Background/Purpose:

Systemic Sclerosis (SSc) is an autoimmune disorder of the connective tissue due to excessive extracellular matrix deposition, leading to fibrosis of the skin and lung. The disease is characterized by the presence of elevated levels of IFN-regulated genes implicating plasmacytoid dendritic cells (PDC) and recently, it was shown that CXCL4 levels correlated with lung and skin fibrosis making this chemokine an interesting biomarker and highlighting the role of PDC in the disease pathogenesis (van Bon et al. NEJM, 2014). Our aim is to better understand what is controlling the activation of PDC in SSc patients.

Methods:

Blood was obtained from 33 SSc patients, 9 classified as early diffuse (edSSc), 15 as late diffuse SSc (ldSSc) and 9 limited (lSSc) and from 13 healthy volunteers (HV). PBMC were isolated and PDC purified either using BDCA4 magnetic beads (Miltenyi) or by cell sorting and purity was routinely >96%. The percentage of immune cells in total PBMC was analyzed by flow cytometry. Gene expression was analyzed in PBMC and PDC by Q-PCR. PDC were cultured for 24h and IFNa and CXCL4 secretion analyzed by ELISA.

Results:

First, we observed that the percentage of PDC and CD8+ T cells were significantly decreased in SSc PBMC as compared to HV (0.25%±0.02 vs 0.51%±0.09; p=0.003 and 13%±0.8 vs 20%±0.9; p<0.001 respectively) while the percentage of monocytes and CD4+ T cells were significantly increased (24%±1.9 vs 13.8%±2.4; p=0.002 and 42%±2.7 vs 33.2%±2.8; p=0.03 respectively). No difference was observed for B cell in SSc PBMC as compared to HV (6.1±0.9 vs 7.9±1.0; NS). As assessed by qPCR, we observed increased level of IFN-regulated genes in SSc patients, as genes such as ISG54, MxB or IP10 were significantly upregulated as compared to HV (in relative Ct to the housekeeping gene Ubiquitin: 641±85 vs 262±69; p=0.02, 421±35 vs 245±39; p=0.03 and 96±12 vs 52±8.5; p=0.005 respectively). Interestingly, this increase was observed in all the patients irrespective of subtypes. We also observed that purified PDC from SSc patients spontaneously secreted CXCL-4 (17,111±1,855 vs 6,903±662 pg/ml; p=0.001). We also noted a disruption in the expression pattern of TLRs in PDC which was confirmed by functional assay using specific agonists or inhibitors of the TLR pathway.

Conclusion:

Taken altogether our data suggests a role of PDC in the pathogenesis of SSc by the involvement of the TLR pathway. This could explain the abnormal activation state of these cells in patients as suggested by the presence of high IFN responses and the migration of PDC to the sites of inflammation such as the skin and lung of the patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-tlrs-in-the-abnormal-secretion-of-cxcl-4-by-plasmacytoid-dendritic-cells-of-patients-with-systemic-sclerosis/