DataSheet_1_IL-40: A New B Cell-Associated Cytokine Up-Regulated in Rheumatoid Arthritis Decreases Following the Rituximab Therapy and Correlates With Disease Activity, Autoantibodies, and NETosis.docx

BackgroundInterleukin 40 (IL-40) is a newly identified B cell-associated cytokine implicated in humoral immune responses and B cell homeostasis. As B cells play a pivotal role in autoimmunity, we investigated the function of IL-40 in rheumatoid arthritis (RA).
MethodsIL-40 expression was determined in the synovial tissue from RA and osteoarthritis (OA) patients. IL-40 was analysed in the serum/synovial fluid of patients with RA (n=50), systemic lupus erythematosus (SLE, n=69), OA (n=44), and healthy controls (HC, n=50). We assessed the changes of IL-40 levels in RA patients following the B cell depletion by rituximab (n=29) or after the TNF inhibition by adalimumab (n=25). We examined the relationship between IL-40, disease activity, autoantibodies, cytokines, and NETosis markers. Effect of IL-40 on synovial fibroblasts was determined.
ResultsIL-40 was overexpressed in RA synovial tissue, particularly by synovial lining and infiltrating immune cells. The levels of IL-40 were up-regulated in the synovial fluid of RA versus OA patients (p<0.0001). Similarly, IL-40 was increased in the serum of RA patients compared to HC, OA, or SLE (p<0.0001 for all) and decreased after 16 and 24 weeks (p<0.01 and p<0.01) following rituximab treatment. No significant effect of adalimumab on IL-40 was observed. IL-40 levels in RA patients correlated with rheumatoid factor-IgM and anti-cyclic citrullinated peptides (anti-CCP) in the serum (p<0.0001 and p<0.01), as well as in the synovial fluid (p<0.0001 and p<0.001). Synovial fluid IL-40 was also associated with disease activity score DAS28 (p<0.05), synovial fluid leukocyte count (p<0.01), neutrophil attractants IL-8 (p<0.01), MIP-1α (p<0.01), and markers of neutrophil extracellular traps externalization (NETosis) such as proteinase 3 (p<0.0001) and neutrophil elastase (p<0.0001). Synovial fibroblasts exposed to IL-40 increased the secretion of IL-8 (p<0.01), MCP-1 (p<0.05), and MMP-13 (p<0.01) compared to the unstimulated cells.
ConclusionsWe show the up-regulation of IL-40 in RA and its decrease following B cell depleting therapy. The association of IL-40 with autoantibodies, chemokines, and markers of NETosis may imply its potential involvement in RA development. Moreover, IL-40 up-regulates the secretion of chemokines and MMP-13 in synovial fibroblasts, indicating its role in the regulation of inflammation and tissue destruction in RA.

背景
白细胞介素40（Interleukin 40，IL-40）是新近发现的一种B细胞相关细胞因子，参与体液免疫应答与B细胞稳态维持。鉴于B细胞在自身免疫性疾病中发挥关键作用，本研究探讨了白细胞介素40在类风湿关节炎（rheumatoid arthritis，RA）中的功能。

方法
本研究检测了类风湿关节炎（RA）与骨关节炎（osteoarthritis，OA）患者的滑膜组织中白细胞介素40的表达水平。同时分析了RA患者（n=50）、系统性红斑狼疮（systemic lupus erythematosus，SLE，n=69）、OA患者（n=44）及健康对照（healthy controls，HC，n=50）的血清/滑膜液中的白细胞介素40水平。我们评估了接受利妥昔单抗（rituximab，n=29）B细胞耗竭治疗，或接受阿达木单抗（adalimumab，n=25）肿瘤坏死因子（tumor necrosis factor，TNF）抑制治疗的RA患者体内白细胞介素40水平的变化。本研究还检测了白细胞介素40与疾病活动度、自身抗体、细胞因子及中性粒细胞胞外陷阱形成（NETosis）标志物之间的相关性，并明确了白细胞介素40对滑膜成纤维细胞的作用。

结果
本研究发现，白细胞介素40在RA患者的滑膜组织中呈高表达，尤其富集于滑膜衬里层及浸润的免疫细胞中。与OA患者相比，RA患者滑膜液中的白细胞介素40水平显著上调（p<0.0001）。类似地，与健康对照、OA患者或SLE患者相比，RA患者血清中的白细胞介素40水平亦显著升高（所有组间比较p<0.0001），且在利妥昔单抗治疗后16周及24周时，该水平明显下降（p<0.01及p<0.01）。阿达木单抗治疗对白细胞介素40水平无显著影响。RA患者血清中的白细胞介素40水平与类风湿因子-IgM及抗环瓜氨酸肽（anti-cyclic citrullinated peptides，anti-CCP）抗体呈显著相关（p<0.0001及p<0.01），滑膜液中的白细胞介素40水平亦与上述两种抗体相关（p<0.0001及p<0.001）。滑膜液中的白细胞介素40水平还与疾病活动度评分DAS28（p<0.05）、滑膜液白细胞计数（p<0.01）、中性粒细胞趋化因子IL-8（p<0.01）、巨噬细胞炎症蛋白1α（MIP-1α，p<0.01）及中性粒细胞胞外陷阱形成标志物（如蛋白酶3（proteinase 3）及中性粒细胞弹性蛋白酶（neutrophil elastase），二者p<0.0001）呈显著相关。与未受刺激的对照组细胞相比，经白细胞介素40处理的滑膜成纤维细胞分泌的IL-8（p<0.01）、单核细胞趋化蛋白1（MCP-1，p<0.05）及基质金属蛋白酶13（MMP-13，p<0.01）水平均显著升高。

结论
本研究证实，白细胞介素40在RA患者体内呈高表达，且其水平在B细胞耗竭治疗后出现下降。白细胞介素40与自身抗体、趋化因子及中性粒细胞胞外陷阱形成标志物的相关性，提示其可能参与RA的发病进程。此外，白细胞介素40可上调滑膜成纤维细胞分泌趋化因子及基质金属蛋白酶13，表明其在RA的炎症调控与组织破坏过程中发挥重要作用。