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The specific mechanisms that mediate CD4(+) T-cell-mediated liver injury have not been fully elucidated. CD4(+) invariant natural killer T (iNKT) cells are required for liver damage in some mouse models of hepatitis, while the chemokine receptors CXCR3 and CCR5 are considered dominant Th1 chemokine receptors involved in Th1 trafficking in inflammatory conditions. BALB/c-Tgfb1(-/-) mice spontaneously develop Th1 hepatitis. Here, we directly test the hypotheses that iNKT cells or the Th1-cell chemokine receptors CXCR3 and CCR5 are required for development of liver disease in Tgfb1(-/-) mice. Tgfb1(-/-) mouse livers exhibited significant increases in iNKT cells and in ligands for CXCR3 or CCR5. Tgfb1(-/-) mice were rendered deficient in iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5, by cross-breeding with appropriate knockout mice. Tgfb1(-/-) mice developed severe liver injury, even in the absence of functional CD1d/iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5. Liver CD4(+) T cells accumulated to high numbers, and spleen CD4(+) T-cell numbers declined, regardless of the functionality of the CXCR3/CCR5 response pathways. Similarly, dendritic cells and macrophages accumulated in Tgfb1(-/-) livers even when CXCR3 and CCR5 were knocked out. Th1-associated cytokines (IFN-γ, TNF-α, IL-2) and chemokines (CXCL9, CXCL10) were strongly overexpressed in Tgfb1(-/-) mice despite knockouts in CD1d, CXCR3, or CCR5. These studies indicate that the cellular and biochemical basis for CD4(+) T-cell-mediated injury in liver can be complex, with myriad pathways potentially involved.

Myeloid derived suppressor cells (MDSC) were first described nearly two decades ago. Until recently, however, descriptions of MDSC populations were found almost exclusively in animal models of cancer or in cancer patients. Over the last few years, an increasing number of reports have been published describing populations of myeloid cells with MDSC-like properties in murine models of autoimmune disease. In contrast to the proposed deleterious role of MDSC in cancer - where these cells likely inhibit tumor immunity - in the context of autoimmunity, MDSC have the potential to suppress the autoimmune response, thereby limiting tissue injury. A logical corollary of this hypothesis is that a failure of endogenous MDSC to appropriately control autoimmune T cell responses in vivo may actually contribute to the pathogenesis of autoimmune disease.

T cells are the proximal agents of parenchymal liver damage in inflammatory liver diseases such as autoimmune hepatitis (AIH) and viral hepatitis. In AIH, CD4+ T cells infiltrate liver parenchyma (1) and release hepatotoxic cytokines such as IFN-γ and TNF-α (2, 3). IFN-γ expression by ex vivo cultured T cells strongly correlates with disease activity (4), implicating type 1 T cell responses in hepatocellular damage. In hepatitis C virus (HCV) infection, liver pathology results from the activity of T cells producing IFN-γ within liver parenchyma, since HCV is not cytopathic (5–7). IFN-γ is essential for parenchymal damage in mouse models of T cell mediated liver injury, including Concanavalin A -induced liver injury (8), and spontaneous liver injury in BALB/c TGF-β1 knockout mice (9). A common theme, therefore, in immune mediated liver injury is pathology associated with activated T cells producing IFN-γ. Given the potential for liver damage by activated Th1 cells, it is important to identify mechanisms that regulate their activity. A variety of liver resident cells participate in the regulation of T cells, including Treg, dendritic cells, Kupffer cells, NK cells, NKT cells, stellate cells, and liver sinusoidal epithelial cells (10). Whether regulatory immunocytes accumulate in liver in response to activated T cells is not known. Such cells may represent an important negative feedback mechanism mitigating pathology mediated by T cell activation. It is reasonable to postulate that inflammatory pathology in liver is attributable both to aberrant activation of T cells and to a deficit in appropriate counter-regulatory mechanisms. Studies emerging from the field of tumor immunity show that tumor-associated inflammation induces the development and accumulation of myeloid-lineage cells with immunomodulatory activity. Termed myeloid derived suppressor cells (MDSC), these pleiomorphic cells are capable of suppressing T cell proliferation and subjugating T cell mediated immunity (11, 12). MDSC comprise a heterogenous group of myeloid cells, employing a variety of mechanisms to inhibit T cell responses. Murine MDSC are operationally defined as CD11b+Gr1+ myeloid cells that suppress T cell proliferation (11, 12). While MDSC have been most extensively described in the context of tumors, recent studies show their involvement in inflammatory responses not associated with tumors (13, 14). MDSC home to liver in tumor-bearing mice (15), and hepatocellular carcinoma, like other solid tumors, exhibit associated populations of MDSC (16, 17), but little is otherwise known about MDSC in liver, particularly in inflammatory pathology. Here, we demonstrate in the BALB/c TGF-β1 knockout mouse model that Th1 cells, through release of IFN-γ, drive accumulation in liver of an MDSC population that can effectively inhibit T cell proliferation through a mechanism involving expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO).

Background/aims BALB/c mice with a homozygous deficiency in the Tgfb1 gene are a model of fulminant autoimmune hepatitis (AIH), spontaneously and rapidly developing Th1-mediated IFN-gamma-dependent necroinflammatory liver disease. We sought to understand the molecular basis for fulminant Th1 liver disease and the specific role of the Ifng gene. Methods Global gene expression in livers from BALB/c Tgfb1(-/-) mice with and without an intact Ifng gene was assessed by microarray analysis. Expression patterns were confirmed by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology clustering analysis was performed to identify altered pathways. The contributions of Ifng to altered expression pathways were quantified. Results Over 100 genes were strongly (>10-fold) upregulated, most encoding proteins involved in immune function/response. Chemokines were the most prominently upregulated group, with eight chemokine genes upregulated >10-fold. Ifng was necessary for the upregulation of CXC chemokines gene, but not of CC chemokine genes. By quantitative analysis, Ifng's role in liver gene upregulation varied greatly among overexpressed genes. Conclusions Gene expression changes indicate a particularly important and heretofore unappreciated role for chemokines in fulminant AIH. Ifng has an important role in expression of some but not all genes. Ifng is dichotomous in the regulation of distinct chemokine subfamilies: specifically, Ifng is critical for overexpression of specific CXCL genes but dispensable for overexpression of specific CCL genes. These results provide a clearer understanding of the role of Ifng in the molecular basis of necroinflammatory liver disease.

Fulminant inflammation in the liver is often accompanied by the accumulation of IFN-gamma-producing T cells. The BALB/c-Tgfb1(-/-) mouse exhibits extensive, spontaneously developing necroinflammation in the liver, accompanied by the accumulation of IFN-gamma-producing CD4(+) and CD8(+) T cells. Liver damage depends on the presence of an intact Ifng gene. We determined the relevant cellular source(s) of IFN-gamma. In Tgfb1(-/-) liver, CD4(+) T cells were more numerous than CD8(+) T cells and NK cells, and produced more IFN-gamma. Depletion of CD4(+) T cells eliminated both the elevation in plasma IFN-gamma and aspartate aminotransferase, whereas depletion of CD8(+) T cells did not. Rag1(-/-)Tgfb1(-/-) mice exhibited neither IFN-gamma elevation nor tissue damage, indicating that NK cells are not sufficient. IFN-gamma was required for strong overexpression of class II genes but not for CD4(+) T cell activation, oligoclonal expansion, or accumulation in the liver. The T cell inhibitory molecule PD-L1 was strongly expressed in Tgfb1(-/-) livers, ruling out a lack of PD-L1 expression as an explanation for aberrant liver T cell activation. Finally, whereas Tgfb1(-/-) CD4(+) T cells overexpressed Fas ligand, hepatocellular damage was observed in Fas(lpr/lpr)Tgfb1(-/-) mice, indicating that liver pathology is Fas independent. We conclude that liver damage in this model of fulminant autoimmune hepatitis is driven by CD4(+) T cell production of IFN-gamma, is independent of both CD8(+) T cells and the Fas ligand/Fas pathway, and is not explained by a lack of PD-L1 expression.

Glycosaminoglycans (GAGs) located on cellular membranes and the extracellular matrix (ECM) are able to interact with chemokines and pro-inflammatory cytokines, leading to local cytokine/chemokine accumulation. The tissue-bound cytokines/chemokines function in promoting leukocyte migration and activation, contributing to local inflammation. Hence, targeting of GAG–cytokine interactions may provide an avenue for the attenuation of inflammatory responses. A cationic peptide (MC2) derived from the heparin-binding sequence of mouse IFN-γ was previously shown by our laboratory to delay allograft rejection in an animal model. In order to further investigate potential anti-inflammatory properties of the MC2 peptide, we have studied its activity in an acute peritoneal inflammation model. Groups of C57Bl/6 mice were injected intraperitoneally with either ConA or thioglycollate and treated with saline (control), the MC2 peptide or two control cationic peptides, poly- l -lysine (PLL) and poly- l -arginine (PLA). Treatment with the MC2 peptide, but not PLA or PLL, resulted in statistically significant reductions in total cell numbers, concentration of total proteins and concentrations of pro-inflammatory cytokines (TNFα, IL-6 or IL-1β) in peritoneal lavage fluids, without alterations to the qualitative cellular composition of the exudate. These results suggest that targeting GAG–cytokine interaction is a viable approach to reduce inflammation.

Summary Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3 , caspase-8 , or fadd . However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1 −/− mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1 −/− tnfr1 −/− mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.