Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular ...mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin IL 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.
Ro52 is a member of the TRIM family of single-protein E3 ligases and is also a target for autoantibody production in systemic lupus erythematosus and Sjögren's syndrome. We previously demonstrated a ...novel function of Ro52 in the ubiquitination and proteasomal degradation of IRF3 following TLR3/4 stimulation. We now present evidence that Ro52 has a similar role in regulating the stability and activity of IRF7. Endogenous immunoprecipitation of Ro52-bound proteins revealed that IRF7 associates with Ro52, an effect which increases following TLR7 and TLR9 stimulation, suggesting that Ro52 interacts with IRF7 post-pathogen recognition. Furthermore, we show that Ro52 ubiquitinates IRF7 in a dose-dependent manner, resulting in a decrease in total IRF7 expression and a subsequent decrease in IFN-alpha production. IRF7 stability was increased in bone marrow-derived macrophages from Ro52-deficient mice stimulated with imiquimod or CpG-B, consistent with a role for Ro52 in the negative regulation of IRF7 signalling. Taken together, these results suggest that Ro52-mediated ubiquitination promotes the degradation of IRF7 following TLR7 and TLR9 stimulation. As Ro52 is known to be IFN-inducible, this system constitutes a negative-feedback loop that acts to protect the host from the prolonged activation of the immune response.
Toll-like receptors (TLRs) are a group of highly conserved molecules that initiate the innate immune response to pathogens by recognizing structural motifs expressed by microbes. We have identified a ...novel TLR, TLR15, by bioinformatic analysis of the chicken genome, which is distinct from any known vertebrate TLR and thus appears to be avian specific. The gene for TLR15 was sequenced and is found on chromosome 3, and it has archetypal TIR and transmembrane domains and a distinctive arrangement of extracellular leucine-rich regions. mRNA for TLR15 was detected in the spleen, bursa, and bone marrow of healthy chickens, suggesting a role for this novel receptor in constitutive host defense. Following in vivo Salmonella enterica serovar Typhimurium infection, quantitative real-time PCR demonstrated significant upregulation of TLR15 in the cecum of infected chickens. Interestingly, similar induction of TLR2 expression following infection was also observed. In vitro studies revealed TLR15 upregulation in chicken embryonic fibroblasts stimulated with heat-killed S. enterica serovar Typhimurium. Collectively, these results suggest a role for the TLR in avian defense against bacterial infection. We hypothesize that TLR15 may represent an avian-specific TLR that has been either retained in chicken and lost in other taxa or gained in the chicken.
The adaptive immune system is not completely developed when chickens hatch, so the innate immune system has evolved a range of mechanisms to deal with early pathogenic assault. Avian β-defensins ...(AvBDs) and cathelicidins (CTHLs) are two major sub-classes of antimicrobial peptides (AMPs) with a fundamental role in both innate and adaptive immune responses. In this study, we demonstrate distinct expression patterns of innate immune genes including – Toll-like receptors (TLRs) (
TLR2,
TLR15 and
TLR21, but not
TLR4), the complete repertoire of AvBDs, CTHLs and both pro- and anti-inflammatory cytokines (
IL1B,
IL8,
IFNG and
IL10) during early chicken embryonic development.
AvBD9 was significantly increased by over 150 fold at day 9; and
AvBD10 was increased by over 100 fold at day 12 in the abdomen of the embryo, relative to day 3 expression levels (
P
<
0.01). In contrast,
AvBD14 was preferentially expressed in the head of the embryo. This is the first study to demonstrate differential patterns of AMP gene expression in the sterile environment of the developing embryo. Our results propose novel roles for AMPs during development and reveal the innate preparedness of developing embryos for pathogenic assault
in ovo, or post-hatching.
In recent years members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to both positively and negatively regulate viral defence and as such are emerging as ...compelling targets for modulating the anti-viral immune response. In this study we identify TRIM68, a close homologue of TRIM21, as a novel regulator of Toll-like receptor (TLR)- and RIG-I-like receptor (RLR)-driven type I IFN production. Proteomic analysis of TRIM68-containing complexes identified TRK-fused gene (TFG) as a potential TRIM68 target. Overexpression of TRIM68 and TFG confirmed their ability to associate, with TLR3 stimulation appearing to enhance the interaction. TFG is a known activator of NF-κB via its ability to interact with inhibitor of NF-κB kinase subunit gamma (IKK-γ) and TRAF family member-associated NF-κB activator (TANK). Our data identifies a novel role for TFG as a positive regulator of type I IFN production and suggests that TRIM68 targets TFG for lysosomal degradation, thus turning off TFG-mediated IFN-β production. Knockdown of TRIM68 in primary human monocytes resulted in enhanced levels of type I IFN and TFG following poly(I:C) treatment. Thus TRIM68 targets TFG, a novel regulator of IFN production, and in doing so turns off and limits type I IFN production in response to anti-viral detection systems.
The concept that viral sensing systems, via their ability to drive pro-inflammatory cytokine and interferon production, contribute to the development of autoimmune and autoinflammatory disease is ...supported by a wide range of clinical and experimental observations. Recently, the tripartite motif-containing proteins (TRIMs) have emerged as having key roles in antiviral immunity - either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. Given their involvement in these pathways, we propose that TRIM proteins contribute to the development and pathology of autoimmune and autoinflammatory conditions, thus making them potential novel targets for therapeutic manipulation.
Antimicrobial peptides (AMPs), essential components of innate immunity, are found in a range of phylogenetically diverse species and are thought to act by disrupting the membrane integrity of ...microbes. In this paper, we used evolutionary signatures to identify sites that are most relevant during the functional evolution of these molecules and introduced amino acid substitutions to improve activity. We first demonstrate that the anti-microbial activity of chicken avian β-defensin-8, previously known as gallinacin-12, can be significantly increased against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella typhimurium phoP- mutant and Streptococcus pyogenes through targeted amino acid substitutions, which confer increased peptide charge. However, by increasing the AMP charge through amino acid substitutions at sites predicted to be subject to positive selection, antimicrobial activity against Escherichia coli was further increased. In contrast, no further increase in activity was observed against the remaining pathogens. This result suggests that charge-increasing modifications confer increased broad-spectrum activity to an AMP, whilst positive selection at particular sites is involved in directing the antimicrobial response against specific pathogens. Thus, there is potential for the rational design of novel therapeutics based on specifically targeted and modified AMPs.
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