Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells.

Janessa L Pickering,Ruth B Thornton,Lea-Ann S Kirkham,Guicheng Zhang,Amy Prosser,Peter C Richmond,Camilla De Gier,Karli J Corscadden

doi:10.3389/fcimb.2016.00050

Abstract

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease.

Highlights

Haemophilus haemolyticus is a respiratory tract commensal that is closely related to the opportunistic pathogen nontypeable Haemophilus influenzae (NTHi)
H. haemolyticus33390 challenge had no impact on Detroit 562 human pharyngeal carcinoma epithelial cells (D562) cell viability at 3 h, the epithelial viability reduced to 50% by 24 h (Figure 1C)
As H. haemolyticus does not grow well in liquid media, it is possible that bacterial death results in release of cytotoxic components over 24 h

Summary

Introduction

Haemophilus haemolyticus is a respiratory tract commensal that is closely related to the opportunistic pathogen nontypeable Haemophilus influenzae (NTHi). The distinction of H. haemolyticus as a true commensal is complicated by the fact that H. haemolyticus can be misidentified as NTHi using routine microbiological tests (reviewed in Pickering et al, 2014b). Whilst in-depth investigations into the genetics of H. haemolyticus and NTHi have been conducted in order to develop discriminatory tests to distinguish these closely related species (McCrea et al, 2008, 2010a; Sandstedt et al, 2008; Norskov-Lauritsen, 2011; Binks et al, 2012; Pickering et al, 2014b), the interaction of H. haemolyticus with host cells has not been previously investigated

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