Interplay between macrophages and non-typeable haemophilus influenzae biofilms on respiratory epithelium

Abstract

Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that causes respiratory infections in patients with pre-existing conditions, for example, it is the most commonly isolated pathogen from patients with Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that is characterised by a reduction or loss of ciliary function. One reason for the persistence of NTHi infection is the formation of biofilms on the airway epithelium. Biofilms are aggregates of bacteria encased in an extracellular matrix that reduces immune clearance and increases antibiotic tolerance. The persistent, yet unresolved nature of the NTHi biofilm leads to the development of chronic inflammation, facilitated in part by tissue resident and recruited macrophages. This project aims to develop an ex vivo model of NTHi biofilms on Primary Nasal Epithelial Cells (PNECs) at the air liquid interface (ALI) to investigate the impact of biofilm formation on macrophage mediated clearance. The characterisation of the inflammatory response and the resulting effect on the respiratory epithelium will increase our understanding of the immune response to NTHi biofilms.
The overarching hypothesis of this work is: Monocyte Derived Macrophages (MDMs) are more effective at clearing established Non-typeable Haemophilus influenzae (NTHi) biofilms on Primary Nasal Epithelial Cell Air-Liquid Interface (PNEC ALI) culture than established biofilms on an abiotic surface.
In order to address this hypothesis, the aim was to first characterise the biofilm formation of a selection of clinical NTHi isolates, both on an abiotic surface and on PNEC ALI cultures. Subsequently, the impact of primary Monocyte Derived Macrophages (MDMs) on NTHi biofilms was investigated in both of these culture systems.
The planktonic growth characteristics of six clinical isolates of NTHi from PCD patients and a GFP expressing strain were investigated using optical density readings and CFU enumeration. Biofilm formation of all seven NTHi strains was then characterised on an abiotic surface. Crystal violet (CV) staining and colony forming units (CFUs) were used as indicators of biomass and viability respectively. Confocal microscopy was used to visualise and quantify biofilm structure. Antibiotic tolerance of GFP-NTHi and PCD-NTHi 4 biofilms was demonstrated to verify biofilm formation.
Following the characterisation of NTHi biofilms on an abiotic surface, PNECs from healthy volunteers were differentiated to a pseudostratified, ciliated phenotype at ALI for 4 weeks prior to infection with GFP-NTHi and PCD-NTHi 4. A range of multiplicities of infections (MOI) were investigated in order to identify a ratio of bacteria to epithelial cells that allows the model to remain viable over 72 h. Transepithelial electrical resistance (TEER) and CFUs were used as indicators of epithelial viability and biofilm viability respectively. Confocal and scanning electron microscopy were used to visualise the co-cultures.
Primary monocyte derived macrophages (MDMs) were then added to PCD-NTHi 4 biofilms grown on an abiotic surface and on ALI cultures. The impact on biofilm size and viability was measured as before as well as the macrophage response in terms of cytokine and Lactate dehydrogenase (LDH) release.
NTHi biofilm and planktonic growth was found to be highly heterogeneous. Following the characterisation of biofilm formation on plastic, PCD-NTHi 4 was identified as a suitable clinical isolate to take forward to ALI culture alongside GFP-NTHi based on viability, biomass and structural uniformity.
Both PCD-NTHi 4 and GFP-NTHi could be recovered from co-cultures with differentiated PNEC ALI cultures after 72 h. MOI and the length of incubation before removing non-attached bacteria were optimised in order to maximise epithelial cell layer integrity and NTHi recovery. GFP-NTHi was found to be unable to form apical biofilms on ciliated ALI cultures, instead invading the cell layer. The strain was therefore deemed unsuitable for this model.
Primary MDMs could be co-cultured with established PCD-NTHi 4 biofilms on plastic. Though a significant pro-inflammatory response was detected, bacterial viability was not affected by the presence of MDMs. Establishing a triple co-culture of PCD-NTHi 4 and MDMs on PNEC ALI cultures showed that all cell types remained viable within this model.
This work suggests the inability of MDMs to reduce NTHi biofilms, both on plastic and on PNEC ALI cultures despite producing a pro-inflammatory response. Instead, an increased number of NTHi is recovered from triple co-cultures than from NTHi on ALI cultures alone. The model developed to investigate this interaction appeared to remain viable for the 96 h co-culture protocol and serves as a basis for further optimisation and development.

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Identifiers

ORCID for Jana Franziska Hueppe: orcid.org/0000-0003-1585-7026

ORCID for Jane Lucas: orcid.org/0000-0001-8701-9975

ORCID for Claire Jackson: orcid.org/0000-0002-1200-0935

ORCID for Karl Staples: orcid.org/0000-0003-3844-6457

ORCID for David Cleary: orcid.org/0000-0003-4533-0700

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