Department of Gastroenterology, The Third Xiangya Hospital, Central South University Changsha, Hunan 410013, PR China; Hunan Key Laboratory of Non-resolving Inflammation and Cancer, Changsha, Hunan 410008, PR China; Cancer Research Institute of Central South University, Changsha, Hunan 410008, PR China.
Department of Gastroenterology, The Third Xiangya Hospital, Central South University Changsha, Hunan 410013, PR China.
EBioMedicine. 2022 Nov;85:104285. doi: 10.1016/j.ebiom.2022.104285. Epub 2022 Sep 28.
Crohn's disease (CD) is a chronic inflammatory disorder characterized by intestinal immune dysfunction. Multiple factors, including gut dysbiosis, are involved in the pathogenesis of CD. However, the effect of commensal bacteria on controlling the inflammatory response in individuals with CD remains unclear.
We detected Toll-like receptor 2 (TLR2), TLR4, and TLR5 expression in Roseburia intestinalis (R. intestinalis)-treated mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Then, we quantified the signs of colonic inflammation, the proportion of regulatory T cells (Tregs) and the expression of thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β in TLR-5-deficient (Tlr5) mice, bone marrow chimera mice (generated using wild-type (WT) and Tlr5 mice), and anti-TSLP/anti-TGFβ-treated C57BL/6 mice with colitis induced by TNBS. In vitro, we used the lipopolysaccharide (LPS)-stimulated human intestinal epithelial cell line Caco-2 as an inflammatory colon cell model treated with or without the TLR5-siRNA intervention in the presence of R. intestinalis and incubated human monocyte-derived dendritic cells (DCs) with the supernatant of Caco-2 cells. Then, we cocultured human CD4 T cells with the aforementioned DCs to determine the differentiation of Tregs. Additionally, samples from patients with CD were collected to analyse the correlation between TLR5/TSLP/TGFβ expression and the percentage of R. intestinalis.
Here, we show that R. intestinalis inhibits the development of CD by increasing the differentiation of anti-inflammatory Tregs. Mechanistically, R. intestinalis stimulates TSLP production in intestinal epithelial cells (IECs) through TLR5 but not TLR2 or TLR4. TSLP produced by IECs specifically induces the secretion of interleukin-10 (IL-10) and TGFβ from DCs, which is necessary for subsequent Treg differentiation. Consequently, the depletion of TLR5 (using Tlr5 mice) or inhibition of TSLP (using anti-TSLP neutralizing antibodies) attenuates the protective effect of R. intestinalis on experimental colitis in mice. Importantly, the expression of TSLP in patients with CD is positively correlated with the level of R. intestinalis.
These findings reveal the previously unknown mechanism of R. intestinalis-mediated intestinal immune regulation, which may provide the basis for new therapeutic strategies for CD.
This work was funded by the National Natural Science Foundation of China (81670504 and 81970494), the Key Project of Research and Development Plan of Hunan Province (2019SK2041) and the Changsha Municipal Natural Science Foundation (kq2014258).
克罗恩病（CD）是一种以肠道免疫功能障碍为特征的慢性炎症性疾病。多种因素，包括肠道菌群失调，参与了 CD 的发病机制。然而，共生菌对控制 CD 患者炎症反应的影响尚不清楚。
我们检测了 2,4,6-三硝基苯磺酸（TNBS）诱导结肠炎的罗斯伯里氏菌（R. intestinalis）处理的 TLR2、TLR4 和 TLR5 在小鼠中的表达。然后，我们在 TLR5 缺陷（Tlr5）小鼠、骨髓嵌合体小鼠（由野生型（WT）和 Tlr5 小鼠生成）和用 TNBS 诱导结肠炎的抗 TSLP/抗 TGFβ 处理的 C57BL/6 小鼠中定量评估结肠炎症的迹象、调节性 T 细胞（Tregs）的比例以及胸腺基质淋巴生成素（TSLP）和转化生长因子（TGF）-β的表达。在体外，我们使用脂多糖（LPS）刺激的人肠道上皮细胞系 Caco-2 作为炎症性结肠细胞模型，在存在 R. intestinalis 的情况下用或不用 TLR5-siRNA 干预，并将人单核细胞衍生的树突状细胞（DCs）与 Caco-2 细胞的上清液共培养。然后，我们将人 CD4 T 细胞与上述 DC 共培养，以确定 Tregs 的分化。此外，还收集了 CD 患者的样本，以分析 TLR5/TSLP/TGFβ 表达与 R. intestinalis 百分比之间的相关性。
在这里，我们表明 R. intestinalis 通过增加抗炎性 Tregs 的分化来抑制 CD 的发展。在机制上，R. intestinalis 通过 TLR5 而不是 TLR2 或 TLR4 刺激肠道上皮细胞（IECs）中 TSLP 的产生。IECs 产生的 TSLP 特异性诱导 DC 分泌白细胞介素-10（IL-10）和 TGFβ，这对于随后的 Treg 分化是必要的。因此，TLR5 的耗竭（使用 Tlr5 小鼠）或 TSLP 的抑制（使用抗 TSLP 中和抗体）减弱了 R. intestinalis 对小鼠实验性结肠炎的保护作用。重要的是，CD 患者 TSLP 的表达与 R. intestinalis 的水平呈正相关。
这些发现揭示了 R. intestinalis 介导的肠道免疫调节的先前未知机制，这可能为 CD 的新治疗策略提供依据。
本工作得到了国家自然科学基金（81670504 和 81970494）、湖南省重点研发计划（2019SK2041）和长沙市自然科学基金（kq2014258）的资助。
