Mammals are defined by their metagenome, a combination of host and microbiome genes. This knowledge presents opportunities to further basic biology with translation to human diseases. However, the now-documented influence of the metagenome on experimental results and the reproducibility of in vivo mammalian models present new challenges. Here we provide the scientific basis for calling on all investigators, editors and funding agencies to embrace changes that will enhance reproducible and interpretable experiments by accounting for metagenomic effects. Implementation of new reporting and experimental design principles will improve experimental work, speed discovery and translation, and properly use substantial investments in biomedical research.
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Brodin, P. et al. Variation in the human immune system is largely driven by non-heritable influences. Cell 160, 37–47 (2015)
Lee, M. N. et al. Common genetic variants modulate pathogen-sensing responses in human dendritic cells. Science 343, 1246980 (2014)
Norman, J. M., Handley, S. A. & Virgin, H. W. Kingdom-agnostic metagenomics and the importance of complete characterization of enteric microbial communities. Gastroenterology 146, 1459–1469 (2014)
Moon, C. & Stappenbeck, T. S. Viral interactions with the host and microbiota in the intestine. Curr. Opin. Immunol. 24, 405–410 (2012)
Hooper, L. V., Littman, D. R. & Macpherson, A. J. Interactions between the microbiota and the immune system. Science 336, 1268–1273 (2012)
Virgin, H. W. The virome in mammalian physiology and disease. Cell 157, 142–150 (2014)
Beck, J. M. et al. Multicenter comparison of lung and oral microbiomes of HIV-infected and HIV-uninfected individuals. Am. J. Respir. Crit. Care Med. 192, 1335–1344 (2015)
Anahtar, M. N. et al. Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract. Immunity 42, 965–976 (2015)
Oh, J. et al. Biogeography and individuality shape function in the human skin metagenome. Nature 514, 59–64 (2014)
MacDuff, D. A. et al. Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection. eLife 4, (2015)
Virgin, H. W. & Todd, J. A. Metagenomics and personalized medicine. Cell 147, 44–56 (2011)
Moon, C. et al. Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. Nature 521, 90–93 (2015).Demonstrates that indigenous intestinal bacteria dominantly influence phenotypes through degradation of fecal IgA and emphasizes the need for control of the metagenome and microbiome in mouse experiments.
Bloom, S. M. et al. Commensal Bacteroides species induce colitis in host-genotype-specific fashion in a mouse model of inflammatory bowel disease. Cell Host Microbe 9, 390–403 (2011)
Cadwell, K. et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell 141, 1135–1145 (2010).With ref. 13 , experimental proof of concept that one cannot only separately consider the effects of host genes or microbiome genes on mouse phenotypes; one must consider the entire metagenome including bacteria and viruses.
Basic, M. et al. Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10-deficient mice. Inflamm. Bowel Dis. 20, 431–443 (2014)
Metchnikoff, O. Life of Elie Metchnikoff 1845–1916. (Houghton Mifflin Company, 1921)
Wang, Z. et al. Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell 163, 1585–1595 (2015).Demonstrates the concept that intestinal metabolites can have dramatic effects on distant organ systems (here blood vessels) and emphasize that the effect of the metagenome and microbiome must be considered in all mouse experiments.
Gevers, D. et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 15, 382–392 (2014)
Reyes, A., Wu, M., McNulty, N. P., Rohwer, F. L. & Gordon, J. I. Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut. Proc. Natl Acad. Sci. USA 110, 20236–20241 (2013)
Norman, J. M. et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 160, 447–460 (2015)
Haberman, Y. et al. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J. Clin. Invest. 124, 3617–3633 (2014)
Handley, S. A. et al. Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome. Cell 151, 253–266 (2012)
Mutlu, E. A. et al. A compositional look at the human gastrointestinal microbiome and immune activation parameters in HIV infected subjects. PLoS Pathog. 10, e1003829 (2014)
Handley, S. A. et al. SIV infection-mediated changes in gastrointestinal bacterial microbiome and virome are associated with immunodeficiency and prevented by vaccination. Cell Host Microbe 19, 323–335 (2016)
Monaco, C. L. et al. Altered virome and bacterial microbiome in human immunodeficiency virus-associated acquired immunodeficiency syndrome. Cell Host Microbe 19, 311–322 (2016)
Scher, J. U. et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife 2, e01202 (2013)
Reyes, A. et al. Gut DNA viromes of Malawian twins discordant for severe acute malnutrition. Proc. Natl Acad. Sci. USA 112, 11941–11946 (2015)
Smith, M. I. et al. Gut microbiomes of Malawian twin pairs discordant for kwashiorkor. Science 339, 548–554 (2013)
Ridaura, V. K. et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341, 1241214 (2013)
Jenq, R. R. et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J. Exp. Med. 209, 903–911 (2012)
Davis-Richardson, A. G. et al. Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes. Front. Microbiol. 5, 678 (2014)
Khosravi, A. et al. Gut microbiota promote hematopoiesis to control bacterial infection. Cell Host Microbe 15, 374–381 (2014)
Hsiao, E. Y. et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463 (2013).With refs 34, 35, 36 , demonstrates the need to consider the effects of the metagenome and microbiome of all organs including the brain.
Yano, J. M. et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161, 264–276 (2015)
Erny, D. et al. Host microbiota constantly control maturation and function of microglia in the CNS. Nat. Neurosci. 18, 965–977 (2015)
Sampson, T. R. & Mazmanian, S. K. Control of brain development, function, and behavior by the microbiome. Cell Host Microbe 17, 565–576 (2015)
Vétizou, M. et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 350, 1079–1084 (2015)
Sjogren, K. et al. The gut microbiota regulates bone mass in mice. J. Bone Miner. Res. 27, 1357–1367 (2012)
De Vlaminck, I. et al. Temporal response of the human virome to immunosuppression and antiviral therapy. Cell 155, 1178–1187 (2013)
Arpaia, N. et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451–455 (2013).With refs 41, 42, 43 and 47 , shows the diverse effects and mechanisms of microbial metabolites (here short chain fatty acids) and emphasizes that mechanisms underlying the metagenome and microbiome require investigation in multiple cell types.
Atarashi, K. et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500, 232–236 (2013)
Sefik, E. et al. Individual intestinal symbionts induce a distinct population of RORγ+ regulatory T cells. Science (2015)
Smith, P. M. et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569–573 (2013)
Kernbauer, E., Ding, Y. & Cadwell, K. An enteric virus can functionally replace the beneficial cues provided by commensal bacteria. Nature 516, 94–98 (2014)
Reinhardt, C. et al. Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling. Nature 483, 627–631 (2012)
Stappenbeck, T. S., Hooper, L. V. & Gordon, J. I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl Acad. Sci. USA 99, 15451–15455 (2002)
Kaiko, G. E. et al. The colonic crypt protects stem cells from microbiota-derived metabolites. Cell 165, 1–13 (2016)
Osborne, L. C. et al. Virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation. Science 345, 578–582 (2014)
Reese, T. A. et al. Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter. Science 345, 573–577 (2014).Emphasizes that phenotypes driven by the metagenome can be polymicrobial and are not exclusively bacterial; here viral and protozoan interactions are important.
Wang, Z. et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 472, 57–63 (2011)
Arrieta, M. C. et al. Early infancy microbial and metabolic alterations affect risk of childhood asthma. Sci. Transl. Med. 7, 307ra152 (2015)
Thorburn, A. N. et al. Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites. Nat. Commun. 6, 7320 (2015).With ref. 51 , shows that intestinal metabolites can affect disease pathogenesis in distant organs (here asthma models in the lung).
Buffie, C. G. et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205–208 (2015)
Reese, T. A. et al. Sequential infection with common pathogens promotes human-like immune gene expression and altered vaccine response. Cell Host Microbe 19, 1–7 (2016).This report emphasizes the history of microbial interactions with an organism additionally shapes phenotypes and this needs to be modelled in mouse experiments.
McCafferty, J. et al. Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model. ISME J. 7, 2116–2125 (2013)
Pantoja-Feliciano, I. G. et al. Biphasic assembly of the murine intestinal microbiota during early development. ISME J. 7, 1112–1115 (2013)
Farkas, A. M. et al. Induction of Th17 cells by segmented filamentous bacteria in the murine intestine. J. Immunol. Methods 421, 104–111 (2015)
Campbell, J. H. et al. Host genetic and environmental effects on mouse intestinal microbiota. ISME J. 6, 2033–2044 (2012)
Yurkovetskiy, L. et al. Gender bias in autoimmunity is influenced by microbiota. Immunity 39, 400–412 (2013)
Markle, J. G. et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 339, 1084–1088 (2013)
Langille, M. G. et al. Microbial shifts in the aging mouse gut. Microbiome 2, 50 (2014)
Mukherji, A., Kobiita, A., Ye, T. & Chambon, P. Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs. Cell 153, 812–827 (2013)
Thaiss, C. A. et al. Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis. Cell 159, 514–529 (2014)
McInnes, E. F. et al. Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period. Lab Anim. (NY) 40, 341–350 (2011)
Karp, C. L. Unstressing intemperate models: how cold stress undermines mouse modeling. J. Exp. Med. 209, 1069–1074 (2012)
Gaskill, B. N. et al. Heat or insulation: behavioral titration of mouse preference for warmth or access to a nest. PLoS One 7, e32799 (2012)
Huang, E. Y. et al. Using corticosteroids to reshape the gut microbiome: implications for inflammatory bowel diseases. Inflamm. Bowel Dis. 21, 963–972 (2015)
Chevalier, C. et al. Gut microbiota orchestrates energy homeostasis during cold. Cell 163, 1360–1374 (2015)
Wolf, K. J. et al. Consumption of acidic water alters the gut microbiome and decreases the risk of diabetes in NOD mice. J. Histochem. Cytochem. 62, 237–250 (2014)
Hall, J. E., White, W. J. & Lang, C. M. Acidification of drinking water: its effects on selected biologic phenomena in male mice. Lab. Anim. Sci. 30, 643–651 (1980)
Carmody, R. N. et al. Diet dominates host genotype in shaping the murine gut microbiota. Cell Host Microbe 17, 72–84 (2015).One example of many cited here that shows in detail the potential rapid effects on environmental influences such as diet on the composition of the microbiome and emphasizes the need to report environmental variables in all experiments.
Ijssennagger, N. et al. Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon. Proc. Natl Acad. Sci. USA 112, 10038–10043 (2015)
Zenewicz, L. A. et al. IL-22 deficiency alters colonic microbiota to be transmissible and colitogenic. J. Immunol. 190, 5306–5312 (2013)
Elinav, E. et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145, 745–757 (2011)
Garrett, W. S. et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 131, 33–45 (2007)
Olszak, T. et al. Microbial exposure during early life has persistent effects on natural killer T cell function. Science 336, 489–493 (2012)
Cox, L. M. et al. Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences. Cell 158, 705–721 (2014).With ref. 76 , emphasizes the early life effects of the microbiota that must be considered in experimental design to control for the metagenome and microbiome.
Trompette, A. et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat. Med. 20, 159–166 (2014)
Sun, J. et al. Pancreatic β-cells limit autoimmune diabetes via an immunoregulatory antimicrobial peptide expressed under the influence of the gut Microbiota. Immunity 43, 304–317 (2015)
Abt, M. C. et al. Commensal bacteria calibrate the activation threshold of innate antiviral immunity. Immunity 37, 158–170 (2012)
Ichinohe, T. et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc. Natl Acad. Sci. USA 108, 5354–5359 (2011)
Ryu, S. H. & Stappenbeck, T. S. Gut–pancreatic axis AMPlified in islets of Langerhans. Immunity 43, 216–218 (2015)
Levy, M. et al. Microbiota-modulated metabolites shape the intestinal microenvironment by regulating NLRP6 inflammasome signaling. Cell 163, 1428–1443 (2015)
Eberl, G. Addressing the experimental variability associated with the microbiota. Mucosal Immunol. 8, 487–490 (2015)
Macpherson, A. J. & McCoy, K. D. Standardised animal models of host microbial mutualism. Mucosal Immunol. 8, 476–486 (2015).Excellent review on the methodology and use of isobiotic mice in experiments.
Goodman, A. L. et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257 (2011)
Atkinson, M. A. & Chervonsky, A. Does the gut microbiota have a role in type 1 diabetes? Early evidence from humans and animal models of the disease. Diabetologia 55, 2868–2877 (2012)
Pozzilli, P., Signore, A., Williams, A. J. & Beales, P. E. NOD mouse colonies around the world–recent facts and figures. Immunol. Today 14, 193–196 (1993)
Turnbaugh, P. J. et al. The human microbiome project. Nature 449, 804–810 (2007)
Wheeler, M. L. & Underhill, D. M. Time to cast a larger net. Nat. Immunol. 15, 1000–1001 (2014)
Kostic, A. D. et al. The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes. Cell Host Microbe 17, 260–273 (2015)
Hajishengallis, G. et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 10, 497–506 (2011).With ref. 93 , shows that microbiome sequence analysis alone cannot always account for mouse phenotypes; these are examples where low abundance microbes and microbial particles drive phenotypes that would both be missed in sequence analysis.
Hickey, C. A. et al. Colitogenic Bacteroides thetaiotaomicron antigens access host immune cells in a sulfatase-dependent manner via outer membrane vesicles. Cell Host Microbe 17, 672–680 (2015)
Segata, N. et al. Metagenomic microbial community profiling using unique clade-specific marker genes. Nat. Methods 9, 811–814 (2012)
Whitman, W. B., Coleman, D. C. & Wiebe, W. J. Prokaryotes: the unseen majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998)
Virgin, H. W., Wherry, E. J. & Ahmed, R. Redefining chronic viral infection. Cell 138, 30–50 (2009)
Young, G. R. et al. Resurrection of endogenous retroviruses in antibody-deficient mice. Nature 491, 774–778 (2012)
Ives, A. et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 331, 775–778 (2011)
Underhill, D. M. & Iliev, I. D. The mycobiota: interactions between commensal fungi and the host immune system. Nat. Rev. Immunol. 14, 405–416 (2014)
Fletcher, S. M., Stark, D., Harkness, J. & Ellis, J. Enteric protozoa in the developed world: a public health perspective. Clin. Microbiol. Rev. 25, 420–449 (2012)
Stelekati, E. & Wherry, E. J. Chronic bystander infections and immunity to unrelated antigens. Cell Host Microbe 12, 458–469 (2012)
Pfeiffer, J. K. & Virgin, H. W. Viral immunity. Transkingdom control of viral infection and immunity in the mammalian intestine. Science 351, 239–245 (2016)
Dantas, G. & Sommer, M. O. Context matters – the complex interplay between resistome genotypes and resistance phenotypes. Curr. Opin. Microbiol. 15, 577–582 (2012)
Stern, A. & Sorek, R. The phage–host arms race: shaping the evolution of microbes. BioEssays 33, 43–51 (2011)
Brüssow, H., Canchaya, C. & Hardt, W.-D. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 68, 560–602 (2004)
Fortier, L.-C. & Sekulovic, O. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 4, 354–365 (2013)
Zhang, B. et al. Viral infection. Prevention and cure of rotavirus infection via TLR5/NLRC4-mediated production of IL-22 and IL-18. Science 346, 861–865 (2014)
Baldridge, M. T. et al. Commensal microbes and interferon-λ determine persistence of enteric murine norovirus infection. Science 347, 266–269 (2015)
Robinson, C. M., Jesudhasan, P. R. & Pfeiffer, J. K. Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus. Cell Host Microbe 15, 36–46 (2014)
Kuss, S. K. et al. Intestinal microbiota promote enteric virus replication and systemic pathogenesis. Science 334, 249–252 (2011)
Kane, M. et al. Successful transmission of a retrovirus depends on the commensal microbiota. Science 334, 245–249 (2011)
Barton, E. S. et al. Herpesvirus latency confers symbiotic protection from bacterial infection. Nature 447, 326–329 (2007)
Furman, D. et al. Cytomegalovirus infection enhances the immune response to influenza. Sci. Transl. Med. 7, 281ra43 (2015)
Oh, J. Z. et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity 41, 478–492 (2014)
Williams, W. B. et al. HIV-1 vaccines. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies. Science 349, aab1253 (2015)
Nam, Y.-D. et al. Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people. J. Microbiol. 46, 491–501 (2008)
Scanlan, P. D. & Marchesi, J. R. Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces. ISME J. 2, 1183–1193 (2008)
Elliott, D. E. & Weinstock, J. V. Helminth–host immunological interactions: prevention and control of immune-mediated diseases. Ann. NY Acad. Sci. 1247, 83–96 (2012)
Hoffmann, C. et al. Archaea and fungi of the human gut microbiome: correlations with diet and bacterial residents. PLoS One 8, e66019 (2013)
Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. J. Pharmacol. Pharmacother. 1, 94–99 (2010)
The authors declare no competing financial interests.
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Stappenbeck, T., Virgin, H. Accounting for reciprocal host–microbiome interactions in experimental science. Nature 534, 191–199 (2016). https://doi.org/10.1038/nature18285
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