Z-DNA binding protein 1 orchestrates innate immunity and inflammatory cell death.

Innate immunity is not only the first line of host defense against microbial infections but is also crucial for the host responses against a variety of noxious stimuli. Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor that can induce inflammatory cell death in both immune and nonimmune cells upon sensing of incursive virus-derived Z-form nucleic acids and self-nucleic acids via its Zα domain. Mechanistically, aberrantly expressed or activated ZBP1 induced by pathogens or noxious stimuli enables recruitment of TANK binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 to drive type I interferon (IFN-I) responses and activation of nuclear factor kappa B (NF-κB) signaling. Meanwhile, ZBP1 promotes the assembly of ZBP1- and absent in melanoma 2 (AIM2)-PANoptosome, which ultimately triggers PANoptosis through caspase 3-mediated apoptosis, mixed lineage kinase domain like pseudokinase (MLKL)-mediated necroptosis, and gasdermin D (GSDMD)-mediated pyroptosis. In response to damaged mitochondrial DNA, ZBP1 can interact with cyclic GMP-AMP synthase to augment IFN-I responses but inhibits toll like receptor 9-mediated inflammatory responses. This review summarizes the structure and expression pattern of ZBP1, discusses its roles in human diseases through immune-dependent (e.g., the production of IFN-I and pro-inflammatory cytokines) and -independent (e.g., the activation of cell death) functions, and highlights the attractive prospect of manipulating ZBP1 as a promising therapeutic target in diseases.

Viral Suppression of RIPK1-Mediated Signaling.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has emerged as a key upstream regulator of cell death and inflammation. RIPK1-mediated signaling governs the outcome of signaling pathways initiated by tumor necrosis factor receptor 1 (TNFR1), Toll-like receptor 3 (TLR3), TLR4, retinoic acid-inducible gene 1 (RIG-I)/melanoma differentiation-associated protein 5 (MDA-5), and Z-binding protein 1 (ZBP1) by signaling for NF-κB activation, mitogen-associated protein kinase (MAPK) and interferon regulatory factor 3/7 (IRF3/7) phosphorylation, and cell death via apoptosis and necroptosis. Both cell death and inflammatory responses play a major role in controlling virus infections. Therefore, viruses have evolved multifaceted mechanisms to exploit host immune responses by targeting RIPK1. This review focuses on the current understanding of RIPK1-mediated inflammatory and cell death pathways and multiple mechanisms by which viruses manipulate these pathways by targeting RIPK1. We also discuss gaps in our knowledge regarding RIPK1-mediated signaling pathways and highlight potential avenues for future research.

RIPK3 Activates MLKL-mediated Necroptosis and Inflammasome Signaling during Streptococcus Infection.

Community-acquired pneumonia is the most common type of pneumonia and remains a leading cause of morbidity and mortality worldwide. Although many different pathogens can contribute to pneumonia, Streptococcus pneumoniae is one of the common bacterial pathogens that underlie community-acquired pneumonia. RIPK3 (receptor-interacting protein kinase 3) is widely recognized as a key modulator of inflammation and cell death. To elucidate a potential role of RIPK3 in pneumonia, we examined plasma from healthy control subjects and patients positive for streptococcal pneumonia. In human studies, RIPK3 protein concentrations were significantly elevated and were identified as a potential plasma marker of pneumococcal pneumonia. To expand these findings, we used an in vivo murine model of pneumococcal pneumonia to demonstrate that RIPK3 deficiency leads to reduced bacterial clearance, severe pathological damage, and high mortality. Our results illustrated that RIPK3 forms a complex with RIPK1, MLKL (mixed-lineage kinase domain-like protein), and MCU (mitochondrial calcium uniporter) to induce mitochondrial calcium uptake and mitochondrial reactive oxygen species(mROS) production during S. pneumoniae infection. In macrophages, RIPK3 initiated necroptosis via the mROS-mediated mitochondrial permeability transition pore opening and NLRP3 inflammasome activation via the mROS-AKT pathway to protect against S. pneumoniae. In conclusion, our study demonstrated a mechanism by which RIPK3-initiated necroptosis is essential for host defense against S. pneumoniae.

Bafilomycin A1 enhances NLRP3 inflammasome activation in human monocytes independent of lysosomal acidification.

The release of interleukin (IL)-1ß from primary human monocytes in response to extracellular LPS occurs through the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. In primary monocytes, in response to LPS, NLRP3 inflammasome activation is characterized by an independence of K+ efflux and ASC speck formation and has been termed the 'alternative' pathway. Here, we report that pharmacological inhibition of V-ATPase with bafilomycin A1 exacerbated LPS-induced NLRP3 inflammasome activation in primary human monocytes. Inhibition of V-ATPase in the presence of extracellular LPS led to NLRP3-dependent, K+ efflux-independent, ASC oligomerization and caspase-1 activation. Although V-ATPases are required for lysosomal acidification, we found that acidic lysosomal pH and protease activity were dispensable for this altered response, suggesting that V-ATPase inhibition triggered alternative signalling events. Therefore, V-ATPases may serve additional roles during NLRP3 inflammasome activation in primary human monocytes.

RIPK3-Dependent Recruitment of Low-Inflammatory Myeloid Cells Does Not Protect from Systemic Salmonella Infection.

Regulated macrophage death has emerged as an important mechanism to defend against intracellular pathogens. However, the importance and consequences of macrophage death during bacterial infection are poorly resolved. This is especially true for the recently described RIPK3-dependent lytic cell death, termed necroptosis. Salmonella enterica serovar Typhimurium is an intracellular pathogen that precisely regulates virulence expression within macrophages to evade and manipulate immune responses, which is a key factor in its ability to cause severe systemic infections. We combined genetic and pharmacological approaches to examine the importance of RIPK3 for S. Typhimurium-induced macrophage death using conditions that recapitulate bacterial gene expression during systemic infection in vivo Our findings indicate that noninvasive S. Typhimurium does not naturally induce macrophage necroptosis but does so in the presence of pan-caspase inhibition. Moreover, our data suggest that RIPK3 induction (following caspase inhibition) does not impact host survival following S. Typhimurium infection, which differs from previous findings based on inert lipopolysaccharide (LPS) injections. Finally, although necroptosis is typically characterized as highly inflammatory, our data suggest that RIPK3 skews the peritoneal myeloid population away from an inflammatory profile to that of a classically noninflammatory profile. Collectively, these data improve our understanding of S. Typhimurium-macrophage interactions, highlight the possibility that purified bacterial components may not accurately recapitulate the complexity of host-pathogen interactions, and reveal a potential and unexpected role for RIPK3 in resolving inflammation.IMPORTANCE Macrophages employ multiple strategies to limit pathogen infection. For example, macrophages may undergo regulated cell death, including RIPK3-dependent necroptosis, as a means of combatting intracellular bacterial pathogens. However, bacteria have evolved mechanisms to evade or exploit immune responses. Salmonella is an intracellular pathogen that avoids and manipulates immune detection within macrophages. We examined the contribution of RIPK3 to Salmonella-induced macrophage death. Our findings indicate that noninvasive Salmonella does not naturally induce necroptosis, but it does so when caspases are inhibited. Moreover, RIPK3 induction (following caspase inhibition) does not impact host survival following Salmonella systemic infection. Finally, our data show that RIPK3 induction results in recruitment of low-inflammatory myeloid cells, which was unexpected, as necroptosis is typically described as highly inflammatory. Collectively, these data improve our understanding of pathogen-macrophage interactions, including outcomes of regulated cell death during infection in vivo, and reveal a potential new role for RIPK3 in resolving inflammation.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Apelin attenuates streptozotocin-induced learning and memory impairment by modulating necroptosis signaling pathway.

Apelin is a neuropeptide that plays an important role in neuronal protection. In this study, we investigated the effects of apelin intracerebroventricular administration on spatial learning and memory-related behaviors, and necroptosis signaling pathways in the hippocampus of streptozotocin (STZ) -injected rats. Apelin treatment was implemented following STZ-induced dementia for 15 days. After conducting a behavioral test (Morris Water Maze), the cellular and molecular aspects were examined to detect the apelin effect on the necroptosis signaling pathway. We demonstrated that STZ administration significantly slowed down the learning capability. However apelin treatment notably reversed this neuroinflammation induced behavioral impairment. Furthermore, molecular investigations showed that apelin treatment reduced the hippocampal RIP1, RIP3, and TNF-α level. Our results suggest that apelin treatment attenuates STZ-induced dementia. This effect may be mediated by inhibition of the necroptosis signaling pathway which seems to be associated with the ability of apelin to reduce central TNF-α level. This data provides evidence of the neuroprotective effect of apelin on STZ-induced learning and memory impairment and characterize some of the underlying mechanisms.

RIPK3 and Caspase-1/11 Are Necessary for Optimal Antigen-Specific CD8 T Cell Response Elicited by Genetically Modified Listeria monocytogenes.

Efficient induction of effector and long-term protective antigen-specific CD8+ T memory response by vaccination is essential to eliminate malignant and pathogen-infected cells. Intracellular infectious bacteria, including Listeria monocytogenes, have been considered potent vectors to carry multiple therapeutic proteins and generate antigen-specific CD8+ T cell responses. Although the role of molecules involved in inflammatory cell death pathways, such as necroptosis (RIPK3-mediated) and pyroptosis (Caspase-1/11-mediated), as effectors of immune response against intracellular bacteria are relatively well understood, their contribution to the adjuvant effect of recombinant bacterial vectors in the context of antigen-specific CD8+ T cell response remained obscure. Therefore, we evaluated the impact of RIPK3 and Caspase-1/11 (Casp-1/11) individual and combined deficiencies on the modulation of antigen-specific CD8+ T cell response during vaccination of mice with ovalbumin-expressing L. monocytogenes (LM-OVA). We observed that Casp-1/11 but not RIPK3 deficiency negatively impacts the capacity of mice to clear LM-OVA. Importantly, both RIPK3 and Casp-1/11 are necessary for optimal LM-OVA-mediated antigen-specific CD8+ T cell response, as measured by in vivo antigen-specific CD8+ T cell proliferation, target cell elimination, and cytokine production. Furthermore, Casp-1/11 and Casp-1/11/RIPK3 combined deficiencies restrict the early initiation of antigen-specific CD8+ T cell memory response. Taken together, our findings demonstrate that RIPK3 and Casp-1/11 influence the quality of CD8+ T cell responses induced by recombinant L. monocytogenes vectors.

RIPK3 Orchestrates Fatty Acid Metabolism in Tumor-Associated Macrophages and Hepatocarcinogenesis.

Metabolic reprogramming is critical for the polarization and function of tumor-associated macrophages (TAM) and hepatocarcinogenesis, but how this reprogramming occurs is unknown. Here, we showed that receptor-interacting protein kinase 3 (RIPK3), a central factor in necroptosis, is downregulated in hepatocellular carcinoma (HCC)-associated macrophages, which correlated with tumorigenesis and enhanced the accumulation and polarization of M2 TAMs. Mechanistically, RIPK3 deficiency in TAMs reduced reactive oxygen species and significantly inhibited caspase1-mediated cleavage of PPAR. These effects enabled PPAR activation and facilitated fatty acid metabolism, including fatty acid oxidation (FAO), and induced M2 polarization in the tumor microenvironment. RIPK3 upregulation or FAO blockade reversed the immunosuppressive activity of TAMs and dampened HCC tumorigenesis. Our findings provide molecular basis for the regulation of RIPK3-mediated, lipid metabolic reprogramming of TAMs, thus highlighting a potential strategy for targeting the immunometabolism of HCC.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism.

Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.

RIPK1 kinase activity has been shown to be essential to driving pyroptosis, apoptosis, and necroptosis. However, here we show a kinase activity-independent role for RIPK1 in these processes using a model of TLR priming in a TAK1-deficient setting to mimic pathogen-induced priming and inhibition. TLR priming of TAK1-deficient macrophages triggered inflammasome activation, including the activation of caspase-8 and gasdermin D, and the recruitment of NLRP3 and ASC into a novel RIPK1 kinase activity-independent cell death complex to drive pyroptosis and apoptosis. Furthermore, we found fully functional RIPK1 kinase activity-independent necroptosis driven by the RIPK3-MLKL pathway in TAK1-deficient macrophages. In vivo, TAK1 inactivation resulted in RIPK3-caspase-8 signaling axis-driven myeloid proliferation and a severe sepsis-like syndrome. Overall, our study highlights a previously unknown mechanism for RIPK1 kinase activity-independent inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis) that could be targeted for treatment of TAK1-associated myeloid proliferation and sepsis.

RIPK1 inhibitor Cpd-71 attenuates renal dysfunction in cisplatin-treated mice via attenuating necroptosis, inflammation and oxidative stress.

Acute kidney injury (AKI) is a destructive clinical condition induced by multiple insults including ischemic reperfusion, nephrotoxic drugs and sepsis. It is characterized by a sudden decline in renal function, in addition to excessive inflammation, oxidative stress and programmed cell death of renal tubular epithelial cells. RIPK1-mediated necroptosis plays an important role in AKI. In the present study, we evaluated the treatment effects of Compound-71 (Cpd-71), a novel RIPK1 inhibitor, by comparing with Necrostatin-1 (Nec-1), a classic RIPK1 inhibitor, which has several drawbacks like the narrow structure-activity relationship (SAR) profile, moderate potency and non-ideal pharmacokinetic properties, in vivo and in vitro Our results showed that pretreatment of Cpd-71 attenuated cisplatin-induced renal injury, restored renal function and suppressed renal inflammation, oxidative stress and cell necroptosis. In addition, Cpd-71 inhibited renal damage while reducing the up-regulated serum creatinine (Cr) and blood urea nitrogen (BUN) levels in established AKI mice model. Consistently, we confirmed that Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1, by physically binding to the allosteric type III ligand binding site of RIPK1, thereby reduced RIPK1 kinase activity, RIPK1/RIPK3 complex formation and phosphor-MLKL membrane translocation by molecular docking, Western blot, co-immunoprecipitation and cellular thermal shift assay (CETSA). Taken together, we currently showed that targeting RIPK1 with Cpd-71 may serve as a promising clinical candidate for AKI treatment.

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity.

Although the signaling events that induce different forms of programmed cell death are well defined, the subsequent immune responses to dying cells in the context of cancer remain relatively unexplored. Necroptosis occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3, whose activation leads to lytic cell death accompanied by de novo production of proinflammatory mediators. Here, we show that ectopic introduction of necroptotic cells to the tumor microenvironment promotes BATF3+ cDC1- and CD8+ leukocyte-dependent antitumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen-presenting cells. 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 adeno-associated viruses induces tumor cell necroptosis, which synergizes with immune checkpoint blockade to promote durable tumor clearance. These findings support a role for RIPK1/RIPK3 activation as a beneficial proximal target in the initiation of tumor immunity. Considering that successful tumor immunotherapy regimens will require the rational application of multiple treatment modalities, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents a beneficial treatment approach that may warrant further clinical development.

Fisetin supplementation prevents high fat diet-induced diabetic nephropathy by repressing insulin resistance and RIP3-regulated inflammation.

Obesity-related renal disease is related to caloric excess promoting deleterious cellular responses. However, a full understanding of the molecular mechanisms involved in progressive kidney disease, as well as a therapeutic strategy, is still absent. Fisetin (FIS), as a natural flavonoid, possesses various bioactivities in a number of disease models. However, its role in obesity-associated kidney injury is still unclear and requires elucidation. In our study, an obesity animal model was established using C57BL/6 mice fed with a normal chow diet (NCD) or high fat diet (HFD) for 16 weeks with or without FIS administration (20, 40 or 80 mg kg-1). Our results indicated that chronic HFD feeding led to a significant body weight gain in mice compared to the normal control group, accompanied by a marked insulin resistance and glucose intolerance, whereas FIS treatment exerted prominently protective effects. In addition, FIS significantly attenuated HFD-induced histological alterations in renal tissue samples. Moreover, FIS treatment down-regulated expression of kidney injury molecule-1 (KIM-1), and up-regulated nephrin and podocin expression levels in the kidneys of HFD-fed mice, improving their renal dysfunction. After HFD feeding, mice treated with FIS exhibited a decrease in phosphorylated IRS1Ser307, and an increase in phosphorylated glycogen synthase kinase 1 (IRS1Tyr608), AKT, forkhead box protein O1 (FOXO1) and glycogen synthase kinase (GSK)-3ß. Furthermore, FIS administration markedly restrained the inflammatory response in the kidneys of HFD-challenged mice, as evidenced by the reduced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1ß and IL-18, which was attributed to the blockage of nuclear factor κB (NF-κB) signaling. Importantly, FIS-treated obese mice exerted a remarkable decrease in RIP3 expressions in the kidneys compared to obese mice in the absence of FIS, along with an evident reduction in the NOD-like receptor protein 3 (NLRP3), an apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1. The protective effects of FIS against HFD-induced renal injury were verified in vitro using palmitate (PAL)-treated HK2 cells, an immortalized proximal tubule epithelial cell line from the adult human kidney. In summary, our results supported the notion that FIS functions as a promising agent to improve insulin resistance and inflammatory response against metabolic stress-induced renal injury.

Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

Is Receptor-Interacting Protein Kinase 3 a Viable Therapeutic Target for Mycobacterium tuberculosis Infection?

The dwindling list of antimicrobial agents exhibiting broad efficacy against clinical strains of Mycobacterium tuberculosis (Mtb) has forced the medical community to redefine current approaches to the treatment of tuberculosis (TB). Host receptor-interacting protein kinase 3 (RIPK3) has been flagged recently as a potential target, given that it is believed to regulate necroptosis-independent signaling pathways, which have been implicated in exacerbating several inflammatory conditions and which reportedly play a role in the necrosis of Mtb-infected macrophages. To examine the therapeutic potential of inhibiting RIPK3, we infected RIPK3-deficient mice with aerosolized Mtb. We found that the loss of RIPK3 did not alter overall disease outcomes, with deficient animals harboring similar bacterial numbers in the lungs and spleens compared to their wild-type counterparts. Mtb-infected macrophages were not rescued from dying by Ripk3 deletion, nor did this affect production of the pro-inflammatory cytokine IL-1ß, both in vitro and in vivo. Infiltration of immune cells into the lungs, as well as the activation of adaptive immunity, similarly was not overtly affected by the loss of RIPK3 signaling. Collectively, our data argue against a role of RIPK3 in mediating pathological inflammation or macrophage necrosis during Mtb disease pathogenesis and thus suggest that this host protein is unlikely to be an attractive therapeutic target for TB.

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.

Necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression.

Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.