Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice

Abstract

Indigenous microbiota have several beneficial effects on host physiological functions; however, little is known about whether or not postnatal microbial colonization can affect the development of brain plasticity and a subsequent physiological system response. To test the idea that such microbes may affect the development of neural systems that govern the endocrine response to stress, we investigated hypothalamic-pituitary-adrenal (HPA) reaction to stress by comparing germfree (GF), specific pathogen free (SPF) and gnotobiotic mice. Plasma ACTH and corticosterone elevation in response to restraint stress was substantially higher in GF mice than in SPF mice, but not in response to stimulation with ether. Moreover, GF mice also exhibited reduced brain-derived neurotrophic factor expression levels in the cortex and hippocampus relative to SPF mice. The exaggerated HPA stress response by GF mice was reversed by reconstitution with Bifidobacterium infantis. In contrast, monoassociation with enteropathogenic Escherichia coli, but not with its mutant strain devoid of the translocated intimin receptor gene, enhanced the response to stress. Importantly, the enhanced HPA response of GF mice was partly corrected by reconstitution with SPF faeces at an early stage, but not by any reconstitution exerted at a later stage, which therefore indicates that exposure to microbes at an early developmental stage is required for the HPA system to become fully susceptible to inhibitory neural regulation. These results suggest that commensal microbiota can affect the postnatal development of the HPA stress response in mice.

Figures

Figure 1. Increased plasma ACTH and corticosterone response to restraint stress, but not to ether exposure in GF mice

A, mice were subjected to a 1 h period of restraint stress (GF, n = 6–11 for each time point, total of 52 animals; SPF, n = 6–11 for each time point, total of 50 animals). The baseline data were obtained by cardiac puncture from mice that were killed by cervical dislocation before stress exposure. The baseline ACTH and corticosterone levels in the GF and SPF mice were 49 ± 12 pg ml−1 and 23 ± 4.2 ng ml−1, in the GF mice, 46 ± 13 pg ml−1 and 19 ± 5.6 ng ml−1, respectively. P < 0.05, **P < 0.01, ***P < 0.001 in Dunnett's post hoc test between GF and SPF. B, GF and SPF mice failed to show any difference in HPA response to ether exposure (n = 6 for each time point, total of 30 animals per group).

Figure 2. NR subunit gene expression and protein BDNF concentration

A, a typical RT-PCR product with NR-1 specific primers revealed PCR products with the predicted 333 bp length of NR-1 mRNA. Total RNA was extracted from a single cerebral cortex of GF and SPF mice. NR-1 (B) and NR-2a transcripts (C) were detected by RT-PCR in the cortex, hypothalamus or hippocampus of GF and SPF mice (9 weeks old). Histograms show the relative band intensities on densitometric analysis as ratios of NR subunit and GAPDH mRNA after 30 cycles of amplification (n = 4–7 per group). D, BDNF protein concentration was measured by ELISA (n = 6–10 per group). *P < 0.05, **P < 0.01 by Mann—Whitney U test.

Figure 3. Effects of restraint stress on plasma ACTH and corticosterone levels in gnotobiotic mice

Plasma ACTH and corticosterone levels were measured before or immediately after 1 h restraint in GF (n = 20), SPF (n = 18) and monoassociated mice (n = 18–24 per group) at 9 weeks of age. *P < 0.05, ***P < 0.001 by Dunnett's test.

Figure 4. Kinetics of cytokine concentration in the plasma and c-Fos gene expression in the paraventricular nucleus upon exposure to Bifidobacterium infantis, EPEC or ΔTir

GF mice at 5 weeks of age received a gavage of either 0.5 ml skimmed milk containing one of the bacterial strains (1 × 109 CFU; Bifidobacterium infantis, EPEC or ΔTir) or skimmed milk alone (control), after which plasma and brain samples were collected before (basal) and 6, 12 or 24 h after inoculation with each bacterium. A, the plasma IL-1β level of the mice exposed to EPEC was measured by ELISA (n = 6–8 for each time point). No significant IL-1β elevation in the plasma was found after inoculation with Bifidobacterium infantis, ΔTir or skimmed milk alone (control). B, plasma IL-6 levels were analysed by the B9 cell bioassay as described in the Methods (n = 6–8 for each time point). C, a typical RT-PCR product with c-Fos specific primers revealed PCR products with the predicted 247 bp length of c-Fos mRNA. D, histogram shows the relative band intensities on densitometric analysis as ratios of c-Fos and GAPDH mRNA after 30 cycles of amplification (n = 4 for each time point). *P < 0.05, **P < 0.01 and ***P < 0.001 were considered to be significantly different from the corresponding basal values.

Figure 5. Effects of anti-IL-6 treatment on the c-Fos expression in the paraventricular nucleus and the corticosterone response in the plasma

GF mice at 5 weeks of age were injected intraperitoneally with either anti-IL-6 antibody (MP5-20F3; 50 or 500 μg) or control rat IgG antibody (control) 1 h before being inoculated with Bifidobacterium infantis. The analysis of c-Fos mRNA expression levels in the paraventricular nucleus was done at 6 or 12 h after the inoculation. A, the results shown are representative of 4 independent experiments. B, histogram shows the relative band intensities on densitometric analysis as ratios of c-Fos and GAPDH mRNA after 30 cycles of amplification (n = 4 for each time point). C, determination of the plasma corticosterone levels was carried out according to the protocol described in the Methods (n = 6 for each time point).

Figure 6. Effects of restraint stress on plasma ACTH and corticosterone levels in mice reconstituted with SPF faeces

SPF flora-reconstituted mice were established by orally introducing fresh SPF mouse faeces into the GF mice at either 1 or 3 weeks before they were subjected to the stress protocol. Restraint stress was applied to these reconstituted mice at 9 (A) and 17 weeks of age (B) (n = 18–24 per group). **P < 0.01, ***P < 0.001 by Dunnett's post hoc test.