The role of intestinal antimicrobial and inflammatory molecules in acute graft-versus-host disease

异基因造血细胞移植(allo-HCT)是治疗不同恶性血液病的有效方法。急性移植物抗宿主病(AGVHD)、复发和感染是allo-HCT后威胁生命的主要并发症。我们报道，天然免疫系统的激活，特别是中性粒细胞，是aGVHD发病的早期步骤。JAK1/2抑制可减少中性粒细胞迁移和MHC-II的表达。我们之前的工作表明，JAK1/2抑制减少了小鼠和患者的aGVHD，这被多中心III期试验证明。虽然在allo-HCT后早期出现的中性粒细胞是促炎的，但我们的初步数据表明，出现在治愈的GVHD小鼠固有层中的中性粒细胞可能促进组织修复和减轻炎症。通过对GVHD愈合时allo-HCT后晚期停留在肠道中的中性粒细胞进行单细胞RNA测序，我们在中性粒细胞中发现了多种抗菌素和炎症分子，包括已知限制细菌生长的抗菌糖蛋白Lipocalin-2(Lcn2)。我们的初步数据表明，Lcn2治疗降低了GVHD相关死亡率，暴露于Lcn2的巨噬细胞产生了包括精氨酸酶-1在内的多种减少T细胞激活的分子。在这项提案中，我们计划确定Lcn2在晚期急性移植物抗宿主病愈合过程中的作用。一个特别的重点将是通过Lcn2诱导肠道巨噬细胞产生炎症、抗菌和免疫抑制分子的调节。我们假设，Lcn2诱导的精氨酸酶-1的产生减少了肠道中同种反应性T细胞的激活，从而允许上皮愈合。此外，我们希望评估Lcn2对肠道微生物组组成、供体T细胞代谢活性和移植物抗白血病作用的影响。为了阐明小鼠实验的结果是否可以应用到人类环境中，我们将测定患有或不患有GVHD的人肠道组织中不同类型细胞中Lcn2的水平，以及修复中的GVHD。此外，我们还将分析接受allo-HCT患者的组织中Lcn2、精氨酸酶-1和24p3R/SLC22A17，以明确这些指标是否与SR-GVHD和无复发死亡率相关。总之，本项目旨在确定Lcn2和其他抗菌素和炎症分子在GVHD中的作用，以更好地了解GVHD的愈合过程，并为GVHD患者开发潜在的新疗法。

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for different hematological malignancies. Acute graft-versus-host disease (aGVHD), relapse and infections are the major life-threatening complications after allo-HCT. We reported that activation of the innate immune system, in particular neutrophil granulocytes (neutrophils), is an early step in the pathogenesis of aGVHD. Neutrophil migration and MHC-II expression were reduced by JAK1/2 inhibition. Our previous work had shown that JAK1/2 inhibition reduced aGVHD in mice and in patients, proven by a multicenter phase III trial. While neutrophils that occurred in the early phase after allo-HCT were pro-inflammatory, our preliminary data indicate that neutrophils that occur in the lamina propria of mice with healing GVHD may promote tissue repair and reduce inflammation. Using single cell RNA sequencing on neutrophils residing in the intestinal tract at late time points after allo-HCT when GVHD is healing, we identified multiple antimicrobial and inflammatory molecules in neutrophils including the antimicrobial glycoprotein Lipocalin-2 (LCN2), that is known to limit bacterial growth. Our preliminary data indicate that LCN2 treatment reduced GVHD-related mortality and macrophages exposed to LCN2 produced multiple molecules that reduce T cell activation including arginase-1. In this proposal we plan to determine the role of LCN2 for the healing process in late acute GVHD. A particular focus will be on the regulation of inflammatory, antimicrobial and immunosuppressive molecules production, induced via LCN2 in intestinal macrophages. We hypothesize that LCN2-induced arginase-1 production reduces allo-reactive T cell activation in the intestines, thereby allowing the epithelium to heal. Additionally we wish to evaluate the impact of LCN2 on the intestinal microbiome composition, on metabolic activity of donor T cells and on graft-versus-leukemia effects. To clarify if the results from the murine setting can be transferred into the human situation, we will determine the level of LCN2 in different cell types residing in human intestinal tissues with or without active GVHD and healing GVHD. Additionally we will analyze the tissues for LCN2, arginase-1 and 24p3R / SLC22A17 in patients undergoing allo-HCT with the prospect to clarify if these correlate with SR-GVHD and non-relapse mortality. Overall this project aims at determining the role of LCN2 and other antimicrobial and inflammatory molecules in GVHD to better understand the GVHD healing process and to develop potential novel treatments for patients with GVHD.