Of bugs, genes, development and intestinal biology

By Nancy Fliesler

The collection of bacteria and other microorganisms living in our intestines – our microbiota – is now understood to play an important role in our physiology. Recent research indicates that it helps regulate our metabolism, immune system and other biological processes, and that imbalances in the microbiota are associated with everything from inflammatory bowel disease to diabetes.

Seth Rakoff-Nahoum, MD, PhD, wants to take this understanding to a new level. An infectious disease clinical fellow at Boston Children’s Hospital, he has systematically probed how genetics interact with environment – including the microbiota – to shape intestinal biology during different stages of development.

His investigations provide interesting clues to disorders that have their origins early in life, ranging from necrotizing enterocolitis in newborns to Hirschsprung’s disease (marked by poor intestinal motility) to food allergies.

We’re all a combination of nature (our genetics) and nurture (our environment). While we’re born with our mothers’ microbiota, our intestines and their complement of bugs change from the moment we enter the world. We begin consuming milk and, later, solid foods. Our immune systems – starting with the innate immune system – are developing all the while and learning to distinguish good bacteria from bad.

In their paper published recently in PNAS, Rakoff-Nahoum and his colleagues took the first comprehensive look at the innate immune system and gene expression in the intestines of mice at birth, after weaning and in adulthood (this included testing some 100 poop samples).

To capture the role of the innate immune system, they silenced certain parts of it (namely, interleukin-1 receptors and Toll-like receptors, an ancient system for recognizing microrganisms) in some of the mice, using genetic manipulation. To control for the microbiota present at birth, they compared mice from the same mother and then tracked the bacteria present at each developmental stage. Finally, they crunched all the data with bioinformatics tools.

The investigators bred genetically altered mice, creating some progeny that lacked both Toll-like receptors and IL-1 receptors – two major components of the innate immune system. They then compared these “double-knockout” (DKO) mice with normal wild-type (WT) mice, looking for increases or decreases in gene expression in the small and large intestines at different stages of development. This gave the researchers a total of 24 pairwise data sets. Since all mice had the same mother, they were exposed to the same maternal microbiota. (Images courtesy Seth Rakoff-Nahoum)

“These were the questions: How important are genes? How important is environment? How important are developmental windows? Breastfeeding? What we eat? And what role does the innate immune system play through normative development?” says Rakoff-Nahoum. “I was crunching data in every spare moment post-call during my medical residency.”

He is not a computational biologist, mind you. He originally trained as an immunologist and mastered the essentials of bioinformatics through a four-day course at Cold Spring Harbor. “I wanted to parse my data in as many ways as possible and used as many free online bioinformatics programs as I could,” he says.

The results establish a new perspective on the biology of the normal newborn intestine and the innate system’s role during development. Overall, the study found that about a quarter of the genes whose expression changed during developmental transitions are influenced by the innate immune system.

There were also specific clues to disease that Rakoff-Nahoum hopes will interest pediatric gastroenterologists, nutritionists, neonatologists, infectious disease specialists and allergists/immunologists. For example, when the innate immune system was silenced, the mice had:

• a defect in smooth muscle development in the colon – perhaps a clue to intestinal motility disorders like Hirschsprung’s disease
• an abundance of mast cells in the colon, which could be relevant to food allergies
• improper regulation of the gene for the enzyme that helps digest milk sugar

But these observations represent just the first part of the equation Rakoff-Nahoum wants to tackle. “In this study, we cut out the way the host recognizes the microbes,” he says. “Now I’m cutting out the genes in the microbes to see, one, how other microbes are affected, and two, how the host is affected.”

This “heatmap” reveals differences in gene activity in the small intestine and colon, comparing wild type mice and double-knockout (DKO) mice, which lacked innate immune systems. The blue-and-white-striped box, for example, indicates genes that were more suppressed (down) in wild-type mice as compared with DKO mice during the newborn (suckling) period and just after weaning. The pink-and-white-striped box shows genes that were induced (up) more in the DKO mice than in the wild-type mice during the same developmental stages. The green squares indicate whose changes (up or down) matched the direction of change in innate immune activity.
Leaving his immunology roots behind, Rakoff-Nahoum is now embracing infectious disease – the discipline he plans to specialize in as a pediatrician – and microbial ecology.

“I was trained as a host biologist and am now training as a microbiologist,” he says. “My goal is to be conversant in the languages of both, to be conversant with this ecosystem. You can either run away from complexity or embrace it.”