Elucidating the roles of systemic and mucosal complement in protection against invasive fungal infections impacting hematopoietic cell transplant

项目摘要 共生真菌白色念珠菌（Ca）每年导致全球约700，000例系统性念珠菌病病例，尽管同时接受抗真菌治疗，相关死亡率仍为40% 1。接受造血细胞移植（HCT）的癌症患者。系统性念珠菌病与显著增加的无复发死亡率相关2，3。更好地了解细胞和分子抗真菌免疫应答有望改善结果。包括接受IICT的癌症患者，因为它可以改善风险分层，以获得更好的预后和鉴定用于抗真菌免疫治疗的新分子。为此，本文提出的工作旨在为补体C3和C5信号传导在局部和全身真菌控制中的作用提供新的机制见解。补体C3和C5对于它们在免疫监视和下游免疫效应子功能调节中的作用是必不可少的。在遇到病原体时，C3快速沉积并裂解成C3 b/iC 3b，导致通过CR 1 -4 4的调理吞噬作用。此外，C3 b形成多聚体转化酶复合物并将C5裂解成有效的免疫效应肽C5 a 5。在该项目的K99阶段，我确定了C5 a在调节中性粒细胞和巨噬细胞依赖性抗真菌效应子功能以抑制真菌过度生长中的关键作用。免疫代谢失调和炎性肾损伤以促进存活。与C5 a一样，较小的C3裂解片段-C3 a是同样重要的免疫调节肽，其通过其受体C3 ar 4发出信号;然而，其在系统性念珠菌病期间的功能仍然完全未被探索。此外，C3/C5的细胞来源，它们在抗真菌细胞网络中的调节和影响，在感染真菌的肾脏或HCT后的胃肠道中的C3/C5尚不清楚。我假设补体C3和C5在系统和粘膜真菌控制中发挥非冗余作用。我将在本奖项的R 00阶段通过三个具体目标系统地评估这一假设。在目标1下，我将描述C5调节的细胞内在机制;为此目的。我将使用C5报告小鼠。来自小鼠/人的原代细胞和一系列离体刺激和细胞内补体活化测定。此外，我将评估的作用，C5在真菌定植控制HCT期间使用小鼠模型的HCT 6（目的1）。我还将评估C3在系统性念珠菌病保护性免疫中的作用。其中使用C3-/-、C3 ar-/-和细胞类型特异性C3/C3 ar敲除小鼠将揭示C3的不同生物活性片段在保护性抗真菌应答中的相对贡献（目的2）。同时，我将开始描绘C3和C5依赖的自/帕拉/内分泌细胞网络，负责灭菌肾抗真菌免疫使用体内抗真菌效应功能测定和细胞类型特异性敲除小鼠（目标3）。因此，通过这个R 00奖的支持将有助于完成这一重要的研究成果。这将为进一步的调查提供坚实的基础。

PROJECT SUMMARY The commensal fungus Candida albicans (Ca) is responsible for ~700,000 annual cases of systemic candidiasis globally, with an associated mortality rate of 40% despite concurrent antifungal therapy 1 . In cancer patients receiving hematopoietic cell transplant (HCT). systemic candidiasis is associated with significantly increased relapse-free mortality 2,3. A better understanding of the cellular and molecular anti-fungal immune responses holds a promise in improving outcomes. including for cancer patients undergoing IICT, as it may allow improved risk stratification for better prognosis and identification of novel molecules for antifungal immunotherapy. To this end the work proposed here aims to provide novel mechanistic insights into the roles of complement C3 and C5 signaling in local and systemic fungal control'. Complement C3 and C5 are essential for their roles in immunosurveillance and downstream immune effector function regulation. Upon pathogen encounter, rapid deposition, and cleavage of C3 into C3b/iC3b leads to opsonophagocytosis via CR1-4 4 • Additionally, C3b forms a multimeric convertase complex and cleaves C5 into the potent immune effector peptide C5a 5 • During the K99 phase of this project, I identified a crucial role for C5a in regulation of neutrophil- and macrophage-dependent antifungal effector functions to curb fungal overgrowth. immunometabolic dysregulation, and inflammatory renal damage to promote survival. Like C5a, the smaller C3 cleavage fragment- C3a is an equally important immunoregulatory peptide which signals via its receptor C3ar 4 ; however, its functions during systemic candidiasis remain completely unexplored. Furthermore, the cellular sources of C3/C5 in Candida-infected kidneys or Candida-colonized gastrointestinal tract post HCT, their regulation and their in1pacts on antifungal cellular network remain unexplored. I hypothesize that complement C3 and C5 play non-redundant roles in systemic and mucosa] fungal control. I will systematically assess this hypothesis during the R00 phase of this award via three specific aims. Under Aim1 I will characterize the cell-intrinsic mechanisms of C5 regulation; to this end. I will use C5 reporter mice. primary cells from mice/humans and a series of ex vivo stimulation and intracellular complement activation assays. Furthermore, I will assess the role of C5 in fungal colonization control during HCT using a mouse model ofHCT6 (Aim 1). I will also assess the role ofC3 in protective immunity during systemic candidiasis. where the use of C3-/-, C3ar-/- and cell-type specific C3/C3ar knockout mice will reveal the relative contributions of different bioactive fragments of C3 in protective antifungal response (Aim 2). Concurrently, I will begin delineating C3 and C5-dependent auto-/para-/endocrine cellular networks responsible for sterilizing renal antifungal immunity using in vivo antifungal effector function assays and cell-type specific knockout mice (Aim 3). Thus, support through this R00 award will be instrumental in completing this important research to fruition. which will provide strong foundation upon which further investigations will he pursued.