Role of Toll-like Receptor 9 in Fibroblastic Reticular Cell-based Therapy for Intra-abdominal Sepsis

总结 脓毒症现在是美国医院死亡的主要原因，目前没有有效的治疗方法。 用于脓毒症的药物治疗。基于基质细胞的疗法已显示出治疗脓毒症的疗效 在实验模型中，并已被批准用于多个国家的各种免疫 失调性疾病成纤维细胞网状细胞（Fibroblasticreticularcells，FRC）是存在于所有组织中的基质细胞亚群。 淋巴器官，包括脂肪组织中的脂肪相关淋巴簇（FAT-Associated Lymphoid Cluster，CCLC）。我们已经证明 TLR 9信号转导抑制FRC中趋化因子的产生。Tlr 9缺陷型FRC的连续转移 与野生型（WT）FRC相比， 盲肠结扎穿孔术（CLP）。在这里，我们在大肠杆菌中鉴定了两种不同的FRC子集（Ly 6Chi和Ly 6Clo）。 基线和CLP后的肠系膜上动脉平滑肌细胞。重要的是，Ly 6Chi FRC亚组表达类似的顶部 作为先前在小鼠和人中描述的CD 55+基质细胞亚群的标记基因 皮下脂肪组织Ly 6Chi FRCs富含先天免疫应答相关基因， CLP，而Ly 6Clo FRC富含体液免疫应答相关基因。而且我们 发现来自Tlr 9-/-小鼠的Ly 6Chi和Ly 6Clo FRC都增加了与以下相关的基因表达： 基线时与WT FRC相比， CLP之后。基于这些发现，我们假设TLR 9在调节肿瘤的生物学过程中起着关键作用。 不同FRC子集，并且以子集特定的方式调制TLR 9信令可以改善不同FRC子集之间的通信。 基于FRC的治疗在脓毒症中的疗效。我们将通过追求两个具体目标来测试我们的假设：目标1： 确定小鼠和人脂肪中TLR 9介导的FRC生物学调节机制 FRC子集。我们将利用单细胞技术（单细胞RNA测序和Mass 流式细胞术）与FRC特异性Tlr 9-/-小鼠组合以确定TLR 9在基因表达中的作用， 细胞命运和体内和体外单个FRC亚群的免疫调节功能。我们还将验证 在人类脂肪FRC中的小鼠FRC的发现。目的2：确定TLR 9抑制的影响 腹腔内脓毒症中FRC治疗的预处理。我们将确定 两种临床相关腹腔内脓毒症模型中的TLR 9抑制预处理FRC子集：（1） CLP诱导的多微生物腹膜炎;和（2）腹腔内感染大肠杆菌的人类菌株 杆菌我们还将使用LPS诱导的腹膜炎模型来确定 基于FRC的治疗的有益作用以及TLR 9对个体FRC亚组治疗的影响。我们 这项研究将促进对FRC亚群的多样性和生物学的理解，以及对 TLR 9在这些不同亚群中的表达，这将发现新的策略来修饰选择性FRC亚群， 改善基于FRC疗法对脓毒症和其它免疫失调疾病的功效。

Summary Sepsis is now the leading cause of death in US hospitals and there are currently no effective pharmacological treatments for sepsis. Stromal cell-based therapies have shown efficacy in treating sepsis in experimental models and have been approved for use in multiple countries for various immune dysregulation diseases. Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells existing in all lymphoid organs including fat associated lymphoid cluster (FALC) in adipose tissue. We have shown that TLR9 signaling suppresses chemokine production in FRCs. Adoptive transfer of Tlr9-deficient FRCs decreased mortality, bacterial load, and systemic inflammation compared with wild type (WT) FRCs after cecal ligation and puncture (CLP). Here, we identified two distinct subsets of FRCs (Ly6Chi and Ly6Clo) in mesenteric FALCs at baseline and after CLP. Importantly, the Ly6Chi FRC subset express similar top marker genes as the CD55+ stromal cell subset that was previously described in both mouse and human subcutaneous adipose tissue. Ly6Chi FRCs are enriched in the innate immune response-related genes after CLP, whereas Ly6Clo FRCs are enriched in the humoral immune response-related genes. Furthermore, we found that both Ly6Chi and Ly6Clo FRCs from Tlr9-/- mice increased gene expression associated with inflammation, proliferation, and extracellular matrix remodeling compared with WT FRCs at baseline and after CLP. Based on these findings, we hypothesize that TLR9 plays critical roles in regulating the biology of distinct FRC subsets, and that modulation of TLR9 signaling in a subset-specific manner may improve the efficacy of FRC-based therapy in sepsis. We will test our hypothesis by pursuing two specific aims: Aim 1: To determine the mechanisms of TLR9-mediated regulation of FRC biology in mouse and human adipose FRC subsets. We will take advantage of single cell technologies (single cell RNA-sequencing and Mass cytometry) combined with the FRC-specific Tlr9-/- mice to determine the role of TLR9 in gene expression, cell fate, and immunoregulatory functions in individual FRC subset in vivo and in vitro. We will also validate the findings from mouse FRCs in human adipose FRCs. Aim 2: To determine the impact of TLR9 inhibition preconditioning on FRC therapy in intra-abdominal sepsis. We will determine the therapeutic efficacy of TLR9 inhibition preconditioned FRC subsets in two clinically relevant intra-abdominal sepsis models: (1) CLP-induced polymicrobial peritonitis; and (2) intra-abdominal infection of a human strain of Escherichia coli. We will also use an LPS-induced peritonitis model to determine the mechanisms underlying the beneficial effects of FRC-based therapy and the impact of TLR9 on individual FRC subset therapies. Our study will advance understanding of the diversity and biology of FRC subsets as well as the regulation of TLR9 in these distinct subsets, which will discover new strategies to modify selective FRC subsets to improve efficacy of FRC-based therapy for sepsis and other immune dysregulation diseases.