DDX41 is a sensor of intracellular double-stranded DNA (dsDNA) in myeloid dendritic cells (mDCs) that triggers a type I interferon response via the signaling adaptor STING. We identified the E3 ligase TRIM21 as a DDX41-interacting protein and found that knockdown of or deficiency in TRIM21 resulted in enhanced type I interferon responses to intracellular dsDNA and DNA viruses. Overexpression of TRIM21 resulted in more degradation of DDX41 and less production of interferon-β (IFN-β) in response to intracellular dsDNA. The SPRY-PRY domain of TRIM21 interacted with the DEADc domain of DDX41. Lys9 and Lys115 of DDX41 were the targets of TRIM21-mediated ubiquitination. TRIM21 is therefore an interferon-inducible E3 ligase that induces the Lys48 (K48)-linked ubiquitination and degradation of DDX41 and negatively regulates the innate immune response to intracellular dsDNA.

The respiratory tract is heavily populated with innate immune cells, but the mechanisms that control such cells are poorly defined. Here we found that the E3 ubiquitin ligase TRIM29 was a selective regulator of the activation of alveolar macrophages, the expression of type I interferons and the production of proinflammatory cytokines in the lungs. We found that deletion of TRIM29 enhanced macrophage production of type I interferons and protected mice from infection with influenza virus, while challenge of Trim29−/− mice with Haemophilus influenzae resulted in lethal lung inflammation due to massive production of proinflammatory cytokines by macrophages. Mechanistically, we demonstrated that TRIM29 inhibited interferon-regulatory factors and signaling via the transcription factor NF-κB by degrading the adaptor NEMO and that TRIM29 directly bound NEMO and subsequently induced its ubiquitination and proteolytic degradation. These data identify TRIM29 as a key negative regulator of alveolar macrophages and might have important clinical implications for local immunity and immunopathology.

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeatcontaining family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA's 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.bacterial RNA | single-stranded RNA | NLRP3 inflammasome | innate immunity | primary macrophages

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.