ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways

The interferon (IFN)–inducible protein Z-DNA binding protein 1 [ZBP1; also known as DNA-dependent activator of IFN regulatory factors (DAI) and DLM-1] was identified as a double-stranded DNA sensor, which instigates innate immune responses. However, this classification has been disputed, and whether ZBP1 functions as a pathogen sensor during an infection has remained unknown. We demonstrated ZBP1-mediated sensing of the influenza A virus (IAV) proteins NP and PB1, triggering cell death and inflammatory responses via the receptorinteracting protein kinase 1 (RIPK1)–RIPK3–caspase-8 axis. ZBP1 regulates NLRP3 (nucleotide and oligomerization domain, leucine-rich repeat–containing protein family, pyrin domain containing 3) inflammasome activation as well as induction of apoptosis, necroptosis, and pyroptosis in IAV-infected cells. ZBP1 deficiency protected mice from mortality during IAV infection owing to reduced inflammatory responses and epithelial damage. Overall, these findings indicate that ZBP1 is an innate immune sensor of IAV and highlight its importance in the pathogenesis of IAV infection.T.-D.K. is supported by the U.S. NIH (AI101935, AI124346, AR056296, and CA163507) and the American Lebanese Syrian Associated Charities; S.M.M. is supported by the National Health and Medical Research Council of Australia R.G. Menzies Early Career Fellowship

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRP ML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell

Detrimental Type I Interferon Signaling Dominates Protective AIM2 Inflammasome Responses during Francisella novicida Infection

Interferons (IFNs) and inflammasomes are essential mediators of anti-microbial immunity. Type I IFN signaling drives activation of the AIM2 inflammasome in macrophages; however, the relative contribution of IFNs and inflammasome responses in host defense is less understood. We report intact AIM2 inflammasome responses in mice lacking type I IFN signaling during infection with F. novicida. Lack of type I IFN signaling conferred protection to F. novicida infection in contrast to the increased susceptibility in AIM2-deficient mice. Mice lacking both AIM2 and IFNAR2 were protected against the infection. The detrimental effects of type I IFN signaling were due to its ability to induce activation of apoptotic caspases and cell death. These results demonstrate the contrasting effects of type I IFN signaling and AIM2 during F. novicida infection in vivo and indicate a dominant role for type I IFNs in mediating detrimental responses despite the protective AIM2 inflammasome responses

RIPK1 Distinctly Regulates Yersinia-Induced Inflammatory Cell Death, PANoptosis

Bacterial pathogens from the genus Yersinia cause fatal sepsis and gastritis in humans. Innate immune signaling and inflammatory cell death (pyroptosis, apoptosis, and necroptosis [PANoptosis]) serve as a first line of antimicrobial host defense. The receptor-interacting protein kinase 1 (RIPK1) is essential for Yersinia-induced pyroptosis and apoptosis and an effective host response. However, it is not clear whether RIPK1 assembles a multifaceted cell death complex capable of regulating caspase-dependent pyroptosis and apoptosis or whether there is cross-talk with necroptosis under these conditions. In this study, we report that Yersinia activates PANoptosis, as evidenced by the concerted activation of proteins involved in PANoptosis. Genetic deletion of RIPK1 abrogated the Yersinia-induced activation of the inflammasome/pyroptosis and apoptosis but enhanced necroptosis. We also found that Yersinia induced assembly of a RIPK1 PANoptosome complex capable of regulating all three branches of PANoptosis. Overall, our results demonstrate a role for the RIPK1 PANoptosome in Yersinia-induced inflammatory cell death and host defense.Y

Inflammatory Cell Death, PANoptosis, Mediated by Cytokines in Diverse Cancer Lineages Inhibits Tumor Growth

Resistance to cell death is a hallmark of cancer. Immunotherapy, particularly immune checkpoint blockade therapy, drives immune-mediated cell death and has greatly improved treatment outcomes for some patients with cancer, but it often fails clinically. Its success relies on the cytokines and cytotoxic functions of effector immune cells to bypass the resistance to cell death and eliminate cancer cells. However, the specific cytokines capable of inducing cell death in tumors and the mechanisms that connect cytokines to cell death across cancer cell types remain unknown. In this study, we analyzed expression of several cytokines that are modulated in tumors and found correlations between cytokine expression and mortality. Of several cytokines tested for their ability to kill cancer cells, only TNF-a and IFN-ｙ together were able to induce cell death in 13 distinct human cancer cell lines derived from colon and lung cancer, melanoma, and leukemia. Further evaluation of the specific programmed cell death pathways activated by TNF-a and IFN-ｙ in these cancer lines identified PANoptosis, a form of inflammatory cell death that was previously shown to be activated by contemporaneous engagement of components from pyroptosis, apoptosis, and/or necroptosis. Specifically, TNF-a and IFN-ｙtriggered activation of gasdermin D, gasdermin E, caspase-8, caspase-3, caspase-7, and MLKL. Furthermore, the intratumoral administration of TNF-a and IFN-ｙ suppressed the growth of transplanted xenograft tumors in an NSG mouse model. Overall, this study shows that PANoptosis, induced by synergism of TNF-a and IFN-g, is an important mechanism to kill cancer cells and suppress tumor growth that could be therapeutically targeted. ImmunoHorizons, 2021, 5: 568–580.Y

Determining distinct roles of IL-1α through generation of an IL-1α knockout mouse with no defect in IL-1β expression

Interleukin 1α (IL-1α) and IL-1β are the founding members of the IL-1 cytokine family, and these innate immune inflammatory mediators are critically important in health and disease. Early studies on these molecules suggested that their expression was interdependent, with an initial genetic model of IL-1α depletion, the IL-1α KO mouse (Il1a-KOline1), showing reduced IL-1β expression. However, studies using this line in models of infection and inflammation resulted in contrasting observations. To overcome the limitations of this genetic model, we have generated and characterized a new line of IL-1α KO mice (Il1a-KOline2) using CRISPR-Cas9 technology. In contrast to cells from Il1a-KOline1, where IL-1β expression was drastically reduced, bone marrow-derived macrophages (BMDMs) from Il1a-KOline2 mice showed normal induction and activation of IL-1β. Additionally, Il1a-KOline2 BMDMs showed normal inflammasome activation and IL-1β expression in response to multiple innate immune triggers, including both pathogen-associated molecular patterns and pathogens. Moreover, using Il1a-KOline2 cells, we confirmed that IL-1α, independent of IL-1β, is critical for the expression of the neutrophil chemoattractant KC/CXCL1. Overall, we report the generation of a new line of IL-1α KO mice and confirm functions for IL-1α independent of IL-1β. Future studies on the unique functions of IL-1α and IL-1β using these mice will be critical to identify new roles for these molecules in health and disease and develop therapeutic strategies

Whole-genome CRISPR screen identifies RAVER1 as a key regulator of RIPK1-mediated inflammatory cell death, PANoptosis

Summary: Transforming growth factor-β-activated kinase 1 (TAK1) is a central regulator of innate immunity, cell death, inflammation, and cellular homeostasis. Therefore, many pathogens carry TAK1 inhibitors (TAK1i). As a host strategy to counteract this, inhibition or deletion of TAK1 induces spontaneous inflammatory cell death, PANoptosis, through the RIPK1-PANoptosome complex, containing the NLRP3 inflammasome and caspase-8/FADD/RIPK3 as integral components; however, PANoptosis also promotes pathological inflammation. Therefore, understanding molecular mechanisms that regulate TAK1i-induced cell death is essential. Here, we report a genome-wide CRISPR screen in macrophages that identified TAK1i-induced cell death regulators, including polypyrimidine tract-binding (PTB) protein 1 (PTBP1), a known regulator of RIPK1, and a previously unknown regulator RAVER1. RAVER1 blocked alternative splicing of Ripk1, and its genetic depletion inhibited TAK1i-induced, RIPK1-mediated inflammasome activation and PANoptosis. Overall, our CRISPR screen identified several positive regulators of PANoptosis. Moreover, our study highlights the utility of genome-wide CRISPR-Cas9 screens in myeloid cells for comprehensive characterization of complex cell death pathways to discover therapeutic targets

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell

NLRP12-PANoptosome activates PANoptosis and pathology in response to heme and PAMPs

Cytosolic innate immune sensors are critical for host defense and form complexes, such as inflammasomes and PANoptosomes, that induce inflammatory cell death. The sensor NLRP12 is associated with infectious and inflammatory diseases, but its activating triggers and roles in cell death and inflammation remain unclear. Here, we discovered that NLRP12 drives inflammasome and PANoptosome activation, cell death, and inflammation in response to heme plus PAMPs or TNF. TLR2/4-mediated signaling through IRF1 induced Nlrp12 expression, which led to inflammasome formation to induce maturation of IL-1b and IL-18. The inflammasome also served as an integral component of a larger NLRP12-PANoptosome that drove inflammatory cell death through caspase-8/RIPK3. Deletion of Nlrp12 protected mice from acute kidney injury and lethality in a hemolytic model. Overall, we identified NLRP12 as an essential cytosolic sensor for heme plus PAMPsmediated PANoptosis, inflammation, and pathology, suggesting that NLRP12 and molecules in this pathway are potential drug targets for hemolytic and inflammatory diseases.Y