Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.Keywords: Caspase-4 r Caspase-5 r LPS r NLRP3 inflammasome r Pyroptosis

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-kB (NF-kB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-kB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IkB kinase ε (IKKε) to drive NF-kB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-kB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations.