Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-Cell Receptor–Positive Cells and Pathogenesis of Cholestatic Liver Disease

Dana Tedesco, Manoj Thapa, Chui Yoke Chin, Yong Ge, Minghao Gong, Jing Li, Sanjeev Gumber, Patrick Speck, Elizabeth J. Elrod, Eileen M. Burd, William H. Kitchens, Joseph F. Magliocca, Andrew B. Adams, David S. Weiss, Mansour Mohamadzadeh, Arash Grakoui

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

Background & Aims: Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells. Methods: We performed studies with Mdr2-/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ+ cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR+ cells. Results: Mdr2–/– mice had collagen deposition around hepatic bile ducts and periportal–bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2–/– mice had increased numbers of IL17A+ γδTCR+ cells—particularly of IL17A+ Vγ6Jγ1 γδ TCR+ cells. Fecal samples from Mdr2–/– mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2–/– mice also had increased intestinal permeability. The γδ TCR+ cells isolated from Mdr2-/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2-/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR+ cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17. Conclusions: In Mdr2-/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR+ cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients.

Original languageEnglish (US)
Pages (from-to)2178-2193
Number of pages16
JournalGastroenterology
Volume154
Issue number8
DOIs
StatePublished - Jun 2018
Externally publishedYes

Bibliographical note

Funding Information:
Funding This project was supported by National Institutes of Health (NIH) grants R01AI070101 , R01AI124680 , R01AI126890 , and R01AI136533 to A.G., and ORIP/OD P51OD011132 (formerly NCRR P51RR000165 ) to the Yerkes National Primate Research Center (AG). M.T. is supported by National Institute of Diabetes, and Digestive and Kidney Diseases Mentored Career Development Award (K01DK109025). This work was facilitated by the Immunology and Flow Cytometry Core of the Center for AIDS Research at Emory University ( P30AI050409 ). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Funding Information:
Commensal microbes drive IL17 responses in the gastrointestinal tract, which has been implicated in maintaining intestinal integrity and homeostasis.41,42> Although beneficial in the gastrointestinal tract, intrahepatic IL17 responses can perpetuate destruction of biliary networks and hepatic architecture via hypersensitization of hepatic stellate cells to profibrotic mediators.12 Because IL17+ γδ T cells are capable of expanding to inflammation,12,43> it is possible that translocation of gut commensals act in concert with other mechanisms to drive the expansion of pathogenic γδ T cells. Furthermore, germ-free mice that lack commensal microflora have a range of immune defects that include ineffective regulatory T cells, and increased Th2 CD4+ T-cell differentiation.44 In combination, these features of the germ-free mouse T-cell compartment would favor perpetuation of liver damage45 and may account for the exacerbation of liver injury reported by Tabibian and colleagues.46 However, our findings revealed that antibiotic treatment in Mdr2-/- mice from 8 weeks to 25 weeks of age does not alter liver disease or IL17 production (unpublished data). Moreover, co-housing of Mdr2-/- with WT mice for 8 weeks revealed no alteration in liver disease and IL17 production by γδ T cells, and a marked enrichment of Lactobacillus was still found in co-housed knockout animals (Supplementary Figure 11). These findings indicate that the genetic deficiency leading to cholestasis is critical in inducing gut dysbiosis and the enrichment of Lactobacillus in these mice; and co-housing is not sufficient to overcome the genetic defect in cholestasis. This is supported by clinical reports that suggest treating PSC patients with metronidazole; an antibiotic that targets anaerobic microbes, such as Lactobacillus47 transiently corrects plasma levels of liver enzymes.40 However, this strategy was not curative, as the study participants still required liver transplantation.48

Publisher Copyright:
© 2018 AGA Institute

Keywords

  • Immune Response
  • Intestinal Microbiota
  • Liver Disease Pathogenesis
  • PSC

Fingerprint

Dive into the research topics of 'Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-Cell Receptor–Positive Cells and Pathogenesis of Cholestatic Liver Disease'. Together they form a unique fingerprint.

Cite this