Natural World versus Laboratory World: Natural Gut Microbiota from Wild Mice Improve Host Fitness in Viral Infection and Carcinogenesis Models

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1 Natural World versus Laboratory World: Natural Gut Microbiota from Wild Mice Improve Host Fitness in Viral Infection and Carcinogenesis Models Barbara Rehermann, MD Liver Diseases Branch, NIDDK, National Institutes of Health Bethesda, Maryland, USA

2 Advantages of of the Laboratory laboratory mouse Mouse model Model Low costs Ease of genetic manipulation wide variety of inbred strains High throughput Standardized environment Discovery of basic immunological mechanisms: T cell receptor recognition, antibody diversification Identification of innate immune receptors and signaling pathways Most Nobel prizes in immunology in the past 30 years have been awarded for work involving mouse models.

3 Advantages of the laboratory mouse model Limitations of the Laboratory Mouse Model High rate of false positive results in preclinical studies Results of 28 (37%) mouse studies replicated in human trials 14 (18%) were contradicted by randomized trials 34 (45%) remain untested. Hackam et al., JAMA 2006 Anti-TNF was developed for treatment of sepsis and protected mice in preclinical studies, but not humans in clinical studies. Abraham et al., Lancet 1998

4 Research in Translation Can Animal Models of Disease Reliably Inform Human St udies? H. Bart van der Worp 1 *, David W. Howells 2, Emily S. Sena 2,3, Michelle J. Porritt 2, Sarah Rewell 2,Victoria O Collins 2, Malcolm R. Macleod 3 1 Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, Utrecht,The Netherlands, 2 National Stroke Research Institute & University of Melbourne Department of Medicine, Austin Health, Melbourne, Australia, 3 Department of Clinical Neurosciences, University of Edinburgh, Edinburgh, United Kingdom

5 Limitations of the laboratory mouse model Advantages of the laboratory mouse model Meta-Genome and Meta-Organism

6 Advantages Limitations of of the Laboratory laboratory mouse Mouse model Model Variation in the microbiome of the laboratory mouse Ivanov et al., Cell 2009 Servick, Science 2016

7 Advantages of the laboratory mouse model Reproducibility Stappenbeck and Virgin, Nature 2016

8 Advantages of the laboratory mouse model Reproducibility Stappenbeck and Virgin, Nature 2016

9 Advantages of the laboratory mouse model Reproducibility Stappenbeck and Virgin, Nature 2016

10 Advantages of the laboratory mouse model Required Information for Publication Stappenbeck and Virgin Nature 2016

11 Advantages of the laboratory mouse model What is the Reference? Germfree or antibiotic treated Lab mice in barrier facility Wild mice Mus musculus domesticus

12 Advantages of the laboratory mouse model Laboratory World

13 Advantages of the laboratory mouse model Normalized and Restrictive Environment

14 Natural World

15 Natural World

16 Advantages of the laboratory mouse model Comparative Immunology of Lab and Wild Mice Serum IgG T cell differentiation Abolins et al., Nat Commun 2016

17 Advantages of the laboratory mouse model Comparative Immunology of Lab and Wild Mice Cytokine production after in vitro stimulation of splenocytes IL-12p40 IL-13 Abolins et al., Nat Commun 2016

18 Hypothesis Laboratory mice lack host-microbe interactions that are physiologically important and found in the natural world. Question Can we learn anything from a naturally co-evolved microbiome?

19 Rosshart et al., Cell 2018

20 Mus Musculus Domesticus from Maryland are Close Relatives to Standard Laboratory Strains Mus musculus castaneus Mus musculus musculus Mus musculus domesticus

21 Mus Musculus Domesticus from Maryland are Close Relatives to Standard Laboratory Strains

22 The Lab Mouse Gut Microbiome Differs from that of their Wild-Living Kin A PC2 (6.67%) C PC1 (19.9%) B D Wild Lab Charles River Jackson Taconic owabundance

23 The Lab Mouse Gut Microbiome Differs from that of their Wild-Living Kin A PC2 (6.67%) PC2 (6.67%) C PC1 0.0 (19.9%) 0.2 PC1 (19.9%) B Wild Lab Charles River Jackson Taconic owabundance D Wild Lab Charles River Jackson Taconic b d

24 The Lab Mouse Gut Microbiome Differs from That of their Wild-Living Kin D 80 High abundance phyla **** **** ** Relative abundance (%) E Relative abundance (%) Order Clostridiales Bacteroidales Campylobacterales Desulfovibironales Deferribacterales Lactobacillales Enterobacteriales Anaeroplasmatales Burkholderiales Verrucomicrobiales Other Relative abundance (%) Low abundance phyla **** **** 0 Location A Location B Location C Taconic Jackson Charles River Wild Lab 0

25 Bio-banking and Selection of Ileocecal Material

26 Transfer of Natural Gut Microbiota into Pregnant Germ-free Mice OFFSPRING Rosshart et al., Cell 2018

27 The Wild Mouse Gut Microbiome can be Maintained in a Laboratory Mouse Colony B 100 Order Relative abundance (%) Wild R F1 F2 F3 F4 WildR Lab R F1 F2 F3 F4 LabR Clostridiales Bacteroidales Campylobacterales Desulfovibironales Deferribacterales Lactobacillales Enterobacteriales Anaeroplasmatales Burkholderiales Verrucomicrobiales Other

28 Machine Learning: Indicator Species Analysis E Lab ind. OTUs Firmicutes Bateriodetes Tenericutes Verrucomicrobia Wild ind. OTUs Proteobacteria Relative abundance (%) Wild Wild mouse microbiota R F1 F2 F3 F4 Lab R F1 F2 F3 F4 Lab mouse microbiota

29 Do Natural Microbiota Promote Host Fitness? Virus Toxins, Mutagens

30 Survival Virus Model

31 Survival The Wild Mouse Gut Microbiome Confers a Survival Advantage Upon Influenza Virus Infection Weight Loss

32 Survival The Wild Mouse Gut Microbiome Confers a Survival Advantage Upon Influenza Virus Infection Weight Loss Virus Titer

33 The Wild Mouse Gut Microbiome Confers Traits that Abrogate Excessive Inflammation Lung Histology Lab LabR WildR

34 The Wild Mouse Gut Microbiome Confers Traits that Abrogate Excessive Inflammation Lung Cytokines and Chemokines Lung Histology Lab LabR WildR Lab LabR WildR Lab LabR WildR

35 Cancer Model AOM G 4 *** ** Inflammation score Lab LabR WildR

36 The Wild Mouse Gut Microbiome Protects from Colorectal Tumorigenesis

37 The Wild Mouse Gut Microbiome Protects from Colorectal Cancer Movat stain H&E stain C 250 μm Lab LabR WildR F Invasiveness score ** * Lab LabR WildR

38 Summary Chimeric Meta-Organism

39 Conclusions Animal models with natural microbiota should enable the discovery of protective mechanisms that are relevant in the natural world and absent in the laboratory. increase the predictive utility of laboratory mice for modeling complex diseases in the natural world.

40 Acknowledgements Immunology Section, Liver Diseases Branch, NIDDK Stephan Rosshart Brian Vassallo Ashli Hunter Collaborators NIAID Davide Angeletti Heather Hickman Jon Yewdell NCI Jonathan Badger Giorgio Trinchieri FDA Kazuyo Takeda University of North Carolina Andrew Morgan F. Pardo-Manuel de Villena Baylor College of Medicine Diane Hutchinson Nadim Ajami Disclosure: NIDDK license to Taconic