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 post-birth. While 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.

Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.

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.

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;)

Fulminant inflammation in the liver is often accompanied by the accumulation of IFN-γ-producing T cells. The BALB/c-Tgfb1−/− mouse exhibits extensive, spontaneously developing necroinflammation in the liver, accompanied by the accumulation of IFN-γ-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-γ. In Tgfb1−/− liver, CD4+ T cells were more numerous than CD8+ T cells and NK cells, and produced more IFN-γ. Depletion of CD4+ T cells eliminated both the elevation in plasma IFN-γ and aspartate aminotransferase, whereas depletion of CD8+ T cells did not. Rag1−/−Tgfb1−/− mice exhibited neither IFN-γ elevation nor tissue damage, indicating that NK cells are not sufficient. IFN-γ 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 Faslpr/lprTgfb1−/− 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-γ, is independent of both CD8+ T cells and the Fas ligand/Fas pathway, and is not explained by a lack of PD-L1 expression.