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.
Section snippets
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).
References (90)
Microglia and cell death in the developing mouse cerebellum
Brain Res. Dev. Brain Res.
(1990)- et al.
Naturally occurring cell death in the cerebral cortex of the rat and removal of dead cells by transitory phagocytes
Neuroscience
(1990) - et al.
Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development
Brain Behav. Immun.
(2013) - et al.
Endocrine immune interactions in human parturition
Mol. Cell. Endocrinol.
(2011) - et al.
Does a prenatal bacterial microbiota exist?
Mucosal Immunol.
(2017) - et al.
Is meconium from healthy newborns actually sterile?
Res. Microbiol.
(2008) - et al.
The hypothalamic arcuate nucleus and the control of peripheral substrates
Best Pract. Res. Clin. Endocrinol. Metab.
(2014) - et al.
Distinct activation profiles in microglia of different ages: a systematic study in isolated embryonic to aged microglial cultures
Neuroscience
(2013) - et al.
Sex and estradiol influence glial pro-inflammatory responses to lipopolysaccharide in rats
Psychoneuroendocrinology
(2012) - et al.
Microglia promote the death of developing Purkinje cells
Neuron
(2004)
Naturally occurring cell death during neural development
Trends Neurosci.
Murine serum cytokines throughout the estrous cycle, pregnancy and post partum period
Anim. Reprod. Sci.
Apoptosis detected in the amygdala following myocardial infarction in the rat
Biol. Psychiatry
Neural-immune interactions: an integrative view of the bidirectional relationship between the brain and immune systems
J. Neuroimmunol.
Dexamethasone induces apoptosis in the developing rat amygdala in an age-, region-, and sex-specific manner
Neuroscience
Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: effects of age and sex
J. Comp. Neurol.
Paradoxical effects of minocycline in the developing mouse somatosensory cortex
Glia
The gut microbiota as an environmental factor that regulates fat storage
Proc. Natl. Acad. Sci. U.S.A.
Entry of blood-borne cytokines into the central nervous system: effects on cognitive processes
Neuroimmunomodulation
The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice
Gastroenterology
Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve
Proc. Natl. Acad. Sci. U.S.A.
Neuroimmune stress responses: reciprocal connections between the hypothalamus and the brainstem
Stress
Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome
Nature
Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4
J. Neurosci.
Age-related differences in neuroinflammatory responses associated with a distinct profile of regulatory markers on neonatal microglia
J. Neuroinflamm.
The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner
Mol. Psychiatry
Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid
Sci. Rep.
Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition
Proc. Natl. Acad. Sci. U.S.A.
The neuroprotective role of inflammation in nervous system injuries
J. Neurol.
Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice
J. Neurosci. Res.
Research review: birth by caesarean section and development of autism spectrum disorder and attention-deficit/hyperactivity disorder: a systematic review and meta-analysis
J. Child. Psychol. Psychiatry
Interleukin-6 in the maternal circulation reaches the rat fetus in mid-gestation
Pediatr. Res.
Dynamics of microglia in the developing rat brain
J. Comp. Neurol.
Maternal intrauterine infection, cytokines, and brain damage in the preterm newborn
Pediatr. Res.
From inflammation to sickness and depression: when the immune system subjugates the brain
Nat. Rev. Neurosci.
Microbiota is essential for social development in the mouse
Mol. Psychiatry
Normal gut microbiota modulates brain development and behavior
Proc. Natl. Acad. Sci. U.S.A.
Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system
Leukoc. Biol.
Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns
Proc. Natl. Acad. Sci. U.S.A.
Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer
Nat. Med.
Host microbiota constantly control maturation and function of microglia in the CNS
Nat. Neurosci.
Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke
J. Neurosci.
Absence of intestinal microbiota does not protect mice from diet-induced obesity
Br. J. Nutr.
Bacterial infection causes stress-induced memory dysfunction in mice
Gut
Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system
FASEB J.
Cited by (56)
Decoding the neurocircuitry of gut feelings: Region-specific microbiome-mediated brain alterations
2023, Neurobiology of DiseaseBirth triggers an inflammatory response in the neonatal periphery and brain
2022, Brain, Behavior, and ImmunityCitation Excerpt :We therefore also examined the phenotype of the microglia in the PVN and CA1 oriens. As previously reported for the perinatal mouse brain (Castillo-Ruiz et al., 2018a; Strahan et al., 2017), most microglia were stout or transitioning (Supplementary Figs. 3 and 4). We also found significant phenotype-by-age interactions for both brain regions and birth modes (vaginal birth: PVN: F12,150 = 3.81, p < 0.0001; CA1 oriens: F12,147 = 3.39, p = 0.0002; Cesarean birth: PVN: F12,138 = 2.06, p = 0.02; CA1 oriens: F12,144 = 8.81, p < 0.0001), due to rapid increases in the number of stout and transitioning microglia between E19 and P3 (Supplementary Figs. 3 and 4).
Animal models for assessing impact of C-section delivery on biological systems
2022, Neuroscience and Biobehavioral ReviewsCitation Excerpt :Consequently, these results indicate that vaginal delivery inhibited cell death in many brain areas, thus having a neuroprotective effect likely to be explained by one or more of the complex processes co-occurring during birth. More importantly, this time-specific cell death has implications well after birth as weaned C-section born mice had less vasopressin-immunoreactive neurons in the PVN compared to vaginally born counterparts (Castillo-Ruiz et al., 2018). Intriguingly, vasopressin is involved in the immune response, the HPA axis as well as in pain and social behaviour (Mavani et al., 2015).
Gut microbiota and neuropsychiatric disorders: Implications for neuroendocrine-immune regulation
2021, Pharmacological Research