Supplementary Material for: Tollip Inhibits IL-33 Release and Inflammation in Influenza A Virus-Infected Mouse Airways

Respiratory influenza A virus (IAV) infection continues to pose significant challenges in healthcare of human diseases including asthma. IAV infection in mice was shown to increase IL-33, a key cytokine in driving airway inflammation in asthma, but how IL-33 is regulated during viral infection remains unclear. We previously found that a genetic mutation in Toll-interacting protein (Tollip) was linked to less airway epithelial Tollip expression, increased neutrophil chemokines, and lower lung function in asthma patients. As Tollip is involved in maintaining mitochondrial function, and mitochondrial stress may contribute to extracellular ATP release and IL-33 secretion, we hypothesized that Tollip downregulates IL-33 secretion via inhibiting ATP release during IAV infection. Wild-type and Tollip knockout (KO) mice were infected with IAV and treated with either an ATP converter apyrase or an IL-33 decoy receptor soluble ST2 (sST2). KO mice significantly lost more body weight and had increased extracellular ATP, IL-33 release, and neutrophilic inflammation. Apyrase treatment reduced extracellular ATP levels, IL-33 release, and neutrophilic inflammation in Tollip KO mice. Excessive lung neutrophilic inflammation in IAV-infected Tollip KO mice was reduced by sST2, which was coupled with less IL-33 release. Our data suggest that Tollip inhibits IAV infection, potentially by inhibiting extracellular ATP release and reducing IL-33 activation and lung inflammation. In addition, sST2 may serve as a potential therapeutic approach to mitigate respiratory viral infection in human subjects with Tollip deficiency.

呼吸道甲型流感病毒（influenza A virus, IAV）感染仍是包括哮喘在内的人类呼吸道疾病临床诊疗中面临的重大挑战。已有研究证实，小鼠感染IAV后可上调白细胞介素33（IL-33）的表达——IL-33是驱动哮喘气道炎症的关键细胞因子，但病毒感染期间IL-33的调控机制仍未明确。我们此前的研究发现，哮喘患者体内的Toll相互作用蛋白（Toll-interacting protein, Tollip）基因突变与气道上皮Tollip表达降低、中性粒细胞趋化因子水平升高以及肺功能下降显著相关。鉴于Tollip参与维持线粒体功能，而线粒体应激可促进细胞外ATP释放与IL-33分泌，我们提出假说：在IAV感染期间，Tollip可通过抑制ATP释放下调IL-33的分泌。我们将野生型与Tollip基因敲除（knockout, KO）小鼠进行IAV感染，并分别给予ATP转化酶腺苷三磷酸双磷酸酶（apyrase）或IL-33诱饵受体可溶性ST2（soluble ST2, sST2）处理。结果显示，Tollip基因敲除小鼠的体重丢失量显著更高，且细胞外ATP水平、IL-33释放量以及中性粒细胞性炎症均有所升高。对Tollip基因敲除小鼠给予腺苷三磷酸双磷酸酶处理后，其细胞外ATP水平、IL-33释放量以及中性粒细胞性炎症均得到有效缓解。经sST2处理后，IAV感染的Tollip基因敲除小鼠体内过度的肺部中性粒细胞性炎症得以改善，同时IL-33的释放量也随之降低。本研究数据表明，Tollip可抑制IAV感染，其潜在机制可能为抑制细胞外ATP释放、减少IL-33活化以及缓解肺部炎症。此外，sST2或可作为一种潜在治疗手段，用于减轻Tollip缺陷人群的呼吸道病毒感染病症。