the gastrointestinal microbiome and probiotics : …...- mechanisms of probiosis should be clearly...
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The Gastrointestinal Microbiome and
Probiotics : Effects on Intestinal
Physiology and Mucosal InflammationJames Versalovic, M.D., Ph.D.
Professor
Department of Pathology &
Immunology
Baylor College of Medicine
Head, Department of Pathology
Chief, Pathology Service
Texas Children’s Hospital
Houston TX USA
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Alexander FlemingIlya Ilyich Mechnikov
Feeding Microbes to Humans Killing Microbes in Humans
PROBIOTICS (1907) ANTIBIOTICS (1928)
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What are Probiotics?
“Probiotics are live micro-organisms which when administered in adequate amounts
confer health benefit on the host” (FAO, 2001)
- Probiotics should be alive in the gastrointestinal tract.
- Mechanisms of probiosis should be clearly defined
before making a health claim.
- The effect of one probiotic strain cannot be
extrapolated to another strain from the same species.
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Context :
The Human Microbiome
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The Human Microbiome Project:
Indigenous Microbiota and Microbiome
Human Colon
> 1000 species
Firmicutes
Bacteroidetes
Firmicutes include Lactobacillus spp.
>60% novel bacteria
>80% nonculturable bacteria
Mixed
Microbial
Communities
(P Eckburg et al. Science
2005;308:1635-38)
(PJ Turnbaugh et al. Nature 2007;449:804-10) (S Macfarlane et al. Appl Environ Microbiol
2005;71:7483-92)
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The Distal Gut Microbiomes of Healthy
Children Differ from Healthy Adults
• Healthy
Children
• Healthy
Adults
V1V3 regions only (1 replicate/sample)
11 samples from healthy adults
(18-40 yo) sequenced
by HGSC at Baylor.
29 samples from healthy children
(7-12 yo) sequenced by HGSC
at Baylor
454 16S metagenomic sequencing
14-16K reads per sample
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454 sequencing reveals strain-
specific changes and increased
microbial diversity after probiotics
Untreated
Strain A
+24 hrs
Strain B
+24 hrs
Strain A
+4 hrs
Strain B
+4 hrs
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Indigenous Lactobacillus of the
Human Gastrointestinal Tract
• Lactobacillus gasseri
• Lactobacillus reuteri
• Lactobacillus ruminis
• Lactobacillus salivarius*
• Autochthonous (Indigenous) versus
Allochthonous (Transient) Microbiota
• Commensal-derived Probiotics
– Derived from the autochthonous microbiota
G. Reuter (2001) Curr. Issues Intest. Microbiol. 2:43-53
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Can we use indigenous
microbes or probiotics to
replenish missing elements or
functions?
Replenish or increase microbial
diversity in intestine. Increased
diversity is beneficial.
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Intestinal Physiology,
Development and Maturation
• Probiotics may stimulate intestinal epithelial cell maturation and migration
• Probiotics may promote villus development – Increased absorptive surface area
• Probiotics may stimulate goblet cells and mucin production
• Probiotics may reduce intestinal permeability and promote tight junctions
• Probiotics enhance expression of anion exchangers (choride) on surfaces of IECs
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Commensal Microbes
and Mechanisms
of Immunomodulation
• Stimulate proliferation of regulatory T cell or
APC populations
• Stimulate expression of anti-inflammatory
cytokines or factors in coordination with TLR
ligation
• Suppress pro-inflammatory cytokine secretion
by innate immune system
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Thomas CM, Versalovic J. Gut Microbes 2010; 1:1-16.
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Chandra Iyer
Can commensal-derived probiotics block immune signaling in
TNF-stimulated myeloid cells?
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NFB
L. reuteri can Inhibit TNF Dependent NF-B Activation
TNFR
TNF
IKK
NFB
NFBIB IB
Nucleus: activate transcription of
inflammatory and anti-apoptotic
proteins
Chandra Iyer, Ph.D.
Phosphorylation
IB
Ub
Ubiquitination
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Pathways Affected by Probiotic L. reuteriModel System: THP-1 cells + TLR2
agonist (PCK)
PCK
TLR2
MAPK Pathway
c-Junc-Fos
AP-1
Nucleus: block
transcription of pro-
inflammatory
cytokines (TNF)
Model System: KBM5 cells + TNFR
agonist (TNF)
TNFR
TNF
IKK
NFB
NFBIB
IB
Nucleus: block
transcription of pro-
inflammatory and anti-
apoptotic proteins
Ub
Degradation
= Probiotic= Probiotic
MAPK
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Carissa Thomas
Can probiotics regulate specific
immune signaling pathways?
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ATCC PTA 6475 Inhibits TNF via Downregulation of TAB1
IL-1 Receptor Antagonist (IL1RN)
Mitogen-activated protein kinase kinase kinase 7 interacting protein 1 (TAB1/MAP3K7IP1)
Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β (IKKB/IKBKB)
Tumor necrosis factor (TNF)
ATCC PTA 6475 ATCC 6475Δfpgs1
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Thomas CM, Versalovic J. Gut Microbes 2010; 1:1-16.
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Thomas CM, Versalovic J. Gut Microbes 2010; 1:1-16.
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Mechanisms of
Immune Enhancement
• Probiotics stimulate Th1 immune responses– Interferon-gamma
• Intestinal bacteria may stimulate Th17 differentiation in the gut mucosa
• Probiotics enhance mucosal IgA and systemic IgA production
• Probiotics-derived CpG –containing DNA activate immune signaling pathways
• Probiotics stimulate epithelial cell proliferation and signaling pathways resulting in cytokine/chemokine production
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Geoff Preidis
Can probiotics reduce
diarrheal disease burden in children?
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0
20
40
60
80
100
1 2 3 4 5 6 7
n = 20Shornikova et al. J Pediatr Gastroenterol Nutr 1997;24(4):399-404.
L. reuteri reduces the duration of
rotaviral diarrhea in infants
% o
f P
ati
en
ts w
ith
Dia
rrh
ea
Placebo
L. reuteri
0 1 2 3 4 5 6
*
*
Days After Therapy Initiation
p = .0005
p = .004
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Intestinal Bacteria in a
Neonatal Mouse Model
FISH probe specific to L. reuteri 16S rRNA / TRITC probe, 200x. Strain DSM 17938 shown.
(Geoff Preidis)
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Experimental Design: Testing
L. reuteri with mouse rotavirus
-3 -2 -1 0 1 2 3 4 5 6 7 dpi
Daily gavage L. reuteri (108 cfu)
Rotavirus
103 ID50
Diarrhea
5 days old 15 days old
Serum
Antibody ELISA
Intestine
Mesenteric Lymph Node
Spleen
Fragment
Culture
Antibody ELISA
FISH Histology
IHC
Score <2 Score 2+
No Diarrhea Diarrhea
Clinical Scoring
(0-4, blinded)
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Total Non-Specific IgA
Mesenteric Lymph Node Ileum
L. reuteri does not enhance mucosal
IgA in uninfected mice
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L. reuteri enhances the earliest
RV-specific mucosal antibody response
p < .001 p < .05p < .05 p < .001
Rotavirus-Specific Antibody
Mesenteric Lymph Node Ileum
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Could L. reuteri enhance virus-
specific antibody production?
• Virus-specific antibody clears rotavirus and protects humans and mice from subsequent infection
• Some probiotics increase fecal IgA in children
Franco et al., Vaccine 2006;24(15):2718-31.
Fukushima et al., Int J Food Microbiol 1998;42(1-2):39-44.
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-3 DPI 4 HPI 1DPI 2DPI 4DPI 6DPI
Daily gavage L. reuteri (108 cfu)
5 days old 14 days old
BrdU30 mg/kg i.p.
Using BrdU to measure
epithelial turnover
Begin
probiotics
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Probiotics increase intestinal
epithelial cell proliferation
No Treatment Strain A Strain B
n = 15 mice/group
***p < 0.001, *p < 0.05 vs untreated
Ileum shown
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Strain-specific differences in IEC
migration are seen at 2 DPI
No Treatment Strain A Strain B
n = 7 mice/group
**p < 0.01, *p < 0.05 vs untreated
#p < 0.01 between strains
Ileum shown
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(GA Preidis and J Versalovic.
Gastroenterology 2009;136:2015-31)
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Blumberg RS and Strober W. 2001. JAMA 285: 645
H. hepaticus L. reuteri
Intestinal Bacterial and Immunological Balance
L. paracaseiSalmonella sp.
PATHOGEN BENEFICIAL
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Future Directions
(GA Preidis and J Versalovic.
Gastroenterology 2009;136:2015-31)
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AcknowledgmentsEntire Versalovic Lab MIT – Division of Comparative MedicineWith special thanks to: James G. Fox and Susan Erdman
Michael Hsieh Zhongming Ge and Arlin Rogers
Delphine Saulnier Jacqueline Fremont and Suresh
Jeremy (Ryan) Pena Michigan St University
Chandra Iyer Robert A. Britton
Jennifer Spinler Kristi Whitehead and Laura Schaefer
Yea Ping Lin
Sara Jones Beckman Research Institute, City of HopeGeoff Preidis Markus Kalkum and Teresa Hong
Sue Venable
Carissa Thomas Biogaia AB (Stockholm, Sweden)Angela Major Eamonn Connolly
Noris Carbajal and Uma Shah
Baylor – Texas Children’s
Milton J. Finegold Swedish Agriculural Univ. (Uppsala, Sweden)George D. Ferry and Mark Gilger Hans Jonsson and Stefan Roos
Carolyn Thibodeaux
Daniel Lacorazza
Doug Burrin and Steven Kim
Sarah Blutt and Margaret Conner
Funding Agencies and Center ResourcesNational Institutes of Health (NIDDK, NCCAM and NIDCR), Crohn’s and Colitis Foundation of
America (CCFA), Texas Medical Center Digestive Diseases Center (NIH NIDDK), U.S. Department of Defense (DARPA, ONR).
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