OP0185 INFLAMMATION-DEPENDENT DECREASED EXPRESSION OF CD52 ON CIRCULATING CD14+ MONOCYTES FACILITATES ADHESION IN SYSTEMIC SCLEROSIS
Authors: Michal Rudnik, Mara Stellato, Przemyslaw Blyszczuk, Karin Klingel, Jörg Henes, Carol Feghali-Bostwick, Oliver Distler, Gabriela Kania
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> During the course of systemic sclerosis (SSc), infiltration of inflammatory cells, including monocytes, into the organs is a major process leading to fatal fibrosis, remodelling and organ dysfunction. CD52 protein is highly expressed on CD4<sup>+</sup> T-cells and plays an important role in the modulation of T-cell receptor signalling. Nevertheless, the function of this protein on monocytes is not completely understood. <h3>Objectives</h3> We aimed to functionally investigate the role of circulating CD14<sup>+</sup> monocytes in the course of SSc with a special focus on monocyte adhesion and the influence of CD52 expression. <h3>Methods</h3> Biopsies from the heart, lungs and skin of SSc patients (n=11, 7, 7 respectively) and healthy controls (HC) (n=10, 7, 9 respectively) were analysed by immunohistochemistry for the presence of CD14<sup>+</sup> cells. PolyA RNA sequencing of CD14<sup>+</sup> monocytes isolated from peripheral blood of lcSSc (n=5, age=54.4±6.7), dcSSc patients (n=5, age=51.8±7.2) and age- and sex-matched HC (n=5, age=50.8±9.7) was performed using Illumina HiSeq 4000 platform. Differentially expressed genes were computed using DeSEQ2 algorithm. Gene ontology and pathway analysis were performed using Metacore software and ShinyApp. Expression of adhesion molecules was confirmed on the protein level using flow cytometry (HC n=8, SSc n =22). Adhesion of CD14<sup>+</sup> monocytes to immobilized ICAM1 and TNFα-stimulated endothelial cells was checked using the 96-well plate adhesion assay (HC n=8, SSc n=22). CD52 regulation in CD14<sup>+</sup> monocytes from HC (n=4) was analysed on mRNA level upon stimulation with different concentrations of LPS, IFNγ, IL-4 and IL-13. THP-1 cell lines with overexpression and silencing of CD52 were generated using lentiviral vectors, and further, their adhesion was investigated (n=4). <h3>Results</h3> Immunochemistry confirmed higher infiltration of CD14<sup>+</sup> cells in the heart (p&lt;0.01), lung (p&lt;0.05), and skin (p&lt;0.001) of SSc patients. We detected 1440 differentially expressed genes between dcSSc vs HC and 225 between lcSSc and HC CD14+ monocytes respectively (p≤0.01; log2 ratio≥0.5). Pathway analysis revealed significant alterations in processes important for tissue infiltration, including adhesion. Flow cytometry analysis showed upregulation of adhesion molecules: integrin α<sub>M</sub> (CD11b) (p&lt;0.01) and integrin β<sub>2</sub> (CD18) (p&lt;0.01On the other hand, expression of CD52 was downregulated in SSc patients (p&lt;0.05). SSc CD14+ monocytes exhibited increased adhesion both to ICAM1-coated plates (p&lt;0.01) and to TNFα-stimulated endothelial cells (p&lt;0.05). CD52 mRNA was significantly increased in a dose-dependent manner after anti-inflammatory stimulation (IL-4, IL-13) and significantly decreased after inflammatory stimulation (LPS, IFN-γ) (p&lt;0.05). Overexpression of CD52 in THP-1 monocytes decreased adhesion to TNFα-stimulated endothelial cells (p&lt;0.01). Accordingly, silencing of CD52 increased adhesion of THP-1 monocytes (p&lt;0.01). <h3>Conclusion</h3> This is the first report pointing to an increased adhesion of peripheral blood CD14<sup>+</sup> monocytes to ICAM1 and endothelial cells in SSc. Our results suggest the primary activation of monocytes in peripheral blood, which translates into higher organ infiltration in SSc patients. Finally yet importantly, we characterised a novel function of CD52 molecule on monocytes and its possible contribution during the course of the disease. <h3>Disclosure of Interests</h3> Michal Rudnik: None declared, Mara Stellato: None declared, Przemyslaw Blyszczuk: None declared, Karin Klingel: None declared, Jörg Henes: None declared, Carol Feghali-Bostwick: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Gabriela Kania: None declared