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

Toll 样受体 9 在基于成纤维网状细胞的腹内脓毒症治疗中的作用

• 批准号: 10678190
• 负责人: Meihong Deng
• 金额: $ 19.33万
• 依托单位: FEINSTEIN INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-24 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678190
• 关键词: Role; Toll; like; Receptor; Fibroblastic

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.

概括 败血症现在是美国医院死亡的主要原因，目前尚无有效的 败血症的药理治疗。基于基质细胞的疗法已显示出在治疗败血症方面的功效 在实验模型中，已被批准用于多个国家 /地区的各种免疫力 失调疾病。成纤维细胞网状细胞（FRC）是所有存在的基质细胞的亚群 脂肪组织中的淋巴器官，包括脂肪相关的淋巴类簇（FALC）。我们已经表明 TLR9信号传导抑制了FRC中的趋化因子产生。 TLR9缺陷型FRC的收养转移 与野生型（WT）FRC相比，死亡率，细菌负荷和全身性炎症降低 盲肠结扎和穿刺（CLP）。在这里，我们确定了FRC的两个不同的子集（Ly6chi和Ly6clo） 肠系膜在基线和CLP之后。重要的是，ly6chi frc子集表达相似的顶部 标记基因作为CD55+基质细胞子集，以前在小鼠和人类中都描述过 皮下脂肪组织。 Ly6chi FRC在与先天免疫反应相关的基因中富集 CLP，而Ly6clo FRC富含与免疫反应相关的基因。此外，我们 发现来自TLR9 - / - 小鼠的Ly6chi和Ly6clo FRC都增加了与 与基线时的WT FRC相比，炎症，增殖和细胞外基质重塑 CLP之后。基于这些发现，我们假设TLR9在调节生物学中起关键作用 不同的FRC子集，以及以亚集特异性方式调制TLR9信号传导可能会改善 基于FRC的疗法在败血症中的功效。我们将通过追求两个具体目标来检验我们的假设：目标1： 确定TLR9介导的小鼠和人脂肪中FRC生物学调节的机制 FRC子集。我们将利用单细胞技术（单细胞RNA-severing和质量 细胞仪）与FRC特异性TLR9 - / - 小鼠结合确定TLR9在基因表达中的作用， 细胞命运以及体内和体外单个FRC子集中的免疫调节功能。我们还将验证 小鼠FRC的发现，在人类脂肪FRC中。目标2：确定TLR9抑制的影响 腹腔内脓毒症的FRC治疗预处理。我们将确定 TLR9在两个临床上相关的腹腔内败血症模型中抑制预处理的FRC子集：（1） CLP诱导的多肌腹膜炎； （2）人体菌株的腹腔内感染 大肠杆菌。我们还将使用LPS诱导的腹膜炎模型来确定该机制 基于FRC的治疗以及TLR9对单个FRC子集疗法的影响。我们的 研究将提高人们对FRC子集的多样性和生物学的了解以及对 TLR9在这些不同的子集中，该子集将发现新的策略将选择性FRC子集修改为 提高基于FRC的疗效对败血症和其他免疫失调疾病的疗效。