Bosentan effect on echocardiographic systolic pulmonary arterial pressure in systemic sclerosis-related pulmonary hypertension: a systematic review and metanalysis.

OBJECTIVES: Bosentan is a dual endothelin receptor antagonist approved for the treatment of SSc digital ulcers (DU) and pulmonary arterial hypertension (PAH). Systolic pulmonary arterial pressure (sPAP) is a relevant parameter for the follow-up and prognosis of SSc-PAH. The therapeutic magnitude of bosentan in SSc-PAH is not fully understood, thus we aim to establish the degree of sPAP reduction in bosentan treated SSc-PAH patients. METHODS: We performed a systematic literature review in three databases from January 2000 to June 2023, involving sPAP measurement at transthoracic echocardiography of SSc patients before and after starting bosentan. Following the study quality assessment and data extraction, we performed random-effects meta-analysis and Egger's test for publication bias. Stratified analysis was performed for mono-/combination therapy, follow up duration (≤1 year), indication for bosentan therapy (PAH or DU/mixed). RESULTS: In the 11 selected manuscripts, sPAP mean difference before and after bosentan therapy was - 5.63mmHg (CI95% -9.79 to -1.48, p=0.0078). In stratified analysis, sPAP mean was significantly different before and after bosentan therapy only for studies considering < 1 year of follow-up (p=0.0020), monotherapy (p=0.0140) and the strict indication for PAH (p=0.0002). CONCLUSIONS: Bosentan significantly decreases sPAP, a relevant prognostic marker, especially in overt SSc-PAH. However, bosentan did not decrease sPAP when started for DU/mixed indication nor for follow-up>1 year. The burden of publication bias was significant. Therefore, further studies are required to assess bosentan's haemodynamic effect in high-risk patients for SSc-PAH.

Increased Prevalence of Headaches and Migraine in Patients with Psoriatic Arthritis and Axial Spondyloarthritis: Insights from an Italian Cohort Study.

BACKGROUND: Psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA) are inflammatory diseases with shared genetic backgrounds and clinical comorbidities. Headache, a common global health issue, affects over 50% of adults and encompasses various types, including migraine, tension-type, and cluster headaches. Migraine, the most prevalent, recurrent, and disabling type, is often associated with other medical conditions such as depression, epilepsy, and psoriasis, but little is known about the relationship between autoimmune disease and the risk of migraine. METHODS: A cross-sectional study was conducted from July to November 2022, enrolling 286 participants, including 216 with PsA, 70 with axSpA, and 87 healthy controls. RESULTS: Headache prevalence was significantly higher in the PsA (39.81%) and axSpA (45.71%) patients compared to the healthy controls. The prevalence of migraine without aura was also significantly higher in both the PsA (18.52%) and axSpA (28.57%) groups compared to the healthy controls. CONCLUSIONS: These findings underscore the high burden of headache and migraine in PsA and axSpA participants, highlighting the need for improved management and treatment strategies for these patients.

Idiopathic and connective tissue disease-associated pulmonary arterial hypertension (PAH): Similarities, differences and the role of autoimmunity.

Pre-capillary pulmonary arterial hypertension (PAH) is hemodynamically characterized by a mean pulmonary arterial pressure (mPAP) ≥ 20 mmHg, pulmonary capillary wedge pressure (PAWP) ≤15 mmHg and pulmonary vascular resistance (PVR) > 2. PAH is classified in six clinical subgroups, including idiopathic PAH (IPAH) and PAH associated to connective tissue diseases (CTD-PAH), that will be the main object of this review. The aim is to compare these two PAH subgroups in terms of epidemiology, histological and pathogenic findings in an attempt to define disease-specific features, including autoimmunity, that may explain the heterogeneity of response to therapy between IPAH and CTD-PAH.

Association between Patient Acceptable Symptom State and disease activity in psoriatic arthritis is disrupted by confounders, including comorbid fibromyalgia.

OBJECTIVES: Due to the prevalence of fibromyalgia in psoriatic arthritis (PsA) patients, any evaluation about PsA-specific patient-reported outcomes (PROs) should take in account the possible bias related to this comorbidity. Patient acceptable symptom state (PASS) is a patient-reported measure evaluating the acceptable and/or satisfactory level of symptoms in rheumatic diseases, which has been proposed as a disease activity index, in patients with PsA. Thus, this study was designed to analyse if the association between PASS and PsA disease activity may be biased by the presence of comorbid fibromyalgia. METHODS: A multi-centre, cross-sectional, observational study enrolling consecutive PsA participants has been conducted from July 2021 to November 2021. The Disease Activity for Psoriatic Arthritis (DAPSA) was collected; the following formulation of PASS question: 'Think about all the ways your PsA has affected you during the last 48 hours. If you were to remain in the next few months as you were during the last 48 hours, would this be acceptable to you?', was submitted to our participants. RESULTS: Multivariable logistic regressions, adjusted for the presence of fibromyalgia, did not show any significant association between PASS and DAPSA low disease activity, DAPSA as nominal variable (remission, low disease activity, moderate disease activity, high disease activity) and DAPSA as continuous variable. CONCLUSIONS: Our data suggest that fibromyalgia influences the patient's perception of the disease and has a negative impact on PASS status independently of disease activity, thus limiting the utility of this Patient reported outcome in real world clinical practice.

Clinical correlates of a subset of anti-fibroblast antibodies in systemic sclerosis.

Anti-fibroblast antibodies (AFA) have been reported in systemic sclerosis (SSc) and are known to promote fibroblast activation. Aim of this study was to characterize the fine specificity of AFA and to analyze any correlations with clinical parameters associated to fibrosis. To this end, AFA were affinity-purified from a patient with diffuse cutaneous SSc (dcSSc) and interstitial lung disease (ILD). Panning of a phage display peptide library with purified AFA identified the motif . The peptide p121, bearing the AFA-specific motif, was used in ELISA to screen sera from 186 SSc patients and 81 healthy donors. Anti-p121 Ab serum levels were statistically higher in SSc than in healthy groups, and directly associated with dcSSc, reduced FVC (FVC < 70), and ILD. Given these clinical correlates, this study lays the groundwork for the identification of the antigen recognized by anti-p121 Ab, which might represent a novel therapeutic target for ILD.

The specialized pro-resolving lipid mediator Protectin D1 affects macrophages differentiation and activity in Adult-onset Still's disease and COVID-19, two hyperinflammatory diseases sharing similar transcriptomic profiles.

Introduction: COVID-19 and autoinflammatory diseases, such as Adult-onset Still's Disease (AOSD), are characterized by hyperinflammation, in which it is observed massive production and uncontrolled secretion of pro-inflammatory cytokines. The specialized pro-resolving lipid mediators (SPMs) family is one the most important processes counteracting hyperinflammation inducing tissue repair and homeostasis restoration. Among SPMs, Protectin D1 (PD1) is able to exert antiviral features, at least in animal models. The aim of this study was to compare the transcriptome of peripheral blood mononuclear cells (PBMCs) from patients with AOSD and COVID-19 and to evaluate the role of PD1 on those diseases, especially in modulating macrophages polarization. Methods: This study enrolled patients with AOSD, COVID-19, and healthy donors HDs, undergoing clinical assessment and blood sample collection. Next-generation deep sequencing was performed to identify differences in PBMCs transcripts profiles. Plasma levels of PD1 were assessed by commercial ELISA kits. Monocyte-derived macrophages were polarized into M1 and M2 phenotypes. We analyzed the effect of PD1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Results: In the transcriptomes from AOSD patients and COVID-19 patients, genes involved in inflammation, lipid catabolism, and monocytes activation were specifically dysregulated in AOSD and COVID-19 patients when compared to HDs. Patients affected by COVID-19, hospitalized in intensive care unit (ICU), showed higher levels of PD1 when compared to not-ICU hospitalized patients and HDs (ICU COVID-19 vs not-ICU COVID-19, p= 0.02; HDs vs ICU COVID-19, p= 0.0006). PD1 levels were increased in AOSD patients with SS ≥1 compared to patients with SS=0 (p=0.028) and HDs (p=0.048). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a significant increase of M2 polarization vs control (p<0.05). Furthermore, a significant release of IL-10 and MIP-1ß from M2 macrophages was observed when compared to controls (p<0.05). Discussion: PD1 is able to induce pro-resolutory programs in both AOSD and COVID-19 increasing M2 polarization and inducing their activity. In particular, PD1-treated M2 macrophages from AOSD and COVID-19 patients increased the production of IL-10 and enhanced homeostatic restoration through MIP-1ß production.

The negative impact of pain catastrophising on disease activity: analyses of data derived from patient-reported outcomes in psoriatic arthritis and axial spondyloarthritis.

OBJECTIVES: Psychosocial factors are recognised as important determinants of pain experience in patients with inflammatory arthritides. Among them, pain catastrophising, a maladaptive cognitive style, observed in patients with anxiety and depressive disorders, garnered specific attention. Here, we evaluated pain catastrophising (PC) and its related domains (Rumination, Magnification, and Helplessness), in psoriatic arthritis (PsA) and axial spondyloarhtiritis (axSpA) participants, to assess its impact on disease activity. Furthermore, we analysed possible correlations of PC-Scale (PCS) with those psychometric domains which have been already related to catastrophisation in patients with chronic pain. Lastly, we aimed to define the relationship between PCS and the different variables included in the composite indices of disease activity. METHODS: A multi-centre, cross-sectional, observational study has been conducted on 135 PsA (age 56 (47-64) years, males/females 40.74/59.26%; Disease Activity in Psoriasic Arthritis (DAPSA) 13.34 (5.21-22.22)) and 71 axSpA (age 49 (37-58) years, males/females 56.34/43.66%; Bath Ankylosing Spondylitis Arthritis Activity (BASDAI) 4.17 (2.1-6.3)) participants. Multivariable regressions and correlations were performed to evaluate the relationship between pain catastrophising and both disease activity and patient-reported outcomes. RESULTS: The adjusted linear regression model showed a positive association between PCS and DAPSA as well as between PCS and BASDAI; PCS negative impacts on the subjective domains of disease activity scores. CONCLUSIONS: This study suggests the role of PC, independently of inflammation, in disease perception and achievement of remission or low disease activity in chronic arthritides.

KLRG1 is reduced on NK cells in SLE patients, inversely correlates with disease activity and is modulated by hydroxychloroquine in vitro.

OBJECTIVES: Killer cell lectin-like receptor G 1 (KLRG1), a transmembrane receptor with inhibitory capacity expressed in human immune cells, emerged as a novel susceptibility gene for systemic lupus erythematosus (SLE). The aim of this study was to investigate the expression of KLRG1 in SLE patients compared to healthy controls (HC) on both NK and T cells and to evaluate its possible involvement in SLE pathogenesis. METHODS: Eighteen SLE patients and twelve healthy controls were enrolled. Peripheral blood mononuclear cells (PBMCs) from these patients were phenotypically characterized by immunofluorescence and flow cytometry. The effect of the hydroxychloroquine (HCQ) in vitro on KLRG1 expression and its signaling mediated functions in NK cells were analyzed. RESULTS: KLRG1 expression was significantly reduced on the analyzed immune cell populations in SLE patients compared to HC, especially on total NK cells. Moreover, expression of KLRG1 on total NK cells inversely correlated with the SLEDAI-2K. A direct association between KLRG1 expression on NK cells and patients' treatment with HCQ was observed. In vitro treatment with HCQ increased KLRG1 expression on NK cells. In HC, KLRG1+ NK cells showed reduced degranulation and IFNγ production, while in SLE patient, this inhibition occurred only for the IFNγ production. CONCLUSION: With this study we revealed a reduced expression and an impaired function of KLRG1 on NK cells in SLE patients. These results suggest a possible role of KLRG1 in the pathogenesis of SLE and as a novel biomarker of this disease.

The similar expression of both ferritin and scavenger receptors activation genes in patients with COVID19 and AOSD support their role in the pathogenesis of these diseases and identify a common mechanism at the basis of the "hyperferritinemic syndromes".

A role for COVID19 in "hyperferritinemic syndromes" has been proposed based on its clinical and serological characteristics and its similarities with AOSD. To better understand the molecular pathways responsible of these similarities, we evaluated in the PBMCs of 4 active AOSD patients, 2 COVID19 patients with ARDS, and 2 HCs the expression of genes associated with iron metabolisms, with monocyte/macrophages activation, and finally with NETs formation.

Fibroblast expression of CD248 may contribute to exacerbation of microvascular damage during systemic sclerosis.

OBJECTIVES: CD248 is a glycoprotein, highly expressed on pericytes and fibroblasts (FBs), that is implicated in the fibrotic process. During angiogenesis, CD248 can promote vessel regression, binding multimerin-2 (MMRN-2). Thus, we investigated the expression of MMRN-2 in systemic sclerosis (SSc)-skin and of CD248 in isolated SSc-FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by co-culturing these cells with healthy control endothelial cells (HC-ECs). The apoptotic effect of CD248 on HC-ECs was evaluated. Finally, the ability of CD248 to prevent activation of VEGF receptor 2 (VEGFR2) was assessed. METHODS: By IF, MMRN-2 was investigated in SSc-skin and CD248 in SSc FBs. The anti-angiogenic property of CD248+ SSc-FBs was evaluated by HC-ECs/SSc-FBs co-cultures. Lentiviral-induced CD248 short-hairpin RNA delivery was employed for loss-of-function studies in SSc-FBs. HC-ECs were cultured in the presence of CD248 to assess apoptosis by IF and VEGFR2 phosphorylation by western blot. RESULTS: MMRN-2 expression was increased in skin SSc-ECs, whereas CD248 expression was increased in SSc-FBs. Functionally, CD248+-SSc-FBs suppressed angiogenesis in the organotypic model, as assessed by the reduction in total tube length of HC-ECs. This anti-angiogenetic behaviour was reversed by CD248 silencing. Furthermore, the presence of CD248 promoted the apoptosis of HC-ECs. Finally, CD248 prevented activation of VEGFR2 by reducing its phosphorylation after VEGF stimulation. CONCLUSION: CD248 was anti-angiogenic in vitro due to a reduction in tube formation and to induction of apoptosis of ECs. Increased expression of CD248 in SSc could contribute to the microvascular rarefaction observed at the tissue level in SSc. Our results suggest a pathogenic role for CD248-MMRN-2 in SSc.

Gut Microbiota Structure and Metabolites, Before and After Treatment in Early Rheumatoid Arthritis Patients: A Pilot Study.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease. Modifications of gut microbiota seem to be associated with the disease, but the impact of gut microbiota on therapies' outcome remains unclear. A role of T cells in RA pathogenesis has been addressed, particularly on the Th17/Treg cells balance. Our study aimed to evaluate in early RA (ERA) patients compared to a control group, fecal gut microbiota composition, short-chain fatty acids concentrations, and the levels of circulating Th17/Treg and their own cytokines, before and after 3 months of standard treatment (Methotrexate (MTX) plus glucocorticoids). Fecal microbiota characterization was carried out on 19 ERA patients and 20 controls matched for sex and age. Significant decreased biodiversity levels, and a partition on the base of the microbiota composition, between the ERA patients at baseline compared to controls, were observed. The co-occurrent analysis of interactions revealed a characteristic clustered structure of the microbial network in controls that is lost in ERA patients where an altered connection between microbes and clinical parameters/metabolites has been reported. Microbial markers such as Acetanaerobacterium elongatum, Cristiansella massiliensis, and Gracilibacter thermotolerans resulted significantly enriched in control group while the species Blautia gnavus emerged to be more abundant in ERA patients. Our results showed an alteration in Th17/Treg balance with higher Th17 levels and lower Treg levels in ERA group respect to control at baseline, those data improved after therapy. Treatment administration and the achievement of a low disease activity/remission appear to exert a positive pressure on the structure of intestinal microbiota with the consequent restoration of biodiversity, of the structure of microbial network, and of the abundance of taxa that became closer to those presented by the subject without the disease. We also found an association between Blautia gnavus and ERA patients characterized by a significant reduction of propionic acid level. Furthermore significant differences highlighted at baseline among controls and ERA patients are no more evident after treatment. These data corroborate the role played by gut microbiota in the disease and suggest that therapy aimed to restore gut microbiota would improve treatment outcome.

2-Arachidonoylglycerol Reduces the Production of Interferon-Gamma in T Lymphocytes from Patients with Systemic Lupus Erythematosus.

Background: the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a pivotal role in immune cells regulation. The plasma levels of 2-AG are increased in patients with systemic lupus erythematosus (SLE) and correlate with disease activity. Moreover, in plasmacytoid dendritic cells from SLE patients, 2-AG is able to control the production of type 1 interferon (IFN) through CB2 activation. The aim of this study was to evaluate the potential role of 2-AG on T lymphocytes from SLE patients. Methods: peripheral blood mononuclear cells (PBMCs) from SLE participants and age- and sex-matched healthy donors (HD) were isolated by Ficoll-Hypaque density-gradient centrifugation. The PBMCs were treated with increasing concentrations of 2-AG, and AM251 and AM630 were used to antagonize CB1 and CB2, respectively. Flow cytometry was used to assess the expression of CD3, CD4, CD8, CD25, IFN-É£, IL-4, and IL-17A. Results: 2-AG (1 µM) decreased IFN-É£ expression (p = 0.0005) in the Th1 lymphocytes of SLE patients. 2-AG did not modulate the cytokine expression of any other T lymphocyte population from either SLE or HD. Treatment with both 2-AG and AM630 increased the IFN-É£ expression in Th1 lymphocytes of SLE patients (p = 0.03). Discussion: 2-AG is able to modulate type 2 IFN production from CD4+ T lymphocytes from SLE patients through CB2 activation.

Autophagy Hijacking in PBMC From COVID-19 Patients Results in Lymphopenia.

Autophagy is a homeostatic process responsible for the self-digestion of intracellular components and antimicrobial defense by inducing the degradation of pathogens into autophagolysosomes. Recent findings suggest an involvement of this process in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the role of autophagy in the immunological mechanisms of coronavirus disease 2019 (COVID-19) pathogenesis remains largely unexplored. This study reveals the presence of autophagy defects in peripheral immune cells from COVID-19 patients. The impairment of the autophagy process resulted in a higher percentage of lymphocytes undergoing apoptosis in COVID-19 patients. Moreover, the inverse correlation between autophagy markers levels and peripheral lymphocyte counts in COVID-19 patients confirms how a defect in autophagy might contribute to lymphopenia, causing a reduction in the activation of viral defense. These results provided intriguing data that could help in understanding the cellular underlying mechanisms in COVID-19 infection, especially in severe forms.

Tofacitinib May Inhibit Myofibroblast Differentiation from Rheumatoid-Fibroblast-like Synoviocytes Induced by TGF-ß and IL-6.

During rheumatoid arthritis (RA), the pathogenic role of resident cells within the synovial membrane is suggested, especially for a population frequently referred to as fibroblast-like synoviocytes (FLSs). In this study, we assess the markers of myofibroblast differentiation of RA-FLSs by ex vivo observations and in vitro evaluations following the stimulation with both TGF-ß and IL-6. Furthermore, we investigated the possible inhibiting role of tofacitinib, a JAK inhibitor, in this context. Myofibroblast differentiation markers were evaluated on RA synovial tissues by immune-fluorescence or immune-histochemistry. RA-FLSs, stimulated with transforming growth factor (TGF-ß) and interleukin-6 (IL-6) with/without tofacitinib, were assessed for myofibroblast differentiation markers expression by qRT-PCR and Western blot. The same markers were evaluated following JAK-1 silencing by siRNA assay. The presence of myofibroblast differentiation markers in RA synovial tissue was significantly higher than healthy controls. Ex vivo, α-SMA was increased, whereas E-Cadherin decreased. In vitro, TGF-ß and IL-6 stimulation of RA-FLSs promoted a significant increased mRNA expression of collagen I and α-SMA, whereas E-Cadherin mRNA expression was decreased. In the same conditions, the stimulation with tofacitinib significantly reduced the mRNA expression of collagen I and α-SMA, even if the Western blot did not confirm this finding. JAK-1 gene silencing did not fully prevent the effects of stimulation with TGF-ß and IL-6 on these features. TGF-ß and IL-6 stimulation may play a role in mediating myofibroblast differentiation from RA-FLSs, promoting collagen I and α-SMA while decreasing E-Cadherin. Following the same stimulation, tofacitinib reduced the increases of both collagen I and α-SMA on RA-FLSs, although further studies are needed to fully evaluate this issue and confirm our results.

Cytokine profile, ferritin and multi-visceral involvement characterize macrophage activation syndrome during adult-onset Still's disease.

OBJECTIVES: To multidimensionally characterize macrophage activation syndrome (MAS) complicating adult-onset Still's disease (AOSD) considering cytokine profile, inflammatory markers and multi-visceral involvement of the disease. To perform a high-dimensional phenotypic analysis of circulating immune cells in AOSD patients with and without MAS. To assess interferon (IFN)-related pathways in AOSD synovial tissues by a bulky RNA sequencing. METHODS: Clinical and biologic data were collected and compared in AOSD patients with and without MAS. Sera biomolecules were analysed by Luminex multiplexing technology. Mass cytometry (CyTOF) was used to characterize circulating immune cells. A bulky RNA sequencing was performed in AOSD synovial tissues. RESULTS: Forty consecutive AOSD patients were assessed, 14 complicated with MAS. Paralleling with increases of systemic score and ferritin, MAS patients showed higher levels of IL-1α, IL-1ß, IL-1Ra, IL-2Ra, IL-6, IL-10, IL-17A, IFN-γ, G-CSF, MCP-1, MIP-1α and SCF. Combining the discriminatory ability of these data in identifying MAS, the best model was composed by systemic score, ferritin, IFN-γ and IL-10. By CyTOF analysis, MAS patients showed an increase of circulating 'classical monocytes' and a reduction of total NK cells. Our assessment showed 3477 IFN-related genes (IRGs) were differently expressed in AOSD synovial tissues. CONCLUSIONS: A multidimensional characterization of AOSD patients suggested that IFN-γ, IL-10, ferritin and systemic score discriminated the occurrence of cytokine storm syndrome associated with MAS. The inflammatory milieu of AOSD and MAS may be related to a signature of circulating immune cells. Finally, our results about IRGs reinforced the role of IFN-γ in these patients.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients' PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors' PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14- (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients' serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.

Up-regulation of autophagy by etanercept treatment results in TNF-induced apoptosis reduction in EA.hy926 endothelial cell line.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease associated with a high prevalence of atherosclerosis. Endothelial dysfunction has emerged as a potentially valuable prognostic tool in predicting the development of atherosclerosis. Tumour necrosis factor (TNF) is the main cytokine involved in RA pathogenesis, exerting a pro-atherogenic role. TNF-inhibitors are effective treatments in RA, also improving endothelial function. Regarding this, no experimental data are known about the involvement of etanercept. We investigated the contribution of TNF to endothelial dysfunction and the effect of in vitro treatment with etanercept, with a special focus on autophagy and apoptosis pathways. METHODS: Autophagy and apoptosis were evaluated by Western blot and flow cytometry in EA.hy926 endothelial cells treated with TNF alone or in combination with etanercept for 24h. RESULTS: Blocking autophagy, TNF was able to induce endothelial cell apoptosis. Co-treatment with etanercept reverted this effect, up-regulating the autophagy pathway. CONCLUSIONS: Our results confirm the protective role of etanercept, by restoring autophagy on TNF-induced endothelial damage.

Increased eryptosis in patients with primary antiphospholipid syndrome (APS): a new actor in the pathogenesis of APS.

OBJECTIVES: Antiphospholipid syndrome (APS) is an autoimmune disease characterised by a hypercoagulable state and the presence of antiphospholipid antibodies (aPL). During the mechanism of red blood cells (RBCs) death, called eryptosis, RBCs can adhere to vascular wall participating in the development of a pro-thrombotic state. It is known that enhanced eryptosis contributes to several pathological conditions but the role of this process in APS has not been investigated yet. We analysed spontaneous eryptosis in a cohort of APS patients and aPL carriers (asymptomatic subjects with positive aPL tests). The effect on eryptosis of antibodies (Abs) purified from serum of APS patients and aPL carriers was also investigated. METHODS: In this study, 30 patients with primary APS (PAPS) and 17 aPL carriers were recruited. Twenty healthy donors (HD) and 13 patients affected by autoimmune haemolytic anaemia (AHIA) were also recruited. RBCs were incubated with PAPS and aPL carriers Abs, purified by ammonium sulfate precipitation. Levels of eryptosis were analysed by flow cytometry. RESULTS: In vitro Abs from APS patients induced eryptosis in RBCs isolated from HD after 4 h of culture. On the contrary, Abs from aPL carriers had no effect on the percentage of phosphatidylserine-exposing RBCs. Ex vivo, APS patients showed higher levels of spontaneous eryptosis compared to HD and aPL carriers. CONCLUSIONS: In this study, we demonstrated a potential new aspect of APS pathogenesis based on the ability of Abs isolated from APS patients, not identified in aPL carriers, to stimulate eryptosis suggesting a possible contribution of this process in the clinical manifestations of APS.

Autophagy occurs in lymphocytes infiltrating Sjögren's syndrome minor salivary glands and correlates with histological severity of salivary gland lesions.

BACKGROUNDS: The organization of minor salivary glands (MSG) infiltrates, in patients with Sjögren's syndrome (SS), associates with disease severity and progression. Aberrant regulation of lymphocyte autophagy is involved in autoimmunity, and in previous work, we provided the first evidence of upregulated autophagy in CD4+ T cells infiltrating SS MSG. The aim of this study was to further explore autophagy in SS infiltrating and circulating lymphocytes and to investigate its role in disease histopathological progression. METHODS: After collection of 20 SS MSG, the presence of lymphocyte aggregates (foci) and the formation of germinal center (GC)-like structures were observed by H&E and confirmed by immunohistochemistry. The expression of autophagy-related genes, Atg5 and MAP1LC3A, was detected by RT-PCR on microdissected salivary gland tissue and control tonsils. In MSG and tonsils, autophagic lymphocytes were identified by the detection of the autophagosome protein LC3B visualized as LC3 puncta staining by immunofluorescence. Peripheral blood autophagy was assessed by flow cytometry in SS and healthy controls (HC). RESULTS: Real-time PCR demonstrated higher expression in the autophagy genes Atg5 and MAP1LC3A in MSG GCs as compared to both small foci (p = 0.0075, p = 0.0002) and GCs from tonsils (p = 0.0001, p = 0.0037). In MSG, LC3 puncta staining was detectable on both CD3+ and CD20+ lymphocytes; in tonsils, LC3 puncta was almost undetectable on all lymphocytes. Compared to HC (n = 20), flow cytometry did not reveal any increase of autophagy in SS circulating lymphocytes (n = 30). CONCLUSIONS: In SS MSG, lymphocytes' autophagy is a feature of infiltrating T and B cells and is associated with histological severity. Interestingly, in MSG aberrant regulation of autophagy is detectable in GC-like structures possibly indicating its involvement in the development and persistence of the autoimmune process within the lesions.

Identification of Subclinical Lung Involvement in ACPA-Positive Subjects through Functional Assessment and Serum Biomarkers.

Lung involvement is related to the natural history of anti-citrullinated proteins antibodies (ACPA)-positive rheumatoid arthritis (RA), both during the pathogenesis of the disease and as a site of disease-related injury. Increasing evidence suggests that there is a subclinical, early lung involvement during the course of the disease, even before the onset of articular manifestations, which can potentially progress to a symptomatic interstitial lung disease. To date, reliable, non-invasive markers of subclinical lung involvement are still lacking in clinical practice. The aim of this study is to evaluate the diagnostic potential of functional assessment and serum biomarkers in the identification of subclinical lung involvement in ACPA-positive subjects. Fifty ACPA-positive subjects with or without confirmed diagnosis of RA (2010 ARC-EULAR criteria) were consecutively enrolled. Each subject underwent clinical evaluation, pulmonary function testing (PFT) with assessment of diffusion lung capacity for carbon monoxide (DLCO), cardiopulmonary exercise testing (CPET), surfactant protein D (SPD) serum levels dosage and high-resolution computed tomography (HRCT) of the chest. The cohort was composed of 21 ACPA-positive subjects without arthritis (ND), 10 early (disease duration < 6 months, treatment-naïve) RA (ERA) and 17 long-standing (disease duration < 36 months, on treatment) RA (LSRA). LSRA patients had a significantly higher frequency of overall HRCT abnormalities compared to the other groups (p = 0.001). SPD serum levels were significantly higher in ACPA-positive subjects compared with healthy controls (158.5 ± 132.3 ng/mL vs 61.27 ± 34.11 ng/mL; p < 0.0001) and showed an increasing trend from ND subjects to LSRD patients (p = 0.004). Patients with HRCT abnormalities showed significantly lower values of DLCO (74.19 ± 13.2% pred. vs 131.7 ± 93% pred.; p = 0.009), evidence of ventilatory inefficiency at CPET and significantly higher SPD serum levels compared with subjects with no HRCT abnormalities (213.5 ± 157.2 ng/mL vs 117.7 ± 157.3 ng/mL; p = 0.018). Abnormal CPET responses and higher SPD levels were also associated with specific radiological findings. Impaired DLCO and increased SPD serum levels were independently associated with the presence of HRCT abnormalities. Subclinical lung abnormalities occur early in RA-associated autoimmunity. The presence of subclinical HRCT abnormalities is associated with several functional abnormalities and increased SPD serum levels of SPD. Functional evaluation through PFT and CPET, together with SPD assessment, may have a diagnostic potential in ACPA-positive subjects, contributing to the identification of those patients to be referred to HRCT scan.