TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril(-/-) mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril(-/-) mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.
The four Toll/IL-1R domain-containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved Toll/IL-1R adaptor, sterile ? and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm(-/-) mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1?, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-?B and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.
Inflammasome activation is gaining recognition as an important mechanism for protection during viral infection. Here, we investigate whether Rift Valley fever virus, a negative-strand RNA virus, can induce inflammasome responses and IL-1? processing in immune cells. We have determined that RVFV induces NLRP3 inflammasome activation in murine dendritic cells, and that this process is dependent upon ASC and caspase-1. Furthermore, absence of the cellular RNA helicase adaptor protein MAVS/IPS-1 significantly reduces extracellular IL-1? during infection. Finally, direct imaging using confocal microscopy shows that the MAVS protein co-localizes with NLRP3 in the cytoplasm of RVFV infected cells.
Synthetic oligodeoxynucleotides (ODNs) comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. In this study, we demonstrate that such suppressive ODN abrogate activation of cytosolic nucleic acid-sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNF-?, and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1? and IL-18 maturation in dendritic cells stimulated with dsDNA and murine CMV. Inhibition was dependent on A151s phosphorothioate backbone, whereas substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
The innate immune response is the first line of defense against infection and relies on the ability of immune cells to detect the presence of infection through germline-encoded pattern recognition receptors. These include the Toll-like receptors, the retinoic acid inducible gene-like receptors, the nucleotide oligomerization domain-like receptors, and a number of DNA-sensing molecules. Members of the PYHIN protein family have recently emerged as sensors of microbial DNA. PYHIN proteins bind microbial DNA and form caspase-1-activating inflammasomes (AIM2) or drive type I IFN gene transcription (IFI16). Here, we review these discoveries and highlight the emerging role of the PYHIN protein family in mammalian host defenses.
TCR signaling leads to the activation of kinases such as inducible tyrosine kinase (Itk), a key regulatory protein in T-lymphocyte activation and function. The homolog of Itk in B cells is Brutons tyrosine kinase, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was twofold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of WT, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itks kinase, pleckstrin-homology, and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T-cell function.
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