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

Interleukin 1α (IL-1α) and IL-1ß 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-1ß 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-1ß expression was drastically reduced, bone marrow-derived macrophages (BMDMs) from Il1a-KOline2 mice showed normal induction and activation of IL-1ß. Additionally, Il1a-KOline2 BMDMs showed normal inflammasome activation and IL-1ß 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-1ß, 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-1ß. Future studies on the unique functions of IL-1α and IL-1ß using these mice will be critical to identify new roles for these molecules in health and disease and develop therapeutic strategies.

Molecular mechanism of RIPK1 and caspase-8 in homeostatic type I interferon production and regulation.

Type I interferons (IFNs) are essential innate immune proteins that maintain tissue homeostasis through tonic expression and can be upregulated to drive antiviral resistance and inflammation upon stimulation. However, the mechanisms that inhibit aberrant IFN upregulation in homeostasis and the impacts of tonic IFN production on health and disease remain enigmatic. Here, we report that caspase-8 negatively regulates type I IFN production by inhibiting the RIPK1-TBK1 axis during homeostasis across multiple cell types and tissues. When caspase-8 is deleted or inhibited, RIPK1 interacts with TBK1 to drive elevated IFN production, leading to heightened resistance to norovirus infection in macrophages but also early onset lymphadenopathy in mice. Combined deletion of caspase-8 and RIPK1 reduces the type I IFN signaling and lymphadenopathy, highlighting the critical role of RIPK1 in this process. Overall, our study identifies a mechanism to constrain tonic type I IFN during homeostasis which could be targeted for infectious and inflammatory diseases.

Caspase-6 promotes activation of the caspase-11-NLRP3 inflammasome during gram-negative bacterial infections.

The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection.

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.

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-α and IFN-γ 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-α and IFN-γ 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-α and IFN-γ triggered activation of gasdermin D, gasdermin E, caspase-8, caspase-3, caspase-7, and MLKL. Furthermore, the intratumoral administration of TNF-α and IFN-γ suppressed the growth of transplanted xenograft tumors in an NSG mouse model. Overall, this study shows that PANoptosis, induced by synergism of TNF-α and IFN-γ, is an important mechanism to kill cancer cells and suppress tumor growth that could be therapeutically targeted.

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.