Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF driven spondyloarthritis [II]. Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF driven spondyloarthritis [II]

Objectives: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn’s-like ileitis and concomitant arthritis. Methods: RNA-sequencing (RNA-seq) and flow cytometry were performed on inflamed gut and joint samples and tissue-derived Tregs from TNF∆ARE mice. In situ hybridization of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble(s) TNFR levels were measured in serum of mice and SpA patients and controls. Treg function was explored by in vitro co-cultures and in vivo by conditional Treg depletion. Results: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 mRNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in SpA patients with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue restricted TNFSF receptor and p38MAPK gene expression. Conclusions: These data point to profound differences in immune-regulation between Crohn’s ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure. Overall design: Ileum and synovium samples from TNFdARE and littermate control WT mice (n=3 per strain) were collected, RNA was isolated and processed for bulk RNA-seq. A comparative gene expression profiling analysis of RNA-seq data of ileum and synovium samples was performed, comparing TNFdARE and WT conditions.

研究目的：肠道与关节炎症常伴发于脊柱关节炎（spondyloarthritis, SpA），该现象极大限制了临床治疗方案的选择。然而，肠道与关节免疫调控差异背后的免疫生物学机制仍尚未明确。本研究旨在评估CD4+FOXP3+调节性T细胞（CD4+FOXP3+ regulatory T, Treg）在克罗恩样回肠炎伴随性关节炎模型中的免疫调控作用。

研究方法：对TNFΔARE小鼠的炎症性肠道与关节样本及组织来源的调节性T细胞开展RNA测序（RNA-sequencing, RNA-seq）与流式细胞术检测。针对人类脊柱关节炎肠道活检标本，实施肿瘤坏死因子（tumor necrosis factor, TNF）及其受体（tumor necrosis factor receptor, TNFR）的原位杂交实验。检测小鼠、脊柱关节炎患者及健康对照血清中的可溶性肿瘤坏死因子受体（soluble tumor necrosis factor receptor, sTNFR）水平。通过体外共培养实验探究调节性T细胞的功能，并借助条件性调节性T细胞清除实验完成体内验证。

研究结果：慢性肿瘤坏死因子暴露以位点特异性方式在滑膜与回肠中诱导了多个肿瘤坏死因子超家族（tumor necrosis factor superfamily, TNFSF）成员（4-1BBL、TWEAK及TRAIL）。在TNFΔARE/+小鼠中检测到肿瘤坏死因子受体2（TNFR2）mRNA水平升高，进而导致可溶性肿瘤坏死因子受体2（sTNFR2）释放增加。同样，伴有肠道炎症的脊柱关节炎患者的sTNFR2水平更高，且与炎症性疾病患者及健康对照存在显著差异。TNFΔARE小鼠的肠道与关节部位均出现调节性T细胞聚集，但滑膜中的调节性T细胞的TNFR2表达与抑制功能显著低于回肠部位的调节性T细胞。与此一致，滑膜与肠道调节性T细胞呈现出独特的转录谱，其肿瘤坏死因子超家族受体及p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38MAPK）基因表达具有组织限制性。

研究结论：本研究数据表明，克罗恩回肠炎与外周关节炎之间的免疫调控存在显著差异。调节性T细胞可调控回肠炎，但无法抑制关节炎症。滑膜驻留的调节性T细胞尤其无法适应慢性肿瘤坏死因子暴露。

实验整体设计：收集TNFΔARE小鼠及同窝野生型（wild type, WT）对照小鼠的回肠与滑膜样本（每组3只），分离RNA并进行批量RNA测序（bulk RNA-seq）。对回肠与滑膜样本的RNA测序数据开展比较基因表达谱分析，对比TNFΔARE与野生型小鼠的实验条件。