Crucial role of CXCL4 and type I interferon in early Systemic sclerosis: ways to block them via new pharmacological intervention by drug repositioning (Hydroxychloroquine) and experimental small molecules

Systemic Sclerosis (SSc) is a rare, chronic, orphan autoimmune disease, characterized by specific autoantibodies, vascular abnormalities or damage and there is no effective treatment against the disease progression. Chemokine (C-X-C motif) ligand 4(CXCL4), is an antimicrobial chemokine with multiple effector functions and is currently considered as a biomarker in systemic sclerosis (SSc). In SSc, the presence of CXCL4 at an early stage, correlates with poor prognosis and severe disease progression. CXCL4 contributes to the interferon-I (IFN-I) signature in the disease by forming pro-inflammatory liquid crystalline complexes with self-DNA/RNA, which activate TLR7/8/9 and induce IFN-a in plasmacytoid dendritic cells (pDCs) and TNF-a by myeloid DC. The study presented in this thesis consists of two parts: in the first part, we evaluate, in vitro, the role of CXCL4 tetramerization on its interferogenic function in complex with DNA. In the second part, we analyze the presence and modulation of CXCL4, CXCL4-DNA/RNA complexes, IFN-I and other pro-inflammatory mediators (e.g. TNF-a), in a cohort of early SSc patients included in an ongoing double blind, randomized, placebo-controlled, add-on trial evaluating efficacy and safety of hydroxychloroquine (HCQ)(HYDROXXY-SSc, Eudract 2001-00230-33). Of note, HCQ is a well-known inhibitor of the IFN-I pathways. By comparing various mutated CXCL4 derived peptides, we find that the physiological pre-assembly of CXCL4 into tetramers, before DNA interaction, is important for stimulation of TLR9 and amplified IFN-I secretion by pDCs (the immune cells that are the main IFN-I producing cells in the body). Most importantly, small molecules that destabilize CXCL4 tetramerization block the pDC-IFN-a response, suggesting new pharmacological interventions in SSc, and possibly in other autoimmune conditions characterized by the presence of high CXCL4 and CXCL4-DNA complexes 1 expression. For the second part of this study, we started to evaluate the expression CXCL4, CXCL4-DNA and CXCL4-RNA complexes as well as IFN-a and TNF-a in a cohort of early SSc patients included in the HYDROXXY-SSc trial. At T0 SSc patients have higher level of CXCL4, complexes of CXCL4 with DNA/RNA, IFN-a and TNF-a compared to healthy donors (HD). This study represents a novel approach to block the CXCL4-driven inflammation: the disruption of the CXCL4 tetrameric structure. This strategy can be used to block IFN-I amplification in SSc. The results of the HYDROXXY-SSc trial will illuminate on the beneficial effect of IFN-I inhibition in early SSc. Since CXCL4 contributes to the IFN- I pathways, HCQ could be an important add on therapy to SSc, blocking IFN-I signature.