Supplementary Material for: Tryptophol acetate and tyrosol acetate, small molecule metabolites identified in a probiotic mixture, inhibit hyperinflammation

Probiotic fermented foods are perceived as contributing to human health, however solid evidence for their presumptive therapeutic systemic benefits is generally lacking. Here we report that tryptophol acetate and tyrosol acetate, small molecule metabolites secreted by the probiotic milk-fermented yeast Kluyveromyces marxianus inhibit hyperinflammation (e.g., “cytokine storm”). Comprehensive in vivo and in vitro analyses, employing LPS-induced hyperinflammation models, reveal dramatic effects of the molecules, added in tandem, on mice morbidity, laboratory parameters, and mortality. Specifically, we observed attenuated levels of the pro-inflammatory cytokines IL-6, IL-1α, IL-1β and TNF-α, and reduced reactive oxygen species. Importantly, tryptophol acetate and tyrosol acetate did not completely suppress pro-inflammatory cytokine generation, rather brought their concentrations back to baseline levels thus maintaining core immune functions, including phagocytosis. The anti-inflammatory effects of tryptophol acetate and tyrosol acetate were mediated through downregulation of TLR4, IL-1R, and TNFR signaling pathways and increased A20 expression, leading to NF-kB inhibition. Overall, this work illuminates phenomenological and molecular details underscoring anti-inflammatory properties of small molecules identified in a probiotic mixture, pointing to potential therapeutic avenues against severe inflammation.

益生菌发酵食品被认为有益于人体健康，但目前普遍缺乏能够证实其推定系统性治疗益处的坚实证据。本研究报道，益生菌牛乳发酵酵母马克斯克鲁维酵母（Kluyveromyces marxianus）分泌的小分子代谢产物乙酸色醇（tryptophol acetate）与乙酸酪醇（tyrosol acetate）可抑制过度炎症反应（如"细胞因子风暴"）。本研究采用脂多糖（LPS，lipopolysaccharide）诱导的过度炎症模型开展全面的体内与体外分析，结果显示这两种代谢物联合使用可对小鼠的发病率、实验室检测指标与死亡率产生显著影响。具体而言，本研究观察到促炎细胞因子白细胞介素6（IL-6）、白细胞介素1α（IL-1α）、白细胞介素1β（IL-1β）与肿瘤坏死因子α（TNF-α）的水平显著下调，活性氧（ROS）的生成也得到抑制。值得注意的是，乙酸色醇与乙酸酪醇并未完全抑制促炎细胞因子的生成，而是将其浓度恢复至基线水平，从而维持包括吞噬作用在内的核心免疫功能。乙酸色醇与乙酸酪醇的抗炎作用通过下调Toll样受体4（TLR4）、白细胞介素1受体（IL-1R）与肿瘤坏死因子受体（TNFR）信号通路，并上调A20表达，进而实现核因子κB（NF-κB）的活性抑制。综上，本研究阐明了益生菌混合物中鉴定出的小分子所具备抗炎特性的现象学与分子细节，为重度炎症的潜在治疗途径提供了新的方向。