TB Research & Training Center, Department of Medicine, University of Washington, Seattle, Washington, United States of America.
Department of Population & Quantitative Health Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America.
PLoS One. 2023 Apr 14;18(4):e0284498. doi: 10.1371/journal.pone.0284498. eCollection 2023.
A mechanistic understanding of uncommon immune outcomes such as resistance to infection has led to the development of novel therapies. Using gene level analytic methods, we previously found distinct monocyte transcriptional responses associated with resistance to Mycobacterium tuberculosis (Mtb) infection defined as persistently negative tuberculin skin test (TST) and interferon gamma release assay (IGRA) reactivity among highly exposed contacts (RSTR phenotype).
Using transcript isoform analyses, we aimed to identify novel RSTR-associated genes hypothesizing that previous gene-level differential expression analysis obscures isoform-specific differences that contribute to phenotype.
Monocytes from 49 RSTR versus 52 subjects with latent Mtb infection (LTBI) were infected with M. tuberculosis (H37Rv) or left unstimulated (media) prior to RNA isolation and sequencing. RSTR-associated gene expression was then identified using differential transcript isoform analysis.
We identified 81 differentially expressed transcripts (DETs) in 70 genes (FDR <0.05) comparing RSTR and LTBI phenotypes with the majority (n = 79 DETs) identified under Mtb-stimulated conditions. Seventeen of these genes were previously identified with gene-level bulk RNAseq analyses including genes in the IFNγ response that had increased expression among LTBI subjects, findings consistent with a clinical phenotype based on IGRA reactivity. Among the subset of 23 genes with positive differential expression among Mtb-infected RSTR monocytes, 13 were not previously identified. These novel DET genes included PDE4A and ZEB2, which each had multiple DETs with higher expression among RSTR subjects, and ACSL4 and GAPDH that each had a single transcript isoform associated with RSTR.
Transcript isoform-specific analyses identify transcriptional associations, such as those associated with resistance to TST/IGRA conversion, that are obscured when using gene-level approaches. These findings should be validated with additional RSTR cohorts and whether the newly identified candidate resistance genes directly influence the monocyte Mtb response requires functional study.
对不常见免疫结果（如抗感染能力）的机制理解导致了新型疗法的发展。我们之前使用基因水平分析方法，发现了与结核分枝杆菌（Mtb）感染抵抗力相关的独特单核细胞转录反应，这种抵抗力表现为高度暴露接触者中持续阴性结核菌素皮肤试验（TST）和干扰素γ释放试验（IGRA）反应（RSTR 表型）。
使用转录本异构体分析，我们旨在确定新的 RSTR 相关基因，假设之前的基因水平差异表达分析掩盖了导致表型的异构体特异性差异。
从 49 名 RSTR 与 52 名潜伏性 Mtb 感染（LTBI）患者的单核细胞中分离出来，在 RNA 分离和测序之前用 M. tuberculosis（H37Rv）或未刺激（培养基）进行感染。然后使用差异转录本异构体分析来鉴定与 RSTR 相关的基因表达。
我们在 RSTR 和 LTBI 表型之间比较了 70 个基因的 81 个差异表达转录本（DETs）（FDR<0.05），其中大多数（n=79 DETs）在 Mtb 刺激条件下被鉴定出来。其中 17 个基因之前在基因水平的批量 RNAseq 分析中被鉴定出来，包括 IFNγ 反应中的基因，这些基因在 LTBI 患者中表达增加，与基于 IGRA 反应的临床表型一致。在 Mtb 感染的 RSTR 单核细胞中具有阳性差异表达的 23 个基因亚集中，有 13 个基因之前未被鉴定出来。这些新的 DET 基因包括 PDE4A 和 ZEB2，它们在 RSTR 患者中都有多个 DET 表达较高，而 ACSL4 和 GAPDH 每个基因都有一个与 RSTR 相关的转录本异构体。
转录本异构体特异性分析确定了转录关联，例如与 TST/IGRA 转换抗性相关的关联，这些关联在使用基因水平方法时被掩盖。这些发现需要用额外的 RSTR 队列进行验证，并且新鉴定的候选耐药基因是否直接影响单核细胞 Mtb 反应需要功能研究。
