Background/Purpose: We have previously shown that synovial fluid mononuclear cells (SFMCs) of patients with Reactive arthritis (ReA) and undifferentiated Spondyloarthropathy (uSpA) proliferated to low molecular weight fractions of outer membrane proteins (OMP) of S typhimurium. Proteomic analysis of the the stimulatory fractions predicted to consist of 10 proteins including OMPA and OMPD . In this study, we examined the in vitroimmunogenecity of recombinant (r ) OMP A and D by measuring frequency of antigen specific CD4+ and CD8+ T cells against rOMP A and D  in the synovial fluid (SF) of patients with ReA/uSpA. Further, induction of proinflammatory cytokines (IFN- γ, IL-17 and IL-23 ) were measured by SFMC stimulated by rOMPA and rOMP D in these patients. 

Methods: Patients presenting with typical  clincial picture of asymmetric lower limb oligoarthritis with a history of  preceding diarrhoea within 4 weeks were labelled as Rea and uSpA patients fulfilled ESSG criteria. Synovial fluid were aspirated from inflammed knee joint prior to steroid injections and monnonuclear cells (SFMC) were isolated and cultured with crude extract of s typhimurium OMP and rOMPA/D for flow cytometry, ELISPOT and cytokines in the culture supernatant.  Intracellular IFN-γ and IL17 producing  SF T (CD4+CD69+ and CD8+ CD69+) cells was enumerated  by Flow cytometry (Beckman Coulter, USA)  Antigen specific IFN-γ producing T cells were also measured  by ELISPOT. A positive response was taken if the ratio of  % cells or mean spots/well with antigen/Control ≥ 3.  IFN-γ (BD,USA) , IL17 and IL23 (ebioscinces, USA) levels were measured by commericial ELISA in the culture supernatant of antigen treated SFMCs.

Results: Out of 20 patients recruited, 17 were males with a median age 27.6 years (range 15-65 year). Eleven patients were ReA. The median duration of current episode was 15 days (range 3-180) days. HLA B27 were positive in 13/19 patients. Thirteen out of 20 patients with ReA/uSpA, showed a CD8+CD69+IFN+ response to crude OMP suggesting  triggered by S. typhimurium. Of these 13, 9 patient’s SF CD8+CD69+T cells positive for intracellular γ-IFN in response to rOMPA as compared to 4 patients who responded to  rOMPD. The mean frequency (%) of antigen specific CD8+CD69+ T cells were 0.56±0.5 to rOMPA and  0.24±0.15 to rOMPD in comparison to blank 0.18±0.17.  Regarding CD4+T cell response, 6 out of 15 were postive against crude OMP and of these 4 responded to rOMPA and only 2 responded to rOMPD. By ELISPOT assay  13, 7 and 3 patients showed increased IFN-γ producing T cells to crude OMP, rOMPA and rOMPD respectively. Only 1 RA patients SF CD8+T cells  showed response to crude OMP. SFMC upon stimulation with rOMP A and not  rOMP D produce significant IFN-γ, IL-17 and Il-23 in comparison to non treated cells (Table)

Table .  Induction of  pro inflammatory cytokines by SFMC stimulated by rOMP A /D

Conclusion: OMPA is a major antigenic target in patients with Salmonella triggered ReA/uSpA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/synovial-fluid-t-cells-of-patients-with-reactive-arthritis-and-undifferentiated-spondyloarthropathy-respond-to-salmonella-typhimurium-outer-membrane-protein-a-and-induces-il17-and-il23-pro/