DataSheet_1_Activation of TLR9 signaling suppresses the immunomodulating functions of CD55lo fibroblastic reticular cells during bacterial peritonitis.csv

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However, FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here, we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore, using single-cell RNA sequencing, we found two subsets of FRCs (CD55hi and CD55lo) in the mesenteric FALC. The CD55hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55lo FRCs were enriched in gene expression related to immune response. Interestingly, we found that TLR9 is dominantly expressed in the CD55lo subset. Activation of TLR9 signaling suppressed proliferation, cytokine production, and retinoid metabolism in the CD55lo FRC, but not CD55hi FRC. Notably, we found that adoptive transfer of Tlr9-/–CD55lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9-/–CD55hi FRC. Furthermore, we identified CD55hi and CD55lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55lo subset. Therefore, inhibition of TLR9 in the CD55lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis.

成纤维网状细胞（Fibroblastic reticular cells, FRCs）是基质细胞的一个亚群，可在健康与疾病状态下调控免疫微环境。本课题组此前的研究表明，脂肪相关淋巴簇（fat-associated lymphoid clusters, FALC）中的成纤维网状细胞的Toll样受体9（TLR9）信号激活，可通过抑制免疫细胞招募与腹膜驻留巨噬细胞（peritoneal resident macrophage, PRM）的存留，调控腹膜免疫功能。
然而，成纤维网状细胞在不同组织与器官中存在显著异质性。目前，各成纤维网状细胞亚群的功能，以及不同亚群中Toll样受体9的调控机制仍未明确。
本研究证实，在成纤维网状细胞中特异性敲除Toll样受体9，可改善腹膜炎感染期间的细菌清除能力并提升宿主存活率。进一步通过单细胞RNA测序，我们在肠系膜脂肪相关淋巴簇中鉴定出两类成纤维网状细胞亚群：CD55高表达（CD55hi）与CD55低表达（CD55lo）亚群。其中，CD55hi成纤维网状细胞富集细胞外基质形成相关的基因表达特征，而CD55lo成纤维网状细胞则富集免疫应答相关的基因表达特征。
有趣的是，我们发现Toll样受体9主要表达于CD55lo亚群中。Toll样受体9信号激活可抑制CD55lo成纤维网状细胞的增殖、细胞因子产生与视黄酸代谢，但对CD55hi亚群无此类调控作用。值得注意的是，与野生型成纤维网状细胞或Toll样受体9敲除的CD55hi成纤维网状细胞相比，过继转移来自肠系膜脂肪相关淋巴簇的Toll样受体9敲除CD55lo成纤维网状细胞，可更有效地改善腹膜炎期间的宿主存活率。
此外，我们在人类脂肪组织来源的成纤维网状细胞中也鉴定出CD55hi与CD55lo两个亚群，并证实了Toll样受体9对CD55lo亚群增殖与细胞因子产生的抑制效应。因此，靶向抑制脂肪组织来源的CD55lo成纤维网状细胞中的Toll样受体9，或可成为提升基于成纤维网状细胞的腹膜炎治疗策略疗效的有效手段。