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

摘要 脓毒症现在是美国医院的主要死亡原因，目前还没有有效的 败血症的药物治疗。以基质细胞为基础的疗法在治疗脓毒症方面显示出有效性。 在实验模型中，并已被批准在多个国家用于各种免疫 调节失调的疾病。成纤维细胞网状细胞(FRC)是ALL中存在的一种基质细胞亚群 淋巴器官包括脂肪组织中的脂肪相关淋巴团(FALC)。我们已经证明了 TLR9信号抑制FRC趋化因子的产生。TLR9缺陷FRCs的过继转移 与野生型(WT)FRC相比，术后死亡率、细菌负荷和全身炎症均降低 盲肠结扎穿孔(CLP)。在这里，我们确定了FRC的两个不同的子集(Ly6chi和Ly6Clo) 基线和CLP后肠系膜FALCs。重要的是，Ly6chi FRC子集表达了类似的Top 作为CD55基质细胞亚群的标记基因，以前在小鼠和人类中都有描述 皮下脂肪组织。Ly6chi FRC富含先天免疫反应相关基因 而Ly6Clo FRC富含体液免疫应答相关基因。此外，我们 发现来自TLR9-/-小鼠的Ly6chi和Ly6Clo FRC都增加了与以下相关的基因表达 炎症、增殖和细胞外基质重构与WT FRC在基线和 在中电之后。基于这些发现，我们推测TLR9在调节肿瘤的生物学过程中起关键作用。 不同的FRC子集，且以特定于子集的方式调制TLR9信令可以改善 以FRC为基础的治疗败血症的疗效。我们将通过追求两个具体目标来验证我们的假设：目标1： TLR9对小鼠和人脂肪FRC生物学调控机制的研究 FRC子集。我们将利用单细胞技术(单细胞RNA测序和质量 细胞仪)与FRC特异性TLR9-/-小鼠相结合，以确定TLR9在基因表达中的作用， 体内和体外单个FRC亚群的细胞命运和免疫调节功能。我们还将验证 这一发现来自人类脂肪FRCs中的小鼠FRCs。目的2：确定TLR9抑制的影响 FRC治疗腹内脓毒症的预适应。我们将确定其治疗效果。 在两种临床相关的腹内脓毒症模型中，TLR9抑制预适应FRC亚群：(1) CLP所致的多菌腹膜炎；及(2)一种人类菌株的腹内感染 Coli.我们还将使用脂多糖诱导的腹膜炎模型来确定 基于FRC的治疗的有益效果以及TLR9对个别FRC亚集治疗的影响。我们的 研究将促进对FRC亚群的多样性和生物学以及对FRC亚群调控的理解 TLR9在这些不同的子集中，它将发现修改选择性FRC子集以 提高基于FRC的治疗脓毒症和其他免疫失调疾病的疗效。

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.