Data Sheet 1_Tuft-cell-derived IL-25 regulates intestinal ILC2 in response to Brucella infection.docx

IntroductionBrucella is a zoonotic pathogen capable of invading the host through the intestinal mucosa. However, the immune mechanisms underlying intestinal infection remain poorly understood. Tuft cells are specialized chemosensory epithelial cells in the intestine that can detect pathogen invasion and secrete IL-25, subsequently activating type 2 innate lymphoid cells (ILC2s) and playing a critical role in anti-parasitic immune responses. Nevertheless, whether the tuft cell-ILC2 circuit participates in immune responses against bacterial infections remains unclear. This study aimed to investigate the dynamic changes of tuft cells and ILC2s following Brucella infection, with a particular focus on elucidating the regulatory role of IL-25 in this process.
MethodsThirty-six mice were divided into six groups: normal control (NC), isotype control (IC), infection groups at different time points (3, 7, and 14 days post-infection, designated as Inf-3d, Inf-7d, and Inf-14d, respectively), and an IL-25 blockade group (IL-25 Blk-7d, in which mice received anti-IL-25 neutralizing antibody treatment prior to Brucella infection and were analyzed at 7 days post-infection). In this study, immunofluorescence assay, flow cytometry, and Western blot were employed to detect the dynamic changes of tuft cells and ILC2s in intestinal tissues, as well as to determine the expression levels of pathway-related proteins.
ResultsThe results showed that the numbers of tuft cells and ILC2s in the mouse intestine increased following Brucella infection, peaking at 7 days post-infection. Pretreatment with IL-25 neutralizing antibody significantly suppressed the proliferation of these two cell populations. Western blot analysis further confirmed that the expression levels of tuft cell-associated proteins (PO2F3, DCLK1, and IL-25) and ILC2-associated proteins (GATA3 and IL-13) were upregulated in infected intestinal tissues, whereas IL-25 blockade treatment inhibited the expression of these proteins. Correlation analysis revealed a remarkably strong positive correlation between the proportions of tuft cells and ILC2s, and this correlation was completely abolished following IL-25 neutralization.
DiscussionThese findings confirm that the activation of the tuft cell-ILC2 circuit is dependent on the regulatory role of IL-25. These findings extend the antimicrobial role of the tuft cell-ILC2 circuit beyond parasitic immunity and identify IL-25 as an essential regulatory mediator in antibacterial defense at the intestinal mucosa.

引言
布鲁氏菌（Brucella）是一种可通过肠黏膜侵入宿主的人畜共患病原菌，然而其引发肠内感染的免疫机制目前仍不甚明确。簇细胞（tuft cells）是肠内特化的化学感应上皮细胞，能够检测病原体入侵并分泌IL-25，进而激活2型天然淋巴细胞（type 2 innate lymphoid cells，ILC2s），在抗寄生虫免疫应答中发挥关键作用。不过，簇细胞-ILC2环路是否参与抗细菌感染的免疫应答仍尚不明确。本研究旨在探究布鲁氏菌感染后肠内簇细胞与ILC2s的动态变化，并重点阐明IL-25在此过程中的调控作用。

方法
将36只小鼠分为6组：正常对照组（normal control，NC）、同型对照组（isotype control，IC）、不同时间点感染组（分别为感染后3、7、14天，记为Inf-3d、Inf-7d、Inf-14d），以及IL-25阻断组（IL-25 Blk-7d，该组小鼠在布鲁氏菌感染前接受抗IL-25中和抗体处理，并于感染后7天进行检测分析）。本研究采用免疫荧光检测法（immunofluorescence assay）、流式细胞术（flow cytometry）与蛋白质印迹法（Western blot），检测肠组织中簇细胞与ILC2s的动态变化，并测定通路相关蛋白的表达水平。

结果
结果显示，小鼠肠内簇细胞与ILC2s的数量在布鲁氏菌感染后显著升高，并于感染后7天达到峰值。经IL-25中和抗体预处理后，这两种细胞的增殖受到显著抑制。蛋白质印迹分析进一步证实，感染的肠组织中簇细胞相关蛋白（PO2F3、DCLK1与IL-25）以及ILC2相关蛋白（GATA3与IL-13）的表达水平均上调，而IL-25阻断处理可抑制这些蛋白的表达。相关性分析显示，簇细胞与ILC2s的占比之间存在显著强正相关，而经IL-25中和后，这种相关性完全消失。

讨论
本研究结果证实，簇细胞-ILC2环路的激活依赖于IL-25的调控作用。上述研究结果将簇细胞-ILC2环路的抗菌作用从寄生虫免疫拓展至细菌免疫，并明确IL-25是肠黏膜抗菌防御中的关键调控介质。