Microbiome in Mechanisms of Asthma Tara F Carr MD, Rhonda Alkatib MD and Monica Kraft MD Department of Medicine, University of Arizona, Tucson AZ Corresponding author: Tara F Carr MD Assistant Professor of Medicine and Otolaryngology University of Arizona 1501 N Campbell Ave Tucson, AZ 85724-5030 tcarr@deptofmed.arizona.edu Rhonda Alkatib MD Fellow, Allergy and Immunology University of Arizona 1501 N Campbell Ave Tucson, AZ 85724-5030 ralkatib@deptofmed.arizona.edu Monica Kraft MD Professor of Medicine University of Arizona 1501 N Campbell Ave Tucson, AZ 85724-5030 mkraft@deptofmed.arizona.edu Disclosure statement: Dr Carr reports royalties from Wolters-Kluwer and has provided consulting services to Sanofi-Regeneron, AstraZeneca, and Boehringer Ingelheim. Dr Alkatib has no disclosures to report. Dr Kraft reports royalties from Elsevier, has received research funds paid to the. University of Arizona by National Institutes of Health, American Lung Association, Sanofi-Regeneron and Chiesi. She has provided consulting services to Astra-Zeneca, Sanofi-Regeneron and TEVA Pharmaceuticals . mailto:ralkatib@deptofmed.arizona.edu 5000 words including key points, text, references, and figure legends (do not embed figures, provide legend for figure) Key Words (5–8) Microbiome Asthma Lung Gut Short chain fatty acids Type 2 inflammation Abstract/Summary [this will be used for indexing services and does not appear with article] (<100 words) The lung and gut microbiome are factors in asthma risk or protection. Relevant elements of the microbiome within both niches include the importance of the early life window for microbiome establishment, the diversity of bacteria, richness of bacteria, and effect of those bacteria on the local epithelium and immune system. Mechanisms of protection include direct anti-inflammatory action or induction of non-type-2 inflammation by certain bacterial colonies. The gut microbiome further impacts asthma risk through the contribution of metabolic products. This chapter will review the mechanisms that connect the lung and gut microbiota to asthma development and severity. Key Points (3–5) • The human microbiome impacts the development of asthma through direct and indirect mechanisms. • There are independent relationships between the different microbiome niches, particularly lung and gut, and the development of asthma. • The mechanisms through which the microbiome relate to asthma involve immune development, immune tolerance, functional metabolites, and relationship to infections. • Further work is necessary to determine how and when manipulation of the microbiome may be implemented toward the prevention or treatment of asthma. Introduction The development and implementation of non-culture-based methods to identify bacteria, commonly 16s ribosomal RNA sequencing, have identified an enormous diversity and quantity of bacterial species living within and on the human host. It is estimated that more than 10,000 different microbial species live in and on humans, representing 100 trillion organisms. Importantly, this so-called human microbiome is in a commensal, symbiotic relationship with the human host, which is thought to be necessary for many host functions, including immune regulation. The microbiome may indeed impact the balance between heath and disease of many organ systems, including the respiratory, cardiovascular, gastrointestinal, and immune systems1-4. Specifically, in asthma, the human microbiome may contribute to both the development and severity of asthma. This chapter will describe the current knowledge of the relationship of human microbiome niches to asthma development and severity, as well as the mechanisms thought to contribute to this relationship. Early life exposures The often-discussed ‘hygiene hypothesis’ harbors the idea that exposure to various bacteria and other microbiota in early infancy and childhood can diminish the risk of subsequent allergy and asthma5. This relationship has been studied in robust population cohorts, some of which leverage geopolitics and population bottlenecking to describe differences in exposures, microbiome development, and health outcomes. Von Mutius and colleagues 6 recognized that growing up in a traditional European farm environment seemed to protect children from developing asthma and other atopic disorders, when compared to children growing up in urbanized areas. This group has subsequently described a variety of environmental exposures to be protective against asthma and allergic sensitization, including exposure to pet dogs and farm animals 7, and measurable differences of endotoxin load in bedding suggested increased diversity of environmental microorganisms in those exposed populations8. Further, ingestion of raw cow’s milk is inversely correlated with asthma in this population9. Murine models of allergic asthma have shown that ingestion of raw cow’s milk can prevent house dust mite-induced airways hyperresponsiveness (AHR) and lung eosinophilia by suppressing airway production of chemokine ligand (CCL)17 and type-2 inflammatory cytokines interleukin(IL)-5 and IL-13, suggesting altered epithelial or innate lymphoid cell responses to the raw milk 10. Individuals living across the Russia-Finland border in the Karelia region, while of similar ancestry, have distinctly different rates of disease and different lifestyles, the Finnish side having higher socioeconomic status and substantial urbanization. Those living on the Finnish side have substantially higher rates of asthma, allergen sensitization, and eczema compared with those living on the Russian side of the border, and the skin and nasal microbial communities differ between populations11. One possible explanation or contributing factor to these epidemiological findings is that those living in Russian Karelia have significantly higher microbial burden in their drinking water compared with those in Finnish Karelia, an exposure which seems to be protective against atopy12. Researchers have determined that health on both sides of that international border relates more to the diversity of overall bacterial environmental exposures than any one exposure. The underlying mechanism may be attributable to immune skewing by less diverse dust to eosinophilic responses, compared with type 1 helper-T cell (Th1)-transformation of naïve T lymphocytes or induction of IL-10 responses by more diverse bacterial exposure 13. The Amish and Hutterite communities of the United States are rural farming populations with closely related European ancestry, who share similarities in diet and family size. However, while the Hutterites have embraced industrialized farming, the Amish use traditional farming practices that exclude mechanization. The prevalence of asthma and allergy in the Amish population is strikingly lower than that of the Hutterites14. Stein et al. made extracts from indoor dust samples collected from representative homes of each of these populations and described marked differences in allergen, endotoxin, and bacterial abundance across the sites with much higher burden in the Amish samples. Using a mouse model of allergic asthma, the authors also showed that the Amish dust extracts were protective against allergic airway inflammation in a manner dependent upon innate immune signaling 15. These data suggest that one or many components of the dust, particularly the bacterial colonies, function through innate immune mechanisms to protect against the development of allergic inflammation and asthma. These and other population studies strongly support that certain environmental exposures, particularly to bacterial organisms, can be protective against the development of asthma and atopic diseases. As the environment can particularly impact the composition of the lung and gut microbiota, the associations between each of these bacterial niches and development of asthma warrant close evaluation for causal relationships, mechanisms, and therapeutic opportunities. Lung Microbiome and Asthma The role of the lung microbiome in the pathogenesis of asthma is not yet fully understood. However, well described are variables during the first years of a child’s life, including home environment, viral infections, and antibiotic exposures, which have been correlated with the onset of atopy and asthma16. As these variables also theoretically relate to differences in respiratory exposures and to the lung microbiome, the need for more careful examination of the relationship between the lung microbiome and asthma has sparked enthusiasm on an international level. The impact of airway bacteria on development of asthma may indeed relate to early life bacterial colony establishment. Bisgaard et al. cultured hypopharyngeal aspirates from 1-month old infants, finding that colonization with the encapsulated bacteria S. pneumoniae, M. catarrhalis, and/or H. influenzae was significantly associated with persistent wheeze, hospitalization for wheeze, and asthma diagnosis at age 5. In addition, colonization with any of these bacteria related to increased blood eosinophil counts and elevated total immunoglobulin (Ig)E at age 417. A recent study of children with asthma showed that coincident infection of Moraxella catarrhalis or Streptococcus pneumoniae and rhinovirus conferred greater severity of respiratory tract illness, including asthma exacerbations, suggesting that these, and possibly other, respiratory bacteria contribute to airway inflammation18. Further, infection or colonization of the airway with atypical bacteria such as Mycoplasma pneumoniae are related to both asthma development and severity in epidemiologic and mechanistic studies, and this type of exposure can be detected with readily available culture-based or serologic techniques 19. For example, M. pneumoniae infection in childhood causes acute wheezing20 and is associated with both structural changes21 of the lung and long term lung function deficits22, as well as to the development of asthma23. For individuals who already have allergic sensitization, M. pneumoniae may contribute to the development of asthma through inducing AHR24 , inflammation25, mucin expression26,27, and remodeling through collagen deposition28. For individuals with asthma, M. pneumoniae may induce pronounced eosinophilic inflammation29 which may relate to severity of disease30 or host innate immune response characteristics31. Treatment of the infection or related inflammation may indeed provide asthma control for the affected patient32 and underscore the importance of bacterial exposures to asthma. Respiratory pathogens, such as those noted above, are relatively easy to identify using culture- based or serologic laboratory techniques that are widely commercially available. However, with the recognition of the myriad bacterial species that are not easily cultured, more recent work has focused on utilizing non-culture-based techniques to examine and characterize the scope of microbiota in human niches, and to relate differences in these populations to clinical outcomes. Researchers and clinicians assumed for many years that the lungs were a sterile environment, however we now recognize a plethora of common bacterial phyla as colonizers of the entire respiratory tract, including in the lungs. Such phyla include Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, with estimates of almost 2,000 bacterial genomes present per square centimeter of a single upper lobe of the lung33. When utilizing non-culture-based techniques to compare bacteria present in the lungs of healthy adults to asthmatics, asthmatics have lower proportions of Bacteroidetes and increased Proteobacteria, particularly Haemophilus34. Marri et al. reported alterations in the microbial composition of induced sputum of asthmatics that were similar among asthmatics suffering from mild or severe disease but distinguished asthmatics from normal controls, and which also noted increased Proteobacteria in samples from asthmatic patients35. Huang et al. performed 16s sequencing for bacterial rRNA on bronchial epithelial brushing from asthmatic and healthy subjects 36. In this study, samples from asthmatic patients had significantly higher bacterial burden and bacterial diversity. Further, AHR in the patients with asthma was related to the relative abundance of members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae phylotypes, and interestingly, increased bacterial diversity was related to improvement in AHR following clarithromycin therapy. Lung microbiota may contribute not only to presence of asthma, but to asthma phenotypes and severity. Durack et al. compared microbiota from bronchial brushings among healthy, asthmatic and atopic patients, showing that subjects with type-2 inflammation had lower bacterial burden than other asthmatics 37. A recent study by Taylor et al. showed that asthma with prominent airway neutrophilia was characterized by lower microbial richness with enrichment of airway colonies for members of the class Gammaproteobacteria, to which Haemophilus and Moraxella belong, and that these microbiota characteristics relate to asthma severity38. Green et al.39 similarly identified bacterial profiles of induced sputum from severe asthmatics, dominated by Moraxella, Haemophilus, or Streptococcal spp., in the airway to be associated with severe airway obstruction and airway neutrophilia. To relate the bronchial microbiome to phenotypic and epigenetic features of asthma, Huang et al. performed bronchial bacterial composition by 16s sequencing in severe asthmatic and healthy control patients. These investigators found that Proteobacteria enrichment related to worsening asthma control, fewer airway eosinophils, and the induction of Th17-related genes40. In contrast, Actinobacteria were prominent in patients with high epithelial gene expression for FK506 binding protein, a marker of steroid responsiveness. These findings suggest that the mechanisms relating the bacterial species enrichment and clinical outcomes relate directly to proinflammatory pathways and treatment responsiveness. Goleva et al. compared 16s sequencing from bronchoalveolar lavage in corticosteroid- resistant and corticosteroid-responsive asthmatics, finding relative increased abundance of Proteobacteria (including Neisseria, Haemophilus spp), Fusobacteria, Firmicutes, and Actinobacteria in the steroid-resistant patients41. The authors supported these findings with in vitro experiments, showing Haemophilus parainfluenzae to activate toll-like receptor 4, subsequently activating transforming growth factor-β–associated kinase-1 (TAK1), which induces transcription of proinflammatory factors like IL-8, while inhibiting glucocorticoid-receptor-mediated mitogen-activated kinase phosphatase 1 production and conferring corticosteroid resistance. As inhaled steroids are a mainstay of asthma therapy, this pathway to steroid resistance may help to explain severe and steroid-unresponsive asthma. Mechanistic evaluations of these airway microbiotic influences on asthma susceptibility are largely reliant upon murine models. Published literature supports the importance of early life lung microbiome development on risk of asthma. Herbst et al. utilized a mouse model of allergic asthma in germ-free and control mice to show that the germ-free mice had elevated airway inflammation, type 2 cytokines, and basophils, and increased AHR upon ovalbumin challenge compared with control mice. However, when the neonatal germ-free mice were recolonized with the diverse microbiota from the control mice prior to allergen sensitization, they exhibited reduced levels of circulating IgE and reduced airway hyperresponsiveness similar to the control mice42. Gollwitzer et al. exposed mice at both neonatal and adult stages to house dust mite antigen to compare the effect of age and microbiome development on reactivity to antigen. Upon antigen exposure, the neonatal mice showed a substantial increase in airway eosinophilia, blood eosinophilia, type 2 cytokine (IL-4, IL-5, and IL-13) production, mucus production, and AHR when compared to mice exposed later in development. The airway microbiome of the neonatal mice was composed of Firmicutes and Gammaproteobacteria, compared with predominance of Bacteroidetes in the adults. The authors concluded that temporal shifts in airway microbiota are associated with decreased responsiveness to aeroallergens, via the induction of T- regulatory cells in a programmed death ligand 1- mediated manner 3. Some bacterial exposures may indeed be protective against a type-2 airway response. For example, in a murine model of ovalbumin-induced allergic asthma, pulmonary administration of E.coli to the lung induced a TLR4-dependent response with induction of γδ-T cells, decreased dendritic cell (DC) activation in the lung, and diminished IL-2 cytokine production, which collectively protected the mice from allergic airway inflammation43. In the aforementioned study by Durack et al., the investigators studied predicted bacterial functions among the differentially expressed bronchial bacteria. Sputum from asthmatic subjects was enriched for members of the Haemophilus, Neisseria, Fusobacterium, and Porphyromonas species and the Sphingomonodaceae family, and depleted in members of the Mogibacteriaceae family and Lactobacillales order, when compared with healthy controls. In silico predicted bacterial functions based on the 16s rRNA sequencing showed enrichment for short-chain fatty acid(SCFA) metabolism37. SCFAs are important for epithelial barrier function and have been implicated as important for immune tolerance in the gut, as will be discussed below. Clearly, patterns of lung microbial dysbiosis are evident among patients with asthma, and Proteobacteria seem to consistently relate to asthma presence, cellular phenotype, and severity. Further, mechanistic work suggests multiple levels of interaction of microbiota and the immune system can confer protection or risk for asthma development. Indeed, the quantity of bacteria, quality of bacteria, time course of exposure and colony establishment, and immunologic function of those bacteria all play a role in the relationship between lung microbiome and asthma (Figure 1). With these findings, the mechanisms by which microbiota determine early-life immunity may have great implications when applied to the prevention of both asthma and allergic diseases. Gut Microbiome and Asthma The gut microbiome is vitally important for health. Evidence underscoring the fundamental need for a gut microbiome includes studies of gnotobiotic mice, in which the mice born in a germ-free environment fail to develop normal intestinal structure or normal immune maturation 44. These findings may simply confirm that an individual’s gut microbiome contributes substantially to the proper digestion of foods, releasing minerals, vitamins, and digestive products such as short chain fatty acids. However, fascinatingly, introduction of different microbial communities into gnotobiotic mice can confer a specific phenotype, such as obesity45, abnormal behavior46, and immune development47. These and other data suggest that manipulation of the gut microbiome may be preventative or therapeutic for many chronic diseases and may indeed be leveraged to address allergic diseases and asthma. To do so, however, will require a better understanding of gut microbiome development and function as pertaining to asthma. The gut microbiome develops in early life, with a rapid increase in diversity that seems necessary for health48. In fact, the microbiome of the gastrointestinal tract, measured by stool sampling, is present even in the first neonatal meconium stool49. The stool microbiome develops with time, and evidence supports critical time periods of development of that microbiome for establishing health- protective bacterial communities. Longitudinal cohorts describe a relationship between establishment of the early life gut microbiome and development of allergic diseases. For example, a cohort from Canada50 revealed that reduced stool bacterial diversity measured from samples obtained at 3 months of life was associated with risk of asthma through age 3. A study from Sweden revealed that lower microbial diversity within stool samples at 1 week and 1 month of life was related to risk for asthma development through age 751. In both of these cohorts, the stool communities among those children at risk or not became more similar with time and by 12 months of age there was no relationship between stool diversity and asthma risk. In contrast, a study of the Copenhagen Prospective Studies on Asthma in Childhood 2010 cohort assessed diversity of the gut and found that relative abundance of species at 1 year of age were related to asthma at age 5, only among children born to asthmatic mothers52. A comparison of cellular responses from neonates born in Papua New Guinea showed that antigen presenting cells from these infants are less active than those from Australia, suggesting that the increased bacterial exposures related to the traditional lifestyle in Papua New Guinea would confer protection against immune responses53. Cox et al. utilized a mouse model of low dose penicillin exposure in early life to show that this exposure transiently affects the microbiota, but has lasting impact on body composition and metabolism54. These, and other studies support that the window for developing and impacting the gut microbiome may be limited to, or crucially important in, early life but may also relate to multifactorial asthma susceptibility and comorbidities49,55. There are many potential contributing factors to the development of stool microbiome (Figure 1). Much of the neonatal microbiome conferred during birth differs by mode of delivery. That is, the microbiome of infants delivered vaginally represent that of the mother’s vagina and gut. In contrast, the microbiome of infants delivered by caesarian section resembles skin flora 56. Caesarian section, particularly when performed prior to membrane rupture, may indeed be a risk factor for the development of atopy and allergic diseases57 however studies of this relationship show conflicting results. Further, early life exposure to livestock or domesticated pets, particularly dogs, decreases the risk of asthma development through mechanisms that may involve the gut microbiome58-60. Early life exposure to traditional, non-mechanized farming environments, such as in the Amish or European cohorts, is protective against asthma in a way that may strongly relate to microbiome through inhaled and ingested exposures15,59. Fujimura and colleagues used a murine model of allergic asthma to show that dog-associated house dust exposure via oral gavage protected against asthmatic changes. Further, this protection specifically related to gut microbiome profile- in particular, enrichment for Lactobacillus johnsonii- and supplementation of this species could confer protection against the asthma phenotype61. Dietary exposure may also contribute to gut microbiome development. Sordillo and colleagues reported that among other factors, breast-feeding, when compared with bottle-feeding, substantially influenced the gut microbiome development within the first six years of life 62. Breastfed infants had relatively lower abundance of stool Clostridales family species in this study. These are findings of interest, as prior work has shown that Clostridium difficile enrichment in early life may actually increase risk of wheeze and asthma in childhood63. Interestingly, Atarashi et al. fed gnotobiotic mice a Clostridium mixture, noting a subsequent expansion of regulatory T cells in the colon as well as lower specific IgE production in a model of ova sensitization, suggesting that this mixture would protect against allergic diseases64. However, Karimi et al. showed a similar pattern of regulatory, non-atopic immune development in a model feeding mice Lactobacillus reuteri65. That multiple bacterial taxa are implicated in immune regulation suggests that the individual taxa of bacterium present in the gut may be less important than the presence of bacteria in general, or perhaps identifies a potential shared metabolic or immunologic function of these bacteria. Further, the administration of macrolide antibiotics in childhood has been associated to both persistent changes in gut microbiome and risk of asthma development in childhood, suggesting the possibility that the changes induced in the gut microbiome- presumably reduction in diversity or of specific dominant colonies- by these antibiotics put individuals at increased risk for asthma 66. Digestive products of the gut microbiome are very important in modulating health. The metabolic functions of the gut microbiota include production of short-chain(SCFAs) and medium-chain fatty acids (MCFAs) by fermentation of dietary fiber and carbohydrates (implicated in colitis, arthritis, and asthma), formation of secondary bile acids (important for reduction in liver disease and metabolic syndrome), generation of metabolites from meat-derived choline and L-carnitine (implicated in cardiovascular disease), production of vitamins K, B12, and folate, and production of indole derivatives (i.e. gamma-aminobutyric acid, the central nervous system neurotransmitter)67. Of these metabolites, SCFAs have been most closely implicated in asthma risk. SCFAs can signal through g protein-coupled receptors, and can inhibit histone deacetylases, controlling gene transcription. Acetate, one SCFA produced by fermentation of fructose by Bifidobacteria, may support colonic epithelial integrity and function68. Additional SCFAs, butyrate and propionate, can induce regulatory T cell development which may play an important role in protection against allergic and inflammatory diseases 69,70. Acetate and proprionate can induce regulatory T cells in the colon, resulting in increased expression of IL-10 and associated regulatory functions71. Consumption of dietary fiber may induce more SCFA production and induction of tolerogenic dendritic cells in the mesenteric lymph nodes72. Evidence supporting SCFA as protective against asthma includes a study of the gut microbiota of infants at high risk for asthma by Arrieta et al. These authors reported a transient gut microbial dysbiosis in the first three months of life, related to lower fecal acetate levels in these high-risk for asthma infants50. Fujimura and colleagues have synthesized this complex relationship of gut microbiome development, metabolic products, and immunologic outcomes related to asthma 49. Neonatal stool samples from a diverse, longitudinal birth cohort underwent 16s ribosomal RNA sequencing and the patterns of stool microbiota were divided into three “neonatal gut microbiota composition states(NGM 1-3)”. These NGM were compositionally different and conferred different risk of atopy by age 2 and asthma by age 4. The highest risk group, NGM3, had lower relative abundance of bacteria, particularly Bifidobacterium, Lactobacillaceae, and Clostridiaceae, and higher abundance of certain fungi, suggesting a substantial dysbiosis in this group. The sterile fecal water from these NGM3 samples induced significantly more IL-4 expression in vitro than the low risk group, and metabolites within that sterile water were enriched for 12,13-DiHOME, a lipid metabolite which can inhibit regulatory T-cell development. In contrast, the sterile fecal water of the low risk groups was enriched for metabolites including the polyunsaturated fatty acids docosapentaenoate and dihomo-γ-linolenate which have anti- inflammatory properties. These findings support that differential production of immunologically active metabolites in the stool by different bacterial species, even very early in life, may be an important mechanism of protection from or risk for asthma (Figure 2). Therapeutics and the microbiome Probiotics are viable microorganisms that potentially confer health benefits on the host, however the safety and efficacy of probiotics as organisms that can prevent or treat disease remains uncertain. Ongoing investigations on probiotic supplementation may quantify and qualify host health benefits and the long-term impact they may have on the host microbiome. If various microbial exposures are related to asthma through direct immune effects or metabolic products, could a probiotic serve a protective or therapeutic role in those diseases through influencing the host microbiota toward disrupting or redirecting immune outcomes? Elazab and colleagues performed a meta-analysis of 25 studies testing prenatal or early life probiotic administration to determine the effects on atopy and asthma in children73. Variable factors among these trials included timing of supplementation and delivery method. The meta-analysis concluded that while some probiotic supplementation may reduce IgE levels and the risk of allergen sensitization, this did not affect the risk of wheezing or asthma. The decreased IgE levels were more evident with longer duration of probiotic administration. The mechanisms underlying this potential benefit against atopy have been addressed in specific trials. One randomized, placebo-controlled clinical trial studying feedings of Lactobacillus paracasei F19 between 4 and 13 months of age to placebo identified more pronounced T-cell IFN-y and IL-17 mRNA expression in the treatment group, suggesting Th1 promoting effects of this probiotic 74. Another trial of prenatal and postnatal supplementation demonstrated a combination of Bifidobacterium bifidum, Bifidobacterium lactis and Lactococcus lactis was associated with a reduction in in vitro lymphocyte IL-5 and IL-13 production by 3 months of age 75. Interestingly, a comparison of prenatal and postnatal supplementation with Bifidobacterium lactis or Lactobacillus rhamnosus showed differential effects of those bacteria, with cord blood IFN-γ higher in the L rhamnosus group. The complex host responses to probiotics will likely therefore vary based on dose, strain, treatment timing and duration. Additionally, probiotic supplementation may contribute to protection from atopic disease include through improved epithelial barrier function 76. Finally, while successfully implemented in severe gastrointestinal disorders 77, the frontier of microbiome transplantation to restore health in asthma has not been well studied but may offer an interesting new approach to microbiome manipulation. Conclusion Risk for asthma is strongly related to both the lung and gut microbiome. Important elements of microbiome development consistent with both niches include the early life window for microbiome establishment, the diversity of bacteria, richness of bacteria, and effect of those bacteria on the local epithelium and immune system. Mechanisms through which the lung microbiome may protect against asthma include early establishment of rich, non-pathogenic bacterial colonies that are anti-inflammatory or less disposed to type-2 inflammation. 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The lung and gut microbiome likely impact risk for asthma through direct immune activation and metabolite effects on the immune system. Microbiome in Mechanisms of Asthma Rhonda Alkatib MD Monica Kraft MD Key Words (5–8) Microbiome Key Points (3–5) Introduction Early life exposures Lung Microbiome and Asthma Gut Microbiome and Asthma Therapeutics and the microbiome Conclusion References