Evaluating the inhibition of IL-17A and TNFα in a cartilage explant model cultured with Th17-derived cytokines

IF 4.7 Q2 IMMUNOLOGY
Solveig Skovlund Groen , Anne-Christine Bay-Jensen , Christian S. Thudium , Morten H. Dziegiel , Marie Skougaard , Simon Francis Thomsen , Signe Holm Nielsen
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引用次数: 0

Abstract

Introduction

T-helper 17 (Th17) cells produce IL-17A playing a critical role in activating the pathogenic chain leading to joint tissue inflammation and destruction. Elevated levels of Th17 cells and IL-17A have been detected in skin lesions, blood, and synovial fluid from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (AS). Moreover, IL-17A inhibitors suppress disease activity in psoriasis, PsA and AS, supporting the evidence of IL-17A contributing to the disease pathogenesis. Although, IL-17A inhibitors are widely approved, it remains unclear how the inhibitory effect of IL-17A alters the extracellular matrix (ECM) of the joint in a Th17-conditioned inflammatory milieu. Therefore, the aim of this study was to establish a cartilage model cultured with conditioned medium from Th17 cells and inhibitors to explore the effect of IL-17A inhibition on joint tissue remodeling.

Methods

Naïve CD4+ T cells from healthy human buffy coat were differentiated into Th17 cells, followed by Th17 cell activation to secrete Th17-related cytokines and molecules into media. The activated Th17 cells were isolated from the conditioned media (CM) and analyzed using flow cytometry to verify Th17 cell differentiation. The CM were assessed with ELISA to quantify the concentrations of cytokines secreted into the media by the Th17 cells. Healthy bovine cartilage explants were cultured with the Th17-CM and treated with IL-17A and TNFα inhibitors for 21 days. In harvested supernatant from the cartilage cultures, MMP- and ADAMTS-mediated biomarker fragments of type II collagen, aggrecan, and fibronectin were measured by ELISA to investigate the ECM remodeling within the cartilage tissue.

Results

Th17-CM stimulated a catabolic response in the cartilage. Markers of type II collagen and aggrecan degradation were upregulated, while anabolic marker of type II collagen formation remained on similar levels as the untreated explants. The addition of IL-17A inhibitor to Th17-CM decreased the elevated type II collagen and aggrecan degradation, however, degenerative levels were still elevated compared to untreated group. The addition of TNFα inhibitor completely reduced both type II collagen and aggrecan degradation compared to untreated explants. Moreover, the TNFα inhibitor treatment did not alter the type II collagen formation compared to untreated group.

Conclusion

This study suggests that inhibition of IL-17A in Th17-conditioned cartilage tissue only partially reduced the MMP-mediated type II collagen degradation and ADAMTS-mediated aggrecan degradation, while the TNFα inhibitor treatment fully reduced both MMP- and ADAMTS-mediated ECM degradation. This exploratory study where ECM biomarkers are combined with Th17-conditioned ex vivo model may hold great potential as output for describing joint disease mechanisms and predicting structural effects of treatment on joint tissue.

在使用 Th17 衍生细胞因子培养的软骨外植体模型中评估 IL-17A 和 TNFα 的抑制作用
导言T-17(Th17)辅助细胞产生的 IL-17A 在激活导致关节组织炎症和破坏的致病链中发挥着关键作用。在银屑病关节炎(PsA)和强直性脊柱炎(AS)患者的皮损、血液和滑液中检测到 Th17 细胞和 IL-17A 水平升高。此外,IL-17A 抑制剂可抑制银屑病、银屑病关节炎和强直性脊柱炎的疾病活动,支持 IL-17A 在疾病发病机制中发挥作用的证据。尽管IL-17A抑制剂已被广泛批准,但目前仍不清楚IL-17A的抑制作用如何改变Th17条件下炎症环境中关节的细胞外基质(ECM)。因此,本研究旨在建立一个用 Th17 细胞和抑制剂的条件培养基培养的软骨模型,以探讨 IL-17A 抑制对关节组织重塑的影响。方法将健康人水包衣中的幼稚 CD4+ T 细胞分化成 Th17 细胞,然后激活 Th17 细胞,使其向培养基中分泌 Th17 相关细胞因子和分子。从条件培养基(CM)中分离出活化的 Th17 细胞,并使用流式细胞术进行分析,以验证 Th17 细胞的分化。用酶联免疫吸附法(ELISA)对条件培养基进行评估,以量化 Th17 细胞分泌到培养基中的细胞因子浓度。用 Th17-CM 培养健康牛软骨外植体,并用 IL-17A 和 TNFα 抑制剂处理 21 天。在软骨培养物收获的上清液中,通过 ELISA 测量 MMP 和 ADAMTS 介导的 II 型胶原、凝集素和纤连蛋白的生物标志物片段,以研究软骨组织内的 ECM 重塑情况。II 型胶原和凝集素降解的标志物上调,而 II 型胶原形成的合成代谢标志物与未处理的外植体保持相似水平。在 Th17-CM 中添加 IL-17A 抑制剂可降低升高的 II 型胶原和凝集素降解,但与未处理组相比,退化水平仍然升高。与未经处理的外植体相比,TNFα抑制剂可完全减少II型胶原和骨凝胶的降解。此外,与未处理组相比,TNFα 抑制剂处理并没有改变 II 型胶原的形成。结论本研究表明,在 Th17 调理的软骨组织中抑制 IL-17A 只部分减少了 MMP 介导的 II 型胶原降解和 ADAMTS 介导的骨凝集素降解,而 TNFα 抑制剂处理则完全减少了 MMP 和 ADAMTS 介导的 ECM 降解。这项探索性研究将 ECM 生物标志物与 Th17 调节的体外模型相结合,在描述关节疾病机制和预测治疗对关节组织结构的影响方面具有很大的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Translational Autoimmunity
Journal of Translational Autoimmunity Medicine-Immunology and Allergy
CiteScore
7.80
自引率
2.60%
发文量
33
审稿时长
55 days
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