Increased inflammatory responsiveness of peripheral blood mononuclear cells (PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis

Background: Ankylosing spondylitis (AS) is a common debilitating rheumatic disease in which the innate immune components especially the Interleukin (IL)-23/IL-17 axis related genes play important role in its pathogenesis. Nucleotide binding oligomerization domain-containing protein (NOD)2, as an innate receptor, is critical for IL-23 production in cells. Therefore, we aimed to stimulate NOD2 signaling and study its effects on cytokine production in peripheral blood mononuclear cells (PBMC) of these patients. Methods: PBMCs from 18 patients with active AS and 18 healthy individuals were separated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of muramyl dipeptide (MDP), as NOD2 ligand. Quantitative expression analysis of NOD1, NOD2, RIPK2, SLC75A4, NLRP1, NLRP3, IL23A, IL17A, IL1B, and TNFA genes was performed using Real-time polymerase chain reaction (PCR). Finally, protein changes of IL23A and IL17A expression were validated using enzyme linked immunosorbent assay (ELISA). Results: Apart from NOD1 that tend to be downregulated in the controls, all the selected genes showed overexpression in response to MDP in cells from the studied groups. Except RIPK2, all the genes had higher expression changes upon MDP stimulation in the AS population. Overexpression of IL23A and IL17A were confirmed at protein levels using ELISA. The strong positive correlation between NLRP3 and NOD2 was decreased after stimulation but new correlations between NLRP3 and IL7B, RIPK2 and SLC15A4 were observed after treatment. Conclusions: This study indicated that AS PBMCs were hyper - responsive to MDP stimulation. This observation implies an important role of NOD2 in the pathogenesis of inflammatory diseases including AS.