Molecular basis for increased susceptibility of Indigenous North Americans to seropositive rheumatoid arthritis.

OBJECTIVE: The pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common motif, the 'shared susceptibility epitope (SE)'. Here, the electropositive P4 pocket of HLA-DRB1 accommodates self-peptide residues containing citrulline but not arginine. HLA-DRB1 His/Phe13ß stratifies with ACPA-positive RA, while His13ßSer polymorphisms stratify with ACPA-negative RA and RA protection. Indigenous North American (INA) populations have high risk of early-onset ACPA-positive RA, whereby HLA-DRB1*04:04 and HLA-DRB1*14:02 are implicated as risk factors for RA in INA. However, HLA-DRB1*14:02 has a His13ßSer polymorphism. Therefore, we aimed to verify this association and determine its molecular mechanism. METHODS: HLA genotype was compared in 344 INA patients with RA and 352 controls. Structures of HLA-DRB1*1402-class II loaded with vimentin-64Arg59-71, vimentin-64Cit59-71 and fibrinogen ß-74Cit69-81 were solved using X-ray crystallography. Vimentin-64Cit59-71-specific and vimentin59-71-specific CD4+ T cells were characterised by flow cytometry using peptide-histocompatibility leukocyte antigen (pHLA) tetramers. After sorting of antigen-specific T cells, TCRα and ß-chains were analysed using multiplex, nested PCR and sequencing. RESULTS: ACPA+ RA in INA was independently associated with HLA-DRB1*14:02. Consequent to the His13ßSer polymorphism and altered P4 pocket of HLA-DRB1*14:02, both citrulline and arginine were accommodated in opposite orientations. Oligoclonal autoreactive CD4+ effector T cells reactive with both citrulline and arginine forms of vimentin59-71 were observed in patients with HLA-DRB1*14:02+ RA and at-risk ACPA- first-degree relatives. HLA-DRB1*14:02-vimentin59-71-specific and HLA-DRB1*14:02-vimentin-64Cit59-71-specific CD4+ memory T cells were phenotypically distinct populations. CONCLUSION: HLA-DRB1*14:02 broadens the capacity for citrullinated and native self-peptide presentation and T cell expansion, increasing risk of ACPA+ RA.

Buprenorphine Alters Inflammatory and Oxidative Stress Molecular Markers in Arthritis.

Buprenorphine is recommended for use as an analgesic in animal models including in murine models of collagen-induced arthritis (CIA). However, the effect of buprenorphine on the expression of disease-associated biomarkers is not well defined. We examined the effect of buprenorphine administration on disease progression and the expression of inflammatory and oxidative stress markers, in a murine model of CIA. Buprenorphine administration altered the expression of cytokines, IFN-γ, IL-6, and MMP-3, and oxidative markers, for example, iNOS, superoxide dismutase (SOD1), and catalase (CAT), in the CIA mice. As buprenorphine is an analgesic, we further monitored the association of expression of these biomarkers with pain scores in a human cohort of early rheumatoid arthritis (RA). Serum MMP-3 levels and blood mRNA expression of antioxidants sod1 and cat correlated with pain scores in the RA cohort. We have demonstrated that administration of buprenorphine alters the expression of inflammatory and oxidative stress-related molecular markers in a murine model of CIA. This caveat needs to be considered in animal experiments using buprenorphine as an analgesic, as it can be a confounding factor in murine studies used for prediction of response to therapy. Furthermore, the antioxidant enzymes that showed an association with pain scores in the human cohort may be explored as biomarkers for pain in future studies.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

NMDA receptors mediate contractile responses in human airway smooth muscle cells.

Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-d-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.

A role for transient receptor potential ankyrin 1 cation channel (TRPA1) in airway hyper-responsiveness?

Airway smooth muscle (ASM) contraction controls the airway caliber. Airway narrowing is exaggerated in obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). The mechanism by which ASM tone is dysregulated in disease is not clearly understood. Recent research on ion channels, particularly transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is uncovering new understanding of altered airway function. TRPA1, a member of the TRP channel superfamily, is a chemo-sensitive cation channel that can be activated by a variety of external and internal stimuli, leading to the influx of Ca(2+). Functional TRPA1 channels have been identified in neuronal and non-neuronal tissues of the lung, including ASM. In the airways, these channels can regulate the release of mediators that are markers of airway inflammation in asthma and COPD. For, example, TRPA1 controls cigarette-smoke-induced inflammatory mediator release and Ca(2+) mobilization in vitro and in vivo, a response tied to disease pathology in COPD. Recent work has revealed that pharmacological or genetic inhibition of TRPA1 inhibits the allergen-induced airway inflammation in vitro and airway hyper-responsiveness (AHR) in vivo. Collectively, it appears that TRPA1 channels may be determinants of ASM contractility and local inflammation control, positioning them as part of novel mechanisms that control (patho)physiological function of airways and ASM.

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.

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.

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.

A bioavailable form of curcumin, in combination with vitamin-D- and omega-3-enriched diet, modifies disease onset and outcomes in a murine model of collagen-induced arthritis.

OBJECTIVE: Curcumin (CUR), vitamin D3 (D3), and omega-3-fatty acids (O3FA) individually modulate inflammation and pain in arthritis. Although these supplements are widely used, their combinatorial effects have not been defined. In this study, we examined the effects of a D3 and O3FA (VO)-enriched diet in conjunction with a highly bioavailable form of CUR (Cureit/Acumin™) in a collagen-induced arthritis (CIA) murine model. METHODS: Male DBA/1J mice were acclimatized to VO-enriched diet and challenged with bovine collagen II (CII). Bioavailable CUR was administered daily by oral gavage from the onset of CII challenge. Disease severity was determined by monitoring joint thickness and standardized clinical score. Cellular infiltration and cartilage degradation in the joints were assessed by histology, serum cytokines profiled by Meso Scale Discovery multiplex assay, and joint matrix metalloproteinases examined by western blots. RESULTS: CUR by itself significantly decreased disease severity by ~ 60%. Administration of CUR in CIA mice taking a VO-enriched diet decreased disease severity by > 80% and maximally delayed disease onset and progression. Some of the disease-modifying effects was mediated by CUR alone, e.g., suppression of serum anti-collagen antibodies and decrease of cellular infiltration and MMP abundance in the joints of CIA mice. Although CUR alone suppressed inflammatory cytokines in serum of CIA mice, the combination of CUR and VO diet significantly enhanced the suppression (> 2-fold compared to CUR) of TNF, IFN-γ, and MCP-1, all known to be associated with RA pathogenesis. CONCLUSION: This study provides proof-of-concept that the combination of bioavailable CUR, vitamin D3, and O3FA substantially delays the development and severity of CIA. These findings provide a rationale for systematically evaluating these widely available supplements in individuals at risk for developing future RA.

Can Studying Genetically Predisposed Individuals Inform Prevention Strategies for RA?

Rheumatoid arthritis (RA) is a prevalent autoimmune disorder in which complex genetic predisposition interacts with multiple environmental factors to precipitate chronic and progressive immune-mediated joint inflammation. Currently, in most affected individuals, ongoing suppression of the inflammation is required to prevent irreversible damage and functional loss. The delineation of a protracted preclinical period in which autoimmunity is initially established and then evolves to become pathogenic provides unprecedented opportunities for interventions that have the potential to prevent the onset of this lifelong disease. Clinical trials aimed at assessing the impact of specific prevention strategies require the identification of individuals who are at high risk of future RA development. Currently, these risk factors include a strong family history of RA, and the detection of circulating RA-associated autoantibodies, particularly anti-citrullinated protein antibodies (ACPA). Yet, even in such individuals, there remains considerable uncertainty about the likelihood and the timeframe for future disease development. Thus, individuals who are approached to participate in such clinical trials are left weighing the risks and benefits of the prevention measures, while having large gaps in our current understanding. To address this challenge, we have undertaken longitudinal studies of the family members of Indigenous North American RA patients, this population being known to have a high prevalence of RA, early age of onset, and familial clustering of cases. Our studies have indicated that the concepts of "risk" and "prevention" need to be communicated in a culturally relevant manner, and proposed prevention interventions need to have an appropriate balance of effectiveness, safety, convenience, and cultural acceptability. We have focused our proposed prevention studies on immunomodulatory/anti-inflammatory nutritional supplements that appear to strike such a complex balance.

Expansion of Alternative Autoantibodies Does Not Follow the Evolution of Anti-Citrullinated Protein Antibodies in Preclinical Rheumatoid Arthritis: An Analysis in At-Risk First Degree Relatives.

OBJECTIVE: Co-occurrence of autoantibodies specific for ≥1 autoimmune disease is widely prevalent in rheumatoid arthritis (RA) patients. To understand the prevalence of polyautoimmunity in preclinical RA, we performed a comprehensive autoantibody assessment in a First Nations cohort of at-risk first-degree relatives (FDR) of RA patients, a subset of whom subsequently developed RA (progressors). METHODS: Venous blood was collected from all study participants (n = 50 RA patients and 64 FDR) at scheduled visits, and serum was stored at -20°C. High-sensitivity C-reactive protein level, anti-citrullinated protein antibody (ACPA) status, and autoantibody status were determined using commercially available enzyme-linked immunosorbent assay kits. Rheumatoid factor (RF) was detected by nephelometry. Antinuclear autoantibodies (ANA) were identified using Hep-2 indirect immunofluorescence assay (IFA) and classified according to international consensus nomenclature as various anti-cell (AC) patterns. RESULTS: Of our study cohort, 78.9% had positive ANA reactivity (≥1:80), which was either a homogenous, fine-speckled (AC-1 and AC-4) or mixed IFA pattern. Importantly, the AC-4 and mixed ANA patterns were also observed in progressors at the time of disease onset. While all of the RA patients showed a high prevalence of arthritis-associated autoantibodies, they also had a high prevalence of extractable nuclear antigen-positive autoantibodies to other autoantigens. In FDR, we did not observe any increase in serum autoreactivity to nonarthritis autoantigens, either cross-sectionally or in samples collected longitudinally from progressors prior to RA onset. CONCLUSION: While alternative autoimmunity and ANA positivity are widely prevalent in First Nations populations, including asymptomatic, seronegative FDR, expansion of alternative autoimmunity does not occur in parallel with ACPA expansion in FDR and is restricted to patients with established RA.

Association of a Serum Protein Signature With Rheumatoid Arthritis Development.

OBJECTIVE: The pathophysiologic events that precede the onset of rheumatoid arthritis (RA) remain incompletely understood. This study was undertaken to identify changes in the serum proteome that precede the onset of RA, with the aim of providing new insights into the pathogenic mechanisms that lead to its development. METHODS: In a cohort of first-degree relatives of Indigenous North American RA patients, the SomaScan proteomics platform was used to determine the levels of 1,307 proteins in multiple longitudinal serum samples from 17 individuals who were followed up prospectively to the time of disease onset. Proteomic signatures from this group of individuals (designated the progressor group) were compared to those in a group of individuals who were considered at risk of developing RA, stratified as either positive (n = 63) or negative (n = 47) for anti-citrullinated protein antibodies (ACPAs) (designated the at-risk group). Machine learning was used to identify a protein signature that could accurately classify those individuals at highest risk of future RA development. RESULTS: A preclinical proteomic signature that differentiated RA progressors from at-risk individuals, irrespective of ACPA status, was identified (area under the curve 0.913, accuracy 91.2%). Importantly, the predictive preclinical proteomic signature was present not only in serum samples obtained close to the onset of RA, but also in serum samples obtained a median of 30.9 months prior to onset. Network analysis implicated the activation of Toll-like receptor 2 and production of tumor necrosis factor and interleukin-1 as key events that precede RA progression. CONCLUSION: Alterations in the serum proteome in the preclinical phase of RA can emerge years prior to the onset of disease. Our findings suggest that the serum proteome provides a rich source of proteins serving both to classify at-risk individuals and to identify molecular pathways involved in the development of clinically detectable RA.

Proteomic Approaches to Defining Remission and the Risk of Relapse in Rheumatoid Arthritis.

Objectives: Patients with Rheumatoid Arthritis (RA) are increasingly achieving stable disease remission, yet the mechanisms that govern ongoing clinical disease and subsequent risk of future flare are not well understood. We sought to identify serum proteomic alterations that dictate clinically important features of stable RA, and couple broad-based proteomics with machine learning to predict future flare. Methods: We studied baseline serum samples from a cohort of stable RA patients (RETRO, n = 130) in clinical remission (DAS28<2.6) and quantified 1307 serum proteins using the SOMAscan platform. Unsupervised hierarchical clustering and supervised classification were applied to identify proteomic-driven clusters and model biomarkers that were associated with future disease flare after 12 months of follow-up and RA medication withdrawal. Network analysis was used to define pathways that were enriched in proteomic datasets. Results: We defined 4 proteomic clusters, with one cluster (Cluster 4) displaying a lower mean DAS28 score (p = 0.03), with DAS28 associating with humoral immune responses and complement activation. Clustering did not clearly predict future risk of flare, however an XGboost machine learning algorithm classified patients who relapsed with an AUC (area under the receiver operating characteristic curve) of 0.80 using only baseline serum proteomics. Conclusions: The serum proteome provides a rich dataset to understand stable RA and its clinical heterogeneity. Combining proteomics and machine learning may enable prediction of future RA disease flare in patients with RA who aim to withdrawal therapy.

Whole Blood Targeted Bisulfite Sequencing and Differential Methylation in the C6ORF10 Gene of Patients with Rheumatoid Arthritis.

OBJECTIVE: Polymorphisms in human major histocompatibility complex (MHC) are the strongest genetic associations with rheumatoid arthritis (RA). Epigenome-wide methylation studies suggest DNA methylation changes within MHC may contribute to disease susceptibility. We profiled MHC-specific methylated CpG (5'-C-phosphate-G-3') in autoantibody-positive patients with RA and matched unaffected anticitrullinated protein antibodies-negative first-degree relatives (ACPA-/FDR) from an indigenous North American (INA) population that is known to have prevalent RA. METHODS: DNA was isolated from whole blood and targeted bisulfite sequencing was used to profile methylated CpG in patients with RA and ACPA-/FDR. Differentially methylated CpG loci (DML) were mapped and gene annotated. Ingenuity pathway analysis (IPA) was used for curating biomolecular networks of mapped genes. Transcript abundance was determined by quantitative (q)PCR. RESULTS: We identified 74 uniquely methylated CpG sites within the MHC region that were differentially methylated in patients with RA (p < 0.05), compared to ACPA-/FDR. Of these, 32 DML were located on 22 genes. IPA showed these genes are involved in regulating the nuclear factor-κB complex and processes involved in antigen presentation, and immune cell crosstalk in autoimmunity. Pearson correlation analysis demonstrated a negative association between differentially methylated CpG in the C6ORF10 gene and risk factors associated with RA. Analysis by qPCR confirmed differential abundance of C6ORF10, TNXB, and HCG18 mRNA in patients with RA compared to ACPA-/FDR. CONCLUSION: Our results confirm the presence of differential methylation at specific gene loci within the MHC region of INA patients with RA. These epigenetic signatures may precede disease onset, or alternatively, may be a result of developing RA.

Circulating levels of free 25(OH)D increase at the onset of rheumatoid arthritis.

OBJECTIVE: Epidemiological studies suggest vitamin D deficiency as a potential risk factor for rheumatoid arthritis (RA) development, a chronic autoimmune disorder highly prevalent in indigenous North American (INA) population. We therefore profiled the circulating levels of 25-hydroxyvitaminD [25(OH)D], an active metabolite of vitamin D, in a cohort of at-risk first-degree relatives (FDR) of INA RA patients, a subset of whom subsequently developed RA (progressors). METHODS: 2007 onward, serum samples from INA RA patients and FDR were collected at the time of a structured baseline visit and stored at -20°C. Anti-citrullinated protein antibodies (ACPA), 25(OH)D, hs-CRP, vitamin-D binding protein (VDBP) and parathyroid hormone (PTH) levels were determined using ELISA and rheumatoid factor (RF) seropositivity was determined by nephelometry. RESULTS: We demonstrate that 25 (OH) D concentrations were lower in winter than summer (P = 0.0538), and that serum 25(OH)D levels were higher in samples collected and stored after 2013 (P<0.0001). Analysis of samples obtained after 2013 demonstrated that 37.6% of study participants were 25(OH)D insufficient (<75nmol/L). Also, seropositive RA patients and FDR had lower 25(OH)D levels compared to ACPA-/FDR (P<0.05, P<0.01 respectively). Linear regression analysis showed 25(OH)D insufficiency was inversely associated with presence of RA autoantibodies. Longitudinal samples from 14 progressors demonstrated a consistent increase in 25(OH)D levels at the time they exhibited clinically detectable joint inflammation, without any significant change in VDBP or PTH levels. Spearman rank correlation analysis showed significant association between 25(OH)D and PTH levels, both in RA patients and progressors at RA onset time. CONCLUSION: We demonstrate that 25(OH)D levels in serum increased at RA onset in progressors. The potential role that vitamin D metabolites and their downstream effects play in RA transition requires further investigation.

A Prospective Study of the Development of Inflammatory Arthritis in the Family Members of Indigenous North American People With Rheumatoid Arthritis.

OBJECTIVE: To determine the incidence of inflammatory arthritis and autoantibody prevalence in Indigenous North American people. METHODS: Unaffected relatives of Indigenous North Americans with rheumatoid arthritis (RA) from central Canada and Alaska were systematically monitored from 2005 to 2017. Rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs) were tested at every visit, and a subset was tested for ACPA fine specificity using a custom multiplex assay. Multistate models based on all available study visits were developed to determine the likelihood of transitioning between autoantibody states, or to inflammatory arthritis. RESULTS: Eighteen of 374 relatives (4.8%) developed inflammatory arthritis during follow-up (after a mean ± SD of 4.7 ± 2.4 years), yielding a transition rate of 9.2 cases/1,000 person-years. Thirty percent of those who developed inflammatory arthritis were seronegative at baseline, but all were seropositive at inflammatory arthritis onset. Although 30% of ACPA/RF double-seropositive individuals developed inflammatory arthritis (after 3.2 ± 2.2 years), the majority of these individuals did not develop inflammatory arthritis. Multistate modeling indicated a 71% and 68% likelihood of ACPA and RF seropositive states, respectively, reverting to a seronegative state after 5 years, and a 39% likelihood of an ACPA/RF double-seropositive state becoming seronegative. Fine specificity testing demonstrated an expansion of the ACPA repertoire prior to the development of inflammatory arthritis. CONCLUSION: Despite a high incidence of inflammatory arthritis in this cohort of at-risk relatives of Indigenous North Americans with RA, a large proportion of autoantibody-positive individuals do not develop inflammatory arthritis and revert back to an autoantibody-negative state.

Whole blood microRNA expression pattern differentiates patients with rheumatoid arthritis, their seropositive first-degree relatives, and healthy unrelated control subjects.

BACKGROUND: Epigenetic mechanisms can integrate gene-environment interactions that mediate disease transition from preclinical to clinically overt rheumatoid arthritis (RA). To better understand their role, we evaluated microRNA (miRNA, miR) expression profile in indigenous North American patients with RA who were positive for anticitrullinated protein antibodies; their autoantibody-positive, asymptomatic first-degree relatives (FDRs); and disease-free healthy control subjects (HCs). METHODS: Total RNA was isolated from whole blood samples obtained from HC (n = 12), patients with RA (n = 18), and FDRs (n = 12). Expression of 35 selected relevant miRNAs, as well as associated downstream messenger RNA (mRNA) targets of miR-103a-3p, was determined by qRT-PCR. RESULTS: Whole blood expression profiling identified significantly differential miRNA expression in patients with RA (13 miRNAs) and FDRs (10 miRNAs) compared with HCs. Among these, expression of miR-103a-3p, miR-155, miR-146a-5p, and miR-26b-3p was significantly upregulated, whereas miR-346 was significantly downregulated, in both study groups. Expression of miR-103a-3p was consistently elevated in FDRs at two time points 1 year apart. We also confirmed increased miR-103a-3p expression in peripheral blood mononuclear cells from patients with RA compared with HCs. Predicted target analyses of differentially expressed miRNAs in patients with RA and FDRs showed overlapping biological networks. Consistent with these curated networks, mRNA expression of DICER1, AGO1, CREB1, DAPK1, and TP53 was downregulated significantly with miR-103a-3p expression in FDRs. CONCLUSIONS: We highlight systematically altered circulating miRNA expression in at-risk FDRs prior to RA onset, a profile they shared with patients with RA. Prominently consistent miR-103a-3p expression indicates its utility as a prognostic biomarker for preclinical RA while highlighting biological pathways important for transition to clinically detectable disease.

Functions of Cationic Host Defense Peptides in Immunity.

Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system.

What's new in asthma pathophysiology and immunopathology?

Research on asthma pathophysiology over the past decade has expanded the complex repertoire involved in the pathophysiology of asthma to include inflammatory, immune and structural cells, as well as a wide range of mediators. Studies have identified a role for connective and other mesenchymal tissues involved in airway remodeling. Recent findings have implicated the innate immune response in asthma and have revealed interesting patterns of interaction between the innate and adaptive immune response and the associated complex chronic inflammatory reaction. New immune cell populations have also been added to this repertoire, including Tregs, natural killer T cells and Th17 cells. The role of the eosinophil, a prominent pathological feature in most asthma phenotypes, has also been expanding to include roles such as tissue modifiers and immune regulators via a number of fascinating and hitherto unexplored mechanistic pathways. In addition, new and significant roles have been proposed for airway smooth muscle cells, fibroblasts, epithelial and endothelial cells. Tissue remodeling is now considered an integral element of asthma pathophysiology. Finally, an intricate network of mediators, released from both immune and inflammatory cells, including thymus stromal lymphopoietin and matrix metalloproteinases, have added to the complex milieu of asthma immunity and inflammation. These findings have implications for therapy and the search for novel strategies towards better disease management. Sadly, and perhaps due to the complex nature of asthma, advances in therapeutic discoveries and developments have been limited. Thus, understanding the precise roles played by the numerous dramatis personae in this odyssey, both individually and collectively within the context of asthma pathophysiology, continues to pose new challenges. It is clear that the next stage in this saga is to embark on studies that transcend reductionist approaches to involve system analysis of the complex and multiple variables involved in asthma, including the need to narrow down the phenotypes of this condition based on careful analysis of the organs (lung and airways), cells, mediators and other factors involved in bronchial asthma.