Functional effects of microbiota in chronic respiratory

disease

Kurtis F Budden*, Shakti D Shukla*, Saima Firdous Rehman, Philip Hugenholtz, Darius PH

Armstrong-James, Ian M Adcock, Sanjay H Chotirmall, Kian Fan Chung, Philip M Hansbro

*Contributed equally

Priority Research Centre for Healthy Lungs, Hunter Medical Research Institute and The

University of Newcastle, Newcastle, New South Wales, Australia (K F Budden, S D Shukla

PhD, S F Rehman, P M Hansbro PhD); Australian Centre for Ecogenomics, School of

Chemistry and Molecular Biology, The University of Queensland, Queensland, Australia (P

Hugenholtz); National Heart & Lung Institute, Imperial College London, London, UK (DPH

Armstrong-James, I M Adcock PhD, K F Chung DSc); Lee Kong Chian School of Medicine,

Nanyang Technological University, Singapore (S H Chotirmall PhD); Centenary Institute and

University of Technology Sydney (P M Hansbro PhD)

Correspondence to:

Prof Philip M Hansbro,

Priority Research Centre for Healthy Lungs,

Hunter Medical Research Institute and The University of Newcastle,

Newcastle, New South Wales, 2300, Australia

1

The composition of the lung microbiome is increasingly well characterised, with dysbiosis

changes in microbial diversity or abundance a key factor in chronic respiratory diseases

(CRDs) such as asthma, cystic fibrosis, bronchiectasis and chronic obstructive pulmonary

disease (COPD). However, the precise effects of dysbiosis the microbiome and the functional

mechanisms by which itthe microbiome regulates host immunity are only now beginning to

be elucidated. Bacteria, viruses and fungi from both the upper and lower respiratory tract

produce structural ligands and metabolites which interact with the host and alter the

development and progression of CRDs. Here, we review recent advances in our

understanding of the composition of the lung microbiome, including the virome and

mycobiome, the mechanisms by which these microbes interact with host immunity, and

their functional effects on the pathogenesis of CRDs, their exacerbations and co-

morbidities., We also describe the current knowledge of interactions between respiratory

microbiota and common therapies of CRDs, and the potential manipulation of the

respiratory microbiome as a therapeutic strategy. Finally, we highlight some of the current

limitations in the field and propose how these may be addressed in future research.

2

Search Strategy and selection Criteria

References for this review were identified through searches of PubMed and Google scholar

for articles published from January, 2007, to June 2018 by use of the terms “lung

microbiome”, “microbiota”, “lung microbiota”, “gut microbiota”, “lung mycobiome”, “lung

virome”, “functional”, “respiratory disease”, “COPD”, “Cystic fibrosis”, “Bronchiectasis” and

“Asthma”. Articles were identified as relevant where studies provided mechanistic insight

into the effects of lung microbiota. Articles resulting from these searches and relevant

references cited in those articles were reviewed. Articles published in English were included.

Key Messages

Although dysbiosis of tThe composition of the respiratory microbiome has been

identified characterised in chronic respiratory diseases (CRDs), yet there is little

available information regarding the mechanisms by which the microbiota regulate

disease development and progression.

Recently, the functional effects of key structural ligands and metabolites from

bacteria, viruses and fungi of the lung microbiota on boht innate and adaptive

immunity have been characterised identified, influencing the development and

progression ofin the context of CRDs, their exacerbations and co-morbidities such as

asthma, cystic fibrosis, bronchiectasis and chronic obstructive pulmonary disease.

Respiratory microbiota may also impact on, or be impacted by, common treatments

for CRDs or extra-pulmonary co-morbidities, resulting in functional consequences in

the lungand may present novel therapeutic targets.

3

Significant limitations remain in our understanding of the functional effects of

respiratory microbiota. We propose that improved understanding in this field can be

driven by improved assessment of microbial function through integrated ‘omics,

targeted study of the virome/mycobiome and their interaction with bacteria,

improved experimental design and emphasis on interventional studies.

Improved understanding of the mechanisms by which lung microbiota interact with

the host may facilitate the development of novel therapeutic strategies to improve

outcomes of CRDs.

Introduction to the respiratory microbiome

The term “microbiome” refers to the collective sum of microorganisms (i.e. bacteria, archea,

viruses, fungi), their genomes, products and environmental conditions in a given

ecosystem.1,2 Subsets of the microbiome are defined as the bacteriome, virome and

mycobiome. “Microbiota” refers to only the microorganisms themselves at a particular

site, , and are classified as pathogenic (causing or contributing to disease

development/progression) or commensal (non-disease causing; interaction neutral or

beneficial for host), although this distinction may differ depending on the disease in

question.2,3 They are detected throughout the upper (URT) and lower respiratory tract (LRT),

with distinct populations and different burdens at different sites.2

Bacteria are not the only microbes constantly present in the respiratory tract, and

our appreciation of the virome and mycobiome is rapidly increasing. The bacteriome and

virome develop soon after birth, and are dynamic and dependent on environmental factors

and genetic background.2,4 They profoundly affect the evolution of innate and adaptive

immune responses,2,5 and can be categorised into pathogenic and commensal components

4

(for viruses; endogenous retroelements and long-term resident persisters).6 Further

complexity occurs with the propensity for rapid genetic evolution of bacteria and viruses.

Bacterial burden in the URT is 100-10,000 times greater than in the LRT, with the

nasal cavity dominated by members of the genera Propionibacterium, Cornynebacterium,

Staphylococcus and Moraxella, and the oral cavity containing primarily Prevotella,

Veillonella, Streptococcus, Haemophilus, Fusobacterium, Neisseria and Corynebacterium.7-9

Microbes from the URT (especially the oral cavity) and external environment enter the LRT,

and the balance of microbial migration (breathing, mucocilliary clearance, microaspiration),

elimination and growth results in a viable bacterial presence without long-term colonisation,

termed a transient or non-resident microbiome.2,10 Though a core microbiome dominated by

Streptococcus, Prevotella, Veillonella, Pseudomonas, Haemophilus and Fusobacterium has

been reported in healthy individuals, 8,9,11Because of the low bacterial load and transient

population, the LRT microbiome has greater variability in community composition and

susceptibility to environmental changes than the URT.,12 This is partially due to the low

bacterial load and transient population, as well as lung architecture (e.g. increasing number

of bronchial branches deeper in the lungs can unevenly disperse inhaled bacteria). Regional

variation in mucus or surfactant secretion, pH, and nutrient (e.g. iron, vitamins) or oxygen

availability (e.g. gas trapping) can also increase biogeographical variability in the LRT

microbiota, and these may be exacerbated during inflammation or structural changes in

CRDs.8but a core microbiome dominated by Streptococcus, Prevotella, Veillonella,

Pseudomonas, Haemophilus and Fusobacterium has been reported in healthy individuals.

The recently discovered diverse vertebrate viral family, the Anelloviridiae family

forms around 70% of the human virome in blood and most organs including the respiratory

5

tract,5 and other significant components include herpes viruses and human papillomavirus.4

Anellovirdiae are currently thought to be apathogenic, but our understanding of how they

influence host immunity is at a nascent stage. There is also a core set of viral predators of

bacteria (bacteriophages) in the URT and LRT, which may alter the microbiome by killing

specific bacterial subpopulations.13 Metagenomic approaches have identified many new

viruses in the respiratory tract with unknown effects, but respiratory syncytial virus (RSV),

influenza A virus (IAV) and rhinovirus are well known to have pathogenic effects.

Fungi lack detailed characterisation in the respiratory tract. Next generation

sequencing, so powerful in profiling bacteria, has substantially increased the understanding

of the pulmonary mycobiome, but is hampered by a dearth of fungal ‘reference’ genes

comparable to bacterial 16S rRNA. The eukaryotic equivalent 18S rRNA does not show good

resolution and the widely used internal transcribed spacer 1 (ITS1) region of the eukaryotic

ribosomal cluster is limited in its ability to uncover fungal diversity.14 The airway is

constantly exposed to fungal spores, but most healthy individuals effectively clear them

with no consequence. Host susceptibility and immune status determines if acute or chronic

disease results. Fungal pathogens exhibit remarkable adaptability to the human lung, partly

due to the expansion of biosynthetic gene clusters, which produce bioactive secondary

metabolites and encompass human toxins (e.g. aflatoxin).15 Given its dominance as the

primary human fungal pathogen, there is sustained interest in how the filamentous

Aspergillus fumigatus exploits these metabolites for virulence and over 30 biosynthetic gene

clusters are known.16

An improved understanding of how bacteria, viruses and fungi of the microbiome

affect the respiratory tract is required to understand their role in disease.

6

Influence on Immunity: Signals from the respiratory microbiome (figure 1)

‘Pneumotypes’, as described by Segal et al., are descriptions of microbiome composition

associated with particular phenotypesphenotypes.17 Enrichment of the LRT microbiome with

oral taxa in healthy individuals is associated with Th17-driven inflammation,17 and lung

transplant recipients with neutrophilic activation profiles or macrophage-dominant,

remodelling profiles have different patterns of dysbiosis (Firmicutes/Proteobacteria- vs

Bacteroidetes-dominant).18 In mice, lung community composition correlates with IL-1α and

IL-4 abundance. However, IL-1 receptor blockade did not alter microbiome composition,

suggesting that, at least in some circumstances, microbiota drive immune phenotypes as

opposed to immunity changing microbiome composition.12 Host immunity is contingent on

symbiosis with the microbiome, and therefore the virome and mycobiome are further important

cofactors in shaping the pulmonary inflammatory response.4,5,19

To define how microbiota shape lung and systemic immunity, it is essential to understand

the signals they present, which are broadly similar between the respiratory tract and gut

microbiota but differ in abundance, composition and localised effects.

Structural ligands

Structural microbial ligands are recognised by host pattern recognition receptors (PRRs),

including Toll-like receptors (TLRs).20 Microbiota stimulation of TLRs improves host defence

through IgA production,21 and the TLR4 agonist lipopolysaccharide (LPS) from Escherichia

coli promotes inflammatory cytokine responses in human alveolar macrophages.17

7

Moreover, intranasal treatment of exopolysaccharide from Bifidobacterium longum to mice

stimulates TLR2 to promote allergic tolerance through IL-10 production and increased

M1/M2 macrophage ratio.22 These studies demonstrate roles for microbiota-derived TLR

agonists in immune regulation yet the ligands were isolated from gut commensals. Whether

ligands from lung microbiota exert similar effects is unclear, although pathogenic members

of the lung microbiome stimulate TLRs more efficiently than commensals,3,8,20 and TLR2/4

are needed for bacteria-mediated protection against allergic airway disease (AAD).23

Furthermore, LRT microbiome composition is associated with the magnitude of TLR

responses, adding further complexity.17

Other host PRRs are involved in host-microbiome interactions, such as the NOD-like

receptor (NLR) NOD2 which is stimulated by peptidoglycan from lung microbiota to promote

alveolar macrophage responses to infection.24 Not all microbiome ligands are derived from

bacteria. RNA from the archaeum Methanosphera stadtmanae promotes antiviral immunity

and activates the inflammasome in monocytes and dendritic cells,25 and torquetenovirus

regulates inflammatory responses through stimulatory CpG (TLR9 agonists),

immunoregulatory miRNAs, and ORF2-mediated suppression of NF-κB translocation.5

Crucially, these effects are species- or strain-specific, and highlight how slight variations in

microbiome composition may have major impacts on immunity. The respiratory virome further

primes and modulates host immune responses and contains bacteriophages, influenced by

environmental exposure and the presence of CRD.4,25

8

MetabolitesMetabolites

Predicting microbial metabolic effects on the lungs from culture-based assays and

community profiling (e.g. 16S rRNA gene sequencing) is unreliable. Pseudomonas

aeruginosa cultured on yeast/malt extract or artificial sputum medium produce vastly

different metabolic profiles, with only the latter producing quinolones, phenazines and

rhamnolipids representative of sputum from cystic fibrosis (CF) patients.26 In contrast,

assessing microbial function through transcriptomics27 and metabolomics28 shows that

metabolic pathways (e.g. fatty acid, sugar and amino acid metabolism) associated with

microbiota have roles in regulating lung nutrient supply. Microbial immunoregulatory

metabolites produced at distal body sites may also affect the lung but have not been

extensively investigated. Changes in intestinal microbiomes reflect that seen in the oropharynx

(including the mycobiome) which through microaspiration directly impacts upon the respiratory

microbiome and host immune response.29 Gut and oral microbiomes reduce dietary nitrate to,

regulate oxidative stress, and produce vasoactive and anti-inflammatory nitrite, nitric oxide

and short chain fatty acids (SCFAs) from fermentation of dietary fibre. These metabolites are

linked to protection against acute and chronic respiratory diseases (CRDs) by inhibiting

histone deacetylases or activating GPR41, GPR43 and GPR109A receptors.2,7 Lung microbiota

such as Pseudomonas species are nitrate reducers7 and others (including Staphylococcus

species) also produce SCFAs.30,31 Whether other gut microbial metabolites, such as anti-

inflammatory trimethylamine or its more toxic product trimethylamine N-oxide are

produced by respiratory microbiota is unclear.32 The tryptophan metabolite indole-3-acteate

is produced by lung microbiota and suppresses macrophage inflammatory responses.31

9

Fungi such as A. fumigatus also synthesise a range of aromatic amino acids

(tryptophan, phenylalanine, tyrosine), which are precursors of toxins such as gliotoxin and

fumigaclavine C.33 Gliotoxin suppresses IFN-γ responses and induces neutrophil apoptosis,34

whilst fumigaclavine C downregulates Th1 cytokines and induces host cell apoptosis.35

Fungal tryptophan is converted by host indolamine 2,3-dioxygenase to kyneurine, which

leads to the expansion of regulatory T-cells and downregulation of Th17-mediated mucosal

inflammation.36 Thus, fungal aromatic amino acids may induce lung inflammation, and

downstream secondary metabolites suppress protective immunity.

Finally, cross-feeding between microbiota may amplify the impacts on host immunity

through synergistic effects. Volatile metabolites from P. aeruginosa or A. fumigatus

stimulate IL-1β production in an organotypic lung model, but co-cultures of both microbes

induced IL-1β but also GM-CSF, CXCL8, IL-6 and IFN-γ.37

However, studies of CRDs through global metabolomics have been limited by technical

difficulties in sample collection (blood contamination of biopsies; sample dilution in BALF),

preparation (inflammatory cells, solute and gel phases in a single sample), metabolite

identification (library development; identification of breakdown products) and data

processing and validation.38 Investigation of microbial metabolites is further complicated, as

low biomass limits the number of unique microbial metabolites detected in dilute samples

and can lead to changes in microbial metabolism being ‘masked’ by more abundant host

metabolites (e.g. amino acids). Moreover, environmental niches in the LRT may result in

dramatic variability in microbial metabolites depending upon the collection technique or

site.8 Additionally, in spite of evidence that viral infections alter the metabolic profile of the

lung,39 the lack of a clearly defined respiratory virome and an inability to reliably manipulate

10

its composition means that its impact on host and microbial metabolism is unknown. Use of

targeted metabolomics to focus on microbial metabolism may overcome some of these

limitations but further improvements in technology and improved analysis of existing

datasets (e.g. integrated ‘omics approaches39) may yield significant information concerning

microbial metabolism in CRDs.

Gut microbiota and the gut-lung axis (figure 2)

Gut and lung microbiota differ in function both as communities and individual microbes,2

and their composition does not always undergo similar changes with similar environmental

challenges.12 The roles of gut microbiomes in respiratory disease and immunity has been

reviewed elsewhere,1,2 yet it warrants mentioning briefly. The impact of bacterial ligands

(e.g. LPS) and metabolites (e.g. SCFAs) that enter the circulation or through shared mucosal

immunity on the lung are potential contributors to CRDs, and additional variables in studies

of respiratory microbiomes. Similarly, the gut virome regulates host immunity, largely

through interactions with host TLRs and NLRs, and lung viral infections can have profound

effects on the composition of the gut microbiome.2,5,40 Gastrointestinal fungi are crucial for

the maturation of distal lymph nodes and regulate dendritic cell function, whilst fungal

dysbiosis contributes to the development of AAD in mice.41 Moreover, regurgitation and

microaspiration may result in upper gastrointestinal microbiota entering the lungs,

particularly in diseases such as asthma and COPD where reflux is a common co-morbidity.9

Distinguishing between the effects of lung and gut microbiota is important when

characterising their roles in disease and in developing effective therapies.

11

Microbiome alterations and implications in disease (figure 3)

Changes in lung microbiota during CRDs are increasingly well characterised. However,

research is now being redirected from observational studies towards elucidating the

functionality of predominant members or communities, and their interactions with host

immunity.2 These are important next steps in developing novel therapeutics.

Asthma

Asthma is a heterogeneous CRD characterised by allergic airway inflammation, remodelling

and hyperresponsiveness (AHR).42-44 The microbiome varies throughout disease progression

and exacerbations. Moraxella catarrhalis, Streptococcus pneumoniae and Klebsiella species

are associated with severe asthma and exacerbations.45,46 Proteobacteria, the most

abundant bacterial phylum in asthmatics, is associated with AHR47 and Th17/IL-17-driven

inflammation, and promote neutrophil recruitment via IL-17A/F in non-eosinophilic/non-Th-

2 asthma.48 In mice, infection with the proteobacterium M. catarrhalis is associated with

neutrophilic infiltrates, high levels of IL-6 and TNF-α, and moderate levels of CD4+ T-cell-

derived IFN-γ and IL-17, which were exaggerated with AAD.49 16S rRNA sequencing of

bronchial microbiota found enrichment of Haemophilus, Neisseria, Fusobacterium, and

Porphyromonas species, and Sphingomonodaceae family members, and low levels of the

Mogibacterium-like bacteria and Lactobacillales in atopic asthmatics.50 Asthmatic lung

microbiota had increased ability to metabolise butyrate and propionate, which may

potentially lead to atopic asthma by limiting the bioavailability of SCFAs. Indeed, SCFA

production by gut bacteria protects against development of AAD in mice.51 H. influenzae is

the commonest potentially pathogenic bacterial species isolated from the airways of severe

12

asthmatics.52 Experimentally this bacterium converts steroid-sensitive Th2/eosinophilic AAD

into a steroid-resistant Th1/neutrophilic phenotype dominated by IL-17 responses and

associated with chronic infection and impaired phagocytosis.53-57 Furthermore, culturing

Haemophilus parainfluenzae with macrophages from bronchoalveolar lavage resulted in p38

MAPK activation, increased CXCL8 and mitogen-activated kinase phosphatase-1, and

inhibited corticosteroid responses, which was not observed with culture with the

commensal Prevotella melaninogenica.58 Staphylococcus species enriched after house dust

mite exposure induced Th2 cytokine production suggesting that microbiomes regulate host

immunity, and thus dysbiosis changes in microbiota may be a cause as well as an effect of

disease development.30 Thus, specific pathogenic bacteria may influence the response of

immune cells to pharmacotherapy, and bacterial colonisation or dysbiosis in the airways of

asthmatics may be potential therapeutic targets. Importantly, early life viral infections are

potentially key triggers for the development of asthma and, Annelloviridae has recently

gained significant attention.5 Fungal exposures are associated with development,

progression, severity and exacerbations in asthma and fungal sensitization inherently results

in poorer functional outcomes.59,60

CF

This disease is primarily caused by mutations in the CF transmembrane conductance

regulator (CFTR) gene.61 In the initial stages of the disease in early childhood, CF microbiota

is dominated by P. aeruginosa, H. influenzae, Staphylococcus aureus, Burkholderia cepacia

complex and Stenotrophomonas maltophilia, although microbial load is negligible.62 As

13

disease progresses (1-2 years), microbiomes become rich in oral taxa (3-5 years), and then a

CF microbiome dominated by P. aeruginosa which correlates with disease features.63

Notably, both oral- and pathogen-dominated microbiomes are associated with increased

inflammation and lung structural changes characteristic of CF.63 Thus, bacteria considered as

URT contaminants may have crucial roles in shaping LRT microbiomes in CF and promoting

inflammatory responses.64 Despite marked differences in lung microbiome profiles in CF

patients, which are affected by multiple factors, the metabolic potential of the whole

microbial community is similar,27 including in including amino acid catabolism, folate

biosynthesis, and lipoic acid biosynthesis pathways.65

Studies employing computational assessments have postulated that inoculation of

bacterial predators (Bdellovibrio, Vampirovibrio) into pulmonary microbiomes at early

disease stages might help control chronic colonisation by CF pathogens.66 This implicates

direct roles for dysbiosis in pathogen-dominated lung microbiota. P. aeruginosa in CF

patients exhibit transition from non-mucoid to mucoid forms over ~11 years,67 which may

facilitate resistance to antibiotics and phagocytosis. Additionally, non-mucoid forms may

have increased survival by negating hydrogen peroxide stress through catalase (KatA)

production.68

Increasing evidence proposes key roles for the virome, particularly Rhinoviruses in

deteriorating lung function, exacerbations and facilitation of bacterial colonisation in CF

while the role of the mycobiome is more established through a range of functional clinical

consequences including colonisation, sensitisation and allergic bronchopulmonary

aspergillosis (ABPA).69-71

14

Improved understanding of inter-microbial interactions and functional and metabolic

effects of lung microbiomes in CF may lead enable the development of novel treatment

strategies.

Non-CF Bronchiectasis

Bronchiectasis is a heterogeneous disease of chronic, irreversible and progressive dilatation

of bronchi.72 A key component is microbial colonisation and infection that drive

pathogenesis,72 and changes from health to disease.73 The core microbiota are comparable

between children with bronchiectasis and CF, but is different to adults.74 This suggests that

there is an ‘early’ bronchiectasis microbiome, which changes over time.74 Adults with

bronchiectasis have Pseudomonas- or Haemophilus-dominant microbiomes or microbiomes

with neither genera dominant, but this classification does not account for the wider

microbial ecosystem, individual patient characteristics, treatments, or inter-species

relationships.75,76 Long-term low-dose erythromycin therapy is associated with greater

microbial diversity and preserved lung function.74 Microbiomes are relatively stable in

individuals despite exacerbations and antibiotic therapy,76,77 but vary considerably between

individuals, influenced by the airway milieu, community composition and degree of immune

dysfunction.78,79 Studies found poorer lung function and more exacerbations were linked to

fucosyltransferase secretors than non-secretors who had less airway P. aeruginosa.80 Other

relationships with microbiota include with clinical phenotype, antibiotic exposure and

exacerbations. Pseudomonas is the only bacterial genus consistently implicated, and is

associated with poor clinical outcomes, exacerbations and mortality.81,82 Antibiotics affect

bronchiectasis microbiota. Carbapenem promotes Stenotrophomonas maltophilia infection

15

and low-dose erythromycin causes displacement of H. influenzae with P. aeruginosa.77 There

are no consistent associations of new pathogens or altered bacterial burden with

exacerbations.75,76,83 The most likely links are with change in microbial behaviour rather than

composition, which is driven by nutrient availability, oxygen tension, bacteriophages, host

immunity and quorum sensing.72,84 Indeed, macrolides reduce exacerbation risk and the

expression of quorum sensing genes despite stable bacterial loads, and thus, potentially act

as quorum sensing inhibitors.85

There are causal roles for the virome in exacerbations, hospitalisations and increased

airway and systemic inflammation. Human T-lymphotropic virus type-1 load was ~100-fold

higher in indigenous patients with bronchiectasis, which correlated with radiological

pulmonary injury scores and serology of the parasitic round worm Strongyloides.86 In adults,

viral exacerbations were significantly associated with increased systemic (IL-6 and TNF-α)

and airway (IL-1β and TNF-α) inflammatory markers.87 Viruses were recovered from

bronchiectasis patients at similar rates to healthy subjects,87 but whether the presence of

viruses in healthy and mild-moderate/stable patients affects overall microbiome

composition and disease progression is unknown. In children, viral exacerbations resulted in

more severe symptoms requiring hospitalisation, hypoxia and chest signs, and increased

systemic CRP and IL-6 levels.88

Aspergillus and Candida are the most frequently isolated fungi in bronchiectasis. The

bronchiectasis mycobiome is characterised by the genera Aspergillus, Cryptococcus, and

Clavispora. Aspergillus is associated with sensitisation and allergic bronchopulmonary

aspergillosis, and A. fumigatus and A. terreus dominate in Asian and European patients,

respectively.19 The latter associates with poorer lung function, fungal-associated IgE and

16

exacerbations. Fungi contribute to pathogenesis through their antigens and proteases, host

genetic susceptibility, and interactions with other microbes notably non-tuberculous

mycobacteria.89 Next-generation sequencing studies are needed to elucidate airway fungal

diversity and clinical relevance.90

COPD

COPD is a heterogeneous disease variably characterised by inflammation-driven bronchitis,

emphysema, fixed airflow obstruction and impaired lung function.91 Microbiota diversity and

relative abundance of members of the lung microbiome in COPD differs substantially from

healthy individuals,10 which is further skewed during exacerbations.92 The dynamics of

change of the lung bacterial microbiome may be attributed to disease heterogeneity,73,93,94

physiological changes with disease stage and progression,95 treatments (e.g. antibiotics and

corticosteroids),31,92 and exacerbations.96 Prevotella and Haemophilus species activate CD83,

CD40 and CD86 in human monocyte-derived dendritic cells, with Haemophilus being 3-fold

more inflammatory. However, in co-culture Prevotella reduced H. influenzae-induced IL-

12p70, highlighting differential interactions of distinct bacteria with immune cells.97 Chronic

airway inflammation in COPD is associated with microbiota dominated by

Gammaproteobacteria,94 and Proteobacteria and Actinobacteria are associated with

infiltrating immune cells in lung tissue from COPD patients, including neutrophils,

eosinophils, and B-cells.98 Enrichment with the oral taxa Veillonella and Prevotella is also

associated with increased lung inflammation.17 This pneumotype was characterised as

having increased levels of the metabolites palmitoleic acid, arachidonic acid, 4-

hydroxybenzoate and glycerol, as well as exhaled nitric oxide, but the precise role of

17

microbial metabolism in these profiles and inflammation is unknown. Conversely,

enrichment of the lung microbiome with oral taxa was also associated with blunted alveolar

macrophage TLR4 responses,17 yet inhibiting TLR activation may be a novel therapeutic

strategy for COPD.99 This suggests that the interactions of lung microbiota and immunity in

COPD are more complex than comparisons of ‘healthy’ and ‘diseased’. Recent studies also

implicate gut microbiome and physiology changes in COPD pathogenesis.100,

The gut microbiomes of smokers have increased abundance of Bacteroidetes, and

reduced Bifidobacteria101,102 and Firmicutes:Bacteroidetes ratio.102 Cigarette smoke-induced

mouse models of COPD also display pathology in the gastrointestinal tract, including

reduced colon length, epithelial barrier dysfunction, hypoxia and increased angiogenesis

and severity of chemical-induced colitis.100 Moreover, in mice, dextran sulfate sodium (DSS)-

induced colitis leads to systemic and pulmonary IL-6 dependent neutrophilia through

bacteraemia (increased endotoxins).103 These smoking-related gastrointestinal pathologies

potentially lead to further dysbiosis in the gut, as well as systemic inflammation and

circulating microbial products (bacterial metabolites, structural ligands) that may reach the

lungs and affect the progression of COPD.

A functional role for the virome in COPD beyond that established in promoting

exacerbations is an ongoing field of investigation however recent data suggest an important

influence of the mycobiome particularly a sensitisation response in COPD-bronchiectasis

overlap.104

Pollution

18

Outdoor environmental pollution with a mixture of fine and ultrafine particles and gases has been

implicated in worsening or inducing many respiratory conditions such as asthma and COPD.

Exposure of mice to ambient air pollution or particles causes gut microbial dysbiosis usually with

altered Firmicutes and Bacteroidetes, depending upon the size of particulate matter (PM;

PM10/PM2.5) and mode of administration (inhalation versus chow), as well as strain of mice

studied.105,106 In rats there was a rapid increase in lung microbiota abundance and diversity,

particularly of Proteobacteria.107 Human studies also support a link between exposure to traffic

pollution and microbiota in the gut or upper respiratory tract.1 The pathophysiological significance of

pollution-induced microbial dysbiosis in the gut was demonstrated in mice exposed to the gaseous

pollutant, ozone, where changes in the gut microbiome contributed to ozone-induced bronchial

hyperresponsiveness through its ability of produce the SCFA metabolite, propionate, providing

support for a gut-lung axis link.108 Further work is needed as to whether the lung microbiome could

produce SCFAs.

Infections and exacerbations

Complex interactions between environmental and host factors and the microbiome exist in

the lungs.109 CRD patients exhibit lung dysbiosis and are prone to bacterial and viral

infections, which further alter microbiomes and induce exacerbations.110-112 This indicates a

feedforward process by which lung dysbiosis that dysregulates host immunity, and leads to

increased risk of pathogenic infections that in turn maintains dysbiosis.2,113 Colonisation with

H. influenzae, S. pneumoniae and M. catarrhalis is associated with high risk of developing

recurrent wheeze and childhood asthma.114 In COPD, 40-50% of exacerbations are caused by

bacteria that increase airway inflammation and obstruction, sputum production and

bronchoconstriction.110,112 This involves high levels of typical Streptococcus, Pseudomonas,

19

Moraxella, Haemophilus, Neisseria, Achromobacter and Corynebacterium genera in COPD,

and atypical bacteria such as Mycoplasma pneumoniae and Chlamydia pneumoniae also

having roles in asthma and COPD exacerbations.115

The respiratory microbiome may also be altered by viral infections, which increase

susceptibility to secondary bacterial infections and/or associated exacerbations. For

instance, acute HRV infection in COPD patients chronically colonized with non-typeable H.

influenzae had greater likelihood of seasonal AECOPD. 116 Notably, significant increases in

16S copy number (6-fold) and numbers of proteobacterial sequence (16%; especially pre-

existing H. influenzae) was reported 15-days post experimental RV infection, though only in

sputum of patients with COPD117 indicating selective outgrowth of newly favored species in

the setting of exacerbations. Importantly, RSV, IAV and HRV infections upregulate bacterial

adhesion molecules (ICAM-1, PAFR, CEACAM-1) on epithelial cells and thereby promote

adherence and growth of specific bacteria in the lung microbiome, including non-typeable

H. influenzae, S. pneumoniae and P. aeruginosa. Viral infections also impair mucociliary

clearance and damage epithelial cells, facilitating host tissue invasion by pathogenic bacteria

(e.g. S. pneumoniae) and thus persistence of pathogens in the lung microbiota. RSV infection

in adults causes exacerbations in CRDs. Binding to nucleolin and CX3CR1 on airway

epithelium and inducing Nox signalling pathways to activate epidermal growth factor

receptor (EGFR) results in CXCL8/10-dependent airway inflammation and mucin production,

and suppressing IFN-lambda responses. Inhibiting EGFR to activate endogenous epithelial

antiviral defences may be a potential treatment in respiratory infections.118 RSV infection

aggravates impaired immune responses from alveolar macrophages involving mitochondrial

dysfunction and suppression of type I IFN responses involving TGF-β1.119 RSV or HRV

20

infection in infants produced different profiles of metabolites and bacterial functional

potential in nasopharangeal samples, although it is unclear whether the virome impacts

host or bacterial metabolism in CRDs or their exacerbations.39 Alternatively, bacterial SCFAs

derived from the gastrointestinal tract protect against respiratory viral infection120, and thus

it is feasible that metabolites produced by the respiratory microbiome an individuals

susceptibility to viral exacerbations. These different responses or susceptibility to infections

and exacerbations (both viral and bacterial) may contribute to heterogeneity in CRDs.

Critically, the role of fungi (and the mycobiome) has emerged as an important contributor to

exacerbations in asthma, COPD, CF and non-CF bronchiectasis with the Aspergillus genus

best studied.19,59,71,104 

It is crucial to better understand the role of infections in disease induction,

progression and exacerbation, which can be achieved with advance omics techniques of

high throughput sequencing, metagenomics, and microbiome analysis. This will assist in

developing new treatments for CRDs and will open new avenues of precision medicine.

Lung microbiome in co-morbidities of CRDs (figure 4)

CRDs are complicated by co-morbidities including cardiovascular disease (CVD),

cerebrovascular diseases, diabetes mellitus, neurological and psychiatric disorders, gut and

renal disorders, musculoskeletal disorders, and malignancies. Although, the direct role of

lung microbiomes in extra-pulmonary co-morbidities has not yet been established, the

respiratory and gut microbiome has been implicated in several gastrointestinal co-

morbidities through the gut-lung axis, as well as other “organ-lung” axes in the heart, brain,

21

muscle, and lymph tissue. These interactions should be fully investigated to dissect the roles

of the respiratory microbiome in co-morbidities of CRDs.

Oral microbiota are altered in smokers and CRD patients (especially COPD and

asthma), which then affects the microbial community composition in the lung and gut

through microaspiration and swallowing, respectively. Furthermore, specific members of

oral microbiota that are increased in COPD121 are directly implicated in rheumatoid arthritis

and CVD. Porphyromonas species increase the production of autoantibodies (anti-

citrullinated protein antibodies and rheumatoid factor) that result in the onset and/or

progression of arthritis.122 Moreover, the combined abundances of Streptococcus and

Veillonella in atherosclerotic plaques correlated with their levels in the oral cavity. Notably,

levels of specific oral taxa implicated in CRDs such as Fusobacterium and Streptococcus

significantly correlated with plasma cholesterol and low- and high-density lipoprotein (LDL

and HDL) and apolipoprotein A1 (ApoA1) levels, respectively, which are major CVD risk

factors.123

Recently, Millares et al., showed that despite similar overall bronchial microbiomes

in stable COPD and during exacerbations, the predictive in silico analysis of functional

metabolic pathways of microbial communities are were significantly altered, especially

those related to tumourigenesis, which may increase the risk of lung cancer in COPD

patients.124 In particular, non-typeable H. influenzae, a major pathogen in CRDs, induced

epithelial IL-17C production in TLR-2/4 dependent pathways that promote tumour-

associated inflammation and tumour proliferation.125

Epidemiological studies show increased risk of inflammatory bowel diseases (IBDs)

associated with CRDs, and vice versa, and dysbiosis is independently linked to the

22

pathogenesis of both. The aberrant immune responses in CRDs accentuated by dysbiotic

lung communities, as well as the transition of CRD-associated microbiota from the URT to

the gastrointestinal tract could play crucial roles in the onset and progression of gut

diseases. Oral-derived Klebsiella pneumoniae ectopically colonize colitis-prone mouse

intestines and elicit TH-1 cell responses and associated gut inflammation.126 IAV infections,

frequent in CRDs, result in migration of lung-derived CCR9+CD4+ T-cells to the small intestine

of mice where they produce IFN-γ leading to dysbiosis and intestinal immune injury.2,127

Thus, recurrent infections with these respiratory pathogens could lead to inflammation in

the gastrointestinal tract that often precedes IBD.

Therapeutics

ThreeTwo classes of medications, antibiotics, and corticosteroids and beta agonists, are

commonly used to treat respiratory conditions where lung dysbiosis ismicrobiota are

involved in pathogenesis. Their effects on the microbiome are only now being investigated,

and extending their efficiency or preventing side-effects might be achieved by regulating gut

and/or lung microbiota. Targeting specific pathogens may also have therapeutic potential.

Antibiotics

Long-term antibiotic therapy, particularly with macrolides, is now established for the

treatment of CRDs including uncontrolled/severe Antibiotics are widely used to treat acute

respiratory infections and long-term antibiotic therapy (especially macrolides) has been

introduced for the treatment of chronic respiratory conditions including

uncontrolled/severe asthma,128 CF,129 bBronchiectasis,130 and COPD.131 They limit both the

23

duration and severity of asthma exacerbations induced by Haemophilus, Chlamydiae and

Mycoplasma.55,128 Despite the undeniablethe therapeutic benefits of antibiotics, prenatal or

early-life use is linked to the development of allergies and asthma through changes in the

gut microbiome.132,133 Infants and adults have reduced microbial richness and abundance in

gut microbiota following azithromycin treatment,134,135 and treated asthmatics have reduced

abundance of lung Prevotella, Staphylococcus and Haemophilus.136 However, in

bronchiectasis divergent effects occur with long-term erythromycin treatment increasing

the levels of H. parainfluenzae and decreasing Streptococcus pseudopneumoniae and

Actinomyces odontolyticus.137 In patients without Pseudomonas-dominated infection and

who had no change in exacerbation rates after azithromycin, there were decreases in H.

influenzae.77 In smokers with emphysema, azithromycin did not suppress lung bacterial

burden, but reduced α-diversity and pro-inflammatory cytokines, and increased anti-

inflammatory bacterial metabolites (glycolic acid, indol-3-acetate, linoleic acid).138 Thus,

antibiotics although reducing diversity may act beneficially on bacterial metabolism to

induce anti-inflammatory effects.

Corticosteroids

Corticosteroids may alter immune responses to bacteria, and thus their pro-inflammatory

effects. Corticosteroid use in asthma increases the abundance of airway Proteobacteria,

including Pseudomonas, and decreases in Bacteroidetes, Fusobacteria and Prevotella

species.139 Similarly, inhaled corticosteroid use in COPD is associated with greater richness

and diversity, while systemic treatment during exacerbations enriched Proteobacteria,

Bacteroidetes and Firmicutes.140 In a mouse model of RV-induced COPD exacerbation,

24

fluticasone propionate impairs both innate (type I IFNs) and adaptive (activated CD4+ and

CD8+ T cells) antiviral responses leading to delayed virus clearance, mucus hypersecretion

and increased lung bacterial loads.141 Moreover, COPD patients on ICS therapy had

suppressed sputum cell IFNβ and IFNλ2/3 expression at exacerbation onset, and

significantly increased bacterial loads 2-weeks post exacerbation onset compared to ICS

non-users.141

Finally, H. parainfluenzae may induce corticosteroid insensitivity suggesting that lung

dysbiosis in asthma might contribute to corticosteroid non-responsiveness.58 Indeed,

experimentally although Chlamydia, H. influenzae, RSV and IAV induce different immune

responses they can all drive steroid-resistant airway inflammation and AHR through miR-

21/PTEN/PI3K/HDAC2 and NLRP3 inflammasome responses.55-57 These effects can be

reversed using miR-21, PI3K and NLRP3 inhibitors.142

Beta Agonists

Data from in vitro studies have demonstrated that salmeterol reduces bacterial adherence

to airway mucosa, as well as bacteria-induced epithelial damage (tight junction leakiness,

epithelial stripping and preventing loss of ciliated cells) caused by both P.

aeruginosa and Haemophilus influenzae.143,144 In contrast, data from cultured mouse

macrophages exhibits that inhalation of beta2-agonist impairs clearance of nontypeable H.

influenzae.145

Probiotics and prebiotics therapies

Probiotics are live microorganisms while prebiotics are non-digestible carbohydrates that

are metabolised by gut bacteria and stimulate the growth and activity of beneficial colonic

25

bacteria. Both change the balance of gut microbiota, interact with innate and adaptive

immunity, promote the release of anti-inflammatory metabolites and secretory products,

and can deliver health benefits.2 Typically, they are taken orally, which implies interference

with the gut-lung axis as being important in maintaining normal microbiota and influencing

immunity in both compartments.2 Randomised clinical trials using single strains of probiotic

Lactobacilli and Bifidobacteria had no effect in preventing allergic asthma.146 However, small

studies showed that Lactobacillus gasseri improved asthma and allergic rhinitis symptoms,

home peak expiratory flow rates, and inflammatory cytokine release from blood

mononuclear cells.147 In children with mild-to-moderate atopic asthma, Lactobacillus

acidophilus and Bifidobacterium bifidum also improved lung function and reduced

exacerbations.148 In CF, probiotics may restore beneficial intestinal microbiomes which have

been altered by frequent antibiotic courses through effects on gut microbiota, including

improved gut motility and intestinal barrier function, inhibition of pathogenic bacteria,

enhanced metabolism and by modulating gut and lung immunity.149 There is evidence for

probiotics reducing pulmonary exacerbations and intestinal inflammation, but studies were

of variable quality.150 An unexplored potential is the administration of probiotics or

prebiotics to the URT to target the lung microbiome directly. Intranasal administration of E.

coli or Acinetobacter iwoffii and Lactococcus lactis strains that promote immunoregulation

reduced allergic inflammation in mice.151,152 Other specific components of gut and lung

bacteria induce anti-inflammatory effects including by promoting regulatory T-cell responses

that may also be harnessed therapeutically.23,153-155 Although these studies are promising,

larger controlled trials are needed to determine whether changes in gut and lung

26

microbiomes occur as a result of prebiotic and probiotic therapies and are potential

treatments.

Pathogen targeting

Other strategies involve targeting major bacterial pathogens to reduce hospitalisations and

mortality. Vaccination against major bacterial pathogens in asthma and COPD patients, may

result in reduced pathogenic burden and associated inflammation.53 Chronic H. influenzae

infection promotes neutrophilic and Th17-driven inflammation, and steroid insensitive AAD,

and thus preventing bacterial colonisation may be beneficial perhaps as adjunct therapies in

asthma and COPD.53 Influenza vaccination may also reduce secondary bacterial infections

with Streptococcus pyogenes.156 Vaccines against S. pneumoniae are strongly recommended

and administered in susceptible populations (children and adults >60 years).157 Nevertheless,

it remains a major bacterial pathogen in susceptible individuals,158 necessitating further

research to improve vaccines to provide sustained protection. The protective effects of

commensal bacteria may also be harnessed. Haemophilus haemolyticus, a common lung

commensal, produces a bacteriocin-like protein that inhibits the growth of pathogenic

nontypeable H. influenzae.159 Also, administration of probiotic Streptococcus salivarius

significantly reduced (~80%) episodes of S. pyogenes-induced pharyngeal infections.160 Thus,

strategies that promote growth of commensal respiratory bacteria may be utilised to

manage infections in CRDs.

Future directions and conclusions (figure 4)

27

The characterisation of dysbiosis microbiome composition in CRDs has helped to understand

the functional effectsfunctions of the respiratory microbiome, but the precise effects of

these functions must be further elucidated to be targeted therapeutically. Increasingly,

‘omics technologies and novel bioinformatics techniques are being employed to further

elucidate the microbial ligands and metabolites that interact with host immunity. Building

on this, future studies must account for the inherent variability in respiratory microbiota,

contributions from the gut-lung axis, and the interaction between different constituents of

the microbiome through shared microbial pathways. In-vivo, bacteriomes co-exist with

viromes and mycobiomes and a collective interpretation of the inter- and intra-kingdom

signalling between them in the context of functional consequence for the host and CRD is

necessary in future work. Most current work assesses the functional consequence of a single

microbiome alone and rarely integrates them, a feature that will be necessary in our current

era of precision medicine. Moreover, the improved ability to discern species- or strain-

specific differences in microbial functions should continue to be explored, including where

purified ligands (e.g. LPS) are administered in animal models. Most importantly, the majority

of studies investigating the respiratory microbiome are observational, and generally fail to

discern the cause-effect relationship between microbial dysbiosis and disease development

or progression. Longitudinal studies in human subjects, and targeted interventions in

validated animal models are crucial to definitively characterise the functional effects of

microbiota on CRDs. Overall, the functional effects of the respiratory microbiota hold

significant potential as therapeutic targets for CRDs, and continued emphasis on the

improved characterisation of these functions is essential to developing such therapies.

28

Contributors

All authors contributed to the design and writing of the manuscript. These are the views of

the senior authors PH, DPHAJ, IMA, SHC, KFC and PMH. KFB, SDS and SFR performed the

literature reviews and drafted text.

Declaration of interests

We declare no competing interests.

Acknowledgements

The authors are supported by fellowships from the National Health and Medical Research

Council (PMH) of Australia, the Australian Research Council (ARC, PH) and the Brawn

Foundation, Faculty of Health and Medicine, University of Newcastle, and grants from the

NHMRC and the Rainbow Foundation (PMH). The authors thank Felicity and Michael

Thomson for their continued support. SHC is supported by the Singapore Ministry of Health’s

National Medical Research Council under its Transition Award (NMRC/TA/0048/2016), the Singapore

Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 1

(2016-T1-001-050), the Lee Kong Chian School of Medicine, Nanyang Technological University

Singapore Start-Up Grant and acknowledges The Academic Respiratory Initiative for

Pulmonary Health (TARIPH). SHC is supported by a Singapore National Medical Research

Council, Ministry of Education, the Lee Kong Chian School of Medicine, Nanyang

Technological University, and acknowledges The Academic Respiratory Initiative for

Pulmonary Health (TARIPH). KFC is a Senior Investigator of the UK National institute for

Health Research (NIHR) and is supported by grants from European Union Horizon 2020 and

National Environmental Research Council grants. PMH is supported by fellowships and

29

grants from the National Health and Medical Research Council of Australia, the Australian

Research Council, the Brawn Foundation, Faculty of Health and Medicine, University of

Newcastle, and the Rainbow Foundation, and thanks Felicity and Michael Thomson for

continued support.

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Figure headings

Figure headings

Figure 1: Signals from the microbiome influence lung immunity. Respiratory microbiota produce

structural ligands (A) or metabolites (B) which influence host immune activity in health and disease.

TLR=Toll-like receptor. IL= Interleukin. MIP=Macrophage inflammatory protein. MDC = Macrophage-

derived chemokine. NOD = Nucleotide-binding oligomerization domain-containing protein. GPR = G

protein-coupled receptor. HDAC = Histone deacetylase. TMA = Trimethylamine. IFN = Interferon.

Figure 2: The gut-lung axis. The gastrointestinal and respiratory tracts are part of a shared mucosal

immune system and interact with the microbiome and each other in numerous ways, including

bacterial ligands (e.g. lipopolysaccharide; LPS) or metabolites (e.g. short chain fatty acids; SCFAs),

migrating immune cells, cytokines, hormones, and microbial migration between sites.

Figure 3: Roles of lung microbiomes in chronic respiratory diseases (CRDs). The abundance and

metabolic potential of specific microbes is increased in CRDs, contributing to immune dysregulation

and lung structural changes leading to progression of asthma, cystic fibrosis, bronchiectasis and

COPD as well as their exacerbations.

AHR= Airway hyperreactivity. CXCL-8= C-X-C Motif Chemokine Ligand 8. DC= Dendritic cell. IFN-γ=

Interferon-gamma. IL= Interleukin. MAPK= Mitogen-activated protein kinase. SCFA= Short chain fatty

acid. TNF-α= Tumor necrosis factor-alpha. TLR=Toll-like receptors.

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Figure 4: Chronic respiratory disease (CRD)-associated extra-pulmonary comorbidities and roles of

bacterial microbiomes. The oral microbiome influences the composition of both lung and gut

microbiomes, and a bi-directional gut-lung axis exists. Oral, lung and gut microbiomes have been

linked to several extra-pulmonary complications that often co-exist with CRDs. The abundance and

metabolic potential of specific microbes in the gut leads to host immune dysregulation and the

production of specific metabolites implicated in various CRD-associated comorbidities.

APCA=Anti-citrullinated protein antibodies. GABA=Gamma-aminobutyric acid. HPA=The

hypothalamic pituitary adrenal. Ig=Immunoglobulin. IL=Interleukin. LPS=Lipopolysaccharide. NF-

kB=Nuclear factor kappa-light-chain-enhancer of activated B cells. NO=Nitric oxide. NLR=Nod-like

receptors. SCFA=Short chain fatty acid. TLR=Toll-like receptors. TMAO=Trimethylamine-N-oxide.

Figure 5: Current understanding of the roles of respiratory microbiomes in chronic respiratory

diseases (CRDs). Most studies of the respiratory microbiome in CRDs have been observational.

Integration of ‘omics’ technologies has improved our understanding of the functions of microbiota.

Future studies must now assess microbial function through longitudinal and interventional studies,

considering emerging concepts in experimental design such as variability of microbiota and species/

strain-specific effects.

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