探討敲除CD4+ T細胞中C1GALT1對類風濕性關節炎小鼠模型的治療效果

Abstract

類風濕性關節炎（Rheumatoid arthritis, RA）是一種慢性自體免疫疾病，由關節滑膜的慢性發炎所引起。RA的發病機制涉及T細胞的活化和促發炎因子的分泌，如GM-CSF、IL-17A、IL-17F和IL-23等，這些促發炎因子促進Th17細胞分化和增值，導致軟骨侵蝕、硬骨侵蝕以及免疫細胞浸潤，最終導致關節結構受損。近年研究發現異常的醣基化與自體免疫疾病的誘發有關，C1GALT1 (Core 1 beta1,3-galactosyltransferase, Core 1 synthase) 是使氧型醣基化中的醣類結構產生分支和複雜度的關鍵酵素。然而，C1GALT1在RA發病機制中的調控機制和影響仍不清楚。本研究旨在探討C1GALT1在RA發病機制中的作用以及對T細胞分化的影響。首先，我們使用免疫組織化學染色法分析了膠原抗體誘導性關節炎（Collagen Antibody-Induced Arthritis, CAIA）小鼠脾臟中C1GALT1的蛋白表達，結果顯示C1GALT1在發炎反應中表達增加。而為了進一步探討C1GALT1對T細胞分化的影響，我們繁殖出了C1GALT1f/f CD4-Cre (C1GALT1 KO)小鼠，其C1GALT1基因在naïve T細胞分化為CD4+ T細胞的過程中被敲除，從而影響CD4+ T 細胞表面醣類結構的修飾。流式細胞儀分析C1GALT1f/f和C1GALT1 KO小鼠的脾臟、胸腺和淋巴結，發現敲除C1GALT1基因後VVA lectin（Vicia Villosa Lectin）的表達增加，表明CD4+ T細胞表面醣類結構發生了改變。此外，在小鼠誘導關節炎的實驗中發現，CD4+ T細胞特異性敲除C1GALT1改善了小鼠的踝關節腫脹、爪力強度以及臨床評分。並在骨表面掃描結果顯示C1GALT1 KO小鼠的腳掌骨表面骨侵蝕減緩。這些研究結果表明，在CAIA關節炎模型中，CD4+ T細胞特異性敲除C1GALT1可能減輕關節發炎所引起的踝關節腫脹，說明C1GALT1或許具有抑制發炎的潛力。瞭解C1GALT1在RA發病機制和T細胞分化中的作用，可以有助於提供醣基化治療RA和其他自體免疫疾病的方法。

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by chronic inflammation targeting synovial membrane in the joints, resulting in arthritis. The pathogenesis of RA involves T cell activation and the secretion of proinflammatory cytokines, such as GM-CSF, IL-17A, IL-17F, and IL-23. These proinflammatory cytokines promote T cell differentiation and proliferation, leading to cartilage and bone erosion, and immune cell infiltration, which ultimately result in damage to joint structures. Abnormal glycosylation has also been implicated in autoimmune diseases, with the glycosyltransferase C1GALT1 playing a critical role in O-glycosylation and glycan complexity. However, the regulatory mechanisms and impact of C1GALT1 on RA pathogenesis remain unclear. This study aimed to investigate the role of C1GALT1 in RA pathogenesis and its effect on T differentiation. Initially, we analyzed the protein expression of C1GALT1 in the spleens of Collagen Antibody-Induced Arthritis (CAIA) induced C57BL/6 mice using immunohistochemistry, revealing increased C1GALT1 expression in response to inflammation. To explore the influence of C1GALT1 on Th17 differentiation, we generated C1GALT1f/f CD4-Cre mice (C1GALT1 KO), in which the C1GALT1 gene was knocked out during the differentiation of naïve T cells into CD4+ T cells, impacting surface glycan modification. Flow cytometry analysis of spleens, thymus, and lymph nodes from C1GALT1f/f and C1GALT1f/f CD4-Cre mice showed increased lectin expression of VVA following C1GALT1 gene knockout, indicating altered O-glycans on the surface of CD4+ T cells. Furthermore, we employed the CAIA model to simulate RA in mice and compared C1GALT1f/f CD4-Cre mice to C1GALT1f/f mice. The results demonstrated that C1GALT1 knockout in T cells alleviated ankle swelling in the CAIA model, with reduced swelling observed from Day 6 to Day 10. Additionally, bone scan analysis revealed a deceleration of bone erosion on the paw bone surface in C1GALT1 KO. These findings suggest that T cell-specific knockout of C1GALT1 may mitigate ankle swelling in the CAIA model, emphasizing the crucial role of C1GALT1 in suppressing inflammation. Understanding the role of C1GALT1 in RA pathogenesis and T cell differentiation could provide insights into potential therapeutic strategies targeting glycosylation pathways for the treatment of RA and other autoimmune diseases.