Periodic Reporting for period 3 - HHMM-Neonates (DEVELOPMENT OF HEALTHY HOST-MICROBIAL MUTUALISM IN EARLY LIFE)
Berichtszeitraum: 2020-09-01 bis 2022-02-28
The body surfaces and the lower intestine of mammals are colonized by vast numbers of micro-organisms. These are generally not harmful to their animal or human host - rather the reverse, as they provide the host with micronutrients, help harvest energy from otherwise indigestible food, and compete with harmful (pathogenic) microbes to limit susceptibility to infections. It has also been established that the presence of these microbes - collectively referred to as the microbiota - has profound effects on almost all body systems.
In this project, we ask how a mother's microbiota affects immunity and the organ systems of her babies. In principle we know that a mother's microbiota has very profound effects on her babies in early life, because we are able to use specially constructed microbes that only temporarily colonise the mother’s intestinal tract in the middle of a pregnancy. The (germ-free) babies born to mothers that have been transiently colonized with intestinal microbes have induced more white blood cells of the sort that immediately respond to microbes or microbial infections, and also express a series of proteins and enzymes in the lining cells of the intestine (the epithelium) that protect against the penetration of microbes and potentially metabolise microbial molecules.
These biological issues are important for society because we are studying a period in early life when cellular decisions are being made that have life-long effect on the mammal.
In this project, we ask how a mother's microbiota affects immunity and the organ systems of her babies. In principle we know that a mother's microbiota has very profound effects on her babies in early life, because we are able to use specially constructed microbes that only temporarily colonise the mother’s intestinal tract in the middle of a pregnancy. The (germ-free) babies born to mothers that have been transiently colonized with intestinal microbes have induced more white blood cells of the sort that immediately respond to microbes or microbial infections, and also express a series of proteins and enzymes in the lining cells of the intestine (the epithelium) that protect against the penetration of microbes and potentially metabolise microbial molecules.
These biological issues are important for society because we are studying a period in early life when cellular decisions are being made that have life-long effect on the mammal.
Progress in the project is reported in its different sub-objectives listed below.
i) Extent of organ-system alterations in neonates driven by the effects of the mother's microbiota
The effects of the mother's microbiota are very widespread in the early life offspring. These include not only the alterations in the cellular composition and function of innate immunity, but also alterations in the cellular composition and function in the intestinal epithelium, liver, placenta and bone marrow. The alterations are currently being defined in mechanistic and epigenetic terms in each responsible tissue, and the range of microbial molecules that drive these compositional and cellular function alterations is being investigated using a combination of stimulation with selected molecules and non-isotopically labelled metabolomic analyses. The underlying principles have recently been reviewed by us in a publication in Science, and the role of nutrition in maternal microbial influences has been described in Nature Reviews Immunology.
The exposure to the maternal microbiota is also able to reshape the B cell repertoire in early life. We have recently had work accepted as an original paper by Nature showing how microbial exposure changes the B cell repertoire in adult animals in specific ways, depending on whether that exposure at central body sites (via the bloodstream) or at intestinal mucous membrane surfaces. We are currently applying these research pipelines to understand why the maternal microbial constituents influence the timing and distribution of B cell development in the early life bone marrow.
We have developed a new transitory colonization microbial model system for this research. L. reuteri VPL3028 expressing recombinase recT was used to enable genetic modification by single-stranded DNA recombineering (SSDR) with an oligonucleotide designed to introduce a 99 nucleotide in-frame deletion into the alanine racemase (alr) gene. This live engineered taxon can be given to germ-free mice but is unable to persist, so the recipient mouse becomes germ-free again. The tool is being used to provide a different way of exposing pregnant female mice to assess the consequences of different sorts of microbes on her offspring and to understand how the new-born mucosal immune system generates responses as it becomes exposed to its own microbiota.
ii) Timing of the window in early life when the effects of the maternal microbiota can be manifest.
We know that the timing of priming of the mother influences the sensitivity of the effect of her microbiota molecular transfer on her offspring, probably through the effect of induction of specific antibodies that enhance microbial molecular uptake via the placenta and the milk. We also have carried out timing effects to establish the dose and persistence of the Lactobacillus reuteri tool in neonatal mice as described above.
iii) How the influences of the mother's microbiota affect early-life colonization of the offspring themselves.
We have carried out long-term experiments to study the transmission of microbial variants between successive generations of mice. This has used a closed system whereby a defined 12-organism microbiota has been followed between different generations without allowing new 'immigrant' taxa to enter the colony. Results show subspecies niche sharing. We have also been able to show that minor taxa that form an almost insignificant percentage of the microbial consortium repertoire in adult mice have major representation in neonatal mice - in other words that there is no detectable microbial bottleneck in early life transmission.
iv) Functional effects on health of the offspring as they grow into adulthood.
This is a relatively early stage in the project for determination of the functional effects on the offspring which will vary according to the organ system that is influenced in early life. No differences have been found with the dextran sodium sulfate colitis model or with the non-obese diabetic mouse model, whether the offspring (on reaching adulthood) had been born to a mother that had transient microbial colonization in pregnancy or not. Nevertheless, we do have evidence that there are durable metabolic consequences of being born to a mother containing a microbiota.
i) Extent of organ-system alterations in neonates driven by the effects of the mother's microbiota
The effects of the mother's microbiota are very widespread in the early life offspring. These include not only the alterations in the cellular composition and function of innate immunity, but also alterations in the cellular composition and function in the intestinal epithelium, liver, placenta and bone marrow. The alterations are currently being defined in mechanistic and epigenetic terms in each responsible tissue, and the range of microbial molecules that drive these compositional and cellular function alterations is being investigated using a combination of stimulation with selected molecules and non-isotopically labelled metabolomic analyses. The underlying principles have recently been reviewed by us in a publication in Science, and the role of nutrition in maternal microbial influences has been described in Nature Reviews Immunology.
The exposure to the maternal microbiota is also able to reshape the B cell repertoire in early life. We have recently had work accepted as an original paper by Nature showing how microbial exposure changes the B cell repertoire in adult animals in specific ways, depending on whether that exposure at central body sites (via the bloodstream) or at intestinal mucous membrane surfaces. We are currently applying these research pipelines to understand why the maternal microbial constituents influence the timing and distribution of B cell development in the early life bone marrow.
We have developed a new transitory colonization microbial model system for this research. L. reuteri VPL3028 expressing recombinase recT was used to enable genetic modification by single-stranded DNA recombineering (SSDR) with an oligonucleotide designed to introduce a 99 nucleotide in-frame deletion into the alanine racemase (alr) gene. This live engineered taxon can be given to germ-free mice but is unable to persist, so the recipient mouse becomes germ-free again. The tool is being used to provide a different way of exposing pregnant female mice to assess the consequences of different sorts of microbes on her offspring and to understand how the new-born mucosal immune system generates responses as it becomes exposed to its own microbiota.
ii) Timing of the window in early life when the effects of the maternal microbiota can be manifest.
We know that the timing of priming of the mother influences the sensitivity of the effect of her microbiota molecular transfer on her offspring, probably through the effect of induction of specific antibodies that enhance microbial molecular uptake via the placenta and the milk. We also have carried out timing effects to establish the dose and persistence of the Lactobacillus reuteri tool in neonatal mice as described above.
iii) How the influences of the mother's microbiota affect early-life colonization of the offspring themselves.
We have carried out long-term experiments to study the transmission of microbial variants between successive generations of mice. This has used a closed system whereby a defined 12-organism microbiota has been followed between different generations without allowing new 'immigrant' taxa to enter the colony. Results show subspecies niche sharing. We have also been able to show that minor taxa that form an almost insignificant percentage of the microbial consortium repertoire in adult mice have major representation in neonatal mice - in other words that there is no detectable microbial bottleneck in early life transmission.
iv) Functional effects on health of the offspring as they grow into adulthood.
This is a relatively early stage in the project for determination of the functional effects on the offspring which will vary according to the organ system that is influenced in early life. No differences have been found with the dextran sodium sulfate colitis model or with the non-obese diabetic mouse model, whether the offspring (on reaching adulthood) had been born to a mother that had transient microbial colonization in pregnancy or not. Nevertheless, we do have evidence that there are durable metabolic consequences of being born to a mother containing a microbiota.
The work on the B cell repertoire in press in Nature (Li et al., 2020) shows that the influences of the microbiota on the B cell repertoire are built up with successive exposures, with both the specificity of the responses and the diversity of the resultant repertoire being dependent on the route of microbial exposure.
Long-term experiments with the genomic evolution of microbial strains using strict gnotobiology show the principles of niche sharing and the existence of sub-strains within taxa during early life development.
In collaboration with Professor Randall Platt (ETH Zürich) we have used a strain of Escherichia coli engineered with a CRISPR-Cas based system to capture records of the transcriptional activity of the test strain during intestinal passage. This is providing insights into the intestinal environment perceived by a microbial recording strain in the early life intestine.
We are now dissecting the mechanisms and responsible microbial metabolites for the different organ effects of the maternal microbiota. We anticipate completing and publishing these results for B and T cell repertoire development, liver and metabolic consequences of maternal microbial colonization and the epigenetic marks in the different cell populations that determine their long-term phenotypes.
Long-term experiments with the genomic evolution of microbial strains using strict gnotobiology show the principles of niche sharing and the existence of sub-strains within taxa during early life development.
In collaboration with Professor Randall Platt (ETH Zürich) we have used a strain of Escherichia coli engineered with a CRISPR-Cas based system to capture records of the transcriptional activity of the test strain during intestinal passage. This is providing insights into the intestinal environment perceived by a microbial recording strain in the early life intestine.
We are now dissecting the mechanisms and responsible microbial metabolites for the different organ effects of the maternal microbiota. We anticipate completing and publishing these results for B and T cell repertoire development, liver and metabolic consequences of maternal microbial colonization and the epigenetic marks in the different cell populations that determine their long-term phenotypes.