Inflammation is a key biological response with essential roles in protective immunity and auto- inflammatory diseases. Necroptosis is a non-apoptotic form of cell death with pro-inflammatory effects due to the release of cellular danger signals from ruptured plasma membranes. Receptor interacting protein kinase 3 (RIPK3) is an essential adaptor for necroptosis downstream of TNF-like death receptors and toll-like receptor 3 (TLR3) and TLR4. Ripk3-deficient mice are protected from many inflammatory diseases driven by inhibition of caspase 8 or the cellular inhibitor of apoptosis (IAP) proteins. These results led to the widely accepted view that RIPK3 drives inflammation through necroptosis. In contrast to these results, we found that Ripk3-deficient mice developed more severe chemical-induced colitis. The increased intestinal inflammation in Ripk3-deficient mice was not due to changes in cell death. Rather, Ripk3-deficient mice were impaired for expression of IL-1?, IL-23 and IL-22. Consistent with the known function of these cytokines in intestinal tissue repair, we found that injury-induced intestinal epithelial cell (IEC) proliferation was defective in Ripk3-deficient mice. These results indicate that immune sentinels in the intestinal lamina propria require RIPK3 to initiate a cytokine-driven tissue repair program. We found that in bone marrow derived dendritic cells (BMDCs), RIPK3 is required for IL-1? and IL-23 expression in response to stimulation with lipopolysaccharide (LPS), a TLR4 agonist. Similar to Ripk3-deficient mice treated with colitis-causing agent, reduced cytokine expression in Ripk3-deficient BMDCs was not due to reduced cell death. Rather, it was caused by defective activation of NF-?B activation and impaired processing of pro-IL-1?. Specifically, RIPK3 is required for optimal activation of RelB-p50 heterodimer to promote IL-23 expression. In contrast, RIPK3 is not required for activation of RelA, c-Rel or p52. In addition, RIPK3 also promotes caspase 1 and caspase 8-mediated processing of pro-IL-1?. These results indicate that RIPK3 critically controls RelB/p50-dependent IL-23 expression and pro-IL-1? processing and that these necroptosis-independent signaling functions are crucial for reparative inflammation. Based on these results, we propose two aims to investigate the molecular mechanisms by which RIPK3 regulates inflammatory cytokine expression in tissue repair responses.
In Aim 1, we will study the biochemical mechanisms that controls RIPK3-mediated RelB-p50 nuclear translocation in BMDCs and CX3CR1+ macrophages from intestinal lamina propria. We hypothesize that RIPK3 and TRIF binding via the RIP homotypic interaction motif (RHIM) is critical for RelB-p50 activation and cytokine gene transcription. Moreover, we hypothesize that unlike necroptosis, this RHIM-mediated interaction does not involve amyloid conversion, RIPK1 or RIPK3 kinase activities. However, it is controlled by reactive oxygen species (ROS). We further hypothesize that upon activation by TRIF, RIPK3 functions as a chaperon to facilitate RelB-p50 nuclear translocation through direct binding to RelB. In addition to RelB-p50, we hypothesize that RIPK3 also promotes pro-IL-1? processing through an atypical RIPK1-RIPK3-FADD-caspase 8 complex. Moreover, cFLIP-long form binding to this complex critically promotes IL-1? secretion by preventing caspase 8-mediated apoptosis.
In Aim 2, we will test the hypothesis that CX3CR1+ inflammatory macrophages in the intestinal lamina propria are the key immune effectors that produce IL-1? and IL-23 in a RIPK3-dependent manner. To test our hypothesis, we will use Cre-mediated recombination to generate mice with specific inactivation of RIPK3 in CD11c+ or CX3CR1+ cells. In addition, we will use mice expressing kinase inactive versions of RIPK1 and RIPK3 to determine the role of kinase activity in RIPK3-dependent cytokine expression and colitis induction. We will further test the hypothesis that correcting the early defect in cytokine expression and tissue repair can minimize chronic inflammation and susceptibility to inflammation-induced colon cancer. Successful completion of these studies will greatly enhance our understanding of the immunobiology of and relationship between RIPK3, dendritic cells/macrophages, necroptosis, tissue repair, inflammatory diseases and cancers.
Inflammation is a central biological response that links many diseases. Apoptosis is a form of cell death that tends to suppress inflammation. On the other hand, inflammatory cell death such as necroptosis promotes inflammation through the release of endogenous adjuvants. The receptor interacting protein kinase 3 (RIPK3) is a key factor in cell death by necroptosis. Unexpectedly, we found that RIPK3 can independently promote inflammation in the absence of necroptosis. In this application, we will explore the underlying molecular mechanism by which RIPK3 facilitates inflammatory cytokine expression and to define the specific immune cell types that are responsible for this necroptosis-independent function of RIPK3. We will further test the physiological significance of this novel function of RIPK3 in experimental models of colitis and tissue repair. These studies will reveal critical information about the basic biology of RIPK3 and provide salient information on the therapeutic potential of targeting RIPK3 in auto-inflammatory diseases.
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