The airway epithelium is the primary site where inhaled and resident microbiota interacts between themselves and the host, potentially playing an important role on allergic asthma development and pathophysiology. With the advent of culture independent molecular techniques and high throughput technologies, the complex composition and diversity of bacterial communities of the airways has been well-documented and the notion of the lungs’ sterility definitively rejected. Recent studies indicate that the microbial composition of the asthmatic airways across the spectrum of disease severity, differ significantly compared with healthy individuals. In parallel, a growing body of evidence suggests that bacterial viruses (bacteriophages or simply phages), regulating bacterial populations, are present in almost every niche of the human body and can also interact directly with the eukaryotic cells. The triptych of airway epithelial cells, bacterial symbionts and resident phages should be considered as a functional and interdependent unit with direct implications on the respiratory and overall homeostasis. While the role of epithelial cells in asthma pathophysiology is well-established, the tripartite interactions between epithelial cells, bacteria and phages should be scrutinized, both to better understand asthma as a system disorder and to explore potential interventions.



Asthma is a chronic inflammatory disease presenting with wheeze, chest tightness and shortness of breath; however these symptoms may vary in appearance, frequency and intensity (1). Underlying mechanisms include genetic and environmental factors acting already from pregnancy and during the first years of life (23), although new cases may appear at any age (4). It is generally accepted that the term “asthma” incorporates a variety of clinical and mechanistic entities (56). Terms such as phenotypes, endotypes, subtypes or clusters are frequently used to subdivide and classify asthma, without however conclusive consensus. Affecting almost 300 million people worldwide, including up to 10% of children (78), asthma has emerged as a significant health problem with huge economic implications on healthcare systems globally (910).

Allergic sensitization of the host and subsequent overreaction to common environmental factors with typical inflammatory and immunological responses are strong determinants for the onset and the development of asthma. The airway epithelium is the primary interphase between inhaled triggers and the host, playing a central role on asthma pathophysiology (development, progression, and exacerbation). The epithelium of the large and small airways of the lungs is constituted by basal and undifferentiated columnar cells as well as ciliated and secretory (large airways) or Clara cells (small airways), forming pseudostratified or columnar and cuboidal structures, respectively. Additionally, distal alveoli are lined by alveolar type I and II epithelial cells (11). The whole spectrum of epithelial lining across the respiratory tract is a physical “tight-sealed” protective barrier against external stressors having also multiple regulating and signaling properties. The epithelial barrier integrity is ensured mainly by the formation of intracellular tight junctions (TJs) and adhesions and secondly by the presence of mucus and periciliary glycocalyx layers produced by adjacent secretory cells. This physical barrier ensures the elimination of noxious agents and their rejection through mucociliary clearance (MCC). This first line of innate immune defense protects the airway tract from incoming pathogens such as bacteria and viruses, as well as other chemical and physical stressors that can act as allergens in the case of atopic asthma.

The asthmatic epithelium has structural and functional abnormalities, probably due to genetic predisposition (12) giving expression of, among other, an “over-reaction” to common environmental allergens. Being under repetitive injury and inflammation, the asthmatic epithelium progressively remodels its structural characteristics (13). Specific allergens or pathogens can induce dysfunction and/or disruption (1417) of the TJs of the cells and access the airway submucosa through the paracellular way, facilitating sensitization against them. Its demonstrative that in-vitro infection of airway epithelial cells, obtained from children with or without asthma, with human rhinovirus, provokes different expression of TJs proteins. In the case of asthmatic cells it was observed permanent reduction of TJs expression and impairment of epithelial barrier integrity (18). In parallel, changes on the mucus composition and production rates in asthmatics have great impact on the MCC (1920). The asthmatic epithelium become more susceptible to infections (21), secreting a cascade of chemokines and cytokines which in turn activate several immune cells (2223). In the case of airway epithelial cells, obtained from asthmatic children and infected with human rhinovirus in Air Liquid Interfase cultures (ALIs), it was observed reduced IFN-β and increased inflammatory cytokine production, compared to healthy control (24). Activation of immune cells due to persistent allergic inflammation in asthma progressively alters the bronchial physiology into a pathological state where airway smooth muscle cells hyperplasia, subepithelial fibrosis, extracellular matrix overproduction, continuous angiogenesis, and mucus hypersecretion are detected (25). As a consequence, bronchial wall inflammation and thickening cause airway obstruction resulting in the typical respiratory symptoms of asthma. Strikingly, it has been suggested that epithelial mediation on allergic and immune responses has an impact on epithelial cell memory, establishing a type of epigenetic imprint on them (21).

The human microbiome is a dynamic and diverse entity, composed of an array of microbial genomes from bacteria, phages, fungi, protozoa and viruses. It is affected by immigration, elimination and growth of microbes within and possibly between different niches of each individual (2628). Despite existing variability, healthy microbial populations share a few common characteristics and abundances that change significantly in the cases of chronic inflammatory diseases, such as asthma (29)where microbial imbalance or “dysbiosis” occurs (3031). Indicatively, it is reported that the airways of healthy individuals are dominated mainly by bacteria of the genera Prevotella (Prevotella melaninogenica, Prevotella nanceiensis, Prevotella salivae) and Veilonella (Veillonella alcalescens, Veillonella parvula, Veillonella dispar) (263234). In contrast, the bacteriome of asthmatic airways is dominated by Haemophilus (Haemophilus influenzae B, non-typable Haemophilus influenzae), Neisseria and Moraxella catarrhalis along with Streptococcus pneumoniae and Staphylococcus aureus (3235). Domination of specific bacterial species in asthma raises several questions about a potentially causal role of microbiome imbalance to this disorder (3637) and the role of epithelial defense to microbial attachment and invasion (3841).

Maintenance of the microbial equilibrium/homeostasis is influenced not only by epithelial responses and host immunity, but also and possibly to a greater extent, by bacterial viruses that colonize, along with bacteria, human mucosal surfaces. Phages are capable of controlling microbial populations, expressing high genetic variation and complexity. They are known as the most abundant biological entities on the planet (42) with many references for their presence in the human body and their direct interactions with eukaryotic cells, organs and tissues (4345). There is a plethora of lytic phages that target bacterial populations, indirectly protecting epithelial surfaces from bacteria colonization and/or overpopulation. On the other hand, there are phages following mainly a lysogenic life cycle integrating their genome on bacterial chromosomes and remaining as prophages in a lethargic state. Induction of prophages may be related with the acquisition of bacterial DNA which includes toxin or antibiotic resistance related genes. Horizontal transfer of these genes (HGT) between bacteria may have an impact on bacterial population fitness across the human body (4648). The human “phageome” imposes important selective pressure on bacterial populations, influencing and determining along with the eukaryotic host the overall health and well-being (49). It seems that microbe-phage interactions along with epithelium responses participate in a co-evolving game of symbiotic and/or antagonistic relationships thus their simultaneous study could add insight into the pathophysiology of asthma.

Within the context of this review we explore the importance of the triptych: phage-bacterium-respiratory epithelium, toward asthma pathogenicity and development. The current literature around this complex biological system is limited and focused mainly on the gut epithelium and its interactions with local bacteria and phages. Undoubtedly, the information gap around asthma development and the contribution of the triptych remains unbridged. We first describe the current understanding about the role of microbiome in asthma development and exacerbation and then assess the interactions of specific bacterial species with the respiratory epithelium. We further elucidate the direct interactions between phages and human epithelial cells and finally identify the few existent in-vitro and in-vivo studies that have assessed the role of the triptych in respiratory disease models.


The Microbiome in Asthma

Healthy adults are breathing ~7000L of air per day (5051) and their upper and lower airways are constantly exposed to a variety and large number of microorganisms. The estimated load of inhaled bacteria, viruses and fungi ranges between thousands to millions of particles per day, with the exact number depending upon the environmental exposure (5254). The nasal cavity, pharynx and paranasal sinuses are part of the upper respiratory tract and their microbial communities along with the oral microbiome determine in a decisive way the lower respiratory tract microbial composition in healthy states (5556). The proposed “Adapted Island Model of Lung Biogeography” describes effectively the immigration rates of microbial communities from the upper respiratory tract to the large and small airways and the distal alveoli of the lungs (2657). It is assumed that the respiratory tract should be considered as a large and unique ecosystem with different environmental niches affecting decisively the immigration rates and the biodiversity levels of microbial communities in healthy states. Undoubtedly, the whole identity of the airways microbiota is drastically changing in the cases of respiratory diseases such as asthma and the overall physiology and airway function is affected (5860). Therefore, the delineation of microbial identities in health and asthma could lay new perspectives for future phenotyping and medical interventions.

The microbiome in the gut may also have a role in asthma pathogenesis by modulating the immune system via the induction of immune cells that circulate and reach the lungs, regulating and influencing lung immunity and microbial equilibrium from the first years of life (276164). The recently emerged lung-gut axis hypothesis was proposed by Schuijt TJ et al. (65), showing that healthy gut microbiota efficiently protect mice lungs from S. pneumoniae infection through the activation of alveolar macrophages and enhanced levels of immunomodulatory cytokines (65). The exact mechanisms by which the gut-lung axis activates innate immune system in health and disease are still unknown. Nevertheless, interactions between respiratory microbiota and the airway mucosa are arguably at least as important in shaping local immunity and dysbiosis in asthma.

In the last decade, culture independent molecular techniques, mostly based on sequencing of the hypervariable regions of the 16S rRNA gene have been used to characterize the airway microbiome in conditions of health and disease (266667). It was shown that the bronchial tree is colonized across its whole spectrum by specific microbial phyla thus having its own microbial identity and being protected from infections and chronic respiratory disease development. Hilty et al. analyzed the airway microbiota of the nose, oropharynx and lungs in asthmatic and chronic obstructive pulmonary disease (COPD) patients and compared the results with healthy individuals (35). Using nasal and oropharyngeal swabs as well as bronchoscopic cytology brushings and broncho-alveolar lavage fluid, they performed cladistic analysis to identify the distribution and abundance of airway microbiota across the spectrum of health and disease. They concluded that pathogenic Proteobacteria, particularly with Haemophilus, Neisseria, and Moraxella genera were more frequent in airways of asthmatic and COPD patients compared to healthy individuals and maybe related to increased risk of early asthma development when found in infant’s pharynxes. In addition, Staphylococcus and Streptococcus dominance was a common characteristic in children with refractory asthma, while in healthy airways, Prevotella and Veillonella were among the prevalent genera (35). Microbial profiles in sputum samples from severe asthmatics were also identified and compared with healthy individuals in another study. The prevalent bacteria in severe asthmatics were amongst the genera of Haemophilus or Streptococcus and M. catarrhalis species (68). Prevalence of specific bacterial species in the respiratory tract of asthmatics compared to healthy controls seems to reduce the microbial diversity and affect in a decisive yet underexplored way the pathophysiology in asthma.

Observations from wider population studies were generally confirmatory and tried to connect early microbiome fluctuations with asthma development in childhood. A Danish birth cohort of 700 children followed until the age of 6 years showed that airway microbiota dominated by S. pneumoniae and subsequent immune responses were correlated with asthma development risk (6970). In another study, nasal samples from 6 to 12 years old asthmatic children had reduced microbiota diversity compared to healthy controls and a notably high abundance of the genus Moraxella. The question raised was whether the loss of abundance in asthmatics is due to prevalence of Moraxella (71). It has been suggested that Moraxella species coming from the upper respiratory system of asthmatic patients are also identified in abundance at the lower airways (7273). In another study, infant’s nasopharynxes were screened during the first year of life where colonization from common respiratory pathogens occurs. It was proposed that early colonization of nasopharynxes with Moraxella, Staphylococcus, Haemophilus, and/or Streptococcus can provoke upper respiratory tract infections and inflammation that possibly spread to the lower airways, predisposing for future asthma onset in childhood (7475). It is apparent that the neonatal airway microbiome in conjunction with genetic predisposition, tissue impairment and/or respiratory infections may act causatively in regard to asthma development from the first years of life and into adulthood. In Table 1 we summarize important clinical studies about the microbiome profile in asthmatic children populations and the role of specific bacterial species found to dominate in their airways.

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