Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.

Influenza-related complications continue to be a major cause of mortality worldwide. Due to unclear mechanisms, a substantial number of influenza-related deaths result from bacterial superinfections, particularly secondary pneumococcal pneumonia. Here, we report what we believe to be a novel mechanism by which influenza-induced type I IFNs sensitize hosts to secondary bacterial infections. Influenza-infected mice deficient for type I IFN-α/β receptor signaling (Ifnar -/-mice) had improved survival and clearance of secondary Streptococcus pneumoniae infection from the lungs and blood, as compared with similarly infected wild-type animals. The less effective response in wild-type mice seemed to be attributable to impaired production of neutrophil chemoattractants KC (also known as Cxcl1) and Mip2 (also known as Cxcl2) following secondary challenge with S. pneumoniae. This resulted in inadequate neutrophil responses during the early phase of host defense against secondary bacterial infection. Indeed, influenza-infected wild-type mice cleared secondary pneumococcal pneumonia after pulmonary administration of exogenous KC and Mip2, whereas neutralization of Cxcr2, the common receptor for KC and Mip2, reversed the protective phenotype observed in Ifnar -/-mice. These data may underscore the importance of the type I IFN inhibitory pathway on CXC chemokine production. Collectively, these findings highlight what we believe to be a novel mechanism by which the antiviral response to influenza sensitizes hosts to secondary bacterial pneumonia.

MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.

Upon recognition of viral infection, RIG-I and Helicard recruit a newly identified adapter termed Cardif, which induces type I interferon (IFN)-mediated antiviral responses through an unknown mechanism. Here, we demonstrate that TRAF3, like Cardif, is required for type I interferon production in response to intracellular doublestranded RNA. Cardif-mediated IFNa induction occurs through a direct interaction between the TRAF domain of TRAF3 and a TRAF-interaction motif (TIM) within Cardif. Interestingly, while the entire N-terminus of TRAF3 was functionally interchangeable with that of TRAF5, the TRAF domain of TRAF3 was not. Our data suggest that this distinction is due to an inability of the TRAF domain of TRAF5 to bind the TIM of Cardif. Finally, we show that preventing association of TRAF3 with this TIM by mutating two critical amino acids in the TRAF domain also abolishes TRAF3-dependent IFN production following viral infection. Thus, our findings suggest that the direct and specific interaction between the TRAF domain of TRAF3 and the TIM of Cardif is required for optimal Cardif-mediated antiviral responses.