Distinct genome-wide DNA methylation and gene expression signatures in classical monocytes from African American patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects women and African Americans. Despite this, African Americans are dramatically underrepresented in SSc research. Additionally, monocytes show heightened activation in SSc and in African Americans relative to European Americans. In this study, we sought to investigate DNA methylation and gene expression patterns in classical monocytes in a health disparity population. METHODS: Classical monocytes (CD14+ + CD16-) were FACS-isolated from 34 self-reported African American women. Samples from 12 SSc patients and 12 healthy controls were hybridized on MethylationEPIC BeadChip array, while RNA-seq was performed on 16 SSc patients and 18 healthy controls. Analyses were computed to identify differentially methylated CpGs (DMCs), differentially expressed genes (DEGs), and CpGs associated with changes in gene expression (eQTM analysis). RESULTS: We observed modest DNA methylation and gene expression differences between cases and controls. The genes harboring the top DMCs, the top DEGs, as well as the top eQTM loci were enriched for metabolic processes. Genes involved in immune processes and pathways showed a weak upregulation in the transcriptomic analysis. While many genes were newly identified, several other have been previously reported as differentially methylated or expressed in different blood cells from patients with SSc, supporting for their potential dysregulation in SSc. CONCLUSIONS: While contrasting with results found in other blood cell types in largely European-descent groups, the results of this study support that variation in DNA methylation and gene expression exists among different cell types and individuals of different genetic, clinical, social, and environmental backgrounds. This finding supports the importance of including diverse, well-characterized patients to understand the different roles of DNA methylation and gene expression variability in the dysregulation of classical monocytes in diverse populations, which might help explaining the health disparities.

Loss-of-function variants in SAT1 cause X-linked childhood-onset systemic lupus erythematosus.

OBJECTIVES: Families that contain multiple siblings affected with childhood onset of systemic lupus erythematosus (SLE) likely have strong genetic predispositions. We performed whole exome sequencing (WES) to identify familial rare risk variants and to assess their effects in lupus. METHODS: Sanger sequencing validated the two ultra-rare, predicted pathogenic risk variants discovered by WES and identified additional variants in 562 additional patients with SLE. Effects of a splice site variant and a frameshift variant were assessed using a Minigene assay and CRISPR/Cas9-mediated knock-in (KI) mice, respectively. RESULTS: The two familial ultra-rare, predicted loss-of-function (LOF) SAT1 variants exhibited X-linked recessive Mendelian inheritance in two unrelated African-American families. Each LOF variant was transmitted from the heterozygous unaffected mother to her two sons with childhood-onset SLE. The p.Asp40Tyr variant affected a splice donor site causing deleterious transcripts. The young hemizygous male and homozygous female Sat1 p.Glu92Leufs*6 KI mice spontaneously developed splenomegaly, enlarged glomeruli with leucocyte infiltration, proteinuria and elevated expression of type I interferon-inducible genes. SAT1 is highly expressed in neutrophils and encodes spermidine/spermine-N1-acetyltransferase 1 (SSAT1), a rate-limiting enzyme in polyamine catabolism. Young male KI mice exhibited neutrophil defects and decreased proportions of Foxp3 +CD4+ T-cell subsets. Circulating neutrophil counts and proportions of Foxp3 +CD4+ T cells correlated with decreased plasma levels of spermine in treatment-naive, incipient SLE patients. CONCLUSIONS: We identified two novel SAT1 LOF variants, showed the ability of the frameshift variant to confer murine lupus, highlighted the pathogenic role of dysregulated polyamine catabolism and identified SAT1 LOF variants as new monogenic causes for SLE.

Conditional knockout of oestrogen receptor alpha in CD11c+ cells impacts female survival and inflammatory cytokine profile in murine lupus.

Oestrogen and oestrogen receptor alpha (ERα) have been implicated in systemic lupus erythematosus pathogenesis. ERα signalling influences dendritic cell (DC) development and function, as well as inflammation and downstream immune responses. We previously reported that ERα modulates multiple Toll-like receptor-stimulated pathways in both conventional and plasmacytoid DCs in lupus-prone mice. For example, CD11chi MHCII+ cell numbers are reduced in mice with global ERα deficiency or when expressing a short variant of ERα. Herein, RNA-seq analysis of CD11chi cells from bone marrow of NZM2410 mice expressing WT ERα versus ERα short versus ERα null revealed differentially expressed complement genes, interferon-related genes and cytokine signalling (e.g., IL-17 and Th17 pathways). To better understand the role of ERα in CD11c+ cells, lupus prone NZM2410 mice with selective deletion of the Esr1 gene in CD11c+ cells were generated. Phenotype and survival of these mice were similar with the exception of Cre positive (CrePos) female mice. CrePos females, but not males, all died unexpectedly prior to 35 weeks. DC subsets were not significantly different between groups. Since ERα is necessary for robust development of DCs, this result suggests that DC fate was determined prior to CD11c expression and subsequent ERα deletion (i.e., proximally in DC ontogeny). Overall, findings point to a clear functional role for ERα in regulating cytokine signalling and inflammation, suggesting that further study into ERα-mediated regulatory mechanisms in DCs and other immune cell types is warranted.

Staphylococcus aureus peptidoglycan (PGN) induces pathogenic autoantibody production via autoreactive B cell receptor clonal selection, implications in systemic lupus erythematosus.

OBJECTIVES: There is an intricate interplay between the microbiome and the immune response impacting development of normal immunity and autoimmunity. However, we do not fully understand how the microbiome affects production of natural-like and pathogenic autoantibodies. Peptidoglycan (PGN) is a component of the bacterial cell wall which is highly antigenic. PGNs from different bacteria can differ in their immune regulatory activities. METHODS: C57BL/6 and MRL/lpr mice were intraperitoneally injected with saline or PGN from Staphylococcus aureus or Bacillus subtilis. Spleen anti-double-stranded DNA (dsDNA) IgG + B cells were sorted for B-cell receptor sequencing. Serum autoantibody levels and kidney damage were analyzed. Further, the association between plasma S. aureus translocation and systemic lupus erythematosus (SLE) pathogenesis was assessed in women. RESULTS: Administration of B. subtilis PGN induced natural-like anti-dsDNA autoantibodies (e.g., IgM, short lived IgG response, and no tissue damage), whereas S. aureus PGN induced pathogenic anti-dsDNA autoantibodies (e.g., prolonged IgG production, low IgM, autoantibody-mediated kidney damage) in C57BL/6 and/or MRL/lpr mice. However, serum total IgG did not differ. S. aureus PGN induced antibodies with reduced clonality and greater hypermutation of IGHV3-74 in splenic anti-dsDNA IgG + B cells from C57BL/6 mice. Further, S. aureus PGN promoted IgG class switch recombination via toll-like receptor 2. Plasma S. aureus DNA levels were increased in women with SLE versus control women and correlated with levels of lupus-related autoantibodies and renal involvement. CONCLUSIONS: S. aureus PGN induces pathogenic autoantibody production, whereas B. subtilis PGN drives production of natural nonpathogenic autoantibodies.

Pre-Clinical Autoimmunity in Lupus Relatives: Self-Reported Questionnaires and Immune Dysregulation Distinguish Relatives Who Develop Incomplete or Classified Lupus From Clinically Unaffected Relatives and Unaffected, Unrelated Individuals.

Systemic lupus erythematosus (SLE) is propelled by pathogenic autoantibody (AutoAb) and immune pathway dysregulation. Identifying populations at risk of reaching classified SLE is essential to curtail inflammatory damage. Lupus blood relatives (Rel) have an increased risk of developing SLE. We tested factors to identify Rel at risk of developing incomplete lupus (ILE) or classified SLE vs. clinically unaffected Rel and healthy controls (HC), drawing from two unique, well characterized lupus cohorts, the lupus autoimmunity in relatives (LAUREL) follow-up cohort, consisting of Rel meeting <4 ACR criteria at baseline, and the Lupus Family Registry and Repository (LFRR), made up of SLE patients, lupus Rel, and HC. Medical record review determined ACR SLE classification criteria; study participants completed the SLE portion of the connective tissue disease questionnaire (SLE-CSQ), type 2 symptom questions, and provided samples for assessment of serum SLE-associated AutoAb specificities and 52 plasma immune mediators. Elevated SLE-CSQ scores were associated with type 2 symptoms, ACR scores, and serology in both cohorts. Fatigue at BL was associated with transition to classified SLE in the LAUREL cohort (p≤0.01). Increased levels of BLyS and decreased levels of IL-10 were associated with type 2 symptoms (p<0.05). SLE-CSQ scores, ACR scores, and accumulated AutoAb specificities correlated with levels of multiple inflammatory immune mediators (p<0.05), including BLyS, IL-2Rα, stem cell factor (SCF), soluble TNF receptors, and Th-1 type mediators and chemokines. Transition to SLE was associated with increased levels of SCF (p<0.05). ILE Rel also had increased levels of TNF-α and IFN-γ, offset by increased levels of regulatory IL-10 and TGF-ß (p<0.05). Clinically unaffected Rel (vs. HC) had higher SLE-CSQ scores (p<0.001), increased serology (p<0.05), and increased inflammatory mediator levels, offset by increased IL-10 and TGF-ß (p<0.01). These findings suggest that Rel at highest risk of transitioning to classified SLE have increased inflammation coupled with decreased regulatory mediators. In contrast, clinically unaffected Rel and Rel with ILE demonstrate increased inflammation offset with increased immune regulation, intimating a window of opportunity for early intervention and enrollment in prevention trials.

Safety and Efficacy of Mesenchymal Stromal Cells and Other Cellular Therapeutics in Rheumatic Diseases in 2022: A Review of What We Know So Far.

Although a number of new immunosuppressive agents and biologics have been approved for treating various autoimmune inflammatory rheumatic diseases, there remains a substantial number of patients who have no clinical response or limited clinical response to these available treatments. Use of cellular therapies is a novel approach for the treatment of autoimmune inflammatory rheumatic diseases, with perhaps enhanced efficacy and less toxicity than current therapies. Autologous hematopoietic stem cell transplants were the first foray into cellular therapies, with proven efficacy in scleroderma and multiple sclerosis. Newer, yet unproven, cellular therapies include allogeneic mesenchymal stromal cells, which have been shown to be effective in graft-versus-host disease and in healing Crohn's fistulas. Chimeric antigen receptor T cells are effective in various malignancies, with possible application in rheumatic diseases, as shown in preclinical studies in murine lupus and recently in human lupus. Treg cells are one of the master controllers of the immune response and are decreased in number and/or effectiveness in specific autoimmune diseases. Expansion of autologous Treg cells is an attractive approach to controlling autoimmunity. There are a number of other regulatory cells in the immune system, including Breg cells, dendritic cells, macrophages, and other T cell types, that are in early stages of development as treatments. In this review, the current evidence for the efficacy and mechanisms of actions of cellular therapies already in use or in clinical trials in human autoimmune diseases will be discussed, including the limitations of these therapies and potential side effects.

A Distinct Plasma Microbiome But Not Gut Microbiome in Patients With Systemic Lupus Erythematosus Compared to Healthy Individuals.

OBJECTIVE: Blood microbiome has been analyzed in cancer patients using machine learning. We aimed to study whether the plasma microbiome represents the microbial community in the gut among patients with systemic lupus erythematosus (SLE) and healthy controls (HCs). METHODS: Paired plasma and stool samples from female patients with SLE and female HCs were assessed for microbiome composition by microbial 16S ribosomal RNA sequencing. RESULTS: Decreased microbial alpha diversity in stool compared to plasma and distinct plasma and gut beta diversity were found in both HCs and patients with SLE. No difference in gut microbial diversity was found; however, plasma alpha diversity was decreased in patients with SLE compared to HCs. The predominant bacteria differed between plasma and stool in both groups. Although the predominant plasma and stool genus bacteria were similar in patients with SLE and HCs, some were clearly different. CONCLUSION: Compared to the gut, the plasma microbiome contained distinct community and greater heterogeneity, indicating that the predominant circulating microbiome may originate from sites (eg, oral or skin) other than the gastrointestinal tract. The decreased plasma but not gut alpha diversity in patients with SLE compared to HCs implies an altered plasma microbiome in SLE, which may be important for systemic immune perturbations and SLE disease pathogenesis.

Human SLE variant NCF1-R90H promotes kidney damage and murine lupus through enhanced Tfh2 responses induced by defective efferocytosis of macrophages.

OBJECTIVES: We previously identified a hypomorphic variant, p.Arg90His (p.R90H) of neutrophil cytosolic factor 1 (NCF1, a regulatory subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 complex), as an putative causal variant for systemic lupus erythematosus (SLE), and established a knock-in (KI) H90 variant in the C57BL/6 background to study how this variant promotes lupus development. METHODS: Wild type (WT) and KI littermates were assessed for immune profiles and lupus-like features. Disease activity and renal damage of patients with SLE were assessed by systemic lupus erythematosus disease activity index (SLEDAI) and renal items of systemic lupus international collaborating clinics (SLICC), respectively. RESULTS: Compared with WT littermates, 5-week-old homozygous KI mice had reduced oxidative burst, splenomegaly, elevated type I interferon (IFN-I) scores, increased ratios of splenic follicular T helper 2 (Tfh2) to either T follicular regulatory (Tfr) or Tfh1 cells, increased ANA+ follicular, germinal centre and plasma cells without spontaneous kidney disease up to 1 year of age. Pristane treatment exacerbated the immune dysregulation and induced IFN-I-dependent kidney disease in 36-week-old H90 KI female mice. Decreased efferocytosis of macrophages derived from KI mice and patients with homozygous H90 SLE promoted elevated ratios of Tfh2/Tfr and Tfh2/Tfh1 as well as dysregulated humoral responses due to reduced voltage-gated proton channel 1 (Hv1)-dependent acidification of phagosome pH to neutralise the decreased electrogenic effect of the H90 variant, resulting in impaired maturation and phagosome proteolysis, and increased autoantibody production and kidney damage in mice and patients with SLE of multiple ancestries. CONCLUSIONS: A lupus causal variant, NCF1-H90, reduces macrophage efferocytosis, enhances Tfh2 responses and promotes autoantibody production and kidney damage in both mice and patients with SLE.

Up-Regulated Interleukin-10 Induced by E2F Transcription Factor 2-MicroRNA-17-5p Circuitry in Extrafollicular Effector B Cells Contributes to Autoantibody Production in Systemic Lupus Erythematosus.

OBJECTIVE: Elevated interleukin-10 (IL-10) levels in patients with systemic lupus erythematosus (SLE) have B cell-promoting effects, contributing to autoantibody production and tissue damage. We aimed to characterize up-regulated IL-10+ B cell subsets and dysregulated IL10 expression in SLE B cells for new therapeutic options. METHODS: Proportions of Th10 and IL-10+ B cell subsets in peripheral blood mononuclear cells (PBMCs) were assessed using flow cytometry. The IL10 3'-untranslated region (3'-UTR) dual-luciferase vector was constructed and cotransfected with small interfering RNA (siRNA), microRNA (miRNA) mimics, or miRNA inhibitors into Raji cells. Transcript levels were quantified using TaqMan assays. RESULTS: Culture conditions that induced IL-10+ Breg cells in healthy controls resulted in expansion of IL-10+ double-negative 2 (DN2; IgD-CD27-CD21-CD11c+) B cells in SLE PBMCs. Proportions of IL-10+ DN2, but not those of IL-10- DN2, correlated with disease activity and levels of antibodies to double-stranded DNA (dsDNA) (r = 0.60, P = 0.03 for cohort 1; r = 0.38, P = 0.03 for cohort 2), and were associated with high levels or seropositivity of anti-Sm (P = 0.03 for cohort 1; P = 0.01 for cohort 2) and IgG anticardiolipin (P < 0.01 for cohort 1; P = 0.02 for cohort 2) in SLE patients from 2 cohorts, of mainly African American subjects (cohort 1) and of Asian subjects (cohort 2). Proportions of Th10 (CD45RA-CXCR5-CXCR3+PD-1high CD4+) cells correlated with IL-10+ DN2 frequencies (r = 0.60, P < 0.01 for cohort 2), antinuclear antibody titers (r = 0.52, P = 0.01 for cohort 2), and proteinuria levels (r = 0.72, P < 0.01 for cohort 2) in SLE patients. Screening of predicted IL10 3'-UTR-targeting miRNAs in SLE B cells identified miRNA-17-5p (miR-17-5p) and miR-20a-5p, with their levels inversely correlated with IL10 (r = -0.47, P < 0.01 for miR-17-5p; r = -0.37, P = 0.03 for miR-20-5p) and transcription factor E2F2 (r = -0.48, P = 0.04 for miR-17-5p; r = -0.45, P = 0.05 for miR-20-5p). In Raji cells, knockdown of E2F2 expression resulted in increased levels of miR-17-5p and miR-20a-5p and decreased IL10 messenger RNA (mRNA) and protein levels, and overexpression and inhibition of miR-17-5p down-regulated and up-regulated, respectively, IL10 mRNA levels, suggesting regulation of IL10 expression by an E2F2-miR-17-5p loop. CONCLUSION: IL-10 promotes extrafollicular autoimmune responses in patients with active SLE, which might be dampened by targeting the E2F2-miR-17-5p circuitry.

Genetic landscape of Gullah African Americans.

OBJECTIVES: Gullah African Americans are descendants of formerly enslaved Africans living in the Sea Islands along the coast of the southeastern U.S., from North Carolina to Florida. Their relatively high numbers and geographic isolation were conducive to the development and preservation of a unique culture that retains deep African features. Although historical evidence supports a West-Central African ancestry for the Gullah, linguistic and cultural evidence of a connection to Sierra Leone has led to the suggestion of this country/region as their ancestral home. This study sought to elucidate the genetic structure and ancestry of the Gullah. MATERIALS AND METHODS: We leveraged whole-genome genotype data from Gullah, African Americans from Jackson, Mississippi, African populations from Sierra Leone, and population reference panels from Africa and Europe to infer population structure, ancestry proportions, and global estimates of admixture. RESULTS: Relative to non-Gullah African Americans from the Southeast US, the Gullah exhibited higher mean African ancestry, lower European admixture, a similarly small Native American contribution, and increased male-biased European admixture. A slightly tighter bottleneck in the Gullah 13 generations ago suggests a largely shared demographic history with non-Gullah African Americans. Despite a slightly higher relatedness to populations from Sierra Leone, our data demonstrate that the Gullah are genetically related to many West African populations. DISCUSSION: This study confirms that subtle differences in African American population structure exist at finer regional levels. Such observations can help to inform medical genetics research in African Americans, and guide the interpretation of genetic data used by African Americans seeking to explore ancestral identities.

Camptothecin and Topotecan, Inhibitors of Transcription Factor Fli-1 and Topoisomerase, Markedly Ameliorate Lupus Nephritis in (NZB × NZW)F1 Mice and Reduce the Production of Inflammatory Mediators in Human Renal Cells.

OBJECTIVE: To examine the therapeutic effects of camptothecin (CPT) and topotecan (TPT), inhibitors of transcription factor Fli-1 and topoisomerase, on lupus nephritis in (NZB × NZW)F1 (NZBWF1) mice, and to examine the effects of CPT and TPT on inflammatory mediators in human renal cells. METHODS: Female NZBWF1 mice were treated with vehicle, cyclophosphamide (CYC), CPT (1 mg/kg or 2 mg/kg), or TPT (0.03 mg/kg, 0.1 mg/kg, or 0. 3 mg/kg) by intraperitoneal injection twice a week, beginning at the age of 25 weeks (n = 8-10 mice per group). Blood and urine were collected for monitoring autoantibodies and proteinuria. Mice were euthanized at 40 weeks, and renal pathology scores were assessed. Human renal endothelial and mesangial cells were treated with CPT or TPT, and cytokine expression was measured. RESULTS: None of the NZBWF1 mice treated with 1 mg/kg or 2 mg/kg of CPT or 0.3 mg/kg of TPT had proteinuria >100 mg/dl at the age of 40 weeks. One of 8 mice treated with 0.1 mg/kg of TPT and 1 of 10 mice treated with CYC had proteinuria >300 mg/dl, whereas 90% of the mice treated with vehicle had proteinuria >300 mg/dl. Compared to vehicle control, mice treated with 1 mg/kg or 2 mg/kg of CPT, 0.1 mg/kg or 0.3 mg/kg of TPT, or CYC had significantly prolonged survival, attenuated renal injury, diminished splenomegaly, reduced anti-double-stranded DNA autoantibody levels, and reduced IgG and C3 deposits in the glomeruli (all P < 0.05). Human renal cells treated with CPT or TPT had reduced expression of Fli-1 and decreased monocyte chemotactic protein 1 production following stimulation with interferon-α (IFNα) or IFNγ. CONCLUSION: Our findings indicate that low-dose CPT and TPT could be repurposed to treat lupus nephritis.

Rigorous Plasma Microbiome Analysis Method Enables Disease Association Discovery in Clinic.

Blood microbiome is important to investigate microbial-host interactions and the effects on systemic immune perturbations. However, this effort has met with major challenges due to low microbial biomass and background artifacts. In the current study, microbial 16S DNA sequencing was applied to analyze plasma microbiome. We have developed a quality-filtering strategy to evaluate and exclude low levels of microbial sequences, potential contaminations, and artifacts from plasma microbial 16S DNA sequencing analyses. Furthermore, we have applied our technique in three cohorts, including tobacco-smokers, HIV-infected individuals, and individuals with systemic lupus erythematosus (SLE), as well as corresponding controls. More than 97% of total sequence data was removed using stringent quality-filtering strategy analyses; those removed amplicon sequence variants (ASVs) were low levels of microbial sequences, contaminations, and artifacts. The specifically enriched pathobiont bacterial ASVs have been identified in plasmas from tobacco-smokers, HIV-infected individuals, and individuals with SLE but not from control subjects. The associations between these ASVs and disease pathogenesis were demonstrated. The pathologic activities of some identified bacteria were further verified in vitro. We present a quality-filtering strategy to identify pathogenesis-associated plasma microbiome. Our approach provides a method for studying the diagnosis of subclinical microbial infection as well as for understanding the roles of microbiome-host interaction in disease pathogenesis.

Association of Epstein-Barr virus serological reactivation with transitioning to systemic lupus erythematosus in at-risk individuals.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with unknown aetiology. Epstein-Barr virus (EBV) is an environmental factor associated with SLE. EBV maintains latency in B cells with frequent reactivation measured by antibodies against viral capsid antigen (VCA) and early antigen (EA). In this study, we determined whether EBV reactivation and single nucleotide polymorphisms (SNPs) in EBV-associated host genes are associated with SLE transition. METHODS: SLE patient relatives (n=436) who did not have SLE at baseline were recontacted after 6.3 (±3.9) years and evaluated for interim transitioning to SLE (≥4 cumulative American College of Rheumatology criteria); 56 (13%) transitioned to SLE prior to the follow-up visit. At both visits, detailed demographic, environmental, clinical information and blood samples were obtained. Antibodies against viral antigens were measured by ELISA. SNPs in IL10, CR2, TNFAIP3 and CD40 genes were typed by ImmunoChip. Generalised estimating equations were used to test associations between viral antibody levels and transitioning to SLE. RESULTS: Mean baseline VCA IgG (4.879±1.797 vs 3.866±1.795, p=0.0003) and EA IgG (1.192±1.113 vs 0.7774±0.8484, p=0.0236) levels were higher in transitioned compared with autoantibody negative non-transitioned relatives. Increased VCA IgG and EA IgG were associated with transitioning to SLE (OR 1.28 95% CI 1.07 to 1.53, p=0.007, OR 1.43 95% CI 1.06 to 1.93, p=0.02, respectively). Significant interactions were observed between CD40 variant rs48100485 and VCA IgG levels and IL10 variant rs3024493 and VCA IgA levels in transitioning to SLE. CONCLUSION: Heightened serologic reactivation of EBV increases the probability of transitioning to SLE in unaffected SLE relatives.

A Link Between Plasma Microbial Translocation, Microbiome, and Autoantibody Development in First-Degree Relatives of Systemic Lupus Erythematosus Patients.

OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by the production of antibodies against self antigens. However, the events underlying autoantibody formation in SLE remain unclear. This study was undertaken to investigate the role of plasma autoantibody levels, microbial translocation, and the microbiome in SLE. METHODS: Plasma samples from 2 cohorts, one with 18 unrelated healthy controls and 18 first-degree relatives and the other with 19 healthy controls and 21 SLE patients, were assessed for autoantibody levels by autoantigen microarray analysis, measurement of lipopolysaccharide (LPS) levels by Limulus amebocyte assay, and determination of microbiome composition by microbial 16S ribosomal DNA sequencing. RESULTS: First-degree relatives and SLE patients exhibited increased plasma autoantibody levels compared to their control groups. Parents and children of lupus patients exhibited elevated plasma LPS levels compared to controls (P = 0.02). Plasma LPS levels positively correlated with plasma anti-double-stranded DNA IgG levels in first-degree relatives (r = 0.51, P = 0.03), but not in SLE patients. Circulating microbiome analysis revealed that first-degree relatives had significantly reduced microbiome diversity compared to their controls (observed species, P = 0.004; Chao1 index, P = 0.005), but this reduction was not observed in SLE patients. The majority of bacteria that were differentially abundant between unrelated healthy controls and first-degree relatives were in the Firmicutes phylum, while differences in bacteria from several phyla were identified between healthy controls and SLE patients. Bacteria in the Paenibacillus genus were the only overlapping differentially abundant bacteria in both cohorts, and were reduced in first-degree relatives (adjusted P [Padj ] = 2.13 × 10-12 ) and SLE patients (Padj = 0.008) but elevated in controls. CONCLUSIONS: These results indicate a possible role of plasma microbial translocation and microbiome composition in influencing autoantibody development in SLE.

Novel mechanism for estrogen receptor alpha modulation of murine lupus.

Female sex is a risk factor for lupus. Sex hormones, sex chromosomes and hormone receptors are implicated in the pathogenic pathways in lupus. Estrogen receptor alpha (ERα) knockout (KO) mice are used for defining hormone receptor effects in lupus. Prior studies of ERα KO in lupus have conflicting results, likely due to sex hormone levels, different lupus strains and different ERα KO constructs. Our objective was to compare a complete KO of ERα vs. the original functional KO of ERα (expressing a short ERα) on disease expression and immune phenotype, while controlling sex hormone levels. We studied female lupus prone NZM2410 WT and ERα mutant mice. All mice (n = 44) were ovariectomized (OVX) for hormonal control. Groups of each genotype were estrogen (E2)-repleted after OVX. We found that OVXed NZM mice expressing the truncated ERα (ERα short) had significantly reduced nephritis and prolonged survival compared to both wildtype and the complete ERαKO (ERα null) mice, but surprisingly only if E2-repleted. ERα null mice were not protected regardless of E2 status. We observed significant differences in splenic B cells and dendritic cells and a decrease in cDC2 (CD11b+CD8-) dendritic cells, without a concomitant decrease in cDC1 (CD11b-CD8a+) cells comparing ERα short to ERα null or WT mice. Our data support a protective role for the ERα short protein. ERα short is similar to an endogenously expressed ERα variant (ERα46). Modulating its expression/activity represents a potential approach for treating female-predominant autoimmune diseases.

An Analytic Approach Using Candidate Gene Selection and Logic Forest to Identify Gene by Environment Interactions (G × E) for Systemic Lupus Erythematosus in African Americans.

Development and progression of many human diseases, such as systemic lupus erythematosus (SLE), are hypothesized to result from interactions between genetic and environmental factors. Current approaches to identify and evaluate interactions are limited, most often focusing on main effects and two-way interactions. While higher order interactions associated with disease are documented, they are difficult to detect since expanding the search space to all possible interactions of p predictors means evaluating 2p - 1 terms. For example, data with 150 candidate predictors requires considering over 1045 main effects and interactions. In this study, we present an analytical approach involving selection of candidate single nucleotide polymorphisms (SNPs) and environmental and/or clinical factors and use of Logic Forest to identify predictors of disease, including higher order interactions, followed by confirmation of the association between those predictors and interactions identified with disease outcome using logistic regression. We applied this approach to a study investigating whether smoking and/or secondhand smoke exposure interacts with candidate SNPs resulting in elevated risk of SLE. The approach identified both genetic and environmental risk factors, with evidence suggesting potential interactions between exposure to secondhand smoke as a child and genetic variation in the ITGAM gene associated with increased risk of SLE.

A plausibly causal functional lupus-associated risk variant in the STAT1-STAT4 locus.

Systemic lupus erythematosus (SLE or lupus) (OMIM: 152700) is a chronic autoimmune disease with debilitating inflammation that affects multiple organ systems. The STAT1-STAT4 locus is one of the first and most highly replicated genetic loci associated with lupus risk. We performed a fine-mapping study to identify plausible causal variants within the STAT1-STAT4 locus associated with increased lupus disease risk. Using complementary frequentist and Bayesian approaches in trans-ancestral Discovery and Replication cohorts, we found one variant whose association with lupus risk is supported across ancestries in both the Discovery and Replication cohorts: rs11889341. In B cell lines from patients with lupus and healthy controls, the lupus risk allele of rs11889341 was associated with increased STAT1 expression. We demonstrated that the transcription factor HMGA1, a member of the HMG transcription factor family with an AT-hook DNA-binding domain, has enriched binding to the risk allele compared with the non-risk allele of rs11889341. We identified a genotype-dependent repressive element in the DNA within the intron of STAT4 surrounding rs11889341. Consistent with expression quantitative trait locus (eQTL) analysis, the lupus risk allele of rs11889341 decreased the activity of this putative repressor. Altogether, we present a plausible molecular mechanism for increased lupus risk at the STAT1-STAT4 locus in which the risk allele of rs11889341, the most probable causal variant, leads to elevated STAT1 expression in B cells due to decreased repressor activity mediated by increased binding of HMGA1.

A Long-Term Follow-Up Study of Allogeneic Mesenchymal Stem/Stromal Cell Transplantation in Patients with Drug-Resistant Systemic Lupus Erythematosus.

Allogeneic mesenchymal stem/stromal cells (MSCs) have been widely studied as an alternative cell source for regenerative medicine. Here, we report a long-term follow-up study of allogeneic bone marrow and/or umbilical cord MSC transplantation (MSCT) in severe and drug-refractory systemic lupus erythematosus (SLE) patients. Eighty-one patients were enrolled, and the 5-year overall survival rate was 84% (68/81) after MSCT. At 5-year follow-up, 27% of patients (22/81) were in complete clinical remission and another 7% (6/81) were in partial clinical remission, with a 5-year disease remission rate of 34% (28/81). In total, 37 patients had achieved clinical remission and then 9 patients subsequently relapsed, with 5-year overall rate of relapse of 24% (9/37). SLE Disease Activity Index scores, serum albumin, complement C3, peripheral white blood cell, and platelet numbers, as well as proteinuria levels, continued to improve during the follow-up. Our results demonstrated that allogeneic MSCT is safe and resulted in long-term clinical remission in SLE patients.

Transancestral mapping and genetic load in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10-8), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.