The microbiome and respiratory inflammation

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1 EARN 3 FREE CPD POINTS Respiratory Leader in digital CPD for Southern African healthcare professionals The microbiome and respiratory inflammation Professor Robin Green Paediatric Pulmonologist Faculty of Health Sciences, University of Pretoria Introduction Over time, humans have evolved a relationship with microorganisms in the environment whereby interactions with these bacteria, viruses and fungi are necessary to normal human immunity. A microbiome in a given organ or system consists of the entire microbial constituents (commensal flora) and their genomes, the surrounding environmental conditions and their interactions with the host. There is a microbiome everywhere in the body. The gastrointestinal (GI) microbiome is the easiest to study and largely influences development of early immunity. Microbiomes are also present in the respiratory tract (including the lower respiratory tract), skin, genitourinary tract and even the blood stream. Much of our immunity from chronic inflammatory disorders hinges upon having a healthy microbiome. Professor Robin Green believes that knowledge and understanding of the microbiomes of the body is still in its early days. He shared his experience of what is known about the microbiome and respiratory inflammation at the recent Cipla Respiratory Symposium (17 March 2018). KEY MESSAGES There is a microbiome everywhere in the body Much of human immunity from chronic inflammatory disorders hinges upon having a healthy microbiome Interactions between the host immune system and intestinal bacteria shape immune system development Many factors of modern living contribute to dysbiosis, creating an inflammatory milieu associated with chronic inflammatory conditions Of the four main drivers of asthma development, three are microbiome interdependent The upper airway microbiome differs between chronic rhinosinusitis (CRS) and allergy The microbiome in CRS is influenced by the presence and type of asthma More research is required for the use of probiotics in airway disease prevention. This module was sponsored by an unrestricted educational grant from Cipla, which had no control over content. The expert participated voluntarily. june 2018 I 1

2 The microbiome and respiratory inflammation Gastrointestinal microbiome No matter what speciality of medicine you are in, the microbiome is going to be the biggest factor in your working life in years to come Professor Green Earn free CPD Points Join our CPD community at and start to earn today! The GI microbiome is inherited orally from the mother during vaginal delivery, to start colonising the infant gut. The largest microbiome in the body, there are more than 100 trillion microbes from over 1000 species in a functional gut microbiome, with influence on health and disease much less dependent on a single species than the entire range of species acting together. It is plastic, adapting to changes in the environment (e.g. diet); with different microbes facilitating particular functions, such as the fermentation of dietary fibre by Bifidobacterium and Faecalibacterium prausnitzii to make Respiratory microbiome By the time humans are a few months old there should be a rich and healthy microbiome in the respiratory tract, allowing for robust and rapid immune responses. The gut microbiome reaches the airway through translocation and micro-aspiration (inhalation/exhalation) and there is also evidence that it is transported through the blood stream to the lung, with continuous recolonising of the lower respiratory tract from the surrounding sites in a dynamic and transient manner. The respiratory microbiome has recently been characterised using culture-independent techniques (16S rrna sequencing) and is dominated by the same major phyla as the GI microbiome (Firmicutes, Gut-airway axis The gut-airway axis refers to the crosstalk between the gut and lung microbiota. The gut microbiota can influence the lung microbiota by modulating lung immunity through production of bacterial ligands, bacterial metabolites and immune cells that circulate through the blood to reach the lungs. These directly influence the lung immune response and composition of the lung microbiome, protecting against chronic inflammatory disorders such as allergy, asthma and infectious diseases. Microbial communities in the lower airways and lungs are shaped by microbes short chain fatty acids (SCFAs) that serve as energy sources and as anti-inflammatory agents. Host-commensal interactions extend beyond the local intestinal environment and shape the repertoires of immune cells at extra-intestinal sites such as the lungs. 1 An increase in gut immune T-Reg cell numbers is seen with increasing microbial diversity. 2 When the gut microbiome goes wrong, many chronic inflammatory conditions such as asthma, allergy, metabolic syndrome, obesity, hypertension and diabetes are associated. Bacteriodetes and Proteobacteria), but with different relative abundances and reduced total bacterial burden. Exposure to commensals in the developmental window of the newborn period directs lung-selective trafficking of group 3 innate lymphoid cells (ILC), a group of sentinel cells that maintain mucosal homeostasis. This is mediated by intestinal dendritic cells, which induce expression of the lung-homing signal CCR4 on ILC3. Lung-selective trafficking of ILC3 promoted the resistance of newborn mice to pneumonia. This may explain the association between widespread use of antibiotics and an increased risk of pneumonia in newborn infants. 1 present in the oral cavity and upper airways, where microbes arrive through inhalation of the surrounding environment or microaspiration of both the upper gut and upper airway microflora. The lung microbiome is likely to be important in shaping the innate and acquired immune responses in the lungs through their interactions with airway epithelium and immune cells. There is also the possibility that innate and acquired immunity could in turn regulate the lung microbiome (Figure 1) I june 2018

3 The microbiome and respiratory inflammation Good clearance mechanism Normal ciliary function Inhalation Normal microbiome Proteobacteria Firmicutes Actinobacteria Fusobacterium Bacteroidetes Microaspiration GUT Innate immune response eg Innate lymphoid cell type 3 (ILC-3) Acquired immune response eg T helper cell-dendritic cell interaction to generate inhibitory Treg cells Bacterial ligands (eg Lipopolysaccharide) Bacterial metabolites (eg Short chain fatty acids) Migratory cells (eg T cells) Figure 1. Gut-airway axis Amended from: Chung KF. J Allerg Clin Immunol 2017; 139(4): Dysbiosis been destroyed. Pathogens bind to different epithelial receptors and start to interact with cells and cytokines that create an inflammatory milieu (Figure 3). The bidirectional relationship between the GI and respiratory microbiomes via the gutairway axis sees many chronic GI diseases having a respiratory component and vice versa. In a healthy microbiome, normal flora attach to specific pattern recognition receptors, inducing changes in the epithelium and submucosa to produce antiinflammatory mechanisms (immune cells and cytokines) that protect us from chronic immune disease (Figure 2). In a disturbed microbiome (dysbiosis), the number or make-up of organisms has SPLUNC1/LPLUNC2 Lysozymes Lactoferrin S. aureus Bacteriodetes bbd-2 S100A7 S100A8/9 Streptococcus Nasal airway Haemophilus TLR2 Mucus barrier Intact epithelial barrier IL-22R IL-22 Mast cell DC T cell MØ Neutrophil Submucosal gland ILC3 Normal nasal mucosa Figure 2. Healthy airway microbiome Mahdavinia M, et al. Clin Exp Allergy 2016; 46(1): june 2018 I 3

4 The microbiome and respiratory inflammation P. aeruginosa Bacterial and fungal proteases PAR2 TSLP IL-33 IL-25 IL-25R IL-33R TSLPR ILC2 Bacteriodetes S. aureus Streptococcus Haemophilus Th2 cell DC IL-13 IL-5 IL-4 Staph superantigen Submucosal gland TLR2 IL-22R IL-22 ILC3 Nasal airway Abundant proteobacteria colonisation Loss of epithelial integrity: Pattern recognition receptors Antimicrobial peptides Glands Il-22R barrier regulation Mucus response (Goblet cell) Basophil responses Alternatively activated MØ Fibrin deposition Eosinophilia Mast cell responses Chronic rhinosinusitis (CRS) Figure 3. Disturbed airway microbiome Mahdavinia M, et al. Clin Exp Allergy 2016; 46(1): Causes of dysbiosis Ethnogeography, the interaction between genetics and the environment, gives rise to different microbiomes in different populations and between individuals. Many factors are implicated in dysbiosis. Early life circumstances contributing to dysbiosis are birth by elective Caesarean section (Box 1), birth in hospital (exposure to resistant microbes), unnecessary use of antibiotics in paediatrics and artificial feeds (replacement of breastmilk with formula). Other influencing factors are urban living, age, diet, nutritional status, cigarette smoking and pollution. Urbanisation has had the consequence, for example, of decreased exposure to healthy microbes from farm animals. Allergy is much less common in those born and having lived in a farming community. Box 1. Associations between elective Caesarean section and future health 4,5 With elective Caesarean section, the newborn is not exposed to the healthy microbiome as occurs with vaginal delivery and it doesn t get the cortisol response required to initiate protection against inflammation. These two factors are associated with increased incidence of: Allergy Asthma Juvenile arthritis Systemic connective tissue disorders Primary immune deficiency Leukaemia Neurological and neuropsychiatric concerns; more likely to become psychotic in later life. Earn free CPD Points Join our CPD community at and start to earn today! Dysbiosis in asthma Epidemiological studies demonstrate a steady increase in incidence of allergic disease, including asthma, in developed countries. The four main drivers of the development of asthma are genetic predisposition and early life allergy, recurrent viral infections of the lower respiratory tract and presence of bacteria in the lower respiratory tract. The early life factors are microbiome interdependent variables, implying that the microbiome is critically important to the development of asthma. Recent experimental data that suggest a lack of proper exposure to varied, mostly gut-associated, microbes in early life may be a contributing factor to dysbiosis and the development of asthma. Factors favouring microaspiration of GI and upper airway secretions into the lungs (ciliary damage, reduced cough reflex and gastroesophageal reflux) could, in combination with environmental factors, contribute to lung dysbiosis in asthma. This is characterised by an increase in bacterial communities such as Proteobacteria, Firmicutes and Actinobacteria. 4 I june 2018

5 The microbiome and respiratory inflammation In the lower respiratory tract, a proposed cycle of dysbiosis leads to increased lung inflammation and immune dysfunction, which contributes to the initiation of allergic asthma and the various traits of severe asthma. Allergic asthma could be initiated through activation of the innate and acquired immune system by components of the bacterial wall or bacterial products within the airways. Induction of a chronic inflammatory process might Air pollution Antibiotics Corticosteroid therapy Infections Cigarette smoke Allergens Breast feeding Exposure to animals Living on a farm form the basis for worsening of established asthma with exacerbations. This inflammatory process can encourage specific bacterial communities that in turn contribute to further microbial dysbiosis (Figure 4). The presence of Haemophilus parainfluenzae has the capacity to directly induce corticosteroid resistance in macrophages, contributing to steroid insensitivity in patients with severe asthma. Environment Temperature Nutrients Selective bacterial growth Selective clearance Microaspiration Proteobacteria: Haemophilus, Kiebsiella, Neisseria, Moraxella Firmicutes: Streptococcus Actinobacter Macrophage T cell ILC Dendritic cell Neotrophil 8 cell Asthma exacerbation Chronic airflow obstruction Corticosteroids insensitivity Mucus production Vascular permeability Cytokines Inflammatory cell recruitment Th17 recruitment Poor clearance Ciliary damage Sedation Reduced cough reflex Laryngeal dysfunction Gastroesophageal reflux Impaired innate immunity Pathogen-associated molecular pattern (PAMPS) Structural components (Flagellin, Lipoteichoic acid) Bacterial products (Peptidoglycan, Polysaccharide A) Lung inflammation Dendritic cell activation Innate lymphoid cell (ILC3) activation Epithelial barrier function Th2 function Initiating allergic asthma Figure 4. Lung microbial dysbiosis in asthma Chung KF. J Allerg Clin Immunol 2017; 139(4): Asthma and allergic rhinosinusitis Asthma and allergic rhinitis frequently coexist. Up to 80% of patients with asthma have allergic rhinitis, 20% of patients with allergic rhinitis have concurrent asthma. Epidemiologic studies support the suggestion that allergic rhinitis should be suspected as a comorbid condition in most patients with asthma. Due to multiple environmental influences, it is difficult to identify and define a normal upper airway microbiome. The microbiome in the nose and sinuses in CRS is different to the microbiome in allergy. CRS specimen culture finds the usually expected bacteria that are treated for with antibiotics (Staphylococcus, Streptococcus, Haemophilus and the anaerobes Prevotella and Peptostreptococcus),3 although there is a large variability between studies. Viral 16S rrna gene sequencing shows different results to culture. The anaerobic bacteria Diaphorobacter (78%) and Peptoniphilus (72%) have been identified june 2018 I 5

6 The microbiome and respiratory inflammation in CRS, but not in control cases, whereas S aureus was found in half of CRS cases, but detected in all controls. 6 Viral sequencing also revealed the presence of Pseudomonas, Citrobacter, Haemophilus, Propionibacterium, Staphylococcus and Streptococcus in the microbiome of CRS. 7 The microbiome in CRS also depends on presence and type of asthma. The sinus microbiome phylum levels in asthmatic CRS cases was more like controls than nonasthmatic CRS. A pattern was observed for Prevotella and Staphylococcus, which had a trend towards a lower abundance in asthmatic CRS compared to controls, but a trend towards higher abundance in the non-asthmatic CRS group. 8 Fungal species associated with CRS vary in different studies and are often found in normal controls. Common are Candida, Aspergillus and Mucur species. Using multiplex PCR for respiratory viruses, viral nucleic acid sequences were found in 64% of sinus scrapings and 50% of nasal lavage samples in the CRS group, significantly higher than healthy controls (30% and 14% in scraping and lavage respectively). Rhinovirus was most frequently detected. 9 Using probiotics for airway disease prevention Earn free CPD Points Are you a member of Southern Africa s leading digital Continuing Professional Development website earning FREE CPD points with access to best practice content? Only a few clicks and you can register to start earning today Visit For all Southern African healthcare professionals Find us at DeNovo The use of probiotics conveys advantage in other medical conditions but use in allergy prevention is still uncertain as we do not yet have the right dose, timing or combination of microbes for the intervention. Overall literature has shown an improvement in asthma and allergic Sepsis Children with sepsis and severe acute respiratory distress syndrome (ARDS) have a pathological lung microbiome resembling that of the gut due to antibiotic use. Gut-associated Bacteroides were common References 1. Gray J, Oehrle K, Worthen G, et al. Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection. Sci Transl Med 2017; 9(376): eaaf Kepert I, Fonseca J, Muller C, et al. D-tryptophan from probiotic bacteria influences the gut microbiome and allergic airway disease. J Allergy Clin Immunol 2017; 139(5): Mahdavinia M, Kesharvarzian A, Tobin MC, et al. A comprehensive review of the nasal microbiome in chronic rhinosinusitis (CRS). Clin Exp Allergy 2016; 46(1): Sevelsted A, Stokholm J, Bonnelykke K, et al. Cesarean section and chronic immune disorders. Pediatrics 2015; 135(1): e O Neill SM, Curran EA, Dalman C, et al. Birth by caesarean section and the risk of adult psychosis: a population-based cohort study. Schizophr Bull 2016; 42(3): Stephenson ME, Mfuna L, Dowd SE, et al. Molecular characterization of the polymicrobial flora I chronic rhinosinusitis. J Otolaryngol Head Neck Surg 2010; 39(2): rhinitis with probiotics and prebiotics. These findings are not consistent and considered preliminary. Administration of pro- and prebiotics through the upper airways to specifically target the lung microbiome might be effective. and abundant in the bronchoalveolar lavage (BAL) fluid of ARDS patients, but not detected in reagent control specimens nor in the BAL fluid of healthy subjects Stressmann FA, Rogers GB, Chan SW, et al. Characterization of bacterial community diversity in chronic rhinosinusitis infections using novel cultureindependent techniques. Am J Rhinol Allergy 2011; 25(4): e Ramakrishnan VR, Hauser LJ, Feazel LM, et al. Sinus microbiota varies among chronic rhinosinusitis phenotypes and predicts surgical outcome. J Allergy Clin Immunol 2015; 136(2): Cho GS, Moon BJ, Lee BJ, et al. High rates of detection of respiratory viruses in the nasal washes and mucosae of patients with chronic rhinosinusitis. J Clin Microbiol 2013; 51(3): Dickson RP, Singer BH, Newstead MW, et al. Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome. Nat Microbiol 2016; 1(10): Chung KF. Airway microbial dysbiosis in asthmatic patients: A target for prevention and treatment. J Allerg Clin Immunol 2017; 139(4): Disclaimer The views and opinions expressed in the article are those of the presenters and do not necessarily reflect those of the publisher or its sponsor. In all clinical instances, medical practitioners are referred to the product insert documentation as approved by relevant control authorities. Published by denovo Medica Reg: 2012/216456/07 70 Arlington Street, Everglen, Cape Town, 7550 Tel: (021) I info@denovomedica.com 6 I june 2018

7 The microbiome and respiratory inflammation CPD Questionnaire Earn FREE CPD points Complete and to or fax to Alternatively, you can complete this questionnaire online at Fill in your details using clear block letters and mark the answers with a tick ( ). I agree that my CPD-accredited certificate will be forwarded to my address. (Signature of healthcare professional) First Name Surname HPC No. Profession Telephone City Sales Representative 1. Much of human immunity from chronic inflammatory disorders hinges upon having a healthy microbiome. A True 2. Which of the following statements is false? B False A The GI microbiome is the largest in the body B There are more than 100 trillion microbes from over 1000 species in a functional gut microbiome C The effect of microbiome on health and disease is more dependent on the dominant species than the entire range acting together D All of the above E None of the above 3. Which of the following statements is false? A Host-microbiome interactions extend beyond the local GI environment and shape the immune response at extra-intestinal sites such as the lungs B Increased gut T-Reg cell numbers are associated with greater microbial diversity C Dysbiosis of the gut microbiome is associated with chronic inflammatory conditions D All of the above E None of the above 4. Culture-independent techniques such as 16S rrna sequencing have indicated that the respiratory microbiome is dominated by the same major phyla as the GI microbiome. A True 5. The gut microbiome reaches the airway via: A Translocation and micro-aspiration C All of the above 6. Which of the following statements is false? B False B Transport through the blood stream D None of the above A The respiratory microbiome is dominated by the same major phyla as the GI microbiome B In newborns, lung-selective trafficking of ILC3 is mediated by intestinal dendritic cells C The lower respiratory tract is sterile 7. The bidirectional relationship between the GI and respiratory microbiomes via the gut-airway axis sees many chronic GI diseases having a respiratory component and vice versa. A True 8. Factors implicated in dysbiosis include: B False A Mode and place of birth, early life nutrition B Use of antibiotics C Urbanisation and pollutants D All of the above 9. Which factor driving the development of asthma is not microbiome interdependent? A Genetics C Early life recurrent viral infection of lower respiratory tract 10. Which of the following statements is true? B Early life allergy D Early life presence of bacteria in lower respiratory tract A Factors favouring microaspiration of GI and upper airway secretions into the lungs could contribute to lung dysbiosis in asthma B Dysbiosis in asthma is characterised by increased communities of Proteobacteria, Firmicutes and Actinobacteria C A cycle of dysbiosis leads to increased lung inflammation and immune dysfunction that may worsen established asthma D All of the above 11. Steroid insensitivity in patients with asthma may be linked to the presence of Rhinovirus. A True 12. What percentage of patients with asthma have co-morbid allergic rhinitis? B False A 20% B 40% C 80% 13. Which bacteria have been identified in CRS but not in healthy control cases? A Diaphorobacter B Peptoniphilus C Staphylococcus D A and B E B and C 14. In what manner does the presence and type of asthma affect the microbiome phylum levels in CRS? A Asthmatic CRS cases resemble healthy controls more than non-asthmatic CRS B Non-asthmatic CRS cases resemble healthy controls more than asthmatic CRS cases 15. The common fungal species Candida, Aspergillus and Mucur are associated with the microbiome in: A CRS C Both of the above B Healthy controls D Neither of the above

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