Department of Gastroenterology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong, China.
Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, Guangdong, China.
Front Immunol. 2024 Apr 15;15:1303356. doi: 10.3389/fimmu.2024.1303356. eCollection 2024.
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined.
Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells.
In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened.
Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.
癌胚抗原相关细胞黏附分子 5（CEACAM5）作为一种典型的肿瘤标志物，在许多疾病中被发现具有免疫调节作用。我们之前报道了支持 SARS-CoV-2 感染胃肠道（GI）的临床和分子证据，并发现 COVID-19 患者粪便中的 CEACAM5 减少，这与肠道菌群失调有关。然而，CEACAM5 在 GI 感染中的作用仍不明确。
通过腹腔注射重组病毒刺突-Fc 建立小鼠模型，模拟肠道炎症。在注射刺突-Fc 或对照-Fc 后 6h、2d、4d 和 7d 收集十二指肠、空肠、回肠和结肠样本，进行蛋白质组学分析。从健康供体采集血液，通过密度梯度离心分离外周血单核细胞（PBMC），然后用磁珠分离 CD4+T 细胞，并与 Caco-2 细胞共培养。
与对照组相比，刺突-Fc 组除了肠道 CEACAM5 表达降低外，紧密连接和 CD4+T 淋巴细胞的百分比也明显降低（p<0.05），同时炎症因子水平升高。KEGG 分析显示差异表达蛋白主要富集在冠状病毒病（COVID-19）、紧密连接、焦点黏附、黏着连接和 PI3K-Akt 信号通路。蛋白质-蛋白质相互作用（PPI）网络分析确定了 CEACAM5 与半乳糖凝集素-9（Galectin-9）之间的相互作用，该相互作用也通过分子对接和 co-IP 实验得到了验证。我们进一步证实，SARS-CoV-2 刺突刺激肠上皮细胞后 CEACAM5 的减少可促进 CD4+T 细胞中 Galectin-9 蛋白的表达。然后通过抑制 PI3K/AKT/mTOR 通路，导致炎症因子释放增加和 CD4+T 细胞凋亡增加，最终导致肠道屏障功能障碍。
我们的研究结果表明，CEACAM5 过表达和 Galectin-9 敲低在刺突-Fc 刺激诱导的肠道屏障损伤中发挥保护作用。总之，我们的研究结果首次表明，SARS-CoV-2 刺突通过 CEACAM5 与 Galectin-9 的相互作用诱导肠道屏障功能障碍。该结果为治疗重症 COVID 患者的肠道屏障功能障碍提供了潜在的治疗靶点。
