Transcript B. fragilis - CLU-IN

Gut Microbiome: Toxicant
Perturbation and Stability
Syed Hashsham
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
Superfund Research Program
Environmental Microbial and Mammalian Biomolecular
Responses to AhR Ligands
Outline
A. Gut Microbiome
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•
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•
Background and Objective
Treg/Th17 system, TCDD, and SFB
Hypothesis and Experimental Details
Results
B. Key Challenges Ahead
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•
Communication channels
Predictive capabilities
Markers/Gut chips
Interventions
C. Summary
Gut Microbiome: Diseases & Interventions
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
Host
Gut Microbiome
Commensals
Keystone Species
Opportunistic Pathogens
Pathobionts
Pathogens
ORGANS
Brain
Mouth
Heart
Kidney
Liver
Immune System
Colon
Tissue
Muscle
INTERVENTIONS
Fecal Transplant
ENVRIONMENAL
Psychobiotics
EXPOSURE
Toxicants Pre- and Probiotics
Antibiotics
Food Habits
CRISPR
Phage
Objective: Characterize the effect of specific gut microbiome
members on Treg/Th17 System with and without TCDD
Treg/Th17
(Host)
2,3,7,8-TCDD
(Toxicant)
Segmented Filamentous Bacteria (SFB)
Bacteroides fragilis
(Key Gut Members)
With TCDD, AhR promotes Treg and suppresses Th17
IL-6
Treg
modulates and
abrogates
autoimmune
disease
Naïve
T cells
IL-6
Treg
Th17
Foxp3+
RORgt
Aryl hydrocarbon
Receptor (AhR)
IL-17
IL-22
IL-10
2,3,7,8-TCDD
Th17
aids in antimicrobial
response;
also causes
inflammation &
autoimmune disease
Why Segmented Filamentous Bacteria (SFB)?
• Obligate symbiont
• Extensive auxotroph
• No genes for amino acids, vitamins/cofactors, nucleic acids
• Host-specific
Candidatus Savagella | Environmental Microbiology 14 (6): 1462-2920 | 2012
SFB cultivation is now possible using TC7 cell lines (BioTechniques, 59 (2):94–98, 2015
SFB in humans?
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 between ages 2 to 3
2013
Hans Jonsson
2009
Ivanov et al., Cell: 2009
Snel et al.
Gut
Clostridia Segmented
Bacteria (SFB)
B. Filamentous
fragilis
SFB
Polysaccharide A
Short Chain
Fatty Acids
Lamina propria
Butyrate
Dendritic cells
IL-6
Naïve
T cells
IL-6
Th17
Treg
RORgt
Foxp3+
IL-10
microRNAs
Aryl Hydrocarbon
Receptor (AhR)
TCDD
IL-21R
Nos2
IL-21
Host
Hypothesis
TCDD exposure disrupts the Treg /Th17 system and specific gut
microbial members are capable of preventing this disruption.
Two Possibilities!
TCDD impacts the gut microbiome
which then impacts host
TCDD
Clostridia
SCFA
B. fragilis
PSA
TCDD impacts the host which then
impacts the gut microbiome
SFB
Experimental Details
C57BL6
Gnotobiotic
Traditional
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
Tim Zacharewski’s Lab
Key measurements
TCDD: 30 µg/kg every 4 d
56 d study
4 per group
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•
•
•
•
•
GF
SFB
B. fragilis
B. fragilis + SFB
UM Germ-Free Facility
•
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)
Fecal pellets, ilium, cecum, blood
•
High Throughput (Wafergen) or qPCR
Fecal pellets, cecum
Ileum
Gnotobiotic C57BL6: Gene Expression
With TCDD
Compared to GF
Up-regulation
Down-regulation
Compared to GF, SFB has
more Up-regulated genes.
Up-regulation
Down-regulation
With TCDD, SFB has more
Down-regulated genes.
Spleen
Gnotobiotic C57BL6: Treg
Colonization p =0.35
cd36
TGF-b
Vehicle
TCDD p<0.0001
TCDD
T r e g c e lls in s p le e n
5000
1000
4000
800
15
GFVehicle B
B+SFB
TCDD SFB
Vehicle
B
S
F
S
F
B
B
+
B
B
F
G
S
+
B
00
F
G
F
1000
200
10
S
2000
400
B
% CD4
+
3000
600
20
F
Normalized count
6000
1200
25
B
7000
1400
TCDD
Parametric two-way ANOVA
Gnotobiotic C57BL6: Th17
Spleen
Ciita was similar!
IL1-b
S
00
GF
B
B+SFB
SFB
Vehicle
Parametric two-way ANOVA
B
S
+
B
F
TCDD
F
B
F
B
S
F
B
+
G
F
200
10
0 .0
G
400
20
0 .5
B
600
30
1 .0
F
+
800
40
1 .5
S
TCDD
1000
50
TCDD p = 0.0196
B
1200
60
2 .0
B
1400
70
T h 1 7 c e lls in s p le e n
Vehicle
% CD4
count
Normalized count
Normalized
1600
80
Colonization p = 0.0004
T
C
D
e
D
6
p=0.038
310 6
210
6
110
6
410
4
110
4
D
410
D
h
0
6
C
500
510
T
1000
6
h
p=0.029
610
e
1500
SFB
V
2000
S F B 1 6 S r R N A g e n e c o p ie s p e r m g ile u m
B. fragilis
V
B . fr a g ilis r p lB g e n e p e r m g c a e c u m
Gnotobiotic C57BL6: B. fragilis and SFB
Traditional C57BL6: Dose Response & Recovery
Day 38
90
120
Overall Interaction of TCDD, B. fragilis, and SFB
Decrease in
B. fragilis &
Clostridia
Clostridia
Gut
Segmented
Filamentous Bacteria (SFB)
B. fragilis
SFB
Polysaccharide A
Increase in
SFB
Butyrate
Lamina propria
Dendritic cells
(Expected change in B.
fragilis & Clostridia was a
DECREASE)
(Expected change in SFB
was an INCREASE)
IL-6
Naïve
T cells
IL-6
Treg
Th17
Foxp3+
RORgt
AhR
IL-21R
Nos2
IL1-b
IL-10
TCDD
TCDD
Host
Traditional C57BL6: Increase in Antimicrobial Resistance Genes
25
20
amphenicol : yidY
MDR : acrF, mdtE, acrR, tolC
beta lactamase: ampC, blaCMY2
other: bacA
tetracycline: tet(32)
sulfonamide: folA
Fold difference
15
10
5
0
0.3
1
3
10
-5
mg/kg TCDD (LS and RS groups)
30
RT-30
B. Key Challenges Ahead
Identity
1. Who is there?
Activity
2. Who is doing what and how?
Prediction
3. Can we predict gut behavior? Quantitatively?
Diagnosis
4. How do we know when something is wrong?
Intervention
5. How to stop or encourage key members?
2. Who is doing what and how?
microRNA expression in SFB-associated mice is
much greater than Germ-free or B. fragilisassociated groups!
Ivanov et al.,
Cell: 2009
Spatial resolution
More sensitive to work with 1-10 µl blood
At all molecular levels
Ileum
MicroRNAs may alter the gut microbiota through fecal
microRNAs, affecting growth and other cellular
processes (Liu et al., 2016).
3. Can we predict the gut behavior – quantitatively?
Perturbation
Response Envelope
Response
Resistance:
Maximum deviation
from the preperturbed
equilibrium
Resilience:
Inverse of time
taken to return to
equilibrium
1
Time
Hashsham et al., Fernandez et al., AEM, 2000
MDSINE
Time series
Biomass
Perturbation
Generalized Lota
Volterra Model
Trajectories
Interactions
Stability
Keystone-ness
Bucci et al. Genome Biology
17:121, 2016
Gut Disruption Index
VRE vs. E. faecium
Halpin et al, Am. J.
Infection Control 44
(2016) 830-6
Disease-specific
Deterministic
Probabilistic
4a. How do we know when something is wrong?
Markers
Carbohydrates
Functions, Guilds
Acetyl-CoA
Butyrate producers
Functional Gene Diversity
Primer Coverage
Roseburia intestinalis
Faecalibacterium prausnitzii
….
Butyryl-CoA
Fish et al., Front. Microbiol. 4: 291 2013
Butyryl-CoA acetate
transferase
BUT
Vital et al., mBio, 2014
Butyrate
Butyrate kinase
BUK
4b. How do we know when something is wrong?
Gut Chips
Illumina
Amplification-based qPCR or Lowdensity Chips
Fluidigm (24 primer sets)
GULDA (Gut Low Density
Array): 31 targets
Hybridization-based Arrays
High Throughput Sequencing
HuMiChip (500 functional genes, 180,000 probes)
HuGChip (66 families, 4000 probes)
IBS/IBD Chip (300 bacteria, 54 probes)
Numerous but most
focused on 16S rRNA
gene based
5. How to stop, encourage, or manage them?
Summary
1. TCDD and SFB/B. fragilis interact through AhR in a predictable manner in
terms of immune cell response. Such interactions may establish the basis for
intervention.
2. Measuring smaller effects of toxicants on gut microbiome members through
the host may be difficult.
3. Gut member activity, mode of communication with the host, quantitative
predictive models, and markers of healthy/sick gut microbiome are some of
the key challenges ahead in gut microbiome research.
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Microbiome
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Feedback:
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Acknowledgements
Research Supported by National Institutes of Environmental Health Sciences (2P42ES004911)
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
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.