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.幼年特发性关节炎（Juvenile Idiopathic Arthritis, JIA）是一种炎症性风湿性疾病。多形核中性粒细胞（Polymorphonuclear Neutrophils, PMNs）可存在于JIA患者的滑液（Synovial Fluid, SF）中，但出现频率存在个体差异。既往研究显示，JIA患者滑液中的多形核中性粒细胞表现出较高的胞吐活性，但吞噬活性与免疫调节活性较低。为进一步评估滑液中性粒细胞增多程度是否与JIA患者体内免疫应答改变相关，本研究纳入16例青少年JIA患者，收集其滑液与血液样本。采用流式细胞术（Flow Cytometry）对滑液及血液中的白细胞进行分析；利用滑液与血浆开展免疫介质定量检测及代谢组学（Metabolomics）分析。将健康供者的血液T细胞在滑液中培养，以评估滑液的免疫调节活性。JIA患者滑液中的中性粒细胞与T细胞频率呈双峰分布，据此可将样本划分为中性粒细胞高/ T细胞低（PMNHigh）与中性粒细胞低/ T细胞高（PMNLow）两类。相较于患者血浆，滑液中的促炎介质水平显著升高；且在PMNHigh组滑液中，促炎与抗炎介质水平进一步提升。与血液样本相比，滑液中的中性粒细胞胞吐活性及PD-1/PD-L1（Programmed Death 1/Programmed Death Ligand 1）表达水平均有所升高；滑液中的中性粒细胞与单核细胞/巨噬细胞的表面结合型精氨酸酶-1（Arginase-1）表达水平亦显著上调。通过SPADE分析发现，滑液中的单核细胞/巨噬细胞亚群可共表达PD-1与PD-L1，且该亚群在PMNHigh组滑液中的表达水平更高。当在PMNHigh组滑液中培养健康供者T细胞时，其共受体表达水平较PMNLow组显著降低。然而，与精氨酸酶-1及IDO-1（Indoleamine 2,3-Dioxygenase 1）通路相关的氨基酸代谢物在两组间无显著差异。综上，部分JIA患者滑液中以中性粒细胞为主的微环境，与免疫介质浓度升高密切相关，并可能改变滑液内单核细胞/巨噬细胞表型及T细胞活化状态，但不会影响免疫调节相关氨基酸的代谢水平。