Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\-)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/-) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/-) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/-) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/-) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

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.

Impression cytology (IC) is an easy and safe technique that has been used in the past for harvesting epithelial cells from the cornea and conjunctiva for various applications including histology, immunohistology and molecular studies. Previous investigations have shown the usage of different types of membranes for the purpose of investigating pathophysiology and staging of diseases. This contributes to a better understanding of ocular surface conditions and helps to provide information for diagnosis, therapeutic options and prognosis. Recently, there has been a shift of focus in research towards understanding the contribution of microRNAs (miRs) to ocular disease. Thus far, impression cytology has been explored for measuring gene expression but not for quantifying miR expression. This study describes how miRs and mRNA can be isolated from conjunctival epithelial cells obtained by impression cytology and determines the optimum membrane and technique for this purpose. The IC technique was optimized using Biopore, Immobilon-P(SQ) and Millicell Hanging Cell Culture Insert membranes on healthy controls. miRs and mRNAs were isolated from the conjunctival epithelial cells (CEC) obtained and measured. Biopore membrane provided the optimum yield of miRs (38.8 ng/μL ± 10.8) and mRNA (155.3 ng/μL ± 20.1) as well as subjectively found to be best tolerated with minimum discomfort. Appreciable levels of miRs and mRNAs were detected from the CEC from healthy controls, confirming that it is possible to isolate miR and mRNA from CEC. Here, we give a detailed description of the application of conjunctival impression cytology to isolate miRs and the convenience of the technique by using the best membrane available. This method can be readily adopted in both clinical and laboratory settings. This technique will facilitate the measurement of miRs to improve our understanding of the pathogenesis of ocular surface conditions as well as potentially identifying novel therapeutic targets.

IRF5 is a member of the Interferon Regulatory Factor (IRF) family of transcription factors activated downstream of the Toll-Like receptors (TLRs). Polymorphisms in IRF5 have been shown to be associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) and other autoimmune conditions, suggesting a central role for IRF5 in the regulation of the immune response. Four different IRF5 isoforms originate due to alternative splicing and to the presence or absence of a 30 nucleotide insertion in IRF5 exon 6. Since the polymorphic region disturbs a PEST domain, a region associated with protein degradation, we hypothesized that the isoforms bearing the insertion might have increased stability, thus explaining the association of individual IRF5 isoforms with SLE. As the E3 ubiquitin ligase TRIpartite Motif 21 (TRIM21) has been shown to regulate the stability and hence activity of members of the IRF family, we investigated whether IRF5 is subjected to regulation by TRIM21 and whether dysregulation of this mechanism could explain the association of IRF5 with SLE. Our results show that IRF5 is degraded following TLR7 activation and that TRIM21 is involved in this process. Comparison of the individual IRF5 variants demonstrates that isoforms generated by alternative splicing are resistant to TRIM21-mediated degradation following TLR7 stimulation, thus providing a functional link between isoforms expression and stability/activity which contributes to explain the association of IRF5 with SLE.

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.

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.

Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.