1. Probiotics and mucosal immunity
interactions in early life
Bruno Pot
Bactéries Lactiques et Immunité des Muqueuses
Institut Pasteur de Lille
Institut de Biologie de Lille
Vrije Universiteit Brussel
The Pharmabiotic Research Institute
2. • The dynamics of the microbiota
• The required signals from the environment
• Not all signals (probiotics!) are alike
• Can probiotics do the job?
• Conclusions
Today’s talk…
3. Aleksandar D. Kostic, Michael R. Howitt and Wendy S. Garrett; Genes Dev. 2013 27: 701-718
Microbiota: the consequence of evolution !
Phylum levels
4. Human Microbiota: a mutual,
evolutionary interplay
• Composition determined by
– Immune system
– Neural system (stress)
– Nutritional status
– External interventions
(infection, drugs, …)
• The µbiome has influence on
– Immune system
– Neural system
– Nutritional status
– …
Fredrik Backhed et al., 2012,
Cell Host & Microbe.
5. Microbiota: a lifetime dynamics
Bacteroidetes
Firmicutes
Acinobacteria
Proteobacteria
Variable/other
Aleksandar D. Kostic, Michael R. Howitt and Wendy S. Garrett; Genes Dev. 2013 27: 701-718
3 Yrs: more diverse
‘‘adult-like’’ structure
adulthood elderly
6. Pfefferle & Renz,
F1000 Prime Reports 2014
Early life-time is very important with life- long impact!
How do we know?
8. • The dynamics of the microbiota
• The required signals from the environment
• Not all signals (probiotics!) are alike
• Can probiotics do the job?
• Conclusions
Today’s talk…
9. Pregnant women get ready for delivery!
Koren et al., Cell, 2012
Fecal bacteria of 91 pregnant women with varying pre-pregnancy BMIs and
gestational diabetes status were compared (16S rRNA gene).
Differences are apparently not related to diet, antibiotics or presence or
absence of gestational diabetes or pre-pregnancy BMI.
First Trimester (T1) Third Trimester (T3)
11. Not only the gut microbiota…
Aagaard et al., PLoS One, 2012
68 vaginal samples from 24 healthy pregnant women (at 18 to 40 weeks) were
compared to 301 samples of 60 non-pregnant control women
Pregnancy reduces diversity of, but with an increase of
streptococci and certain lactobacilli.
Pregnant
Non-pregnant
12. • Breast milk itself is also a source of bacteria and contains up to
10 exp 9 bacteria / L in healthy mothers.
• The most frequently encountered bacterial groups include
staphylococci, streptococci, corynebacteria, lactobacilli, micrococci,
propionibacteria and bifidobacteria.
• Lactobacilli were isolated in 55.3% of colostrum samples of 116
Chilean mothers, with concentrations of 3.33 ± 0.55 (log CFU/ml).
• The predominant species were L. plantarum (64%), L. fermentum
(16%) and L. pentosus (9%).
.. and there is more…
R Wall et al., 2009 Clinical Medicine: Pediatrics
13. A programmed change throughout?
Lactobacilli and streptococci are well-known commensal lactic acid producing
bacteria, some of which are capable to
(i) selectively interact with the innate and the adaptive immune system of
their host, able to
(ii) prevent growth of pathogenic bacteria and
(iii) assist in digestion and are
(iv) less anaerobic, more acid resistant
(v) their growth is promoted by breast milk
Suggesting a natural increase of levels of these bacteria, early in life, and
Suggesting the possibility of ‘artificially’ increasing levels through food
supplements or adapted nutritional interventions, to
(i) promote a healthy development of a balanced microbiota
(ii) protect against accidental dysbiosis in early life phases.
14. Can it really work? How and when?
• Also a group of pregnant mice were orally inoculated with a genetically
labelled E. faecium strain previously isolated from breast milk of a healthy
woman.
• The labelled strain could be isolated and PCR-detected from meconium of
the inoculated animals obtained by caesarean section one day before the
predicted date of labor.
PCR detection of E. faecium JLM3 among colonies
isolated from meconium.
Lane 1, 100 bp ladder (Bioline, London, UK); lane 2,
PCR positive control (genomic DNA obtained from
transgenic soy); lanes 3e7, colonies obtained from
group A mice that grew on MRS-Cm agar plates;
lanes 8e12, colonies obtained from group B mice;
lane 13, PCR-negative control (E. faecium HA1).
15. In the mean time…
Bacteria were isolated from
- umbilical cord blood of healthy neonates, and
- placenta from mothers
- amniotic fluid and meconium obtained of mice (after caesarean section)
Dasanayake, A.P., Li, Y., Wiener, H., Ruby, J.D., Lee, M.J. (2005) Salivary
Actinomyces naeslundii genospecies 2 and Lactobacillus casei levels
predict pregnancy outcomes. Journal of Periodontology 76, 171e177
Jimenez, E., Fernandez, L., Marın, M.L., Martın, R., Odriozola, J.M.,
Nueno-Palop, C., et al. (2005) Isolation of commensal bacteria from umbilical
cord blood of healthy neonates born by caesarean section. Current
Microbiology 51, 270e274.
Pettker CM, Buhimschi IA, Magloire LK, et al. Value of placental microbial evaluation in diagnosing intra-
amniotic infection. Obstet Gynecol. 2007;109(3):739–49.
Prenatal mother-to-child efflux of commensal bacteria exists
Likely an active strategy to prepare the infant for future
contact with commensals (and pathogens)
16. • The dynamics of the microbiota
• The required signals from the environment
• Not all signals (probiotics!) are alike
• Can probiotics do the job?
• Conclusions
Today’s talk…
17. Aleksandar D. Kostic, Michael R. Howitt and Wendy S. Garrett; Genes Dev. 2013 27: 701-718
The co-evolutionary aspect!
Phylum levels
18. Not all bacteria / signals are alike!
Germ-free (GF) mice were colonized with mouse microbiota (MMb) or human
microbiota (HMb) to test whether immune maturation depends on a host-specific
microbiota relationship (co-evolution driven).
Chung et al., Cell, 2012
Bacterial numbers and
phylum abundance were
similar in MMb and HMb
mice, but bacterial
genera / species differed
Mouse Microbiota Human Microbiota
family
19. Not all signals are alike!
HMb mouse intestines had
many characteristics of GF
mice
- low levels of CD4+ and
CD8+ T cells
- few proliferating T-cells
- few dendritic cells
- low antimicrobial
peptide expression
Chung et al., Cell, 2012
More similar
20. Not all signals are alike!
Mouse-segmented filamentous bacteria (SFB) partially restored
the T-cell numbers in HMb mice, suggesting that SFB and other
host specific MMb organisms are required for full immune
maturation in mice.
Chung et al., Cell, 2012
21. • The dynamics of the microbiota
• The required signals from the environment
• Not all signals (probiotics!) are alike
• Can probiotics do the job?
• Conclusions
Today’s talk…
22. Data from clinical trials that included either prenatal or the prenatal and postnatal or
postnatal intervention phase of probiotics, prebiotics, or both.
PFEFFERLE, PRESCOTT, AND KOPP, 2013, J ALLERGY CLIN IMMUNOL
Prenatal
Pre & post
natal
25. Continued …
PFEFFERLE, PRESCOTT, AND KOPP, 2013, J ALLERGY CLIN IMMUNOL
In about 1 out of 2 studies, some positive parameters could be measured…
Probably not only the pro- / prebiotic ALONE is responsible, but other factors
will determine the effect they can have.
26. PFEFFERLE, PRESCOTT AND KOPP, 2013 J ALLERGY CLIN IMMUNOL
Possible mechanisms of prenatal and postnatal induction of tolerance by
microbial components
(1) Prebiotics and probiotics can influence the
mother’s gut microbiome, potentially inducing
regulatory cytokines that might pass the
placenta and prime the fetal immune system
toward tolerance.
(2) Through vaginal birth, the newborn’s gut
acquires the maternal vaginal microbiome.
(3) Oral application of prebiotics/probiotics might
support a homeostatic colonization of the
infant’s gut.
27. PFEFFERLE, PRESCOTT AND KOPP, 2013 J ALLERGY CLIN IMMUNOL
Possible mechanisms of prenatal and postnatal induction of tolerance by
microbial components
(1) Prebiotics and probiotics can influence the
mother’s gut microbiome, potentially inducing
regulatory cytokines that might pass the
placenta and prime the fetal immune system
toward tolerance.
(2) Through vaginal birth, the newborn’s gut
acquires the maternal vaginal microbiome.
(3) Oral application of prebiotics/probiotics might
support a homeostatic colonization of the
infant’s gut.
28. Antibiotics can disturb the signaling!
Sebastian Zeissig & Richard S Blumberg, 2014, Nature
CORRECT
COMMENSAL
COLONIZATION
= REQUIRED FOR
MATURATION
Windows of opportunity
Immune development
is only during certain
window of opportunity
29. J-F Bach et al. N Engl J Med, Vol. 347, No. 12 September 19, 2002
30. Antibiotics can disturb the signaling!
Sebastian Zeissig & Richard S Blumberg, 2014, Nature
CORRECT
COMMENSAL
COLONIZATION
= REQUIRED FOR
MATURATION
Windows of opportunity
During window
of opportunityProbiotics
Prebiotics
31. After: Susan L Prescott, Current Allergy & Clinical Immunology, June 2008
Logical approaches to promote tolerance:
* Optimise colonisation and gut maturation / permeability (probiotics / prebiotics)
* Allergen exposure - optimise timing, dose, interval, route (rather than avoidance)
* Breast feeding - promotes tolerogenic conditions during allergen encounter
* Immunomodulatory factors (omega-3 fatty acids; requires further studies)
Birth 3-4? 6-7? >12 months
? ?
Tolerance
induction
window
risk risk resolution
Window of opportunity?
Be sure not to miss it…
33. Epigenetic changes?
• Durable epigenetic changes might contribute to persistent
effects of microbial regulation of host immunity
• Strategies to reverse epigenetic changes could suggests a
possible therapeutic manipulation of epigenetic modifications,
providing a suitable approach for restoring immunological
defects acquired as a consequence of missed opportunities for
host-microbe interactions
• An interesting story: the Quebec Ice Storm Babies
Sebastian Zeissig & Richard S Blumberg, 2014, Nature
Coa-Lei et al. 2014. PLoS One.
34. • The dynamics of the microbiota
• The required signals from the environment
• Not all signals (probiotics!) are alike
• Can probiotics do the job?
• Conclusions
Todays talk…
35. Conclusions
• Already in utero, the mother, her microbiota and the baby coexist, as a
kind of interconnected meta-organism (the co-evolution aspect).
• The purpose is to deliver to the baby, in a timely matter, a commensal
composition of bacteria able to assist in the healthy maturation of its
immune- and metabolic system.
• There is some evidence that this involves epigenetic changes that will
take place during specific time slots (limited!).
• Although underlying mechanisms are unknown in detail, we can learn
from observed defects how the microbiota and immune systems co-
evolve and how metabolic phenotypes reflect the myriad of functions
encoded in the human genome and the gut microbiome.
• These findings underscore the need to systematically consider the
microbiome when evaluating human development, nutritional needs,
physiological variations.
• Further insghts and research may reveal how to reverse epigenetic
changes to restore a healthy situation.
36. Can probiotics do ‘the’ job?
Yes, some, and probably not ALONE
Within a (limited?) window of opportunity
which unfortunately is not yet well defined
37. Thanks to my team in Lille!
Jeanne Agard
Jérôme Breton
Denise Boutillier
Benoit Foligné
Catherine Daniel
Véronique Dennin
Joëlle Dewulf
Corinne Grangette
Sabine Poiret
Coline Plé
(Elise Macho-Fernandez)
(Véronique Valenti)
&
Marie-Christine Renaud
39. Epigenetic changes: The Ice Storm
Babies
• “There’s quite a large body of literature suggesting that early
life experiences are registered in methylation changes,”
• “The main problem in humans is that you can’t easily test
whether the changes in methylation are caused by genetics or
by experiences.”
• “ When you have a natural disaster like the 1998 Quebec ice
storm, it’s random; it doesn’t select who it affects based on
genetics”
Moshe Szyf, McGill University
Coa-Lei et al., 2014, PLoS One.
40. Ice Storm Babies experiment
• Five months after the 1998 Quebec ice storm,
women who were pregnant during the storm or had
conceived shortly afterward were recruited.
• The scientific question: What is the effect on the
babies of extreme stress during pregnancy …?
• In 2011, the Ice Storm Babies—now in their teens
provided blood samples for analysis.
• A total of 36 agreed to participate in the follow-up,
33 of whom had also provided saliva samples at age
8 for genetic analysis.
Coa-Lei et al. PLoS One.
41. Results
• 1675 methylation sites in the genomes of Ice Storm
Babies were associated with levels of maternal
stress during the storm:
– The longer a mother was without power or the higher
she ranked her stress levels, the more likely (or less
likely, in some cases) these sites would be methylated in
her child.
• It was verified whether the genes had been
methylated in the saliva provided five years before
to exclude that the methylation changes happened
later in life.
42. The methylations were strong and
organized, concentrated in functional
genes, mostly related to the immune
system.
The top pathway the genes fed into was
the CD28 signaling pathway in T helper
cells, required for T cell activation.
Coa-Lei et al. 2014. PLoS One.
Genes with altered methylation
patterns were clustered in immune
pathways.
43. Follow up and conclusion
• What the methylation changes mean for the children’s
immune systems was not yet studied.
• The hypothesis: “An exposure to stress in utero may prepare
to be exposed to stress during later life”.
• and “It makes sense that these changes would impact the
immune system, one of the body’s front lines against stress”.
• Further studies may pave the way toward understanding how
methylation is established throughout fetal development.
• Focus will be on how stress hormones that cross the placenta
into an embryo’s bloodstream interact with pathways altering
methylation.
What about the importance of the Microbiota in stress conditions?
Coa-Lei et al. 2014. PLoS One.
Notas do Editor
Figure 1. The structure of the microbiota across species. Although there can be significant inter-individual variation in the
composition of the microbiota, broad trends exist within a given species, particularly at the phylum level. Phyla are represented by color, and the relative abundance of the lower taxonomic levels is indicated by font size. This figure was produced with data adapted from Arumugam et al. (2011), Brinkman et al. (2011), Chandler et al. (2011), and Roeselers et al. (2011).
Figure 2. The structure of the human intestinal microbiota across the life cycle. The composition of the gut microbiome changes throughout the course of life. The infant microbiome shows great inter-individual variability and relatively low diversity but becomes more diverse and converges into an ‘‘adult-like’’ structure by 3 yr after birth. Pregnancy is associated with an increase in Actinobacteria and Proteobacteria and increased diversity, but the gut microbiota returns to its original structure sometime after delivery. Old age (>65 yr) is associated with a number of changes in the microbiota, including an increase in the abundance of Bacteroidetes.
Figure 1. 16S rRNA Gene Surveys Reveal
Changes to Microbial Diversity during Pregnancy
(A–G) Microbial communities clustered using
PCoA of the weighted UniFrac matrix. The
percentage of variation explained by the principal
coordinates is indicated on the axes. The same
plots are shown for (A)–(G), except 1 month
postpartum samples are additionally included in
(A). Each point corresponds to a community
colored by T1, T3, or 1 month postpartum (A);
prepregnancy BMI (B); gestational diabetes
(GDM; C); trimester and birth order of expected
child (D); abundance gradient of Bacteroidetes (E);
abundance gradient of Firmicutes (F); and abundance
gradient of Proteobacteria (G). Arrows in (D)
point to samples from women who received antibiotics
in T1 (orange arrows) and T2 (not T3, gray
arrows). (E–G) Gradients are colored from low
abundance (blue) to high abundance (red).
(H) Boxplots for community richness (a-diversity)
for T1 and T3 samples. For both T1 and T3, data
shown are Faith’s phylogenetic diversity (PD)
for 100 iterations of 790 randomly selected
sequences/sample. ***p < 0.0001.
See Figure S1.
Heatmap of OTU abundances found to
discriminate between T1 and T3 by machine
learning. Counts were standardized (Z score,
shown in legend) prior to unsupervised hierarchical
clustering of samples (columns). The color
bar indicates the origin of the samples (T1, orange;
T3, gray). The taxonomic assignment of each OTU
is indicated to the right of the rows (OTUs; note
several OTUs may share the same taxonomic
assignment).
See Figure S2.
Figure 1. The structure of the microbiota across species. Although there can be significant inter-individual variation in the
composition of the microbiota, broad trends exist within a given species, particularly at the phylum level. Phyla are represented by color, and the relative abundance of the lower taxonomic levels is indicated by font size. This figure was produced with data adapted from Arumugam et al. (2011), Brinkman et al. (2011), Chandler et al. (2011), and Roeselers et al. (2011).
FIG 1.. Possible mechanisms of prenatal and postnatal induction of tolerance by microbial components
1, Prebiotics and probiotics can influence the mother’s gut microbiome, potentially inducing regulatory
cytokines that might pass the placenta and prime the fetal immune system toward tolerance. 2, Through
vaginal birth, the newborn’s gut acquires the maternal vaginal microbiome. 3, Oral application of prebiotics/
probiotics might support a homeostatic colonization of the infant’s gut.
FIG 1.. Possible mechanisms of prenatal and postnatal induction of tolerance by microbial components
1, Prebiotics and probiotics can influence the mother’s gut microbiome, potentially inducing regulatory
cytokines that might pass the placenta and prime the fetal immune system toward tolerance. 2, Through
vaginal birth, the newborn’s gut acquires the maternal vaginal microbiome. 3, Oral application of prebiotics/
probiotics might support a homeostatic colonization of the infant’s gut.
Microbial colonization, development of the immune system and their perturbation by treatment with antibiotics early in life. Microbial colonization during early postnatal development represents a dynamic process, which evolves toward an adult-like configuration within 3 years after birth. Colonization by commensals is associated with and required for the maturation of host immunity (blue boxes, bottom); this leads to immunological and metabolic homeostasis of the host. The influences of microbes on specific pathways of host immunity are often confined to windows of opportunity, with a particular role for specific microbes whose effects may be persistent and durable in their presence or absence during neonatal life. Treatment with antibiotics is associated with alterations in the host-associated microbiota that, even if transient, may lead to persistent alterations in immunological function if they occur during such windows of opportunity. Consequently, such events may regulate susceptibility later in life to atopic, immune-mediated, metabolic and potentially neoplastic diseases, as well as neuropsychiatric disorders. Durable epigenetic changes probably contribute to persistent effects of microbial regulation of host immunity. The proposal of strategies to reverse epigenetic changes, well illustrated by the ability to convert differentiated cells into pluripotent stem cells, thereby suggests that the therapeutic manipulation of epigenetic modifications may provide a suitable approach for restoring immunological defects acquired as a consequence of missed opportunities for host-microbe interactions. Distance of microbial communities between children and adults (vertical axis) is reported according to ref. 10. AMPs, antimicrobial peptides; iLF, isolated lymphoid follicle; sIgA, secretory IgA.
Microbial colonization, development of the immune system and their perturbation by treatment with antibiotics early in life. Microbial colonization during early postnatal development represents a dynamic process, which evolves toward an adult-like configuration within 3 years after birth. Colonization by commensals is associated with and required for the maturation of host immunity (blue boxes, bottom); this leads to immunological and metabolic homeostasis of the host. The influences of microbes on specific pathways of host immunity are often confined to windows of opportunity, with a particular role for specific microbes whose effects may be persistent and durable in their presence or absence during neonatal life. Treatment with antibiotics is associated with alterations in the host-associated microbiota that, even if transient, may lead to persistent alterations in immunological function if they occur during such windows of opportunity. Consequently, such events may regulate susceptibility later in life to atopic, immune-mediated, metabolic and potentially neoplastic diseases, as well as neuropsychiatric disorders. Durable epigenetic changes probably contribute to persistent effects of microbial regulation of host immunity. The proposal of strategies to reverse epigenetic changes, well illustrated by the ability to convert differentiated cells into pluripotent stem cells, thereby suggests that the therapeutic manipulation of epigenetic modifications may provide a suitable approach for restoring immunological defects acquired as a consequence of missed opportunities for host-microbe interactions. Distance of microbial communities between children and adults (vertical axis) is reported according to ref. 10. AMPs, antimicrobial peptides; iLF, isolated lymphoid follicle; sIgA, secretory IgA.
STRATEGIES TO PROMOTE TOLERANCE
The aim of intervention in early life is to promote
favorable conditions for normal tolerance during critical
periods of immune development (Fig. 2). Successful
tolerance is likely to depend on other conducive exposures
(such as favourable gut colonisation,4 breast milk9
and/or other nutritional immunomodulatory factors24).
Thus, logical approaches can be considered broadly in
four areas:
1. Promoting optimal colonisation and gut maturation
2. Promoting optimal allergen exposure (timing, dose,
interval and route)
3. Using immunomodulatory factors such as n-3 fatty
acids and breast milk that may promote tolerogenic
conditions during allergen encounter and processing
4. Avoiding known irritants such as smoking that predispose
to persistent disease.
At present, there are very few formal recommendations
on these points as good evidence is still not available.
Challenging many long-held concepts, there are now studies (in progress and in design) that will examine
the hypothesis that earlier introduction and regular
exposure to ‘allergenic’ foods (rather than avoidance)
may reduce the risk of specific allergies to these foods.
To promote early colonisation, there is preliminary evidence
that supplementation with some probiotic
strains may reduce atopic dermatitis (but not other
allergy outcomes)77 (and now several other researchers).
However, more studies are needed before
recommendations can be made. Similarly, while exposure
to fish oil in early life may have some beneficial
effects, the role in allergy prevention is still unclear and
large-scale trials are still in progress. While the role of
breastfeeding in allergy prevention is unclear (with many contradicting studies), this is still recommended
for other reasons and there is some evidence that continued
breastfeeding during introduction of complementary
foods promotes tolerance.
