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Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program Environmental Microbial and Mammalian Biomolecular Responses to AhR Ligands Department of Civil and Environmental Engineering Center for Microbial Ecology Risk e-Learning Webinar Series The Interplay Between Environmental Exposures and Infectious Agents October 31, 2016

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Page 1: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

GutMicrobiome: Toxicant Perturbation and Stability

Syed Hashsham

Superfund Research Program Environmental Microbial and Mammalian Biomolecular

Responses to AhR Ligands

Department of Civil and Environmental Engineering Center for MicrobialEcology

Risk e-Learning Webinar Series The Interplay Between Environmental Exposures and Infectious Agents

October 31, 2016

Page 2: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

         

     

Outline

A. GutMicrobiome • Background and Objective • Treg/Th17 system, TCDD, and SFB • Hypothesis and Experimental Details • Results

B. Key Challenges Ahead • Communication channels • Predictive capabilities • Markers/Gutchips • Interventions

C. Summary

Page 3: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

INTERVENTIONS Fecal Transplant Psychobiotics

Pre- and Probiotics Antibiotics Food Habits

CRISPR Phage

Gut Microbiome: Diseases & Interventions ORGANS Brain Mouth Heart Kidney Liver

Immune System Colon Tissue Muscle

Toxicants

Commensals Keystone Species

Opportunistic Pathogens Pathobionts Pathogens

Host

Gut Microbiome ENVRIONMENAL

EXPOSURE

DISEASES Allergies Asthma Anxiety Autism

Autoimmune Cardiovascular

Crohn’s Depression

IBD Mood Disorder

Migraine Multiple Sclerosis

NAFLD Obesity

Parkinson's Spinal Cord Injury

Stroke Type 2 Diabetes

Page 4: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Objective: Characterize the effect of specific gut microbiome members on Treg/Th17 System with and without TCDD

Segmented Filamentous Bacteria (SFB) Bacteroides fragilis 2,3,7,8-TCDD

Treg/Th17 (Host)

(Toxicant) (Key Gut Members)

Page 5: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

IL-6

Treg Foxp3+

IL-6

Th17 RORγt

With TCDD, AhR promotes Treg and suppresses Th17

Naïve T cells

Th17 Treg aids in antimicrobial

modulates and response; abrogates also causes

autoimmune inflammation& disease autoimmune disease

Arylhydrocarbon Receptor (AhR)

IL-17 IL-22 IL-10

2,3,7,8-TCDD

Page 6: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

   

   

Why Segmented Filamentous Bacteria (SFB)? • Obligate symbiont • No genes for amino acids, vitamins/cofactors, nucleic acids • Extensive auxotroph • Host-specific

SFB in humans? Candidatus Savagella | Environmental Microbiology 14 (6): 1462-2920 | 2012

SFB cultivation is nowpossibleusing TC7celllines (BioTechniques, 59 (2):94–98, 2015 2015 Dig Dis Sci. 60(10): 2953-62

SFB in patients by qPCR.Less in IBD constipated, and more in IBD diarrhea.

2013 Yin et al., ISME Journal

251 humans: majority colonized betweenages2 to3

2013 Hans Jonsson

2009 Snel et al. Ivanov et al., Cell: 2009

Page 7: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Segmented Filamentous Bacteria (SFB)

Clostridia B. fragilis Polysaccharide A

Short Chain Fatty Acids

Lamina propria Butyrate

Dendritic cells

Gut

SFB

ArylHydrocarbon Receptor (AhR)

Th17 TregFoxp3+ RORγt

IL-10

Naïve Tcells IL-6 IL-6

IL-21R Nos2 IL-21

microRNAs

TCDD Host

Page 8: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Hypothesis TCDD exposure disrupts the Treg /Th17 system and specific gut microbialmembersarecapableofpreventingthisdisruption.

Page 9: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

SCFAPSA

SFBB.fragilisClostridia

TCDD

TCDDimpactsthehostwhichthenimpactsthegutmicrobiome

TwoPossibilities!TCDDimpactsthegutmicrobiome

whichthenimpactshost

Page 10: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

ExperimentalDetails

Traditional Gnotobiotic

C57BL6

TCDD: 0.01 to 30 µg/kg every 4 d 30 d study 120 d study (90 d + 30 d recovery) 8 per group Cage separation

TCDD: 30 µg/kg every 4 d 56 d study 4 per group • GF • SFB • B. fragilis • B. fragilis + SFB

TimZacharewski’sLab UMGerm-FreeFacility

•  mRNA expression of ileal immunology genes (nCounter: 547 Immunology gene targets)

•  T-cells in blood/spleen (Flow cytometry)

•  microRNA expression in ileum (nCounter: 600 mouse microRNAs)

•  High Throughput (Wafergen) or qPCR

Fecalpellets,cecum

Fecalpellets,ilium,cecum,blood

Keymeasurements

Page 11: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

IleumGnotobioticC57BL6:GeneExpression

ComparedtoGF,SFBhasmoreUp-regulatedgenes.

WithTCDD,SFBhasmoreDown-regulatedgenes.

WithTCDDComparedtoGF

Up-regulation Down-regulation Up-regulation Down-regulation

Page 12: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

0

200

400

600

800

1000

1200

1400

Vehicle TCDD

cd36

Spleen

0

1000

2000

3000

4000

5000

6000

7000

GF B B+SFB SFB

TGF-β

TCDDVehicle

Nor

mal

ized

cou

nt

GnotobioticC57BL6:Treg

GF B

B +S F BS F B

GF B

B + S F B S F B

1 0

1 5

2 0

2 5

% C

D4

+ T

reg

ce

lls

in

sp

lee

n

Vehicle TCDD

p<0.0001

p=0.35Colonization

TCDD

Parametrictwo-wayANOVA

Page 13: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

0

10

20

30

40

50

60

70

80

GF B B+SFB SFB

Nor

mal

ized

cou

nt

IL1-β

0

200

400

600

800

1000

1200

1400

1600

GF B B+SFB SFB

Vehicle

TCDD

Ciitawassimilar!

Nor

mal

ized

cou

nt

SpleenGnotobioticC57BL6:Th17

GF B

S F B +BS F B G

F B

S F B +BS F B

0 .0

0 .5

1 .0

1 .5

2 .0

% C

D4

+ T

h1

7 c

ell

s i

n s

ple

en

Vehicle TCDD

p=0.0004

p=0.0196

Colonization

TCDD

Parametrictwo-wayANOVA

Page 14: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

GnotobioticC57BL6:B.fragilisandSFB

B.fragilis SFB

V e h

T C D D

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

B.

fra

gil

is r

plB

ge

ne

pe

r m

g c

ae

cu

m

p=0.029

V e h

T C D D

1 1́ 0 4

4 1́ 0 4

1 1́ 0 6

2 1́ 0 6

3 1́ 0 6

4 1́ 0 6

5 1́ 0 6

6 1́ 0 6

SF

B 1

6S

rR

NA

ge

ne

co

pie

s p

er

mg

ile

um

p=0.038

Page 15: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

TraditionalC57BL6:DoseResponse&Recovery

Day38 90120

Page 16: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

TCDD

IncreaseinSFB

DecreaseinB.fragilis&Clostridia

(ExpectedchangeinSFBwasanINCREASE)(ExpectedchangeinB.

fragilis&ClostridiawasaDECREASE)

SegmentedFilamentousBacteria(SFB)

TCDD

AhR

Th17TregFoxp3+ RORγt

IL-10

NaïveTcells

IL-6 IL-6

IL-21R Nos2 IL1-β

SFB

Laminapropria

Gut

Host

Dendriticcells

B.fragilis

PolysaccharideA

Clostridia

Butyrate

OverallInteractionofTCDD,B.fragilis,andSFB

Page 17: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

TraditionalC57BL6:IncreaseinAntimicrobialResistanceGenes

-5

0

5

10

15

20

25

0.3 1 3 10 30 RT-30

Folddifferen

ce

µg/kgTCDD(LSandRSgroups)

amphenicolMDRbetalactamaseothertetracyclinesulfonamide

:acrF,mdtE,acrR,tolC:yidY

:tet(32):bacA

:ampC,blaCMY2

:folA

Page 18: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

1.  Who is there?

2.  Who is doing what and how?

3.  Can we predict gut behavior? Quantitatively?

4.  How do we know when something is wrong?

5.  How to stop or encourage key members?

Prediction

Diagnosis

Intervention

B.KeyChallengesAheadIdentity

Activity

Page 19: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

microRNAexpressioninSFB-associatedmiceismuchgreaterthanGerm-freeorB.fragilis-

associatedgroups!

2.Whoisdoingwhatandhow?

MicroRNAs may alter the gut microbiota through fecal microRNAs, affecting growth and other cellular

processes (Liu et al., 2016). Ileum

Spatial resolution Moresensitivetoworkwith1-10µlbloodAtallmolecularlevels

Ivanov et al., Cell: 2009

Page 20: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Resistance: Maximum deviation from the pre-perturbed equilibrium

Resilience: Inverse of time taken to return to equilibrium

Res

pons

e

1

ResponseEnvelopePerturbation

Time

3.Canwepredictthegutbehavior–quantitatively?

Hashshametal.,Fernandezetal.,AEM,2000

Trajectories Interactions

Stability Keystone-ness

Buccietal.GenomeBiology17:121,2016

Time series Biomass

Perturbation

GeneralizedLotaVolterraModel

Disease-specific

GutDisruptionIndexMDSINE

DeterministicProbabilistic

Halpinetal,Am.J.InfectionControl44

(2016)830-6

VREvs.E.faecium

Page 21: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Vital et al., mBio, 2014 Butyrate

Carbohydrates

Acetyl-CoA

ButyratekinaseBUK

Butyryl-CoA

Butyrateproducers

Roseburiaintestinalis

Faecalibacteriumprausnitzii….

Butyryl-CoAacetatetransferase

BUT

Markers

4a.Howdoweknowwhensomethingiswrong?

FunctionalGeneDiversityPrimerCoverage

Fishetal.,Front.Microbiol.4:2912013

Functions, Guilds

Page 22: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

GutChips

HighThroughputSequencing

4b.Howdoweknowwhensomethingiswrong?

Amplification-basedqPCRorLow-densityChips

Hybridization-basedArrays

Illumina

HuMiChip(500functionalgenes,180,000probes)HuGChip(66families,4000probes)IBS/IBDChip(300bacteria,54probes)

Fluidigm(24primersets)GULDA(GutLowDensity

Array):31targets

Numerousbutmostfocusedon16SrRNA

genebased

Page 23: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

5.Howtostop,encourage,ormanagethem?

Page 24: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Summary

1.   TCDDandSFB/B.fragilisinteractthroughAhRinapredictablemannerintermsofimmunecellresponse.Suchinteractionsmayestablishthebasisforintervention.

2.  Measuringsmallereffectsoftoxicantsongutmicrobiomemembersthroughthehostmaybedifficult.

3.   Gutmemberactivity,modeofcommunicationwiththehost,quantitativepredictivemodels,andmarkersofhealthy/sickgutmicrobiomearesomeofthekeychallengesaheadingutmicrobiomeresearch.

Page 25: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Gut Microbiome

PlayStore

AppStore

Feedback:[email protected]

Page 26: Gut Microbiome: Toxicant Perturbation and Stability · Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham Superfund Research Program ... GULDA (Gut Low Density Array):

Acknowledgements

Research Supported by National Institutes of Environmental Health Sciences (2P42ES004911)

Gnotobiotic study was conducted at the University of Michigan’s Germ-Free facility with Dr. Kathryn Eaton.

SFB source: Candidatus Arthromitus SFB-mouse-Japan was provided by Dr. Tomomi Kuwahara under MTA.

Co-PIs&Collaborators

James Tiedje, Norb Kaminski, Tim Zacharewski, Gerben Zylstra, James Cole, Benli Chai, and Brad Upham

Robert Stedtfeld, Maggie Williams, Robert Crawford, Tiffany Stedtfeld, Shao Xiangwen, Prianca Bhaduri, and Kelly Fader