Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13(-/-) mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13(-/-) mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13(-/-) murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I . C) also show a significant increase in beta IFN (IFN-beta) levels, but, in contrast, IFN-beta responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection. IMPORTANCE: The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.

Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the "sepsis syndrome," a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.

Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair - Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM) - to TNF-alpha expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-alpha protein expression even at times when TNF-alpha mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-alpha 3' untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-alpha translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-alpha 3' untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-alpha protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-alpha protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-alpha translation.

Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1beta (IL-1beta) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1beta and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell-dependent production of interferon-gamma, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.