the microbiome and respiratory inflammation · 2018-06-25 · and respiratory inflammation...

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Professor Robin GreenPaediatric PulmonologistFaculty of Health Sciences, University of Pretoria

This module was sponsored by an unrestricted educational grant from Cipla, which had no control over content. The expert participated voluntarily.

The microbiome and respiraTory inflammaTionIntroductionOver 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

• Thereisamicrobiomeeverywhereinthebody

• Muchofhumanimmunityfromchronicinflammatorydisordershingesuponhavingahealthymicrobiome

• Interactionsbetweenthehostimmunesystemandintestinalbacteriashapeimmunesystemdevelopment

• Many factors of modern living contribute to dysbiosis, creating an inflammatory milieuassociatedwithchronicinflammatoryconditions

• Ofthefourmaindriversofasthmadevelopment,threearemicrobiomeinterdependent

• Theupperairwaymicrobiomediffersbetweenchronicrhinosinusitis(CRS)andallergy

• ThemicrobiomeinCRSisinfluencedbythepresenceandtypeofasthma

• Moreresearchisrequiredfortheuseofprobioticsinairwaydiseaseprevention.

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The microbiome and respiraTory inflammaTion

“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

Gastrointestinal microbiomeThe 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 micro-biome, 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 par-ticular functions, such as the fermenta-tion of dietary fibre by Bifidobacterium and Faecalibacterium prausnitzii to make

short chain fatty acids (SCFAs) that serve as energy sources and as anti-inflamma-tory 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 con-ditions such as asthma, allergy, metabolic syndrome, obesity, hypertension and dia-betes are associated.

Respiratory microbiomeBy the time humans are a few months old there should be a rich and healthy micro-biome in the respiratory tract, allowing for robust and rapid immune responses. The gut microbiome reaches the airway through translocation and micro-aspi-ration (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-inde-pendent techniques (16S rRNA sequenc-ing) and is dominated by the same major phyla as the GI microbiome (Firmicutes,

Bacteriodetes and Proteobacteria), but with different relative abundances and reduced total bacterial burden.

Exposure to commensals in the devel-opmental 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 intesti-nal dendritic cells, which induce expres-sion 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 asso-ciation between widespread use of antibi-otics and an increased risk of pneumonia in newborn infants.1

Gut-airway axis The gut-airway axis refers to the cross-talk 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

present in the oral cavity and upper air-ways, where microbes arrive through inha-lation 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).11

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Dysbiosis In a healthy microbiome, normal flora attach to specific pattern recognition receptors, inducing changes in the epi-thelium and submucosa to produce anti-inflammatory 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

been destroyed. Pathogens bind to differ-ent epithelial receptors and start to inter-act with cells and cytokines that create an inflammatory milieu (Figure 3). 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.

Figure 1. Gut-airway axisAmended from: Chung KF. J Allerg Clin Immunol 2017; 139(4): 1071-1081.11

Normal microbiomeProteobacteriaFirmicutes

ActinobacteriaFusobacteriumBacteroidetes

innate immune responseegInnatelymphoidcelltype3(ILC-3)

acquired immune responseegThelpercell-dendriticcellinteraction

togenerateinhibitoryTregcells

Bacterial ligands (eg Lipopolysaccharide)Bacterial metabolites (eg short chain fatty acids)Migratory cells (eg t cells)

Good clearance mechanismNormal ciliary function

Microaspiration

inhalation

GUt

Figure 2. Healthy airway microbiomeMahdavinia M, et al. Clin Exp Allergy 2016; 46(1): 21-41.3

Normal nasal mucosa

Nasal airway

t cell

MØ

DC

Neutrophil

Bacteriodetes

spLUNC1/LpLUNC2LysozymesLactoferrin

bBD-2s100a7

s100a8/9

s. aureus streptococcus Haemophilus

Mast cell

iLC3

iL-22R

tLR2

submucosal gland

Mucus barrierintact epithelial barrier

iL-22

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Causes of dysbiosisEthnogeography, the interaction between genetics and the environment, gives rise to different microbiomes in different popula-tions and between individuals. Many fac-tors 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 (replace-ment 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 health4,5

With elective Caesarean section, the newborn isnotexposedtothehealthymicrobiomeasoccurswithvaginaldeliveryanditdoesn’tgetthecortisolresponse required to initiate protection againstinflammation. These two factors are associatedwithincreasedincidenceof:

• Allergy

• Asthma

• Juvenilearthritis

• Systemicconnectivetissuedisorders

• Primaryimmunedeficiency

• Leukaemia

• Neurological and neuropsychiatric concerns;morelikelytobecomepsychoticinlaterlife.

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 pre-disposition 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 combi-nation with environmental factors, con-tribute to lung dysbiosis in asthma. This is characterised by an increase in bacte-rial communities such as Proteobacteria, Firmicutes and Actinobacteria.

Figure 3. Disturbed airway microbiomeMahdavinia M, et al. Clin Exp Allergy 2016; 46(1): 21-41.3

Chronic rhinosinusitis (CRs)

Nasal airway

DC

iL-13 iL-5 iL-4

staph superantigen

th2 cell

iLC2

paR2

tsLpiL-33iL-25

iL-25R iL-33RtsLpR

Bacterial and fungal proteases

Bacteriodetesp. aeruginosas. aureus

streptococcus Haemophilus

Mast cell responses

eosinophiliaFibrin deposition

alternatively activated MØ

Basophil responses

Mucus response (Goblet cell)

iLC3

iL-22R

tLR2

submucosal gland

abundant proteobacteria colonisationLoss of epithelial integrity:pattern recognition receptorsantimicrobial peptidesGlandsil-22R barrier regulation

iL-22

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In the lower respiratory tract, a pro-posed cycle of dysbiosis leads to increased lung inflammation and immune dysfunc-tion, 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 compo-nents of the bacterial wall or bacterial products within the airways. Induction of a chronic inflammatory process might

form the basis for worsening of estab-lished asthma with exacerbations. This inflammatory process can encourage spe-cific 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 mac-rophages, contributing to steroid insensi-tivity in patients with severe asthma.

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 concur-rent asthma. Epidemiologic studies sup-port the suggestion that allergic rhinitis should be suspected as a comorbid condi-tion in most patients with asthma.

Due to multiple environmental influ-ences, it is difficult to identify and define a ‘normal’ upper airway micro-biome. The microbiome in the nose

and sinuses in CRS is different to the microbiome in allergy. CRS specimen culture finds the usually expected bac-teria that are treated for with antibi-otics (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 anaero-bic bacteria Diaphorobacter (78%) and Peptoniphilus (72%) have been identified

environment

Microaspiration

AirpollutionAntibioticsCorticosteroidtherapyInfectionsCigarettesmokeAllergensBreastfeedingExposuretoanimalsLivingonafarm

PoorclearanceCiliarydamageSedationReducedcoughreflexLaryngealdysfunctionGastroesophagealrefluxImpairedinnateimmunity

Pathogen-associatedmolecularpattern(PAMPS)Structuralcomponents(Flagellin,Lipoteichoicacid)Bacterialproducts(Peptidoglycan,PolysaccharideA)

LunginflammationDendriticcellactivationInnatelymphoidcell(ILC3)activationEpithelialbarrierfunction↑ Th2function

↑ Mucusproduction↑ Vascularpermeability↑ Cytokines↑ Inflammatorycellrecruitment↑ Th17recruitment

↑ Temperature↑ NutrientsSelectivebacterialgrowthSelectiveclearance

initiating allergic asthma

↑ proteobacteria:Haemophilus,Kiebsiella,Neisseria,Moraxella

↑ Firmicutes:Streptococcus

↑ actinobacter

asthma exacerbationChronic airflow obstructionCorticosteroids insensitivity

Figure 4. Lung microbial dysbiosis in asthmaChung KF. J Allerg Clin Immunol 2017; 139(4): 1071-1081.11

DendriticcellMacrophage

NeotrophilTcell

8cellILC

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DisclaimerThe 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.

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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 non-asthmatic 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 spe-cies. Using multiplex PCR for respira-tory 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 preventionThe use of probiotics conveys advan-tage in other medical conditions but use in allergy prevention is still uncertain as we do not yet have the right dose, tim-ing or combination of microbes for the intervention. Overall literature has shown an improvement in asthma and allergic

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.

SepsisChildren with sepsis and severe acute res-piratory distress syndrome (ARDS) have a pathological lung microbiome resem-bling that of the gut due to antibiotic use. Gut-associated Bacteroides were common

and abundant in the bronchoalveolar lav-age (BAL) fluid of ARDS patients, but not detected in reagent control specimens nor in the BAL fluid of healthy subjects.10

References1. 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): eaaf9412.

2. 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): 1525-1535.

3. 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): 21-41.

4. Sevelsted A, Stokholm J, Bonnelykke K, et al. Cesarean section and chronic immune disorders. Pediatrics 2015; 135(1): e92-8.

5. 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): 633-41.

6. 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): 182-7.

7. Stressmann FA, Rogers GB, Chan SW, et al. Characterization of bacterial community diversity in chronic rhinosinusitis infections using novel culture-independent techniques. Am J Rhinol Allergy 2011; 25(4): e133-40.

8. 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): 334-42.

9. 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): 979-84.

10. 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): 16113.

11. Chung KF. Airway microbial dysbiosis in asthmatic patients: A target for prevention and treatment. J Allerg Clin Immunol 2017; 139(4): 1071-1081.

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1. Much of human immunity from chronic inflammatory disorders hinges upon having a healthy microbiome.

a True B False

2. Which of the following statements is 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


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 B False

5. the gut microbiome reaches the airway via:

a Translocation and micro-aspiration B Transport through the blood stream C All of the above 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 B False

8. Factors implicated in dysbiosis include:

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 B Early life allergy C Early life recurrent viral infection of lower respiratory tract D Early life presence of bacteria in lower respiratory tract

10. Which of the following statements is true?

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 B False

12. What percentage of patients with asthma have co-morbid allergic rhinitis?

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 B Healthy controls C Both of the above D Neither of the above

the microbiome and respiratory inflammation · 2018-06-25 · and respiratory inflammation...

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