Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state ...that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.
Significance
Pathogenic autoantibodies are a feature of systemic lupus erythematosus (SLE), of which 85% of patients are women. Multiple X chromosomes increase the risk for SLE, suggesting an ...important role for X-linked gene expression for the female sex bias. X-chromosome inactivation (XCI) regulates X-linked gene expression on the inactive X. This study examines XCI maintenance across multiple human B cell subsets from healthy individuals and SLE patients. Importantly, we found that both pediatric and adult SLE patient B cells have significant reductions with epigenetic modifications on the inactive X and aberrant X-linked gene expression. Our findings will be instrumental for future investigations of disease mechanisms underlying the female bias of SLE and abnormal autoantibody production in B cells.
Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.
Abstract
Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease characterized by the presence of circulating autoreactive anti-nuclear antibodies (ANAs). ANAs form immune complexes ...(ICs) upon binding to nuclear antigens and these ANA-ICs promote a feedforward loop by enhancing immune responses in cells that recognize ANA-ICs via Fc receptors (FcRs). Plasmacytoid dendritic cells (pDCs) are FcR expressing cells that promote SLE pathology. ANA-ICs deliver nucleic acids to endosomal TLR7 and TLR9. These TLRs induce type I IFN secretion and immune activation upon nucleic acid recognition. Type I IFNs, including IFNα, and interferon stimulated genes are correlated with SLE disease activity. IgG isotype facilitated ANA-IC internalization via FcgRIIA has been the most studied antibody-mediated route for IFNα production in pDCs. The importance of IgA isotype ANAs remains under investigated despite these autoantibodies being present in half of SLE patients. We show here the novel result that human pDCs express the IgA-specific FcαR (CD89) by flow cytometry, that pDCs from SLE patients express increased FcαR compared to pDCs from healthy controls, and that increased pDC FcαR expression in SLE correlates with hallmark IFNα gene set. Additionally, we found that IgA autoantibodies were a critical component of pDC ANA-IC activation when these ANA-IC were generated with serum from IgA autoantibody positive SLE donors complexed with smRNP nuclear antigen. Therefore, our study shows that IgA-containing immune complexes and FαRI are important contributors to pDC type I IFN production in SLE.
Supported by LRA Lupus Mechanisms and Targets Award (JAH), R21AI154841 (JAH), and ITHS TL1 training grant (HRW).
Abstract
Studies of the frequencies of myelin-specific T cells in peripheral blood mononuclear cells (PBMCs) from MS patients compared to healthy controls (HCs) have varied in their results, but no ...previous work has considered the potential influence of lesion localization on these frequencies. While the majority of relapse-remitting MS (RRMS) patients have lesions localized predominantly in the brain, a subset of RRMS patients (~5%) has neuroinflammation localized predominantly in the spinal cord. Importantly, spinal cord localized lesions are associated with a worse prognosis for patients due to their effect on motor function. Animal models of MS suggest that distinct T cell effector functions correlate with brain versus spinal cord lesions. Therefore, we analyzed PBMCs from MS patients with lesions predominantly in the brain or predominantly in the spinal cord and HCs using ELISPOT to assess the frequency of IFN-γ+ and IL-17+ cells responding to two myelin antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Significant differences in T cell frequencies were determined in some, but not all, comparisons, with MBP-specific IFN-γ+ frequencies showing the greatest separation between our three groups. Strikingly, combining the IFN-γ+ and IL-17+ responses to both MBP and MOG using logistic regression defined distinct T cell signatures that distinguished MS patients with predominantly brain versus predominantly spinal cord lesions. Collectively, this suggests that different myelin-specific T cell responses may influence neuroinflammatory patterns in RRMS patients. Such patterns of peripheral T cell responses may be useful in generating more tailored therapies for patients with MS.
OBJECTIVE:We investigated T cell responses to myelin proteins in the blood of healthy controls and 2 groups of patients with relapsing-remitting multiple sclerosis (RRMS) who exhibited lesions either ...predominantly in the brain or predominantly in the spinal cord in order to assess whether distinct neuroinflammatory patterns were associated with different myelin protein–specific T cell effector function profiles and whether these profiles differed from healthy controls.
METHODS:Peripheral blood mononuclear cells were obtained from patients with brain-predominant RRMS, patients with spinal cord–predominant RRMS, and age-matched healthy controls and analyzed by enzyme-linked immunosorbent spot assays to quantify interferon gamma–secreting (Th1) and interleukin 17–secreting (Th17) cells responding directly ex vivo to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG).
RESULTS:Although MBP and MOG elicited different responses, patients with multiple sclerosis (MS) who had spinal cord–predominant lesions exhibited significantly higher Th17:Th1 ratios in response to both MBP and MOG compared to patients with brain-predominant MS. Incorporating the cytokine responses to both antigens into logistic regression models showed that these cytokine responses were able to provide good discrimination between patients with distinct neuroinflammatory patterns.
CONCLUSIONS:Our findings suggest that the localization of lesions within the brain vs the spinal cord in patients with MS is associated with different effector T cell responses to myelin proteins. Further investigation of the relationship between T cell effector function, antigen specificities, and lesion sites may reveal features of pathogenic pathways that are distinct to patients with different neuroinflammatory patterns.
Residence-time-dependent changes in fibrinogen after its adsorption to Biomer were examined by measuring platelet adhesion and antibody binding to the adsorbed protein, and the amount of adsorbed ...fibrinogen which could be eluted by sodium dodecyl sulfate (SDS). Baboon fibrinogen was first adsorbed (from either pure solution or dilute plasma) to Biomer, which was then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the adherent protein layer was either probed for fibrinogen capable of mediating platelet adhesion using 111In radiolabeled, washed platelet suspensions under both static and shearing conditions, or for fibrinogen capable of binding antibody using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed protein layer was soaked in a 3% SDS solution, and the amount of 125I radiolabeled fibrinogen retained was measured. Decreases in platelet and antibody binding, and in the SDS elutability of the adsorbed fibrinogen after it was stored in buffer were detected, although different rates of decrease were observed for each method. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen was prevented. While each of the three assays measures a different property of adsorbed fibrinogen, this study suggests that the adherent protein undergoes time dependent conformational changes which render it less reactive toward platelets and antibodies, and more resistant to elution by SDS.
Pre-existing (chronic) atrial fibrillation (AF) has been identified as a risk factor for cardiovascular complications and mortality in patients with COVID-19; however, evidence in Latin America ...(LATAM) is scarce. This prospective and multicenter study from the CARDIO COVID 19–20 database includes hospitalized adults with COVID-19 from 14 countries in LATAM. A parsimonious logistic regression model was used to identify the main factors associated with mortality in a simulated case-control setting comparing patients with a history of AF to those without. In total, 3260 patients were included, of which 115 had AF. The AF group was older, had a higher prevalence of comorbidities, and had greater use of cardiovascular medications. In the model, AF, chronic kidney disease, and a respiratory rate > 25 at admission were associated with higher in-hospital mortality. The use of corticosteroids did not reach statistical significance; however, an effect was seen through the confidence interval. Thus, pre-existing AF increases mortality risk irrespective of other concomitant factors. Chronic kidney disease and a high respiratory rate at admission are also key factors for in-hospital mortality. These findings highlight the importance of comorbidities and regional characteristics in COVID-19 outcomes, in this instance, enhancing the evidence for patients from LATAM.
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