Asthma and the Microbiome

Monica Kraft, M.D. Robert and Irene Flinn Professor of Medicine Chair, Department of Medicine Unieversity of Arizona Health Sciences Center

1 Disclosures Research: NIH, Chiesi (Funds paid to University of Arizona) Consulting: TEVA, Genentech, Astra-Zeneca

Royalties: Elsevier

2 Objectives To discuss the current understanding of the microbiome in asthma

To discuss how specific environmental exposures can influence the microbiome and asthma development

To discuss how manipulation of the microbiome can alter asthma pathobiology

3 The Microbiome

The totality of microbes and their genetic elements (genomes), in an environmental niche; usually includes microbiota and their complete genetic elements. Microbes: organisms too small to be visible to the unassisted human eye (viruses, bacteria, archea, fungi, parasites). Environmental niche: The ecological space occupied by a species. A geographically limited region sustaining characteristic life-forms in steady state (in humans: mouth, gastrointestinal tract, sinuses, vagina).

Exploration of the Bacterial World: Gene for 16S rRNA

• Culture-independent identification of bacteria relies on the fact that the genes for ribosomal RNA genes are well conserved across species

• Structurally conserved regions permits universal primer design and thus amplification of 16S from all bacterial species • Variable region • Taxon specific • Permits identification by sequencing or microarray. L Weng, EM Rubin, J Bristow. Genome Research, 2006;16:316 Dimensions of the Microbial World

Standard medical culture techniques fail to document the presence of up to 99% of environmental microbes. Even under growth conditions thought to mimic environmental conditions, ~ 80% of organisms do not grow in culture.

L Weng, EM Rubin, J Bristow. Genome Research, 2006;16:316 16S rRNA-based tools characterize microbial ecology in human niches

Structure of bacterial communities are associated with states of health or disease:

• Periodontitis (Sakamoto, 2004)

• Inflammatory bowel disease

• Obesity (Ley, Nature 2006)

• Cystic fibrosis (Harris, PNAS 2007) Major bacterial • Ventilator-associated pneumonia phyla identified in (Flanagan, JCM 2007) human niches. (Dethlefsen et al., Nature 2007) Outline

History: Reasons for interest in airway microbiota in asthma. Direct study of airway microbiota: “disordered” in asthma. Mechanisms of protection or harm from airway microbes. Epidemiologic studies of environmental associations with protection against allergy and asthma – the Hygiene Hypothesis.

Mechanisms of protection or harm from environmental microbiota – animal studies and human studies Reasons for Interest: Antibiotic Treatment Might be Beneficial for Asthma

The Use of Triacetyloleandomycin in Chronic Infectious Asthma

“Patients with chronic asthma usually have an infectious component. The signs of cough, wheezing, and attacks of dyspnea are always associated with the amount and type of sputum produced.”

38 of 44 patients reported to improve after course of TAO therapy.

Kaplan MA, Goldin M, in Welch H, Marti-Ibanez, editors, Antibiotics Annual, 1958-59, New York, 1959, Medical Encyclopedia, Inc, pp. 273-6. 2 Reasons for Interest: Antibiotic Treatment Might be Beneficial for Asthma

ARJCCM 1998; 158:998-1001

JACI 2002; 121:1782-1788 2 Reasons for Interest: Childhood Asthma after Neonatal Airway Colonization by H. influenzae, Moraxella, or Streptococcus.

Bisgaard H et al. N Engl J Med 2007;357:1487-1495.11 Outline

Mechanisms of protection or harm from environmental microbiota – animal studies and human studies Healthy vs. Asthma, Bacterial burden, and Airway hyperresponsiveness #

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§ Higher bacterial burden observed in asthmatics compared to healthy control (16S rRNA amplicon concentration as proxy)

§ Generally higher bacterial burden in those with “more hyperresponsive” vs. “less hyperresponsive” airways by methacholine challenge

Huang Y, J Allergy Clin Immunol 2011 Airway Microbiome in Mild-Moderate Asthma

Higher baseline bacterial diversity in asthmatics with improved bronchial reactivity after 6 weeks of clarithromycin

Huang Y, J Allergy Clin Immunol 2011 Specific bacterial taxa (~100) strongly correlated with asthma and airway hyperresponsiveness

)

) Bacterial taxa of interest: unclassif. (

Alteromonadaceae unclassif. ( Shewanellaceae Enterobacteriaceae* *

Alteromonadaceae

Piscirickettsiaceae ibrionaceae * Gammaproteobacteria Francisellaceae •Species with functional potential to Methylococcaeae

Symbionts Alcanivoraceae Gammaproteobacteria Chromatiaceae Alteromonadaceae onadaceae Gammaproteobacteria degrade steroid compounds

A AB094597.1

AJ132226.1

B AF172840.1

0 AJ845014.1

5 AJ233429.1

Chromatiaceae AB055791.1 2 uranium waste clones( 6 Pseudom unclassif.) AJ295715.1 4

. AF384373.1 1

X95229.1

AB053127.1

AF406527.1

AY102612.1 AF482686.1

AF513454.1 AF307138.1 AF420370.1 Thiotrichaceae AF448515.1 •Glycosphingolipid species – iNKT AJ242772.1 (unclassif.) AF405328.1 Rhodocyclaceae AJ419674.1 AJ401211.1 AF238494.1 AF523888.1 GAO cluster AF181576.1 AB046997.1 MND1 clone group M96395.1 AF502233.1 AJ536870.1 cell interactions? Y17591.1 Betaproteobacteria L40998.1 AY221081.1 Methylophilaceae AY098909.1 AF293006.1 AJ519624.1 Rhodocyclaceae * Verrucomicrobia subdivision 7 AF289159.1 AY013692.1 •Macrolide-susceptible species other AY123800.1 Nitrosomonadaceae AY114314.1 AY123811.1 AY114322.1 AY123795.1 AF454310.2 BX321856.1 than M. or C. pneumoniae Verrucomicrobia AF419674.1 AY538706.1 Comamonadaceae

AJ401113.1 AY218566.1 Burkholderiaceae LD1PA group AY114324.1 AY218644.1 Comamonadaceae AF526930.2 AJ532721.1 Neisseriaceae •Species possessing functional nitric Acidobacteria10 AY169431.1 AY114325.1 AY899912.1 WS3 AJ532725.1 AF336312.1 Comamonadaceae AM AF282254.1 SP AF371864.1 oxide reductase activity AF481216.1 X67039.1 Firmicutes * AJ518790.1 AF280433.1 Alcaligenaceae AF085226.1 AF523979.1 Burkholderiaceae Synergistes AF312022.1 AJ534634.1 AF361023.1 • AF529322.1 Specific noteworthy taxa: Polyangiaceae AY593479.1 Ralstoniaceae AF529350.1 AB021375.1

Z95722.1 AB074523.1

Y08845.1 AF366270.1 U54470.1 AF530136.1 AF355039.1 * AF523879.1 Acidobacteriaceae AJ347046.1 U70723.1 AY048657.1 • Neisseriaceae U87784.1 Oxalobacteraceae D16144.1 AF091110.1 AY785128.1 AY167834.1

Y09639.1

AF425996.1 AJ294416.1 AB021415.1

Plectonema . AJ011759.1 3

8 D88524.1

6 NC 005955.1

3 Bdellovibrionales AF143830.1 U87765.1

5 AY007683.1 J Alphaproteobacteria AF530467.1 A • Sphingomonadaceae Sphingomonadaceae

Flavobacteriaceae Sphingomonadales

Sphingomonadaceae * CrenotrichaceaeChloroplasts Caulobacteraceae Plectonema

(unclassif.) • Pseudomonadaceae

Bartonellaceae Phyllobacteriaceae* (unclassif.)

Phyllobacteriaceae

Bradyrhizobiales Phyllobacteriaceae * Bradyrhizobiaceae • Enterobacteriaceae * * p < 0.01, * q-val < 0.02 Alphaproteobacteria Airway Bacterial Microbiota – Healthy vs. Asthma or COPD

24 adults: asthma (11), COPD (5), healthy (8). Traditional 16S rRNA clone library-serial sequencing approach (34-63 sequenced clones/ subject) Proteobacteria

LUL brushings BAL (adults) (children) Asthma patients: more pathogenic Proteobacteria, e.g. Haemophilus spp. Healthy controls: more Bacteroidetes, esp. Prevotella species Hilty et al., PLoS One 2010 Summary of Findings

§ Differences from healthy controls have been found in the bacterial microbiome of airway samples of asthmatics not taking as well as taking an inhaled corticosteroid. § Bacterial bioburden is greater in asthmatics taking than in healthy subjects. § The bacterial populations increased in the airway samples from asthmatic subjects are polymicrobial. § In asthmatics, Trend toward greater abundance of Proteobacteria (Pseudomonas, Hemophilus) Outline

Mechanisms of protection or harm from environmental microbiota – animal studies and human studies. Mechanisms of Protection or Harm from Airway Microbes

Microbial communities normally present in a host niche inhibit the outgrowth of pathogens.

Examples of antibiotic-associated diseases due to outgrowth of microbes present in health.

Oral and vaginal candidiasis.

C. difficile colitis. C. tuberculostericum sinusitis.

Sinus Microbiome

• Surgical samples obtainedfrom 10 CRS patients, 10 healthy controls undergoing open maxillary sinus surgery

• Varying pre-operative antimicrobial administration

Abreu et al., Sci Transl Med. 2012 Sep 12;4(151):151ra124. CRS is associated with Microbiome Collapse

• Loss of bacterial richness, evenness, and diversity in CRS

Abreu et al., Sci Transl Med. 2012 Sep 12;4(151):151ra124. Discrete taxa that characterize health status

• Known pathogenic members of the Pseudomonadaceae, Lachnospiraceae, Ralstoniaceae, Mycobacteriaceae and Helicobacteriaceae detected in both groups

• Taxon-based comparative analyses of CRS vs healthy:

• 1,482 taxa significantly lower relative abundance in CRS ptx

• Lactobacillales • Phylogenetically distinct LAB • Carnobacterium, Enterococcus, Pediococcus spp. • Lactobacillus sakei (t value = 7.059; p < 0.00002; q < 0.00014)

• Single taxon significantly increased in relative abundance in CRS ptx • Actinobacteria: Corynebacterium tuberculostearicum (t value = -2.652 p < 0.034; q < 0.003)

Abreu et al., Sci Transl Med. 2012 Sep 12;4(151):151ra124. Sinus Mucosal Pathogenesis and Protection

Abreu et al., Sci Transl Med. 2012 Sep 12;4(151):151ra124. Summary of Findings and Conclusions

• Innoculation of Corynebacterium tuberculostearicum into the sinonasal track of mice:

• Causes no changes of sinusitis in untreated mice • Causes inflammation and mucus hyperplasia in mice pretreated with antibiotics. • In antibiotic treated mice, coincident inoculation of Lactobacillus sakei prevents induction of sinusitis-like histopathology from inoculation with C. tuberculostearicum

• C. tuberculostearicum is a pathobiont.

• L. sakei protects mucosal surface from pathogenesis.

Abreu et al., Sci Transl Med. 2012 Sep 12;4(151):151ra124. Outline

Mechanisms of protection or harm from environmental microbiota – animal studies and human studies. Features of Epidemiologic Studies of Environmental Exposures and Asthma

Similar impacts from farm animals, household pets, unpasteurized milk, contaminated water. Risk reduction is not allergen specific. Impact of exposure greatest in 1st year of life. Impacts reported for other diseases of immune dysregulation including Th1 diseases – e.g. IBD. Impact does not correlate well with environmental sample concentrations of animal allergen, endotoxin, muramic acid, egosterol. Detroit Childhood Allergy-Asthma Study:

Birth cohort of 833 middle-class children living in Detroit suburbs Yearly questionnaires concerning home environments plus home visits at 2 and 4 years Evaluation between 6 and 8 years for asthma and allergy

Ownby DR et al JAMA 2002;288:963-972 First Year Pet Exposure And Allergic Sensitivity

P = .003 P = .005

P = .02 P = .04 PercentChildrenof

Ownby DR et al JAMA 2002;288:963-972 Early-life Exposure to Dogs and Cats Protects Against Childhood Allergic Sensitization

. Analysis of Microbial Content of House Dust

D C NP

Fujimura, K. et al., JACI. 2010 126(2):410-2 Early-life Exposure to Dogs and Cats Protects Against Childhood Allergic Sensitization

. Analysis of Microbial Content of House Dust

Fujimura, K. et al., JACI. 2010 126(2):410-2 Impact of Dog Ownership on House Dust Microbiota

Taxa significantly increased in dog-associated house dust:

• Proteobacteria • Actinobacteria • Firmicutes • Bacteroidetes • Spirochaetes • Verrucomicrobia

Key phyla of the human gastrointestinal microbiome

31 Mechanism of Protection?

• NIAID PO1 (AI089473-01) Christine Cole-Johnson and Dennis Ownby Pets & the Infant Microbiome: Effects on Immune Maturation & Atopic Asthma

Dog vs No pets

Fujimura K,. PNAS 2014;111(2):805-10. House Dust Exposure Modulates Airway Response to Allergen Challenge

Fujimura ,. PNAS 2014;111(2):805-10. Lactobacillus johnsonii isolation

• Cecal contents of 4 mice > Lactobacillus isolation media • Sequenced 16S rRNA of twenty one isolates • 99% coverage and 99% homology to expected Lactobacillus species – L. johnsonii • Large-scale batch culture • Standardized (1 x 107 CFU) supplements

Daily gavage Lactobacillus sp. 1 x 107

Lactobacillus gavage 2 X per week

Fujimura K, PNAS 2014;111(2):805-10. Lactobacillus Supplementation Protects against RSV Challenge

Fujimura K, PNAS 2014;111(2):805-10. Microbial Diversity, Living on a Farm, and Asthma

Ege et al, N Engl J Med 2011;364:701-9. Children Raised on Animal Farms Are Protected Against All Forms of Early Wheezing

Fuchs et al, JACI 2012,382–388 Intestinal Microbes and Allergic Asthma

One of the most convincing hygiene-hypothesis-based associations is that reported between exposure to food-borne microbes and respiratory allergy

Matricardi et al. showed that subjects with a history of infection with several food-borne pathogens were protected against the development of allergic asthma

Von Mutius and coworkers showed that children fed with unpasteurized milk in early life were less likely to develop subsequent allergic asthma Matricardi et al, BMJ. 2000;320:412-7 Braun-Fahrländer C, von Muus E. Clin Exp Allergy. 2011;41:29-35 The Common Mucosal Immune System

These studies suggest that the gut and gut-associated lymphoid tissue may shape the immune response against environmental antigens in distant mucosae

This hypothesis is based on the “common mucosal immune system” theory: lymphoid cells can be “programmed” in the gut and then homed to other mucosal sites Intestinal Microbes and Allergic Asthma Several investigators have used live oral probiotics to treat and prevent many diseases including eczema and asthma

Results of a recent review: “We found no evidence to support a protective association between perinatal use of probiotics and doctor diagnosed asthma or childhood wheeze” (Azad et al, BMJ 2014; 347:f6471).

Biochemical complexity of live probiotics poses great challenges re: drug regulation/standardization

Investigators have thus explored the possibility that bacterial extracts could be used as a surrogate Overarching Hypothesis

• That gut microbial community composition in the first years of life is shaped by what the infant puts in his mouth– breast milk, food, dirt, dust, and their associated microbes. • That the gut microbiome in infancy shapes the development of immune function. • That the differences in immune function related to differences in gut microbiome are expressed In the lungs and airways, shaping the response to inhalation of allergen and to respiratory viral infection.

Bacterial Extracts Bacterial extracts (OM-85BV) are lyophilized fractionated alkaline extracts of H. influenzae, D. pneumonia, K. ozaenae, K. pneumoniae, S. aureus, S. pyogenes, S. viridans, and N. catarrhalis

It is a mixture of acidic proteins, peptides and amino acids, with minor components of detoxified LPS and lipoteichoic acids

Clinical Use of Bacterial Extracts Bacterial extracts have been widely used in Europe for the last 2-3 decades in children and adults as oral medicines to reduce the frequency and duration of URIs

They have also been used in the prevention of exacerbations in CF and COPD Forest plot of comparison of OM-85BV trials for URI

The reducon in the proporon of ARTIs was -35.90% (95% CI -49.46 to -22.35)

Del Rio Navarro et al, Cochrane Database Syst Rev. 2006 Oct 18;(4):CD004974. Conclusions of Cochrane Review “After reviewing all available studies on the prevention of ARTIs in children, we empathize with the skepticism of many physicians”

Low quality and reporting of the trials on this topic, the heterogeneity of the included trials and the possibility of publication bias

Few papers complied with standards for methodological quality and reporting of clinical trials

Thus, the global quality of the evidence on the effect of immunostimulants to reduce the incidence of ARTIs is regarded as moderate.

Del Rio Navarro et al, Cochrane Database Syst Rev. 2006 Oct 18;(4):CD004974. The Study by Razi et al. This study was a randomized, double-blind, placebo- controlled, parallel group study with OM-85 BV in patients with recurrent wheezing in Ankara, Turkey

75 (40 OM-85BV, 35 placebo) 1-6 yr old children with recurrent wheezing

Participants were randomly assigned to groups given either OM-85 BV or placebo (1 capsule per day for 10 days each month for 3 consecutive months) at the start of the trial.

J Allergy Clin Immunol 2010;126:763-9 Cumulative Yearly Number of Wheezing Episodes per Participant

p<0.001

J Allergy Clin Immunol 2010;126:763-9 Duration (Days±SD) of Each Episode of Wheezing

p<0.001

J Allergy Clin Immunol 2010;126:763-9 Adverse Events The overall incidence of adverse effects in clinical trials was between 3 and 4%. Gastrointestinal troubles (nausea, abdominal pain, vomiting), dermatologic reactions (rash, urticaria), and respiratory disorders (coughing, dyspnea, asthma), as well as generalized problems (fever, fatigue, allergic reactions) are the most frequent complaints reported

Only GI effects were more frequent in the OM-85BV group in most studies

Prevention of BHR by Oral Administration of Bacterial Extracts

Navarro et al, Mucosal Immunol 2011; 4:43 Decreased Airway Inflammation by Oral Administration of Bacterial Extracts

Navarro et al, Mucosal Immunol 2011; 4:43 Decreased BAL Cytokines by Oral Administration of Bacterial Extracts

Navarro et al, Mucosal Immunol 2011; 4:43 Increased Tracheal Treg Cells by Oral Administration of Bacterial Extracts

Navarro et al, Mucosal Immunol 2011; 4:43 Activation of Treg in Airway Mucosa by Oral OM-85BV in Rats Oral pretreatment of sensitized rats with OM-85BV strikingly accelerated the resolution of AHR that is triggered by aeroallergen exposure.

The kinetics of the AHR response in treated animals closely mirrored associated CD86 expression on airway mucosal dendritic cells

The principal feature distinguishing OM-85BV treated rats from controls was their markedly increased (2 × ) baseline numbers of airway mucosal Tregs.

Strickland and Holt, Mucosal Immunol. 2011;4:43-52 ORBEX Trial Schemata WLRI outcome “Asthma” outcome

OM-85BV Usual Rx

Run-in

Placebo Usual Rx

1062 6-18 mo olds 2 yrs 1 yr at high risk for asthma Implications for Strategies for Prevention

• Enable proper development of G-I microbiome and induction of immune function by changing what infants puts in their mouths. • Drugs – high cultural, ethical, and legal hurdles. • Diet – breast feeding, unpasteurized milk, high-fiber foods, field-picked fresh foods, especially produce. • Animal exposures – something about dogs.