Recently, we have proposed the possibility of controlling dysbiotic bacterial populations in asthma through phage interventions (162). New therapeutic modalities for asthma could potentially include targeted and customized re-colonization of the upper airway with lytic phages against key bacteria, including the ability of phages to act as anti-biofilm agents (76). Isolation and characterization of naturally occurring phages against M. catarrhalis, S. aureus, S. pneumoniae and H. influenzae may have prominent importance in this context.

It is well-documented that co-evolution phenomena are taking place between phages and their respective hosts (163). Understanding the molecular and ecological characteristics of M. catarrhalis and its phages is an important target (164, 165). Until now, isolation of lytic phages for M. catarrhalis has not been achieved. Nevertheless, several prophages have been identified and characterized which could be indicative of the microbial ecology and evolution of the species and especially gram-negative bacteria that pre-occupy the respiratory niche as referred previously (166). Davie et al. described phage elements in a M. catarrhalis comparative genomic study of clinical isolates (167), while Ariff et al. also described the presence of at least 32 putative whole prophages harbored in the genomes of 95 M. catarrhalis clinical isolates (168). Although it is yet to be determined if these prophages could be active and able to infect the host if induced, these studies provide evidence of a large variety of prophages and prophage elements, which have interacted and fused their genome with Moraxella hosts over the years. Moreover, host molecular anti-phage strategies, such as CRISPR (169), against lytic phages had been well-documented, a strong indication of ancestral lytic phage-host interaction (167, 170). It is plausible that the large abundances of prophages and prophage elements (171) coupled with the significant presence of CRISPR systems harbored in M. catarrhalis genomes, could act as strong anti-phage strategies, making lytic phage isolation a challenging process.

Haemophilus influenzae is central in the basic phage research for many years (172–174). The binary models of H. influenzae and temperate phages HP1 and HP2 is a valuable tool for researchers to understand the lysis-lysogeny decision of temperate phages and the genes involved in it (175, 176). However, the literature still lacks a well-characterized virulent phage against this genus. Only recently researchers have reported a number of four possible isolates against H. influenzae with the complete characterization of these phages and their lytic efficacy remaining an open question (177).

Staphylococcal species colonize and dominate the human hypopharynx microbiome from early life (178). S. aureushas been suggested to be a serious threat for asthmatic incidents in adult life, through its superantigens (179). Contrary to M. catarrhalis and H. influenzae, staphylococcal lytic phages have been extensively studied as antimicrobial agents for many years against human and non-human bacterial infections. Systematic isolation and characterization of staphylococcal lytic phages has been taking place since 1950s. S. aureus was in the center of attention for the research community being a highly virulent agent with common antibiotic resistance, making it a high priority for discovering novel antimicrobial solutions against it. Interestingly, apart from the phages, lysozymes and endolysins were also tested against antibiotic-resistant strains of S. aureus, especially in strains colonizing skin tissue. Phage treatment has a much better background against S. aureus than other bacteria, offering promising results already (180, 181). Especially, Sb1 and ISP phages, expressing high virulence amongst antibiotic resistant S. aureus strains are used intravenously as therapeutics for many years now by the Eliava Institute of Bacteriophage, Microbiology and Virology (182, 183).

In regard to S. pneumoniae, lytic phages have also been isolated for many years now (184). S. pneumoniae demonstrates severe antibiotic resistance and apart from asthma-related incidents, it is a major causative agent for several respiratory diseases worldwide. Thus, novel approaches such as phage therapy could potentially have important role against it. Just as in S. aureus, potential therapeutic candidates are phage-originated murein hydrolases, which are able to degrade the outer thick peptidoglycan layer, the first line of defense for gram-positive bacteria, and induce lysis, without the use of phages (185–187).

Phages are part of the human virome; collectively, also referred as “phageome,” they regulate the populations of bacteria through prey-predator interactions (44). Recent studies show that phages are present in every niche of the human body including oral (188, 189), pulmonary (190, 191) and intestinal (192, 193) cavities and also the urinary tract (194), the skin (195, 196) and the blood (43, 197). The phageome constitutes the majority of viruses in a healthy lung and play a potentially crucial role to lung health and immunity, framing the bacterial population (45, 198). Bacteriophages have also been shown to reprogram the eukaryotic microbiome (199) and play an important role, as part of the immune system of mammals (200). The microbial load in healthy airways appears to be reduced as we proceed from the upper to the lower respiratory tract and except from the anatomical differences, it is also affected by the presence of different phage communities in different niches (26).

The surface of the nasal and bronchial epithelium is covered, as previously mentioned by mucus, while the surface of the alveolar epithelium is covered by pulmonary surfactant secreted from alveolar type II cells. Mucus contains mucin glycoproteins and nutrients that provide a favorable environment for commensal bacteria and phage symbionts. The most abundant types of mucin glycoproteins found in human airways are MUC5AC and MUC5B. In-vivo experiments in mice showed that MUC5B play a critical role in airway defense and bacterial clearance through MCC (30). In-vitro experiments with ALI differentiated bronchial epithelial cells from asthmatics, COPD and healthy control individuals, infected with rhinovirus-A1, showed no alteration of MUC5AC protein levels in any group (23). Nevertheless, the alteration of mucus patterns and production rates in many cases of clinical asthma provoke lung inflammation and multiple bacterial species colonization. In parallel, it is established that phage communities are more abundant in mucosal surfaces compared to adjacent non-mucosal areas (201, 202). The overall dysfunction of mucus secretions in asthma may influence not only the MCC mechanism but also the phage communities that regulate and control bacterial populations.

Mucus is an important intermediate in phage-bacteria interactions. In one related study they used T4 phages expressing or not an Ig-like protein (Hoc) in their capsid that binds to mucin glycoproteins. They performed in-vitro testing on mucus producing and mucus knockdown A549 lung epithelial cells. The cells were pretreated with hoc+ and hoc– T4 phages and then infected with Escherichia coli strains. They observed that mucus-producing cells pretreated with hoc+ phages, were protected from bacterial infection and cell death, in contrast to the mucus knockdown cells. In a microfluidic device simulating the constant air flow and mucin secretion dynamics, they showed the persistence of T4 phages and their subdiffusive motion on mucosal surfaces contributes to efficient phage-host encounters (201, 203). Although, recent literature about the interaction of phages with mucus is still limited, it is possible that future studies may unravel important mediation of these viruses in microbial equilibrium at the healthy airways.

During the last decade it was shown that several phages from the Myoviridae, Siphoviridae, and Podoviridae families, approximately one quarter of them, are carrying capsids with Ig-like proteins on their surfaces thus having the ability to complementary bind with mucin glycoproteins (204–206). In addition, it has been reported that there are large and diverse phage populations in mucosal surfaces compared with resident bacteria populations and with adjacent non-mucosal areas (201). We can assume that the direct association of phages with mucosal surfaces is providing a non-host-mediated mechanism of immunity (201) or an enhanced mechanism of host innate immunity that may induce a long-lasting adaptive immune response. In case of an infection, epithelial cells respond by secreting antimicrobial agents and mucin glycoproteins, thus giving the possibility for more phages to be attached and protect the surfaces from further bacterial colonization. The presence of phages in mucosal surfaces may also stimulate weak immune responses from the host thus inducing the continuous production of low concentrations of cytokines that may act protectively in the case of an infection (207, 208). Of note, it has been demonstrated in-vitro that Ig-like domains facilitate phage adsorption to the bacterial host and give a proliferation advantage against bacterial symbionts in mucosal areas (205, 206). It is becoming increasingly apparent that phages are key players for the overall homeostasis of human mucosal surfaces; further research may unravel new mechanisms and models for this complex symbiosis.

The direct implication of phages with epithelial cells is a recently proposed concept, enhanced by several in-vitro studies. Lehti et al. propose that E. coli phage PK1A2, belonging to Podoviridae family, could directly interact with eukaryotic neuroblastoma epithelial cells. They used fluorescent and electron microscopy to observe the specific binding of phages with mammalian cells and follow the internalization process through the endolysosomal mechanism. Internalized phages remained intact for 24 h into the cytoplasm without affecting the viability of cells and then appeared in the lysosomes where their proteins and nucleic acids degraded from the cell (209). Recent reports, explain also the pivotal interactions that phages can develop with mammalian cells, including entering mammalian cells similarly to mammalian viruses, killing bacteria not only in the periplasmic space, but also both in the phagosome and the cytosol areas and finally express their genetic information in the nucleus (210). In-vitro experiments with bronchial primary epithelial cells, obtained from asthmatic children attempt to understand, as previously mentioned, the mechanisms of epithelial cell repairing and migration after wounding treatment (127). Future in-vitro and in-vivo studies could be focused on asthmatic epithelial repair during bacterial infection and/or phage presence. These hypotheses emphasize the critical role of phages in human environments and raise many questions about their undefined role, as protective, anti-inflammatory, biocontrol or nutrient agents for the cells.

Neuroblastoma cells express polysialic acid on their surfaces that shows high homology with polysaccharide of the bacterial host E. coli K1 of the phage. Additional molecules in airway epithelial surfaces have molecular homology with bacterial components, such as hyaluronan capsule of group A (211) and sialylated capsular polysaccharide of group B (212) S. pneumoniae and sialylated lipooligosaccharide of Neisseria meningitides (213). It appears that eukaryotic and prokaryotic cells exchange genetic material through HGT mainly after internalization of bacteria and/or phages in human cells. In addition, phages appear to have intrinsic tropism not only to bacterial cells but also to eukaryotic cells (214, 215). It is proposed that the molecular mimicry between bacteria and host cells may have an impact on phage's interaction with human cells.

Considering the enormous diversity of phage symbionts, a relevant number of epithelial epitopes may be capable to induce efficient intake of phages into the epithelial human cells. Specifically, it has been assumed that the human body can absorb ~10¹⁰ phage particles per day which can be transported through epithelial surfaces and reach the blood circulation (209, 216). Internalization and deposition of phages to the lymphatic and circulatory systems may also contribute to the remission of strong intracellular immunity signals that potentially make the cells resistant against microbial invaders (217). Typically, antigen recognition is a well-characterized ability of specific immune cells of the adaptive system; nevertheless Charles A. Janeway proposed that germline-encoded receptors on cells can sense bacterial conserved patterns and activate intracellular pathways for bacterial destruction after the cell invasion (218). We can assume that endocytosed phages in epithelial cells could be implicated in similar intracellular pathways that give the advantage for rapid response in the case of bacterial invasion.

Phages pass through epithelial mucosal surfaces via transcytosis (216), adhere to specific cell receptors and be endocytosed from the apical epithelial surface (215, 219). If they successfully escape lysosome degradation, phages are finally exocytosed from the basal side of the human cells and getting into the bloodstream from where they can reach distal areas (216, 220). This phenomenon was described for several phages such as T4, T5, T7, P22, SP01, and SPP1 (216). It was also reported that phages provided intraperitoneally or intranasally can be found even in the brain (221), suggesting the high motility and utterly different dynamic properties characterizing these viruses.

In an in-vitro study they used a series of different human epithelial cells from the colon (CaCo2 and T84), lung (A549), liver (Huh7), kidney (MDCK), and brain (hBMec) to monitor different phage families (Podoviridae, Myoviridae, and Siphoviridae) and to assess the ability of transcytosis across the confluent epithelial cell monolayers. Approximately 10% of epithelial cells expressed specific phage receptors and contained membrane-bound vesicles that allowed the internalization and transcytosis of phages (216). In another in-vitro study they used bovine mammary epithelial cells infected with S. aureus JYG2 in order to test the ability of virulent vB_SauM_JS25 phage to enter and lyse intracellular bacteria. After 12 h incubation with phages, they observed via confocal microscopy the presence of phages near the nuclei of infected cells and they established the progressive elimination of intracellular S. aureus in a time and dose dependent manner (222). In addition, It is reported that several phages such as Φ29, Bam35, Cp-1, Nf, PRD1 possess terminal proteins with inherent nuclear transmission signals (47) that enable the phage absorption to the eukaryotic nuclei and the effective delivery of proteins and genes into the eukaryotic genome (46). Transcytosis of phages enables the effective HGT between phages and eukaryotic cells and raise the question of whether endocytosed phages can replicate inside the cells and/or protect them from inflammation and cell death after bacterial invasion (210). Results from further experiments showed that phages can reduce inflammation in MAC-T bovine mammary epithelial cells suppressing the LPS-induced phosphorylation of NF-kB p65 protein from S. aureus (223).

The ability of phages to enter the cells and pass through internal organs is a new and yet not fully described phenomenon. The plethora of phages and the variety of human cells that can be used render the aforementioned experiments just the beginning for a new field of research. The characterization of the phenomenon demands the expansion of the related in-vitro and in-vivo studies in order to establish a good predictive model of the phage addition in the human body. Phages could play a significant role to host immunity and microbial balance and can be used as a novel therapeutic and anti-inflammatory tool in many chronic airway diseases. The future introduction of phages in clinical trials will be a new and promising step for the whole medical community and pharmaceutical industry, dealing with chronic and refractory respiratory diseases.

Although airway inflammation differs between asthma and cystic fibrosis (CF) important lessons can be learnt from the latter. Phage populations within the lungs of healthy individuals differ significantly compared with those suffering from CF (190). Different areas of explanted lungs of late-stage CF patients were examined with metagenomic analysis in order to identify and quantify different viral populations. They interestingly observed the absence of phages related to CF common pathogen P. aeruginosa, S. aureus, and H. influenzae at the left and right upper lobes (224), suggesting that phage communities play an important role in the homeostasis and overall health of the airways. Phage communities express similarities between CF patients and have a specific metabolic profile adapted to CF pathophysiology that differs significantly from the healthy state (225).

Recently, it has been reported that phage populations within the lungs of healthy individuals differ significantly compared with those suffering from asthma (162) and/or CF (190). The absence of phages related to common pathogens in asthma (162) and CF (224) suggests an important role of phage symbionts in airways homeostasis and healthy state. In an in-vitro study primary bronchial epithelial cells were isolated from children with CF and healthy controls and different concentrations of purified P. aeruginosa phage preparations were tested on submerged cell cultures in order to investigate the cytotoxic and/or inflammatory effect of phages. Results showed that the viability of cells was maintained, while no production of inflammatory cytokines IL-6 or IL-8 was observed. Of note, the apoptosis of cells was reduced in the presence of phages compared to untreated control cells (225). In contrast, in-vivo induction of filamentous phage Pf4 from P. aeruginosa biofilms enhances bacteria persistence in CF patient's lungs, promoting adhesion of the bacteria to mucus and inhibiting cell invasion (226). Therefore, in-vivo production of Pf4 phage leads to reduced lung injury, giving the fitness benefit to bacterial population to escape from phagocytosis and immune response. It is apparent that induction of prophage in this case changes the inflammation human response and lead to chronic persistence of bacterial population in CF lungs (227). From the aforementioned experiments we can expect that phages related to common pathogens in asthma may induce diverse behaviors in-vitro and in-vivo, favoring or eliminating bacterial populations.

Co-culture of S. pneumoniae with human cells can provoke the induction of streptococcal pyrogenic exotoxin C (SpeC), a well-characterized phage gene product. Interestingly this induction occurred without the addition of any phage induction reagents, like mytomycin C or application of UV radiation and just occurred during the co-culture. Broudy T. et al. examined the supernatants of the culture with electron microscopy to validate the existence of phage particles and they identified whole phage viruses during the co-culture of group A streptococci with Detroit 562 human pharyngeal cells (228). These results suggest the existence of a soluble yet uncharacterized phage inducing factor secreted from epithelial cells. This induction may occur independently from bacterial contact with human cells leading to the lysis of some streptococci and the subsequent release of cytoplasmic virulence factors. Subsequent in-vitro and in-vivo experiments in mice showed that the presence of mammalian cells may play a significant role in phage induction, implying that phages have obtained through natural selection a system where induction occurred at eukaryotic niches where bacteria are becoming more virulent (229).

The presence of prophage genetic elements with phage-encoded lysins in ~70% of clinically isolated S. pneumoniae strains (230) is a well-described phenomenon but the role of prophages during bacterial colonization is mainly undefined. In an in-vivo study, it was shown that the presence of Spn1 prophage element at S. pneumoniae provoked changes on bacterial cell wall and had a negative effect on nasopharyngeal colonization in mice (231). In another study, SV1 prophage elements were correlated with S. pneumoniae biofilm formation and promotion of chronic lung infection (232). Adherence of another nasopharyngeal human commensal N. meningitides to the epithelial surfaces was enhanced by the existence of filamentous prophage MDAΦ in an in-vitro study. This finding suggests that MDAΦ colonization of its host bacterium may increase occurrence of meningitis. Interestingly, this effect was not observed on endothelial cells, but only on epithelial cells tested (233).

In another in-vitro study, phiCDHS1 lytic phages appeared more active against Clostridium difficile in the presence of HT-29 epithelial cells than without them. They performed subsequent experiments to audit whether there are specific HT-29 secretions that enhance phage activity, culturing phages with fresh or spend HT-29 culture medium. They concluded that phage lysis activity is not affected by the substances on cell culture media but it is promoted by the adherence of phages to the epithelial monolayer facilitating subsequent adherence with C. difficile (234).

An interesting example of nasopharyngeal microbiome regulation by phages has been reported regarding S. pneumoniae and S. aureus interactions. It was reported that S. aureus may be restricted from the nasopharynx in the presence of S. pneumoniae (235). H₂O₂ produced by S. pneumoniae promotes the production of hyperoxides via Fenton chemical reaction. Hyperoxides are able to induce SOS response in S. aureus that is typically a reaction that promotes DNA restoration, causing the activation of resident prophages from S. aureus lysogenic strains. Subsequent lysis of S. aureus promotes the S. pneumoniae prevalence at the nasopharynx. In contrary, S. pneumoniae lysogenic strains do not undergo SOS-response thus having a prevalence asset against S. aureus symbionts. The induction of prophages in neighboring organisms is a strategy for releasing active phages into the human body. This effect can cause subsequent spread of virulence genes and mobile genetic elements into different cells thus reshaping the existent equilibrium (236).

From the aforementioned literature it is clear that phages possess high immune modulatory capacities (207, 237) in CF (238, 239), COPD (240, 241), and other respiratory disease conditions. To the extent of our knowledge there are limited relevant published studies about the role that phages own on asthmatic epithelium. It cannot be excluded that microbial imbalance in asthma may be related and/or caused by underlying phage imbalance (162). In the near future, with the continuous research on that field, we expect the establishment of new mechanisms of phages-bacteria-eukaryote cells interactions in order to better orchestrate the triptych symbioses model (Figure 1).