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Systemic lupus erythematosus (SLE) patients are 90% women and over three times more likely to die of cardiovascular disease than women in the general population. Chest pain with no obstructive cardiac disease is associated with coronary microvascular disease (CMD), where narrowing of the small blood vessels can lead to ischemia, and frequently reported by SLE patients. Using whole blood RNA samples, we asked whether gene signatures discriminate SLE patients with coronary microvascular dysfunction (CMD) on cardiac MRI (n = 4) from those without (n = 7) and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD. RNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and healthy control (HC) groups, with virus defense and interferon (IFN) signaling being the key pathways identified as enriched in SLE as expected. We next conducted a comparative analysis of genes differentially expressed in SLE-CMD and SLE-non-CMD relative to HC samples. Our analysis highlighted differences in IFN signaling, RNA sensing and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD. This is the first study to investigate possible gene signatures associating with CMD in SLE, and our data strongly suggests that distinct molecular mechanisms underly vascular changes in CMD and non-CMD involvement in SLE., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: The experiments were approved by the institutional review board at Cedars-Sinai Medical Center, which oversees the approval of experiments, specifically in the realm of medical ethics. We confirm that all experiments were conducted in accordance with relevant guidelines and regulations, including the Principles of Human Experimentation Ethics and Good Clinical Practice., (© 2025. The Author(s).)

The role of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) is well documented, but the role of interleukin (IL)-1β remains elusive. In this issue of Immunity, Caielli et al. identified an SLE monocyte population coproducing IL-1β and IFN-I and described how mitochondrial nucleic-acid-containing RBCs engage cGAS/STING, RIG-I, MDA5, and NLRP3 for unconventional IL-1β release., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Fetal sex and placental development impact pregnancy outcomes and fetal-maternal health, but the critical timepoint of placenta establishment in first trimester is understudied in human pregnancies., Methods: Pregnant subjects were recruited in late first trimester (weeks 10-14) at time of chorionic villus sampling, a prenatal diagnostic test. Leftover placenta tissue was collected and stored until birth outcomes were known, then DNA and RNA were isolated from singleton, normal karyotype pregnancies resulting in live births. DNA methylation was measured with the Illumina Infinium MethylationEPIC BeadChip array (n = 56). Differential methylation analysis compared 25 females versus 31 males using a generalized linear model on 743,461 autosomal probes. Gene expression sex differences were analyzed with RNA-sequencing (n = 74). An integrated analysis was performed using linear regression to correlate gene expression and DNA methylation in 51 overlapping placentas., Results: Methylation analysis identified 151 differentially methylated probes (DMPs) significant at false discovery rate < 0.05, including 89 (59%) hypermethylated in females. Probe cg17612569 (GABPA, ATP5J) was the most significant CpG site, hypermethylated in males. There were 11 differentially methylated regions affected by fetal sex, with transcription factors ZNF300 and ZNF311 most significantly hypermethylated in males and females, respectively. RNA-sequencing identified 152 genes significantly sexually dimorphic at false discovery rate < 0.05. The 151 DMPs were associated with 18 genes with gene downregulation (P < 0.05) in the direction of hypermethylation, including 2 genes significant at false discovery rate < 0.05 (ZNF300 and CUB and Sushi multiple domains 1, CSMD1). Both genes, as well as Family With Sequence Similarity 228 Member A (FAM228A), showed significant correlation between DNA methylation and sexually dimorphic gene expression, though FAM228A DNA methylation was less sexually dimorphic. Comparison with other sex differences studies found that cg17612569 is male-hypermethylated across gestation in placenta and in human blood up to adulthood., Conclusions: Overall, sex dimorphic differential methylation with associated differential gene expression in the first trimester placenta is small, but there remain significant genes that may be regulated through methylation leading to differences in the first trimester placenta., (© 2024. The Author(s).)

The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal-fetal health. The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes (SEGs) not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background. Placenta expresses 14,979 polyadenylated genes above sequencing noise (transcripts per million > 0.66), with 10.7% SEGs across gestation. Differentially expressed genes (DEGs) account for 86.7% of genes in the full cohort [false discovery rate (FDR) < 0.05]. Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR < 0.001, fold change > 1.5), there remains 50.1% DEGs (3353 upregulated in first and 4155 upregulated in third trimester). This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and SEGs may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers for maternal-fetal health., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Fetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes., Objectives: We utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation., Study Design: We analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences., Results: Placenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3., Discussion: This is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes., Competing Interests: Declaration of competing interest The authors report no conflict of interest, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Systemic lupus erythematosus (SLE) patients are 90% women and over three times more likely to die of cardiovascular disease than women in the general population. Chest pain with no obstructive cardiac disease is associated with coronary microvascular disease (CMD), where narrowing of the small blood vessels can lead to ischemia, and frequently reported by SLE patients. Using whole blood RNA samples, we asked whether gene signatures discriminate SLE patients with coronary microvascular dysfunction (CMD) on cardiac MRI (n=4) from those without (n=7) and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD. RNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and healthy control (HC) groups, with virus defense and interferon (IFN) signaling being the key pathways identified as enriched in SLE as expected. We next conducted a comparative analysis of genes differentially expressed in SLE-CMD and SLE-non-CMD relative to HC samples. Our analysis highlighted differences in IFN signaling, RNA sensing and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD. This is the first study to investigate possible gene signatures associating with CMD in SLE, and our data strongly suggests that distinct molecular mechanisms underly vascular changes in CMD and non-CMD involvement in SLE., Competing Interests: Competing Interest Statement: Authors declare that there are no competing interests.

Objective: We aimed to investigate the hypothesis that interferon (IFN)-stimulated gene (ISG) expression in systemic lupus erythematosus (SLE) monocytes is linked to changes in metabolic reprogramming and epigenetic regulation of ISG expression., Methods: Monocytes from healthy volunteers and patients with SLE at baseline or following IFNα treatment were analyzed by extracellular flux analysis, proteomics, metabolomics, chromatin immunoprecipitation, and gene expression. The histone demethylases KDM6A/B were inhibited using glycogen synthase kinase J4 (GSK-J4). GSK-J4 was tested in pristane and resiquimod (R848) models of IFN-driven SLE., Results: SLE monocytes had enhanced rates of glycolysis and oxidative phosphorylation compared to healthy control monocytes, as well as increased levels of isocitrate dehydrogenase and its product, α-ketoglutarate (α-KG). Because α-KG is a required cofactor for histone demethylases KDM6A and KDM6B, we hypothesized that IFNα may be driving "trained immune" responses through altering histone methylation. IFNα priming (day 1) resulted in a sustained increase in the expression of ISGs in primed cells (day 5) and enhanced expression on restimulation with IFNα. Importantly, decreased H3K27 trimethylation was observed at the promoters of ISGs following IFNα priming. Finally, GSK-J4 (KDM6A/B inhibitor) resulted in decreased ISG expression in SLE patient monocytes, as well as reduced autoantibody production, ISG expression, and kidney pathology in R848-treated BALB/c mice., Conclusion: Our study suggests long-term IFNα exposure alters the epigenetic regulation of ISG expression in SLE monocytes via changes in immunometabolism, a mechanism reflecting trained immunity to type I IFN. Importantly, it opens the possibility that targeting histone-modifying enzymes, such as KDM6A/B, may reduce IFN responses in SLE., (© 2023 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

During transfusion of red blood cells (RBCs), recipients are exposed to both ABO and non-ABO 'minor' antigens. RBC donor units and recipient RBCs are not routinely matched for non-ABO antigens. Thus, recipients are exposed to many RBC alloantigens that can lead to RBC alloantibody production and subsequent clinically significant hemolysis. RBC alloantibodies also significantly limit the provision of compatible RBC units for recipients. Prior studies indicate that the frequency of RBC alloimmunization is increased during inflammatory responses and in patients with autoimmune diseases. Still, mechanisms contributing to alloimmune responses in patients with autoimmunity are not well understood. More than half of adult patients with systemic lupus erythematosus (SLE) produce type 1 interferons (IFNα/β) and express IFNα/β stimulated genes (ISGs). Previously, we reported that IFNα/β promote RBC alloimmune responses in the pristane mouse model, which develops a lupus-like phenotype that is dependent on IFNα/β signaling. However, it is unclear whether IFNα/β or the lupus-like phenotype induces alloimmunization in lupus models. Therefore, we tested the hypothesis that IFNα/β promotes RBC alloimmune responses in lupus by examining alloimmune responses in IFNα/β-independent (MRL- lpr ) and IFNα/β-dependent (pristane) lupus models. Whereas pristane treatment significantly induced interferon-stimulated genes (ISGs), MRL- lpr mice produced significantly lower levels that were comparable to levels in untreated WT mice. Transfusion of murine RBCs that express the KEL antigen led to anti-KEL IgG production by pristane-treated WT mice. However, MRL- lpr mice produced minimal levels of anti-KEL IgG. Treatment of MRL-lpr mice with recombinant IFNα significantly enhanced alloimmunization. Collectively, results indicate that a lupus-like phenotype in pre-clinical models is not sufficient to induce RBC alloantibody production, and IFNα/β gene signatures may be responsible for RBC alloimmune responses in lupus mouse models. If these findings are extended to alternate pre-clinical models and clinical studies, patients with SLE who express an IFNα/β gene signature may have an increased risk of developing RBC alloantibodies and may benefit from more personalized transfusion protocols., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Paul, Hernández-Armengol, Lee, Chang, Shibata, Yamashita, Jefferies and Gibb.)

Background: To analyze individually and interactively critical risk factors, which are closely related to low bone mineral density (BMD) in patient with ankylosing spondylitis (AS)., Methods: A total of 249 AS patients who visited China-Japan Friendship Hospital were included in this training set. Patients with questionnaire data, blood samples, X-rays, and BMD were collected. Logistic regression analysis was employed to identify key risk factors for low BMD in different sites, and predictive accuracy was improved by incorporating the selected significant risk factors into the baseline model, which was then validated using a validation set. The interaction between risk factors was analyzed, and predictive nomograms for low BMD in different sites were established., Results: There were 113 patients with normal BMD, and 136 patients with low BMD. AS patients with hip involvement are more likely to have low BMD in the total hip, whereas those without hip involvement are more prone to low BMD in the lumbar spine. Chest expansion, mSASSS, radiographic average grade of the sacroiliac joint, and hip involvement were significantly associated with low BMD of the femoral neck and total hip. Syndesmophytes, hip involvement and higher radiographic average grade of the sacroiliac joint increases the risk of low BMD of the femoral neck and total hip in an additive manner. Finally, a prediction model was constructed to predict the risk of low BMD in total hip and femoral neck., Conclusions: This study identified hip involvement was strongly associated with low BMD of the total hip in AS patients. Furthermore, the risk of low BMD of the femoral neck and total hip was found to increase in an additive manner with the presence of syndesmophytes, hip involvement, and severe sacroiliitis. This finding may help rheumatologists to identify AS patients who are at a high risk of developing low BMD and prompt early intervention to prevent fractures., Competing Interests: The authors declare that they have no competing interests., (© 2023 Sun et al.)

Background: Women with SLE have an elevated risk of cardiovascular disease. Many women with SLE frequently report chest pain in the absence of obstructive coronary artery disease (CAD) due to coronary microvascular dysfunction (CMD), a form of ischemia with no obstructive CAD. Echocardiographic studies have shown that SLE patients have reduced left ventricular (LV) function, which may also correlate with higher SLE disease activity scores. As such, we used cardiac magnetic resonance imaging (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers, and cardiac function in female SLE patients., Methods: We performed stress cMRI in women with SLE and chest pain with no obstructive CAD (n=13, all met ACR 1997 criteria,) and reference controls (n=22) using our published protocol. We evaluated LV function, tissue characterization (T1 mapping, ECV), and delayed enhancement, using CV142 software (Circle Cardiovascular Imaging Inc, Calgary, AB, Canada). Myocardial perfusion reserve index (MPRI) was calculated using our published protocol. SLEDAI and SLICC Damage Index (DI) were calculated per validated criteria. Serum samples were analyzed for inflammatory markers and autoantibodies. Wilcoxon rank-sum test was performed on clinical values with CMD and no CMD SLE subjects, and on cMRI values with all SLE subjects and controls. Correlation analysis was done on clinical values, and cMRI values on all SLE subjects., Results: Overall, 40% of SLE subjects had MPRI values < 1.84, consistent with CMD. Compared to controls, SLE subjects had significantly lower LVEF, and higher LVESVi and LVMi. Corresponding to this, radial, longitudinal, and circumferential strain were significantly lower in the SLE subjects. In correlation analysis of serum inflammatory biomarkers to cMRI values in the SLE subjects, SLICC DI was related to worse cardiac function (lower radial, circumferential and longitudinal strain) and higher T1 time. Additionally, fasting insulin and ESR were negatively correlated with LVMi. Fasting insulin also negatively correlated with ECV. CRP had a positive association with LVESV index and CI and a negative association with longitudinal strain., Conclusions: Among women with SLE with chest pain and no obstructive CAD, 40% have CMD. While evaluations of known inflammatory markers (such as CRP and ESR) predictably correlated with decreased cardiac function, our study found that decreased fasting insulin levels as a novel marker of diminished LV function. In addition, low insulin levels were observed to correlate with increased LVMi and ECV, suggesting a cardioprotective effect of insulin in SLE patients. We also noted that SLICC DI, an assessment of SLE damage, correlates with cardiac dysfunction in SLE. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE, particularly in patients with high SLICC DI, ESR and CRP and low fasting insulin levels., Competing Interests: The authors have no disclosures to report.

Background: The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal- fetal health., Methods: The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background., Results: Placenta expresses 14,979 mRNAs above sequencing noise (TPM>0.66), with 1,545 stably expressed genes across gestation. Differentially expressed genes account for 86.7% of genes in the full cohort (FDR<0.05). Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR<0.001, fold change>1.5), there are 6,941 differentially expressed protein coding genes (3,206 upregulated in first and 3,735 upregulated in third trimester)., Conclusion: This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and stably expressed genes may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers in maternal-fetal disease.

Metabolic rewiring underlies the effector functions of macrophages 1-3 , but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Women with SLE have an elevated risk of CVD morbidity and mortality and frequently report chest pain in the absence of obstructive CAD. Echocardiographic studies often demonstrate reduced LV function, correlating with higher disease activity. We used cardiac MRI (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers and cardiac function in female SLE patients., Methods: Women with SLE reporting chest pain with no obstructive CAD (n=13) and reference controls (n=22) were evaluated using stress-rest cMRI to measure LV structure, function, tissue characteristics, and myocardial perfusion reserve index (MPRI). Coronary microvascular dysfunction (CMD) was defined as MPRI <1.84. Serum samples were analyzed for inflammatory markers. Relationships between clinical and cMRI values of SLE subjects were assessed, and groups were compared., Results: 40% of SLE subjects had MPRI < 1.84 on cMRI. Compared to controls, SLE subjects had higher LV volumes and mass and lower LV systolic function. SLICC DI was related to worse cardiac function and higher T1. CRP was related to higher cardiac output and a trend to better systolic function, while ESR and fasting insulin were related to lower LV mass. Lower fasting insulin levels correlated with increased ECV., Conclusions: Among our female SLE cohort, 40% had CMD, and SLICC DI correlated with worse cardiac function and diffuse fibrosis. Higher inflammatory markers and low insulin levels may associate with LV dysfunction. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE patients.

Rationale: Effective therapies to reduce the severity and high mortality of pulmonary vasculitis and diffuse alveolar hemorrhage (DAH) in patients with systemic lupus erythematosus (SLE) is a serious unmet need. We explored whether biologic neutralization of eNAMPT (extracellular nicotinamide phosphoribosyl-transferase), a novel DAMP and Toll-like receptor 4 ligand, represents a viable therapeutic strategy in lupus vasculitis., Methods: Serum was collected from SLE subjects (n = 37) for eNAMPT protein measurements. In the preclinical pristane-induced murine model of lung vasculitis/hemorrhage, C57BL/6 J mice (n = 5-10/group) were treated with PBS, IgG (1 mg/kg), or the eNAMPT-neutralizing ALT-100 mAb (1 mg/kg, IP or subcutaneously (SQ). Lung injury evaluation (Day 10) included histology/immuno-histochemistry, BAL protein/cellularity, tissue biochemistry, RNA sequencing, and plasma biomarker assessment., Results: SLE subjects showed highly significant increases in blood NAMPT mRNA expression and eNAMPT protein levels compared to healthy controls. Preclinical pristane-exposed mice studies showed significantly increased NAMPT lung tissue expression and increased plasma eNAMPT levels accompanied by marked increases in alveolar hemorrhage and lung inflammation (BAL protein, PMNs, activated monocytes). In contrast, ALT-100 mAb-treated mice showed significant attenuation of inflammatory lung injury, alveolar hemorrhage, BAL protein, tissue leukocytes, and plasma inflammatory cytokines (eNAMPT, IL-6, IL-8). Lung RNA sequencing showed pristane-induced activation of inflammatory genes/pathways including NFkB, cytokine/chemokine, IL-1β, and MMP signaling pathways, each rectified in ALT-100 mAb-treated mice., Conclusions: These findings highlight the role of eNAMPT/TLR4-mediated inflammatory signaling in the pathobiology of SLE pulmonary vasculitis and alveolar hemorrhage. Biologic neutralization of this novel DAMP appears to serve as a viable strategy to reduce the severity of SLE lung vasculitis., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joe G.N. Garcia, MD reports financial support and equipment, drugs, or supplies were provided by Aqualung Therapeutics, LLC. Joe G.N. Garcia, MD reports a relationship with Aqualung Therapeutics, LLC that includes: equity or stocks. Joe G.N. Garcia, MD has patent pending to Aqualung Therapeutics, LLC., (© 2022 Published by Elsevier B.V.)

SARS-CoV-2 vaccines have unquestionably blunted the overall impact of the COVID-19 pandemic, but host factors such as age, sex, obesity, and other co-morbidities can affect vaccine efficacy. We identified individuals in a relatively healthy population of healthcare workers (CORALE study cohort) who had unexpectedly low peak anti-spike receptor binding domain (S-RBD) antibody levels after receiving the BNT162b2 vaccine. Compared to matched controls, "low responders" had fewer spike-specific antibody-producing B cells after the second and third/booster doses. Moreover, their spike-specific T cell receptor (TCR) repertoire had less depth and their CD4 + and CD8 + T cell responses to spike peptide stimulation were less robust. Single cell transcriptomic evaluation of peripheral blood mononuclear cells revealed activation of aging pathways in low responder B and CD4 + T cells that could underlie their attenuated anti-S-RBD antibody production. Premature lymphocyte aging may therefore contribute to a less effective humoral response and could reduce vaccination efficacy., Competing Interests: Authors declare that they have no competing interests., (© 2022 The Author(s).)

Acknowledging sex differences in immune response is particularly important when we consider the differences between men and women in the incidence of disease. For example, over 80% of autoimmune disease occurs in women, whereas men have a higher incidence of solid tumors compared to women. In general women have stronger innate and adaptive immune responses than men, explaining their ability to clear viral and bacterial infections faster, but also contributing to their increased susceptibility to autoimmune disease. The autoimmune disease systemic lupus erythematosus (SLE) is the archetypical sexually dimorphic disease, with 90% of patients being women. Various mechanisms have been suggested to account for the female prevalence of SLE, including sex hormones, X-linked genes, and epigenetic regulation of gene expression. Here, we will discuss how these mechanisms contribute to pathobiology of SLE and how type I interferons work with them to augment sex specific disease pathogenesis in SLE., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 The Author(s), Published by Wolters Kluwer Health, Inc.)

Chest pain is a common symptom in patients with systemic lupus erythematosus, an autoimmune disease that is associated with increased cardiovascular morbidity and mortality. While chest pain mechanisms can be multifactorial and often attributed to non-coronary or non-cardiac cardiac etiologies, emerging evidence suggests that ischemia with no obstructive coronary arteries (INOCA) is a prevalent condition in patients with chest pain and no obstructive coronary artery disease. Coronary microvascular dysfunction is reported in approximately half of SLE patients with suspected INOCA. In this mini review, we highlight the cardiovascular risk assessment, mechanisms of INOCA, and diagnostic approach for patients with SLE and suspected CMD., Competing Interests: CB has served as consultant for Sanofi, Abbott Diagnostics, and iRhythm. DB has served as a consultant for Bayer and received software royalties from Cedars-Sinai Medical Center. JW has served as consultant and advisory board member for Abbott Vascular. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Manchanda, Kwan, Ishimori, Thomson, Li, Berman, Bairey Merz, Jefferies and Wei.)

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR < 0.05) miRNAs were identified in the first and 4 in the third trimester, all upregulated in females, including miR-361-5p, significant in both trimesters. Sex-specific analyses across gestation identified 677 differentially expressed (DE) miRNAs at FDR < 0.05 and baseMean>10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b + Ly6G + neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip . Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tumurkhuu, Laguna, Moore, Contreras, Santos, Akaveka, Montano, Wang, Ishimori, Venuturupalli, Forbess, Stripp, Wallace and Jefferies.)

Objectives: A cross-sectional study was conducted in 270 Chinese patients with ankylosing spondylitis (AS) in order to identify potential risk factors for severity of spinal structural damage., Methods: Two hundred seventy AS patients fulfilled the Modified New York Criteria. Computed tomography (CT) was used to scan sacroiliac and hip joints, and radiography was used to scan anteroposterior and lateral lumbar spine, as well as lateral cervical spine. Bath Ankylosing Spondylitis Radiology Index and modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) were scored in duplicate., Results: One hundred eighty-three patients had low mSASSS (mSASSS, <10), and 87 patients had high mSASSS (mSASSS, ≥10). Univariate analysis revealed that AS age of onset, body mass index (BMI), smoking duration, duration of symptoms, diagnostic delay, hip involvement, and sacroiliitis grade were significantly associated with the risk of having high mSASSS after adjustment (all p's < 0.05). Hip involvement interacted significantly with BMI and smoking duration in a graded manner. Particularly, relative to patients with low BMI-negative hip involvement, those with high BMI-negative hip involvement, low BMI-positive hip involvement, and high BMI-positive hip involvement had a 1.94-fold, 3.29-fold, and 5.07-fold increased risk of high mSASSS (95% confidence interval, 0.84-4.47, 1.37-7.89, and 1.97-13.06, p = 0.118, 0.008, and 0.001, respectively). Finally, a nomogram graph based on 7 significant risk factors was generated with substantial prediction accuracy (concordance index, 0.906)., Conclusions: We have identified 7 potential risk factors for the severity of spinal structural damage in Chinese AS patients. Importantly, positive hip involvement, combined with high BMI or long smoking duration, was associated with a remarkably increased risk of having severe spinal structural damage., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Type I interferon (IFN-I) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and the closely associated monogenic autoinflammatory disorders termed the "interferonopathies." Recently, the cytosolic DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and its downstream signaling adaptor stimulator of interferon genes (STING) have been identified as having important, if not central, roles in driving IFN-I expression in response to self-DNA. This review highlights the many ways in which this pathway is regulated in order to prevent self-DNA recognition and underlines the importance of maintaining tight control in order to prevent autoimmune disease. We will discuss the murine and human studies that have implicated the cGAS-STING pathway as being an important contributor to breakdown in tolerance in SLE and highlight the potential therapeutic application of this knowledge for the treatment of SLE., Competing Interests: Conflict of Interest There is no conflict of interest declared., (© 2021 Audrey M. Hagiwara et al., published by Sciendo.)

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.

Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that promote clinically significant hemolytic events. Multiple studies have reported an increased frequency of RBC alloimmunization in patients with autoimmunity. However, cellular and molecular mechanisms that underlie autoimmunity-induced alloimmunization have not been reported. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature. Thus, we utilized the pristane-induced lupus mouse model to test the hypothesis that inflammation in lupus promotes RBC alloimmunization, and to examine the potential role of IFNα/β. Intraperitoneal injection of pristane, a hydrocarbon oil, led to autoantibody production, glomerulonephritis, and pulmonary hemorrhage in wild type (WT) mice. Pristane treatment significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISGs) in peripheral blood and peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL glycoprotein, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane induced comparable levels of inflammatory cells and cytokines in mice lacking the IFNα/β receptor (IFNAR1 -/- ) or the IFNα/β-inducing transcriptions factors (IRF3/7 -/- ), compared to WT mice. However, pristane-treated IFNAR1 -/- and IRF3/7 -/- mice failed to produce ISGs and produced significantly lower levels of transfusion-induced anti-KEL IgG, compared to WT mice. Thus, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. To our knowledge, this is the first examination of molecular mechanisms contributing to RBC alloimmunization in a model of autoimmunity. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of the IFNα/β gene signature to the elevated frequency of alloimmunization in patients with SLE., (Copyright © 2020 Lee, Madany, El Kadi, Pandya, Ng, Yamashita, Jefferies and Gibb.)

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease in which type I interferons (IFN) play a key role. The IFN response can be triggered when oxidized DNA engages the cytosolic DNA sensing platform cGAS-STING, but the repair mechanisms that modulate this process and govern disease progression are unclear. To gain insight into this biology, we interrogated the role of oxyguanine glycosylase 1 (OGG1), which repairs oxidized guanine 8-Oxo-2'-deoxyguanosine (8-OH-dG), in the pristane-induced mouse model of SLE. Ogg1 -/- mice showed increased influx of Ly6C hi monocytes into the peritoneal cavity and enhanced IFN-driven gene expression in response to short-term exposure to pristane. Loss of Ogg1 was associated with increased auto-antibodies (anti-dsDNA and anti-RNP), higher total IgG, and expression of interferon stimulated genes (ISG) to longer exposure to pristane, accompanied by aggravated skin pathology such as hair loss, thicker epidermis, and increased deposition of IgG in skin lesions. Supporting a role for type I IFNs in this model, skin lesions of Ogg1 -/- mice had significantly higher expression of type I IFN genes ( Isg15, Irf9 , and Ifnb ). In keeping with loss of Ogg1 resulting in dysregulated IFN responses, enhanced basal and cGAMP-dependent Ifnb expression was observed in BMDMs from Ogg1 -/- mice. Use of the STING inhibitor, H151, reduced both basal and cGAMP-driven increases, indicating that OGG1 regulates Ifnb expression through the cGAS-STING pathway. Finally, in support for a role for OGG1 in the pathology of cutaneous disease, reduced OGG1 expression in monocytes associated with skin involvement in SLE patients and the expression of OGG1 was significantly lower in lesional skin compared with non-lesional skin in patients with Discoid Lupus. Taken together, these data support an important role for OGG1 in protecting against IFN production and SLE skin disease., (Copyright © 2020 Tumurkhuu, Chen, Montano, Ercan Laguna, De Los Santos, Yu, Lane, Yamashita, Markman, Blanco, Kaplan, Shimada, Crother, Ishimori, Wallace, Jefferies and Arditi.)

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.

The Interferon regulatory factors (IRFs) are a family of transcription factors that play pivotal roles in many aspects of the immune response, including immune cell development and differentiation and regulating responses to pathogens. Three family members, IRF3, IRF5, and IRF7, are critical to production of type I interferons downstream of pathogen recognition receptors that detect viral RNA and DNA. A fourth family member, IRF9, regulates interferon-driven gene expression. In addition, IRF4, IRF8, and IRF5 regulate myeloid cell development and phenotype, thus playing important roles in regulating inflammatory responses. Thus, understanding how their levels and activity is regulated is of critical importance given that perturbations in either can result in dysregulated immune responses and potential autoimmune disease. This review will focus the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation, with particular emphasis on how regulation of their levels and activity by ubiquitination and microRNAs may impact autoimmune disease.

Using an in vivo model of tolerance to TLR7-induced skin inflammation, we found a critical role for macrophage-derived MMP10 in mediating immune hypo-responsiveness. Cutaneous exposure to Imiquimod (IMQ), a TLR7 agonist, induced acute expression of pro-inflammatory factors (IL1β, IL6, CXCL1) and neutrophil influx equally in both wildtype and Mmp10 -/- mice. However, whereas subsequent exposure (11 and 12 days later) to IMQ led to marked abrogation of pro-inflammatory factor expression in wildtype mice, Mmp10 -/- mice responded similarly as they did to the first application. In addition, the second exposure led to increased expression of negative regulators of TLR signaling (TNFAIP3, IRAK3) and immunosuppressive cytokines (IL10, TGFβ1) in wildtype mice but not in Mmp10 -/- mice. In vitro studies demonstrated that prior exposure of IMQ to bone marrow-derived macrophages (BMDM) made wildtype cells refractory to subsequent stimulation but did not for Mmp10 -/- macrophages. These findings expand the critical roles MMP10 plays in controlling macrophage activation to indicate that the development of immune tolerance to TLR7 ligand is dependent on this macrophage-derived proteinase.

Severe lung inflammation and alveolar hemorrhage can be life-threatening in systemic lupus erythematosus (SLE) patients if not treated early and aggressively. Neutrophil influx is the driver key of this pathology, but little is known regarding the molecular events regulating this recruitment. Here, we uncover a role for IL-16/mir-125a in this pathology and show not only that IL-16 is a target for miR-125a but that reduced miR-125a expression in SLE patients associates with lung involvement. Furthermore, in the pristane model of acute "SLE-like" lung inflammation and alveolar hemorrhage, we observed reduced pulmonary miR-125a and enhanced IL-16 expression. Neutrophil infiltration was markedly reduced in the peritoneal lavage of pristane-treated IL-16-deficient mice and elevated following i.n. delivery of IL-16. Moreover, a miR-125a mimic reduced pristane-induced IL-16 expression and neutrophil recruitment and rescued lung pathology. Mechanistically, IL-16 acts directly on the pulmonary epithelium and markedly enhances neutrophil chemoattractant expression both in vitro and in vivo, while the miR-125a mimic can prevent this. Our results reveal a role for miR-125a/IL-16 in regulating lung inflammation and suggest this axis may be a therapeutic target for management of acute lung injury in SLE.

La/SS-B (or La) is a 48 kDa RNA-binding protein and an autoantigen in autoimmune disorders such as systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). La involvement in regulating the type I interferon (IFN) response is controversial - acting through both positive and negative regulatory mechanisms; inhibiting the IFN response and enhancing viral growth, or directly inhibiting viral replication. We therefore sought to clarify how La regulates IFN production in response to viral infection. ShRNA knockdown of La in HEK 293 T cells increased Sendai virus infection efficiency, decreased IFN-β, IFN-λ1, and interferon-stimulated chemokine gene expression. In addition, knockdown attenuated CCL-5 and IFN-λ1 secretion. Thus, La has a positive role in enhancing type I and type III IFN production. Mechanistically, we show that La directly binds RIG-I and have mapped this interaction to the CARD domains of RIG-I and the N terminal domain of La. In addition, we showed that this interaction is induced following RIG-I activation and that overexpression of La enhances RIG-I-ligand binding. Together, our results demonstrate a novel role for La in mediating RIG-I-driven responses downstream of viral RNA detection, ultimately leading to enhanced type I and III IFN production and positive regulation of the anti-viral response.

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.)

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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Carriers are often an essential element of drug delivery, bestowing attributes to their cargo such as biocompatibility, enhanced delivery, extended half-life and efficacy as well as mediating specific targeting at a tissue, cell or intracellular level. Liposomes and lipid-based carriers have been investigated for decades for this purpose, many achieving clinical approval including products such as Doxil® and Myocet™. Large-scale compound screens are routinely carried out in the field of drug discovery; however, less work has been done on harnessing high-throughput methods for carrier material screening. Screening the interaction of drug carriers and materials with cells is particularly critical for the development of emerging therapies, including biomedicines, in order to facilitate the development of safe and efficient drug products. Herein, a range of liposomes of neutral, anionic and cationic charge and others that are surface-modified with mannose residues were screened for cell interaction, toxicity and immune reactivity in THP-1-derived macrophages using a high-throughput format. Liposomes were seen to be efficacious in a concentration-dependent and, for mannosylated liposomes, mannosylated cholesterol linker length-dependent manner.

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.)

Innate immune detection and subsequent immune responses rely on the initial recognition of pathogen specific molecular motifs. Foreign nucleic acids are key structures recognised by the immune system, recognition of which occurs mainly through the use of nucleic acid receptors including members of the Toll-like receptors, AIM2-like receptors, RIG-I-like receptors and intracellular DNA receptors. While the immune system is critically important in protecting the host from infection, it is of utmost importance that it is tightly regulated, in order to prevent recognition of self-nucleic acids and the subsequent development of autoimmunity. Defects in the mechanisms regulating such pathways, for example mutations in endonucleases that clear DNA, altered expression of nucleic acid sensors and defects in negative regulators of these signalling pathways involved in RNA/DNA sensing, have all been implicated in promoting the generation of autoimmune responses. This evidence, as reviewed here, suggests that novel therapeutics targeting these sensors and their downstream pathways may be of use in the treatment of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and primary Sjögren's syndrome., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to explore the role of cytokines in the pathogenesis of SLE in a genetically homogeneous Caucasian SLE patient population., Methods: Serum levels of the following cytokines were determined by ELISA in SLE patients (diagnosed as per ACR diagnostic criteria): IL-1β, IL-10, IL-12p70 and TNF-α. Demographic data, disease activity as per the SLEDAI and damage scores (SLICC) at the 5-year follow-up were calculated., Results: Enhanced production of TNF-α, IL-1 and IL-10 were observed in SLE patients compared with controls. A strong positive correlation was seen between levels of IL-12p70 and IL-10. In addition, IL-10, TNF-α and IL-1 demonstrated a significant relationship with disease activity. Interestingly, elevated levels of IL-10 were observed in SLE patients with CNS involvement while patients with elevated levels of TNF-α were more likely to have renal involvement and sustain damage over the follow-up period. Additionally, the ratio of all cytokines assayed to IL-12p70 levels were significantly higher in SLE patients when compared with controls, with an association seen between damage accrual and the IL-1β/IL-12p70 ratio (r = 0.431, P = 0.003), IL-10/IL-12p70 ratio (r = 0.351, P = 0.018) and TNF-α/IL-12p70 ratio (r = 0.33, P = 0.028). When the respective ratios were analysed for organ-specific disease, significant differences were observed for the IL-1β/IL-12p70 ratio (0.79 vs 0.47, P = 0.036), IL-10/IL-12p70 ratio (4.29 vs 1.87, P = 0.018) and TNF-α/IL-12p70 ratio (7.49 vs 5.21, P = 0.018) with respect to renal involvement., Conclusion: Increased levels of a number of immunomodulatory cytokines relative to IL-12p70 in this Caucasian SLE patient population are seen in patients with renal involvement and are associated with increased accrual of damage at the 5-year follow-up., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Transcription of the type I IFN genes is regulated by members of the Interferon Regulatory Factor (IRF) family of transcription factors, composed in humans of 9 distinct proteins. In addition to IRF3 and IRF7, the transcription factor IRF5 has been shown to be involved in type I IFN production and interestingly, polymorphisms of the IRF5 gene in humans can result in risk or protective haplotypes with regard to SLE susceptibility. In addition to regulation of type I IFN expression, IRF5 is involved in other signaling pathways, including IgG switching in B cells, macrophage polarization and apoptosis, and its role in SLE pathogenesis may therefore not be limited to dysregulated control of IFN expression. In this review we will comprehensively discuss the role of IRF5 in immune-mediated responses and its potential multifaceted role in conferring SLE susceptibility., (Copyright © 2014. Published by Elsevier Inc.)

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.

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.

Objective: To examine the role of 17β-estradiol in the regulation of the autoantigen tripartite motif-containing protein 21 (TRIM-21) in patients with systemic lupus erythematosus (SLE)., Methods: Monocytes isolated from healthy control subjects and patients with SLE were stimulated with 17β-estradiol and/or the estrogen receptor α (ERα) antagonist methyl-piperidino-pyrazole dihydrochloride. TRIM-21, ERα, and CREMα expression was determined by real-time polymerase chain reaction (PCR) analysis. MatInspector software was used to identify putative binding sites within the TRIM-21 promoter. ERα binding to the TRIM-21 gene promoter region in monocytes was analyzed by chromatin immunoprecipitation (ChIP) assay. TRIM-21 and interferon regulatory factor 3 protein levels were analyzed by Western blotting., Results: Real-time PCR analysis demonstrated a role of estrogen in the regulation of TRIM-21 expression in monocytes, which correlated positively with ERα gene expression in patients with SLE. Investigations into the human TRIM-21 promoter revealed the presence of an estrogen response element, with ChIP assays confirming ERα binding to this site. Studies into estrogen-induced TRIM-21 expression revealed a hyperresponsiveness of SLE patients to 17β-estradiol, which led to the enhanced levels of TRIM-21 observed in these individuals., Conclusion: Our results demonstrate a role of estrogen in the regulation of TRIM-21 expression through an ERα-dependent mechanism, a pathway that we observed to be overactive in SLE patients. Treatment of monocytes with an ERα antagonist abrogated estrogen-induced TRIM-21 expression and, as a consequence, decreased the expression of interleukin-23. These findings identify TRIM-21 as a novel ERα-regulated gene and provide novel insights into the link between estrogen and the molecular pathogenesis of SLE., (Copyright © 2014 by the American College of Rheumatology.)

Objective: The overall aim of this study is to identify clinical and serological features that are associated with B lymphocyte stimulator (BLyS) elevation in a homogeneous Caucasian SLE population and thereby identify patients who are most likely to benefit from BLyS blockade., Methods: Patients with SLE (as per ACR criteria) were recruited. Clinical history, disease activity measures and laboratory measures of disease were recorded. BLyS levels were determined by ELISA., Results: BLyS elevation was defined as being higher than the 95th percentile of BLyS levels measured in controls. Patients were divided into two groups: those with elevated BLyS levels (group 1, n = 23) and those with normal BLyS levels (group 2, n = 22). Elevated BLyS levels were significantly associated with patients of younger age and shorter disease duration. In keeping with previous reports, patients with elevated BLyS levels had more active disease (SLEDAI 5.1 vs 0.86, P < 0.001); however, our analysis also demonstrates that BLyS elevation was significantly associated with increased organ damage at 5-year follow-up [Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/ACR DI) 0.53 vs 0.13, P = 0.012]. Furthermore, the presence of Sm autoantibody significantly predicted elevated BLyS levels in a Caucasian population. BLyS levels were significantly higher in those with musculoskeletal involvement, malar rash, renal disease and evidence of immunological activity., Conclusion: BLyS blockade may be most beneficial if introduced early in the course of disease in young Caucasian patients presenting with renal, musculoskeletal and skin disease in an effort to reduce long-term damage.

In addition to regulating B cell development and activation, Bruton's tyrosine kinase (Btk) functions downstream of multiple TLRs, including TLR7, to regulate innate immune responses in myeloid cells. Although critical for defense against RNA viruses such as influenza and Sendai virus, recognition of self-RNA by TLR7 also has been shown to be an important contributor to the pathophysiology of systemic lupus erythematosus. To date, the role of Btk in regulating TLR7-mediated responses is poorly understood. In the current study, we have demonstrated a hitherto undiscovered role for Btk in apoptotic cell uptake, identifying the molecular chaperone calreticulin (CRT) as a novel substrate for Btk in regulating this response. CRT together with the transmembrane receptor CD91 function at the cell membrane and regulate uptake of C1q-opsonised apoptotic cells. Our results show that Btk directly phosphorylates CRT and that in the absence of Btk, CRT fails to localize with CD91 at the cell surface and at the phagocytic cup. Critically, a blocking Ab against CRT in wild-type macrophages mimics the inability of Btk-deficient macrophages to phagocytose apoptotic cells efficiently, indicating the critical importance of Btk in regulating CRT-driven apoptotic cell uptake. Our data have revealed a novel regulatory role for Btk in mediating apoptotic cell clearance, with CRT identified as the critical component of the CRT/CD91/C1q system targeted by Btk. Given the importance of clearing apoptotic cell debris to prevent inappropriate exposure of TLRs to endogenous ligands, our results have important implications regarding the role of Btk in myeloid cell function.

Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses., (Copyright © 2013 Elsevier Inc. All rights reserved.)