Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

Highlights d PfPK9-binding compounds were discovered d PfPK9-binding compounds inhibit K63-linked ubiquitination in Plasmodium d Takinib and PfPK9-selective HS220 inhibit liver-stage Plasmodium d Takinib and PfPK9-selective HS220 increase liver-stage parasite size

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Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in Pstpip2cmo mice, which carry a missense mutation in the proline–serine–threonine phosphatase–interacting protein 2 (Pstpip2) gene. As cmo disease in mice, the experimental model analogous to human CRMO, is mediated specifically by IL-1β and not by IL-1α, delineating the molecular pathways contributing to pathogenic IL-1β production is crucial to developing targeted therapies. In particular, our earlier findings support redundant roles of NLR family pyrin domain-containing 3 (NLRP3) and caspase-1 with caspase-8 in instigating cmo. However, the signaling components upstream of caspase-8 and pro-IL-1β cleavage in Pstpip2cmo mice are not well-understood. Therefore, here we investigated the signaling pathways in these mice and discovered a central role of a nonreceptor tyrosine kinase, spleen tyrosine kinase (SYK), in mediating osteomyelitis. Using several mutant mouse strains, immunoblotting, and microcomputed tomography, we demonstrate that absent in melanoma 2 (AIM2), receptor-interacting serine/ threonine protein kinase 3 (RIPK3), and caspase recruitment domain–containing protein 9 (CARD9) are each dispensable for osteomyelitis induction in Pstpip2cmo mice, whereas genetic deletion of Syk completely abrogates the disease phenotype. We further show that SYK centrally mediates signaling upstream of caspase-1 and caspase-8 activation and principally up-regulates NF-κB and IL-1β signaling in Pstpip2cmo mice, thereby inducing cmo. These results provide a rationale for directly targeting SYK and its downstream signaling components in CRMO.

SummaryPrior to initiating symptomatic malaria, Plasmodium parasites infect and develop within hepatocytes. We performed a forward genetic, genome-wide CRISPR-Cas9 screen to identify host regulators of Plasmodium liver infection. Single guide RNAs targeting genes involved in vesicle trafficking, cytoskeleton organization and lipid biogenesis altered Plasmodium liver development. We observed a redistribution of Golgi-derived vesicles and fragmented Golgi stacks with the parasitophorous vacuolar membrane (PVM). The host microtubule network and non-centrosomal microtubule organizing centers (ncMTOC) also re-localized following infection, closely associating with the parasite. Knocking out the centrosomal MTOC protein CENPJ exasperated the re-localization of MTOCs to the parasite and increased infection, suggesting that the parasite relies on ncMTOC assembly. Thus, we have uncovered a mechanism by which parasites sequester host material for survival and development. Our data provide a wealth of yet untested hypotheses about the elusive biology of the liver stage parasite and serves as a foundation for future investigation.