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The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys48-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.

Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.

When properly activated, macrophages can be tumoricidal, thus making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-;-activated macrophages, produced in large scale for clinical use (MAK cells), have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IFN regulatory factor (IRF)-3 and IRF-7, with subsequent type

The immunoregulatory transcriptional modulators -IFN-regulatory factor (IRF)-3 and IRF-7 -possess similar structural features but distinct gene-regulatory potentials. For example, adenovirus-mediated transduction of the constitutively active form of IRF-3 triggered cell death in primary human MU, whereas expression of active IRF-7 induced a strong anti-tumoral activity in vitro. To further characterize target genes involved in these distinct cellular responses, transcriptional profiles of active IRF-3-or IRF-7-transduced primary human MU were compared and used to direct further mechanistic studies. The pro-apoptotic BH3-only protein Noxa was identified as a primary IRF-3 target gene and an essential regulator of IRF-3, dsRNA and vesicular stomatitis virus-induced cell death. The critical role of IRF-7 and type I IFN production in increasing the immunostimulatory capacity of MU was also evaluated; IRF-7 increased the expression of a broad range of IFNstimulated genes including immunomodulatory cytokines and genes involved in antigen processing and presentation. Furthermore, active IRF-7 augmented the cross-presentation capacity and tumoricidal activity of MU and led to an anti-tumor response against the B16 melanoma model in vivo. Altogether, these data further highlight the respective functions of IRF-3 and IRF-7 to program apoptotic, immune and anti-tumor responses.Key words: Anti-tumor immunotherapy . Apoptosis . Cross-presentation .IFN-regulatory factor . Microarray Supporting Information available online IntroductionType I interferons (IFN-a/b) induce the expression of hundreds of IFN-stimulated genes (ISG), which mediate a broad range of anti-viral, growth-regulatory and immunomodulatory effects. In virus-infected cells, these cytokines inhibit many RNA and DNA viruses at various stages of their replication cycles [1,2]. During tumor-cell development, type I IFN exert direct cytotoxic or antiproliferative functions and negatively regulate angiogenesis [3,4]. Furthermore, direct anti-viral and anti-tumor properties [14,15] leading to their dimerization, nuclear translocation and binding to the promoters of type I IFN genes [16][17][18][19]. Although IRF-3 and IRF-7 share highly related structural and functional characteristics [13,20,21], major biological and functional distinctions can be identified between the two. While IRF-3 is ubiquitously expressed [22], the basal expression of IRF-7 is low in most cells (with the notable exception of plasmacytoid DC [23]) and is strongly increased by type I IFN-mediated signaling [24]. IRF-3 and IRF-7 present both redundant and distinct transcriptional profiles. Qualitative and quantitative differences exist in type I IFN induction capacities of IRF-3 and IRF-7 upon activation: IRF-7 can induce the efficient activation of IFNB genes and multiple IFNA subtypes, whereas IRF-3 is a potent activator of IFNB genes but not of IFNA genes with the exception of human IFNA1 and mouse Ifna4 genes [21,[24][25][26]. This observation may account for the increased vulnerability to ...

The major group of human immunodeficiency virus type 1 (HIV-1) strains that comprise the current global pandemic have diversified during their worldwide spread into at least 10 distinct subtypes, or clades. Subtype C predominates in sub-Saharan Africa and is responsible for the majority of worldwide HIV-1 infections, subtype B predominates in North America and Europe, and subtype E is prevalent in Southeast Asia. Significant amino acid variations have been observed among the clade-specific Tat proteins. For the present study, we examined clade-specific interactions between Tat, transactivation-responsive (TAR) element, and P-TEFb proteins and how these interactions may modulate the efficiency of HIV-1 transcription. Clade-specific Tat proteins significantly modified viral gene expression. Tat proteins derived from HIV-1 clades C and E were strong transactivators of long terminal repeat (LTR) activity; Tat E also had a longer half-life than the other Tat proteins and interacted more efficiently with the stem-loop TAR element. Chimeric Tat proteins harboring the Tat E activation domain were strong transactivators of LTR expression. While Tat B, C, and E were able to rescue a Tat-defective HIV-1 proviral clone, Tat E was significantly more efficient at rescue than Tat C, possibly due to the relative stability of the Tat protein. Swapping the activation domains of Tat B, C, and E identified the cyclin T1 association domain as a critical determinant of the transactivation efficiency and of Tat-defective HIV-1 provirus rescue.

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