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Ajay S. Gulati, MD Associate Professor of Pediatrics and Pathology Division of Pediatric Gastroenterology University of North Carolina at Chapel Hill Critical.

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Presentation on theme: "Ajay S. Gulati, MD Associate Professor of Pediatrics and Pathology Division of Pediatric Gastroenterology University of North Carolina at Chapel Hill Critical."— Presentation transcript:

1 Ajay S. Gulati, MD Associate Professor of Pediatrics and Pathology Division of Pediatric Gastroenterology University of North Carolina at Chapel Hill Critical Design Issues Involved in Mouse Studies of the Intestinal Microbiota

2 Functions of the Intestinal Microbiota  Modulating host innate and adaptive immune function  Regulating energy utilization and metabolism  Enhancing epithelial responses to injury and repair  Regulating brain development and behavior “The Microbial Cloud” - Jonathan Eisen Sekirov et al. (2010) Physiol Rev 90:

3 Functions of the Intestinal Microbiota  Modulating host innate and adaptive immune function  Regulating energy utilization and metabolism  Enhancing epithelial responses to injury and repair  Regulating brain development and behavior “The Microbial Cloud” - Jonathan Eisen Sekirov et al. (2010) Physiol Rev 90: Demonstrated using mouse models

4 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism”  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Study mechanisms by which exposures regulate bacterial communities  Could anything else explain the results?  Control confounders such as breeding strategy and environmental conditions QuestionsMouse Studies

5 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism” QuestionsMouse Studies  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Study mechanisms by which exposures regulate the gut microbiota  Could anything else explain the results?  Control confounders such as breeding strategy and environmental conditions

6 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism” QuestionsMouse Studies  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Could anything else explain the results?

7 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism” QuestionsMouse Studies  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Study mechanisms by which exposures regulate the gut microbiota  Could anything else explain the results?

8 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism” QuestionsMouse Studies  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Study mechanisms by which exposures regulate the gut microbiota  Could anything else explain the results?  Control confounders such as breeding strategy and environmental conditions

9 … a healthy dose of scepticism? Hanage WP (2014) Nature 512:247-8 “Microbiome science needs a healthy dose of scepticism”  Do microbiome studies show causation or just correlation?  Subject mice to defined exposures and measure direct effects on microbial communities  What is the mechanism proposed?  Study mechanisms by which exposures regulate the gut microbiota  Could anything else explain the results?  Control confounders such as breeding strategy and environmental conditions QuestionsMouse Studies

10 Talk Outline  Intrinsic − Mouse genetic background  Legacy effect ‾ Heritability of the intestinal microbiota  Extrinsic ‾ Cage effects ‾ Housing conditions RALPH STEADMAN

11 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:  Profiled the cecal microbiota of 10 distinct mouse strains  All mice were purchased from same vendor  Analyzed using 454 sequencing of the 16S rRNA gene

12 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:

13 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:  Effect of strain was found to be significant (P = )  Accounted for 41.1% of all variation

14 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:  Effect of strain was found to be significant (P = )  Accounted for 41.1% of all variation  Effect of gender was also significant (P = 0.01)  Accounted for only 0.7% of variation in the data

15 Reciprocal Colonization of Germ-Free Mice Gulati et al (2012) PLoS One 7:e32403

16 Reciprocal Colonization of Germ-Free Mice  Stool was collected at 2, 4, and 12 weeks after colonization  Mice were sacrificed at 12 weeks for tissue procurement  Microbiota profiled using 454 sequencing of 16S rRNA gene Gulati et al (2012) PLoS One 7:e32403

17 Reciprocal Colonization of Germ-Free Mice 1.Ensured that mice from different backgrounds are exposed to the same initial bacteria at colonization 2.Allowed us to characterize the establishment of bacterial communities over time Gulati et al (2012) PLoS One 7:e32403

18 Mouse Background Drives Composition of the Intestinal Microbiota GF C57BL/6 Mice GF 129/SvEv Mice GF 129/SvEv Mice GF C57BL/6 Mice (B6→B6)(B6→129)(129→129) Stool From SPF C57BL/6 Mice Stool From SPF 129/SvEv Mice (129→B6) ILEAL MICROBIOTA (12 weeks post-colonization)  If donor stool is driving microbial composition, the solid squares will cluster independently from the open squares  If recipient background strain is driving microbial composition, the red squares will cluster independently from the blue squares Gulati et al (2012) PLoS One 7:e PC2 PC1

19 Mouse Background Drives Composition of the Intestinal Microbiota GF C57BL/6 Mice GF 129/SvEv Mice GF 129/SvEv Mice GF C57BL/6 Mice (B6→B6)(B6→129)(129→129) Stool From SPF C57BL/6 Mice Stool From SPF 129/SvEv Mice (129→B6) ILEAL MICROBIOTA (12 weeks post-colonization)  If donor stool is driving microbial composition, the solid squares will cluster independently from the open squares  If recipient background strain is driving microbial composition, the red squares will cluster independently from the blue squares Gulati et al (2012) PLoS One 7:e P = Recipients B6 Recipients PC2 PC1

20 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC1 Donor B6 129 B6 129 Recip 129 B6 129 Recipients Fecal Microbiota (2 weeks)

21 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC1 Fecal Microbiota (2 weeks) Donor B6 129 B6 129 Recip 129 B6 129 Recipients B6 Recipients 129 stool B6 stool

22 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC PC2 PC1 Fecal Microbiota (4 weeks) Donor B6 129 B6 129 Recip 129 B6 129 Recipients B6 Recipients 129 stool B6 stool B6 Fecal Microbiota (2 weeks)

23 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC PC2 PC1 Donor B6 129 B6 129 Recip 129 B6 129 Recipients B6 Recipients 129 stool B6 stool B PC2 PC1 Fecal Microbiota (12 weeks) 129 Recipients B6 Recipients Fecal Microbiota (4 weeks) Fecal Microbiota (2 weeks)

24 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC PC2 PC1 Donor B6 129 B6 129 Recip 129 B6 129 Recipients B6 Recipients 129 stool B6 stool B PC2 PC1 Fecal Microbiota (12 weeks) 129 Recipients B6 Recipients Fecal Microbiota (4 weeks) Fecal Microbiota (2 weeks)  Mouse background dictates the rate at which microbial homeostasis is achieved after colonization

25 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC PC2 PC1 Donor B6 129 B6 129 Recip 129 B6 129 Recipients B6 Recipients 129 stool B6 stool B PC2 PC1 Fecal Microbiota (12 weeks) 129 Recipients B6 Recipients Fecal Microbiota (4 weeks) Fecal Microbiota (2 weeks)  Mouse background dictates the rate at which microbial homeostasis is achieved after colonization  Mouse background influences the individual variability within a given strain

26 Maternal Effects on the Gut Microbiota Friswell MK et al (2010) PLoS One 5:e8584 BDF1 Female Agouti Embryos C57BL/6 Embryos

27 Maternal Effects on the Gut Microbiota Friswell MK et al (2010) PLoS One 5:e8584 BDF1 Female Agouti Embryos C57BL/6 Embryos Fecal bacterial communities of offspring were indistinguishable from one another

28 Legacy Effect × WT × KO WT KO Homozygous Breeding

29 Legacy Effect × WT × KO WT KO Homozygous Breeding  Differences between WT and KO mice may be due to the gene of interest  Random differences between the founders that are transmitted to their progeny by the legacy effect OR

30 Legacy Effect × WT × KO WT KO Homozygous Breeding  Differences between WT and KO mice may be due to the gene of interest  Random differences between the founders that are transmitted to their progeny by the legacy effect OR

31 Legacy Effect Het × WT KO Het Heterozygous Breeding  Ensures that littermates are exposed to the same microbiota  Differences between WT and KO mice are likely due to the gene of interest

32 The Role of Nod2 in Regulating the Intestinal Microbiota Rehman et al (2011) Gut 60: Nod2 KO Wild-type × WT × KO WT KO Homozygous Breeding Nod2 genotype had a profound influence on fecal microbial composition

33 The Role of Nod2 in Regulating the Intestinal Microbiota PCo2 PCo1 Shanahan et al (2014) Gut 63: Nod2 KO Wild-type PCo1 versus PCo2 Nod2 genotype had no effect on fecal microbial composition Het × WT KO Het Heterozygous Breeding

34 Similar findings have been shown for other bacterial ligand receptors including TLR- 2,4,5,9 (Ubeda et al, JEM, 2012) The Role of Nod2 in Regulating the Intestinal Microbiota PCo2 PCo1 Shanahan et al (2014) Gut 63: Nod2 KO Wild-type PCo1 versus PCo2 Het × WT KO Het Heterozygous Breeding Nod2 genotype had no effect on fecal microbial composition

35 PCo2 PCo1 Nod2 KO Wild-type PCo1 versus PCo2 Cage Effects Have a Stronger Impact on the Intestinal Microbiota than Nod2 Genotype Shanahan et al (2014) Gut 63: Het × WT KO Het Heterozygous Breeding Mice colored by the cage they were housed in

36 PCo2 PCo1 Nod2 KO Wild-type PCo1 versus PCo2 Cage Effects Have a Stronger Impact on the Intestinal Microbiota than Nod2 Genotype Shanahan et al (2014) Gut 63: Principal Coordinate P Value *2.35x *1.87x *2.41x One-Way ANOVA * Cage was a significant driver for 3 of the first 5 principal coordinates

37 Cage Effects on Gut Microbial Composition McCafferty et al (2013) ISME J 7: Germ-Free Specific Pathogen Free (SPF)  At least 4 cages were used for each group  Stool samples were collected at 1, 2, 4, 8 weeks after transfer  Processed for 16S rRNA sequencing Gavaged with the fecal microbiota pooled from 4 SPF mice Allowed to naturally acquire a microbiota over time Gavaged (n = 12) Acquired (n = 12 )  Cage effects developed in both groups by 4 weeks post- colonization  Even after being gavaged with the same microbiota, cage effects still develop over time

38  In addition to the micro-environment of an individual cage, the macro-environment that mice are housed in can also profoundly impact the intestinal microbiota Impact of Housing Conditions on the Intestinal Microbiota Liu et al (2013) Am J Physiol GI Liv 305:G573-G584 Hemoccult Index DSS Duration (days) * Irgm1 KO Wild-type Conventional Housing

39  In addition to the micro-environment of an individual cage, the macro-environment that mice are housed in can also profoundly impact the intestinal microbiota Impact of Housing Conditions on the Intestinal Microbiota Liu et al (2013) Am J Physiol GI Liv 305:G573-G584 Hemoccult Index DSS Duration (days) * Irgm1 KO Wild-type Conventional Housing Hemoccult Index DSS Duration (days) Specific Pathogen Free Housing Irgm1 KO Wild-type

40 Impact of Housing Conditions on the Intestinal Microbiota  No differences in fecal microbiota between wild-type (WT) and Irgm1 knock-out (KO) mice Irgm1 KO vs. WT Fecal Microbiota

41 Impact of Housing Conditions on the Intestinal Microbiota  No differences in fecal microbiota between wild-type (WT) and Irgm1 knock-out (KO) mice  Clear separation between mice raised in conventional (CV) versus specific pathogen free (SPF) conditions Irgm1 KO vs. WT Fecal Microbiota

42 Impact of Housing Conditions on the Intestinal Microbiota Irgm1 KO vs. WT Fecal Microbiota * p = 2.61x10 -9 Helicobacter genus

43 Impact of Housing Conditions on the Intestinal Microbiota Irgm1 KO vs. WT Fecal Microbiota * p = 2.61x10 -9 Helicobacter genus  Now in the process of inoculating WT and KO SPF mice with H. hepaticus to determine if this induces DSS-susceptibility H. hepaticus

44 Summary of Key Issues That Can Impact Mouse Studies of the Intestinal Microbiota 1.Mouse genetic background profoundly influences the gut microbiota  Bacterial composition  Establishment of bacterial communities over time  Individual microbial variability between mice

45 Summary of Key Issues That Can Impact Mouse Studies of the Intestinal Microbiota 1.Mouse genetic background profoundly influences the gut microbiota 2.The intestinal microbiota is heritable  Homozygous versus heterozygous breeding strategies can influence how the microbiota develops within a given mouse colony

46 Summary of Key Issues That Can Impact Mouse Studies of the Intestinal Microbiota 1.Mouse genetic background profoundly influences the gut microbiota 2.The intestinal microbiota is heritable 3.The local cage environment strongly impacts the gut microbiota  Relevant even in experiments which expose mice to the same initial microbial colonization

47 1.Mouse genetic background profoundly influences the gut microbiota 2.The intestinal microbiota is heritable 3.The local cage environment strongly impacts the gut microbiota 4.Housing conditions can significantly affect mouse phenotype and intestinal microbial composition  Offers the ability to identify specific organisms relevant to disease pathogenesis Summary of Key Issues That Can Impact Mouse Studies of the Intestinal Microbiota

48 Considerations for Experimental Design 1.Control what is feasible to control  Includes genetic background, housing conditions, age, sex, diet  Many of these are difficult to control in human studies, but can be regulated in mouse experiments 2.Record metadata for factors that are difficult to control  Cage, litter, mother – these can be adjusted for statistically in downstream analyses  Initial power analyses should consider not only the numbers of mice per group, but also the number of cages and litters per group 3.Attempt to control initial microbial exposure  Heterozygous breeding  Colonization of germ-free mice  Co-housing/co-fostering

49 Considerations for Experimental Design 1.Control what is feasible to control  Includes genetic background, housing conditions, age, sex, diet  Many of these are difficult to control in human studies, but can be regulated in mouse experiments 2.Record metadata for factors that are difficult to control  Cage, litter, mother – these can be adjusted for statistically in downstream analyses  Initial power analyses should consider not only the numbers of mice per group, but also the number of cages and litters per group 3.Attempt to control initial microbial exposure  Heterozygous breeding  Colonization of germ-free mice  Co-housing/co-fostering

50 Considerations for Experimental Design 1.Control what is feasible to control  Includes genetic background, housing conditions, age, sex, diet  Many of these are difficult to control in human studies, but can be regulated in mouse experiments 2.Record metadata for factors that are difficult to control  Cage, litter, mother – these can be adjusted for statistically in downstream analyses  Initial power analyses should consider not only the numbers of mice per group, but also the number of cages and litters per group 3.Attempt to control initial microbial exposure  Heterozygous breeding  Colonization of germ-free mice  Co-housing/co-fostering

51 Conclusions  Because we can address causation and study mechanism, mouse models are a powerful tool to study the intestinal microbiota  However, we do have to consider careful experimental design and control confounders to draw appropriate conclusions from these studies

52 Conclusions  Because we can address causation and study mechanism, mouse models are a powerful tool to study the intestinal microbiota  However, we do have to consider careful experimental design and control confounders to draw appropriate conclusions from these studies  Numerous thorough reviews have been published on this topic: − Spor et al. (2011) Nat Rev Microbiol, 9: − Kostic et al. (2013) Genes Dev, 369:

53 Acknowledgements Balfour Sartor Alexi Schoenborn Michael Shanahan Greg Taylor Raad Gharaibeh Anthony Fodor Ian Carroll Allison Rogala Bo Liu Richard von Furstenberg Susan Henning Christian Jobin Janelle Arthur Maureen Bower Lisa Holt Funding K08DK (Gulati) KL2RR (Gulati) CCFA/CDHNF Career Development Award (Gulati) Global Probiotics Council Young Investigator Grant for Probiotic Research (Gulati) P30 DK34987 (CGIBD) National Gnotobiotic Rodent Resource Center P40R (Sartor) Cores UNC Gnotobiotic Core (CGIBD) Cell Service & Histology Core (CGIBD) Microbiome Core (CGIBD)

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56 The Role of Nod2 in Regulating the Intestinal Microbiota Rehman et al (2011) Gut 60: Nod2 KO Wild-type × WT × KO WT KO Homozygous Breeding  Nod2 genotype had a profound influence on fecal microbial composition

57 WT Nod2 -/- B. vulgatus cfu N.D B. vulgatus cfu WT Nod2 -/- Days post separation **** ** The Role of Nod2 in Regulating the Intestinal Microbiota Ramanan et al (2014) Immunity 41:311-24

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59 The Microbial Cloud

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61 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:  Variability of the microbiota from mouse-to-mouse also depends on background strain  Circled strains have tight clustering of biological replicates (BL6J, C3HRI, DBAJR, PWK, WSB)  Others have greater individual variation (129S1, AJ, CAST, NOD, NZO)

62 Reciprocal Colonization of Germ-Free Mice GF C57BL/6 Mice GF 129/SvEv Mice GF 129/SvEv Mice GF C57BL/6 Mice (B6→B6)(B6→129)(129→129) Stool From SPF C57BL/6 Mice Stool From SPF 129/SvEv Mice (129→B6) CECAL CONTENTS (12 weeks post-colonization)  If donor stool is driving microbial composition, the solid squares will cluster independently from the open squares  If recipient background strain is driving microbial composition, the red squares will cluster independently from the blue squares  Samples segregate based on background strain of recipient mice (p < 0.05)

63 Mouse Background Directs Ecological Succession of Gut Microbiota After Colonization PC2 PC1 2 weeks Post-colonization Donor B6 129 B6 129 Recip 129 B6

64 Mouse Background Drives Composition of the Intestinal Microbiota GF C57BL/6 Mice GF 129/SvEv Mice GF 129/SvEv Mice GF C57BL/6 Mice (B6→B6)(B6→129)(129→129) Stool From SPF C57BL/6 Mice Stool From SPF 129/SvEv Mice (129→B6) FECAL MICROBIOTA (12 weeks post-colonization)  If donor stool is driving microbial composition, the solid squares will cluster independently from the open squares  If recipient background strain is driving microbial composition, the red squares will cluster independently from the blue squares  Samples segregate based on background strain of recipient mice (p < ) PC2 PC1 129 Recipients B6 Recipients Gulati et al (2012) PLoS One 7:e32403

65 Legacy Effect × WT × KO WT KO Offspring can be re-bred to generate additional mice for downstream analysis Het × WT KO Het Homozygous Breeding Heterozygous Breeding Hets re-bred to generate additional littermates WT and KO mice used for analysis

66 Cage Effects on Gut Microbial Composition Germ-Free Specific Pathogen Free (SPF) Gavaged (n = 12) Acquired (n = 12 )  Cage effects developed in both groups by 4 weeks post- colonization  Even after being gavaged with the same microbiota, cage effects still develop over time McCafferty et al (2013) ISME J 7:

67 1.Mouse genetic background profoundly influences the gut microbiota  Includes composition, but also the development of bacterial communities over time, and the individual microbial variability between mice 2.The intestinal microbiota is heritable  Homozygous versus heterozygous breeding strategies can dictate how the microbiota develops within a given mouse colony 3.The local cage environment strongly impacts the gut microbiota  Relevant even in experiments which expose mice to the same initial microbial colonization 4.Housing conditions can significantly affect phenotype and intestinal microbial composition  Offers the ability the identify specific organisms relevant to disease pathogenesis Summary of Key Issues That Can Impact Mouse Studies of the Intestinal Microbiota

68 Conclusions “When aware of the environmental and legacy effects in mouse models and their implications for microbiome experimental design, mice can still be a stalwart tool for unraveling mechanisms of host- microbiota interactions relevant to humans.”

69 Impact of Mouse Genetic Background on the Gut Microbiota Campbell JH et al (2012) ISME J 6:  Analyzed the cecal microbiota of 10 distinct mouse strains using 454 sequencing of the 16S rRNA gene  8 of these strains are currently being used to develop the Collaborative Cross  All are derived from these original 8 lines, which have their genomes sequenced  Will allow investigators to link specific genetic loci to phenotypic traits, and patterns of microbial colonization


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