Targeting mitochondrial double-stranded RNAs ameliorates autoimmune characteristics of Sjӧgren’s syndrome

Sjӧgren’s syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. Here, we investigate the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating type I IFN response in SS. We find that mt-dsRNAs are elevated in the saliva and tear of SS patients and in salivary glands of non-obese diabetic mice with salivary dysfunction. Using the in-house- developed 3D culture, we show that dsRNA stimulation increases mt-dsRNAs expression via JAK1/STAT pathway and facilitates their cytosolic export, which is accompanied by autoimmune signatures observed in SS. We further show that muscarinic receptor ligand acetylcholine or antioxidant resveratrol ameliorates autoimmune characteristics by preventing mt-dsRNA- mediated immune activation. In addition, direct suppression of mt-dsRNAs reverses the autoimmune signatures of SS. Altogether, our study underscores the significance of mt-dsRNA upregulation and suggests mt-dsRNAs as an important key to the puzzle of SS.SummaryMitochondrial double-stranded RNA levels are elevated in the tear and saliva of SS patients. These RNAs promote type I interferon signature, as well as other autoimmune phenotypes in SS. Downregulation of mitochondrial dsRNAs alleviates autoimmune signatures of SS in salivary gland epithelial cells.Graphical Abstractmt-dsRNAs as the molecular mediator of autoimmune phenotypes in SS.mt-dsRNAs are elevated in both human samples and mouse model of SS and function to exacerbate dsRNA-induced autoimmune phenotypes in SS. Countering the accumulation of mt- dsRNAs alleviates the autoimmune phenotypes in SGECs.

Fibroblasts in Sjögren’s Syndrome

The Sjögren’s syndrome is an autoimmune disease characterized by chronic inflammation of the exocrine glands, leading to dryness of mucosal surfaces, and often to severe systemic manifestations. Here, the immunomodulatory function of fibroblasts derived from salivary glands, a primary site affected by the Sjögren’s syndrome, is discussed. Specific subsets of these fibroblasts drive the formation of tertiary lymphoid structures, which are associated with severe disease and which constitute a risk factor for the development of lymphoma in Sjögren’s syndrome. Single cell RNA-sequencing has provided new insights into subsets of fibroblasts in inflamed salivary glands and has provided evidence for the existence of shared inflammation-associated fibroblasts across chronically inflamed tissues. These findings support the concept of targeting the fibroblast compartment in Sjögren’s syndrome and other chronic inflammatory diseases. In addition to the immunomodulatory role of fibroblasts, the interaction of the epithelium with fibroblasts is essential for salivary gland homeostasis. Fibroblasts provide essential signals for the regeneration of salivary gland epithelial cells, which is disturbed in Sjögren’s syndrome, and leading to the loss of saliva secreting cells and subsequent hyposalivation.

Salivary dysbiosis in Sjögren’s syndrome and a commensal-mediated immunomodulatory effect of salivary gland epithelial cells

Salivary gland epithelial cells (SGECs) have been implicated in the pathogenesis of Sjögren’s syndrome due to aberrant antigen-presentation function. This study examined the hypothesis that oral dysbiosis modulates the antigen-presentation function of SGECs, which regulates CD4 T cell proliferation in primary Sjögren’s syndrome (pSS). Saliva samples from 8 pSS patients and 16 healthy subjects were analyzed for bacterial 16S ribosomal DNA. As a result, 39 differentially abundant taxa were identified. Among them, the phylum Proteobacteria comprised 21 taxa, and this phylum was mostly enriched in the healthy controls. The proteobacterium Haemophilus parainfluenzae was enriched in the healthy controls, with the greatest effect size at the species level. Treatment of A253 cells in vitro with H. parainfluenzae upregulated PD-L1 expression, and H. parainfluenzae-pretreated A253 cells suppressed CD4 T cell proliferation. The suppression was partially reversed by PD-L1 blockade. Among low-grade xerostomia patients, salivary abundance of H. parainfluenzae decreased in pSS patients compared to that in non-pSS sicca patients. Our findings suggest that H. parainfluenzae may be an immunomodulatory commensal bacterium in pSS.

Immortalization of Salivary Gland Epithelial Cells of Xerostomic Patients: Establishment and Characterization of Novel Cell Lines

Primary Sjögren’s Syndrome (pSS) is an autoimmune disease mainly affecting salivary and lacrimal glands. Previous pSS studies have relied on primary cell culture models or cancer cell lines with limited relevance to the disease. Our objective was to generate and characterize immortalized salivary gland epithelial cells (iSGECs) derived from labial salivary gland (LSG) biopsies of pSS patients (focus score > 1) and non-Sjögren’s Syndrome (nSS) xerostomic (i.e., sicca) female patients. To characterize iSGECs (n = 3), mRNA expression of specific epithelial and acinar cell markers was quantified by qRT-PCR. Protein expression of characterization markers was determined by immunocytochemistry and Western blot. Secretion of α-amylase by iSGECs was confirmed through colorimetric activity assay. Spheroid formation and associated alterations in expression markers were determined using matrigel-coated cell culture plates. Consistent mRNA and protein expressions of both epithelial and pro-acinar cell markers were observed in all three iSGEC lines. When cultured on matrigel medium, iSGECs formed spheroids, secreted α-amylase after β-adrenergic stimulation, and expressed multiple acinar cell markers at late passages. One iSGEC line retained adequate cell morphology without a loss of SV40Lt expression and proliferation potential after over 100 passages. In conclusion, our established iSGEC lines represent a viable model for salivary research due to their passaging capacity and maintenance of pro-acinar cell characteristics.

Salivary gland epithelial cells from patients with Sjögren’s syndrome induce B-lymphocyte survival and activation

ObjectivePrimary Sjögren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes.MethodsPatients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed.ResultsGene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors.ConclusionsSGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.

THU0220 AUTOPHAGY IN SJOGREN’S SYNDROME SALIVARY GLAND EPITHELIAL CELLS (SGECS) IS ASSOCIATED WITH THE SEVERITY OF INFLAMMATION AND EPITHELIAL CELLS ACTIVATION.

Background:Sjögren’s Syndrome (SS) is characterized by chronic inflammation supported by intrinsic activation of salivary gland epithelial cells (SGECs). Eventually, apoptosis of SGECs ensues, which leads to salivary gland dysfunction and exposition of autoantigens. Autophagy is a stress coping mechanisms of cells implicated in both survival and exposition of autoantigens, and is thereby plausibly implicated in the pathogenesis of SS. At present, the exact relationship between apoptosis and autophagy in SS SGECs is unclear, as is the link between these mechanisms and SGECs activation.Objectives:To explore autophagy in SGECs from patients with SS and to evaluate its relationship with apoptosis and SGECs activation.Methods:Consecutive patients with suspected SS referring to our “Sjogren Clinic” were enrolled, and minor salivary gland (MSG) biopsies were collected for: (1) SGECs culture, (2) PCR analysis, (3) IFI analysis. In SGECs cultures, the expression of autophagy (LC3II), apoptosis (annexin V/PI) and adhesion molecules (ICAM) was investigated by flow cytometry (results expressed as mean % ± SD). The expression of the autophagy gene MAP1LC3II was evaluated by PCR (expressed as 2^deltaCT normalized to GADPH) on both MSG sections and MSG acinar and ductal epithelium samples obtained by laser capture microdissection. Tissue expression of LC3II was evaluated by IFI on SS MSG.Results:Primary SGECs cultures were established from 14 MSG obtained for diagnostic purposes (SS n=8, Sicca n=6). These cells exhibited an inverse correlation between apoptosis and autophagy (p=0.007, r=-0.784), with lower levels of apoptosis (19.7±6.5 vs 24.5±8.5, p=ns) and higher levels of autophagy (59.7±13.1 vs 54.19±19.4, p=ns) in SS compared to Sicca. In SS, MAP1LC3 was positively correlated with Focus Score (p=0.021 r=0.478); however, PCR studies did not reveal significant differences in MAP1LC3 expression between SS (n=26) and Sicca (n=15) (0.024±0.010 vs 0.022±0.008, p=ns). Ductal SGECs (n=4) isolated by laser microdissection of MSG revealed a higher expression of MAP1LC3 (0.005±0.0005 vs 0.003±0.0008; p=0.057) compared to normal acinar epithelium (n=5); a major expression of LC3II in ducts was confirmed by IFI (Image).In SS, a higher expression of ICAM compared to sicca was observed (11.1±3.8 vs 6.9±6.9, p=0.006) and autophagy and apoptosis showed a trend of positive and negative correlation with this molecule, respectively (p=0.683 r=0.118 and p=0.106 r=-0.446).Figure.LC3-II staining in SS MSG [LC3-II+ (green) and Hoechst stain (blue); 60x magnification].Conclusion:In SS, autophagy is upregulated in SGECs and inversely correlated with apoptosis, thus supporting a role of this process in cells’ death prevention during inflammatory process. Indeed, the degree of msg inflammation is correlated more with the activation of autophagy than apoptosis. Interesting, in SS, SGECs autophagy is mainly observed at ductal level and is correlated with higher expression of adhesion molecules suggesting a link between this pathway and changes in SGECs immune phenotype.Disclosure of Interests: :Serena Colafrancesco: None declared, cristiana barbati: None declared, Valentina Iannizzotto: None declared, Linda Mastromanno: None declared, Saba Nayar: None declared, Elena Pipi: None declared, angelica gattamelata: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie, cristiano alessandri Grant/research support from: Pfizer, Francesca Barone: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Roberta Priori: None declared

Alpha 6 integrins promote cytokinesis by regulating the expression of RSK2 and MKLP1

ABSTRACTThe integrin-mediated interaction of cells with components of the extracellular matrix (ECM) regulates many cellular processes including cell division. Cytokinesis is the last step of cell division and is critical for normal development and tissue homeostasis as it ensures the proper segregation of genetic and cytoplasmic material between daughter cells. Cytokinesis failure leads to defects in development and tissue differentiation, as well as tumorigenesis. Abscission of intercellular bridge that connects presumptive daughter cells is the last step of cell division. The mitotic kinesin-like protein 1 (MKLP1) plays a central role in positioning the abscission machinery. Here, we show that α6 integrins promote successful cytokinesis in salivary gland epithelial cells by regulating the expression of MKLP1. RNAi-mediated depletion of α6 integrins inhibits cytokinesis and the expression of MKLP1 and p90 ribosomal-S6-kinase 2 (RSK2). Depletion of RSK2 results in similar defects in cytokinesis and also inhibits the expression of MKLP1, suggesting that the expression of RSK2 is required downstream of integrins to promote MKLP1 expression and successful cytokinesis. RNAi-mediated depletion of RSK2 in embryonic salivary glands in organ culture also results in the inhibition of cytokinesis and MKLP1 expression, indicating the physiological significance of this pathway.