Data_Sheet_1_Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-κB-Dependent Mechanism.PDF

Introduction: Adipose-derived mesenchymal stem cells (ADSC) have been shown to have remarkable immune-modulating effects. However, their efficacy in clinical trials has yet to be fully demonstrated. This could be due to a lack of a proper inflammatory environment in vivo that primes ADSC. Here, we define how the articular microenvironment of rheumatoid arthritis (RA) patients modulates the therapeutic efficiency of ADSC.
Methods: Synovial fluids (SF) were collected from 8 RA patients, 2 Spondyloarthritis patients and one control synovial fluid from a patient undergoing traumatic-related surgery. SF inflammatory status was determined by routine analysis and quantification of pro-inflammatory cytokines. ADSC were first treated with SF and ADSC proliferation and gene expression of immunomodulatory factors was evaluated. In order to determine the mechanisms underlying the effect of SF on ADSC, tumor necrosis factor (TNF), interleukin-6 (IL-6), and NF-κB neutralization assays were performed. To evaluate the effect of SF on ADSC functions, ADSC were pre-treated with SF and then co-cultured with either macrophages or T cells. The modulation of their phenotype was assessed by flow cytometry.
Results: Pro-inflammatory RASF maintained the proliferative capacity of ADSC and upregulated the gene expression of cyclooxygenase-2 (COX2), indoleamine-1,2-dioxygenase (IDO), interleukin-6 (IL-6), tumor-necrosis factor stimulated gene 6 (TSG6), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and programmed death-ligand 1 (PD-L1), all factors involved in ADSC immunomodulatory potential. The RASF-induced gene expression was mainly mediated by TNF alone or in combination with IL-6 and signaled through the NF-κB pathway. Conditioning ADSC with pro-inflammatory RASF enhanced their ability to induce CD4+Foxp3+CD25high regulatory T cells (Tregs) and inhibit pro-inflammatory markers CD40 and CD80 in activated macrophages.
Conclusions: Inflammatory synovial fluids from RA patients had the capacity to modulate ADSC response, to induce Tregs and modulate the phenotype of macrophages. The clinical use of ADSC in affected joints should take into account the influence of the local articular environment on their potential. Having a sufficient pro-inflammatory microenvironment will determine whether optimal immunoregulatory response should be expected. Direct ADSC intra-articular delivery to patients could be a potential strategy to properly prime their immunomodulatory potential and enhance their clinical benefits.

**引言**：脂肪间充质干细胞（Adipose-derived mesenchymal stem cells，ADSC）已被证实具有显著的免疫调节作用，但其在临床试验中的疗效尚未得到充分验证。这一现象可能源于体内缺乏预激活ADSC的适宜炎症环境。本研究阐明了类风湿关节炎（rheumatoid arthritis，RA）患者的关节微环境如何调控ADSC的治疗效能。

**方法**：本研究从8名RA患者、2名脊柱关节炎患者以及1名接受创伤相关手术的患者的对照滑液样本中采集滑液（Synovial fluids，SF）。通过常规分析及促炎细胞因子定量检测评估滑液的炎症状态。首先用滑液处理ADSC，随后评估ADSC的增殖能力及免疫调节因子的基因表达水平。为明确滑液调控ADSC的潜在机制，本研究开展了肿瘤坏死因子（tumor necrosis factor，TNF）、白细胞介素-6（interleukin-6，IL-6）及核因子κB（NF-κB）的中和实验。为评估滑液对ADSC功能的影响，本研究先用滑液预处理ADSC，随后将其分别与巨噬细胞或T细胞共培养，通过流式细胞术检测二者表型的变化。

**结果**：促炎性类风湿关节炎滑液可维持ADSC的增殖能力，并上调环氧合酶-2（cyclooxygenase-2，COX2）、吲哚胺2,3-双加氧酶（indoleamine-1,2-dioxygenase，IDO）、白细胞介素-6（IL-6）、肿瘤坏死因子诱导基因6（tumor-necrosis factor stimulated gene 6，TSG6）、细胞间黏附分子1（intercellular adhesion molecule 1，ICAM-1）、血管细胞黏附分子1（vascular cell adhesion molecule 1，VCAM-1）及程序性死亡受体配体1（programmed death-ligand 1，PD-L1）的基因表达，上述因子均与ADSC的免疫调节潜能相关。促炎性类风湿关节炎滑液诱导的基因表达主要由TNF单独介导或TNF与IL-6联合介导，并通过NF-κB通路传递信号。用促炎性类风湿关节炎滑液预处理ADSC，可增强其诱导CD4+Foxp3+CD25高表达调节性T细胞（Tregs）的能力，并抑制活化巨噬细胞中促炎标志物CD40与CD80的表达。

**结论**：RA患者的炎性滑液可调控ADSC的应答反应，诱导Tregs生成并调控巨噬细胞表型。在受累关节中临床应用ADSC时，应考虑局部关节微环境对其功能潜能的影响。充足的促炎性微环境是获得最佳免疫调节应答的关键。对患者直接实施ADSC关节腔内给药，或是适当预激活ADSC的免疫调节潜能，有望提升其临床获益。