Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

Paul T King,Philip G Bardin,Belinda Thomas,Naghmeh Radhakrishna,Roleen Sharma,Ama-Tawiah Essilfie,Stephen R Holdsworth,Stavros Selemidis,James Ngui,Steven Lim,Peter Mclaughlin,Michael Farmer,Camden Lo,Daniel P Steinfort ,Bradley Randal Scott Broughton ,Jyotika D Prasad ,Peter Holmes ,Judith Mary Callaghan ,Philip M Hansbro ,Kim M O’sullivan ,Bethany Jennings ,Michael J Hickey

doi:10.1371/journal.pone.0120371

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

Highlights

Nontypeable Haemophilus influenzae (NTHi) lacks a capsule and is present as a commensal in the pharynx of healthy adults
NTHi induces sustained and extracellular production of ROS by lung macrophages As macrophages are the first cells involved in host defence against airway bacterial infection and express high levels of ROS to NTHi, we investigated their function in more detail
In this study we examined the effect of the prevalent bacterium nontypeable Haemophilus influenzae on lung inflammation

Summary

Introduction

Nontypeable Haemophilus influenzae (NTHi) lacks a capsule (which distinguishes it from typeable forms such as HiB) and is present as a commensal in the pharynx of healthy adults. It may move down into the lower respiratory tract where it is the most common bacterium isolated in subjects with chronic bronchitis and chronic obstructive airway disease (COPD) and is an important cause of pneumonia [1]. It is prevalent in bronchiectasis, cystic fibrosis and upper respiratory tract conditions including otitis media, conjunctivitis, tonsillitis and sinusitis. It has been described as “an innocent bystander” [4] but there is increasing evidence that it causes lung inflammation in the context of COPD [5,6,7]

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Methods

Conclusion