Immunometabolic profiling of juvenile idiopathic arthritis patients reveals arginase-independent blockade of synovial T cells
Background: Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic disorder featuring inflamed joints and degradation of synovial tissue. Autoimmune complications are seen in 30-50% of JIA patients who develop anti-nuclear antibodies (ANA), which can result in uveitis and vision loss. Although systemic pro-inflammatory signals have been implicated in JIA pathogenesis, relatively little is known about the local immunometabolic poise in the inflamed synovium itself. Approach: To shed light into immunopathogenesis of JIA, we collected synovial fluid (SF) from the knee (n = 17) and venous blood (n = 11) of adolescent JIA patients ( n = 16) at 20 individual clinic visits. SF and blood leukocytes were analyzed by flow cytometry, and SF fluid and plasma used for 20-plex mediator quantification and metabolomics. Healthy donor PBMCs were cultured with anti-CD3/CD28 and IL-2 with or without SF to assess the impact of the fluid on T-cell activation. Results: Compared to blood, SF neutrophils showed increased degranulation, and both SF neutrophils and macrophages had increased surface-bound arginase-1 (Arg1), a myeloid cell-derived T-cell inhibitory enzyme. In addition, the neutrophil: T-cell ratio in SF was higher in ANA-negative patients, suggesting that neutrophil dominance in inflamed JIA joints may impede autoantibody production. In vitro, T cells cultured in SF showed impaired proliferation and upregulation of activation markers CD25 and CD69. Interestingly, T-cell blockade was similar in SF from patients who were neutrophil-high and ANA-negative and those who were neutrophil-low and ANA-positive. Furthermore, levels of arginine and ornithine (substrate and product of Arg1) did not differ in SF from ANA-positive and negative patients. Similarly, levels of tryptophan and kynurenine, the substrate and product of indolamine deoxygenase-1 (IDO-1, another T-cell inhibitory enzyme) did not differ in SF from ANA-positive and negative patients. Conclusion: Collectively, our findings show that conventional T-cell activation is blocked in SF from JIA patients. Even though ANA-negative JIA patients tend to present with neutrophil- and Arg1-rich SF, T-cell blockade therein is not mediated by immunometabolic routes controlled by Arg1. Future studies should investigate alternative pathways that may control the balance between neutrophils and T cells in the complex joint microenvironment of JIA patients.
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FLOW CYTOMETRY DATA Data acquisition: Cells were stained with antibodies following blocking of Fc receptors and application of live/dead stain. Cells were fixed using BD Phosflow Lyse/Fix and acquired on a BD LSRII or Symphony. Data analysis: FCS files were analyzed using FlowJo v9.9.5 and data was exported to be organized in Microsoft Excel The excel file contains 4 sheets with data: patient samples, in vitro_pooled SF, in vitro_neutrophil low vs high, and in vitro_SF washout Sheet 1, Patient samples -in section 1 for leukocyte frequencies, plot the blood and SF frequencies for each cell type. compare blood vs SF for each using Mann-Whitney test -in section 2 for Neutrophil:T cell ratio, separate the ratios by ANA status (+ or -). compare by Mann Whitney test -in section 3 for surface markers, plot blood and SF for each marker with each cell type. compare by wilcoxon matched pairs signed rank test Sheet 2, in vitro pooled SF for each marker with each cell type, plot the 4 culture conditions: media, media stim, SF, and SF stim perform the following comparisons using wilcoxon matched pairs signed rank test: media vs media stim, SF vs SF stim, media vs SF, and media stim vs SF stim Sheet 3, in vitro neutrophil low vs neutrophil high -for each marker with each cell type, plot the 4 culture conditions: PMN low unstim, PMN low stim, PMN high unstim, PMN high stim perform comparisons in the same way as for sheet 2 sheet 4, in vitro SF washout -for each marker with each cell type, plot the 4 culture conditions: control unstim and stim, and SF washout unstim and stim -statistical comparison was not performed due to limited number of replicates CYTOKINE DATA Data acquisition: A 20-plex cytokine detection kit (UPLEX, Meso Scale Diagnostics) was used to quantify cytokines in plasma and SF Data analysis: Plates were read using a SQ120 plate reader. Files were analyzed using Discovery Workbench and data was exported to Excel The excel file contains 4 sheets with data: correlations_plasma, correlations_SF, ANA, and paired plasma+SF Sheet 1 and Sheet 2 -conduct Spearman's correlation between cell type frequencies and cytokine concentration Sheet 3 compare concentrations of the included cytokines for ANA- vs ANA+ subjects by Mann Whitney test Sheet 4 compare concentrations of each cytokine between plasma and SF by wilcoxon matched pairs signed rank test. Exclude statistical comparison for IL-1a, IL-1b, and GM-CSF due to many points being below limit of detection (shown in red) MASS SPECTROMETRY DATA Data acquisition: Targeted Orbitrap MS with stable isotope dilution was performed on plasma and SF metabolite extracts Data analysis: plot data for ANA- and ANA+ subjects in both plasma and SF for each analyte compare concentration between ANA- and ANA+ in plasma and in SF by Mann Whitney test