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. 2016 Jan;63(1):185-96.
doi: 10.1002/hep.27927. Epub 2015 Aug 10.

Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis (V体育平台登录)

James H Tabibian  1   2 , Steven P O'Hara  1   2 , Christy E Trussoni  1   2 , Pamela S Tietz  1   2 , Patrick L Splinter  1   2 , Taofic Mounajjed  3 , Lee R Hagey  4 , Nicholas F LaRusso  1   2
Affiliations

Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis

James H Tabibian et al. Hepatology. 2016 Jan.

Abstract

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ-free (GF) mutltidrug resistance 2 knockout (mdr2(-/-) ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2(-/-) mice. Mdr2(-/-) mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age-matched CV mdr2(-/-) mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM) VSports手机版. Cholangiocyte senescence was assessed by p16(INK4a) in situ hybridization in liver tissue and by senescence-associated β-galactosidase staining in a culture-based model of insult-induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2(-/-) (P < 0. 01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2(-/-) mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2(-/-) mice (P < 0. 01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2(-/-) mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. .

Conclusions: GF mdr2(-/-) mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro V体育安卓版. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC. .

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Disclosures, conflicts of interest: none

Figures

Figure 1
Figure 1
Germ-free (GF) mdr2−/− mice have significantly more severe serum biochemical abnormalities. Serum A) alkaline phosphatase, B) aspartate aminotransferase, C) bilirubin, and D) bile acids, are all significantly higher in GF compared to CV mdr2−/− mice (*p<0.001), and E) alanine aminotransferase shows a trend toward being significantly higher in GF mdr2−/− mice (**p=0.07). These differences are indicative of more severe hepatobiliary disease in GF mdr2−/− mice.
Figure 2
Figure 2
Germ-free (GF) mdr2−/− mice demonstrate more hepatic fibrosis histologically, morphometrically, and biochemically. Representative images of Masson’s Trichrome-stained liver sections of A) CV mdr2−/− and B) GF mdr2−/− mice. C) Based on histopathologic evaluation of Trichrome-stained liver sections, a significantly higher proportion of GF compared to CV mdr2−/− mice exhibit advanced fibrosis (≥ Stage 3) by 60 days (p<0.001). D) Morphometric assessment of Picrosirius red-stained liver sections demonstrates a significantly higher percentage of fibrotic area in GF compared to CV mdr2−/− mice (p=0.016). E) Biochemical assessment of hepatic fibrosis confirms significantly increased hydroxyproline concentration in GF compared to CV mdr2−/− mice (p=0.035). Taken together, these data provide multimodal evidence of more advanced hepatic fibrosis in GF mdr2−/− mice.
Figure 3
Figure 3
Germ-free (GF) mdr2−/− mice exhibit significantly more ductular reaction. Representative images of H&E-stained liver sections of A) CV mdr2−/− and B) GF mdr2−/− mice showing periportal regions and ductular reaction. C) Based on histopathologic evaluation, there appears to be significantly more ductular reaction (arrows) in GF compared to CV mdr2−/− mice (p<0.001), with all but two GF mdr2−/− mice demonstrating grade 4 ductular reaction, an indicator of severe biliary injury.
Figure 4
Figure 4
Germ-free (GF) mdr2−/− mice demonstrate significantly more ductopenia by light microscopy and confocal immunofluorescence microscopy (IFM). Representative light microscopy images of H&E-stained A) CV mdr2−/− and B) GF mdr2−/− mouse liver sections; black arrowheads denote bile duct branches (absent in GF mdr2−/− mouse liver image). Histopathologic evaluation of C) CV and D) GF mdr2−/− liver sections reveals a significantly greater proportion of ductopenia in GF mdr2−/− mice (p=0.004), with all but one being ductopenic. E & F. IFM of CK-19 (red) of mouse liver sections confirms markedly increased ductular reaction and ductopenia in GF compared to CV mdr2−/− mice.
Figure 5
Figure 5
CD4+ T-cells are present in CV and GF mdr2−/− mice, while GF mice exhibit significantly increased numbers of neutrophils. A. Representative light microscope images of IHC for CD4+ T-cells in CV and GF mdr2−/− mice. CD4+ cells accumulated in portal tracts of both groups of mice. B. Quantitative analysis (5 mice per group, 10 portal tracts per mouse liver) demonstrated similar numbers of CD4+ cells/20x field. C. Representative light microscope images of IHC for neutrophils. The neutrophils also aggregated in portal tracts in both CV and GF mdr2−/− mice. D. Quantitative analysis demonstrated increased numbers of neutrophils in the GF mdr2−/− mice (*, p< 0.001).
Figure 6
Figure 6
Cholangiocyte senescence is increased in germ-free (GF) mdr2−/− mice and can be abrogated in vitro by the commensal bacterial metabolite, ursodeoxycholic acid. Representative images of A) WT, B) CV mdr2−/−, and C) GF mdr2−/− mouse liver p16INK4a FISH, a well-established marker of cellular senescence used in tissue section assessment (p16INK4a, green; DAPI, blue). D. Quantitation p16INK4a fluorescence intensity demonstrates significantly more cholangiocyte senescence in GF mdr2−/− mice than both CV mdr2−/− and WT mice (p<0.001). E. In vitro model of cholangiocyte senescence (exposure to 50 nM H202, a known inducer of cellular senescence) demonstrates that secondary bile acids (known metabolites of commensal bacteria) can protect against cholangiocyte injury and senescence. Cells were treated every 48 hours with or without deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), or ethanol vehicle (Veh). Representative bright field and fluorescence microscopy images following 10 days of treatment demonstrating increased SA-β-gal stain (blue) positivity (black arrows), a well-established in vitro marker of cellular senescence. F. Quantitation of SA-β-gal stain-positive cells demonstrates that treatment with UDCA, but not DCA, results in significant abrogation of H202-induced NHC senescence (41% reduction, p=0.02).
Figure 7
Figure 7
Conceptual framework for the role of the microbiota in the etiopathogenesis of PSC. In this working model, the cholangiocyte is exposed to a biliary insult, to which it responds through increased expression of proinflammatory mediators and induction of epithelial repair processes (e.g. proliferation), i.e. a phenotype known as the reactive cholangiocyte. Intricate cellular crosstalk between resident and recruited hepatobiliary cells, including hepatocytes, progenitor cells, fibroblasts, leukocytes, and/or cholangiocytes themselves, is subsequently increased in an attempt to resolve biliary injury. The dynamics and outcomes of these signaling processes are modified by and depend on the composition of the microbiota and its metabolites. In the setting of inadequate cytoprotective commensal microbial metabolites (coupled with host immunogenetic susceptibility), there is impaired resolution of biliary injury and consequent progression to chronic hepatobiliary disease. Chronic hepatobiliary disease, most notably PSC, is characterized and may be driven by increased cholangiocyte senescence, a pathobiologial cellular phenotype which can progress to a state of hypersecretion of pro-fiboinflammatory and oncogenic mediators known as the senescence-associated secretory phenotype (SASP). The SASP may be associated may lead to the clinico-pathologic sequelae of PSC, including liver failure and cholangiocarcinoma, and is an area of ongoing investigation.
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