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 Overall design: 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.