Towards understanding the susceptibility of primary ciliary dyskinesia airway epithelia to non-typeable haemophilus influenzae biofilm infection

Abstract

Primary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia. Global prevalence of PCD is 1 in 7554, with individuals of African ancestry being mostly commonly affected. Reduced mucociliary clearance by defective motile cilia in the airways results in neonatal respiratory distress, chronic sinusitis, persistent daily wet cough, recurrent lower respiratory tract infections and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. Biofilms are notoriously difficult to eradicate and they act as persistent sources of infection, perpetuating the bronchiectasis “vortex”.
Besides the characteristic lack of mucociliary clearance and low nasal nitric oxide in PCD, intrinsic factors contributing to chronic respiratory infections and bronchiectasis remain largely unknown. The hypothesis for this study is that there are dysregulated biological processes in PCD airway epithelia that could contribute to increased susceptibility to NTHi biofilm infection. To address this, an in vitro air-liquid-interface (ALI) model of primary nasal epithelial cells and 72-hour NTHi (PCD clinical isolate) biofilm infection was optimised and characterised. The model was first used as a basis to investigate early NTHi infection in the upper and lower respiratory tracts by flow cytometry. Then, differing biological processes present at baseline and in response to 72-hour NTHi infection in PCD airway epithelia (compared with healthy donors) were assessed by liquid chromatography mass spectrometry analysis of proteins.Systematic characterisation of ALI-cultured nasal epithelia evidenced maximal differentiation by day 28 with widespread ciliation, motile cilia, goblet cells, tight junctions and maintained epithelial barrier integrity until the last experimental time-point of 63 days. Cultures at 28+ days of ALI-culture were then used for co-culture optimisation work. Based on maintained epithelial health and high bacterial recoverability following72-hours of infection, multiplicity of infection 50 was selected and biofilm aggregate formation was confirmed. Flow-cytometric analysis of major epithelial cellular subtypes (basal, ciliated and secretory) suggests that secretory cells are the most abundant subtype and that upper respiratory tract cells are more readily infected by NTHi than lower. Due to low sample number, respiratory disease type comparisons were not conclusive, but in the one PCD sample used there were a high number of internalised NTHi. Proteomic analysis revealed several dysregulated biological processes in PCD when compared with healthy epithelia. In PCD there was heightened oxidative stress and metabolic state, more cell death, actin-related cytoskeletal dysfunction and protein S- nitrosylation. Of note, PCD epithelia lacked HSP90 which is associated with nitric oxide production, worsened ciliary beating and decreased bacterial killing. Several S100 proteins (with diverse functions) were differentially expressed at baseline in PCD epithelia and S100-A4 increased in expression with infection. In response to infection, ICAM-1 was not upregulated by PCD cells but was in healthy. One actin-related protein (filamin A) was upregulated in response to infection in PCD but downregulated in healthy. In summary, interrogation of host-pathogen interactions using the ALI-co-culture platform has highlighted that several biological processes could be contributing to PCD airway susceptibility to NTHi biofilm infection. High NTHi infectivity rate of PCD nasal cells (albeit in one sample) detected by flow cytometry supports the concept that PCD epithelium is more susceptible to NTHi colonisation/infection, warranting further investigation. Given that upper airway cells were found to be more readily infected than lower airway cells using the flow cytometric method, extrapolation of proteomics findings in nasal epithelia to bronchial should be avoided. Dysregulation of individual proteins (detected by proteomic analysis) including members of the S100 family, ICAM-1, actin- related cytoskeleton organisation and the relationship between HSP90 and NO in PCD merits further study. Likewise, further validation of dysregulated biological processes and differentially expressed proteins in PCD epithelia needs to be conducted.

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Identifiers

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

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

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