Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Implementation of automated behavior metrics to evaluate voluntary wheel running effects on inflammatory-erosive arthritis and interstitial lung disease in TNF-Tg mice.

BACKGROUND: Although treatment options and algorithms for rheumatoid arthritis (RA) have improved remarkably in recent decades, there continues to be no definitive cure or pharmacologic intervention with reliable long-term efficacy. For this reason, the combination of medications and healthy lifestyle modifications are essential for controlling joint disease, and extra-articular manifestations of RA, such as interstitial lung disease (ILD) and other lung pathologies, which greatly impact morbidity and mortality. Generally, exercise has been deemed beneficial in RA patients, and both patients and clinicians are motivated to incorporate effective non-pharmacologic interventions. However, there are limited evidence-based and specific exercise regimens available to support engagement in such activities for RA patients. Here, we provided the continuous opportunity for exercise to mice and implemented automated recording and quantification of wheel running behavior. This allowed us to describe the associated effects on the progression of inflammatory-erosive arthritis and ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA. METHODS: Wild-type (WT; males, n=9; females, n=9) and TNF-Tg (males, n=12; females, n=14) mice were singly housed with free access to a running wheel starting at 2 months until 5 to 5.5 months of age. Measures of running included distance, rate, length, and number of run bouts, which were derived from continuously recorded data streams collected automatically and in real-time. In vivo lung, ankle, and knee micro-computed tomography (micro-CT), along with terminal micro-CT and histology were performed to examine the association of running behaviors and disease progression relative to sedentary controls. RESULTS: TNF-Tg males and females exhibited significantly reduced running distance, rate, length, and number of run bouts compared to WT counterparts by 5 months of age (p<0.0001). Compared to sedentary controls, running males and females showed increased aerated lung volumes (p<0.05) that were positively correlated with running distance and rate in female mice (WT: Distance, ρ=0.705/rate, ρ=0.693 (p<0.01); TNF-Tg: ρ=0.380 (p=0.06)/ρ=0.403 (p<0.05)). Talus bone volumes were significantly reduced in running versus sedentary males and negatively correlated with running distance and rate in TNF-Tg mice (male: ρ=-903/ρ=-0.865; female: ρ=-0.614/ρ=-0.594 (p<0.001)). Histopathology validated the lung and ankle micro-CT findings. CONCLUSIONS: Implementation of automated wheel running behavior metrics allows for evaluation of longitudinal activity modifications hands-off and in real-time to relate with biomarkers of disease severity. Through such analysis, we determined that wheel running activity increases aerated lung volumes, but exacerbates inflammatory-erosive arthritis in TNF-Tg mice. To the end of a clinically relevant model, additional functional assessment of these outcomes and studies of pain behavior are warranted.

Alterations of the Primary Cilia Gene SPAG17 and SOX9 Locus Noncoding RNAs Identified by RNA-Sequencing Analysis in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by immune activation, vasculopathy, and unresolving fibrosis in the skin, lungs, and other organs. We performed RNA-sequencing analysis on skin biopsy samples and peripheral blood mononuclear cells (PBMCs) from SSc patients and unaffected controls to better understand the pathogenesis of SSc. METHODS: We analyzed these data 1) to test for case/control differences and 2) to identify genes whose expression levels correlate with SSc severity as measured by local skin score, modified Rodnan skin thickness score (MRSS), forced vital capacity (FVC), or diffusing capacity for carbon monoxide (DLco). RESULTS: We found that PBMCs from SSc patients showed a strong type I interferon signature. This signal was found to be replicated in the skin, with additional signals for increased extracellular matrix (ECM) genes, classical complement pathway activation, and the presence of B cells. Notably, we observed a marked decrease in the expression of SPAG17, a cilia component, in SSc skin. We identified genes that correlated with the MRSS, DLco, and FVC in SSc PBMCs and skin using weighted gene coexpression network analysis. These genes were largely distinct from the case/control differentially expressed genes. In PBMCs, type I interferon signatures negatively correlated with the DLco. In SSc skin, ECM gene expression positively correlated with the MRSS. Network analysis of SSc skin genes that correlated with clinical features identified the noncoding RNAs SOX9-AS1 and ROCR, both near the SOX9 locus, as highly connected, "hub-like" genes in the network. CONCLUSION: These results identify noncoding RNAs and SPAG17 as novel factors potentially implicated in the pathogenesis of SSc.

Circulating CTRP9 Is Associated With Severity of Systemic Sclerosis-Associated Interstitial Lung Disease.

OBJECTIVE: While interstitial lung disease (ILD) is the leading cause of morbidity and mortality in systemic sclerosis (SSc), there remains a paucity of predictive markers to assess disease progression. We previously demonstrated that adipose tissue metabolism and adipokine homeostasis is dysregulated in SSc. The present study was undertaken to determine the association and predictive ability of the novel adipokine C1q/tumor necrosis factor-related protein 9 (CTRP9) for SSc-associated ILD. METHODS: We performed a retrospective longitudinal study utilizing the Northwestern Scleroderma Program Patient Registry and Biorepository. Serum levels of CTRP9 were measured in 110 SSc patients at baseline, and demographic, clinical, and pulmonary function test data were collected in 12-month intervals to 48 months. Longitudinal trajectory of forced vital capacity percent predicted (FVC%) was used as a primary outcome measure. We utilized a mixed model to compare trajectories of lung function by CTRP9 groups and performed latent trajectory analysis to accommodate for heterogeneity. RESULTS: In cross-sectional analysis, elevated circulating CTRP9 was associated with significantly lower FVC% at baseline (72% ± 17 versus 80% ± 18; P = 0.02) and 48 months (68 ± 19 versus 84 ± 18; P = 0.001). In mixed model analysis, high CTRP9 was associated with worse lung function but not with a different trajectory (P = 0.23). In contrast, low CTRP9 identified patients with stability of lung disease with reasonable accuracy (sensitivity 73%). Latent trajectory analysis confirmed the association of lower CTRP9 with higher FVC%. CONCLUSION: Higher circulating CTRP9 associated with worse pulmonary function, while low CTRP9 identified patients with lung disease stability over time. These findings suggest that CTRP9 may be a potential biomarker in SSc-associated ILD.

Tumor Necrosis Factor Induces Obliterative Pulmonary Vascular Disease in a Novel Model of Connective Tissue Disease-Associated Pulmonary Arterial Hypertension.

OBJECTIVE: Connective tissue disease (CTD)-associated pulmonary arterial hypertension (PAH) is the second most common etiology of PAH and carries a poor prognosis. Recently, it has been shown that female human tumor necrosis factor (TNF)-transgenic (Tg) mice die of cardiopulmonary disease by 6 months of age. This study was undertaken to characterize this pathophysiology and assess its potential as a novel model of CTD-PAH. METHODS: Histologic analysis was performed on TNF-Tg and wild-type (WT) mice to characterize pulmonary vascular and right ventricular (RV) pathology (n = 40 [4-5 mice per group per time point]). Mice underwent right-sided heart catheterization (n = 29) and micro-computed tomographic angiography (n = 8) to assess vascular disease. Bone marrow chimeric mice (n = 12), and anti-TNF-treated mice versus placebo-treated mice (n = 12), were assessed. RNA sequencing was performed on mouse lung tissue (n = 6). RESULTS: TNF-Tg mice displayed a pulmonary vasculopathy marked by collagen deposition (P < 0.001) and vascular occlusion (P < 0.001) with associated RV hypertrophy (P < 0.001) and severely increased RV systolic pressure (mean ± SD 75.1 ± 19.3 mm Hg versus 26.7 ± 1.7 mm Hg in WT animals; P < 0.0001). TNF-Tg mice had increased α-smooth muscle actin (α-SMA) staining, which corresponded to proliferation and loss of von Willebrand factor (vWF)-positive endothelial cells (P < 0.01). There was an increase in α-SMA-positive, vWF-positive cells (P < 0.01), implicating endothelial-mesenchymal transition. Bone marrow chimera experiments revealed that mesenchymal but not bone marrow-derived cells are necessary to drive this process. Treatment with anti-TNF therapy halted the progression of disease. This pathology closely mimics human CTD-PAH, in which patient lungs demonstrate increased TNF signaling and significant similarities in genomic pathway dysregulation. CONCLUSION: The TNF-Tg mouse represents a novel model of CTD-PAH, recapitulates key disease features, and can serve as a valuable tool for discovery and assessment of therapeutics.

HLA and autoantibodies define scleroderma subtypes and risk in African and European Americans and suggest a role for molecular mimicry.

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

Brief Report: Association of Elevated Adipsin Levels With Pulmonary Arterial Hypertension in Systemic Sclerosis.

OBJECTIVE: Adipose tissues secrete adipokines, peptides with potent effects modulating fibrosis, inflammation, and vascular homeostasis. Dysregulated adipose tissue biology and adipokine balance have recently been implicated in systemic sclerosis (SSc). This study was undertaken to determine whether altered circulating adipokine levels correlate with SSc disease subsets or clinical manifestations. METHODS: Multiplex assays were used to measure circulating adipokine levels in 198 patients with SSc and 33 healthy controls. Data were evaluated for correlations between serum adipokine levels and demographic and clinical features, including pulmonary arterial hypertension (PAH). To assess the relevance of adipsin, an adipokine involved in complement pathway activation, in SSc, we analyzed publicly available genetic and transcriptomic data. RESULTS: Levels of adiponectin and adipsin differed significantly between controls and patients. Adipsin was significantly elevated in patients with limited cutaneous SSc (odds ratio [OR] 28.3 [95% confidence interval (95% CI) 7.0-113.8]; P < 0.0001), and its levels were associated with serum autoantibody status, pulmonary function and cardiovascular parameters, and PAH (OR 3.3 [95% CI 1.3-8.7]; P = 0.02). Elevated adipsin was more strongly associated with PAH than B-type natriuretic peptide was. Moreover, in SSc patients, adipsin gene single-nucleotide polymorphisms were associated with PAH. Transcriptome data set analysis demonstrated elevated adipsin expression in patients with SSc-related PAH. CONCLUSION: We identify adipsin as a novel adipose tissue-derived marker of SSc-related PAH. Circulating adipsin levels might serve as predictive biomarkers in SSc. Mechanistically, adipsin might represent a pathogenic link between adipocyte dysfunction and complement pathway activation and play an important role in the pathogenesis of SSc-related PAH.

Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis.

INTRODUCTION: Our objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes' expression is related to atherosclerosis in SLE. METHODS: Monocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined. RESULTS: Gene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors. CONCLUSIONS: Many genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.

Common variants at CD40 and other loci confer risk of rheumatoid arthritis.

To identify rheumatoid arthritis risk loci in European populations, we conducted a meta-analysis of two published genome-wide association (GWA) studies totaling 3,393 cases and 12,462 controls. We genotyped 31 top-ranked SNPs not previously associated with rheumatoid arthritis in an independent replication of 3,929 autoantibody-positive rheumatoid arthritis cases and 5,807 matched controls from eight separate collections. We identified a common variant at the CD40 gene locus (rs4810485, P = 0.0032 replication, P = 8.2 x 10(-9) overall, OR = 0.87). Along with other associations near TRAF1 (refs. 2,3) and TNFAIP3 (refs. 4,5), this implies a central role for the CD40 signaling pathway in rheumatoid arthritis pathogenesis. We also identified association at the CCL21 gene locus (rs2812378, P = 0.00097 replication, P = 2.8 x 10(-7) overall), a gene involved in lymphocyte trafficking. Finally, we identified evidence of association at four additional gene loci: MMEL1-TNFRSF14 (rs3890745, P = 0.0035 replication, P = 1.1 x 10(-7) overall), CDK6 (rs42041, P = 0.010 replication, P = 4.0 x 10(-6) overall), PRKCQ (rs4750316, P = 0.0078 replication, P = 4.4 x 10(-6) overall), and KIF5A-PIP4K2C (rs1678542, P = 0.0026 replication, P = 8.8 x 10(-8) overall).

Combination immunosuppressive therapies: the promise and the peril.

BACKGROUND: Targeted immunotherapeutic agents (TIs), also known as biological agents, are efficacious treatments for many immunologically mediated disorders, including psoriasis. In several of these diseases, including rheumatoid arthritis, Crohn's disease, and multiple sclerosis, certain TIs have been studied in combination with nonspecific immunosuppressive agents and with other TIs. OBSERVATIONS: Recently, the rheumatology, neurology, and gastroenterology literature has reported several examples of possible associated toxic effects when certain TIs are used in combination with other immunosuppressive agents. These toxic effects have included an increased risk of infection and malignancy. CONCLUSIONS: Combination therapies are often used by dermatologists. Several TIs have been approved for psoriasis; however, clinical trials using these drugs in combination with other immunosuppressive agents have not yet been performed. The implications for dermatologists of the toxic effects associated with TI combination therapy are unclear. However, combination therapy with certain TIs should be used with caution until more data are available.