Pulmonary manifestations in systemic lupus erythematosus (SLE) are associated with significant morbidity and mortality and all compartments of the thorax can be impacted, either as part of the systemic disease or due to pulmonary infection, as a consequence of the immunosuppressive effects of the disease itself or the drugs used to treat it. Inflammation and dysregulation of immune responses are important drivers of lung pathology in autoimmune disease. This chapter focuses on immune-mediated mechanisms contributing to pulmonary manifestations in SLE, the clinical presentation and best management of such presentations and how novel pathways that regulate immune dysfunction in SLE may present new opportunities for treatment.

Aims Type I interferons (IFNs) are linked to an increased risk of cardiovascular disease and are chronically elevated in systemic autoimmune diseases such as systemic lupus erythematosus (SLE). We evaluated the effect of chronic IFNa exposure on cardiac and cardiomyocyte function in order to better understand IFN-driven cardiac disease from the perspective of SLE. Methods and Results Administration of the TLR7/8 agonist, resiquimod, to C57BL6 mice, drove chronic IFN induction and reduced ejection fraction and fractional shortening compared to mice treated with vehicle control. Multiomic analysis demonstrated increased IFN signature in resiquimod treated hearts (transcriptomic and proteomic) and a decrease in genes and proteins representing electron transport chain (ETC) and cytochrome complex assembly. Integration of metabolomics with transcriptomics revealed pathways representing chemokine signaling, Toll-like receptor signaling, HIF-1 signaling and Dilated cardiomyopathy. As our in vivo model of IFN-driven SLE-like heart disease showed both proteomic and transcriptomic changes that reflected changes in mitochondrial and potentially cardiac function, we conducted an in vitro analysis of the effects of acute and chronic IFN on cardiomyocytes, the most energetically demanding cell type in the heart, and the cells most susceptible to stress and inflammation. As with our in vivo findings, proteomic and transcriptomic analysis of AC16 cardiomyocytes exposed to chronic IFNα indicated perturbation of mitochondrial pathways. Extracellular flux analysis of chronically exposed AC16 cells showed impaired basal respiration, maximal respiration, ATP production and non-glycolytic acidification, glycolysis and glycolytic capacity, indicating increased mitochondrial stress in response to chronic exposure to IFNa. MitoTracker green staining showed increased fragmentation and perinuclear localization in AC16 chronically exposed to IFN and an increase in mtDNA release and expression of proteins known to contribute to mtDNA release and detection. Conclusions Our results directly support a role for chronic IFN in driving cardiac dysfunction in both a mouse model of IFN-driven disease that mimics SLE and in cardiomyocytes, though enhanced mitochondrial stress, mtDNA release and potentially exacerbation of cGAS-STING-IFN axis. Translational Perspectives Many immune features of SLE such as elevated type I interferons, chronic inflammation, and persistent autoantibody production are strongly correlated with increased cardiovascular risk, but it remains difficult to prove direct biological causation. This study demonstrates that chronic IFN exposure induces mitochondrial dysfunction and mtDNA release in cardiomyocytes, directly complementing an in vivo model of SLE-cardiac disease. It suggests that targeting IFNs may reduce CVD risk in IFN-driven autoimmunity. ### Competing Interest Statement The authors have declared no competing interest. NIH Common Fund, https://ror.org/001d55x84, R01AI164504 Congressionally Directed Medical Research Programs, W81XWH-18-1-0709 Californa institute of regenerative medicine

OBJECTIVE:Patients with systemic lupus erythematosus (SLE) experience photosensitivity, with exposure to UVB light driving lupus flares and triggering symptoms like joint pain, fatigue, and cutaneous lesions. Although the mechanism(s) linking UVB exposure to systemic effects are unclear, type I interferons (IFNs) are known to play a role. Our previous work has shown that TRIM21, an autoantigen in SLE, functions as a negative regulator on the pathways driving IFN expression. Here we explore how TRIM21 functions to regulate both local and systemic inflammation following UVB exposure and how altered expression may drive cutaneous inflammation and photosensitivity in SLE. METHODS:Wild-type (WT; C57BL/6) and Trim21-/- mice were irradiated with UVB (100 mJ/cm2) on the shaved dorsal region on consecutive days for 1 and 3 weeks, and UVB-induced local cutaneous manifestations and systemic inflammation in blood, spleen, and kidney were examined by messenger RNA expression of inflammatory and type I IFN response genes, histology, and flow cytometry. Mechanistic studies were performed in bone marrow-derived macrophages (BMDMs) and murine dermal fibroblasts (MDFs) from WT and Trim21-/- mice and TRIM21-/- THP-1 cells. RESULTS:Infiltration of inflammatory cells and induction of type I IFN developed in UVB-exposed areas in both sets of mice. Most notably after UVB exposure, we observed splenomegaly and enhanced expression of IFN-stimulated genes in the blood and spleen of Trim21-/- mice. Inflammatory chemokines CXCL10 and CXCL12 were also detected at significantly higher levels in serum of Trim21-/- mice after UVB exposure. Trim21-/- mice exposed to UVB also demonstrated enhanced total IgG levels in serum accompanied by increased skin and kidney deposition of IgG and increased glomerular cellularity and size. To determine the mechanism, we assessed UVB- and cyclic GMP-AMP-dependent Ifnb1 expression in Trim21-/- BMDMs and MDFs, noting increased responses compared with WT cells. This effect was lost in BMDMs from Trim21/Sting1 double knockout mice and skin explants, in keeping with the ability of TRIM21 to regulate cytoplasmic DNA sensing. In keeping with previous reports, we found that degradation of both DDX41 and STING levels were affected in stimulated Trim21-/- BMDMs. CONCLUSION:Taken together, our results indicate that TRIM21 protects against IFN induction at both local and systemic levels by restricting STING signaling.

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.

PV107 / #499 Poster Topic:AS12 - Genetics, Epigenetics, Transcriptomics Lupus nephritis (LN) is one of the most common and severe manifestations of systemic lupus erythematosus (SLE). We performed genome-wide association studies (GWAS) for lupus nephritis and kidney function measures over time. We hypothesized that analyzing a person’s eGFR variability over time would be a good proxy for LN and improve power for detecting genetic loci for LN. We also used local ancestry estimation to facilitate inclusion of admixed individuals. We included SLE patients from several child and adult dedicated lupus databases and the Systemic Lupus International Collaborating Clinics (SLICC) cohort. All met American College of Rheumatology and/or SLICC SLE criteria and were genotyped on a multi-ethnic Illumina array. Ungenotyped SNPs were imputed to the Trans-Omics for Precision Medicine program (TopMed), and local ancestry of chromosomal information was estimated using RFMix and Tractor software. LN was defined by SLE criteria, with a subset confirmed by kidney biopsy. Kidney function (estimated glomerular filtration rate, eGFR) was calculated using the Schwartz formula for measures <18 years and CKD-EPI for >18 years of age. Wilcoxon rank-sum or Chi-square tests were used to compare characteristics between LN and Non-LN patients. We completed separate GWAS for the outcomes of LN, mean eGFR and eGFR variability over time (log of the mean absolute deviation from mean eGFR per participant), in marginal and multivariable-adjusted regression models with sex, site and local principal components using Regenie. Local ancestry analysis was restricted to individuals of European, African and East Asian ancestry using Tractor. We meta-analyzed ancestry-specific results with METAL software (significancep<5x10^-8). We studied 2981 individuals with SLE, 88% female, 46% of European ancestry, 27% childhood-onset SLE, and 45% with LN (Table). Kidney failure was observed in 25 patients over a median follow-up time of 8.9 years (IQR: 4.1,14.8). Within-person eGFR was similar between people with and without LN, but eGFR variability was significantly greater in people with LN (P-value= 2.2e-16). Variability was calculated using a median of 16 [IQR: 8,35] eGFR measurements per person. GWAS of LN did not identify a significant LN locus, yet GWAS of eGFR variability demonstrated a significant peak on chromosome 15, downstream ofSCH4and intronic toSECISBP2L(Figure). The variant was found in a genomic region of African ancestry. Table. Figure. We completed GWAS of LN, eGFR mean and variability over time, generating ancestry-specific estimates and identified a genome-wide significant locus for variability in measures of renal function over time, in a multiethnic cohort of children and adults with SLE. This locus was only found in an African ancestry portion of the genome. Variability in measures of renal function is correlated with LN, yet GWAS of LN did not identify significant loci. Future work includes repeating analyses to include all global ancestries, and to investigate the biologic link between the loci and LN.

Abstract Extracellular vesicles (EVs) have been recognized as key players in cancer progression and metastasis, particularly large oncosomes (LO), secreted by invasive cancer cells, represent a distinct class of EVs with unique features. Previous research by the Di Vizio lab revealed LO's ability to alter recipient cell behavior, induce vascular morphogenesis, and carry cancer-specific biomolecules. Our preliminary studies point to a new mechanism of intercellular communication that involves the release of LO-containing DNA from PC cells. Notably, LO from prostate cancer exhibits enhanced potency in reprogramming bone marrow-derived mesenchymal stem cells (BM-MSCs) compared to exosomes (Exo) as observed by RNAseq. When evaluating the response to LO and Exo of BM-MSC, which are emerging as important drivers of bone metastasis, LO induces a robust inflammatory response, including metastasis-relevant chemokines and transcription factors involved in type I IFN response such as IRF7 and RASD2. Further, REACTOME pathway analysis of the differentially expressed genes revealed enrichment of the cGAS-STING pathway in BM-MSCs treated with LO, indicating a potential link between DNA/RNA sensors and the inflammatory response. BM cells isolated from WT and mutant Sting1gt/J mice demonstrated that up-regulation of IRF3 and IFNA1 genes induced by LO is inhibited when STING is absent. Suggesting that nucleic acid sensors engage in the LO-induced immune response in BM cells. Gene ontology analysis (GO) also identified several chemokines involved in neutrophil chemotaxis. In line with the observation from GO, neutrophil chemotaxis was significantly promoted by PC-derived LO-treated BM-MSC. Interestingly, human blood-derived neutrophils exposed to a conditioned medium from EVs-treated BM-MSC significantly increased the percentage of a population of LOX-1high and CD62Llow/− after 6h of culture. This was observed in concert with increased expression of Arginase1 and IL-10, suggesting their differentiation into polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSC). An experimental orthotopic model using the breast cancer bone metastatic 4T1.2 cell line, with increased capacity to shed LO, demonstrated an increase in the presence of PMN-MDSC in the bone marrow of tumor-bearing mice in addition to a complete lack of lymphocyte populations, indicating their immunosuppressive activity that could lead to a pre-metastatic niche formation. This comprehensive study unveils the molecular pathways underlying the immune response to LO in the bone marrow, shedding light on mechanisms that could influence metastatic progression in prostate cancer. The findings underscore the unique role of LO in modulating the BM microenvironment, providing potential targets for therapeutic intervention and biomarkers for early disease detection. Citation Format: Taylon F. Silva, Diana Kitka, Blandine Victor, Chen Qian, Minhyung Kim, Tatyana Vagner, Giorgia Guerra, Catherine Grasso, Sungyong You, Fayyazz Sutterwala, Caroline Jefferies, Paola de Candia, Michael Freeman, Helen Goodridge, Karen Cavassani, Jlenia Guarnerio, Dolores Di Vizio. Activation of the cGAS-STING pathway by large oncosomes induces type I IFN inflammation in mesenchymal stem cells that triggers an immunosuppressive phenotype in neutrophils [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1382.

Objective To investigate whether gene signatures discriminate systemic lupus erythematosus (SLE) patients with coronary microvascular dysfunction (CMD) from those without and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD.Methods This study collected whole blood RNA samples from female subjects aged 37 to 57, comprising 11 SLE patients (4 SLE-CMD, 7 SLE-non-CMD) and 10 HC. Total RNA was then used for library preparation and sequencing. Differential gene expression analysis was performed to identify gene signatures associated with CMD in SLE patients using DEseq2 v1.42.0. Gene Set Enrichment Analysis were performed by ClusterProfiler v4.10.0 and pathfindR v2.3.1.Results RNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and HC groups. GO analysis indicated associations with virus defense and interferon signaling in SLE. 14 DE genes were identified from comparison between SLE-CMD and SLE-non-CMD with adjusted parameters (padj < 0.1). Notably, SLE-CMD exhibited elevated levels of genes associated with RNA sensing, while downregulated genes in SLE-non-CMD were associated with blood coagulation and cell-cell junction. Further investigation highlighted differences in IFN signaling and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD, suggesting distinct molecular mechanisms underlying vascular changes in CMD and reduced left ventricular function in non-CMD.Conclusion Our study identified a unique gene signature in SLE-CMD compared to the HC group, highlighting the significant involvement of type 1 interferon, RIG-I family proteins, and chronic inflammation in the progression of SLE-CMD. The intricate relationship between SLE-CMD and these factors underscores their probable role in initiating and advancing SLE-CMD.### Competing Interest StatementThe authors have declared no competing interest.

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 (Manchanda et al, 2022). Echocardiographic studies have shown that SLE patients have reduced left ventricular (LV) function, which may also correlate with higher SLE disease activity scores (Gegenava et al, 2020). 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, table 1) and reference controls (n=22) using our published protocol (Aldiwani et al, 2022). 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 (Thomson et al, 2015). SLEDAI and SLICC Damage Index (DI) were calculated per validated criteria (Bombardier et al, 1992, Gladman et al, 1997). Serum samples were analyzed for inflammatory markers and autoantibodies (table 1). Independent two-tailed t 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 (table 2). 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 (table 3). 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 (Jha et al, 2022), our study found that decreased fasting insulin levels as a novel marker of diminished LV function. In addition, although studies have used SLEDAI as a marker of disease activity in cardiac dysfunction, we are the first to demonstrate that SLICC DI, an assessment of SLE damage, is also correlated with cardiac dysfunction in SLE. This indicates that SLE patients with higher SLICC DI and increased SLE-related damage could potentially have silent involvement in their cardiac tissue, and as such using SLICC DI is another tool that should be used to evaluate the association between SLE and LV dysfunction. [694]

Background/Purpose Systemic lupus erythematosus (SLE) is an autoimmune disease often characterized by multiorgan involvement with pain as a prevalent symptom. However, pain severity can differ greatly in each presentation.1 While multi-factorial, one mechanism is related to inflammatory processes which cause nociceptive receptor activation and central nervous system alteration.2 Given the multitude of possible signaling pathways, there are still additional contributing mechanisms and factors that have yet to be fully determined. To add to the growing literature, we analyzed the inflammatory markers of SLE patients to explore their relation to varying degrees of pain. Methods Data from 83 patients was obtained including clinical measurements of: erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), complement component 3 (C3), complement component 4 (C4), antinuclear antibody (ANA), and anti-double stranded DNA (anti-dsDNA). Whole blood analysis of 6 interferon stimulated genes (ISG) from RNA Paxgene tubes was conducted by qPCR. Pain frequency was also recorded using the Thrive Quality of Life Questionnaire, a clinically validated QOL assessment tool.3Patients were stratified into a low-pain group (n = 44) if they reported pain 'none of the time' or 'a little of the time', or into a high-pain group (n = 39) if they reported pain 'some of the time' or 'all the time'. Analysis was done using unpaired t-tests or chi-squared test as appropriate. Demographics including age of SLE onset and smoking history were also analyzed. Results Analysis showed significant differences in ISG score, CRP, C4, and C3 mean levels between patients reporting high pain compared to low pain. ISG (11.53 vs 8.516, P value 0.0003), CRP (3.710 vs 1.150, P value < 0.0001), and C3 (113.8 vs 95.50, P value 0.0233) mean levels were higher in the high-pain group. C4 (3.710 vs 20.97, P value <0.0001) mean level was lower in the high-pain group. ESR (27.39 vs 27.56, P value 0.9712), ANA (97.80 vs 119.0, P value 0.7350), anti-dsDNA (22.07 vs 16.97, P value 0.2664), and age of onset (26.10 vs 24.46, P value 0.5521). Interestingly, smoking history (ever smoked) was associated with pain (relative risk = 1.74, 95% CI 1.04 to 2.72, P value 0.0136) (figure 1 and table 1). Conclusion Our data shows that higher pain levels were associated with higher ISG scores, a surrogate of immunologic and type I IFN activity in SLE. Studies show that IFNs may cause both analgesia and hyperalgesia by nervous system regulation.4 While looking at other diseases, IFN inhibition correlates with improved pain. For example, in the RA-BEAM trial, baricitinib, a Janus kinase inhibitor which causes known IFN inhibition, had improved pain relief with evidence possibly suggesting at least some effect may be independent of inflammation activity.5 Another notable point is that other markers of disease activity such as high CRP and low C4, were also associated with pain scores. In other studies unrelated to SLE, both smoking and higher CRP levels were seen with increased pain sensitivity.6 Our findings are consistent with data from existing non-SLE pain literature. With the significant difference seen in ISG score, C4, and CRP, it indicates that there is a role for inflammation and potentially type I IFNs in pain experienced by SLE patients. Future work will further explore IFNs as well as additional markers and their relation to SLE pain. References Waldheim, Eva, et al. 'Variation in Pain Related to Systemic Lupus Erythematosus (SLE): A 7-Year Follow-up Study.' Clinical Rheumatology, 2018:37(7);1825–1834, https://doi.org/10.1007/s10067-018-4079-1. Nichilatti, Louise P, et al. 'Physiopathology of Pain in Systemic Erythematosus Lupus.' Lupus, 2020:29(7);721–726, https://doi.org/10.1177/0961203320919872. Wicks P, McCaffrey S, Goodwin K, Black R, Hoole M, Heywood J. A modular health-related quality of life instrument for electronic assessment and treatment monitoring: web-based development and psychometric validation of core thrive items. J Med Internet Res. 2019 Jan 25:21(1);e12075. doi: 10.2196/12075. PMID: 30681962; PMCID: PMC6367664. Tan, Ping-Heng, et al. 'Interferons in Pain and Infections: Emerging Roles in Neuro-Immune and Neuro-Glial Interactions.' Frontiers in Immunology, 2021:12, https://doi.org/10.3389/fimmu.2021.783725. Taylor, Peter C, et al. 'Achieving pain control in rheumatoid arthritis with baricitinib or adalimumab plus methotrexate: results from the RA-Beam Trial.' Journal of Clinical Medicine, 2019:8(6);831, https://doi.org/10.3390/jcm8060831. Afari, Niloofar, et al. 'C-reactive protein and pain sensitivity: findings from female twins.' Annals of Behavioral Medicine, 2011:42(2);277–283, https://doi.org/10.1007/s12160-011-9297-6.

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.

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.

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.

Abstract 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.

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.

IntroductionFetal 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.ObjectivesWe 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 designWe 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.ResultsPlacenta 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.DiscussionThis 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.

ABSTRACT Background Exposure of systemic lupus erythematosus (SLE) patients to ultraviolet light B (UVB) triggers local and systemic inflammation, with cytosolic DNA sensing and induction of type I interferons (IFNs) known to play a role. We previously identified TRIM21 as a negative regulator of DNA sensing and IFN expression. Here we explore the role of TRIM21 in regulating local and systemic responses following UVB exposure. Methods WT (C57BL/6) and Trim21 -/- mice were irradiated with UVB (100mJ/cm 2 ) daily for 1 and 3 weeks, and UVB-induced inflammation in skin, blood, and spleen were analyzed by qPCR, histology, RNA sequencing and flow cytometry. Mechanistic studies were performed in bone marrow-derived macrophages (BMDMs) and mouse skin fibroblasts (MDF) from WT and Trim21 -/- mice, and TRIM21 -/- THP-1 cells. Results Infiltration of inflammatory cells and induction of type I IFN developed in UVB-exposed areas in both sets of mice, however Trim21 -/- mice developed splenomegaly, enhanced total IgG levels and IFN-stimulated genes (ISG) in the blood and spleen. Enhanced basal and UVB-dependent Ifnb1 expression was observed in Trim21 -/- BMDMs and MDFs, which was dependent on the cytosolic DNA sensing cGAS-STING pathway. Mechanistically, we found both degradation of DDX41 and STING levels were impaired in stimulated Trim21 -/- BMDMs. Conclusion Taken together, our results indicate that TRIM21 protects against IFN induction at local and systemic levels through restricting STING signaling. Our finding that reduced levels of TRIM21 are observed in SLE patients with cutaneous involvement indicates a potential role for TRIM21 in guarding against systemic flare in SLE patients.