{
  "abstract": "Introduction Viral infections trigger exacerbations of chronic obstructive pulmonary disease (COPD) through poorly defined mechanisms. Perturbation of the airway microbiome with expansion of Haemophilus genus is reported in COPD. Haemophilus influenzae colonisation is strongly associated with exacerbation susceptibility but roles played by other resident Haemophilus commensals are unknown. We sought to investigate airway microbiome-immune interactions and define roles played in driving COPD viral susceptibility.Methods We used metagenomics to profile microbiota in subjects with COPD undergoing rhinovirus challenge (n=21) and analysed relationships between commensals and immunological/clinical responses to infection. We employed mouse models to study the specific effects of Haemophilus species upon immune responses to rhinovirus infection with experimental manipulations to define functional relationships.Results Sputum metagenomics indicated that baseline Haemophilus genus abundance correlated with rhinovirus induction of antiviral mediators (sputum interferon-λ1, r=0.4846, P<0.05), and other immunological readouts including sputum% neutrophils (r=0.8108, P<0.05). Linear discriminant analysis effect size (LEfSe) analysis comparing susceptible (i.e. developed exacerbation) versus resistant (i.e. no clinical/immunological response to rhinovirus challenge) COPD indicated that H.influenzae and several Streptococci were associated with viral susceptibility whilst H.parainfluenzae and other Haemophilus_D species were associated with resistance.Using mouse models of Haemophilus dysbiosis, we found that H.influenzae potently exacerbated early airway antiviral (type-I/III interferon) and subsequent pro-inflammatory responses (neutrophilic inflammation, exhibiting more activated/pathogenic phenotype) to rhinovirus. Conversely, H.parainfluenzae administration attenuated neutrophilic responses to rhinovirus whilst other Haemophilus species tested had minimal immunomodulatory effects.Blockade of immune signalling using an anti-IFNAR1 antibody abrogated H.influenzae-mediated pro-inflammatory responses to rhinovirus, indicating that the exacerbating effect is dependent on type-I interferon. This effect was retained when heat-killed H.influenzae was administered, suggesting a mechanism via pattern-recognition receptor (PRR) stimulation. Comparison of relative PRR stimulation using a HEK293 cell reporter assay indicated that H.influenzae more potently activated TLR2/NOD2 than other Haemophilus species. Given that TLR2 activation can promote type-I interferon, ongoing studies are examining effects of TLR2 inhibition within these models.Abstract T2 Figure 1(A) Sputum bacterial species associated with susceptibility to or resistance from rhinovirus challenge in COPD. Linear Discriminant Analysis Effect Size (LEfSe) of shotgun metagenomics, calculated with LDA alpha score 0.05 and LDA threshold 2.0. (B) Haemophilus species-specific augmentation of rhinovirus-induced neutrophil recruitment in mouse lung is driven by type-I interferon. Flow cytometry showing numbers of neutrophils in lung from BALB/c mice treated with intranasal H.influenzael H.parainfluenzae (1x104 CFU) and subsequently infected with RV-A1B (MOI1) for 24 hours; anti-IFNAR1 antibody administered intranasally one hour prior to Haemophilus treatment. Data generated from two independent experiments (n=5–10), represented as mean ± SEM. Statistical significance analysed by Mann-Whitney U, where *P<0.05 and **P<0.01. (C) Comparison of PRR stimulation by Haemophilus species in HEK293-reporter assay. NF-κB activation (OD630nm) of HEK derived reporter cell lines, TLR2/TLR4/NOD2 or Null (control) by Haemophilus species (MOI1). Statistical significance analysed by Mann-Whitney U, where **P<0.01 and ****P<0.0001 difference between H.influenzae and other speciesConclusion Haemophilus commensals within the airway microbiome exhibit differing pro-inflammatory and anti-inflammatory effects which may influence COPD viral susceptibility. Further studies are needed to define the optimum approaches for therapeutically targeting Haemophilus dysbiosis in COPD.",
  "authors": [
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "MM Jackson"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "O Katsoulis"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "M Kusumoto"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "C Chen"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "K Sanghavi"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "J Aniscenko"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "P Mallia"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "KT Mincham"
    },
    {
      "affiliations": [
        "University of Warwick, Coventry, UK"
      ],
      "name": "A Decout"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "SL Johnston"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "TB Clarke"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "RJ Snelgrove"
    },
    {
      "affiliations": [
        "Imperial College London, London, UK"
      ],
      "name": "A Singanayagam"
    }
  ],
  "title": "T2 Haemophilus commensals within the airway microbiome modulate immune responses and dictate viral susceptibility in COPD",
  "uid": "c1302e64-cd09-5975-ba06-7e65b415668f"
}