Background/Purpose: Systemic sclerosis (SSc) patients exhibit alterations in innate and acquired cellular responses including an enhanced inflammatory response to toll like receptor (TLR) agonists that lead to increased production of Th1, Th2 and Th17-associated cytokines and may be associated to disease progression and phenotype. Pathogenic mechanisms of these alterations are not fully understood. The aim of this study was to determine the effect of TLR3, TLR4 and TLR9 stimulation of monocyte derived dendritic cells (Mo-DCs) in the production of Th17-related molecules in SSc patients.

Methods: We  included 16 SSc patients (4 early limited cutaneous lcSSc, 4 late lcSSc, 4, early diffuse cutaneous dcSSc and 4 late dcSSc patients) and 5 non-related healthy controls. We isolated peripheral blood mononuclear cells (PBMCs) from 55 ml of venous blood. Mo-DCs were differentiated using a commercially available and validated medium containing GM-CSF and IL-4 from ex vivo purified CD14+ monocytes from ex-vivo purified CD14+ monocytes, stimulated with TLR agonists (LPS/TLR4, poly:IC/TLR3, CpG/TLR9) and co-cultured with PBMCs. Cytokine levels in supernatants were measured after 96 h, by Luminex. Differences were evaluated by Mann-Whitney U test.

Results: All SSc patients were females (mean age 48+/-14.4 years). We found significantly higher levels (p<0.05) of IL-6, IL-10, IL-17F, IL-22, IL-23, IL-31, IL-1b and IFN-g in LPS/TLR4-stimulated Mo-DCs-PBMC co-culture supernatants from lcSSc patients when compared to those from dcSSc patients. In contrast, Mo-DCs-PBMC co-cultures from dcSSc patients produced higher levels of IL-33 with and without TLR agonist stimulation than lcSSc patients’ co-cultures. When SSc patients were categorized as early and late SSc we found that LPS/TLR4-stimulated Mo-DCs-PBMC co-cultures from early SSc patients produced higher amounts of IL-6, IL-10, IL-17F, IL-22, IFN-g and TNF-a when compared to those from late SSc patients. Interestingly, IL-33 secretion was increased with or without stimulation with TLR agonists (p<0.05) in Mo-DCs-PBMC co-cultures from late SSc in comparison with those from early SSc patients. 

Conclusion: Mo-DCs stimulation with TLR4 ligand potently induces cytokines involved with the pro-inflammatory Th17 phenotype. TLR4-stimulated Mo-DCs from lcSSc patients produce higher amounts of Th17 (IL-6, IL-22 and IL-23) and Th1 cytokines. Early SSc patients exhibit enhanced Th17 responses. Mo-DCs from dcSSc and late SSc patients exhibit enhanced production of IL-33. Our findings suggest that functional variations in the production of Th17 cytokines by Mo-DCs are associated with different SSc clinical subsets and phases

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differential-production-of-th17-related-cytokines-by-toll-like-receptor-tlr-ligands-stimulated-monocyte-derived-dendritic-cells-mo-dcs-from-systemic-sclerosis-ssc-patients-relevance-of-il-22-an/