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Final Report Summary - MICROBIOTA-NEONATE (Characterization of maternal microbiota-dependent imprinting of the neonatal immune system)

MICROBIOTA-NEONATE (#627206) - Characterization of maternal microbiota-dependent imprinting of the neonatal immune system
Researcher: Dr. Stephanie Ganal-Vonarburg
Scientist in charge: Prof. Dr. Andrew Macpherson
Host institution: Department for Clinical Research, University of Berne, Switzerland

Trillions of bacteria inhabit the inner and outer body surfaces of healthy mammals, such as the skin, the airways and the intestine. Their entity is known as the commensal microbiota. Most of these bacteria can be found in the lower intestine where they importantly contribute to host physiology. The intestinal bacteria help us to digest food, produce essential vitamins and protect us from infection. During the past few years it has become clear that the intestinal microbiota is also essential for shaping a healthy immune system within the gut. Alterations in the composition of the gut microbiota are associated with an increased risk to develop food allergies or inflammatory bowl disease (e.g. Crohn’s disease and ulcerative colitis). The unborn child lives in a sterile environment within the uterus and is not yet colonized by a microbiota. Colonization with beneficial bacteria starts only with birth and the first bacteria to colonize the newborn will be those present in the immediate environment. For example, babies born by natural delivery have a different initial microbiota than those born through caesarian section, and during the first month of life breast or formula-fed infants have different gut bacteria. We also know that children that grow up on a countryside farm have a lower risk to come down with hay fever or asthma compared to children raised in the more sterile environment of a city. It seems to be this early stage of life that is of great importance to the shaping of the intestinal microbiota and can influence a child’s later risk to develop allergies or autoimmune disorders. Researchers often call this period the “window of opportunities”. However, it was not known whether the unborn child within the uterus is already exposed to signals from the gut microbiota of its mother, and if such signals could also contribute to the development of a healthy immune system. These questions were central to the Marie Curie IEF project MICROBIOTA-NEONATE. Our research focused on two main scientific objectives, which we addressed using the model organism mouse. First, we aimed to interrogate the phenotypic and functional effects of the immune alterations induced by maternal microbiota on immunity in the newborn. Second, we were interested in the mediators and their route of transfer by which maternal microbiota can modify the newborn immune system.
In order to uncouple colonization of the mother’s intestine from colonization of the offspring’s own intestine, we made use of Escherichia coli HA107, a commensal bacterium strain that was generated in our laboratory several years ago and that can reversibly colonize the murine intestine of germ-free mice that do not harbor any microbiota (Hapfelmeier et al., 2010). This bacterial strain is deficient in the synthesis of two bacteria-specific amino acids and can therefore only proliferate in culture supplemented with the two amino acids, but not within the murine intestine. For our experiments, we exposed pregnant germ-free mice to E. coli HA107 but ensured their return to the germ-free status before the delivery of the pups in order to prevent any direct contact of the offspring to the bacteria. The offspring of these gestationally colonized dams were compared to the offspring of mice that had remained germ-free throughout pregnancy (Fig. 1).
Of note, two immune cell populations of the innate immune system present in the intestinal mucosa of the offspring were significantly enriched in absolute cell numbers in the pups born to the gestationally colonized compared to germ-free control mothers (Gomez de Agüero, Ganal-Vonarburg et al., 2016). Among these were a subset of type 3 innate lymphoid cells (ILC3), namely the NKp46-expressing ILC3, and F4/80+CD11c+ intestinal mononuclear cells (iMNC). Interestingly, this phenotype was long-lasting and could still be observed in adult age. Both ILC3s and iMNCs are important players in maintaining homeostasis at the host-microbial interface and in the first defense against invading pathogens (Sonnenberg, 2014). Accordingly, we have tested the ability of the neonatal immune system to react to such challenges. In order to mimic colonization with commensal bacteria, we have exposed 14 day-old offspring born from either germ-free control or HA107-colonized females with a commensal bacterium. 18 hours after the challenge, we measured translocation of bacteria to the mesenteric lymph nodes (MLN). Offspring born and raised by gestationally treated mothers were protected from translocation to the MLN whereas the offspring from germ-free dams showed obvious bacterial translocation to the MLN (Gomez de Agüero, Ganal-Vonarburg et al., 2016). These data were underlined by results from an RNA sequencing experiment in which small intestinal gene expression of 14 day-old pups from gestationally treated mothers showed upregulation of genes involved in the antimicrobial defense and intestinal homeostasis (Gomez de Agüero, Ganal-Vonarburg et al., 2016), supporting our previous notion that maternal microbiota-derived signals prepare the neonatal immune system for the first encounter with commensal bacteria.
The second aim of the project was to understand the molecular mechanism underlying the maternal microbiota-induced alterations in the immune system of the offspring. Using mice deficient in B cells, we observed that a large fraction of the observed alterations in the offspring immune system introduced by gestational colonization was dependent on the presence of maternal antibodies (Gomez de Agüero, Ganal-Vonarburg et al., 2016). Maternal-fetal/neonatal transfer of antibodies occurs prenatally via the placenta as well as postnatally via the maternal milk. Cross-fostering experiments, in which half of the litter from germ-free or gestationally colonized dams was swapped at the time of birth, revealed that exposure to a reversibly colonized mother both before and after birth is required to induce full maturation of the early postnatal immune system (Gomez de Agüero, Ganal-Vonarburg et al., 2016). In a next set of experiments, we aimed to identify microbiota-derived factors that are transferred from the maternal intestine to the offspring. For this purpose, we labeled E. coli HA107 with the heavy carbon isotope [13]C and used this to colonize pregnant germ-free mice. Using mass spectrometry, we were able to follow metabolites of bacterial origin as fully 13C-labeled. When analyzing the maternal milk of mothers at day 2 after birth, we identified a great number of significantly enriched metabolites in the milk of gestationally colonized wild-type mice compared to germ-free controls. Among these were more than 70 fully 13C-labeled metabolites, proving that bacterial-derived factors are present in the maternal milk and can reach the offspring. Interestingly, these metabolites were absent in the milk of antibody-deficient mothers (Gomez de Agüero, Ganal-Vonarburg et al., 2016). These results and additional data on the binding capacity of these bacterial metabolites to serum antibodies, led us hypothesize that maternal antibodies are important carriers that mediate an efficient transport of metabolites derived from the maternal intestinal microbiota to the offspring. Last, we tried to mimic the effect of gestational colonization on postnatal immunity, by feeding purified bacterial ligands to pregnant dams. Among these was indole-3-carbinol, a ligand for the arylhydrocarbon (AhR) receptor, a ligand-dependent transcription factor that has previously been reported to be involved in the development and homeostasis of ILC3 (Kiss et al., 2011). The reason for choosing to feed pregnant mice an AhR ligand was, that our mass spectrometry analysis had revealed several 13C-labeled (bacterial-derived) AhR ligands that were enriched in the milk of HA107-colonized wild-type compared to antibody-deficient mice. Of note, the delivery of I3C during pregnancy was sufficient to induce immune maturation in the offspring.
Our study shows for the first time that microbial shaping of our immune system begins already before birth by the maternal microbiota. There are still 6 million deaths each year worldwide in children under the age of 5, many of which are caused by intestinal infections and malnutrition. One of the biggest problems of a newborn child after birth is that the intestine must become colonized by microbes without allowing body tissues to become infected or damaging the ability of the intestine to absorb nutrients. We now showed that signals derived from the maternal microbiota during pregnancy or during lactation are very important to shape the immune system of the offspring and to prepare him for colonization with its own intestinal microbiota after brith.
The work was published with Stephanie Ganal-Vonarburg as a shared first author in Science (Gomez de Agüero, Ganal-Vonarburg et al., 2016).

M. Gomez de Agüero*, S. C. Ganal-Vonarburg* et al., Science 351, 1296-302 (2016) - *equal contribution
S. Hapfelmeier et al., Science 328, 1705-1709 (2010)
E. A. Kiss et al., Science 334, 1561–1565 (2011)
G. F. Sonnenberg et al., Science 336, 1321–1325 (2012)

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