Search

BackgroundGiant cell arteritis (GCA) is a large-vessel vasculitis mainly involving the aorta and cranial arteries. It is the most frequent vasculitis in adults over 50 years. When they are stimulated by interferon-gamma (IFN-γ), vascular smooth muscle cells (VSMC) contribute to GCA pathogenesis by producing chemokines triggering the recruitment of pro-inflammatory T cells and monocytes (1).ObjectivesCurrent knowledge about the interaction between resident cells of the vascular wall (VSMC, myofibroblasts [MF]) and immune cells is limited. The aim of our research was to better characterize the interactions between VSMC, MF and T cells in GCA.MethodsFresh fragments of temporal artery biopsies (TAB) performed at Dijon university hospital (France) were prospectively sent to our research unit. Fresh sections of positive and negative TAB were fixed and embedded in optimal cutting temperature OCT and stored at -80°C. Then, cryostat sections were fixed, permeabilized, blocked and incubated with primary antibodies (anti-alpha smooth muscle actin [α-SMA], anti-myosin heavy chain 11 [MHC11], anti-Desmin, anti CD90, anti-CD45, anti-HLA-DR, anti-phospho STAT1 [pSTAT1] and anti-pSTAT3) and secondary antibodies for confocal microscopy analyses. Fresh sections of healthy TAB were embedded in MATRIGEL and covered by DMEM to obtain vascular cells in culture. Cells were treated with trypsina-EDTA between each passage. Vascular cells were used after 4-7 doubling passages. Cells were analyzed by immunofluorescence, flow cytometry and RT-PCR and their proliferation was evaluated by impedancemetry (iCELLigence system). Peripheral blood mononuclear cells (PBMC) and vascular cells thus obtained were co-cultured for 7 days in different conditions. Vascular cells were cultured in the presence or absence of IFN-γ and tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) and soluble receptor of IL-6 for 72 hours. When cells reached confluence, they were cultured alone or with allogenic PBMC activated with anti-CD3/CD28 microbeads. After 7 days of culture, cells were separated with a treatment with EDTA and studied by flow cytometry.ResultsConfocal microscopy analyses of GCA arteries showed that neointima was mainly composed of myofibroblasts (MF) (α-SMA+Desmin+MHC11lowCD90+) in contact with CD45+ cells and that MF expressed HLA-DR, the phosphorylated form of STAT1 (pSTAT1) and in a lesser extent pSTAT3, strongly suggesting the activation of the IFN-γ signaling pathway rather than the IL-6 pathway. The phenotype of cultured vascular cells isolated from fresh TAB was consistent with MF. When MF were exposed to IFN-γ and TNF-α in vitro, their proliferation capacity decreased and their levels of expression of HLA-DR and CD86 increased (median fluorescence intensity [MFI] from 0 to 57 [p=0.03] and from 34 to 103 [p=0.03], respectively). In addition, co-cultures of MF and activated PBMC revealed that MF maintained the polarization of T cells into Th1 and Tc1 cells (p≤0.001) and to a lesser extent into Th17 and Tc17 cells (p=0.03). This effect was even more significant when MF were previously exposed to IFN-γ and TNF-α but not when they were exposed to IL-6.ConclusionOur results show that myofibroblasts are present in the neointima of GCA patients and that these MF activate signaling pathways indicative of IFN-γ exposure. Moreover, these MF, especially when exposed to IFN-γ, maintain the polarization of T cells into Th1 and Tc1 cells, which contributes to amplify the production of IFN-γ and thus initiate a pro-inflammatory amplification loop that likely participates in vascular inflammation and remodelling.References[1]Corbera-Bellalta M, Planas-Rigol E, Lozano E, Terrades-Garcia N, Alba MA, Prieto-Gonzalez S, et al. Blocking interferon gamma reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis. Ann Rheum Dis 2016;75:1177-86.Disclosure of InterestsNone declared

Introduction La maladie de Rendu-Osler (MRO) est une maladie genetique vasculaire rare caracterisee par une neo-angiogenese deregulee aboutissant a des epistaxis anemiantes parfois associees a malformations arterio-veineuses (MAV) pulmonaires, hepatiques ou cerebrales. La MRO est egalement associee a une lymphopenie T predominant sur les CD4+ dont les causes et les consequences sont mal connues. L’objectif de ce travail est de decrire les polarisations lymphocytaires Th1, Th2, Th17, Th1-17 et Treg au cours de la MRO. Patients et methodes Les patients atteints de MRO confirmee genetiquement ont ete prospectivement inclus lors de leur suivi habituel. Le score de severite des epistaxis (ESS), la presence de MAV, le traitement martial en cours, le taux d’hemoglobine et de ferritine ont ete recueillis. La duree mensuelle moyenne des epistaxis (DMM) a ete prospectivement etablie grâce a des grilles quotidiennement remplies pendant 3 mois apres l’inclusion. Les sujets presentant une pathologie impliquant l’immunite (infection, maladie auto-immune, cancer) ou traites par des molecules modulant l’angiogenese ou l’immunite (y compris l’acide tranexamique et le bevacizumab) ont ete exclus. Les populations lymphocytaires B, T cytotoxiques, T auxilliaires (Th) et T regulateurs (Treg) ont ete quantifies par cytometrie en flux a partir d’echantillons sanguins grâce aux marqueurs CD19, CD3, CD8, CD4, CD25 et Foxp3 (respectivement). Les polarisations Th1, Th2, Th17, Th1-17 ont ete evaluees apres marquage intra-cytoplasmique de l’interferon (IFN) -γ, l’interleukine (IL)-4 et l’IL-17 apres 4 h de stimulation par PMA-Ionomycine. Resultats Quarante patients et 20 sujets controles ont ete inclus avec respectivement un âge median de 52 ans (min : 19 ans max : 74 ans) et 50 ans (min : 24 ans, max : 80 ans). Le sex-ratio etait de 20/40 chez les MRO et 10/20 chez les temoins. L’endogline etait mutee chez 17 patients, ACVRL1 chez 22 patients et SMAD4 chez 1 patient. Dix patients recevaient un traitement martial, 20 avaient une atteinte pulmonaire et 10 une atteinte hepatique de la MRO. Les taux medians d’hemoglobine et de ferritine etaient respectivement de 14,5 g/dl (min : 9,3, max : 17,1) et 31 μg/l (5–487) chez les patients MRO. Le score ESS et la DMM etaient respectivement de 1,72 (0–5,47) et 18 min (0–585). Le groupe MRO avait un taux de lymphocytes T auxiliaires significativement plus bas que celui des temoins (medianes des CD3 + CD4+ : 724/mm3 vs 1157/mm3, p = 0,0031) alors que les taux de lymphocytes T cytotoxiques (CD3 + CD8 + ) et de lymphocytes B (CD19 + ) etaient comparables. Comparativement aux temoins les sujets MRO presentaient une augmentation significative des polarisations Th2 (mediane : 1,34 % vs 0,91 %, p = 0,0014), Th17 (mediane : 0,32 % vs 0,20 %, p = 0,0069), Th1-17 (mediane : 0,0305 % vs 0,0140 %, p = 0,0003) et Treg (mediane : 5,13 % vs 3,19 %, p = 0,0002). Le sexe, l’âge et le gene mute n’avaient pas d’influence sur ces modifications de polarisation. Le pourcentage de polarisation Th17 etait positivement correle avec la duree moyenne mensuelle des epistaxis (r = 0,448, p = 0,0042) et le score ESS (r = 0,336, p = 0,0365). Conclusion Notre etude decrit pour la premiere fois une augmentation significative des polarisations Th2, Th17, Th1-17 et Treg au cours de la MRO. L’environnement pro-angiogenique de la MRO pourrait etre a l’origine d’une modification de la reponse immunitaire via un remodelage specifique des populations lymphocytaires T auxiliaires par un ou plusieurs mecanismes qui restent a determiner.