Supplementary Material for: Mesenchymal stromal cells from human Wharton’s jelly modulate the intraocular immune response in a glucocorticoid hypertension model: an exploratory analysis

Introduction: Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells. Recent research suggests that immunological changes such as cytokine imbalance may play a role in its pathophysiology. This implies that immunomodulation, similar to that produced by mesenchymal cells, could be a potential therapeutic avenue for this disease. However, the effects of intravitreal injections of human Wharton’s jelly derived mesenchymal stromal cells(hWJ-MSCs) on intraocular immune response have not been assessed in ocular hypertension (OH) models. Methods: We measured explored this by measuring cytokine levels and expression of other markers, such as glial fibrillary acidic protein (GFAP) and T cells, in 15 randomly divided New Zealand rabbits: G1: OH; G2: hWJ-MSCs; and G3: OH+hWJ-MSCs. We analyzed the aqueous humor (IL-6, IL-8, and TNF-α) and vitreous humor (IFN-γ, IL-10, and TGF-β) using ELISA and flow cytometry (cell populations), as well as TCD3+, TCD3+/TCD4+, and TCD3+/TCD8+ lymphocytes, and GFAP in the retina and optic nerve through immunohistochemistry. Results: We found a decrease in TNF-α, IL-6, IFN-γ, IL-10, and IL-8 in G3 compared to G1 and an increase in TGF-β in both G2 and G3. TCD3+ retinal infiltration in all groups was primarily TCD8+ rather than TCD4+ cells, and strong GFAP expression was observed in both the retina and optic nerves in all groups. Conclusion: Our results suggest that cellular and humoral immune responses may play a role in glaucomatous optic neuropathy, and that intravitreal hWJ-MSCs can induce an immunosuppressive environment by inhibiting proinflammatory cytokines and enhancing regulatory cytokines.

引言：青光眼是一类以视网膜神经节细胞丢失为特征的神经退行性疾病。近期研究表明，细胞因子失衡等免疫变化可能在其病理生理过程中发挥作用。这提示，类似间充质细胞介导的免疫调节，或可成为该疾病的潜在治疗策略。然而，针对人沃顿胶源间充质基质细胞（human Wharton’s jelly derived mesenchymal stromal cells，hWJ-MSCs）玻璃体内注射对高眼压症（ocular hypertension，OH）模型眼内免疫应答的影响，目前尚未有相关评估。方法：本研究以15只随机分组的新西兰兔为研究对象，分为三组：G1组（高眼压模型组，OH）、G2组（人沃顿胶源间充质基质细胞注射组，hWJ-MSCs）及G3组（高眼压+人沃顿胶源间充质基质细胞注射组，OH+hWJ-MSCs），通过检测细胞因子水平及其他标志物表达探究上述影响。具体检测内容包括：采用酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）与流式细胞术（flow cytometry，分析细胞群）检测房水白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、肿瘤坏死因子-α（TNF-α）以及玻璃体干扰素-γ（IFN-γ）、白细胞介素-10（IL-10）、转化生长因子-β（TGF-β）的水平；通过免疫组织化学法检测视网膜与视神经中的CD3+T淋巴细胞、CD3+/CD4+淋巴细胞、CD3+/CD8+淋巴细胞，以及胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）的表达。结果：与G1组相比，G3组的TNF-α、IL-6、IFN-γ、IL-10及IL-8水平均有所降低；而G2组与G3组的TGF-β水平均有所升高。各组视网膜浸润的T淋巴细胞以CD8+细胞为主，而非CD4+细胞；且所有组的视网膜与视神经中均观察到较强的GFAP表达。结论：本研究结果表明，细胞免疫与体液免疫应答可能参与青光眼性视神经病变的发生发展；玻璃体内注射hWJ-MSCs可通过抑制促炎细胞因子、上调调节性细胞因子，诱导产生免疫抑制微环境。