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Aim: To understand miRNA changes across gestation in healthy human placentae. This is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy. Materials & methods: Using next-generation sequencing, we characterize the normative human placenta miRNome in first (n = 113) and third trimester (n = 47). Results & conclusion: There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (p ≥ 0.05, fold change ≤2) and 180 significantly different (false discovery rate <0.05, fold change >2). Of placenta-specific miRNA clusters, chromosome 14 miRNA cluster decreases across gestation and chromosome 19 miRNA cluster is overall highly expressed. Chromosome 13 clusters are upregulated in first trimester. This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

Herpes simplex keratitis (HSK), caused by herpes simplex virus type 1 (HSV-1) infection, is the commonest cause of infectious blindness in the developed world. Following infection the virus is initially suspended in the tear film, where it encounters a multi-pronged immune response comprising enzymes, complement, immunoglobulins and crucially, a range of anti-viral and pro-inflammatory cytokines. However, given that HSV-1 can overcome innate immune responses to establish lifelong latency throughout a susceptible individual's lifetime, there is significant interest in understanding the mechanisms employed by HSV-1 to downregulate the anti-viral type I interferon (IFN) mediated immune responses. This study aimed to investigate the interactions between infected cell protein (ICP)0 and key elements of the IFN pathway to identify possible novel targets that contribute to viral immune evasion. Reporter gene assays demonstrated the ability of ICP0 to inhibit type I IFN activity downstream of pathogen recognition receptors (PRRs) which are known to be involved in host antiviral defences. Further experiments identified interferon regulatory factor (IRF)7, a driver of type I IFN, as a potential target for ICP0. These findings increase our understanding of the pathogenesis of HSK and suggest IRF7 as a potential therapeutic target.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation 1-3 . Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells 4-6 . Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72 -/- mice show marked early activation of the type I interferon response, and C9orf72 -/- myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein-a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72 -/- myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72 -/- immune cells as well as splenomegaly and inflammation in C9orf72 -/- mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.

Innate immune sensors that detect nucleic acids are attractive targets for therapeutic intervention because of their diverse roles in many disease processes. In detecting RNA and DNA from either self or non-self, nucleic acid sensors mediate the pathogenesis of many autoimmune and inflammatory conditions. Despite promising pre-clinical data and investigational use in the clinic, relatively few drugs targeting nucleic acid sensors are approved for therapeutic use. Nevertheless, there is growing appreciation for the untapped potential of nucleic acid sensors as therapeutic targets, driven by the need for better therapies for cancer, infectious diseases, and autoimmune disorders. This review highlights the diverse mechanisms by which nucleic acid sensors are activated and exert their biological effects in the context of various disease settings. We discuss current therapeutic strategies utilizing agonists and antagonists targeting nucleic acid sensors to treat infectious disease, cancer, and autoimmune and inflammatory disorders., Competing Interests: Declaration of Interests S.M.M. is an employee and shareholder at Aduro Biotech and an inventor on Aduro Biotech patents and patent applications. This review was authored in part by S.M.M. in her personal capacity. The information and opinions expressed in the text are the author’s own and do not represent the views of Aduro Biotech., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In primary Sjögren's syndrome (pSS) the exocrine glands become infiltrated with lymphocytes instigating severe damage to the salivary and lacrimal glands causing dry eyes and dry mouth. Previous investigations have suggested that dysregulated localized and systemic inflammation contributes to the development and pathogenesis of pSS. A miR microarray performed in primary human conjunctival epithelial cells (PECs) demonstrated significant differences in miR expression at the ocular surface between pSS patients and healthy controls. MicroRNA-744-5p (miR-744-5p) was identified as being of particular interest, as its top predicted target is Pellino3 (PELI3), a known negative regulator of inflammation. Validation studies confirmed that miR-744-5p expression is significantly increased in PECs from pSS patients, whilst PELI3 was significantly reduced. We validated the miR-744 binding site in the 3' untranslated region (UTR) of PELI3 and demonstrated that increasing PELI3 levels with a miR-744-5p antagomir in an inflammatory environment resulted in reduced levels of IFN dependent chemokines Rantes (CCL5) and CXCL10. These results reveal a novel role for miR-744-5p in mediating ocular inflammation via Pellino3 expression in pSS patients and suggest that miR-744-5p may be a potential therapeutic target for the management of severe dry eye disease and ocular inflammation in pSS patients.

This study sought to identify potential therapeutic targets in herpes simplex keratitis (HSK) patients with active and inactive infection by investigating peripheral cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum were prepared from healthy controls and HSK patients during active infection or following treatment (inactive infection). Serum antibody titres were determined by ELISA. Protein expression levels were analysed by Western blot. Cytokine levels were determined by multiplex ELISA. Active corneal herpes simplex virus type 1 (HSV-1) infection resulted in significantly elevated peripheral levels of IL-1β in HSK patients compared to healthy controls, and remained significantly increased following treatment. Elevated production of IL-1β in inactive patients was associated with significantly increased levels of IRF3 and STAT1, key proteins involved in promoting anti-viral immune responses. Our data suggest that inflammation persists beyond the period that it is clinically evident and that enhanced peripheral production of IL-1β may have implications for HSV-1 viral clearance in active and inactive HSK patients.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-β. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING , TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Purpose of Review: The systemic inflammatory nature of systemic lupus erythematosus (SLE) is patent not only in the diverse clinical manifestations of the disease but also in the increased risk of premature cardiovascular diseases (CVD). In this review, we discuss the latest findings on the key factors of the innate immune system known to play critical roles in the pathogenesis of accelerated CVD in patients with SLE and discuss the potential that immunometabolism may play a key role in this respect., Recent Findings: Recent studies exploring the association between SLE and premature CVD clearly showed that alterations of specific immune functions play a pivotal role in the increased cardiovascular morbidity and mortality in the SLE patients. Novel molecular factors such as type I interferons (IFN), dysregulated neutrophil function, and changes to cellular metabolism and metabolites are emerging as important regulators of systemic immune dysfunction and as strong risk factors for premature CVD in SLE. Although corticosteroids and cytotoxic agents can be used to effectively manage and control various lupus-related complications, to date, no drug has been proven to prevent the development of premature atherosclerosis in SLE. However, as new mechanisms underlying this complication of SLE are uncovered, such as the role of metabolism and neutrophil-driven inflammation, new avenues for therapeutic intervention are being discovered.

Ward JB, Lajczak NK, Kelly OB, O'Dwyer AM, Giddam AK, Ní Gabhann J, Franco P, Tambuwala MM, Jefferies CA, Keely S, Roda A, Keely SJ. Ursodeoxycholic acid and lithocholic acid exert anti-inflammatory actions in the colon. Am J Physiol Gastrointest Liver Physiol 312: G550-G558, 2017. First published March 30, 2017; doi:10.1152/ajpgi.00256.2016.-Inflammatory bowel diseases (IBD) comprise a group of common and debilitating chronic intestinal disorders for which currently available therapies are often unsatisfactory. The naturally occurring secondary bile acid, ursodeoxycholic acid (UDCA), has well-established anti-inflammatory and cytoprotective actions and may therefore be effective in treating IBD. We aimed to investigate regulation of colonic inflammatory responses by UDCA and to determine the potential impact of bacterial metabolism on its therapeutic actions. The anti-inflammatory efficacy of UDCA, a nonmetabolizable analog, 6α-methyl-UDCA (6-MUDCA), and its primary colonic metabolite lithocholic acid (LCA) was assessed in the murine dextran sodium sulfate (DSS) model of mucosal injury. The effects of bile acids on cytokine (TNF-α, IL-6, Il-1β, and IFN-γ) release from cultured colonic epithelial cells and mouse colonic tissue in vivo were investigated. Luminal bile acids were measured by gas chromatography-mass spectrometry. UDCA attenuated release of proinflammatory cytokines from colonic epithelial cells in vitro and was protective against the development of colonic inflammation in vivo. In contrast, although 6-MUDCA mimicked the effects of UDCA on epithelial cytokine release in vitro, it was ineffective in preventing inflammation in the DSS model. In UDCA-treated mice, LCA became the most common colonic bile acid. Finally, LCA treatment more potently inhibited epithelial cytokine release and protected against DSS-induced mucosal inflammation than did UDCA. These studies identify a new role for the primary metabolite of UDCA, LCA, in preventing colonic inflammation and suggest that microbial metabolism of UDCA is necessary for the full expression of its protective actions. NEW & NOTEWORTHY On the basis of its cytoprotective and anti-inflammatory actions, the secondary bile acid ursodeoxycholic acid (UDCA) has well-established uses in both traditional and Western medicine. We identify a new role for the primary metabolite of UDCA, lithocholic acid, as a potent inhibitor of intestinal inflammatory responses, and we present data to suggest that microbial metabolism of UDCA is necessary for the full expression of its protective effects against colonic inflammation., (Copyright © 2017 the American Physiological Society.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, result from a loss of tolerance to self-antigens and immune-mediated injury precipitated by the overproduction of type I IFN and inflammatory cytokines. We have identified the inositol 5' phosphatase SHIP-1 as a negative regulator of TLR3-induced type I IFN production. SHIP-1-deficient macrophages display enhanced TLR-induced IFN-beta production, and overexpression of SHIP-1 negatively regulates the ability of TLR3 and its adaptor, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta, to induce IFN-beta promoter activity, indicating that SHIP-1 negatively regulates TLR-induced IFN-beta production. Further dissection of the IFN-beta pathway implicates TANK-binding kinase 1 (TBK1) as the target for SHIP-1. Critically, in the absence of SHIP-1, TBK1 appears to be hyperphosphorylated both in unstimulated cells and following TLR3 stimulation. In addition, TBK1 appears to be constitutively associated with Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta and TNFR-associated factor 3 in SHIP-1 deficient cells, whereas in wild-type cells this association is inducible following TLR3 stimulation. In support of a role for SHIP-1 in regulating complex formation, confocal microscopy demonstrates that TBK1 distribution in the cell is significantly altered in SHIP-1-deficient cells, with more prominent endosomal staining observed, compared with wild-type controls. Taken together, our results point to SHIP-1 as a critical negative regulator of IFN-beta production downstream of TLR3 through the regulation of TBK1 localization and activity.

Two-dimensional (2-D) gel electrophoresis concerted with protein identification by mass spectrometry (MS) is an extremely powerful method for comparative expression profiling of complex protein samples such as cell lysates. The highly resolutive 2-D electrophoresis allows the separation of heterogeneous protein samples on the basis of isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance ((1) J Biol Chem 250: 4007-4021, 1975; (2) Electrophoresis 14: 1067-1073, 1993). Consequently, it provides a comprehensive view of a proteome state ((3) Electrophoresis 21: 1037-1053, 2000), where variations in protein expression levels, isoforms, or post-translational modifications (e.g. phosphorylation) can be highlighted and investigated ((4) Electrophoresis 21: 2196-2208, 2000). Furthermore, this allows the identification of biological markers that characterize a specific physiological or pathological background of a cell or a tissue ((5) Proteomics 1: 397-408, 2001; (6) J Bacteriol 179: 7595-7599, 1997). In this way one can compare the effects of a stimulus or drug on cells or tissue, or more importantly, analyse the effects of disease on the expression level of proteins. Relatively recently, conventional 2-D gel electrophoresis has been combined with protein labelling strategies using up to three different fluorescent dyes to allow comparative analysis of different protein samples within a single 2-D gel platform. In this technique, termed differential in-gel electrophoresis (DIGE), samples are labelled separately then combined and run on the same 2D gel minimizing experimental variation and greatly facilitating spot matching. When three CyDyes (Cy2, Cy3, and Cy5) have been used, three images of the gel are captured then superposed to localize the differentially regulated spots on the 2-D gel using image analysis software. This is an extremely powerful tool in comparative proteomics as these dyes provide a linear response to protein concentration up to five orders of magnitude and great sensitivity with detection down to 125 pg of a single protein, which is less than needed for MS identification. In this chapter, we describe the basic methods for protein labelling, optimization of the isoelectrofocusing parameters for the first dimension (where proteins are separated according to their isoelectric point (pI)), sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation for the second dimension (based on molecular weight (MW)), and different post-staining protocols of the 2-D gel and protein preparation for mass spectrometry identification.

Purification of protein complexes and identification of the constituent components therein have been made relatively simple by the recent advances in proteomics. Uniting good biochemical and protein chemistry techniques with protein identification by mass spectrometry (MS) has resulted in advances in this field that are unprecedented. Our knowledge of Toll-like receptor (TLR) biology has been considerably advanced through the use of such techniques, with key intermediates such as TRAF3, TANK, RIP1 all being identified using proteomic strategies. Applying these techniques to key questions in TLR -biology will undoubtedly serve to further advance the field.

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-beta response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-beta promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-beta production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.