21Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete 22 understanding of the molecular mechanisms underlying fibrosis has severely hampered effective 23 drug development. CXCL4 is associated with the onset and extent of fibrosis development in 24 systemic sclerosis, a prototypic inflammatory and fibrotic disease. Here, we integrated 65 paired 25 longitudinal transcriptional and methylation profiles from monocyte-derived cells with/without 26 2 CXCL4 exposure. Using data-driven gene regulatory network analyses, we demonstrate that 27 CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-28 inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including 29 CIITA. CXCL4 exposed monocyte-derived cells directly trigger a fibrotic cascade by producing 30 ECM molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked 31 CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of 32 the gene regulatory network. Our study unveils CXCL4 as a key secreted factor driving innate 33 immune training and forming the long-sought link between inflammation and fibrosis. 34 35 106 antigen processing and presentation (Figure 1-figure supplement 1E). For instance, CXCL4-107 moDCs, in the absence of further stimulation, up-regulate expression of several inflammatory 108 molecules such as CTSL, FLT1, CD86, LAMP1, CHI3L1, and down-regulate signaling receptors 109 such as CLEC10A, IL1R1, IL1R2 compared to moDCs (Figure 1G-I and Figure 1-figure 110 supplement 1A-D). Strikingly, CXCL4 exposure also leads to dramatic changes in expression of 111 genes regulating metabolism and transcription (Figure 1-figure supplement 1E), reminiscent 112 of changes previously observed in myeloid cells undergoing immune training (Saeed et al., 2014). 113 Thus, CXCL4 orchestrates a differentiation process dramatically different than that of the 114 conventional moDCs. 115 116 Mature CXCL4-moDCs are functionally distinct from conventional moDCs 117 5To study the effects of CXCL4 on moDC maturation, we stimulated the cells with polyI:C on 118 day 7. This perturbed the expression of 8,949 and 7,767 genes in CXCL4-moDCs and 119 conventional moDCs, respectively, compared to the day 7 transcriptional profiles of their 120 unstimulated counterparts (Figure 1C and 1E, left and middle panels). 2,397 genes responded 121 differently to polyI:C stimulation in CXCL4-moDCs compared to conventional moDCs ( Figure 122 1C and Figure 1-figure supplement 2). Several pathways involved in inflammatory responses 123 such as TLR signaling, interferon signaling, and cytokine signaling, were significantly 124 upregulated in CXCL4-moDCs compared to conventional moDCs (Figure 1-figure 125 supplement 2E). Confirming our previous findings (Silva-Cardoso et al., 2017), these 126 transcriptional changes were followed by increased production of pro-inflammatory mediators 127 such as IL-1β, IL-6, IL-12, IL-23, IL-27, TNF and...

gelatinase expressed in three major forms: dimer, monomer and a complex with neutrophil gelatin-associated lipocalin (NGAL). Interleukin-(IL)-6 is a pleiotropic cytokine expressed by a variety of immune and non-immune cells. However, the mechanisms by which IL-6 contributes to the pathogenesis of chronic arthropathies are not fully understood. Objectives: The purpose of the present work was to perform a comparative study of the IL-6 production and MMP-9 activity in FLS stimulated with SF from patients with osteoarthritis (OA), rheumatoid arthritis (RA) or spondyloarthritis (SpA). In addition, the effect of IL-6 blockade on MMP-9 activity was evaluated. Methods: Primary FLS were obtained from SF of the RA patients. Furthermore, the SW982 human synovial cell line was used. The SF of patients with OA (n=11), RA (n=11) or SpA (n=9) patients were pooled. The FLS were stimulated with OA, RA or SpA SF pools and supernatants (SN) were collected after 24, 48 and 72 h. The IL-6 levels were assessed in the SN by ELISA. The gelatinase activity of the SN was determined by zymography. The IL-6 function was blocked with the anti-IL-6 receptor antagonist tocilizumab (TCZ) (200μg/ml). Results: Earlier induction of IL-6 in SW982 cell line was observed by RA and SpA SF stimulation since significant levels were detected at 24 h (p<0.001 and p<0.01 compared with non-stimulated cells, respectively), whilst OA SF induced significant IL-6 secretion at 72 h (p<0.01). Similar results were observed in primary FLS. In contrast to SF of OA patients, SF of patients with RA or SpA induced increased and sustained secretion of active MMP-9. Moreover, the molecular weight band corresponding with NGAL-MMP-9 complex, considered a protected form of MMP-9, was detected with higher intensity in the SN of FLS stimulated with RA or SpA SF compared with OA SF (p<0.001). In the presence of TCZ, significant inhibition in the gelatinase activity of all MMP-9 forms was observed at 48h of stimulation with RA or SpA SF (p<0.001 for MMP-9 dimer and NGAL-MMP-9 complex; p<0.01 for MMP-9 monomer, compared with FLS stimulated in absence of TCZ). Conclusions: We conclude that SF of patients with inflammatory arthritis recreate a differential microenvironment for FLS that impacts on early phenotypic changes of these cells. The IL-6 provokes augmented and persistent MMP-9 activity in FLS stimulated with RA or SpA SF. This work identifies TCZ as an inhibitor of all forms of MMP-9.