1812: Inflammasome and UPR activation in monocytes of HLA B27 positive children with Enthesitis related arthritis category of JIA

Background/Purpose: Enthesitis related arthritis (ERA) is a chronic immune-inflammatory disease with unknown etiology. HLA-B27 is the strongest risk factor predisposing to ERA like Spondyloarthritis (SpA). In adult SpA, HLA-B27 has been associated with unfolded protein response (UPR) and inflammasome activation in monocytes. However, no data is available in ERA.

Methods: Patients with ERA and healthy controls (HC) were enrolled after informed consent. Monocytes from ERA patients and HC were separated from PBMCs/SFMCs using MACS columns. Purity and viability were checked by flow cytometry. Monocytes were stimulated with LPS (100ng/ml) and mRNA levels of TLR4, NLRP3, IL-1β, IL-18, BiP, sXBP1, CHOP, IRE1α and IL-23R were measured by qPCR. Protein levels of IL-1β and IL-18 in the culture supernatant were measured by ELISA. The caspase-1 activity was measured by flow cytometry using the Caspase 1 Kit.

Results: 12 HLA B27 positive, 6 HLA B27 negative ERA patients and 6 HLA B27 negative HC were included. Clinical features such as arthritis, enthesitis and sacroiliitis were more common in HLA-B27 positive ERA patients than in B27 negative patients (Table1). HLA B27 positive patients had higher disease activity compared with HLA B27 negative patients.



Monocytes of HLA-B27 positive ERA patients had more than 1.5-fold increase in mRNA expression of IL-1β, NLRP3, and TLR4 than HC. On the other hand, monocytes from HLA-B27 positive ERA patients also had more than 1.5-fold increase in mRNA expression of IL-18, NLRP3, and TLR4 as compared to monocytes from HLA-B27 negative patients. Monocytes from HLA B27 positive ERA patients produce more IL-1β than HLA B27 negative HC (p< 0.01). After LPS stimulation, there was further increase in mRNA expression of all the 4 genes and IL-1β production (p< 0.001). In comparison to HLA-B27 negative HC (p< 0.05) and HLA -B27 negative ERA patients (p< 0.05), the caspase expression was greater in HLA B27 positive ERA patients (Figure1). Expression of mRNA related to UPR activation (BiP and sXBP1, IRE1α) were similar in all 3 groups.



Paired analysis of monocytes from SFMCs of HLA B27 positive patients revealed 1.5-fold higher expression of IL-18, NLRP3, TLR4, BiP, sXBP 1, and IRE1α as compared to PBMCs. IL23R mRNA expression was only present in the monocytes isolated from SFMCs.

Conclusion: Activation of inflammasome pathway in the circulating monocytes and activation of both ER stress and inflammasome pathway at local site may contribute to higher inflammation burden and more severe disease in HLA-B27 positive ERA patients.