Autoantibodies from Patients with Scleroderma Renal Crisis Promote PAR-1 Receptor Activation and IL-6 Production in Endothelial Cells

Background. Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). Methods. Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. Results. Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. Conclusions. Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC

α1A-Adrenergic Receptor-Directed Autoimmunity Induces Left Ventricular Damage and Diastolic Dysfunction in Rats

BACKGROUND: Agonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. METHODS/PRINCIPAL FINDINGS: We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A)-adrenergic receptor and maintained them for one year. Alpha(1A)-adrenergic antibodies (alpha(1A)-AR-AB) were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A)-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max) demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min). Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. CONCLUSIONS/SIGNIFICANCE: We show that alpha(1A)-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A)-AR-AB could contribute to cardiovascular endorgan damage

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1RAbs- induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis

Increase of angiotensin II type 1 receptor auto-antibodies in Huntington’s disease

Background In the recent years, a role of the immune system in Huntington’s disease (HD) is increasingly recognized. Here we investigate the presence of T cell activating auto-antibodies against angiotensin II type 1 receptors (AT1R) in all stages of the disease as compared to healthy controls and patients suffering from multiple sclerosis (MS) as a prototype neurologic autoimmune disease. Results As compared to controls, MS patients show higher titers of anti-AT1R antibodies, especially in individuals with active disease. In HD, anti-AT1R antibodies are more frequent than in healthy controls or even MS and occur in 37.9% of patients with relevant titers ≥ 20 U/ml. In a correlation analysis with clinical parameters, the presence of AT1R antibodies in the sera of HD individuals inversely correlated with the age of onset and positively with the disease burden score as well as with smoking and infection. Conclusions These data suggest a dysfunction of the adaptive immune system in HD which may be triggered by different stimuli including autoimmune responses, infection and possibly also smoking

Beta-1-Adrenergic Receptor Antibodies in Acute Coronary Syndrome: Is Less Sometimes More?

Background: Anti-beta-1-adrenergic receptor antibodies (anti-β1AR Ab) are associated with ischemic cardiomyopathies (ICM). Evidence continues to emerge supporting an autoimmune component to various cardiac diseases. This study compares anti-β1AR Ab concentrations in patients with different entities of acute coronary syndromes (ACS) to asymptomatic non-ACS patients with positron-emission computed tomography (PET/CT)-proven atherosclerosis, and healthy controls.Methods: Serum anti-β1AR Ab IgG concentrations were measured in 212 ACS patients, 100 atherosclerosis patients, and 62 controls using ELISA. All ACS patients underwent coronary angiography. All 374 patients participating completed a structured questionnaire regarding traditional cardiovascular risk factors. ACS patients were followed up for 6 months.Results: Patients with ACS exhibited lower anti-β1AR Ab levels compared to patients with atherosclerosis or healthy controls (both p < 0.001). No differences in the ab levels were evident between healthy controls and patients with atherosclerosis. In the ACS groups, lower concentrations were found in patients with ST-elevation myocardial infarction (STEMI) (0.67 μg/ml) compared to patients with angina pectoris (AP) and non-ST elevation myocardial infarction (NSTEMI) (both 0.76 μg/ml, p = 0.008). Anti-β1AR Ab levels ≤ 0.772 μg/ml were predictive for death and reinfarction (AUC 0.77, p = 0.006). No significant correlations between anti-β1AR Ab levels and atherosclerotic burden or traditional cardiovascular risk factors were identified.Conclusions: Lower anti-β1AR Ab concentrations appear to characterize ACS phenotypes and could serve as diagnostic and prognostic markers independent from traditional risk factors for atheroscle. The prognostic predictive value of anti-β1AR Ab in ACS remains to be confirmed in larger studies

Transfer of PBMC From SSc Patients Induces Autoantibodies and Systemic Inflammation in Rag2-/-/IL2rg-/- Mice

ObjectiveThe contribution of sustained autologous autoantibody production by B cells to the pathogenesis of systemic sclerosis (SSc) and granulomatosis with polyangiitis (GPA) is not fully understood. To investigate this, a humanized mouse model was generated by transferring patient-derived peripheral blood mononuclear cells (PBMC) into immunocompromised mice.MethodsPBMC derived from patients with SSc and GPA as well as healthy controls (HD) were isolated, characterized by flow cytometry, and infused into Rag2-/-/IL2rg-/- mice. In addition, PBMC from SSc patients treated with rituximab were transferred into mice. Twelve weeks later, human autoantibodies were determined in blood of the recipient mice and affected tissues were analyzed for pathological changes by histology and immunohistochemistry.ResultsMice engrafted with PBMC derived from SSc patients developed autoantibodies such as antinuclear antibodies (ANA) mimicking the pattern of the respective donors. Moreover, cellular infiltrates dominated by B cells were observed in lung, kidney and muscles of the recipient mice. By contrast, PBMC derived from HD or GPA patients survived in recipient mice after transfer, but neither human autoantibodies nor inflammatory infiltrates in tissues were detected. Furthermore, these pathological changes were absent in mice transferred with PBMC from rituximab-treated SSc patients.ConclusionThis humanized mouse model is indicative for cross-reactivity of human lymphocytes to murine autoantigens and argues for a pivotal role of B cells as well as of sustained autoimmunity in the pathogenesis of SSc. It provides a powerful tool to study interstitial lung disease and so far, under-recognized disease manifestations such as myositis and interstitial nephritis

Low avidity circulating SARS-CoV-2 reactive CD8+ T cells with proinflammatory TEMRA phenotype are associated with post-acute sequelae of COVID-19

The role of adaptive SARS-CoV-2 specific immunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although a growing population of convalescent COVID-19 patients with manifestation of PASC is observed. We analyzed the SARS-CoV-2-specific immune response, via pseudovirus neutralizing assay and multiparametric flow cytometry in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. Although frequencies of SARS-CoV-2-reactive CD4+ T cells were similar between the studied cohorts, a stronger SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRA phenotype but low functional TCR avidity was detected in PASC patients compared to controls. Of interest, high avidity SARS-CoV-2-reactive CD4+ and CD8+ T cells were comparable between the groups demonstrating sufficient cellular antiviral response in PASC. In line with the cellular immunity, neutralizing capacity in PASC patients was not inferior compared to controls. In conclusion, our data suggest that PASC may be driven by an inflammatory response triggered by an expanded population of low avidity SARS-CoV-2 reactive pro-inflammatory CD8+ T cells. These pro-inflammatory T cells with TEMRA phenotype are known to be activated by a low or even without TCR stimulation and lead to a tissue damage. Further studies including animal models are required for a better understanding of underlying immunopathogensis. Summary: A CD8+ driven persistent inflammatory response triggered by SARS-CoV-2 may be responsible for the observed sequelae in PASC patients

Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling

Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses

Dysregulation of circulating autoantibodies against VEGF-A, VEGFR-1 and PlGF in preeclampsia ? A role in placental and vascular health?

Objectives: Preeclampsia is a state of antiangiogenesis, with high levels of maternal circulating sVEGFR-1 (soluble vascular endothelial growth factor receptor 1, also named sFlt1) and low levels of PlGF (placenta growth factor). Various autoantibodies have been detected in preeclamptic patients. We hypothesize that circulating autoantibodies against VEGF-A (AA-VEGF-A), VEGFR-1 (AA-VEGFR-1) and PlGF (AA-PlGF) are present in preeclamptic women, with different levels from pregnant women with normotensive pregnancies. Secondly, we wanted to analyze if autoantibody levels are associated to sFlt1 or PLGF levels. Study design: Retrospective cross sectional study of 88 women with singleton pregnancies who delivered at Oslo University Hospital of whom 46 had preeclampsia and 42 had uncomplicated normotensive pregnancies. Novel immunoassays for IgG-autoantibodies against VEGFA, VEGFR-1 and PlGF were developed and serum samples were assayed. Main outcome measures and results: AA-VEGF-A, AA-VEGF-R1 and AA-PlGF were significantly lower in preeclamptic pregnancies (n = 42) compared to normotensive pregnancies (n = 46) (p < 0.05). On unadjusted analysis, only AA-VEGFA and AA-VEGFR-1 were predictors of PE, but none were independent predictors after adjusting for BMI (body mass index) and parity. In the subgroup of normotensive and PE women with overlapping sVEGFR-1/PlGF-ratios, AA-VEGF was a significant predictor of PE with AUC: 0.735. Conclusion: IgG autoantibodies against VEGF-A VEGFR-1 and PlGF can be found in pregnant women. They are dysregulated in preeclampsia. The roles of these autoantibodies are unknown, but this study suggests they play a protective role in pregnancy. The levels of AA against VEGF-A, VEGFR-1 and PlGF might be important factors contributing to anti-angiogenesis regulation