Host-Microbe Interactions: Gut Microbiota and its Effects on Developmental Programming of the Brain, Placenta and testis

Abstract: Reproduction of a mammalian organism involves inherited genetic programming and environmental factors that collectively shape organ development and function in the new offspring. One such factor are the indigenous microbiota and their interactions with the host. In mammals, the placenta ensures the supply of nutrition and oxygen to the fetus in utero. Microbes are thought to contribute to establishment of barrier functions, activation of the immune system and supply of nutrients to the host. The objectives of my thesis were to assess whether microbes can modulate barrier functions connected to the placenta, brain, and testis, as well as influence the physiological functions of these organs. All three have distinctive tissue barriers that control the passage of molecules between the blood and tissue in order to optimize function. Paper I – Maternal microbes influence placental development and reduce maternal metabolic stress during pregnancy. Germ-free (GF) pregnant mice exhibited elevated glucocorticoids levels and increased gluconeogenesis and ketone body production. As a result, these dams showed marked impairment of placental development and establishment of the blood-placental barrier (BPB), with impaired capillary microstructure and reduced expression of tight junction proteins (TJPs). Metabolically, GF dams showed altered lipid and carbohydrate metabolism and drastically reduced hepatic levels of glycogen, as well as elevated levels of angiopoietin-4 (ANGPTL4), which is known to inhibit lipoprotein lipase and thus lipogenesis. Paper II – Maternal microbes contribute to the establishment and integrity of the bloodbrain barrier (BBB). During intra-uterine life, the BBB in GF mice was more permeable than that of specific-pathogen-free (SPF) animals, a difference that persisted into adulthood and was associated with reduced expression of TJPs. Exposure of adult GF mice to the gut microbiota of SPF animals reduced this permeability and up-regulated the expression of some TJPs. Furthermore, perfusion with Evans blue revealed that monocolonization of the intestine of adult GF mice with either Clostridium tyrobutyricum, a bacterial strain that produces butyrate, or Bacteroides thetaiotaomicron, which produces mainly acetate and propionate, was sufficient to reduce BBB permeability. Moreover, oral administration of the bacterial metabolite butyrate mimicked this effect. This effect of gut microbiota and butyrate may be mediated by an epigenetic mechanism, since administration of butyrate or monocolonization with Clostridium tyrobutyricum elevated levels of histone acetylation in brain lysates. Paper III – Gut microbes modulate the permeability of the blood-testis barrier (BTB) and regulate endocrine functions of the testis. Establishment of the BTB, which normally occurs 16 days postpartum, was delayed in GF mice. Perfusion with Evans blue demonstrated increased BTB permeability associated with reduced expression of TJPs in these same mice during adulthood. The testis- pituitary axis was also affected by the lack of gut microbiota, since GF mice exhibited lower serum levels of gonadotropins (LH and FSH) and lower intratesticular levels of testosterone than the SPF animals. Interestingly, exposure of GF mice to Clostridium tyrobutyricum restored the integrity of the BTB and normalized testosterone levels. In conclusion, the present work documents the influence of indigenous microbiota on the functions of the murine BPP, BBB and BTB, as well as their ability to support the mother during pregnancy. These findings suggest that microbes contribute to programming during critical windows of development.