NHNE DEGs.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, ‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

流感嗜血杆菌（Haemophilus influenzae）是人类呼吸道致病菌，仅以人类呼吸道作为其唯一生存生态位。尽管如此，目前学界对流感嗜血杆菌建立人类上皮持续性感染的分子机制仍知之甚少。本研究利用一种高度模拟人类鼻上皮的原代细胞感染模型（正常人类鼻上皮细胞，NHNE），探究了流感嗜血杆菌如何适应宿主环境并触发宿主免疫应答。通过生理学实验联合双转录组测序（dualRNAseq）分析发现，5名健康供体来源的正常人类鼻上皮细胞在感染流感嗜血杆菌后，均会先呈现初始的“无效性”炎症应答——该应答伴随显著的缺氧特征，但并未产生促炎细胞因子。后续研究观察到，宿主细胞逐渐对大量细胞外及上皮内的流感嗜血杆菌载量产生耐受，此时正常人类鼻上皮细胞的转录组图谱恢复至感染前状态。这一过程与上皮内细菌种群的形成同步发生，且似乎涉及核因子κB（NFκB）信号通路的阻断。这是首次在感染过程中观测到流感嗜血杆菌可通过大规模免疫调节作用促进自身持续定植；同时本研究证实，仅活的（而非热灭活的）流感嗜血杆菌感染可引发免疫调节，并下调受核因子κB调控的促炎细胞因子，如白细胞介素1β（IL-1β）、白细胞介素36γ（IL-36γ）及肿瘤坏死因子α（TNFα）的表达。有趣的是，在14天感染周期的末期，正常人类鼻上皮细胞可重新激活促炎应答，最终释放白细胞介素8（IL-8）与肿瘤坏死因子α。本研究不仅首次揭示了流感嗜血杆菌在宿主内实现持续定植的分子机制，同时数据还提示存在感染阶段特异性的基因表达模块，凸显了正常人类鼻上皮细胞免疫应答与经典免疫细胞免疫应答之间的核心相似性，该发现值得进一步深入研究。