Lim Rayne R, Thomas Anju, Ramasubramanian Aparna, Chaurasia Shyam S
bioRxiv. 2024 Oct 31:2024.10.30.621160. doi: 10.1101/2024.10.30.621160.
We established S100A9 as a myeloid-derived damage-associated molecular pattern (DAMPs) protein associated with increasing severity of diabetic retinopathy (DR) in type 2 diabetic subjects. The present study investigates the retinal localization, expression, and mechanisms of action for S100A9 in the young obese Ossabaw pig retina.
Retinae from Ossabaw pigs fed a Western diet for 10 weeks were evaluated for S100 and inflammatory mediator expression using quantitative PCR and Western blot. Double immunohistochemistry was performed to identify the cellular sources of S100A9 in the pig retina. Primary pig retinal microglial cells (pMicroglia) were examined for S100A9 production. S100A9-induced responses were also investigated, and inhibitor studies elucidated the mechanism of action via the NLRP3 inflammasome. A specific inhibitor, Paquinimod (ABR-215757), was administered to assess the rescue of S100A9-induced NLRP3 inflammasome activation in pMicroglia.
The expression of the S100 family in the obese Ossabaw pig retina showed a significant elevation of S100A9, consistent with increased levels of circulating S100A9. Moreover, the retina had elevated levels of inflammatory mediators IL-6, IL-8, MCP-1, IL-1β and NLRP3. Retinal microglia in obese Ossabaw were activated and accompanied by an increased expression of intracellular S100A9. pMicroglia isolated from pig retina transformed from ramified to amoeboid state when activated with LPS and produced high S100A9 transcript and protein levels. The S100A9 protein, in turn, further activated pMicroglia by heightened production of S100A9 transcripts and secretion of pro-inflammatory IL-1β protein. Inhibition of TLR4 with TAK242 and NLRP3 with MCC950 attenuated the production of IL-1β during S100A9 stimulus. Finally, pre-treatment with Paquinimod successfully reduced S100A9-driven increases of glycosylated-TLR4, NLRP3, ASC, Caspase-1, and IL-1β production.
We demonstrated that microglial-derived S100A9 perpetuates pro-inflammatory responses via the NLRP3 inflammasome in the retina of young Western-diet-fed Ossabaw pigs exhibiting diabetic retinopathy.
我们确定了S100A9作为一种源自骨髓的损伤相关分子模式（DAMPs）蛋白，与2型糖尿病患者糖尿病视网膜病变（DR）严重程度增加相关。本研究调查了S100A9在年轻肥胖奥萨巴猪视网膜中的定位、表达及作用机制。
对喂食西方饮食10周的奥萨巴猪视网膜进行评估，采用定量PCR和蛋白质印迹法检测S100及炎症介质的表达。进行双重免疫组织化学以确定猪视网膜中S100A9的细胞来源。检测原代猪视网膜小胶质细胞（pMicroglia）中S100A9的产生情况。还研究了S100A9诱导的反应，抑制剂研究阐明了通过NLRP3炎性小体的作用机制。给予特异性抑制剂帕喹莫德（ABR-215757）以评估其对pMicroglia中S100A9诱导的NLRP3炎性小体激活的挽救作用。
肥胖奥萨巴猪视网膜中S100家族的表达显示S100A9显著升高，与循环中S100A9水平升高一致。此外，视网膜中炎症介质白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、单核细胞趋化蛋白-1（MCP-1）、白细胞介素-1β（IL-1β）和NLRP3水平升高。肥胖奥萨巴猪的视网膜小胶质细胞被激活，并伴有细胞内S100A9表达增加。从猪视网膜分离的pMicroglia在用脂多糖（LPS）激活时从分支状转变为阿米巴样状态，并产生高水平的S100A9转录本和蛋白。反过来，S100A9蛋白通过增加S100A9转录本的产生和促炎IL-1β蛋白的分泌进一步激活pMicroglia。用TAK242抑制Toll样受体4（TLR4）和用MCC950抑制NLRP3可减弱S100A9刺激期间IL-1β的产生。最后，用帕喹莫德预处理成功降低了S100A9驱动的糖基化TLR4、NLRP3、凋亡相关斑点样蛋白（ASC）、半胱天冬酶-1（Caspase-1）和IL-1β产生的增加。
我们证明，在表现出糖尿病视网膜病变的年轻西方饮食喂养的奥萨巴猪视网膜中，小胶质细胞衍生的S100A9通过NLRP炎性小体使促炎反应持续存在。
