Full-length ArticleThe microbiota influences cell death and microglial colonization in the perinatal mouse brain
Introduction
Birth is an inflammatory event. The onset of labor depends on a state of ‘sterile inflammation,’ marked by a surge of maternal inflammatory cytokines (Golightly et al., 2011, Kobayashi, 2012, Thomson et al., 1999) that may reach the fetus (Dahlgren et al., 2006, Zaretsky et al., 2004) and stimulate brain cytokine expression (Dammann and Leviton, 1997). At parturition the fetus transitions from the relatively sterile environment of the womb to one teeming with microorganisms. Neonatal blood leukocyte populations expand rapidly following birth, likely due to both the stress of birth and sudden antigenic stimulation from maternal and environmental microbes (Marchini et al., 2000, Steinborn et al., 1999, Yektaei-Karin et al., 2007). Whether and how this microbial colonization affects the perinatal brain remains to be determined. However, effects of the microbiota on adult brain physiology and behavior have been reported in a variety of species including humans (Bravo et al., 2011, Clarke et al., 2013, Messaoudi et al., 2011).
One approach to address the question of the effects of the microbiota on perinatal brain development is to examine neonates born into germ-free (GF) conditions, because any role of microbial exposure would be absent in these animals. Neuronal cell death is a major neurodevelopmental event occurring around the time of birth in mice. Roughly 50% of the neurons that are initially produced are eliminated by apoptosis (Oppenheim, 1991). This large-scale pruning of neurons occurs primarily during the first week of life (Ahern et al., 2013) and is crucial for sculpting neuronal circuits. Although the importance of neuronal cell death is widely recognized, some surprisingly basic questions remain, such as what initiates the cell death period, or what accounts for the large regional differences in the magnitude of cell death. We recently found that cell death peaks just after birth in most forebrain regions of C57BL/6 mice (Mosley et al., 2017), suggesting that birth triggers or amplifies cell death.
Microglia are the resident immune cells of the brain and have been causally linked to developmental neuronal cell death (Marín-Teva et al., 2011). Microglia respond to perturbations by initiating an immune response involving the release of pro-inflammatory cytokines such as interleukin (IL) -6, IL-1β and tumor necrosis factor α (TNF-α; Olson and Miller, 2004), and are also activated by peripherally produced cytokines that reach the brain (Chen et al., 2012, Dantzer et al., 2008, Dilger and Johnson, 2008, Godbout et al., 2005, Qin et al., 2007). Microglial number increases during the first few weeks of postnatal life (Crain et al., 2013, Dalmau et al., 2003, Sharaf et al., 2013), and perinatal microglia have a relatively activated morphology and gene expression profile (Christensen et al., 2014, Crain et al., 2013, Lai et al., 2013, Schwarz et al., 2012, Strahan et al., 2017). Microglia may actively promote developmental cell death in the hippocampus and cerebellum (Marín-Teva et al., 2004, Wakselman et al., 2008), but enhance neuronal survival in the cerebral cortex (Arnoux et al., 2014, Ueno et al., 2013).
Adult GF mice have increased microglial numbers and altered microglial morphology (Erny et al., 2015), but whether effects of the microbiota on microglia are present early in life is unknown. Here, we investigated whether the normal exposure to microorganisms that occurs at birth influences cytokine expression, cell death, or microglial colonization of the newborn brain. Compared to conventionally colonized (CC) mice, we found markedly reduced levels of pro-inflammatory cytokine expression in the brains of GF mice on the day of birth and three days later. This was associated with brain-region specific changes in developmental neuronal cell death, and increased microglial density in GF mice. None of these changes were seen in the brains of GF embryos 12 h prior to expected birth. Together, our results suggest that the microbiota plays an important, region-specific role in brain development, and does so within hours of birth.
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Animals
Swiss Webster mice (GF and CC) were obtained from our breeding program at Georgia State University. GF mice were kept under sterile conditions in a Park Bioservices isolator, as previously described (Chassaing et al., 2015), and CC mice were housed under conventional conditions. Mice were maintained in a 12:12 light dark cycle with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at Georgia State University and followed the
The microbiota increases the expression of inflammatory cytokines in the neonatal brain
We examined the expression of anti- (IL-10) and pro-inflammatory (IL-1β, IL-6, and TNF-α) cytokines in the mid- and hind-brain on P0 and P3. While there was no effect of microbiota status on the expression of IL-10 (F1, 76 = 0.18, p = 0.67; Fig. 1A), expression of the pro-inflammatory cytokines was markedly suppressed in the neonatal GF brain: IL-1β was reduced by 87% (F1, 76 = 11.38, p < 0.002; Fig. 1B) and TNF-α by 90% compared to CC mice (F1, 76 = 11.06, p < 0.002; Fig. 1C). There was also an 83%
Discussion
We find that lack of a microbiota alters neonatal brain development. Previously, altered brain physiology and behavior have been reported in adult GF mice. Compared to CC mice, GF adults exhibit a behavioral phenotype including reduced anxiety (Diaz Heijtz et al., 2011, Neufeld et al., 2011), a hyper-responsive stress response (Clarke et al., 2013, Sudo et al., 2004), impaired social behavior (Desbonnet et al., 2014), and impaired memory consolidation (Gareau et al., 2011). The production of
Conclusions
Taken together, our study highlights the importance of microbial exposure for neonatal brain development. Recently, differential exposure to microbiota at birth has been associated with the development of psychological disorders, such as autism (Curran et al., 2015). Millions of years of evolution have shaped the symbiotic relationship between mammalian species and their microbiota, so it is not surprising that our microbial symbionts influence key developmental processes. Moreover, because
Acknowledgments
We thank Geert de Vries, Mary Holder, Carla Cisternas, Nicole Peters, and Laura Cortes for critical comments on earlier versions of this manuscript. We also thank Daniel Cox and Atit Patel for RT-PCR training and Lucie Etienne-Mesmin for technical assistance. Supported by the National Science Foundation IOS-1743673 and the National Institutes of Health R21-MH108345 (to N.G.F). B.C. is a recipient of the Research Fellowship award from the Crohn’s and Colitis Foundation of America (CCFA).
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