Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham - - PowerPoint PPT Presentation

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Gut Microbiome: Toxicant Perturbation and Stability Syed Hashsham - - PowerPoint PPT Presentation

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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

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SLIDE 1

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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SLIDE 2

Outline

  • A. Gut Microbiome
  • Background and Objective
  • Treg/Th17 system, TCDD, and SFB
  • Hypothesis and Experimental Details
  • Results
  • B. Key Challenges Ahead
  • Communication

channels

  • Predictive

capabilities

  • Markers/Gut chips
  • Interventions

C. Summary

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SLIDE 3

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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SLIDE 4

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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SLIDE 5

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

Aryl hydrocarbon Receptor (AhR)

IL-17 IL-22 IL-10

2,3,7,8-TCDD

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SLIDE 6

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 now possible using TC7 cell lines (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 between ages 2 to 3

2013

Hans Jonsson

2009

Snel et al.

Ivanov et al., Cell: 2009

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SLIDE 7

Segmented Filamentous Bacteria (SFB)

Clostridia

  • B. fragilis

Polysaccharide A

Short Chain Fatty Acids

Lamina propria Butyrate Dendritic cells

Gut

SFB

Aryl Hydrocarbon Receptor (AhR)

Th17 Treg

Foxp3+ RORγt

IL-10

Naïve T cells

IL-6 IL-6 IL-21R Nos2 IL-21

microRNAs

TCDD

Host

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SLIDE 8

Hypothesis TCDD exposure disrupts the Treg /Th17 system and specific gut microbial members are capable of preventing this disruption.

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SLIDE 9

SCFA PSA

SFB

  • B. fragilis

Clostridia

TCDD

TCDD impacts the host which then impacts the gut microbiome

Two Possibilities!

TCDD impacts the gut microbiome which then impacts host

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SLIDE 10

Experimental Details

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

Tim Zacharewski’s Lab 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)

  • High Throughput (Wafergen) or qPCR

Fecal pellets, cecum Fecal pellets, ilium, cecum, blood

Key measurements

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SLIDE 11

Ileum

Gnotobiotic C57BL6: Gene Expression

Compared to GF, SFB has more Up-regulated genes. With TCDD, SFB has more Down-regulated genes.

With TCDD Compared to GF

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

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SLIDE 12

200 400 600 800 1000 1200 1400 Vehicle TCDD

cd36

Spleen

1000 2000 3000 4000 5000 6000 7000

GF B B+SFB SFB

TGF-β

TCDD Vehicle

Normalized count

Gnotobiotic C57BL6: Treg

G F B B + S F B S F B G F B B + S F B S F B 1 0 1 5 2 0 2 5

% C D 4

+ T re g c e lls in s p le e n

Vehicle TCDD p<0.0001 p =0.35 Colonization TCDD Parametric two-way ANOVA

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SLIDE 13

10 20 30 40 50 60 70 80 GF B B+SFB SFB Normalized count

IL1-β

200 400 600 800 1000 1200 1400 1600

GF B B+SFB SFB

Vehicle TCDD

Ciita was similar!

Normalized count

Spleen Gnotobiotic C57BL6: Th17

G F B S F B + B S F B G F B S F B + B S F B 0 .0 0 .5 1 .0 1 .5 2 .0

% C D 4

+ T h 1 7 c e lls in s p le e n

Vehicle TCDD p = 0.0004 p = 0.0196 Colonization TCDD Parametric two-way ANOVA

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SLIDE 14

Gnotobiotic C57BL6: B. fragilis and SFB

  • B. fragilis

SFB

V e h T C D D 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0

B . fra g ilis rp lB g e n e p e r m g c a e c u m

p=0.029

V e h T C D D 1´ 1 0 4 4´ 1 0 4 1´ 1 0 6 2´ 1 0 6 3´ 1 0 6 4´ 1 0 6 5´ 1 0 6 6´ 1 0 6

S F B 1 6 S rR N A g e n e c o p ie s p e r m g ile u m

p=0.038

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SLIDE 15

Traditional C57BL6: Dose Response & Recovery

Day 38 90 120

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SLIDE 16

TCDD Increase in SFB Decrease in

  • B. fragilis &

Clostridia

(Expected change in SFB was an INCREASE) (Expected change in B. fragilis & Clostridia was a DECREASE) Segmented Filamentous Bacteria (SFB)

TCDD

AhR

Th17 Treg

Foxp3+ RORγt

IL-10

Naïve T cells

IL-6 IL-6 IL-21R Nos2 IL1-β

SFB

Lamina propria

Gut Host

Dendritic cells

  • B. fragilis

Polysaccharide A

Clostridia

Butyrate

Overall Interaction of TCDD, B. fragilis, and SFB

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SLIDE 17

Traditional C57BL6: Increase in Antimicrobial Resistance Genes

  • 5

5 10 15 20 25 0.3 1 3 10 30 RT-30 Fold difference µg/kg TCDD (LS and RS groups) amphenicol MDR beta lactamase

  • ther

tetracycline sulfonamide

: acrF, mdtE, acrR, tolC : yidY : tet(32) : bacA : ampC, blaCMY2 : folA

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SLIDE 18
  • 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. Key Challenges Ahead

Identity Activity

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SLIDE 19

microRNA expression in SFB-associated mice is much greater than Germ-free or B. fragilis- associated groups!

  • 2. Who is doing what and how?

MicroRNAs may alter the gut microbiota through fecal microRNAs, affecting growth and other cellular processes (Liu et al., 2016).

Ileum Spatial resolution

More sensitive to work with 1-10 µl blood At all molecular levels

Ivanov et al., Cell: 2009

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SLIDE 20

Resistance: Maximum deviation from the pre- perturbed equilibrium Resilience: Inverse of time taken to return to equilibrium

Response

1

Response Envelope Perturbation

Time

  • 3. Can we predict the gut behavior – quantitatively?

Hashsham et al., Fernandez et al., AEM, 2000 Trajectories Interactions Stability Keystone-ness Bucci et al. Genome Biology 17:121, 2016 Time series Biomass Perturbation Generalized Lota Volterra Model

Disease-specific Gut Disruption Index

MDSINE Deterministic Probabilistic Halpin et al, Am. J. Infection Control 44 (2016) 830-6 VRE vs. E. faecium

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SLIDE 21

Vital et al., mBio, 2014

Butyrate Carbohydrates

Acetyl-CoA Butyrate kinase BUK Butyryl-CoA Butyrate producers

Roseburia intestinalis Faecalibacterium prausnitzii ….

Butyryl-CoA acetate transferase BUT

Markers

  • 4a. How do we know when something is wrong?

Functional Gene Diversity Primer Coverage

Fish et al., Front. Microbiol. 4: 291 2013 Functions, Guilds

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SLIDE 22

Gut Chips

High Throughput Sequencing

  • 4b. How do we know when something is wrong?

Amplification-based qPCR or Low- density Chips Hybridization-based Arrays Illumina HuMiChip (500 functional genes, 180,000 probes) HuGChip (66 families, 4000 probes) IBS/IBD Chip (300 bacteria, 54 probes) Fluidigm (24 primer sets) GULDA (Gut Low Density Array): 31 targets Numerous but most focused on 16S rRNA gene based

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SLIDE 23
  • 5. How to stop, encourage, or manage them?
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SLIDE 24

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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SLIDE 25

Gut Microbiome

Play Store App Store Feedback: hashshamlab@gmail.com

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SLIDE 26

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