THU0356 STAPHYLOCOCCUS AUREUS REGULATES FIBROBLAST FUNCTIONS – IMPLICATIONS FOR TISSUE REPAIR IN CHRONIC DIGITAL ULCERS IN SYSTEMIC SCLEROSIS
Authors: Masaya Yokota, Matthew Kassier, Nicola Häffner, Matthias Brunner, Janine Schniering, Oliver Distler, Annelies S. Zinkernagel, Britta Maurer
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Chronic digital ulcers (DU) are a major complication in systemic sclerosis (SSc). Non-healing wounds are characterized by persistent inflammation, defective re-epithelialization and impaired matrix remodeling, and are often accompanied by bacterial colonization. In DU, the breach of the basement membrane exposes tissue and cell layers to commensal skin bacteria. <h3>Objectives:</h3> To investigate whether interactions between commensal skin bacteria and dermal fibroblasts affect tissue repair mechanisms. <h3>Methods:</h3> Dermal fibroblasts isolated from healthy controls (HC) and patients with diffuse cutaneous SSc (dcSSc) (n=3, each) were co-cultured with <i>Staphylococcus aureus</i> (SA) for 3 h at using a lower inoculum rather representing colonization (1 x 10<sup>5</sup> CFU/ml) and a higher inoculum reflecting infection (1 x 10<sup>7</sup> CFU/ml). Thereafter, fibroblasts were cultured with flucloxacillin-containing medium to kill extracellular and adherent bacteria. For mechanistic studies, a fibroblast cell line (BJ-5ta) was used. IL-6, IL-8, pro-collagen Iα, and interferon (IFN)-β proteins in culture supernatants were measured by ELISA. Gene expression was assessed by quantitative PCR. Contractility was analyzed by collagen gel contraction assay. Wound closure assay was performed on a confluent cell layer with a uniform cell-free gap, generated by a cell culture insert. Expression of alpha-smooth muscle actin (α-SMA) was evaluated by Western blotting, cell proliferation with a colorimetric assay. Cell death was analyzed by live cell real time assay. Bacterial invasion was assessed by immunofluorescence staining using confocal microscopy and CFU recovery. Gene knockdown was performed using siRNA. <h3>Results:</h3> Exposure to 1 x 10<sup>7</sup> CFU/ml of SA influenced fibroblast function. In brief, exposure to SA increased the secretion of IL-6 and IL-8 in dermal fibroblasts by 1571/455-fold (HC/dcSSc; p=0.08/&lt;0.05) and by 325/1402-fold (HC/dcSSc; p&lt;0.05 each) respectively. In addition we observed upregulation of the expression of MMP1 by 8.2/5.7-fold (HC/dcSSc; p&lt;0.05 each) and MMP3 by 5.1/5.7-fold (HC/dcSSc; p=0.06/&lt;0.05). There was a trend towards decreased secretion of pro-collagen Iα. TGF-β-induced α-SMA expression was completely inhibited. Cell contraction was impaired by 22.0/31.6% (HC/dcSSc; p=0.08/&lt;0.05), wound closure by 18.8/14.9% (HC/dcSSc; p&lt;0.05 each), and proliferation by 30.5/44.3% (HC/dcSSc; p=0.24/&lt;0.05). SA induced apoptosis and necrosis. SA invaded fibroblasts via endocytosis (25-50% of inoculated live SA). IFN-β secretion was induced 8.4/31.7-fold (HC/dcSSc; p&lt;0.05 each). The genes of cytosolic dsDNA sensor molecules such as cyclic GMP-AMP synthase (cGAS) were upregulated 11.9/8.6-fold (HC/dcSSc; p&lt;0.05 each). TLR9, an endosomal DNA sensor, was constitutively expressed. Knockdown of STING or MyD88, downstream mediators of cGAS and TLR9 respectively, reduced the induction of IFN-β (down to 68.2/66.2%; p=0.20/0.16), IL-6 (down to 57.2/55.2%; p&lt;0.05 each), and IL-8 (down to 45.3/36.0%; p&lt;0.05 each). Furthermore, inhibition of endocytosis with cytochalasin D decreased the expression of IFN-β (down to 24.6%; p&lt;0.05), IL-6 (down to 34.9%; p&lt;0.05), and IL-8 (down to 27.6%; p&lt;0.05). In contrast, inhibition of endocytosis did not affect induction of apoptosis. This suggests that the invasion of fibroblasts by SA and the subsequent intracellular DNA sensing are crucial for the induction of these genes independently of apoptosis. <h3>Conclusion:</h3> Invasion of dermal fibroblasts by SA with activation of the STING and TLR9-MyD88 pathways is a key element in the impairment of tissue repair responses.