The Microbiome in Asthma

Immunology of asthma

Asthma is a complex and heterogeneous disease initially thought to be mediated by eosinophils and Th2 immune cells as evidenced by elevated numbers of interleukin 4 (IL-4) and IL-5 producing CD4⁺ T cells in bronchoalveolar lavage (BAL) fluid collected from allergic asthmatic patients [12]. Asthma heterogeneity was further characterized by gene expression analysis and the identification of two human endotypes, Th2^high and Th2^low, which differ mainly by their Th2 cytokine expression pattern and their response to medical treatment [13] (Figure 1). The Th2^high endotype is associated with increased activation of dendritic cells (DCs), CD4⁺ Th2 T cells, innate lymphoid cells type 2 (ILC2) and B cells resulting in heightened allergen-specific IgE and Th2 cytokine production as well as elevated numbers of lung infiltrating and circulating eosinophils. On the other hand, the Th2^low endotype display reduced levels of Th2 cells activation, Th2 cytokine production and eosinophil accumulation in the lungs (Figure 1). Strategies aiming to block the α chain of the IL-4 receptor and thereby disrupting both IL-4 and IL-13 signaling (Dupilumab) [14] or targeting directly IL-13 (Lebrikizumab) [15] improve asthma clinical aspects for the Th2^high phenotype. However, those therapeutical approaches appear to be less successful for patients with a Th2^low profile. Recent studies demonstrate that intrinsic non-allergic, non-IgE dependent, late-onset asthma triggered by environmental factors such as respiratory airway infection, air pollution and/or smoking, involves a Th1/Th17 immune response and intense neutrophils recruitment in the lungs [16–18] (Figure 1). The secretion of IL-8, IL-17, IL-21, IL-22, and monocyte colony stimulatory factor by either CD4⁺ Th17 T cells or ILC3s will promote the recruitment of neutrophils and airway inflammation [19,20]. This Th17-driven asthma phenotype is linked to a more severe pathology and a poor response to corticosteroid treatment [21–23] (Figure 1). Asthma endotype classification and disease heterogeneity are not fixed and exclusive. While each endotype is characterized by specific immune responses and pathological patterns, the possibility that those pathways concur and act synergistically in asthmatic patients cannot be excluded.

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Figure 1

Asthma heterogeneity and immunological patterns involved in disease pathology

(A) The Th2^high endotype is characterized by increased activation of DCs, Th2 CD4⁺ T cells, ILC2s and B cells which result in heightened allergen-specific IgE and Th2 cytokine production as well as elevated numbers of lung infiltrating and circulating eosinophils. On the other hand, the Th2^low endotype display reduced activation of Th2 cells and Th2 cytokine production as well as decreased numbers of eosinophils. (B) Intrinsic non-allergic and steroid resistant asthma is IgE and Th2 independent, associated with a Th1/Th17 immune response and the participation of ILC3s as well as intense neutrophils recruitment in the lungs.

The microbiome

The advancement and the development of affordable next generation high-throughput sequencing techniques has expanded our knowledge on the composition of the human microbiome as well as its relation to disease [24]. Recently, it has been estimated that the human body is composed of 3x10¹³ eukaryotic cells and colonized by 4 x10¹³ bacteria, roughly representing a 1:1 ratio [25]. The microbiome is constituted of bacteria, fungi, and viruses that colonize body surfaces exposed or opened to the outside environment such as the skin [26], the lung [27,28], the oral cavity [29] and the gastrointestinal tract [30]. Most of the current studies focus on the characterization of the bacterial microbiota and omit the presence of the virome and the mycobiome. Fungi are often considered as pathogenic, however many fungal species do not trigger disease and coexist with the bacterial microbiota. Compared to the bacterial microbiome, the characterization of the mycobiome composition is rendered more complicated by the lack of a complete fungal database; it is currently estimated that only 1% of fungal species are present in the NCBI GenBank database and among those sequences a large part are not classified correctly or of uncharacterized origin [31]. Currently, up to 75 different fungal genera have been described and are present at different body sites such as the oral cavity [32], intestine [33], skin [34] and lungs [35]. Commensal fungi also regulate immune responses and host physiology as fungal dysbiosis is associated with changes in the commensal bacteria composition that could potentially fuel further inflammation and disease [31,36,37].

Early microbial exposure influences the atopic status

The continuous rise in asthma incidence in industrialized societies cannot be attributed to genetic factors alone and implies that some environmental factors resulting from the modern lifestyle promotes asthma [38] (Figure 2). The “hygiene hypothesis” states that personal hygiene improvement, declining family size and decreased infection burden result in reduced early-life microbial exposure and promotion of atopic diseases [39]. The current dogma is that microbial colonization begins at birth from exposure to the mother’s vaginal and fecal microbial flora, although recent studies demonstrate that microbial exposition may start earlier, in utero, as indicated by the presence of bacterial DNA in the placenta and meconium of pre-term babies [40,41]. Maternal farming microbial exposure during pregnancy is correlated with elevated levels of regulatory T cells (T_Reg) and decreased Th2 cytokine secretion [42]. Those studies suggest the possibility that in utero bacterial exposure could initiate colonization at different fetal surfaces during pregnancy. Alternatively, it is the maternal exposure that directly influences the fetal immune development. The mode of delivery is also known to strongly influence the infant gut bacterial colonization [43]. Infants born by Caesarian section (C-section) are at higher risks of developing atopic diseases [44,45] and are principally colonized by microbial communities similar to the mother’s skin, such as Staphylococcus species [43,46]. Early colonization patterns of the neonatal gut, but not the airways, is also affected by the mode of delivery; C-section promotes the emergence of Citrobacter, Klebsiella, Enterobacter, and Enterococcus species whereas E. coli colonization was associated with natural delivery [47]. Right after birth, inhalation of environmental microorganisms will result in lung and airways colonization [48,49]. In mice neonates, the main bacterial phyla of the lung microbiome, Proteobacteria and Firmicutes, will stabilize during the first month of life and aging is correlated with an outgrowth of Bacteroidetes [49]. Similar bacterial clusters were detected in the gut and respiratory airways of young infants diagnosed with cystic fibrosis, thereby suggesting the possibility of dynamic crosstalk and interactions between the intestinal and the respiratory microbiota [48].

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Figure 2

Environmental factors influencing asthma development

The continuous rise in asthma incidence in “westernized” societies cannot be only attributed to a genetic component alone; environmental factors resulting from a modern lifestyle are involved in this increased prevalence. The composition of the microbial flora is constantly fluctuating and strongly influenced by environmental factors. Microbial dysbiosis can be triggered by stress, the modern-lifestyle diet, antibiotic use, birth and feeding mode. By promoting the growth of pathogenic bacteria, microbial dysbiosis will also prevent early exposure to health-promoting bacteria. In genetically predisposed hosts, this alteration in microbial communities will promote the development of asthma.

Breast- versus formula-feeding is another factor that influences the infant microbiome composition and development. Breast milk shapes the infant gut microbial communities and promotes intestinal homeostasis through diverse mechanisms; it is a source of bacterial communities transmitted to the infant [50], it provides maternal antibodies, such as secretory IgA, that further promotes gut homeostasis [51], and supplies nutrients that shape the microbial flora composition during the first year of life [46]. Consequently, compared to formula-fed infants, breast-fed infants harbor a more uniform commensal flora composition associated with higher abundance of Bifidobacteria and Lactobacillus, whereas formula-fed infants display higher abundances of Bacteroides, Clostridium, Streptococcus, Enterobacteria, and Veillonella species (reviewed in [52]). Compared to formula-fed infants, breast-feeding is also associated with decreased risks of developing asthma and this trend is even higher in infants with family history of asthma [53–55]. However, whether infant feeding mode can be correlated with increased risks to develop atopic diseases still remains uncertain and will require further studies in order to identify the specific microbial communities present in breast-versus formula-fed infants.

Early microbial exposure and bacterial colonization are important for the maturation and the generation of a normal immune system. Compared to specific pathogen-free (SPF) mice, Germ-free (GF) mice which are devoid of any microbiota harbor distinct physical differences such as increased size cecum, decreased gastrointestinal motility, abnormal morphology, and smaller Peyer’s patches and mesenteric lymph nodes [56]. Absence of microbial flora in GF mice is linked to a Th2-skewed immune response and elevated levels of serum IgE [57,58]. Mono-colonization of GF mice with either Bacteroides fragilis or segmented filamentous bacteria will redirect and balance the T cell immune response towards stable Th1/Th2 or Th17 phenotypes respectively [59,60]. There is a critical “time window of opportunity” in early-life for an appropriate education of the immune system by the microbiota [56]. Therefore, early-life events able to alter the microbiota composition or disrupt microbiota-immune interactions can be deleterious and promote immune deviation towards atopy. In mice, interruption of microbial sensing by the immune system promotes the development of allergic inflammation and food allergy development has been associated with the emergence of a specific microbial signature [61–63]. In humans, the use of antibiotics during the pre- or post-natal period is associated with increased development of atopic dermatitis and asthma [64,65]. Early-life intestinal microbial flora dysbiosis in children is correlated with increased asthma development and a reduction of four specific bacterial genera: Faecalibacterium, Lachnospira, Veillonella and Rothia [66]. Supplementation of these 4 bacterial species in GF mice reduced asthma incidence in their adult offspring by decreasing neutrophil recruitment in the lungs and reducing the Th1/Th17 immune response associated with severe human asthma [66]. Whether early-life microbial dysbiosis triggered by environmental factors elicit allergic disease or is a consequence of allergic disease development remains unknown and will require further investigations.

Asthma and the lung microbiome

Until recently, healthy lungs and respiratory airways were considered sterile. The use of 16S rDNA sequencing techniques demonstrated the presence of a lung microbiota in healthy subjects as well as its specific topographical distribution since bacterial biomass decreases from upper to lower respiratory airways [27]. The characterization of the lung microbiome is rendered more difficult due to its low microbial load compared to other organs such as the skin, the oral cavity and the gut, and the possible presence of bacterial contaminants from either the upper respiratory airways during sample collection by bronchoscopy or contaminants present in DNA extraction kit [67].

In mice, absence of lung microbial colonization in OVA-sensitized and intranasally challenged GF mice results in enhanced allergic airway inflammation with increased levels of Th2 cytokines and serum IgE as well as augmented lymphocytes and eosinophils infiltrations. Colonization of GF mice by SPF flora before allergen exposure protects from allergic airway inflammation development [68]. Furthermore, SPF mice intra-nasally treated with either Staphylococcus sciuri or Escherichia coli were protected from allergic airway development [69,70].

In humans, 16s rDNA sequencing of various type of specimens, including bronchial brushing [71,72], BAL [73], and induced sputum samples [74] allowed the detection in the lower airways of bacterial species belonging to the 5 major phyla (Proteobacteria, Firmicutes, Actinobacteria, Fusobacterium and Bacteroidetes). The asthmatic airway microbiota composition differs from healthy subjects and is characterized by a higher bacterial load and diversity as well as increased abundance of species belonging to the Proteobacteria phylum such as Comamonadaceae, Nitrosomonadaceae, Oxalobacteraceae, Pastereurellaceae and Pseudomonadaceae families [71,72,74]. On the other hand, members from the Bacteroidetes and Firmicutes phyla have been found more abundantly in the airways of healthy controls subjects [71,72,74]. The microbial airway composition also differs based on clinical features and the type of immune response triggered by the aeroallergens. For instance, the airways of corticosteroid-resistant and severe asthmatic patients are predominantly colonized by pathogenic organisms such as M. catarrhalis or members of the Haemophilus or Streptococcus genera [75]. Th17 immune-mediated asthma exhibit a predominance of Proteobacteria belonging to the Pasteurellaceae, Enterobacteriaceae, and Bacillaceae families [76]. This specific bacterial signature was only observed in Th17-driven inflammation and did not overlap with a Th2-driven airway inflammation [76]. Studies linking the metabolic activities of those dysbiotic bacteria to asthma severity and treatment prognosis are further required. For example, steroid-resistance treatment could be related to the presence and expansion in the airways of bacterial species that are capable of degrading steroids.

Fungi are also present in the sputum of allergic and healthy subjects [77]. Malassezia pachydermatis; a fungus already associated with the development of atopic dermatitis, was present in the sputum of asthmatic patients and absent from healthy controls [77]. The last couple of years have seen huge improvement in high-throughput fungal rDNA sequencing techniques and refinement of fungal sequences databases [31]. Therefore, in order to gain a better understanding of the lung mycobiome function during asthma, further studies taking advantage of those advances are required to define the role of fungal colonization in asthma.

Financial support and sponsorship.

This work was supported by the following grants: R01AI117968-01A1 (to M.A.) and R21AI112826-02 (to T.R.C.).