gut microbiome: toxicant perturbation and stability · gut microbiome: toxicant perturbation and...
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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
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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
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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
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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)
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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
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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
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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
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Hypothesis TCDD exposure disrupts the Treg /Th17 system and specific gut microbialmembersarecapableofpreventingthisdisruption.
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SCFAPSA
SFBB.fragilisClostridia
TCDD
TCDDimpactsthehostwhichthenimpactsthegutmicrobiome
TwoPossibilities!TCDDimpactsthegutmicrobiome
whichthenimpactshost
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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
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IleumGnotobioticC57BL6:GeneExpression
ComparedtoGF,SFBhasmoreUp-regulatedgenes.
WithTCDD,SFBhasmoreDown-regulatedgenes.
WithTCDDComparedtoGF
Up-regulation Down-regulation Up-regulation Down-regulation
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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
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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
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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
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TraditionalC57BL6:DoseResponse&Recovery
Day38 90120
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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
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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
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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
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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
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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
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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
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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
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5.Howtostop,encourage,ormanagethem?
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Summary
1. TCDDandSFB/B.fragilisinteractthroughAhRinapredictablemannerintermsofimmunecellresponse.Suchinteractionsmayestablishthebasisforintervention.
2. Measuringsmallereffectsoftoxicantsongutmicrobiomemembersthroughthehostmaybedifficult.
3. Gutmemberactivity,modeofcommunicationwiththehost,quantitativepredictivemodels,andmarkersofhealthy/sickgutmicrobiomearesomeofthekeychallengesaheadingutmicrobiomeresearch.
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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