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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.

frontiers in cellular and infection microbiology

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