RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Dendritic cell CD83 homotypic interactions regulate inflammation and promote mucosal homeostasis.

Dendritic cells (DCs) form an extensive network in the intestinal lamina propria, which orchestrates the mucosal immune response. Alterations in DC function can predispose to inflammatory bowel disease, although by unknown mechanisms. We show that CD83, a highly regulated DC cell surface protein, modulates the immune response to prevent colitis. Mice with a conditional knockout of CD83 in DCs develop exacerbated colitis following dextran sodium sulfate challenge, whereas mucosal overexpression of CD83 inhibits DC inflammatory response and protects against colitis. These CD83 perturbations can be modeled in vitro where we show that CD83 homotypic interaction occurs via cell-cell contact and inhibits pro-inflammatory responses. CD83 knockdown or cytoplasmic truncation abrogates the effects of homotypic binding. We demonstrate that CD83 homotypic interaction regulates DC activation via the mitogen-activated protein kinase pathway by inhibiting p38α phosphorylation. Our findings indicate that CD83 homotypic interactions regulate DC activation and promote mucosal homeostasis.

Conditional activation of Pik3ca(H1047R) in a knock-in mouse model promotes mammary tumorigenesis and emergence of mutations.

Oncogenic mutations in PIK3CA, which encodes the phosphoinositide-3-kinase (PI3K) catalytic subunit p110α, occur in ∼25% of human breast cancers. In this study, we report the development of a knock-in mouse model for breast cancer where the endogenous Pik3ca allele was modified to allow tissue-specific conditional expression of a frequently found Pik3ca(H1047R) (Pik3ca(e20H1047R)) mutant allele. We found that activation of the latent Pik3ca(H1047R) allele resulted in breast tumors with multiple histological types. Whole-exome analysis of the Pik3ca(H1047R)-driven mammary tumors identified multiple mutations, including Trp53 mutations that appeared spontaneously during the development of adenocarinoma and spindle cell tumors. Further, we used this model to test the efficacy of GDC-0941, a PI3K inhibitor, in clinical development, and showed that the tumors respond to PI3K inhibition.

Opposing consequences of IL-23 signaling mediated by innate and adaptive cells in chemically induced colitis in mice.

The interleukin-23 (IL-23) pathway has emerged as a promising therapeutic target for inflammatory bowel disease. Although the pathogenic role of IL-23 receptor (IL-23R) on T lymphocytes is well established, its function on innate immune cells has not been thoroughly examined. Here we investigate the consequence of IL-23R deletion in dextran sulfate sodium (DSS)-induced colitis. In IL23R(-/-) and IL23p19(-/-) mice, we observed decreased weight loss and reduced leukocyte infiltrate following DSS exposure. Surprisingly, when the IL-23R(-/-) allele was crossed into Rag2(-/-) mice, we observed exacerbated disease, increased epithelial damage, reduced pSTAT3 in the epithelium, and delayed recovery of IL23R(-/-)Rag2(-/-) mice. This phenotype was rescued with exogenous IL22-Fc, and epithelial pSTAT3 was restored. Depletion of Thy1(+) innate lymphoid cells eliminated the majority of IL-22 production in the colon lamina propria of DSS-treated Rag2(-/-) mice, suggesting that these are the major IL-23 responsive innate cells in this context. In summary, we provide evidence for opposing consequences of IL-23R on innate and adaptive lymphoid cells in murine colitis.

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor.

Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin-mediated fibrin degradation by removing the C-terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completely heal the cutaneous wounds was slightly delayed in TAFI-deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI-deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re-epithelialization and scar formation, occurs in TAFI-deficient mice, TAFI appears to be important for appropriate organization of the healing process.

Activation and accumulation of B cells in TACI-deficient mice.

The tumor necrosis factor (TNF)-related ligand B lymphocyte stimulator (BLyS) binds two TNF receptor family members, transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI) and B cell maturation molecule (BCMA). Mice that are transgenic for BLyS show B cell accumulation, activation and autoimmune lupus-like nephritis. The existence of at least two distinct BLyS receptors raises the question of the relative contribution of each to B cell functions. We therefore generated mice that were deficient in TACI. TACI-/- mice showed increased B cell accumulation and marked splenomegaly. Isolated TACI-/- B cells hyperproliferated and produced increased amounts of immunoglobulins in vitro. In vivo antigen challenge resulted in enhanced antigen-specific antibody production. Thus, TACI may play an unexpected inhibitory role in B cell activation that helps maintain immunological homeostasis.