Interaction of B cells with targeted cells in SSc: fibroblasts, keratinocytes and endothelium

Several studies have shown that B cells could infiltrate the skin, especially the dermis in SSc patients. Consequently, B cells and dermal FB can interact through direct contact and/or paracrine secretion. Our hypothesis is that infiltrating B cells interact with targeted cells in SSc. Our goal is to characterize these interactions using cultures of B cells with an innovative 3-dimensional reconstituted skin mode, which we developed recently (Le Maitre et al. Front Immunol 2024, under revision). In this project, we will develop a 3D coculture model of pathological skin using keratinocytes and skin FB from SSc patients, thanks to bioprinting/organ-on-chip (collaboration with CPER Tecsanté). Then, 3D reconstituted SSc skin will be co- cultured with B cells to assess the activation status of fibroblasts and B cells, as well as functional programs related to the coculture process (ScRNAseq), in order to identify common activation pathways. A validation step of the identified pathways will be performed using biobanked skin collections (FHU Precise). We will also target the signaling pathways identified in B cells and fibroblasts and validate the best targeting strategy using animal models (HOCl, bleomycin, TOPO-1 immunization).

We will complete the study of the interaction between the immune system and fibroblasts in SSc by (i) characterizing the heterogeneity of FB in SSc. Along the line of the FRM team project (portfolio item 10), our objective is to study the different subpopulations of FB deriving from the single-cell RNAseq analysis that we have carried out on different tissues (skin for patients with SSc, already available within the frame of FHU PRECISE database & biobanking) deriving from different chronic inflammatory diseases including SSc. The goal is to isolate one or some populations common to the different pathologies and potentially to define one or more molecular signature(s) associated with this(these) population(s) with the ultimate aim of selecting new therapeutic targets. We will particularly focus on JAK-STAT pathway, as our preliminary analysis and data suggest an activation in SSc patients. (ii) Assess the interactions between inflammation, metabolism and fibrosis through the VATFIB project (visceral adipose tissue dysfunction: a new hallmark of progressive fibrosis). Beginning with the hypothesis that visceral fat directly associates with end-organ inflammation and fibrosis, this project aims to elucidate whether a causal relationship exists between inflammation, metabolism, and fibrosis in SSc, and to identify the molecular mechanisms involved. Initially, we plan to identify circulating mediators common to SSc and control groups, including obesity, metabolic dysfunction associated steatohepatitis, and IBD (in collaboration with WP2), or specific to each disease. We intend to employ wide-scale proteomics and high- throughput computational biology to establish an adipokine-based signature linked to inflammation and fibrosis in these diseases. Furthermore, we aim to characterize the molecular crosstalk between visceral adipose tissue and inflamed/fibrotic tissues, identifying molecular changes associated with pro-fibrotic and/or adipogenic cellular activation. Leveraging extensive retrospective cohorts and biobanks containing blood and paraffin- embedded tissues (including skin and as controls colon, liver, and abdominal fat tissue), we will characterize the circulating and in situ expression of key proteins identified as potential targets. This endeavour also aims to develop new anti-fibrotic strategies, addressing a significant unmet need in the management of IMIDs (collaboration with WP4). Ultimately, VATFIB seeks to establish a diagnostic and prognostic biomarker signature for fibrosis in IMID, supported by CPER Resist omics (Portfolio item 11).

Accumulating evidence have linked B cells anomalies with the vasculopathy of SSc and pulmonary arterial hypertension (PAH), but their direct pathogenic role remains controversial, as described in our review (Sanges et al; Eur Resp Journal 2024). The objective of our work is to better decipher the functional role played by B cells in the pathogenesis of SSc-PAH, by performing an exhaustive characterization of the B cells compartment at the single-cell level in an optimized animal model of the disease: Fra2Tg mice. We will conduct a thorough investigation of the lung vasculopathy of Fra2Tg mice that will be acquired through our collaboration with Inserm Unit 1016 (Prof. F. Batteux) at Cochin Hospital. We will try to determine how the Fra2Tg mouse model can be adapted to reproduce the lung characteristics of SSc-PAH. We will characterize B cells compartment in the lungs of Fra2Tg mice at the single-cell level. B cells from the lungs of Fra2Tg mice will be sorted and undergo an extensive multi-omics characterization at the single-cell level (Chromium Single Cell Immune Profiling, 10X Genomics granted by the CPER Resist-omics, with analysis of transcriptome, BCR repertoire, and membrane phenotype data. To better elucidate the complex interrelations between B cells and other cellular players involved in the generation of lung vascular lesions, other immune cells, EC and FB will also be sorted and undergo single-cell transcriptomic analysis, with receptor-ligand analysis and elaboration of interaction maps. Finally, we will assess the interactions between B cells and EC in human SSc-PAH. Peripheral mononuclear blood cells (PBMC) from SSc-PAH patients will be collected. Total B cells, as well as pathogenic B cell subsets identified in Fra2Tg mice will be sorted and cultured for 48h. B cell culture supernatants will be added to the culture media of human pulmonary artery EC commercial cell-line, with or without specific inhibitors of the interactions identified. The capacity of B cells supernatant to induce EC activation, proliferation and mesenchymal transition will be assessed. Complex 3D models using sorted SSc-PAH B cells and pulmonary vessels-on-chip will then be used to further explore any relevant result.