背景：

       系统性红斑狼疮（Systemic lupus erythematosus，SLE）是一种可导致多器官损伤的自身免疫性疾病，免疫细胞的异常活化及自身抗体的产生是SLE发病的特点。SLE的发病机制仍未完全阐明，尚无根治SLE的有效方法。因此，深入探究SLE发病的机制，并探索新的治疗策略，仍然是该领域的核心挑战。

      肠道作为人体重要的免疫器官，其上皮层内富集的免疫细胞与上皮细胞共同构成肠道屏障，抵御病原体入侵。肠道免疫微环境紊乱已被证实参与乳糜泻等多种自身免疫性疾病的发生，但其在SLE发病中的作用及机制仍不明确。

目的：

       解析SLE模型鼠肠道上皮层免疫细胞的组成及功能改变，阐明肠道免疫微环境的紊乱在SLE模型鼠发病中的作用及机制。

方法：

       1. 基于SLE与乳糜泻患者全基因组关联分析数据，采用孟德尔随机化（Mendelian Randomization，MR）分析SLE与乳糜泻之间发病的关联。

       2. 采用葡聚糖硫酸钠（Dextran sodium sulfate，DSS）预先破坏小鼠肠道屏障，

并建立BM12诱导狼疮鼠模型，观察肠道屏障的破坏对小鼠发病的影响。

       3. 使用单细胞测序（Single-cell RNA sequencing，scRNA-seq）解析自发性SLE模型MRL/lpr（Lpr）鼠肠道上皮内淋巴细胞（Intraepithelial lymphocytes，IEL）及基质细胞的组成及功能改变，并分析细胞间通讯网络。流式细胞术用于验证scRNA-seq分析结果。

       4. 基于scRNA-seq分析Lpr鼠肠道γδT细胞中差异表达基因，饮水补硒观察抗氧化应激对Lpr鼠肠道γδT细胞、肠道屏障及小鼠发病的影响。联合干扰素γ（interferon γ，IFNγ）刺激及芳香烃受体（Aryl hydrocarbon receptor，AHR）抑制剂处理，体外验证AHR过度活化的分子机制。Zz

结果：

       1. 基于人群数据的MR分析表明SLE显著增加了乳糜泻的发病风险（比值比（Odds Ratio，OR）=1.24，95%可信区间（Confidence Interval，CI）=1.03-1.49，P=0.022）。反之，乳糜泻也显著增加了SLE的发病风险（OR=1.51，95%CI=1.40-1.64，P=4.9×10^-25）。

       2. 与对照组相比，DSS引起BM12诱导狼疮鼠肠道屏障破坏后，脾脏中CD4⁺ 效应T细胞及记忆性T细胞、Th1细胞、记忆性B细胞显著扩增，小鼠肾脏损伤加重，而野生对照鼠经DSS破坏肠道屏障未出现类似表型。

       3. Lpr鼠肠上皮内CD8⁺ IEL比例失调，总的CD8αα⁺ IEL显著减少，CD8αβ⁺ IEL显著增多，并鉴定出效应功能增强的扩增新亚群KLRC⁺ CD8αα，拟时序分析表明该亚群的分化可能受转录因子ZEB2调控；γδT细胞在多个SLE模型鼠肠道上皮层中显著减少；此外，Lpr鼠肠上皮细胞屏障维护功能减弱，且免疫细胞-基质细胞互作网络发生改变。

       4. Lpr鼠肠道γδT细胞中AHR抑制因子AHRR表达下调，与肠道IFNγ增多有关。AHRR下调导致AHR过度活化并引起氧化应激，进而导致Lpr鼠肠道γδT细胞减少，肠道屏障破坏。补硒抗氧化应激可以有效抑制γδT细胞减少，并改善肠道屏障及Lpr鼠发病。

结论：

       1. SLE与肠道自身免疫性疾病乳糜泻之间存在双向发病风险关联。

       2. DSS诱导肠道屏障破坏加剧了BM12诱导狼疮鼠肾脏组织损伤，而野生对照鼠在诱导肠道屏障破坏后不会引起类似损害。

       3. Lpr鼠肠道CD8⁺ T细胞亚群比例显著失调，细胞间通讯改变，肠道上皮细胞屏障维护功能减弱；肠道γδT细胞在多个SLE模型鼠中显著减少，与IFNγ-AHRR/AHR轴介导的氧化应激有关，补硒抗氧化应激可以有效抑制γδT细胞减少、改善肠道屏障及Lpr鼠发病。

       总之，本研究通过对SLE模型鼠肠道免疫微环境的初步解析与探索，为揭示肠道免疫失调在SLE发病中的作用奠定重要基础。

Background:

       Systemic lupus erythematosus (SLE) is an autoimmune disease that can lead to multi-organ damage, characterized by abnormal activation of immune cells and the production of autoantibodies. The pathogenesis of SLE remains incompletely understood, and there is currently no effective cure for the disease. Therefore, further exploration of the mechanisms underlying SLE pathogenesis and the development of new therapeutic strategies remain central challenges in the field.

       The gut, as a crucial immune organ in the human body, houses immune cells within its epithelial layer, which, together with epithelial cells, form the intestinal barrier that defends against pathogen invasion. Disruption of the intestinal immune microenvironment has been confirmed to play a role in the onset of several autoimmune diseases, such as Coeliac disease, but its involvement and mechanisms in SLE pathogenesis remain unclear.

Objective:

       To analyze the composition and functional alterations of immune cells in the intestinal epithelial layer of SLE model mice and elucidate the role and mechanisms of intestinal immune microenvironment disruption in SLE pathogenesis.

Methods:

       1. Based on the genome-wide association analysis data of SLE and Coeliac disease patients, Mendelian randomization (MR) analysis was used to investigate the association between SLE and Coeliac disease onset.

       2. Dextran sodium sulfate (DSS) was used to pre-damage the intestinal barrier of mice, and the BM12-induced lupus mouse model was established to observe the impact of intestinal barrier damage on disease development.

       3. Single-cell RNA sequencing (scRNA-seq) was used to analyze the composition and functional changes of intraepithelial lymphocytes (IEL) and structural cells in the intestinal epithelium of SLE model MRL/lpr (Lpr) mice, and intercellular communication networks were analyzed. Flow cytometry was used to validate the scRNA-seq results.

       4. Differentially expressed genes in the γδT cells from the Lpr mouse intestine were analyzed based on scRNA-seq data. Selenium supplementation was used to observe the effects of antioxidant on γδT cells, intestinal barrier function, and disease development in Lpr mice. In vitro validation of AHR overactivation was performed using IFNγ stimulation and AHR inhibitor treatment.

Results:

       1. MR analysis based on population data showed that SLE significantly increased the risk of developing Coeliac disease (Odds Ratio [OR] = 1.24, 95% Confidence Interval [CI] = 1.03-1.49, P = 0.022), and vice versa, Coeliac disease also significantly increased the risk of SLE (OR = 1.51, 95% CI = 1.40-1.64, P = 4.9×10^-25).

       2. Compared with the control group, DSS treatment-induced destruction of the intestinal barrier in BM12-induced lupus mice significantly increased the expansion of CD4⁺ effector T cells, memory T cells, Th1 cells, and memory B cells in the spleen, exacerbating renal damage, whereas no similar phenotype was observed in healthy mice after DSS-induced intestinal barrier damage.

       3. In the Lpr mouse intestine, the proportion of CD8⁺ IELs was significantly imbalanced, with a notable decrease in CD8αα⁺ IELs and an increase in CD8αβ⁺ IELs. A new subpopulation of KLRC⁺ CD8αα with enhanced effector function was identified. Pseudo-time analysis revealed that differentiation of this subpopulation was regulated by the transcription factor ZEB2. γδT cells were significantly reduced in the intestines of multiple SLE model mice. Additionally, intestinal epithelial cells in Lpr mice showed weakened intestinal barrier maintenance, and the immune cell-structural cell interaction network was altered.

       4. In Lpr mouse intestinal γδT cells, the expression of AHR repressor AHRR was downregulated, which was associated with increased intestinal IFNγ levels. Downregulation of AHRR led to excessive activation of AHR and induced oxidative stress, resulting in a reduction in intestinal γδT cells and disruption of the intestinal barrier. Selenium supplementation alleviated the reduction of γδT cells, improved intestinal barrier function, and alleviated disease in Lpr mice.

Conclusion:

       1. There is a bidirectional risk association between SLE and the intestinal autoimmune disease Coeliac disease.

       2. DSS-induced intestinal barrier disruption exacerbates renal tissue damage in BM12-induced lupus mice, whereas no similar damage occurs in healthy mice after intestinal barrier disruption.

       3. The proportion of CD8⁺ T cell subpopulations is significantly imbalanced in the Lpr mouse intestine, with changes in intercellular communication and weakened intestinal epithelial barrier maintenance. Intestinal γδT cells are significantly reduced in multiple SLE model mice, which is associated with IFNγ-AHRR/AHR-mediated oxidative stress. Selenium supplementation can effectively inhibit the reduction of γδT cells, improve intestinal barrier function, and alleviate disease in mice.

       In summary, this study provides new insights into the pathogenesis of SLE mediated by intestinal immune dysregulation through the analysis and exploration of the intestinal immune microenvironment in SLE model mice.