Immunometabolic analysis of synovial fluid from juvenile idiopathic arthritis patients
Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic disorder. Polymorphonuclear neutrophils (PMNs) are present in JIA synovial fluid (SF), but with variable frequency. Besides, SF PMNs in JIA were previously shown to display high exocytic but low phagocytic and immunoregulatory activities. To further assess whether the degree of SF neutrophilia associated with altered immune responses in JIA, we collected SF and blood from 16 adolescent JIA patients. SF and blood leukocytes were analyzed by flow cytometry. SF and plasma were used for immune mediator quantification and metabolomics. Healthy donor blood T cells were cultured in SF to evaluate its immunoregulatory activities. PMN and T-cell frequencies were bimodal in JIA SF, delineating PMN high / T-cell low (PMNHigh) and PMN low / T-cell high (PMNLow) samples. Pro-inflammatory mediators were increased in SF compared to plasma across patients, and pro- and anti-inflammatory mediators were further elevated in PMNHigh SF. Compared to blood, SF PMNs showed increased exocytosis and PD-1 / PD-L1 expression, and SF PMNs and monocytes/macrophages had increased surface-bound arginase-1. SPADE analysis revealed SF monocyte/macrophage subpopulations coexpressing PD-1 and PD-L1, with higher expression in PMNHigh SF. Healthy donor T cells showed reduced coreceptor expression when stimulated in PMNHigh vs. PMNLow SF. However, amino acid metabolites related to arginase-1 and IDO-1 pathways did not differ between the two groups. Hence, PMN predominance in the SF of a subset of JIA patients is associated with elevated immune mediator concentration and may alter SF monocyte/macrophage phenotype and T-cell activation, without altering immunoregulatory amino acids.
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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 2 sheets with data: patient samples and in vitro_neutrophil low vs high 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 neutrophil low vs neutrophil high -for each marker with each cell type, plot the freshly isolated PBMC data, the 2 control culture conditions (unstimulated and stimulate) and the 4 SF culture conditions: PMN low unstim, PMN low stim, PMN high unstim, PMN high stim -compare columns in the following pairs using the wilcoxon matched pairs signed rank test: 4+5, 6+7, 4+6, 5+7 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 5 sheets: workflow, correlations table_plasma, correlations table_SF, neutrophil low vs high, and paired plasma+SF Sheet 2 and Sheet 3 -conduct Spearman's correlation between cell type frequencies and cytokine concentration. The calculated p values and coefficients are provided in the separate excel file called "cytokine correlations_pvalues and coefficients" Sheet 4 compare concentrations of the included cytokines for neutrophil low vs high subjects by Mann Whitney test Sheet 5 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: The excel file contains 2 sheets: Neutrophil low vs high and total plasma vs SF Sheet 1 plot data for neutrophil low vs high subjects in both plasma and SF for each analyte Then compare concentration between neutrophil low vs high in plasma and in SF using Mann Whitney test Sheet 2 plot the total plasma vs SF concentrations and compare by Mann-Whitney test. Then plot the paired plasma and SF for Arginine and compare by Wilcoxon matched pairs test