Single-cell RNA sequencing analysis of vestibular schwannoma reveals functionally distinct macrophage subsets.

BACKGROUND: Vestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown. OBJECTIVES: The objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq). METHODS: scRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules. RESULTS: scRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1ß. AREG and PLAUR were expressed in the CD68+CD163+IL-1ß+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1ß- subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163-IL-1ß+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1ß, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume. CONCLUSIONS: Macrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.

CXCL13 as biomarker for histological involvement in Sjögren's syndrome.

OBJECTIVES: SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. METHODS: 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control. RESULTS: CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. CONCLUSION: Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

Immunofibroblasts are pivotal drivers of tertiary lymphoid structure formation and local pathology.

Resident fibroblasts at sites of infection, chronic inflammation, or cancer undergo phenotypic and functional changes to support leukocyte migration and, in some cases, aggregation into tertiary lymphoid structures (TLS). The molecular programming that shapes these changes and the functional requirements of this population in TLS development are unclear. Here, we demonstrate that external triggers at mucosal sites are able to induce the progressive differentiation of a population of podoplanin (pdpn)-positive stromal cells into a network of immunofibroblasts that are able to support the earliest phases of TLS establishment. This program of events, that precedes lymphocyte infiltration in the tissue, is mediated by paracrine and autocrine signals mainly regulated by IL13. This initial fibroblast network is expanded and stabilized, once lymphocytes are recruited, by the local production of the cytokines IL22 and lymphotoxin. Interfering with this regulated program of events or depleting the immunofibroblasts in vivo results in abrogation of local pathology, demonstrating the functional role of immunofibroblasts in supporting TLS maintenance in the tissue and suggesting novel therapeutic targets in TLS-associated diseases.

Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.