Single-cell RNA-seq analysis reveals cell subsets and gene signatures associated with Rheumatoid Arthritis Disease Activity

Rheumatoid arthritis (RA) management leans toward achieving remission or low disease activity. In this study, we conducted single-cell RNA sequencing (scRNA-Seq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 patients with RA and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFN-induced transmembrane 3-overexpressing (IFITM3-overexpressing) IFN-activated monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate-high disease activity (DAS28-CRP ≥ 3.2) and a decrease in nonclassical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of proinflammatory genes in the γδ T cell subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and nonclassical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of proinflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, and G0S2 and downregulation of genes including HLA-DQB1, HLA-DRB5, and TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity

Large-Scale Imputation of KIR Copy Number and HLA Alleles in North American and European Psoriasis Case-Control Cohorts Reveals Association of Inhibitory KIR2DL2 With Psoriasis

Killer cell immunoglobulin-like receptors (KIR) regulate immune responses in NK and CD8+ T cells via interaction with HLA ligands. KIR genes, including KIR2DS1, KIR3DL1, and KIR3DS1 have previously been implicated in psoriasis susceptibility. However, these previous studies were constrained to small sample sizes, in part due to the time and expense required for direct genotyping of KIR genes. Here, we implemented KIR*IMP to impute KIR copy number from single-nucleotide polymorphisms (SNPs) on chromosome 19 in the discovery cohort (n=11,912) from the PAGE consortium, University of California San Francisco, and the University of Dundee, and in a replication cohort (n=66,357) from Kaiser Permanente Northern California. Stratified multivariate logistic regression that accounted for patient ancestry and high-risk HLA alleles revealed that KIR2DL2 copy number was significantly associated with psoriasis in the discovery cohort (p ≤ 0.05). The KIR2DL2 copy number association was replicated in the Kaiser Permanente replication cohort. This is the first reported association of KIR2DL2 copy number with psoriasis and highlights the importance of KIR genetics in the pathogenesis of psoriasis

Differential genetic associations for systemic lupus erythematosus based on anti-dsDNA autoantibody production

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti-dsDNA autoantibody production, a SLE-related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti-dsDNA autoantibody positive (anti-dsDNA +, n = 811) and anti-dsDNA autoantibody negative (anti-dsDNA -, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti-dsDNA + SLE. Far fewer and weaker associations were observed for anti-dsDNA - SLE. For example, rs7574865 in STAT4 had an OR for anti-dsDNA + SLE of 1.77 (95% CI 1.57-1.99, p = 2.0E-20) compared to an OR for anti-dsDNA - SLE of 1.26 (95% CI 1.12-1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti-dsDNA + SLE and were not associated with anti-dsDNA - SLE. In conclusion, we identified differential genetic associations with SLE based on anti-dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti-dsDNA - SLE

High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions

A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9.99×10−16). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2.21, p = 2.53×10−12), *1401 (OR = 0.50, p = 0.0002), and *1501 (OR = 1.39, p = 0.0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2.44 and p = 2.80×10−13. Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE–associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0.006), CREBL1 (class III, rs8283, p = 0.01), and DQB2 (class II, rs7769979, p = 0.003, and rs10947345, p = 0.0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0.006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation

Increased alloreactive and autoreactive antihuman leucocyte antigen antibodies associated with systemic lupus erythematosus and rheumatoid arthritis.

ObjectivesRheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) disproportionately affect women during and following childbearing years. Antihuman leucocyte antigen (HLA) alloantibody responses are common in healthy parous women, and as these diseases are both linked with HLA and immune dysregulation, we sought to evaluate anti-HLA antibodies in RA and SLE.MethodsAnti-HLA antibodies were measured among parous SLE cases (n=224), parous RA cases (n=202) and healthy parous controls (n=239) and compared with each other as well as with nulliparous female and male controls. Antibody specificities were identified and compared against subject HLA types to determine autoreactivity versus alloreactivity. The association of anti-HLA antibodies with clinical outcomes was evaluated.ResultsLevels and frequencies of anti-HLA antibodies were significantly higher among parous females with SLE (52%) or RA (46%) compared with controls (26%), and anti-HLA antibodies were also found among nulliparous females and males with SLE and RA. Autoreactive anti-HLA antibodies were observed among SLE and RA antibody-positive subjects, but not healthy controls, with the highest frequency of autoreactive anti-HLA antibodies found in the SLE subjects. Higher levels of anti-HLA antibodies were associated with nephritis among the nulliparous SLE cases (p&lt;0.01). The presence of anti-class I HLA antibodies was associated with younger age at diagnosis among both the RA and SLE nulliparous cases.ConclusionsBoth autoreactive and alloreactive anti-HLA antibodies were found at high levels in RA and SLE subjects. These occurred even in the absence of alloexposure, particularly among SLE subjects and may be linked with disease severity

Differentiation of follicular helper T cells by salivary gland epithelial cells in primary Sjögren's syndrome.

Follicular helper T cells (Tfh), which play a pivotal role in B cell activation and differentiation in lymphoid structures, secrete IL-21 whose augmented secretion is a hallmark of several autoimmune diseases. To decipher the cellular and molecular interactions occurring in salivary glands of patients suffering from primary Sjögren's syndrome (pSS), we investigated whether salivary gland epithelial cells (SGECs) were capable to induce Tfh differentiation. Co-cultures of naïve CD4(+) T cells and SGECs from both patients with pSS and controls were performed. Here, we report that IL-6 and ICOSL expression by SGECs contributes to naïve CD4(+) T differentiation into Tfh cells, as evidenced by their acquisition of a specific phenotype, characterized by Bcl-6, ICOS and CXCR5 expression and IL-21 secretion, but also but by their main functional feature: the capacity to enhance B lymphocytes survival. We demonstrated an increase of serum IL-21 with systemic activity. Finally, we analyzed the potential occurrence of a genetic association between IL-21 or IL-21R gene polymorphisms and pSS or elevated IL-21 secretion. This study, which demonstrates a direct induction of Tfh differentiation by SGECs, emphasizes a yet unknown pathogenic role of SGECs and suggests that Tfh and IL-21 might be relevant biomarkers and/or therapeutic targets in primary Sjögren's syndrome.journal articleresearch support, non-u.s. gov't2014 Jun2014 01 09importe

Transcriptomic analysis of immune cells in a multi-ethnic cohort of systemic lupus erythematosus patients identifies ethnicity- and disease-specific expression signatures.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which outcomes vary among different racial groups. We leverage cell-sorted RNA-seq data (CD14+ monocytes, B cells, CD4+ T cells, and NK cells) from 120 SLE patients (63 Asian and 57 White individuals) and apply a four-tier approach including unsupervised clustering, differential expression analyses, gene co-expression analyses, and machine learning to identify SLE subgroups within this multiethnic cohort. K-means clustering on each cell-type resulted in three clusters for CD4 and CD14, and two for B and NK cells. To understand the identified clusters, correlation analysis revealed significant positive associations between the clusters and clinical parameters including disease activity as well as ethnicity. We then explored differentially expressed genes between Asian and White groups for each cell-type. The shared differentially expressed genes across cells were involved in SLE or other autoimmune-related pathways. Co-expression analysis identified similarly regulated genes across samples and grouped these genes into modules. Finally, random forest classification of disease activity in the White and Asian cohorts showed the best classification in CD4+ T cells in White individuals. The results from these analyses will help stratify patients based on their gene expression signatures to enable SLE precision medicine

Large-Scale Imputation of KIR Copy Number and HLA Alleles in North American and European Psoriasis Case-Control Cohorts Reveals Association of Inhibitory KIR2DL2 With Psoriasis.

Killer cell immunoglobulin-like receptors (KIR) regulate immune responses in NK and CD8+ T cells via interaction with HLA ligands. KIR genes, including KIR2DS1, KIR3DL1, and KIR3DS1 have previously been implicated in psoriasis susceptibility. However, these previous studies were constrained to small sample sizes, in part due to the time and expense required for direct genotyping of KIR genes. Here, we implemented KIR*IMP to impute KIR copy number from single-nucleotide polymorphisms (SNPs) on chromosome 19 in the discovery cohort (n=11,912) from the PAGE consortium, University of California San Francisco, and the University of Dundee, and in a replication cohort (n=66,357) from Kaiser Permanente Northern California. Stratified multivariate logistic regression that accounted for patient ancestry and high-risk HLA alleles revealed that KIR2DL2 copy number was significantly associated with psoriasis in the discovery cohort (p ≤ 0.05). The KIR2DL2 copy number association was replicated in the Kaiser Permanente replication cohort. This is the first reported association of KIR2DL2 copy number with psoriasis and highlights the importance of KIR genetics in the pathogenesis of psoriasis

Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjogren's syndrome.

International audienceSeveral autoimmune diseases, including primary Sjögren's syndrome (pSS), are associated with an increased risk for lymphoma. Polymorphisms of TNFAIP3, which encodes the A20 protein that plays a key role in controlling nuclear factor κB activation, have been associated with several autoimmune diseases. Somatic mutations of TNFAIP3 have been observed in the mucosa-associated lymphoid tissue lymphoma subtype frequently associated with pSS. We studied germline and somatic abnormalities of TNFAIP3 in 574 patients with pSS, including 25 with lymphoma. Nineteen additional patients with pSS and lymphoma were available for exome sequence analysis. Functional abnormalities of A20 were assessed by gene reporter assays. The rs2230926 exonic variant was associated with an increased risk for pSS complicated by lymphoma (odds ratio, 3.36 [95% confidence interval, 1.34-8.42], and odds ratio, 3.26 [95% confidence interval, 1.31-8.12], vs controls and pSS patients without lymphoma, respectively; P = .011). Twelve (60%) of the 20 patients with paired germline and lymphoma TNFAIP3 sequence data had functional abnormalities of A20: 6 in germline DNA, 5 in lymphoma DNA, and 1 in both. The frequency was even higher (77%) among pSS patients with mucosa-associated lymphoid tissue lymphoma. Some of these variants showed impaired control of nuclear factor κB activation. These results support a key role for germline and somatic variations of A20 in the transformation between autoimmunity and lymphoma