Background/Purpose: Dendritic cells (DC) are a heterogeneous population of antigen presenting cells which link both innate & adaptive immunity. To date their classification within blood & skin has been well characterised however evidence identifying DC within other tissues, in particular in the context of autoimmunity is limited. In this study we aim to characterise the DC populations within the inflamed synovium & to elucidate the effect of the inflamed joint microenvironment on DC characterisation & function & subsequent effects on T cell activation.

Methods: Synovial tissue was obtained from RA patients through arthroscopy. For characterisation of tissue DC, biopsies were digested & gated on CD45⁺ cells. DC were defined as HLADR⁺, Lineage^– &  cell surface expression of CD11c, CD1c, CD141, CD123, CD80, CD83 & CD40 was assessed by flow cytometry.  In parallel, RA synovial fluid (SF) & peripheral blood (PB) DC characterisation was also analysed in comparison to synovial tissue.  To assess the effect of the inflamed microenvironment on DC activation & function, RA synovial tissue explants were cultured for 24hr allowing the spontaneous release of proinflammatory cytokines & mediators into the culture medium.  MoDC were then cultured in the presence of RA explant conditioned media (ECM), RA SF under normoxia or hypoxia & the expression of CD80, CD83 & CD40 assessed.  Furthermore MoDC were treated with RA SF & cocultured with CD4⁺ T cells for 5 days, supernatants harvested & T cells restimulated with PMA (50ng/ml) & Ionomycin (500ng/ml) to examine intracellular expression of IFNγ.

Results: Phenotypic characterization of mDC in PB, SF & synovial tissue from RA patients demonstrated a significant increased gradient in DC maturation markers CD40 & CD80 as DC migrate from blood, to fluid & finally to the synovial tissue (p<0.05; p<0.05). This is consistent with our data showing that RA patients have a significant decrease (p<0.05) in CD11c mDC circulating in PB compared to age matched HC. A significant increase in CD141⁺ DC in the SF compared to PB (p<0.005) was demonstrated. This DC population has not been previously identified in the joint.  Furthermore analysis of RA synovial tissue DC demonstrated subpopulations of DC. CD1c DC were identified in the tissue however no CD141 DC were present which is in contrast to a strong CD141 population in the fluid. To mimic the joint microenvironment, MoDC were cultured in the presence of ECM or SF under normoxic or hypoxic conditions. A significant increase in CD80 expression in response to RA ECM or RASF was demonstrated (p<0.05; p<0.0001 respectively), furthermore hypoxia significantly potentiated the effect of ECM on CD80, CD40 & CD83 compared to basal (all p<0.0001). Finally, enhanced IFNγ production (p<0.05) & T cell proliferation was demonstrated in SF MoDC & CD4⁺ T cells co-cultures compared media control.

Conclusion: In this study we identified differential DC subpopulations in the inflamed joint compared to systemic circulation. These cells display a more activated phenotype & can induce T cell function, effects of which are influenced by the RA joint microenvironment, through increased proinflammatory mediators & through the hypoxic nature of the joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/investigating-the-role-of-dendritic-cell-maturation-t-cell-activation-within-the-inflamed-synovium-in-rheumatoid-arthritis/