Systemic Lupus Erythematosus Monocytes Research Articles

Mx1-ing it up—Mitochondrial relay for interferon-dependent, unconventional IL-1β release in SLE monocytes

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 etal. 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.

Regulation of monocyte apoptosis and DNA extrusion in monocyte extracellular traps by PSGL-1: Relevance in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by severe organ damage and lacking curative treatment. While various immune cell types, especially dysfunctional B and T cells and neutrophils, have been related with disease pathogenesis, limited research has focused on the role of monocytes in SLE. Increased DNA extracellular traps, apoptosis and necrosis have been related to lupus pathogenesis. Our goal is to analyze the contribution of P-selectin glycoprotein ligand 1 (PSGL-1) in SLE monocytes to disease pathogenesis by investigating the control exerted by PSGL-1 on monocyte apoptosis and DNA extrusion in extracellular traps (METs). Monocytes from active disease patients (aSLE) exhibited reduced levels of PSGL-1. Importantly, lower PSGL-1 levels in SLE monocytes associated with several clinical characteristics, including anti-dsDNA autoantibodies, lupus anticoagulant, clinical lung involvement, and anemia. Monocytes from SLE patients showed higher susceptibility to apoptosis than healthy donors (HD) monocytes and PSGL-1/P-selectin interaction decreased secondary necrosis in HD but not in aSLE monocytes. Regarding METs, aSLE monocytes exhibited higher susceptibility to generate METs than HD monocytes. The interaction of HD monocytes with P-selectin induced Syk activation and reduced the levels of DNA extruded in METs. However, in aSLE monocytes, PSGL-1/P-selectin interaction did not activate Syk or reduce the amount of extruded DNA. Our data suggest a dysfunctional PSGL-1/P-selectin axis in aSLE monocytes, unable to reduce secondary necrosis or the amount of DNA released into the extracellular medium in METs, potentially contributing to lupus pathogenesis.

Macrophage colony-stimulating factor receptor/CD115+ non-classical monocytes are expanded in systemic lupus erythematosus and associated with lupus nephritis

Objective In systemic lupus erythematosus (SLE), the non-classical monocyte compartment is expanded, but its phenotype and association with clinical disease manifestations have not been explored. Method Monocyte subsets from 39 SLE patients, 32 healthy age-matched controls, and 16 patients from a disease control (autoimmune connective tissue disease other than SLE) were determined based on CD14 and CD16 surface expression. Cell surface expression of the receptors for macrophage colony-stimulating factor (M-CSF) (CD115) and granulocyte–macrophage colony-stimulating factor (GM-CSF) (CD116), as well as 6-Sulpho LacNAc (slan), were analysed by flow cytometry. The association of monocyte populations with disease manifestations, disease activity markers, and current medication of each patient was analysed by chart review. Results Non-classical monocytes displayed a cell-type specific signature of high M-CSF receptor CD115 and low GM-CSF receptor CD116 expression that separated them from the other two monocyte subsets. In healthy individuals, the M-CSF receptor on non-classical monocytes was an age-dependent surface marker, with lower expression in young adults. However, SLE monocytes were characterized by a marked expansion of M-CSF receptor/CD115+ non-classical monocytes in patients of all ages. The expanded population of M-CSF receptor/CD115+ non-classical monocytes was associated with lupus nephritis but not with disease activity, and coexpressed slan. Conclusion The non-classical monocyte subset in SLE is characterized by an expansion of M-CSF receptor/CD115+ cells that are associated with lupus nephritis and coexpress slan.

Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

α-Ketoglutarate-Dependent KDM6 Histone Demethylases and Interferon-Stimulated Gene Expression in Lupus.

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. 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. 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. 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.

Identification of monocyte-associated biomarkers in systemic lupus erythematosus and their pan-cancer analysis.

Immune dysregulation is not only a pathogenic mechanism in systemic lupus erythematosus (SLE) but also a potential cause of the link between SLE and cancer. The current understanding of SLE monocyte-associated biomarkers is limited, and the precise mechanism behind the link between SLE and cancer is uncertain. By using WGCNA and immune infiltration to analyze the GSE72326 dataset, we determined the most pertinent modules for monocytes and discovered eight candidate hub genes from them. The limma software was used to find genes that were differently expressed in SLE. The genes that overlapped between the two were chosen using a Venn diagram as the essential genes related to monocytes in SLE, and the essential genes were verified by several datasets. Correlation analysis and GSEA analysis were used to examine the probable immunological pathways connected to key genes. We examined the expression of hub genes in cancer and their interaction with monocytes using the GEPIA and TIMER databases to understand the significance of essential genes in tumorigenesis. In addition, we performed transcription factor identification. We discovered three biomarkers (IFI30, BLVRA, and RIN2) that are mostly involved in interferon-related signaling pathways and are associated with monocyte-mediated immune responses in SLE. The three important genes are also strongly expressed in a number of malignancies and have a relationship with monocytes. As a result, IFI30, BLVRA, and RIN2 may act as SLE-associated biomarkers of monocytes and as a bridge between SLE and tumors. We proposed that interferon-related signaling pathways might function as possible mediators of cancer risk in SLE.

Transcriptomic and proteomic profiling reveals distinct pathogenic features of peripheral non-classical monocytes in systemic lupus erythematosus

Peripheral blood monocytes propagate inflammation in systemic lupus erythematosus (SLE). Three major populations of monocytes have been recognized namely classical (CM), intermediate (IM) and non-classical monocytes (NCM). Herein, we performed a comprehensive transcriptomic, proteomic and functional characterization of the three peripheral monocytic subsets from active SLE patients and healthy individuals. Our data demonstrate extensive molecular disruptions in circulating SLE NCM, characterized by enhanced inflammatory features such as deregulated DNA repair, cell cycle and heightened IFN signaling combined with differentiation and developmental cues. Enhanced DNA damage, elevated expression of p53, G0 arrest of cell cycle and increased autophagy stress the differentiation potential of NCM in SLE. This immunogenic profile is associated with an activated macrophage phenotype of NCM exhibiting M1 characteristics in the circulation, fueling the inflammatory response. Together, these findings identify circulating SLE NCM as a pathogenic cell type in the disease that could represent an additional therapeutic target.

Restoration of aberrant gene expression of monocytes in systemic lupus erythematosus via a combined transcriptome-reversal and network-based drug repurposing strategy

BackgroundMonocytes -key regulators of the innate immune response- are actively involved in the pathogenesis of systemic lupus erythematosus (SLE). We sought to identify novel compounds that might serve as monocyte-directed targeted therapies in SLE.ResultsWe performed mRNA sequencing in monocytes from 15 patients with active SLE and 10 healthy individuals. Disease activity was assessed with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K). Leveraging the drug repurposing platforms iLINCS, CLUE and L1000CDS2, we identified perturbagens capable of reversing the SLE monocyte signature. We identified transcription factors and microRNAs (miRNAs) that regulate the transcriptome of SLE monocytes, using the TRRUST and miRWalk databases, respectively. A gene regulatory network, integrating implicated transcription factors and miRNAs was constructed, and drugs targeting central components of the network were retrieved from the DGIDb database. Inhibitors of the NF-κB pathway, compounds targeting the heat shock protein 90 (HSP90), as well as a small molecule disrupting the Pim-1/NFATc1/NLRP3 signaling axis were predicted to efficiently counteract the aberrant monocyte gene signature in SLE. An additional analysis was conducted, to enhance the specificity of our drug repurposing approach on monocytes, using the iLINCS, CLUE and L1000CDS2 platforms on publicly available datasets from circulating B-lymphocytes, CD4+ and CD8+ T-cells, derived from SLE patients. Through this approach we identified, small molecule compounds, that could potentially affect more selectively the transcriptome of SLE monocytes, such as, certain NF-κB pathway inhibitors, Pim-1 and SYK kinase inhibitors. Furthermore, according to our network-based drug repurposing approach, an IL-12/23 inhibitor and an EGFR inhibitor may represent potential drug candidates in SLE.ConclusionsApplication of two independent - a transcriptome-reversal and a network-based -drug repurposing strategies uncovered novel agents that might remedy transcriptional disturbances of monocytes in SLE.

NLRP12 is an innate immune checkpoint for repressing IFN signatures and attenuating lupus nephritis progression.

Signaling driven by nucleic acid sensors participates in interferonopathy-mediated autoimmune diseases. NLRP12, a pyrin-containing NLR protein, is a negative regulator of innate immune activation and type I interferon (IFN-I) production. Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients expressed lower levels of NLRP12, with an inverse correlation with IFNA expression and high disease activity. NLRP12 expression was transcriptionally suppressed by runt-related transcription factor 1-dependent (RUNX1-dependent) epigenetic regulation under IFN-I treatment, which enhanced a negative feedback loop between low NLRP12 expression and IFN-I production. Reduced NLRP12 protein levels in SLE monocytes was linked to spontaneous activation of innate immune signaling and hyperresponsiveness to nucleic acid stimulations. Pristane-treated Nlrp12-/- mice exhibited augmented inflammation and immune responses; and substantial lymphoid hypertrophy was characterized in NLRP12-deficient lupus-prone mice. NLRP12 deficiency mediated the increase of autoantibody production, intensive glomerular IgG deposition, monocyte recruitment, and the deterioration of kidney function. These were bound in an IFN-I signature-dependent manner in the mouse models. Collectively, we reveal a remarkable link between low NLRP12 expression and lupus progression, which suggests the impact of NLRP12 on homeostasis and immune resilience.

The inflammatory signature in monocytes of Sjögren's syndrome and systemic lupus erythematosus, revealed by the integrated Reactome and drug target analysis.

The innate immune regulation, especially by thetype I IFN signature in the CD14+ monocytes, is known to be critical in the pathogenesis of autoimmune Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). Since patients with one condition can be overlapped with another, this study is to identify shared differentially expressed genes (DEGs) in SjS and SLE compared to healthy controls (HCs) and refine transcriptomic profiles with the integrated Reactome and gene-drug network analysis for an anti-inflammation therapy. CD14+ monocytes were purified from whole blood of SjS and SLE patients (females, ages from 32 to 62) and subject to bulk RNA-sequencing, followed by data analyses for comparison with HC monocytes (females, ages 30 and 33). Functional categorizations, using Gene Ontology (GO) and the Reactome pathway analysis, were performed and DEGs associated with therapeutic drugs were identified from the Drug Repurposing Hub (DHUB) database. The GO analysis revealed that DEGs in the inflammatory response and the cellular response to cytokine were highly enriched in both conditions. A propensity toward M1 macrophage differentiation appears to be prominent in SjS while the Response to Virus was significant in SLE monocytes. Through the Reactome pathway analysis, DEGs in the IFN signaling and the cytokine signaling in immune system were most significantly enriched in both. Upregulation of NGF-induced transcription activity in SjS and the complement cascade activity in SLE were also noted. Multiple anti-inflammatory drugs, such as prostaglandin-endoperoxide synthase and angiotensin-I-converting- enzyme were associated with the DEGs in these conditions. Taken together, our analysis indicates distinct inflammatory transcriptomic profiles shared in SjS and SLE monocytes. Comprehensive characterizations of the data from these conditions will ultimately allow differential diagnosis of each condition and identification of therapeutic targets.

Analyzing the pathogenesis of systemic lupus erythematosus complicated by atherosclerosis using transcriptome data

BackgroundAccumulating evidence supports the predisposition of systemic lupus erythematosus (SLE) to atherosclerosis (AS). However, the common pathogenesis of these two diseases remains unclear. This study aimed to explore the mechanisms of SLE complicated by AS.MethodsGene expression profiles of SLE (GSE50772) and AS (GSE100927) were downloaded from the Gene Expression Omnibus. We analyzed differentially expressed genes (DEGs) of SLE and AS and performed enrichment analyses separately. After analyzing the common DEGs (CDEGs), we performed functional enrichment analysis, protein-protein interaction (PPI) network analysis, and hub genes (HGs) identification of CDEGs. Then, we performed a co-expression analysis of HGs and verified their expression and diagnostic value. We further explored immune cell infiltration and analyzed the correlation between HGs and infiltrating immune cells (IICs). Finally, we verified the reliability of the screening pathway.ResultsWe obtained 530 DEGs from the GSE50772 dataset and 448 DEGs from the GSE100927 dataset. The results of the enrichment analysis showed that there were many similar immune- and inflammation-related processes between the two diseases. We analyzed 26 CDEGs (two downregulated genes and 24 upregulated genes) and enrichment analysis highlighted the important role of the IL-17 signaling pathway. We identified five HGs (CCR1, CD163, IL1RN, MMP9, and SIGLEC1) using the CytoHubba plugin and HGs validation showed that the five HGs screened were reliable. Co-expression networks showed that five HGs can affect mononuclear cell migration. Immune cell infiltration analysis indicated monocytes in SLE and M0 macrophages in AS accounted for a high proportion of all IICs, and the difference in infiltration was obvious. We also found a significant positive correlation between CCR1, CD163, IL1RN, and MMP9 and monocytes in SLE, and a significant positive correlation between CCR1, IL1RN, MMP9, and SIGLEC1 and M0 macrophages in AS. Pathway validation also demonstrated that the IL-17 signaling pathway was a key pathway for the differentiation of monocytes into macrophages.ConclusionsThe five HGs may promote the differentiation of monocytes into macrophages by influencing the IL-17 signaling pathway, leading to SLE complicated by AS. Our study provides insights into the mechanisms of SLE complicated by AS.

Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes.

BACKGROUNDFasting and NAD+-boosting compounds, including NAD+ precursor nicotinamide riboside (NR), confer antiinflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined.METHODSWe explored the underlying biology in myeloid cells from healthy volunteers following in vivo placebo or NR administration and subsequently tested the findings in vitro in monocytes extracted from patients with systemic lupus erythematosus (SLE).RESULTSRNA-Seq of unstimulated and LPS-activated monocytes implicated NR in the regulation of autophagy and type I IFN signaling. In primary monocytes, NR blunted LPS-induced IFN-β production, and genetic or pharmacological disruption of autophagy phenocopied this effect. Given that NAD+ is a coenzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased the inosine level. Inosine supplementation similarly blunted autophagy and IFN-β release. Finally, because SLE exhibits type I IFN dysregulation, we assessed the NR effect on monocytes from patients with SLE and found that NR reduced autophagy and IFN-β release.CONCLUSIONWe conclude that NR, in an NAD+-dependent manner and in part via inosine signaling, mediated suppression of autophagy and attenuated type I IFN in myeloid cells, and we identified NR as a potential adjunct for SLE management.TRIAL REGISTRATIONClinicalTrials.gov registration numbers NCT02812238, NCT00001846, and NCT00001372.FUNDINGThis work was supported by the NHLBI and NIAMS Intramural Research divisions.

Anti-dsDNA Antibodies Increase the Cardiovascular Risk in Systemic Lupus Erythematosus Promoting a Distinctive Immune and Vascular Activation.

Systemic lupus erythematosus (SLE) is associated to boosted atherosclerosis development and a higher cardiovascular disease risk. This study aimed to delineate the role of anti-double stranded DNA (anti-dsDNA) antibodies on the molecular profile and the activity of immune and vascular cells, as well as on their enhanced cardiovascular risk. Eighty SLE patients were included. Extensive clinical/analytical evaluation was performed, including cardiovascular disease parameters (endothelial function, proatherogenic dyslipidemia, and carotid intima-media thickness). Gene and protein expression profiles were evaluated in monocytes from patients diagnosed positive or negative for anti-dsDNA antibodies by using NanoString and cytokine arrays, respectively. NETosis and circulating inflammatory profile was assessed in both neutrophils and plasma. Positivity and persistence of anti-dsDNA antibodies in SLE patients were associated to endothelial dysfunction, proatherogenic dyslipidemia, and accelerated atherosclerosis. In parallel, anti-dsDNA antibodies were linked to the aberrant activation of innate immune cells, so that anti-dsDNA(+) SLE monocytes showed distinctive gene and protein expression/activity profiles, and neutrophils were more prone to suffer NETosis in comparison with anti-dsDNA(−) patients. Anti-dsDNA(+) patients further displayed altered levels of numerous circulating mediators related to inflammation, NETosis, and cardiovascular risk. In vitro, Ig-dsDNA promoted NETosis on neutrophils, apoptosis on monocytes, modulated the expression of inflammation and thrombosis-related molecules, and induced endothelial activation, at least partially, by FcR (Fc receptor)-binding mechanisms. Anti-dsDNA antibodies increase the cardiovascular risk of SLE patients by altering key molecular processes that drive a distinctive and coordinated immune and vascular activation, representing a potential tool in the management of this comorbidity.

Ultrastructural Alterations of Platelets and Monocytes Associated with Autoantibodies in Systemic Lupus Erythematosus

Introduction. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high titer of autoantibodies, and SLE patients are predisposed to thrombosis. Monocytes and platelets are potentially involved in the pathogenesis of SLE. Monocytes are a source of immune modulators for T and B lymphocytes and they increase the risk of thrombosis through exposing tissue factor on their cell surface. Platelets are activated in SLE and may promote thrombotic events by expressing the procoagulant phosphatidylserine. The aim of this work was to investigate the ultrastructural changes of monocytes and platelets in SLE patients in association with titers of autoantibodies.Methods. SLE patients were included in the study based on the diagnostic criteria of the American College of Rheumatology. Platelets from 5 SLE patients and 3 healthy donors were isolated by gel-filtration on Sepharose 2B from citrated platelet-rich plasma. Monocytes of 6 SLE patients and 3 healthy donors were isolated using density gradient centrifugation of whole citrated blood followed by negative immune-separation. The >96% purity of the isolated monocytes was confirmed using flow cytometry and the viability of the isolated monocytes assessed by exclusion of trypan blue was 95-99%. The ultrastructure of platelets and monocytes from SLE patients and healthy donors was studied with transmission electron microscopy. A total 192 individual platelets and 154 monocytes from SLE patients were analyzed and compared with 60 individual platelets and 22 monocytes from healthy donors.Results . Most platelets from SLE patients and healthy donors had a normal discoid shape without or with 1-2 short filopodia. Intracellular components (α-granules, dense granules, open canalicular system, lysosomes and mitochondria) were well visualized, albeit somewhat less clear in the SLE platelets. The biggest morphological difference between platelets from healthy subjects and SLE patients was revealed in the platelet plasma membrane. All platelets from healthy donors had normal ultrastructure of the plasma membrane, while the membrane of SLE platelets was very often (in 70% of the cells) shaggy and rough and not as smooth as in normal platelets. About 38% of the SLE platelets were fully bordered with a shaggy plasma membrane, while 32% of the platelets had only partially abnormal plasma membrane. Cells from a patient with anti-cardiolipin and anti-beta-2-glypoprotein antibodies had the most striking membrane abnormalities that were found in 100% of platelets analyzed.Monocytes with normal ultrastructure (ruffled cell surface, formation of filopodia, an indented nucleus, and uniform chromatin distribution) comprised 64% of the total in the SLE patients and 95% in the healthy donors. Unlike monocytes from healthy subjects, 19% of monocytes from the SLE patients had ultrastructural signs of apoptosis with a smooth cell surface, electron-dense cytoplasm, mitochondrial swelling, and a spherical nucleus with perinuclear accumulation of condensed chromatin or condensation of chromatin into dense spherical particles. About 17% of monocytes from the SLE patients and 5% from the healthy donors had necrotic features, such as smooth cell surface without or with quite a few filopodia, low electron-density cytoplasm, small electron-dense mitochondria, and spherical pyknotic nucleus with more diffuse euchromatin. The SLE patients with a high titer of anti-dsDNA antibodies (>200 IU/ml) had significantly more apoptotic (25%, p<0.01, χ2-test) and necrotic (20%, p<0.05) monocytes than those of the SLE patients with a normal titer of anti-dsDNA antibodies (<25 IU/ml) (6% and 10%, respectively).Conclusions . In SLE patients, most platelets have abnormal ultrastructure of the plasma membrane (loss of smoothness, shagginess), likely due to deposition of pathological immune complexes. A substantial fraction of monocytes in SLE undergo morphological alternations of apoptotic or necrotic types. Such alterations were more pronounced in patients with an increased level of anti-cardiolipin, anti-beta-2-glypoprotein, and anti-dsDNA autoantibodies. The results confirm the involvement of both platelets and monocytes in the pathogenesis of SLE, including functional and structural alterations associated with increased production of various autoantibodies.The work was supported by the Program for Competitive Growth at Kazan Federal University. DisclosuresNo relevant conflicts of interest to declare.

Differential expression of the inflammasome complex genes in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder involving heterogeneous clinical manifestations and numerous susceptibility genes. Several findings evidence the critical role of inflammasomes in the predisposition to autoimmune diseases and in SLE. We investigated whether inflammasome polymorphins could affect susceptibility to develop and/or severity SLE. Moreover, differences in inflammasome activation in peripheral blood were also evaluated in SLE patients and controls. The distribution of 13 SNPs in eight inflammasome genes was evaluated. To assess inflammasome priming in peripheral blood monocytes of SLE and controls, differential expression of selected inflammasome genes and IL-1ß production was analyzed in resting condition as well as after LPS and ATP stimulation. Results showed that the gain-of-function variant rs10754558 (NLRP3) was significantly more frequent in SLE patients with nephritis, reinforcing the concept of a key role of NLRP3 inflammasome not only in SLE but also especially in kidney disease. SLE monocytes in resting condition showed a higher level of IL-1ß expression and produced higher levels of IL-1ß when stimulated with LPS+ATP comparing to controls. The stimulation induced a significant expression of NLRP1, AIM2, CASP1, and IL1B genes, suggesting that the NLRP1 inflammasome is responsible for the IL-1ß production observed in monocytes. These data emphasized once more the important contribution of inflammasome in SLE-associated inflammation.

DNA-Mediated Interferon Signature Induction by SLE Serum Occurs in Monocytes Through Two Pathways: A Mechanism to Inhibit Both Pathways.

A primary mechanism for activation of innate immunity is recognition of damage or pathogen associated molecular patterns by pattern recognition receptors (PRRs). Nucleic acid is a damage associated molecular pattern molecule that when internalized into a monocyte and recognized by intracellular nucleic acid sensing toll like receptors will cause production of type 1 interferon. The process by which DNA or RNA is delivered into the cytosol of monocytes in systemic lupus erythematosus remains incompletely understood, and therapeutic approaches to prevent DNA-mediated monocyte activation are needed. We identified two mechanisms for internalization of DNA by monocytes. IgG-bound DNA was internalized by interacting with Fc gamma receptor IIa, while high-mobility group box-1 protein-bound DNA was internalized by interacting with the receptor for advanced glycation end products. Both pathways contribute to an inflammatory phenotype in monocytes exposed to serum from patients with SLE. Moreover, both of these pathways can be inhibited by a pentapeptide, DWEYS, which is a DNA mimetope. In one instance DWEYS directly competes with DNA for antibody binding and in the other DWEYS binds high-mobility group box-1 and blocks its interaction with RAGE. Our data highlight distinct pathways involved in nucleic acid enters monocytes in SLE, and identify a potential therapeutic to prevent nucleic acid internalization in SLE.

Association of Abnormal Elevations in IFIT3 With Overactive Cyclic GMP-AMP Synthase/Stimulator of Interferon Genes Signaling in Human Systemic Lupus Erythematosus Monocytes.

Increasing evidence indicates that the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) signaling pathway has a critical pathogenic role in systemic lupus erythematosus (SLE). Expression levels of the interferon (IFN)-inducible gene IFIT3 are elevated in SLE patients. However, it is still not clear how IFIT3 contributes to the pathogenesis of SLE. This study was undertaken to investigate the activation of the cGAS/STING signaling pathway in human SLE monocytes, and to determine how elevated expression of IFIT3 could contribute to overactive cGAS/STING signaling in patients with SLE. Monocytes from SLE patients or healthy controls were examined for activity of the cGAS/STING signaling pathway and expression levels of IFIT3. Correlations between cGAS/STING signaling activity and SLE clinical features were analyzed. Gain- or loss-of-function experiments were used to determine the role of IFIT3 in cGAS/STING signaling. Coimmunoprecipitation assays were used to identify the interaction between IFIT3 and other proteins. The cGAS/STING signaling pathway was found to have enhanced activity in monocytes from SLE patients compared to healthy controls, as indicated by the higher expression of IFNβ downstream. Levels of IFIT3 were significantly elevated in human SLE monocytes, and this was positively correlated with the activity of the cGAS/STING signaling pathway. In vitro, the expression of VACV70-induced IFNβ was reduced by knockdown of IFIT3, whereas overexpression of IFIT3 produced an opposite effect. Finally, IFIT3 was found to interact with both STING and TANK-binding kinase 1. These findings suggest that IFIT3 is one of the genes that contributes to the overactive cGAS/STING signaling pathway in human SLE monocytes. IFIT3 may therefore serve as a novel therapeutic target for blocking the production of type I IFN and other proinflammatory cytokines by the cGAS/STING signaling pathway in patients with SLE.

Overall Downregulation of mRNAs and Enrichment of H3K4me3 Change Near Genome-Wide Association Study Signals in Systemic Lupus Erythematosus: Cell-Specific Effects.

This study was designed to define gene expression and H3K4me3 histone modifications in T cells, B cells, and monocytes in systemic lupus erythematosus (SLE). Array studies of total peripheral blood mononuclear cells have demonstrated gene expression signatures related to neutrophils, interferon, and other inflammatory pathways. It is not clear how consistent these effects are across different cell types. In this study, RNA-seq and chromatin immunoprecipitation-seq were utilized to identify gene expression patterns and H3K4me3 histone modifications related to promoter activation in SLE. Across the three cell types, there was 55% concordance for gene expression changes related to SLE. Key conserved pathways were ribosome biogenesis among upregulated genes and heat shock response among downregulated genes. ETS family transcription factors (TFs) and STAT1 were revealed as common regulators by position weight matrices. When epigenetic changes were leveraged with gene expression, the pivotal TFs ATF3 and FOS were defined with ATF3 also cross-referencing with gene expression-identified TFs. Genome-wide association study (GWAS) single nucleotide polymorphisms associated with SLE were cross-referenced with both mRNA and H3K4me3 changes in SLE. Baseline mRNA expression and H3K4me3 peak height was higher at sites that cross-referenced with GWAS signals, however, all three cell types exhibited an overall decrease in expression of GWAS-associated RNAs differentially expressed in SLE. H3K4me3 changes in SLE were also enriched in GWAS-associated sites. In summary, the SLE disease process is associated with both shared and cell-specific changes in gene expression and epigenetics. Surprisingly, GWAS-associated RNAs were overall markedly decreased across all three cell types. TF analysis identified ATF3, FOS, STAT1, and ETS family members as critical, all pathways with a recognized relationship to the SLE disease process. GWAS signals clearly mark both cell-type specific changes in SLE as well as concordant changes across all three cell types. Interpretation of single nucleotide polymorphism effects in SLE will require tissue-specific mechanistic studies and therapeutics will require mechanistic studies in multiple cell types.

Structural Alterations of Monocytes in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the immune system mistakenly attacks multiple organs and tissues of the body. To elucidate the involvement of blood cells in the pathogenesis of SLE, we used transmission electron microscopy to study ultrastructure of monocytes isolated from the blood of SLE patients. We found that in the SLE patients, a substantial fraction of monocytes had abnormal morphology that corresponded to the structural signs of either necrosis or apoptosis. The number of altered monocytes in the SLE patients was significantly higher than in healthy subjects and related directly with the level of anti-dsDNA autoantibodies in the blood. Our results suggest that monocytes are involved in the pathogenesis of SLE and undergo adverse necrotic and/or apoptotic changes, likely induced by autoantibodies.

Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus

ObjectiveDefective or inefficient DNA double-strand break (DSB) repair results in failure to preserve genomic integrity leading to apoptotic cell death, a hallmark of systemic lupus erythematosus (SLE). Compelling evidence linked...