NETched in Stone.

Gallstone formation in adults is a common, yet incompletely understood disease process. In this issue, Muñoz et al. (2019) report a pathogenic link between neutrophil extracellular traps (NETs) and the formation of gallstones.

Rheumatoid Arthritis: The Continuum of Disease and Strategies for Prediction, Early Intervention, and Prevention.

Rheumatoid arthritis (RA) is known to include a pre-RA stage that can be defined as the presence of familial or genetic risk factors, biomarker abnormalities (eg, anticitrullinated protein antibodies [ACPA]), symptoms, and even abnormal imaging findings prior to the development of the onset of clinical RA with inflammatory arthritis that is apparent on physical examination. Indeed, there are multiple completed or ongoing retrospective case-control as well as prospective observational studies to identify the key biologic drivers of disease. Further, building on the predictive ability of combinations of biomarkers, symptoms, and imaging for future RA, there are multiple clinical trials completed, underway, or in development to identify approaches that may prevent, delay, or ameliorate future clinical RA in at-risk individuals. Importantly, however, although an effective preventive intervention has not yet been identified, at-risk individuals are being increasingly identified in clinical care; this presents a challenge of how to manage these individuals in clinical practice. This review will discuss the current understanding of the biology and natural history of RA development, nomenclature, and current models for prediction of future RA, as well as evaluate the current and ongoing clinical prevention trials with the overall goal to provide insights into the challenges and opportunities in the field of RA prevention. Moreover, this review will provide up-to-date options for clinical management of individuals at risk for RA.

Perceived Access to Healthcare of Indigenous Peoples in Canada With Rheumatoid Arthritis and Their First-Degree Relatives.

OBJECTIVE: There are complex and interrelated factors that lead to inequitable healthcare delivery in Canada. Many of the factors that underlie these inequities for Canada's geographically dispersed Indigenous peoples remain underexamined. METHODS: A cohort of 831 First Nations (FN) individuals from urban and remote communities were recruited into a longitudinal study of rheumatoid arthritis (RA) risk from 2005 to 2017. Data from each participant's initial enrollment visit were assessed using a survey that captured concerns with healthcare access. RESULTS: We found that remote participants with RA reported poor access compared to remote first-degree relatives (FDRs; P < 0.001); this difference was not observed for urban participants with RA. We observed substantial differences based on sex; female participants perceived access to care to be more difficult than male participants in both urban and remote cohorts (P < 0.001). We also observed that male participants with RA reported poor access to care compared to male FDRs. Importantly, access to care in remote communities appeared to improve over the duration of the study (P = 0.01). In a logistic regression analysis, female sex, remote location, and older age were independent predictors of poor access to care. Predictors of poor access in participants with RA also included female sex, remote location, and older age. CONCLUSION: FN peoples living in remote communities, particularly those with an established RA diagnosis, report more problems accessing health care. Sex-based inequities exist, with FN female individuals reporting greater difficulties in accessing appropriate health care, regardless of RA diagnosis. Addressing these sex-based inequities should be a high priority for improving healthcare delivery.

Single-Cell Analysis Reveals the Range of Transcriptional States of Circulating Human Neutrophils.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.

Neutrophils in Inflammatory Bone Diseases.

PURPOSE OF REVIEW: In this review, we summarize the current evidence that suggests that neutrophils play a key role in facilitating damage to local bone structures. RECENT FINDINGS: Neutrophil infiltration is a hallmark of inflammatory bone diseases such as rheumatoid arthritis (RA) and periodontitis disease (PD). Both of these human diseases are marked by an imbalance in bone homeostasis, favoring the degradation of local bone which ultimately leads to erosions. Osteoclasts, a multinucleated resident bone cell, are responsible for facilitating the turnover of bone and the bone damage observed in these diseases. The involvement of neutrophils and neutrophil extracellular trap formation have recently been implicated in exacerbating osteoclast function through direct and indirect mechanisms. We highlight a recent finding that NET proteins such as histones and elastase can generate non-canonical, inflammatory osteoclasts, and this process is mediated by post-translational modifications such as citrullination and carbamylation, both of which act as autoantigens in RA. It appears that NETs, autoantibodies, modified proteins, cytokines, and osteoclasts all ultimately contribute to local and permanent bone damage in RA and PD. However, more studies are needed to fully understand the role of neutrophils in inflammatory bone diseases.

Neutrophil extracellular trap-associated carbamylation and histones trigger osteoclast formation in rheumatoid arthritis.

OBJECTIVE: Neutrophil infiltration into the synovial joint is a hallmark of rheumatoid arthritis (RA), a disease characterised by progressive bone erosion. However, the mechanisms by which neutrophils participate in bone destruction remain unclear. Carbamylation is a posttranslational modification linked to increased bone erosion in RA and we previously showed that carbamylation is present in RA neutrophil extracellular traps (NETs). However, it remains unclear whether NETs and their carbamylated protein cargo directly promote bone destruction and alter osteoclast biology. METHODS: NETs and carbamylated NETs (cNETs) were assessed for their capacity to induce osteoclast formation in CD14+ monocytes. Chemical inhibitors and neutralising antibodies were used to elucidate the pathway by which NETs induce osteoclastogenesis. HLA-DRB1*04:01 mice received intra-articular injection of cNETs for 4 weeks. Joints were isolated and assessed for osteoclast formation. Plasma and synovial fluid samples from patients with RA (n=32) were assessed for the presence of carbamylated histone, and correlations to disease specific outcomes were performed. RESULTS: We found that NETs, when cNETs, instruct monocytes to undergo rapid osteoclast formation. NET-mediated osteoclastogenesis appears to depend on Toll-like receptor 4 signalling and NET-associated proteins including histones and neutrophil elastase. In vivo, we identified that the number of osteoclasts increased following immunisation with cNETs in HLA-DRB1*04:01 transgenic mice. Furthermore, carbamylated histones are increased in plasma and synovial fluid from patients with RA and correlate with active bone resorption and inflammatory markers. CONCLUSIONS: Our results suggest that NETs have a direct role in RA-associated bone erosion by promoting osteoclast formation.

HAL-X: Scalable hierarchical clustering for rapid and tunable single-cell analysis.

Data clustering plays a significant role in biomedical sciences, particularly in single-cell data analysis. Researchers use clustering algorithms to group individual cells into populations that can be evaluated across different levels of disease progression, drug response, and other clinical statuses. In many cases, multiple sets of clusters must be generated to assess varying levels of cluster specificity. For example, there are many subtypes of leukocytes (e.g. T cells), whose individual preponderance and phenotype must be assessed for statistical/functional significance. In this report, we introduce a novel hierarchical density clustering algorithm (HAL-x) that uses supervised linkage methods to build a cluster hierarchy on raw single-cell data. With this new approach, HAL-x can quickly predict multiple sets of labels for immense datasets, achieving a considerable improvement in computational efficiency on large datasets compared to existing methods. We also show that cell clusters generated by HAL-x yield near-perfect F1-scores when classifying different clinical statuses based on single-cell profiles. Our hierarchical density clustering algorithm achieves high accuracy in single cell classification in a scalable, tunable and rapid manner.

Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus.

Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.

Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism.

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus.

Neutrophil dysregulation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). SLE is characterized by elevated levels of a pathogenic neutrophil subset known as low-density granulocytes (LDGs). The origin and phenotypic, functional, and pathogenic heterogeneity of LDGs remain to be systematically determined. Transcriptomics and epigenetic assessment of lupus LDGs, autologous normal-density neutrophils, and healthy control neutrophils was performed by bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. Functional readouts were compared among neutrophil subsets. SLE LDGs display significant transcriptional and epigenetic heterogeneity and comprise 2 subpopulations of intermediate-mature and immature neutrophils, with different degrees of chromatin accessibility and differences in transcription factor motif analysis. Differences in neutrophil extracellular trap (NET) formation, oxidized mitochondrial DNA release, chemotaxis, phagocytosis, degranulation, ability to harm the endothelium, and responses to type I interferon (IFN) stimulation are evident among LDG subsets. Compared with other immune cell subsets, LDGs display the highest expression of IFN-inducible genes. Distinct LDG subsets correlate with specific clinical features of lupus and with the presence and severity of coronary artery disease. Phenotypic, functional, and pathogenic neutrophil heterogeneity are prevalent in SLE and may promote immune dysregulation and prominent vascular damage characteristic of this disease.

Deficiency of adenosine deaminase 2 triggers adenosine-mediated NETosis and TNF production in patients with DADA2.

Reduction of adenosine deaminase 2 (ADA2) activity due to autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1) results in a systemic vasculitis known as deficiency of ADA2 (DADA2). Neutrophils and a subset of neutrophils known as low-density granulocytes (LDGs) have been implicated in the pathogenesis of vasculitis, at least in part, through the formation of neutrophil extracellular traps (NETs). The study objective was to determine whether neutrophils and NETs play a pathogenic role in DADA2. In vivo evidence demonstrated NETs and macrophages in affected gastrointestinal tissue from patients with DADA2. An abundance of circulating LDGs prone to spontaneous NET formation was observed during active disease in DADA2 and were significantly reduced after remission induction by anti-tumor necrosis factor (TNF) therapy. Increased circulating LDGs were identified in unaffected family members with monoallelic ADA2 mutations. Adenosine triggered NET formation, particularly in neutrophils from female patients, by engaging A1 and A3 adenosine receptors (ARs) and through reactive oxygen species- and peptidylarginine deiminase-dependent pathways. Adenosine-induced NET formation was inhibited by recombinant ADA2, A1/A3 AR antagonists, or by an A2A agonist. M1 macrophages incubated with NETs derived from patients with DADA2 released significantly greater amounts of TNF-α. Treatment with an A2AAR agonist decreased nuclear translocation of NF-κB and subsequent production of inflammatory cytokines in DADA2 monocyte-derived macrophages. These results suggest that neutrophils may play a pathogenic role in DADA2. Modulation of adenosine-mediated NET formation may contribute a novel and directed therapeutic approach in the treatment of DADA2 and potentially other inflammatory diseases.

Lipoxygenase-derived oxylipins are enriched in anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is typically preceded by an extended preclinical period where circulating autoantibodies, particularly anti-citrullinated protein antibodies (ACPA), are detectable in the absence of clinical arthritis. Increased dietary intake of anti-inflammatory omega-3 (ω3) polyunsaturated fatty acids (PUFA) has been shown to be associated with a lower the risk of developing incident RA in large epidemiological studies. It is currently not known how changes in fatty acid (FA) metabolism may impact on the progression towards RA in at-risk individuals. To begin to address this question, we profiled serum FAs and oxylipins in an established cohort of at-risk ACPA-positive first-degree relatives (FDR) of RA patients (N = 31), some of whom developed RA (N = 4), and compared their profile to ACPA-negative FDR from the same population (N = 10). METHODS: Gas chromatography (GC) was used for FA quantitation. Oxylipins were extracted and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). RESULTS: Although we did not detect any meaningful differences in overall FA content between ACPA + and ACPA - FDR, the levels of oxylipins derived from FA metabolism demonstrated significant differences between the two groups, with the ACPA + group demonstrating enrichment in circulating arachidonic acid- and eicosapentaenoic acid-derived molecules. Compared with the ACPA - FDR group, the ACPA + FDR, including those who progressed into inflammatory arthritis, displayed higher levels of LOX-derived oxylipins. CONCLUSION: ACPA seropositivity in otherwise unaffected individuals at-risk for developing future RA based on family history (FDR) is associated with alterations in the serum oxylipin profile that suggests dysregulated LOX activity.

Modifiable risk factors linked to the development of rheumatoid arthritis: evidence, immunological mechanisms and prevention.

Rheumatoid Arthritis (RA) is a common autoimmune disease that targets the synovial joints leading to arthritis. Although the etiology of RA remains largely unknown, it is clear that numerous modifiable risk factors confer increased risk to developing RA. Of these risk factors, cigarette smoking, nutrition, obesity, occupational exposures and periodontal disease all incrementally increase RA risk. However, the precise immunological mechanisms by which these risk factors lead to RA are not well understood. Basic and translational studies have provided key insights into the relationship between inflammation, antibody production and the influence in other key cellular events such as T cell polarization in RA risk. Improving our general understanding of the mechanisms which lead to RA will help identify targets for prevention trials, which are underway in at-risk populations. Herein, we review the modifiable risk factors that are linked to RA development and describe immune mechanisms that may be involved. We highlight the few studies that have sought to understand if modification of these risk factors reduces RA risk. Finally, we speculate that modification of risk factors may be an appealing avenue for prevention for some at-risk individuals, specifically those who prefer lifestyle interventions due to safety and economic reasons.

Deep learning-based joint detection in Rheumatoid arthritis hand radiographs.

Advancements in technology have enabled diverse tools and medical devices that are able to improve the efficiency of diagnosis and detection of various health diseases. Rheumatoid arthritis is an autoimmune disease that affects multiple joints including the wrist, hands and feet. We used YOLOv5l6 to detect these joints in radiograph images. In this paper, we show that training YOLOv5l6 on joint images of healthy patients is able to achieve a high performance when used to evaluate joint images of patients with rheumatoid arthritis, even when there is a limited number of training samples. In addition to training joint images from healthy individuals with YOLOv5l6, we added several data augmentation steps to further improve the generalization of the deep learning model.

Effectiveness and Safety of Immune Checkpoint Inhibitors in Cancer Patients With Autoimmune Disease: A Retrospective Cohort Study.

Although immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, patients with pre-existing autoimmune diseases (PADs) have largely been excluded from clinical trials evaluating this drug class. This study evaluates the effectiveness and safety of ICI therapy in individuals with PAD in a real-world setting. A retrospective study of patients exposed to ICI therapy between 2012 and 2019 was conducted. Patients with PAD were identified and matched to an ICI-exposed group without PAD based on age, sex, and cancer type. Primary outcomes included toxicity, time to treatment failure, overall survival, and objective response rate. The association between PAD status and outcomes was determined using Cox and logistic regression modeling. A total of 813 patients exposed to ICI therapy were identified, of which 8.2% (N=67) had a PAD. When compared with a matched cohort without PAD (N=132), there was no significant difference in the rates of new immune-related adverse events (irAEs, 42.4% in the non-PAD group vs. 47.8% in the PAD group, P=0.474). After controlling for the type of ICI, there was no significant association between PAD status and irAE (odds ratio 1.67, 95% CI: 0.9-3.21 P=0.1). There was no significant association between overall survival and PAD status (hazard ratio 1.12, 95% CI: 0.76-1.66. P=0.56) or between time to treatment failure and PAD status (hazard ratio 0.82, 95% CI: 0.6-1.12, P=0.22). There was an association between PAD status and objective response rate (odds ratio 3.28, 95% CI: 1.28-8.38, P=0.013). In summary, PAD status was not associated with enhanced toxicity when compared with patients without PAD, with similar oncologic effectiveness between these 2 groups.

Functional Disability to Evaluate the Risk of Arthritis in First-degree Relatives of Patients With Rheumatoid Arthritis.

OBJECTIVE: The events that occur prior to the onset of rheumatoid arthritis (RA) continue to be delineated. We examined the relationship between self-reported joint symptoms, functional disability, and anticitrullinated protein antibody (ACPA) status in a cohort of first-degree relatives (FDR) of patients with RA who are at risk of future disease development. METHODS: We studied a cohort of 279 FDR of First Nations (FN) patients with RA who are at increased risk for future RA development, and analyzed data collected at their enrollment study visit. In parallel, we analyzed data from 279 FN subjects with no family history of RA. A subset of FDR developed inflammatory arthritis and we analyzed longitudinal data in this group. RESULTS: The prevalence of joint symptoms and functional disability was higher in FDR compared to non-FDR (all P < 0.001). Difficulty walking (37.3% vs 18.0%) and modified Health Assessment Questionnaire (HAQ) results were higher in ACPA-positive FDR compared to ACPA-negative FDR, and HAQ was independently associated with ACPA seropositivity (OR 2.79, 95% CI 1.56-5.00). Longitudinally, in individuals who developed ACPA-positive RA, ACPA level and HAQ score were significantly associated (R = 0.45, P < 0.001) in the preclinical period. CONCLUSION: Compared to population-based controls, FDR have a high burden of joint symptoms and functional disability. Functional disability was most closely associated with ACPA seropositivity in the FDR, suggesting a direct role for ACPA outside of the context of clinically detectable synovitis. HAQ appears to be particularly valuable in the assessment of individuals at risk for future RA development.

Serum proteomic networks associate with pre-clinical rheumatoid arthritis autoantibodies and longitudinal outcomes.

Objectives: The development of autoantibody directed towards citrullinated proteins (ACPA) are predictive of RA in at-risk individuals. The biological events that underpin loss of immune tolerance and progression into inflammatory arthritis are not known. We sought to identify serum proteomic alterations that drive autoantibody formation, persistence and progression into inflammatory arthritis in a cohort of first-degree relatives (FDR) of RA patients. Methods: We studied baseline serum samples from a cohort of Indigenous FDR (n = 147) and quantified serum proteins using a 48-plex platform. Longitudinal outcomes were defined on the basis of ACPA status and progression into inflammatory arthritis (IA). K-means clustering, differential expression, and principal components analyze group differences. A co-expression module analysis was used to identify enriched networks. Random forest was used to classify ACPA positive samples, while network analysis was used to understand underlying biological processes based on protein expression. Results: We defined 6 proteomic clusters, with enrichment of ACPA positive samples in one of the clusters. 23 of 24 differentially expressed proteins in ACPA positive samples were upregulated. A co-expression network was enriched in ACPA positive sera and individuals who progressed into IA. Random Forest achieved an area under the curve of 0.767 to classify ACPA positive sera in a test dataset. Network analysis revealed upregulation of JAK-STAT signalling as being activated in those at highest risk to develop future IA. Conclusions: The serum proteome provides a rich dataset to understand biological processes in ACPA seropositive individuals. A combination of serum biomarkers, including ACPA, may predict future arthritis onset in at-risk individuals.

Utility of Baseline Transcriptomic Analysis of Rheumatoid Arthritis Synovium as an Indicator for Long-Term Clinical Outcomes.

Objective: Rheumatoid arthritis is a chronic inflammatory autoimmune disease that can lead to synovial damage, persistent joint pain, and functional disability. Our objective was to evaluate baseline synovial transcriptome from early inflammatory arthritis patients (EIA) and identify pretreatment biomarkers that could potentially provide insights into long-term functional outcomes of rheumatoid arthritis (RA). Methods: Synovial biopsies from clinically inflamed knee joints were procured from either 17 EIA patients before initiation of disease modifying anti-rheumatic drug (DMARD) therapy (DMARD-naïve EIA) using the minimally invasive closed needle biopsy technique or advanced RA patients undergoing arthroplasty. Affymetrix Human Genome U133 Plus 2 microarray platform was used to profile the synovial transcriptome. The cohort was followed clinically for a median of 12.3 years, and patient data was collected at each visit. Short-term and long-term clinical outcomes were determined by assessing RA-associated clinical parameters Statistical adjustments were made to account for asynchronous clinical visits and duration of follow up. Results: Based on the transcriptomic analysis, we identified 5 differentially expressed genes (DEGs), including matrix metalloproteinase (MMP)-1 (fibroblast collagenase) and MMP-3 (stromelysin-1) in DMARD-naïve EIA patients, relative to advanced RA patients (q < 0.05). Dichotomous expression of MMP-1 and MMP-3 mRNA and protein was confirmed by qPCR and immunohistochemistry respectively, based on which DMARD-naïve EIA subjects were classified as MMP-high or MMP-low. Hierarchical clustering of transcriptomic data identified 947 DEGs between MMP-high and MMP-low cohorts. Co-expression and IPA analysis of DEGs in the MMP-high cohort showed an enrichment of genes that participated in metabolic or biochemical functions and intracellular immune signaling were regulated through NF-κB and ß-catenin complexes and correlated with markers of systemic inflammation. Analysis of short-term clinical outcomes in MMP-high cohort showed a significant reduction in the DAS-CRP scores relative to baseline (P <0.001), whereas area under the curve analyses of modified HAQ (mHAQ) scores correlated negatively with baseline MMP-1 (R = -0.59, P = 0.03). Further, longitudinal mHAQ scores, number of swollen joints, number of DMARDs and median follow-up duration appeared to be higher in MMP-low cohort. Conclusion: Overall, our results indicate that the gene expression profiling of synovial biopsies obtained at the DMARD-naive stage in patients with EIA categorizes them into subsets with varying degrees of inflammation and can predict the future of long-term clinical outcome.

Increased frequency of TIGIT+ CD4 T Cell subset in autoantibody-positive first-degree relatives of patients with rheumatoid arthritis.

Background: Despite immune cell dysregulation being an important event preceding the onset of rheumatoid arthritis (RA), the phenotype of T and B cells in preclinical RA is less understood. The aim of this study was to characterize T and B cell populations in RA patients and their autoantibody (aAb) negative and positive first-degree relatives (FDR). Methods: Cryopreserved peripheral blood mononuclear cells (PBMCs) collected at scheduled visits from aAb-(n=25), and aAb+ FDR (n=10) and RA patients (n=13) were thawed and stained using optimized antibody cocktails as per a specific 13-color T or B cell panel. Immunophenotyping was performed using a Cytoflex LX (Beckman-Coulter) flow cytometer and FlowJo software was used for analyzing the frequency of immune cell populations. Results: Multicolor flow cytometry experiments identified an increased TIGIT expression in circulating lymphocytes of aAb+ FDR and RA patients, relative to aAb- FDR (P<0.01). These TIGIT+ T cells exhibited a memory phenotype and expressed high levels of PD-1, ICOS, HLA-DR, CXCR3 and CXCR5. Moreover, increased TIGIT+ CD4 T cell frequency correlated with the frequency of PD-1+ CD4 T cells (r = 0.4705: P = 0.0043) and circulating levels of ACPA and RF. We also identified a decreased frequency of CD27+IgD- switched memory B cells in RA patients (P < 0.01), while increased frequency of TIGIT+ CD4 T cells in FDR correlated with the frequency of PD1+PTEN+ B cells (r = 0.6838, P = 0.0004) and autoantibody positivity (P = 0.01). Conclusion: We demonstrate TIGIT as a distinct CD4 T cell marker for differentiating aAb- FDR from aAb+FDR and might play a critical role in regulating T and B cell crosstalk in preclinical RA.

Autoantibodies Present in Hidradenitis Suppurativa Correlate with Disease Severity and Promote the Release of Proinflammatory Cytokines in Macrophages.

Hidradenitis suppurativa (HS), also known as acne inversa, is a debilitating inflammatory skin disorder that is characterized by nodules that lead to the development of connected tunnels and scars as it progresses from Hurley stages I to III. HS has been associated with several autoimmune diseases, including inflammatory bowel disease and spondyloarthritis. We previously reported dysregulation of humoral immune responses in HS, characterized by elevated serum total IgG, B-cell activation, and antibodies recognizing citrullinated proteins. In this study, we characterized IgG autoreactivity in HS sera and lesional skin compared with those in normal healthy controls using an array-based high-throughput autoantibody screening. The Cy3-labeled anti-human assay showed the presence of autoantibodies against nuclear antigens, cytokines, cytoplasmic proteins, extracellular matrix proteins, neutrophil proteins, and citrullinated antigens. Most of these autoantibodies were significantly elevated in stages II‒III in HS sera and stage III in HS skin lesions compared with those of healthy controls. Furthermore, immune complexes containing both native and citrullinated versions of antigens can activate M1 and M2 macrophages to release proinflammatory cytokines such as TNF-α, IL-8, IL-6, and IL-12. Taken together, the identification of specific IgG autoantibodies that recognize circulating and tissue antigens in HS suggests an autoimmune mechanism and uncovers putative therapeutic targets.