2/13/2018. What s bugging you? Relevance of the Human Microbiome in Health and Disease Heidi H. Kong, MD, MHSc, FAAD Dermatology Branch

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1 What s bugging you? Relevance of the Human Microbiome in Health and Disease Heidi H. Kong, MD, MHSc, FAAD Dermatology Branch DISCLOSURE OF RELATIONSHIPS WITH INDUSTRY Heidi H Kong, MD, MHSc, FAAD U017: Relevance of the Human Microbiome in Health & Disease DISCLOSURES No relevant conflicts to disclose U017: Relevance of the Human Microbiome in Health & Disease - Learning Objectives Discuss current literature on human microbiome research Recognize potential clinical relevance of human microbiome research to human health and disease Human Microbiome Defining microbiome Microbiome in health & disease Non-dermatologic Cardiometabolism, Cancer Skin Potential applications Clostridium difficile colitis Humans microbes are complex and multi-faceted Microbes are more often considered pathogenic Mycobacterium tuberculosis Herpes simplex virus Microbes perform functions important for human health Vitamin synthesis Development and activity of immune system Inhibition of skin colonization by pathogens Many microbial-host and microbial-microbial interactions remain unknown 1

2 Definition of Microbiome Pipeline for skin microbiome studies Collection of microbes including bacteria, viruses, and eukaryotes (fungi) and their collective genomes Collect superficial skin sample Isolate DNA Amplify DNA with primers (bacteria = 16S rrna gene) DNA sequencing/analysis Kong. Trends Molec Med Microbiome sequencing: amplicon vs. whole genome shotgun metagenomics Amplicon sequencing Amplify marker regions with universal primers (16S/bacteria, ITS1/fungi) Fungi Viruses Archaea Bacteria Shotgun metagenomic sequencing Fragment genomic DNA Direct sequencing vs. culture-based methods or Identify bacterial (or fungal) members of the microbial communities Identify members of the global microbial communities Determine full genetic potential of microbial communities Diversity from direct sequencing Diversity from culture NIH Common Fund: Human Microbiome Project Longitudinally assess microbial diversity of 250 healthy subjects at 5 major body sites HMP Consortium. Nature 2012 Science

3 Defining the Human Microbiome Collection of microbes including bacteria, viruses, and eukaryotes (fungi) and their collective genomes Human microbes more diverse that observed with traditional culturing Integrating genomics into microbial investigations is advancing our knowledge about our microbes Different body sites contain distinctive microbial communities Human Microbiome Defining microbiome Microbiome in health & disease Non-dermatologic Cardiometabolism, Cancer Skin Potential applications Clostridium difficile colitis Germ-free mice gain little weight with high-fat diet Obesity-associated fecal transfer results in obese phenotype Obesity-associated fecal bacteria Indigestible dietary complex carbohydrates Efficiently harvest energy Facilitate absorption of shortchain fatty acids Turnbaugh P at al. Nature Aron-Wishewsky A, Clement K. Nature Rev Nephrol Turnbaugh P at al. Nature Aron-Wishewsky A, Clement K. Nature Rev Nephrol Linking diet, microbiota & cardiovascular/kidney disease Linking diet, microbiota & cardiovascular/kidney disease Meat, fish, eggs Meat, fish, eggs Aron-Wishewsky A, Clement K. Nature Rev Nephrol observations debated TMAO = trimethylamine N-oxide TMAO = trimethylamine N-oxide MACE = major adverse cardiac events Aron-Wishewsky A, Clement K. Nature Rev Nephrol observations debated 3

4 Gut microbiota influences efficacy of PD-1 inhibitors Specific microbiota were associated with responders vs non-responders Gopalakrishnan et al. Science Matson et al. Science Routy et al. Science Jobin. Science 2018 Human Microbiome Defining microbiome Microbiome in health & disease Non-dermatologic Cardiometabolism, Cancer Skin Potential applications Clostridium difficile colitis Long History of Research in Skin Microbes J Invest Dermatol 1950 J Invest Dermatol 1962 J Invest Dermatol

5 The Skin Microbiome Skin Microbes Skin bacterial communities demonstrate topographical diversity and site-specificity Oily 20 skin sites Moist Dry Host factors influence skin microbial Host influence host skin Bacterial skin communities are site-specific Skin microenvironments are associated with differences in the relative abundances of predominant bacteria Grice, Kong Segre. Science Life Stages Bacterial communities in skin and nares shift dramatically between Tanner stages 3 and 4 Appropriate selection of healthy pediatric controls is important for studies in children Tanner stages Infant Child Adult Elderly Oh Segre*, Kong*. Genome Med Microbial communities on skin vary in diversity and distribution Do individual strain signatures persist longitudinally? Multiple strains of bacterial species may be present on human skin Strain differences may be individual-specific and/or site-specific Biological differences observed among distinct strains? Grice et al. Science Findley, Oh et al. Nature Oh et al. Nature

6 IFN-g Parasites per 10 3 cells 2/13/2018 How stable is the skin microbiome? The skin microbiome is relatively stable within healthy individuals HV 5 HV 6 HV 12 Oh, Byrd et al. (2016)Cell. Anatomical site Skin microenvironments Sexual maturity Host factors influence skin microbial Skin Microbes Host influence host skin How can we modify human skin microbes? Can we sufficiently modify microbes to promote human health? Host factors **What influence is the skin microbial landscape?** skin microbial Healthy individuals influence host skin Patients with skin diseases Skin commensals restore immunity to Leishmania major in germ-free mice SPF L. major GF Parasite Burden In mice, skin microbes can: 750 Tune level of activation & function of skin-resident 500 T cells CD4 GF+S. epidermidis Promote immunity to pathogens SPF GF S.epi Naik S et al. Science 2012 Anatomical site Skin microenvironments Sexual maturity Host factors influence skin microbial Skin Microbes Host influence host skin S. epidermidis How can we modify human skin microbes? Can we sufficiently modify microbes to promote human health? Host factors **What influence is the skin microbial landscape?** skin microbial Healthy individuals influence host skin Patients with skin diseases Anatomical site Skin microenvironments Sexual maturity Host factors influence skin microbial Skin Microbes Host influence host skin S. epidermidis How can we modify human skin microbes? Can we sufficiently modify microbes to promote human health? Host factors **What influence is the skin microbial landscape?** skin microbial Healthy individuals influence host skin Patients with skin diseases Bacterial and viral skin infections Common treatments: Atopic Dermatitis (AD) Topical/systemic antimicrobials and steroids Incidence doubled in last 3 decades in industrialized countries Possible external factor Mice: skin exposure to antigens mucosal sensitization Understanding triggers of AD may allow us to modify the development of AD/atopic diseases and develop therapeutic targets 6

7 Staphylococcus spp increase during AD flares Staphylococcus spp increase during AD flares Age-matched controls Children with atopic dermatitis Age-matched controls Children with atopic dermatitis?? Kong, Oh Segre. Genome Res Kong, Oh Segre. Genome Res Hypothesis integrating AD flare, increase in Staphylococcus and decrease in microbial diversity Itchy dermatitis in Adam17 deficient mice Kong, Oh Segre. Genome Res ADAM17 (A disintegrin and metalloproteinase 17): Cell surface protein that cleaves other surface proteins (pro-tnf-a and pro-egfr) Adam17 fl/fl Sox9- Cre Kobayashi Nagao. Immunity Patients with ADAM17 deletion exhibit AD-like disease Wk 3 Adam17 deficient mice exhibit atopic dermatitis-like disease and bacterial dysbiosis WT Adam17 fl/fl Sox9-Cre Eczema Pustules S. aureus infections Wk 8 Wk 14 WT Adam17 fl/fl Sox9-Cre Blobel et al. NEJM Kobayashi et al. Immunity

8 Mean relative abundance Relative abundance 2/13/2018 Antibiotics reverse dysbiosis in Adam17 deficient mice Week 10 Week 13 No abx + abx Staphylococcus aureus exacerbates skin disease in Adam17 deficient mice + abx No abx IL-4 dermatitis Family-level: Decrease in bacterial diversity, driven by Staphylococcaceae *AcPc combined Control Baseline Flare Postflare Individual Actinobacteria Corynebacteriaceae Propionibacteriaceae Bacteroidetes Firmicutes Staphylococcacea Streptococcaceae Proteobacteria Betaproteobacteria Gammaproteobacteria Other Bacteria Which staphylococcal species were relatively increased in Flares? Byrd et al. Sci Trans Med Species-level: Staphylococci that change between flare & postflare S. epidermidis and S. aureus *AcPc combined S. aureus S. capitis S. epidermidis S. hominis F PF F PF What differed between patients with S. aureus- vs S. epidermidisassociated Flares? 46 Species-level: Predominant staphylococcal species differed based on severity of AD flares More severe flares (objscorad 45 ± 3) S. aureus-predominant Less severe flares (objscorad 31 ± 1.9) S. epidermidispredominant Are the flares driven by clonal vs proportional increases in staphylococcal strains? More severe AD flares Less severe Controls AD flares S. aureus S. epidermidis 47 Strain-level: S. aureus strains monoclonal in more severe AD flares; S. epidermidis is polyclonal S. aureus strains (based on total bacteria) Less severe AD flares More severe AD flares S. epidermidis strains (based on total S. epi) Are there distinct biological effects associated with these staphylococcal strains? 8

9 } } 2/13/2018 Primary isolates used in murine studies Some S. aureus strains induce epidermal thickening & immune cell infiltration Staphylococcal Staphylococcal topical topical association application Tested isolates 8 S. aureus from AD flares (more/less severe) 1 S. aureus control from healthy child 1 S. aureus lab strain (USA300/CA-MRSA) 3 S. epidermidis from less severe flare patients Day WT B6 Intact ear skin TSB = tryptic soy broth 8 Some S. aureus strains induce epidermal thickening & immune cell infiltration Commensal staphylococci can produce AMPs to selectively kill S. aureus Staphylococcal topical association Day Strain-level differences in epidermal alterations & immune cell infiltrate Nakatsuji T et al. Sci Transl Med 2017 Acne association with Propionibacterium acnes Similar relative abundance of P. acne in healthy individuals (n=52) vs. acne patients (n=49) Many different P. acnes strains Identified some genetic differences between healthassociated vs acne-associated Possible therapeutic strategy? Liu J et al. ISME J Tomida S. et al. mbio Fitz-Gibbon S et al. J Invest Dermatol Anatomical site Skin microenvironments Sexual maturity Disease state Host factors influence skin microbial Skin Microbes Host influence host skin S. epidermidis S. aureus P. acnes How can we modify human skin microbes? Can we sufficiently modify microbes to promote human health? Host factors **What influence is the skin microbial landscape?** skin microbial Healthy individuals influence host skin Patients with skin diseases 9

10 Human Microbiome Defining microbiome Microbiome in health & disease Non-dermatologic Cardiometabolism, Cancer Skin Potential applications Clostridium difficile colitis C. difficile colitis can follow antibiotic treatment This infectious diarrhea may be mild to severe, with 20% relapse rate Fecal Microbiota Transplant FMT = Transfer of stool/bacteria from healthy donor to another individual First reported use: 4 th century China 1950s in US without significant regulation Recent increase in use (C. difficile colitis) 80% remission rate 61 yo female Fecal bacterial after FMT 8 month h/o of diarrhea after course of antibiotics Recalcitrant Bowel movements q 15 minutes Wheelchair bound Husband was donor Solid stool within 2 days Clostridium spp. Khoruts et al. J Clin Gastroenterol FDA evaluating FMT as a drug How to ensure safety? Variability of stool Screening donors Stool preparation methods Active components? Need controlled studies Other GI disorders Fecal Microbiota Transplant Human Microbiome Defining microbiome Microbiome in health & disease Non-dermatologic Cardiometabolism, Cancer Skin Potential applications Clostridium difficile colitis 10

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