Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1^–/–(and Casp1/11^–/–) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4^–/–mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11^–/–and Casp8/1/11^–/–mice recapitulate the full susceptibility of Nlrc4^–/–mice. Gsdmd^–/–mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd^–/–Casp7^–/–mice are as susceptible as the Nlrc4^–/–mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila. Inflammasomes are multi-protein complexes that detect infection and other stimuli and activate the Caspase-1 (CASP1) protease. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Active CASP1 cleaves and activates the pore-forming protein gasdermin D (GSDMD) to induce inflammation and cell death. We have previously shown that CASP8 is activated by the NAIP5/NLRC4 inflammasome independently of CASP1 and functions to restrict replication of the intracellular bacterium Legionella pneumophila. Here, we show that CASP7 is activated downstream of CASP8 and is required for CASP8-dependent restriction of L. pneumophila replication in macrophages and in vivo. In addition, CASP7 is also activated by CASP1. Taken together, these results imply that CASP7 and GSDMD are the two key caspase substrates downstream of NAIP5/NLRC4. In support of this hypothesis, we found that mice double deficient in CASP7 and GSDMD are more susceptible than the single knockouts and are as susceptible as the Nlrc4 deficient mice for restriction of L. pneumophila replication in vivo. Collectively, our data indicate that GSDMD and CASP7 are activated by CASP1 and induce cell death and restriction of bacterial infection. Therefore, GSDMD and multiple caspases (CASP1, CASP7 and CASP8) operate downstream of the NAIP5/NLRC4 inflammasome for restriction of infection by pathogenic bacteria.

The latent viral reservoir represents one of the major barriers to curing HIV-1. Focus on the “kick and kill” (also called “shock and kill”) approach, in which virus expression is reactivated, and then cells producing virus are selectively depleted, has led to the discovery of many latency-reversing agents (LRAs) that have furthered our understanding of the mechanisms driving HIV-1 latency and latency reversal. Thus far, individual compounds have yet to be robust enough to work as a therapy, highlighting the importance of identifying new compounds that target novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ~4250 compounds. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events and across different latency models. Co-treating cells with NSC95397 and known LRAs demonstrated that NSC95397 synergizes with different drugs under both standard normoxic and physiological hypoxic conditions. NSC95397 does not globally increase open chromatin, and bulk RNA sequencing revealed that NSC95397 does not greatly increase cellular transcription. Instead, NSC95397 downregulates pathways key to metabolism, cell growth, and DNA repair—highlighting the potential of these pathways in regulating HIV-1 latency. Overall, we identified NSC95397 as a novel LRA that does not largely alter global transcription, shows potential for synergy with known LRAs, and may act through novel pathways not previously recognized for their ability to modulate HIV-1 latency.