Association between Altered Tryptophan Metabolism, Plasma Aryl Hydrocarbon Receptor Agonists, and Inflammatory Chagas Disease

Chagas disease causes a cardiac illness characterized by immunoinflammatory reactions leading to myocardial fibrosis and remodeling. The development of Chronic Chagas Cardiomyopathy (CCC) in some patients while others remain asymptomatic is not fully understood, but dysregulated inflammatory responses are implicated. The Aryl hydrocarbon receptor (AhR) plays a crucial role in regulating inflammation. Certain tryptophan (Trp) metabolites have been identified as AhR ligands with regulatory functions. We investigated AhR expression, agonist response, ligand production, and AhR-dependent responses, such as IDO activation and regulatory T (Treg) cells induction, in two T.cruzi-infected mouse strains (B6 and Balb/c) showing different polymorphisms in AhR. Furthermore, we assessed the metabolic profile of Trp catabolites and AhR agonistic activity levels in plasma samples from patients with chronic Chagas disease (CCD) and healthy donors (HD) using a luciferase reporter assay and liquid chromatography-mass spectrophotometry (LC-MS) analysis. T. cruzi-infected B6 mice showed impaired AhR-dependent responses compared to Balb/c mice, including reduced IDO activity, kynurenine levels, Treg cell induction, CYP1A1 up-regulation, and AhR expression following agonist activation. Additionally, B6 mice exhibited no detectable 34 AhR agonist activity in plasma and displayed lower CYP1A1 up-regulation and AhR expression upon agonist activation. Similarly, CCC patients had decreased AhR agonistic activity in plasma compared to HD patients and exhibited dysregulation in Trp metabolic pathways, resulting in altered plasma metabolite profiles. Notably, patients with severe CCC specifically showed increased N-acetylserotonin levels in their plasma. The methods and findings presented here contribute to a better understanding of CCC development mechanisms and may identify potential specific biomarkers for T. cruzi infection and the severity of associated heart disease. These insights could be valuable in designing new therapeutic strategies. Ultimately, this research aims to establish the AhR agonistic activity and Trp metabolic profile in plasma as an innovative, non-invasive predictor of prognosis for chronic Chagas disease. Bone marrow macrophages (BMDM) from C57BL/6 mice were cultured for 24h with Trypanosoma cruzi (Tulahuen Strain, TcVI). Cell to parasite ratio was set to 1:3. Total RNA was harvested according to the Qiagen RNeasy Mini Kit protocol. Only Samples that passed the purity cutoff (Abs 260/Abs 280 = 1,7-2) were selected for downstream processing. Libraries were processed by the Broad Institute's Broad Technology Labs and the Broad Genomics Platform. Expression profiling analysis by bulk-RNAseq was carried out across 2 experimental groups, each consisting of four replicates: Ctrl (non-infected BMDM) and Tc-infected BMDM.

查加斯病（Chagas disease）可引发以免疫炎症反应为特征的心脏疾病，此类反应最终会导致心肌纤维化与心肌重塑。部分患者会进展为慢性查加斯心肌病（Chronic Chagas Cardiomyopathy，CCC），而其余患者则保持无症状，该表型差异的具体机制尚未完全阐明，但失调的炎症反应被认为参与其中。芳香烃受体（Aryl hydrocarbon receptor，AhR）在炎症调控中发挥关键作用，部分色氨酸（tryptophan，Trp）代谢产物已被证实为具有调控功能的AhR配体。本研究针对两种在AhR基因上存在多态性差异的克氏锥虫（Trypanosoma cruzi，T. cruzi）感染小鼠品系——C57BL/6（B6）与BALB/c小鼠，开展了AhR表达、激动剂应答、配体生成以及AhR依赖型应答（包括吲哚胺2,3-双加氧酶[indoleamine 2,3-dioxygenase，IDO]活化、调节性T细胞[regulatory T cells，Treg]诱导）相关的检测。此外，本研究通过荧光素酶报告基因检测（luciferase reporter assay）与液相色谱-质谱联用法（liquid chromatography-mass spectrophotometry，LC-MS）分析，对慢性查加斯病（Chronic Chagas disease，CCD）患者与健康供体（healthy donors，HD）的血浆样本，开展了Trp代谢产物谱与AhR激动活性水平的评估。相较于BALB/c小鼠，感染T. cruzi的B6小鼠AhR依赖型应答受损，具体表现为IDO活性降低、犬尿氨酸水平下降、Treg细胞诱导减少、CYP1A1上调受阻以及激动剂激活后AhR表达量降低。此外，B6小鼠血浆中未检测到34种AhR激动剂活性，且在激动剂激活后CYP1A1上调与AhR表达的幅度均更低。类似地，与健康供体相比，CCC患者血浆中的AhR激动活性显著降低，同时其Trp代谢通路出现失调，导致血浆代谢物谱发生改变。值得注意的是，重度CCC患者的血浆中N-乙酰血清素水平特异性升高。本研究的方法与发现有助于进一步阐明CCC的发病机制，有望为T. cruzi感染及其相关心脏疾病的严重程度识别潜在的特异性生物标志物。上述研究结果可为新型治疗策略的开发提供参考。本研究最终旨在确立血浆AhR激动活性与Trp代谢谱作为慢性查加斯病无创预后预测指标的创新应用价值。将C57BL/6小鼠的骨髓源性巨噬细胞（bone marrow-derived macrophages，BMDM）与克氏锥虫（Trypanosoma cruzi，Tulahuen株，TcVI）共培养24小时，细胞与寄生虫的比例设置为1:3。总RNA提取按照凯杰（Qiagen）RNeasy迷你试剂盒（RNeasy Mini Kit）的操作流程进行。仅选取纯度达标（Abs260/Abs280比值为1.7~2.0）的样本进行后续实验。文库构建由博德研究所（Broad Institute）的博德技术实验室与博德基因组平台完成。本研究通过批量RNA测序（bulk RNA-seq）对两组实验样本开展表达谱分析，每组均包含4个生物学重复：对照组（未感染的BMDM）与T. cruzi感染的BMDM组。