Immune‐mediated liver injury in hepatitis is due to activated T cells producing interferon‐γ (IFN‐γ). It is important to identify negative feedback immune mechanisms that can regulate T cell activity. In this study, we demonstrate that liver inflammation mediated by type 1 T helper (Th1) cells can induce the accumulation of myeloid‐derived suppressor cells (MDSCs), pleiomorphic cells capable of modulating T cell–mediated immunity, that heretofore have been studied almost exclusively in the context of tumor‐associated inflammation. Mice deficient in the gene encoding transforming growth factor‐β1 (Tgfb1−/− mice) acutely develop liver necroinflammation caused by IFN‐γ–producing clusters of differentiation 4–positive (CD4+) T cells. Liver Th1 cell accumulation was accompanied by myeloid cells expressing CD11b and Gr1, phenotypic hallmarks of MDSCs. Isolated Tgfb1−/− liver CD11b+Gr1+ cells were functional MDSCs, readily suppressing T cell proliferation in vitro. Pharmacologic inhibitors of inducible nitric oxide (NO) synthase completely eliminated suppressor function. Suppressor function and the production of NO were dependent on cell–cell contact between MDSCs and T cells, and upon IFN‐γ, and were specifically associated with the “monocytic” CD11b+Ly6G− Ly6Chi subset of liver Tgfb1−/− CD11b+ cells. The rapid accumulation of CD11b+Gr1+ cells in Tgfb1−/− liver was abrogated when mice were either depleted of CD4+ T cells or rendered unable to produce IFN‐γ, showing that Th1 activity induces MDSC accumulation. Conclusion: Th1 liver inflammation mobilizes an MDSC response that, through the production of NO, can inhibit T cell proliferation. We propose that MDSCs serve an important negative feedback function in liver immune homeostasis, and that insufficient or inappropriate activity of this cell population may contribute to inflammatory liver pathology. (HEPATOLOGY 2010;)

Neither the early postnatal development of the liver Treg compartment nor the factors that regulate its development has been characterized. We compared the early developmental patterns of Treg cell accumulation in murine liver, thymus, and spleen. A FoxP3EGFP reporter mouse was employed to identify Treg cells. Mononuclear cells were isolated from organs postnatally, stained for CD4, and examined by flow cytometry to enumerate FoxP3+CD4hi cells. To assess roles for TGF-β1, MyD88, and TLR2, gene-specific knockout pups were generated from heterozygous breeders. To test the role of commensal bacteria, pregnant dams were administered antibiotics during gestation and after parturition. The pattern of appearance of Treg cells differed in liver, spleen, and thymus. Notably, at 1-2 weeks, the frequency of CD4hi FoxP3+ T cells in liver exceeded that in spleen by 1.5- to 2-fold. The relative increase in liver Treg frequency was transient and was dependent upon TGF-β1 and MyD88, but not TLR2, and was abrogated by antibiotic treatment. A relative increase in liver Treg frequency occurs approximately 1-2 weeks after parturition that appears to be driven by colonization of the intestine with commensal bacteria and is mediated by a pathway that requires TGF-β1 and MyD88, but not TLR2.

Th1 responses in the liver are the basis for parenchymal damage in autoimmune hepatitis (AIH). The cellular pathways that inhibit liver Th1 responses are not well understood. BALB/c mice lacking TGF-β1 spontaneously and rapidly develop acute Th1 cell-mediated necroinflammatory hepatitis. Liver damage requires interferon gamma (IFN-γ) production by CD4+ T cells. We hypothesized that the Th1 response would also activate pathways capable of down-regulating T cell responses, specifically, a myeloid derived suppressor cell (MDSC) population. Myeloid cells were indeed abundant in liver parenchyma in histological sections of Tgfb1KO livers. Characterization of this cell type showed that they were CD11b+Gr-1+ cells that strongly suppressed T cell proliferation in vitro, thus MDSCs. By contrast, Tgfb1WT liver CD11b+Gr-1+ cells were much less abundant and exhibited no T cell suppression capabilities. Suppression required nitric oxide (NO) and cell-cell contact, but not arginase, PDL1, or H2O2. Tgfb1KO MDSCs suppressed T cell proliferation by inducing apoptosis. Further studies using Tgfb1KO/IfngKO mice showed that IFN-γ was required both for MDSC accumulation in liver as well as for MDSC suppressor activity. These results demonstrate that, during hepatitis, Th1 cells activate through IFN-γ production an MDSC negative feedback pathway that can inhibit T cell responses through NO. Why this negative feedback pathway ultimately fails in AIH remains critical to elucidate.

Regulatory T cells (Treg) play a major role in the maintenance of immune tolerance and homeostasis. The liver microenvironment promotes immune tolerance, but very little is known about the development of the Treg compartment in liver. We examined the kinetics of Treg (FoxP3+CD4+ T cells) development in the post-natal mouse liver. Liver Tregs were first detected in liver at post-natal day 2 (P2). The Treg frequency in liver rapidly surged thereafter, peaking to 20-25% of liver CD4+ T cells at P8, over twice as frequent as in spleen (10%), and declined thereafter to reach an adult frequency of ~3% CD4+ T cells. The liver receives a large supply of its blood through the portal vein, which drains the intestine. We hypothesized that the early “surge” in liver Treg frequency occurs in response to colonization of the gut by commensal bacteria. In support of this hypothesis, the surge was partially abrogated in Myd88+/- mice and completely abrogated in Myd88-/- mice. The cytokine TGF-β1 has been previously shown to be important for several aspects of the Treg response pathway; the liver Treg surge was abrogated in Tgfb1-/- mice. We conclude that the early post-natal surge of Treg in liver is dependent on both MyD88 and TGF-β1. We suggest the following model: colonization of the newborn gut drives a surge in the liver Treg compartment, likely mediated by one or more TLR pathways, and TGF-β1 is an important effector cytokine of this response.

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