Dual RNASeq Reveals NTHi-Macrophage Transcriptomic Changes During Intracellular Persistence.

Jodie Ackland,Ashley I. Heinson,Tom M. A. Wilkinson,David W. Cleary,Myron Christodoulides,Karl J. Staples

doi:10.3389/fcimb.2021.723481

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a pathobiont which chronically colonises the airway of individuals with chronic respiratory disease and is associated with poor clinical outcomes. 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, we 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. Analysis of the macrophage response to NTHi identified temporally regulated transcriptomic profiles, with a specific ‘core’ profile displaying conserved expression of genes across time points. Gene list enrichment analysis identified enrichment of immune responses in the core gene set, with KEGG pathway analysis revealing specific enrichment of intracellular immune response pathways. NTHi persistence was facilitated by modulation of bacterial metabolic, stress response and ribosome pathways. Levels of NTHi genes bioC, mepM and dps were differentially expressed by intracellular NTHi compared to planktonic NTHi, indicating that the transcriptomic adaption was distinct between the two different NTHi lifestyles. Overall, this study provides crucial insights into the transcriptomic adaptations facilitating NTHi persistence within macrophages. Targeting these reported pathways with novel therapeutics to reduce NTHi burden in the airway could be an effective treatment strategy given the current antimicrobial resistance crisis and lack of NTHi vaccines.

Highlights

Haemophilus influenzae is a human-restricted pathobiont (Erwin and Smith, 2007) and is commonly isolated from the nasopharynx, middle ear and respiratory tract (King, 2012; Swords, 2012; Ahearn et al, 2017)
monocyte-derived macrophages (MDM) were challenged with a clinical isolate of nontypeable Haemophilus influenzae (NTHi) (ST14) for 2 h, 6 h or 24 h followed by a 90 min gentamicin wash to kill and remove extracellular NTHi, resulting in only intracellular NTHi present in the model
Macrophages may act as a protected niche within the airway, promoting NTHi colonisation, especially in chronic respiratory diseases that already have evidence of macrophage dysfunction

Summary

Introduction

Haemophilus influenzae is a human-restricted pathobiont (Erwin and Smith, 2007) and is commonly isolated from the nasopharynx, middle ear and respiratory tract (King, 2012; Swords, 2012; Ahearn et al, 2017). Encapsulated strains are NTHi-Macrophage Transcriptomic Changes classified into six serotypes (a-f), with strains not in possession of a capsule unable to be serotyped and are designated as nontypeable Haemophilus influenzae (NTHi). NTHi is implicated in exacerbations of chronic respiratory diseases, NTHi has been isolated from the airway during stable periods of disease (Wood et al, 2010; Iikura et al, 2015; Zhang et al, 2016; Wilkinson et al, 2017; Mayhew et al, 2018). The duration of NTHi airway colonisation varies, with longitudinal studies suggesting persistence ranges from months up to as long as 7 years (Murphy et al, 2004; Román et al, 2004; Gallo et al, 2018)

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