Investigating macrophage-pathogen interactions in asthma

The pathobiont, Nontypeable Haemophilus influenzae (NTHi), colonises the airway of individuals with chronic respiratory disease and is particularly associated with severe, neutrophilic, steroid-resistant asthma. Although NTHi has been implicated in asthma, respiratory tract viral infections remain the main aetiological agent of asthma exacerbations. However, it is now becoming clear that the presence of potentially pathogenic bacteria, such as NTHi, are present in the airway prior to respiratory tract viral infections. The macrophage is the predominant immune cell in the airway, yet accumulating evidence suggests NTHi is able to infect and persist within macrophages, which are also a target of the influenza A virus (IAV). It is unclear whether NTHi infection and persistence modulates macrophage responses to respiratory tract viral infections.The aim of this thesis was to investigate modulation of macrophage gene expression during intracellular NTHi infection and how this interaction impacts on the response of these cells to subsequent infection with IAV. In addition, this thesis aimed to investigate transcriptomic alterations of NTHi during intracellular infection of macrophages, in order to identify mechanisms of persistence.To achieve these aims, dual RNASeq analysis of an NTHi-monocyte-derived macrophage (MDM) infection model was performed. Transcriptomic analysis of NTHi-infected MDM identified enrichment of macrophage intracellular immune response pathways. Use of WGCNA identified CASP4, PNRC1 and SGPP2 to be the central MDM genes in the gene module most significantly associated with NTHi infection. Despite activation of MDM innate immune responses, NTHi was still able to persist within these cells. NTHi adaptation to persistence was associated with modulation of bacterial pathways involved in metabolic and stress responses, and downregulation of NTHi ribosomal protein genes. However, validation of the top NTHi differentially expressed genes bioC, mepM and dps, found strain-dependent expression of NTHi genes. Validation of select macrophage intracellular immune response genes demonstrated conservation of the MDM transcriptomic response when challenged with additional clinical strains of NTHi. Furthermore, NTHi presence was detected by FISH in 56% of severe asthma bronchoalveolar lavage (BAL) samples, which was associated with increased neutrophil inflammation (p=0.0462) and asthma duration (p=0.0436). Elevated IL1B (p=0.0041), GBP1 (p=0.0477) and SGPP2 (p=0.0221) gene expression was detected in samples determined as NTHi positive compared to NTHi negative, indicating modulation of airway inflammation by NTHi. Adaptation of the MDM model to incorporate the IAV following NTHi infection resulted in further modulation of the infection process; IAV replication levels decreased (p=0.0049), whereas NTHi load increased (p=0.0313). Decreased IAV levels was suggested to be due to NTHi-mediated upregulation of macrophage anti-viral immunity, specifically the type I IFN pathway, prior to IAV infection. Increased NTHi presence was associated with transcriptomic changes in NTHi genes previously identified to be involved in NTHi adaptation to intracellular persistence, bioC and mepM. Consequently, NTHi-infected macrophages exhibited a sustained inflammatory response, compared to MDM infected with IAV-alone.The data in this thesis indicate the ability of NTHi to adapt in order to persist within macrophages, despite activation of macrophage intracellular immune response pathways. The subsequent modulation of IAV infection and NTHi colonisation during co-infection resulted in sustained macrophage inflammation that was not sufficient to completely clear either pathogen. Modulation of macrophage responses prior to and during bacterial-viral co-infection could have important implications for designing future studies to better our understanding of multiple host-pathogen interactions in the lung