ROLE OF MICROBIOME IN THE PATHOPHYSIOLOGY AND DISEASE COURSE OF ASTHMA

Dr Aran Singanayagam MRCP. PhD.

Dr Andrew I Ritchie MRCP.

Professor Sebastian L Johnston FRCP. PhD.

Airway Disease Infection Section. National Heart and Lung Institute. Imperial College. St Mary’s Campus. London W2 1PG

Corresponding author:

Professor Sebastian L Johnston

s.johnston@imperial.ac.uk

Keywords: Asthma; bacteria; microbiome; pathophysiology

Acknowledgements: NIL

Conflicts of Interest: NIL

Funding: N/A

Abstract

Purpose of review: The emergence of next-generation 16S rRNA sequencing techniques has facilitated a more detailed study of the body’s microbiota and led to renewed interest in the association between microbial exposure and asthma inception. In this review, we evaluate the evidence that the respiratory tract and intestinal microbiota contribute to asthma pathogenesis and progression.

Recent findings: Human studies have revealed associations between the presence of potentially pathogenic bacteria in the respiratory tract in early life and subsequent risk of allergic sensitization and asthma. Similarly, alterations in the intestinal microbiota of neonates have also been shown to precede the development of asthma. Emerging evidence suggests that the lung microbiota is dysregulated in asthma with specific changes in bacterial diversity and community composition according to severity and phenotype. Studies using germ-free mice have been invaluable in moving our understanding from correlation to causation by demonstrating a mechanistic link between the neonatal microbiota and the development of allergic airway inflammation.

Summary: An expanding body of literature supports the hypothesis that early life microbial exposures and bacterial communities within the lung and/or intestine play an important role in shaping early immunological development. Perturbations in the microbiota may play an important role in promoting immune defects associated with the development of asthma and allergic sensitization.

Introduction

Overview of asthma pathophysiology and link between environmental exposures and development of disease

Asthma is a chronic inflammatory airway disease that can be sub-divided into the disease-specific endotypes which arise through different pathophysiological pathways[1]. Atopic asthma is characterized by a T helper cell type (Th)-2 dominant inflammatory process associated with enhanced expression of cytokines interleukins(IL)-4, -5 and -13 leading to IgE class-switching, airway eosinophilia and mucus production[2]. By contrast, non-atopic ‘neutrophilic’ asthma may be mediated by Th17 lymphocytes with enhanced expression of cytokines IL-17 and IL-22[3]. Numerous other methods of sub-phenotyping asthma have been described, based on age of onset, severity, precipitating factors and response to treatment. The diverse and heterogeneous nature of the disease presents a considerable challenge to our understanding of the underlying pathophysiology.

The increasing prevalence of asthma over the last few decades, particularly in industrially developed countries, has led to belief that lifestyle factors contribute to development of the disease. The extensively described hygiene hypothesis postulates that a reduction in exposure to environmental microbes during childhood impairs healthy immune system maturation and contributes to immune dysfunction and development of allergic diseases including asthma later in life. This hypothesis is supported by numerous studies that have demonstrated that individuals that grow up in environments with high microbiological diversity (such as children raised on farms) have a significantly lower risk of developing asthma[4-6], with recent evidence suggesting that bacterial endotoxin exposure leads to suppression of Th2-immunity through induction of the ubiquitin-modifying enzyme A20[7]. Increasing unrestricted use of antibiotics, in combination with changes in lifestyle and diet may be associated with alterations in early microbial composition potentially contributing to the immune system abnormalities associated with specific asthma endotypes.

Modern molecular techniques have allowed detailed characterization of the complex bacterial communities that exist in the human intestine and, more recently, the lung. This has led to an expansion in the hygiene hypothesis concept with increasing belief that the dynamic changes in the airway and/or intestinal microbiota early in life could be critical determinants for subsequent asthma risk by shaping early immunological development. In this paper, we aim to review evidence that composition of the microbiome is an important factor contributing to development of asthma.

Next-generation sequencing techniques have revealed a complex and diverse microbiota in the human intestine and airway

Historically, studies of microbial colonization in the human intestine and lung were based on culture-dependent techniques. However, a large proportion of bacteria cannot be cultured by standard microbiological techniques so previous insights into the microbiota were limited in their scope. Recent advances in molecular sequencing and bioinformatic techniques have facilitated more detailed characterization of the full spectra of resident bacterial communities in the body. The 16S rRNA gene is ubiquitously present in all prokaryotes and can be targeted by PCR-based methods to allow sequencing of all bacteria present in a given sample. Application of these techniques to the study of the human microbiome has paved the way for a re-evaluation of the effect of pathogen exposure on immune system development and pathogenesis of asthma. The intestinal microbiome is more densely populated with bacteria (to the order of around 104-1013 cells/ml)[8] than the lung microbiome and therefore has been historically more amenable to being studied. Previously, it was widely believed that the lower respiratory tract was sterile in health but in 2010 Hilty et al published the first study describing the microbiota composition of the lower airways[9]. An ever expanding body of literature now supports these observations.

Evidence that the respiratory tract microbiome contributes to development of asthma

Cross sectional studies of the asthmatic lung microbiome in adults

Relationships between the microbiome and clinical parameters have been examined in cross sectional studies of asthmatic patients and these studies offer some indirect insight into the influence of the microbiota on asthma pathogenesis and progression. The importance of the airway microbiota in asthmatics has been suggested by intervention studies in which improved airway hyper-responsiveness and measures of airway inflammation were associated with modulation of the microbiota in response to prophylactic macrolide antibiotic treatment [10,11].

Community characteristics including both burden and diversity of the bacterial communities colonizing the airways have been found to be greater in asthmatic compared with healthy subjects[12]. Studies also suggest that the lung microbiota changes according to disease severity. In patients with mild disease, bacterial diversity is inversely associated with the degree of airway hyper-responsiveness [12], whereas as asthma becomes more severe, lower bacterial diversity is, conversely, associated with increased airflow obstruction[13]. These studies raise speculation that the microbiota might contribute to disease progression in asthma but such cross-sectional study designs cannot offer definitive insight into causation.

Some studies have observed variations in microbial communities according to phenotype of asthma. For example, the microbiota in sputum samples from poorly controlled neutrophilic asthmatics has been shown to be less diverse compared to non-neutrophilic asthma[14]. Huang et al reported that subjects with poor asthma control, despite high-dose ICS therapy, demonstrate significant relationships between different bacterial community profiles and clinical features such as symptom scores [15]. Additionally, the authors demonstrated that, in the bronchial brushings of more severe asthmatics, an expansion of dominant bacteria and associated lower community diversity correlated with neutrophilic airway inflammation. In steroid-treated severe asthmatics, Th17-mediated inflammation correlates with relative airway abundance of H. influenzae [12] and expansion of this species has also been observed in induced sputum samples from asthmatics with poorly controlled, severe neutrophilic asthma[16] and steroid-resistant severe asthma [17].

The composition of bronchial microbial communities associated with asthma reported in studies to date has led to the emergence of the concept of expansion of Proteobacteria in asthma across a range of severities. Proteobacteria are observed in higher numbers in the lower airways of moderately severe asthma patients, primarily dominated by Haemophilus [9]. In more severe disease, Huang et al describe enrichment of Klebsiella[18]. Furthermore, the relative abundance of a range of phylogenetically distinct Proteobacteria members has been reported to correlate strongly with the degree of airway hyper-responsiveness [12]. Table 1 summarises the findings of published studies that have evaluated the lung microbiota according to severity and/or phenotype in asthma.

Combined, these studies provide evidence for contribution of the lower airways microbiota to asthma pathophysiology, by demonstrating that specific bacterial taxa are associated with disease severity, response to therapy and host immune responses. Longitudinal studies to evaluate changes in the lung microbiota during disease progression are now required to more comprehensively investigate the role of the microbiota in asthma.

Neonatal culture-based studies

To date, there are no existing cohort studies that have carried out a detailed longitudinal evaluation of how the lower respiratory microbiota develops from birth through infancy and related this to how specific changes in community composition and/or diversity might correlate with subsequent development of asthma. However, evidence does exist from studies that have demonstrated that early life airway colonization with specific bacteria assessed by culture is associated with increased risk of asthma development. Using a prospective cohort study in Copenhagen, Bisgaard et al reported that hypopharyngeal colonization with potentially pathogenic microorganisms S. pneumoniae, H. influenzae and/or M. catarrhalis at one month of age was associated with significantly increased risk of asthma by 5 years. [19] Similar associations were also reported in two retrospective studies [20,21]. A recent study using culture-independent molecular methods on upper airway samples reported that high relative abundance of Streptococcus reads on nasopharyngeal swabs taken at < 9 weeks of age was associated with increased risk of frequent wheezing episodes at 5 years of age[22].

The Copenhagen prospective cohort was further utilized to demonstrate that children who develop asthma by 7 years of age have aberrant ex vivo immune responses to pathogenic bacteria in peripheral blood mononuclear cells taken at age 6 months including enhanced IL-5 and IL-13 induction by S. pneumoniae, H. influenzae and M. catarrhalis and enhanced 1L-17 induction by H. influenzae and M. catarrhalis[23]. Although these studies raise intriguing speculation that bacteria might directly contribute to development of the atopic and neutrophilic asthma endotypes, cause and effect cannot be inferred. It is equally plausible that bacterial colonization may simply represent a surrogate marker of an early life immune dysfunction that subsequently causes development of asthma rather than being a direct causative factor.

Further indirect evidence that the composition of respiratory tract bacteria contributes to asthma pathogenesis is provided by studies that have evaluated the effects of early life antibiotic use on subsequent risk of disease development. In a large US cohort, Risnes et al demonstrated an association between antibiotic exposure in the first six months of life and development of asthma by 6 years of age. [24] Mai et al reported similar findings in a Norwegian cohort study[25].Semic-Jusufagic et al reported higher frequency of severe wheeze and asthma exacerbations if antibiotics were taken in infancy[26]. It is unclear whether antibiotic use in these studies is simply a surrogate marker of treatment for presence of the potentially pathogenic bacteria that have been linked to asthma pathogenesis or if antibiotic use itself directly promotes development of asthma by inducting early alterations in composition of the microbiota. Evidence exists to suggest that antibiotic use can induce changes in bacterial community composition of the lungs[27] and intestine[28] but proving that these perturbations directly lead to asthma is challenging and currently unclear.

Insights from ‘Germ free’ mouse models

A limitation to the human studies discussed above is that the observed associations cannot be used to make definitive conclusions regarding cause and effect. Therefore, to further evaluate the direct contribution of the microbiota on asthma pathogenesis, mouse model studies have been utilized and have provided some important mechanistic insight. Axenic or ‘germ free’ mice are reared under strict conditions to ensure they lack microbes. This model provides a powerful tool to study whether absence of a regular microbiome affects asthma pathogenesis. Herbst et al demonstrated that germ-free mice have exaggerated Th-2 responses in an ovalbumin-induced model of allergic airway inflammation with increased airway eosinophilia, enhanced airway hyper-responsiveness and mucus hypersecretion compared to control mice reared in standard conditions[29]. Intriguingly, these effects could be overcome by recolonization of germ free animals with a normal microbiota ( by introducing a non germ-free animal into the same housing cage) prior to allergic sensitization[29]. Gollwitzer et al demonstrated that immediately after birth, neonatal mice displayed enhanced allergic airway inflammation to house dust mite, associated with a predominance of Gammaproteobacteria and Firmicutes in the lungs. During the first two weeks after birth, profile changes to predominance of Bacteroidetes with an associated reduction in aeroallergen responsiveness[30]. Further evidence from animal models that the lung microbiota may play a causal role in asthma inception is provided in a study by Nembrini et al in which intranasal exposure to E coli showed protection against ovalbumin-induced Th2 responses and allergic airway inflammation[31].

Combined, these studies provide convincing evidence that the microbiota may play a causal role in the development of asthma. However, these studies focus primarily on atopic asthma and do not inform whether the effects observed in germ-free mice are due to an absence of microbiota in the lungs or in the intestine as the absence of microbes induced is not tissue-specific.

Evidence that the intestinal microbiome contributes to development of asthma

Human intestinal microbiota studies

In contrast to the relative paucity of longitudinal data on the lung microbiota, the longer duration that 16S rRNA technology has been available for sequencing intestinal bacterial communities has allowed sufficient time for cohort studies to be carried out examining early gut microbiota composition and association with development of asthma. A number of studies have reported microbial dysbiosis in the intestine of neonates preceding asthma development including reduced E.coli[32], Bifidobacteria[33,34] and increased Clostridia[33,35]. In keeping with the Bifidobacteria signal observed in neonatal studies, Helvia et al also demonstrated that the abundance of faecal Bifidobacteria is reduced in adult patients with long-standing atopic asthma compared to healthy control subjects.[36] Other studies have focused on overall diversity of gut microbiota with contrasting findings. Abrahamsson et al reported that reduced overall bacterial diversity was associated with increased risk of asthma [37] but conversely Bisgaard et al found no association[38].

Mouse model studies

A number of studies have used manipulations targeted at specifically altering the intestinal microbiota in mice and examined effects on Th2 responses following experimental induction of asthma. As with the association between asthma and early life culture of potentially pathogenic bacteria in the airways, the human studies described in the previous section cannot be used to inform about cause and effect. In a recent study, Arrieta et al used a combination of human correlation studies and mouse models to more comprehensively investigate causation[39]. They found that the relative abundance of Lachnospira, Veillonella, Faecalibacterium and Rothia was reduced in a large cohort of children at risk of asthma. The authors then proceeded to use mouse models to test causation by inoculating germ-free mice with each of these four bacterial taxa which led to attenuation of airway inflammation in a mouse model of ovalbumin-induced asthma[39].

Several studies have also utilized dietary interventions to alter the intestinal microbiota and examined subsequent effects on allergic airway inflammation. Thorburn et al showed that mice fed on a high-fibre diet had alterations in the gut microbiota and also had suppressed allergic airways disease due to enhancement of T-regulatory cell numbers and function[40]. Interestingly, they also showed that high-fibre feeding of pregnant mice reduces allergic airway disease in their offspring[40]. These findings were supported in a study by Trompette et al in which high-fibre diet in mice led to an increased relative abundance of Bacteroidaceae and Bifidobacteriaceae and suppressed allergic airway inflammation following house dust mite exposure[41].

Potential role of probiotics in asthma prevention and/or treatment

The studies described above demonstrate that the gut microbiota may be an important determinant of asthma development and raise speculation that interventions such as oral administration of live microorganisms in the form of probiotics to beneficially modulate the intestinal microbiota might represent a promising strategy for prevention and/or treatment of asthma. Such approaches have been extensively studied in gastrointestinal disorders.[42] There is evidence from mouse models that oral probiotic administration ameliorates allergic airway inflammation.[43] However, human studies to date have reported conflicting findings with two studies showing no effect of Lactobacillus supplementation on markers of Th2 inflammation[44,45] but two other studies of 105 and 46 patients respectively demonstrated beneficial improvements in clinical symptoms[46], lung function[46,47] and exacerbation frequency[47] associated with probiotic administration in asthma.

Longitudinal studies of the respiratory tract and intestinal microbiota

To date, studies evaluating the longitudinal changes in the microbiota leading up to development of asthma and during progression of the disease are lacking. However, a previous study described dynamic changes in the upper respiratory tract microbiota from birth up to two years of age with specific bacterial profiles identifiable by 18 months of age[48]. Additionally, studies have also demonstrated that the gut microbiota varies according to factors and comorbidities relevant to asthma including age[49], obesity[50] and allergic rhinitis[51]. These studies suggest that the microbiota is highly likely to change as asthma develops and progresses and a number of intrinsic and extrinsic factors may influence this.

Conclusions and Future directions

The emergence of next generation molecular sequencing techniques to characterize the microbiota has facilitated renewed interest in the role of microbes in pathogenesis and progression of asthma. To date, human association studies and mouse models provide strong supportive evidence for the hypothesis that the lung and/or gut microbiota influences the developing immune system and predisposes to atopy and asthma. However, few studies to date have focused on the interactions between bacteria and the immune system in driving development of other non-atopic asthma phenotypes and this represents an important area for future studies to focus on. Longitudinal studies that characterize the changes in lower respiratory tract microbiota from birth up to development of asthma are currently unavailable. Additionally, longitudinal microbiome studies in adult asthmatics will offer important insight into the specific changes that occur during the natural progression of the disease. There is also considerable interest in the gut-lung axis and future studies should be targeted at understanding the complex interactions that occur between the bacterial communities in these distinct anatomical locations.

SUMMARY POINTS

- Human studies have revealed associations between the presence of potentially pathogenic bacteria in the respiratory tract or alterations of the gut microbiota in early life and subsequent risk of asthma.

- Emerging evidence suggests that the lung microbiota is dysregulated in asthma with specific changes in community composition according to severity and phenotype.

- Studies using germ-free mice have demonstrated a mechanistic link between the neonatal microbiota and development of allergic airway inflammation.

-Bacterial communities within the lung and/or intestine play an important role in shaping early immunological development and may contribute to development and progression of asthma

-Future work should be targeted towards longitudinal sampling to examine the effect of external and internal factors including therapies on the intestinal and lung microbiome and associations with disease outcomes.

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