Dual RNASeq reveals NTHi-macrophage transcriptomic changes during intracellular persistence

Nontypeable Haemophilus influenzae (NTHi) is a pathobiont which chronically colonises the airway of individuals with chronic respiratory disease. It is unclear how NTHi persists in the airway, however accumulating evidence suggests that NTHi can invade and persist within macrophages. To better understand the mechanisms of NTHi persistence within macrophages, this work developed an in vitro model of NTHi intracellular persistence using human monocyte-derived macrophages (MDM). Dual RNA Sequencing was used to assess MDM and NTHi transcriptomic regulation occurring simultaneously during NTHi persistence. This work demonstrates that NTHi can invade and persist within human macrophages. Macrophages activate innate immune responses, whereas NTHi persistence was facilitated by transcriptomic adaptations in bacterial metabolic, stress response and ribosome pathways. This research provides transcriptomic insights into NTHi-macrophage interactions, enhances our understanding of how NTHi can utilise host immune cells to chronically colonise the airway and identifies potential bacterial gene pathways that may be attractive therapeutic targets Monocyte derived macrophages (MDM) were infected with NTHi ST14 at MOI 100, or left uninfected (controls) for 6 h, washed with gentamicin for 90 min and incubated in antibiotic free infection media until 24 h. RNA from five biological repeats was harvested at 7.5 h (following 90 min gentamicin wash, indicated as the 6h timepoint) and at 24 h using Trizol and total RNA was extracted using miRNeasy kit. Samples were supplied to Novogene (Hong Kong) for sequencing. Briefly, ribosomal RNA (rRNA) was removed from all samples using Illumina® Ribo-Zero Plus rRNA Depletion Kit. Libraries were generated using the NEBNext®Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). Sequencing was performed using NovaSeq 6000 Illumina platform and 150-base pair (bp) paired end reads were generated using a sequencing depth of 90 million reads.

不可分型流感嗜血杆菌（Nontypeable Haemophilus influenzae，NTHi）是一种致病共生体，可慢性定植于慢性呼吸道疾病患者的气道中。目前学界尚未明确NTHi在气道内的存活机制，但越来越多的研究证据表明，该菌可侵入并存活于巨噬细胞内。为深入阐明NTHi在巨噬细胞内的存活机制，本研究构建了基于人单核细胞衍生巨噬细胞（monocyte-derived macrophages，MDM）的体外感染模型，用于模拟NTHi的胞内存活过程。本研究采用双转录组测序（Dual RNA Sequencing）技术，同步分析NTHi存活期间宿主巨噬细胞与细菌自身的转录调控变化。

本研究证实，NTHi可侵入并存活于人巨噬细胞内：巨噬细胞会激活先天免疫应答通路，而NTHi则通过调控自身代谢、应激反应及核糖体相关通路的转录组适应性改变，实现胞内存活。本研究为解析NTHi与巨噬细胞的互作机制提供了转录组层面的全新视角，加深了学界对NTHi如何利用宿主免疫细胞实现气道慢性定植的认知，并鉴定出若干潜在的细菌基因通路，可作为后续治疗干预的靶点。

具体实验流程如下：以感染复数（multiplicity of infection，MOI）为100的NTHi ST14菌株感染人单核细胞衍生巨噬细胞（MDM），设置未感染组作为空白对照；感染6小时后，使用庆大霉素冲洗细胞90分钟以清除胞外游离细菌，随后更换无抗生素的感染培养基继续培养至24小时。分别于7.5小时（即庆大霉素冲洗结束后，对应本研究标注的6小时时间点）和24小时收集样本，共设置5次生物学重复。采用TRIzol试剂裂解细胞提取总RNA，再通过miRNeasy试剂盒纯化总RNA。样本交由香港诺禾致源（Novogene）开展测序工作。具体测序流程为：使用Illumina® Ribo-Zero Plus rRNA去除试剂盒移除所有样本中的核糖体RNA（rRNA）；采用NEBNext®Ultra™ Directional RNA Library Prep Kit for Illumina®（美国NEB公司）构建测序文库；依托Illumina NovaSeq 6000测序平台进行测序，生成150碱基对（bp）的双端读段，测序深度达9000万条读段。