RIPK3 restricts viral pathogenesis via cell death-independent neuroinflammation

Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3(-/-) mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3(-/-) mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS

Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells.

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current (ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation

RIPK3 activation leads to cytokine synthesis that continues after loss of cell membrane integrity

Necroptosis is a form of programmed cell death that is defined by activation of the kinase RIPK3 and subsequent cell membrane permeabilization by the effector MLKL. RIPK3 activation can also promote immune responses via production of cytokines and chemokines. How active cytokine production is coordinated with the terminal process of necroptosis is unclear. Here, we report that cytokine production continues within necroptotic cells even after they have lost cell membrane integrity and irreversibly committed to death. This continued cytokine production is dependent on mRNA translation and requires maintenance of endoplasmic reticulum integrity that remains after plasma membrane integrity is lost. The continued translation of cytokines by cellular corpses contributes to necroptotic cell uptake by innate immune cells and priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells

Potentiation of anti-tumor immunity by RIPK1/RIPK3-dependent necroptosis

Thesis (Ph.D.)--University of Washington, 2019Programmed cell death (PCD) represents a set of signaling processes evolved as a mechanism to eliminate cells, both during turnover under homeostatic conditions as well as removal of cells that have been compromised by insults such as infection or injury. Distinct cell death modalities, such as apoptosis, necroptosis, and pyroptosis, differ with respect to their signaling requirements, morphological characteristics, and molecular species produced or released by dying cells. The immune system has evolved to recognize various types of signals associated with cell death, allowing it to distinguish between physiological cell death at equilibrium and potential threats to the host such as infection. The immune system can then respond to these signals appropriately in order to either (a) dampen responses to promote immune tolerance in the context of homeostatic cellular turnover, or (b) promote inflammation that potentiates pathogen clearance in the context of infection. The mechanisms underlying the downstream immune responses mounted against dying cells have been primarily studied in settings of autoimmunity or pathogenic infection. However, characterization of these responses has been relatively poorly defined in models of tumor immunology, as these studies are complicated by the evasion of cell death signaling mechanisms commonly exhibited by transformed tumor cells. Tumor immunotherapy encompasses a repertoire of clinical treatments that aim to stimulate immune cells such that they recognize and eliminate tumor cells, and has shown remarkable efficacy in patients. Many immunotherapeutic agents function by promoting inflammatory immune responses, either by stimulating innate immune signaling pathways or by enhancing the activation and expansion of tumor-specific cytotoxic CD8+ T cells. Therefore, strategies to manipulate the immunogenicity of tumor cell death to more potently stimulate tumor-specific immunity constitutes an important target that could potentially synergize with existing immunotherapy treatments. Inflammatory forms of PCD are of particular interest in the context of tumor immunity, as the tumor microenvironment (TME) is typically viewed as a highly immunosuppressive tissue niche. Necroptosis is one such inflammatory form of PCD which occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3. The activation of this complex leads to lytic cell death via MLKL-mediated pore formation, accompanied by de novo production of pro-inflammatory mediators such as chemokines and cytokines. Considering the potently inflammatory nature of necroptosis in comparison to other cell death modalities such as apoptosis, we sought to test how specific induction of necroptosis within the TME instructs anti-tumor immunity. In this dissertation, we show that ectopic administration of necroptotic cells to the TME promotes BATF3+ cDC1- and CD8+ leukocyte-dependent anti-tumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen presenting cells. Tumor control by necroptotic cells requires the activity of the RIPK1/RIPK3 signaling complex and its subsequent activation of NF-B, but not the release of damage-associated molecular patterns (DAMPs) or their ability to stimulate innate pattern recognition receptors (PRRs), highlighting a critical role for death-independent functions of the RIPK1/RIPK3 necrosome in determining the immunogenicity of necroptotic cells. Additionally, immune stimulation by necroptotic cells synergizes with co-administration of the immune checkpoint blockade (ICB) reagent -PD-1, conferring durable tumor clearance in animals that received dual therapy. Furthermore, we report the development of constitutively-active forms of the necroptosis-inducing enzyme RIPK3, and show that delivery of a gene encoding this enzyme to tumor cells using recombinant adeno-associated viruses (AAVs) induces tumor cell necroptosis which synergizes with immune checkpoint blockade to promote durable tumor clearance. Collectively, these findings define a beneficial role for RIPK1/RIPK3 activation as a proximal target in the initiation of tumor-specific immune responses. Although monotherapy with existing immunotherapy modalities such as ICB has shown great efficacy in a subset of cancer patients, restoration of neoangiten recognition alone is often insufficient to eliminate tumors in most individuals. Therefore, successful tumor immunotherapy regimens will likely require the rational selection of multiple treatment modalities aimed at orthogonal immune targets in order to achieve optimal clinical outcomes. Based on our findings, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents one such beneficial treatment approach that may warrant further clinical development. This work has yielded additional insights regarding the relationship between PCD modalities and anti-tumor immune responses, and provides evidence that induction of alternate death pathways such as necroptosis could improve therapeutic outcomes in the context of tumor immunity

Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD

Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS