Sylvain Dubucquoi

Antinuclear antibodies (ANAs) are a hallmark of connective tissue diseases (CTDs) and serve as robust diagnostic biomarkers. Because their cognate antigens are intracellular, ANAs have long been considered non-pathogenic in CTDs. Here, using systemic sclerosis (SSc) associated anti-topoisomerase I antibodies (ATAs) as a model, we provide data challenging this view. We show that ANAs enter living cells, accumulate in nuclei, and engage their intracellular antigen. Nuclear ATAs inhibit topoisomerase I enzymatic activity, induces DNA damage, fibrosis, and, through the STING pathway, activates type I interferon production. We further identify neonatal Fc receptor (FcRn)-dependent intracellular trafficking as a key determinant of ANAs nuclear access and demonstrate that pharmacological FcRn blockade impairs ATAs functionality. These findings reveal a previously unrecognized intracellular effector function of ANAs and establish a mechanistic framework by which ANAs may directly contribute to tissue injury in CTDs.

Dans le cadre du diagnostic biologique des maladies auto-immunes, les autoanticorpssont généralement recherchés par des méthodes qui utilisent des antigènes(ou peptides) présélectionnés. De telles techniques ont des limites qui pourraient êtrecontournées par de nouvelles approches. Dans le premier temps de ce travail, nousrapportons l'intérêt d’évaluer les réactivités d'auto-anticorps vis-à-vis de ciblesmodifiées par des processus post traductionnels, comme la citrullination dans lecadre diagnostique de la polyarthrite rhumatoïde. Cette approche ne peut toutefoispas être transposée à toutes les pathologies auto-immunes, et notamment lasclérose en plaques. Dans un deuxième temps, notre démarche s’est élargie àl’analyse globale de la réactivité des immunoglobulines G sériques dirigée contredifférents extraits d’antigènes tissulaires, notamment issus du cerveau. Malgré lagrande hétérogénéité des réponses interindividuelles, il a été possible d’observer desprofils de réactivités distinguant les sujets sains de patients souffrant de différentespathologies à composantes auto-immunes. Cette signature sérologique permetégalement de distinguer les profils auto-immuns associés à différentes maladies etmême de distinguer leurs formes cliniques. Dans le modèle expérimental développéau laboratoire, nous avons observé que des modifications de ces profils apparaissentde façon précoce et peuvent être liés à une évolution péjorative ou favorable de lamaladie auto-immune. Ces résultats ont été confirmés dans la sclérose en plaques,où l’étude menée chez des patients ne présentant qu’un syndrome clinique isolé, amontré que les profils de réactivité sérique des IgG sont déjà marqués de l’empreintede la maladie qui se révèlera cliniquement en moyenne trois ans plus tard. De façonintéressante, d'autres travaux ont montré que le répertoire des IgM pouvaitégalement être perturbé au cours d’un processus auto-immun dont la pathogénierepose sur un dysfonctionnement des lymphocytes T (syndrome APECED). Cesrésultats suggèrent que des mécanismes T dépendants mais aussi T indépendantspèsent sur la constitution et l’entretien du répertoire auto-immun pathologique.Pour identifier les cibles des réactivités discriminantes révélées par cette premièreapproche, nous avons développé une technique de caractérisation moléculairefaisant appel à la technique immunoprotéomique. Alors que les pathologieshumaines et les modèles animaux étudiés sont principalement des pathologiesspécifiques d’organe, les antigènes tissulaires qui ont été identifiés comme cibles desréactivités spécifiquement associées à ces pathologies sont des antigènesubiquitaires, et non des antigènes spécifiques d’organe. Ces résultats posent laquestion de l’implication réelle de ces cibles dans la physiopathologie des maladiesauto-immunes. Ils illustrent également l’impérative nécessité de connaître les limitesdes résultats apportés par les méthodes d’immunoprotéomique.La caractérisation de la signature sérologique d’un processus pathologique, autravers de l’analyse des perturbations globales des réactivités sériques qui lui sontassociées, offre des perspectives intéressantes tant en termes de prise en charge dupatient qu’en termes de compréhension physiopathologique des maladies autoimmunes.Elle pourrait aboutir à d’utiles débouchés thérapeutiques. Ces attentesjustifient pleinement l’investissement qui a été mis en place par notre laboratoiredans le cadre de sa validation méthodologique.

Purpose: High throughput technologies have identified molecular patterns in colorectal cancer (CRC) cells, aiding in modeling responses to anti‐cancer treatments. The different responses observed depend on the type of cancer, the tumour grade and the functional programme of the cancer cells. Recent studies suggest that the unfolded protein response (UPR), autophagy and apoptosis could be involved in treatment resistance mechanisms by interacting with the tumour microenvironment (TME).Experimental Design: We analysed by LC‐MS/MS the proteome of two representative colon adenocarcinoma epithelial cell lines from different tumour grades (CCL‐233 and CCL‐221) at the basal state or after the UPR induction.Results: Cell lines expressed a different proteome on about 10% of their total proteins identified, especially on UPR, autophagy and apoptosis pathways proteins at basal state. After UPR induction, the proteome of the cells was modified with a greater adaptive response to cellular stress in CCL‐221 cells where the UPR was strongly activated at the basal state.Conclusions and Clinical Relevance: CRC cell lines at different tumour grades expressed different functional programmes at the proteomic level and were characterised by different responses to the UPR induction. This study suggests that baseline cancer cell stress status could have an impact on the efficiency of cancer therapies.

Systemic sclerosis (SSc) is the most severe systemic autoimmune disease with currently no cure. Intravenous immunoglobulins (IVIg) are an attractive candidate in this disease to counteract inflammation and fibrosis but data are scarce and conflicting. This study, assessed the effects of IVIg in a murine HOCl-induced model of SSc. We showed that IVIg prevented skin inflammation and fibrosis, by mitigating the immune cell infiltration (p = 0.04), proinflammatory cytokines gene overexpression (IL1β, p = 0.04; TNFα, p = 0.04; IL6, p = 0.05), skin and dermal thickening (p = 0.003 at d21 and p = 0.0003 at d42), the expression markers of fibrosis, such as αSMA (p = 0.031 for mRNA and p = 0.05 for protein), collagen (p = 0.05 for mRNA and p = 0.04 for protein, p = 0.05 for the hydroxyproline content) and fibronectin (p = 0.033 for mRNA). Moreover, IVIg prevented HOCl-induced alterations in splenic cell homeostasis. When administered in curative mode, despite their ability to reduce skin and dermal thickness (p < 0.0001 and p = 0.0002), IVIg showed partial or more mixed effects on skin inflammation and established fibrosis. These data favor further clinical trials in SSc patients on the potential efficiency of early and/or repeated IVIg administration.

Severe COVID-19 infections are at high risk of causing thromboembolic events (TEEs). However, the usual exams may be unavailable or unreliable in predicting the risk of TEEs at admission or during hospitalization. We performed a retrospective analysis of two centers (n = 124 patients) including severe COVID-19 patients to determine the specific risk factors of TEEs in SARS-CoV-2 infection at admission and during stays at the intensive care unit (ICU). We used stepwise regression to create two composite scores in order to predict TEEs in the first 48 h (H0–H48) and during the first 15 days (D1–D15) in ICU. We then evaluated the performance of our scores in our cohort. During the period H0–H48, patients with a TEE diagnosis had higher D-Dimers and ferritin values at day 1 (D1) and day 3 (D3) and a greater drop in fibrinogen between D1 and D3 compared with patients without TEEs. Over the period D1-D15, patients with a diagnosis of a TEE showed a more marked drop in fibrinogen and had higher D-Dimers and lactate dehydrogenase (LDH) values at D1 and D3. Based on ROC analysis, the COVID-related acute lung and deep vein thrombosis (CALT) 1 score, calculated at D1, had a diagnostic performance for TEEs at H0–H48, estimated using an area under the curve (AUC) of 0.85 (CI95%: 0.76–0.93, p < 10⁻³). The CALT 2 score, calculated at D3, predicted the occurrence of TEEs over the period D1-D15 with an estimated AUC of 0.85 (CI95%: 0.77–0.93, p < 10⁻³). These two scores were used as the basis for the development of the CALT protocol, a tool to assist in the decision to use anticoagulation during severe SARS-CoV-2 infections. The CALT scores showed good performances in predicting the risk of TEEs in severe COVID-19 patients at admission and during ICU stays. They could, therefore, be used as a decision support protocol on whether or not to initiate therapeutic anticoagulation.

Systemic sclerosis (SSc) is an autoimmune disease associated with endothelial activation and fibrosis. Non-O blood group patients carry an increased risk of thrombosis, fibrosis and autoimmune diseases. The aim of our work was to evaluate the distribution of ABO groups in SSc patients and their association with the disease’s characteristics. ABO groups were determined in 504 SSc patients (with 131 completed by a genotypic analysis). The distribution of ABO groups and their diplotypes in SSc patients was comparable to that of the general population, except for haplotypes O1 and B (65.6% vs. 61.6% and 8.8% vs. 5.8% in SSc patients vs. the general population, respectively, p = 0.01). The frequency of interstitial lung disease, pulmonary hypertension, calcinosis, digital ulcers, digestive diseases and venous thrombosis, and the Medsger score, were higher in non-O than in O-SSc patients, although they did not display statistical significance. Patients in the non-O group had higher levels of inflammation and endothelial activation biomarkers. In conclusion, the ABO blood group distribution of SSc patients did not differ significantly from that of the general population, but non-O blood groups were associated with inflammation and endothelial activation, and with a non-significant higher frequency of pulmonary and vascular complications in SSc.

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF‐β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF‐β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF‐β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF‐β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter‐data comparisons in future in vitro studies, especially when using “omics” approaches.

Background: The detection of additional autoantibodies is of great concern in systemic sclerosis (SSc) when those included in the ACR/EULAR classification are negative. In this context, the interest of antifibrillarin (anti-U3RNP) autoantibodies (AFAs) in the routine evaluation of SSc remains unclear. We aimed to assess the relevance of AFAs and their clinical association in SSc patients. Methods: In a multicenter observational retrospective study, we collected immunological and clinical data associated with AFA positivity in SSc (n = 42) and non-SSc patients (n = 13). Patients with SSc negative for AFAs (n = 83) were considered as a control group. AFAs were detected by indirect immunofluorescence (IIF) using HEp-2 cells, EliA or immunoblot techniques. Results: We confirmed a typical nuclear IIF pattern and showed that AFAs are mostly exclusive towards SSc conventional autoantibodies. Although also observed in non-SSc patients, high levels of AFAs with the ELiA technique allowed the diagnosis of SSc. Compared to AFA-negative SSc patients, AFA-positive SSc patients more frequently exhibited visceral involvements. They more frequently suffered from the diffuse cutaneous form and had a higher global severity of the disease. Conclusions: We demonstrate the usefulness of quantifying AFAs in the immunological exploration of SSc, especially when patients are seronegative for SSc conventional autoantibodies and display a typical IIF pattern. AFAs might constitute an interesting marker of SSc severity.