by FUJIFILM Wako Pure Chemical Corporation). The
resulting DNA samples were treated with Ribonuclease A
(manufactured by FUJIFILM Wako Pure Chemical Corporation) for
purification and were then precipitated with 20% polyethylene
glycol solution (PEG 6000, 2.5 M sodium chloride aqueous solution).
Each precipitate was collected by centrifugation and was then
washed with 75% ethanol and dissolved in a Tris-EDTA buffer.
(4) 16S rRNA Metagenomic Analysis
[0076] The hypervariable region V3-V4 of 16S rRNA gene of each of
the DNAs obtained in the above (3) was amplified with TaKaRa Ex
Taq.RTM. Hot Start Version (manufactured by Takara Bio Inc.), and
the amplicon was purified with AMPure XP (manufactured by Beckman
Coulter, Inc.). A mixture sample was prepared from the resulting
DNA, and sequencing was performed with Miseq Reagent Kit V3 and
Miseq Sequencer (manufactured by Illumina, Inc.) in accordance with
the product manual. The sequence analysis was performed with QIIME
software package ver. 1.9.1. Paired-end sequences were joined using
a fastq-join tool in the EA-Utils software package. From the
quality filter-passed sequences, 15,000 high-quality sequences were
chosen for each sample. Chimera sequences were detected by the
USEARCH, and the primer sequences were removed using cutadapt.
Assignment of OTUs was performed using the UCLUST algorism with a
sequence similarity of 96%. The assignment of OTUs was performed
using the GLSEARCH program by similarity searching against the 16S
(RDP version 10.27 and CORE update 2 Sep. 2012) and the NCBI genome
database. The data were rarefied to 10,000 sequences per sample, as
determined by the rarefaction curves, and the relative abundances
of the bacteria were determined. The unweighted UniFrac analysis
was performed in accordance with the method described in the
document (Tsuda, A., et al., Influence of Proton-Pump Inhibitors on
the Luminal Microbiota in the Gastrointestinal Tract, Clin. Transl.
Gastroenterol., 6, e89 (2015)). The results are shown in FIGS. 1a
and 1b. The results demonstrated that the main bacterial flora was
conserved in human and humanized gnotobiotic mice.
[Example 2] Measurement of Gene Expression Levels of Serum Amyloid
Protein A (SAA) and IL-1.beta. in Organ
[0077] The liver, colon, and spleen of the humanized gnotobiotic
mouse produced in Example 1 were homogenized, and total RNA of each
organ was extracted using RNeasy Mini Kit (manufactured by QIAGEN
N.V.). Complementary DNA was synthesized from 1 .mu.g of the
resulting total RNA by reverse transcription, and each target gene
was amplified by PCR using AmpliTaq Gold Fast PCR MasterMix
(manufactured by Applied Biosystems, Inc.) and the following
designed primers (manufactured by Thermo Fisher Scientific
Inc.).
[0078] Glyceraldehyde-3-phosphate dehydrogenase (Gapdh):
Mm03302249_g1
[0079] Saa1: Mm00656927_g1
[0080] Saa2: Mm04208126_mH
[0081] Saa3: Mm00441203_m1
[0082] IL-1.beta.: Mm01336189_g1
[0083] The amplicons were quantitatively measured by real-time PCR
using TaqMan Universal Master Mix and StepOne Plus systems
(manufactured by Applied Biosystems, Inc.). In each sample, the
target gene expression level was standardized using Gapdh. The
results are shown in FIGS. 2a to 2c. The results demonstrated that
in the liver (FIG. 2a) and the colon (FIG. 2b) of humanized
gnotobiotic mice (PSCUC in the graphs) administered a fecal sample
derived from Psc/Uc, the gene expression of serum amyloid protein A
and IL-1.beta. was increased and that bacteria involved in hepatic
and large intestinal diseases were present in the intestinal
bacterial flora of Psc/Uc.
[Example 3] Production of Mouse Model Suffering from Both Primary
Sclerosing Cholangitis and Ulcerative Colitis
(1) Measurement of Serum Alkaline Phosphatase and Serum Total
Bilirubin
[0084] GF mice and the humanized gnotobiotic mice produced in
Example 1 (2) by orally administering feces of Hc or Psc/Uc 21 days
after the administration were given free access to a diet
containing 0.05% 3,5-dicarbetoxy-1,4-dihydrocollidine (DDC) for 14
days, and serum alkaline phosphatase (ALP) and total bilirubin (TB)
were measured by an LDH-UV kinetic method (manufactured by SRL,
Inc.). The results are shown in FIG. 3a. The humanized gnotobiotic
mice administered the fecal sample derived from Psc/Uc (PSCUC in
the graphs) showed high expression levels of both ALP (left in FIG.
1a) and TB (right in FIG. 1a), compared to those in GF mice (GF in
the graphs) and the humanized gnotobiotic mice administered the
fecal sample derived from Hc (HC in the graphs).
(2) Histochemical Observation of Liver
[0085] Livers were collected from the DDC-treated humanized
gnotobiotic mice and were fixed in 10% formalin and embedded in
paraffin according to a usual method to produce paraffin blocks.
Sections were cut from the paraffin blocks and were stained with
hematoxylin-eosin and Masson-trichrome or stained with Sirius red
to prepare specimen samples. The results of microscopic observation
of the specimen samples are shown in FIG. 3b. The upper part of
FIG. 3b shows the results of Sirius red staining, and the lower
part of FIG. 3b shows the results of co-staining with
hematoxylin-eosin and Masson-trichrome. In the liver of the
humanized gnotobiotic mice administered the fecal sample derived
from Psc/Uc, hepatic disorder due to fiber formation in the liver
was observed.
(3) Immunological Analysis
[0086] Livers were collected from the DCC-treated mice and were
perfused with PBS from the portal vein. After the perfusion, the
livers were chopped and passed through a 100-.mu.m nylon mesh to
obtain cells of the livers.
[0087] The resulting cells were suspended in 40% Percoll solution
and then overlaid in 75% Percoll fraction, followed by density
gradient centrifugal separation at 840.times.g for 20 minutes at
room temperature. Mononuclear cells were collected from the
intermediate layer. The resulting mononuclear cells were washed and
were suspended in FACS buffer. The mononuclear cells were subjected
to blocking treatment using an anti-Fc antibody (CD16/32,
manufactured by BD Pharmingen) and were then subjected to
intracellular staining with an anti-CD4 antibody (APC-cy7, BV510)
and an anti-CD11b antibody (APC-cy7). The intracellular stained
cells were treated with Ionomycine (500 ng/mL) or Golgistop (10
.mu.g/mL, manufactured by BD Biosciences) in the presence of
lipopolysaccharide (derived from Escherichia coli B5, manufactured
by Sigma-Aldrich Co. LLC) or PMA (50 ng/mL, manufactured by
Sigma-Aldrich Co. LLC) and brefeldin A (10 .mu.g/mL, manufactured
by BD Biosciences).
[0088] Subsequently, an anti-IFN-.gamma. antibody, an
anti-TNF-.alpha. antibody, an anti-IL-1.beta. antibody, and an
anti-IL-17 antibody (manufactured by BD Pharmingen) were added
thereto, followed by co-culture at 4.degree. C. for 20 minutes. The
cells after the culture were washed with PBS and were measured with
a cell sorter ("FACS CantoII," manufactured by Becton, Dickinson
and Company) and analyzed using FlowJo software (manufactured by
Tree Star, Inc.). The results are shown in FIG. 3c. The results
demonstrated that in DDC-treated mice administered a fecal sample
derived from Psc/Uc, the development of hepatic disorder and
recruitment of Th17 cells and IL-1.beta..sup.+ CD11b.sup.+
macrophages were observed, and a mouse model suffering from both
primary sclerosing cholangitis and ulcerative colitis was
provided.
[Example 4] Identification of Intestinal Bacterial Flora Derived
from Psc/Uc
(1) Analysis of Intestinal Bacterial Flora in Mouse Fecal
Sample
[0089] In order to identify the bacteria that induce hepatitis, the
livers, mesenteric lymph nodes, and spleens were collected from
humanized gnotobiotic mice orally administered a fecal sample of Hc
or Psc/Uc and SPF mice (6- to 8-week old C57BL/6 mice, available
from CLEA Japan, Inc.) as a control group on the 21st day from the
administration, and the bacteria were cultured on agar plates. As a
result, no colonies were observed in the liver and spleen of any of
the mice. In the mesenteric lymph nodes, colonies were observed in
only that from the humanized gnotobiotic mice administered a fecal
sample derived from Psc/Uc. Bacterial flora analysis of 16S rRNA of
the colonies demonstrated that the bacteria of the colonies were
Klebsiella pneumoniae (KP), Proteus mirabilis (PM), and
Enterococcus gallinarum (EG).
(2) Analysis of Intestinal Bacterial Flora in Human Fecal
Sample
[0090] Fecal samples derived from Hc, Psc/Uc, and Uc were
anaerobically cultured in BHI medium (manufactured by Becton,
Dickinson and Company), and the KP, PM, and EG cells were counted.
The results are shown in FIG. 4a. It was demonstrated that in the
fecal sample derived from Psc/Uc, KP was remarkably present, and PM
and EG were also present. The KP included a KP strain (deposit
number: NITE BP-02879) having a DNA consisting of the nucleotide
sequence registered in the National Center for Biotechnology
Information (NCBI) under Assembly Name: ASM385182v1 and a KP strain
having a DNA consisting of the nucleotide sequence registered in
the National Center for Biotechnology Information (NCBI) under
Assembly Name: ASM386511v1. The PM included a strain whose
accession number is NITE ABP-02923, and the EG included a strain
whose accession number is NITE ABP-02922.
(3) Correlation Analysis of KP in Fecal Samples of Human and
Humanized Gnotobiotic Mice
[0091] As in Example 2 (3), correlations between the proportion of
Th17 cells or Th1 cells in the liver of the humanized gnotobiotic
mice administered the fecal sample derived from Psc/Uc and the
number of KP cells in the fecal sample were verified by flow
cytometric analysis using an anti-CD3e antibody (FITC), an anti-CD4
antibody (APC-cy7, BV510), an anti-IFN-.gamma. antibody, and an
anti-IL-17 antibody, and the significance was verified by a
Spearman rank-order correlation test. The results are shown in FIG.
4b. A correlation was found between KP and Th17 cells (left in FIG.
4b), but no correlation was found between KP and Th1 cells (right
in FIG. 4b). The results demonstrated that KP derived from Psc/Uc
contributed to induction of Th17 cells in the liver.
[Example 5] Evaluation of Properties of KP, PM, and EG
(1) Evaluation of Induction of Th17 Cell in Liver by Single Strain
and Mixed Strain Administration
[0092] In order to investigate contribution of the above-mentioned
three strains to Th17 cell induction, 1.times.10.sup.8 CFU/200
.mu.L of KP, PM, and EG were orally administered to GF mice by (1)
KP alone (KP), (2) combination of PM and EG (PM+EG), or (3)
combination of three strains (3-mix) each twice per week. On the
21st day from the administration, as in Example 2 (3), the liver,
colon, and mesenteric lymph node were analyzed by flow cytometry
using an anti-CD4 antibody (APC-cy7, BV510), an anti-IL-17
antibody, and an anti-ROR.gamma.t antibody (manufactured by BD
Pharmingen). The results are shown in FIG. 5a. In all organs of the
liver (left in FIG. 5a), the colon (center in FIG. 5a), and the
mesenteric lymph node (right in FIG. 5a), Th17 cell induction was
recognized in the KP administration groups, in particular, in the
3-mix administration group, compared to those in GF mice (GF) and
SPF mice (SPF) not administered the strains.
(2) Histochemical Observation of Large Intestine
[0093] Ileum tissues including fecal materials of humanized
gnotobiotic mice prepared by administering KP alone, PM and EG, and
three strains of KP, PM, and EG to GF mice (KP, PM+EG gnoto, and
3-mix gnoto) were fixed in Carnoy's solution for 3 hours and
embedded in paraffin to produce paraffin blocks. Tissue sections
were cut from the blocks and were hybridized at 50.degree. C.
overnight using EUB338 probe (Alexa555 label), hybridizing with the
DNA of the above-mentioned strains, prepared such that the final
concentration in hybridization buffer (20 mM Tris-HCl (pH 7.4), 0.9
M NaCl, 0.1% SDS, 20% formamide) was 10 .mu.g/mL.
[0094] The tissue sections were washed with washing buffer (20 mM
Tris-HCl (pH 7.4), 0.9 M NaCl) for 10 minutes and with PBS for 10
minutes three times and were then stained with Phalloidin-iFluor
(manufactured by Abcam plc.). After washing with PBS for 10 minutes
three times, the sections were mounted in Prolong anti-fade
mounting media with DAPI (manufactured by Life Technologies).
Microscopic observation was performed with a BIO-REVO BZ-9000
fluorescence microscope (manufactured by Keyence Corporation). The
results are shown in FIG. 5b. It was observed that the bacteria
stained in red in the micrographs invaded the large intestinal
epithelium mucous membrane in the 3-mix gnoto and KP gnoto, and
some of the bacteria further invaded the large intestinal
epithelium. In particular, the invasion was remarkable in the 3-mix
gnoto. In contrast, the invasion was only slight in the PM+EG gnoto
and was not observed in the control SPF.
[Example 6] Evaluation 1 of Ability to Form Pore on Large
Intestinal Epithelium of KP Stain
(1) Production of Monolayer Organoid Co-Culture System
[0095] Healthy human colon organoids were embedded in Matrigel
(Corning) and were three-dimensionally cultured in Advanced
DMEM/F12 culture solution (containing penicillin/streptomycin, 10
mM HEPES, 2 mM GlutaMAX, 1.times.B27 (manufactured by Life
Technologies), 1 mM N-acetylcysteine (manufactured by FUJIFILM Wako
Pure Chemical Corporation), 10 nM Gastrin I (manufactured by
Sigma-Aldrich Co. LLC), 50 ng/mL human recombinant EGF, 0.5 .mu.M
A83-01, 3 .mu.M SB202190, 50% Afamin-Wnt3a (Afm-W) complex
condition medium (CM, v/v), R-spondin 1 (R)-CM (10% v/v), and
Noggin (N)-CM (10% v/v)).
[0096] The three-dimensionally cultured human colon organoids were
cultured for at least one day in a medium containing 10 .mu.M
Y-27632 and containing Afm-Wnt, R-spondin 1, Noggin, EGF, A83-01,
and SB202190 and were then separated and seeded in a ThinCert
24-well plate (manufactured by Greiner Bio-One International GmbH)
coated with 10% Cellmatrix type I-C (manufactured by Nitta Gelatin
Inc.) and having a pore size of 0.4 .mu.m. 2 to 3 days after the
seeding, the medium was replaced by a condition medium not
containing Afm-Wnt and SB202190Y-27632. The colonic epithelium was
washed with Advanced DMEM/F12 culture solution before seeding of
the strain, and antibiotic-free DM was added thereto to produce a
monolayer organoid co-culture system.
(2) Co-Culture of Strain and Large Intestinal Epithelial Cell and
Morphological Analysis
[0097] KP strains (KP-P1 and KP-P5) isolated from the mesenteric
lymph nodes of mice administered a fecal sample derived from
Psc/Uc, enterohemorrhagic Escherichia coli O-157:H7, and a KP
strain (KP JCM1662) obtained from Riken BioResource Research Center
were added to the monolayer organoid co-culture systems each at a
concentration of 1.times.10.sup.5 CFU, followed by culturing for 8
hours. As a control, PBS was used. The co-cultured large intestinal
epithelium was collected and pre-fixed in 5% glutaraldehyde
(manufactured by TCI Chemicals) dissolved in PBS at 4.degree. C.
overnight. The pre-fixed sample was post-fixed in 1% osmium
tetraoxide dissolved in PBS for 1 hour. The post-fixed sample was
dehydrated with ethanol and coated by gold sputtering and was then
observed with a scanning electron microscope ("VHX-D510 scanning
electron microscope," manufactured by Keyence Corporation)" in a
high-vacuum mode. The results are shown in FIG. 6a.
[0098] In addition, the pores having a pore size of 10 .mu.m or
more on the large intestine were counted by observation of four
independent positions of the large intestinal epithelium. The
experiment was repeated 3 to 6 times. The results are shown in FIG.
6b. The results demonstrated that the KP strains (KP-P1 and KP-P5)
of mice receiving a fecal sample derived from Psc/Uc formed pores
on the large intestinal epithelium, but the JCM1662 strain formed
no pores. In addition, a same test using other KP strains
demonstrated that seven KP strains (JCM20034, ATCC BAA1705, ATCC
BAA2552, ATCC700721, JCM20348, JCM20694, and JCM20507) formed pores
on the large intestinal epithelium, but four strains (JCM1662,
JCM1663, JCM1664, and ATCC700603) formed no pores on the large
intestinal epithelium.
[0099] Furthermore, the co-cultured large intestinal epithelial
cells were triple-stained with an anti-cleaved caspase-3 antibody
(manufactured by Cell Signaling Technology, Inc.), a filamentous
actin stain phalloidin (manufactured by Thermo Fisher Scientific
Inc.), and a DNA stain Hoechst 33324 (manufactured by Thermo Fisher
Scientific Inc.), and apoptosis cells were observed with a confocal
laser microscope (SP5, manufactured by Leica). The results are
shown in FIG. 6c. It is inferred from the results that KP-P1 comes
into direct contact with the large intestinal epithelium and
induces apoptosis.
(3) Genome Sequencing of KP Strain
[0100] The whole genome sequencing of the KP strain having or not
having an ability to form a pore on the large intestinal epithelium
was performed by whole-genome shotgun sequencing using PacBio RSII
and Illumina MiSeq sequencers, and comparative analysis was
performed. The results are shown in FIG. 7. The results
demonstrated that in 97 orthologous genes, the KP strain having an
ability to form a pore on the large intestinal epithelia includes
genes involved in a type 6 secretion system (T6SS) and reactive
oxygen species (ROS) decomposition.
[Example 7] Evaluation 2 of Ability to Form a Pore on the Large
Intestinal Epithelium of KP Stain
(1) Histochemical Observation of Large Intestine
[0101] Humanized gnotobiotic mice (3-mix gnoto) were produced by
administering to GF mice three strains: PM, EG, and KP-P1 which is
a KP strain forming pores on the large intestinal epithelia, and
humanized gnotobiotic mice (m3-mix gnoto) were produced by
administering to mice three strains: PM, EG, and KP JCM1662 which
is a KP strain not forming pores on the large intestinal epithelia.
On the 21st day from the administration, the ileum was collected
from each mouse and was subjected to histochemical observation in
the same manner as in Example 5 (2). The results are shown in FIG.
8b. The results demonstrated that 3-mix gnoto invaded the large
intestine mucous membrane and the epithelial tissue, whereas m3-mix
gnoto only slightly invaded the large intestine mucous
membrane.
(2) Measurement of Serum LPS Level
[0102] Blood was collected from GF mice, SPF mice, 3-mix gnoto, and
m3-mix gnoto, and samples were prepared using ToxinSensor
Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit
(manufactured by GenScript Biotech Corporation) in accordance with
the manual of the product. The absorbance at 540 nm was measured
using FilterMax F3 Multi-Mode Microplate Reader (manufactured by
Molecular Devices, LLC.). The results are shown in FIG. 8c. The
results demonstrated that the serum LPS level in 3-mix
significantly increased compared to those in the GF mice, SPF mice,
and m3-mix groups.
(3) Evaluation of Induction of Th17 Cells
[0103] The livers of GF mice, 3-mix gnoto, and m3-mix gnoto were
analyzed by flow cytometry using an anti-CD4 antibody (APC-cy7,
BV510), an anti-IL-17 antibody, and an anti-ROR.gamma.t antibody
(manufactured by BD Pharmingen) in the same manner as in Example 2
(3). The results are shown in FIG. 8d. The results demonstrated
that 3-mix gnoto significantly induced Th17 cells compared to GF
mice and m3-mix gnoto.
[Example 8] Evaluation of Pathogenicity of KP Strain-Induced Th17
Cells
(1) Evaluation of Induction of Th17 Cells by ROR.gamma.t Inverse
Agonist Treatment
[0104] 3-mix gnoto was fed on a diet containing an ROR.gamma.t
inverse agonist (ROR.gamma.t IA) or water as a control and DDC
every day for 2 weeks 14 days after the administration of the
strain (FIG. 9a). Subsequently, the liver of each mouse was
subjected to flow cytometric analysis using an anti-CD4 antibody
(APC-cy7, BV510), an anti-IL-17 antibody, an anti-IFN-.gamma.
antibody, and an anti-ROR.gamma.t antibody (manufactured by BD
Pharmingen) in the same manner as in Example 2 (3). The results are
shown in FIG. 9b.
[0105] In addition, serum alkaline phosphatase and serum total
bilirubin were measured. The results are shown in FIG. 9c in the
same manner as in Example 3 (1). Furthermore, sections of the liver
were stained with hematoxylin-eosin and Masson-trichrome and were
subjected to microscopic observation in the same manner as in
Example 3 (2). The results are shown in FIG. 10.
[0106] It is inferred from these results that Th17 cells induced by
strains in the liver play a pathogenetic role in the development of
bile duct disorder induced by DDC.
[0107] The contents of Japanese Patent Application No. 2018-082192
(application date: Apr. 23, 2018), including the specification,
claims, drawings, and abstract, are incorporated herein by
reference in its entirety.
* * * * *