Microbiota influences neuronal development

One of the hallmarks of brain development is neuronal differentiation with permanent exit from the mitotic cycle. NeuN is a 46/48-kD nuclear protein antigen used widely to identify postmitotic mature neurons in both research and diagnostics49. Increased expression of neurofilament proteins (NFs), which provide support for axonal growth, is closely associated with continued growth of axons and axon diameter50. To examine whether microbial colonization impacts early neuronal development, we measured the expression levels of NeuN and NF, markers of early development in the brain. In our study, we colonized pregnant GF mice at E15 with the human microbiome of interest, and the newborn pups were not separated from the colonized mother until weaning thus the offspring acquired a microbiota reflecting the human microbiome of interest from contact with the mother15. The fecal samples were from the first two postnatal weeks in human preterm infants with two different growth rates (M PI -H and M PI -L) during their NICU course, and resulted in corresponding high and low growth phenotypes in the pup offspring15. In this study, western blot analysis of cerebral cortex homogenates with anti-NeuN antibody showed significantly increased levels of NeuN expression in M PI -H mice colonized with microbiota from a preterm infant with good growth compared with GF pups at two weeks of age (Fig. 1A,B). M PI -L pups colonized with microbiota from a preterm donor with poor growth demonstrated significantly lower NeuN expression compared to M PI -H mice colonized with a preterm donor with a good growth at four weeks of age (Fig. 1C,D). These observations were further demonstrated by immunostaining (Fig. 1E,F) at both two and four weeks of age.

Figure 1 Regulation of neuronal development by gut microbiota. Neuron development was evaluated by western blot and immunohistochemistry. (A, B) Representative images and quantification of western blots of the expression of NeuN at two weeks of age in GF, M PI -L and M PI -H mice (all n = 3). (C, D) Representative images and quantification of western blots of the expression of NeuN at four weeks of age in GF (n = 5), M PI -L (n = 3) and M PI -H mice (n = 4). (E, F) Representative images of immunofluorescence labeling of NeuN (Red) specific for neurons, counterstaining with DAPI (blue) to visualize all cells, showing decreased NeuN in mouse cortex sections in the M PI -L pups compared to M PI -H mice at both two and four weeks of age. (G, H, I, J) Representative images and quantification of western blots showing the expression of NFL at two and four weeks of age, respectively (for two weeks n as indicated for previous data; for four weeks GF (n = 7), M PI -L (n = 5) and M PI -H mice (n = 4)). One-way ANOVA was used to detect the difference among groups. Bars with indicates a significant difference between the two bars (at least p < 0.05). Blots were cropped and the original blots are presented in Supplementary Figure S1. Full size image

There was no significant difference in NFL expression among the experimental mice at two weeks of age (Fig. 1G,H). At four weeks of age, M PI -L mice had significantly lower expression of NFL when compared to M PI -H mice (Fig. 1I,J). Taken together, these data demonstrate a microbiota-dependent delay of neuronal development in the brains of M PI -L mice compared to M PI -H mice.

Microbiota influences oligodendrocyte development

Nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte precursor cells (OPC) but not by any other mature neural cell-type51. OPCs under defined regulation first differentiate into premyelinating oligodendrocytes (OLs) where OL-specific transcription factors such as Olig2 drive the transcription of genes required for differentiation52. Differentiated OLs myelinate axons of neurons to ensure rapid propagation of action potentials and provide metabolic support for axons. Myelin basic protein (MBP) is the major structural element of myelin and is essential for axon myelination, compacting, and wrapping and is developmentally regulated53. Protein expression levels tested by western blot demonstrated that cerebral cortex expression of NG2 was not statistically different among the three experimental groups at two weeks of age (Fig. 2A,B). Expression of Olig2 was higher in M PI -H compared to GF mice at 2 weeks of age (Fig. 2A,C). NG2 and Olig2 were only minimally detected at four weeks of age in all groups (data not shown).

Figure 2 Regulation of cortex myelination by gut microbiota. Development of oligodendrocytes was evaluated by western blot and immunohistochemistry. (A, B, C) Representative images and quantification of western blots showing the expression of NG2, an oligodendrocyte progenitor cell marker, and Olig2, a marker for pre-myelinating oligodendrocytes at two weeks of age in GF, M PI -L and M PI -H mice (all n = 3). Representative images and quantification of western blots showing the expression of MBP (D, E) at two weeks of age in GF (n = 5), M PI -L (n = 5) and M PI -H mice (n = 5) and (F, G) at four weeks of age in GF (n = 7), M PI -L (n = 5) and M PI -H (n = 4) mice. (H, I) Representative images of immunostaining of MBP (green), counterstaining by DAPI (blue) for nuclei, showing the decreased MBP in mouse cortex sections in the M PI -L pups compared with M PI -H mice at both two and four weeks of age. One-way ANOVA was used to detect the difference among groups. Bars with indicates a significant difference between the two bars (at least p < 0.05). Blots were cropped and the original blots are presented in Supplementary Figure S2. Full size image

Immunoblot analysis of cerebral cortex samples from two-week old mice revealed that M PI -L demonstrated significantly reduced MBP expression when compared to M PI -H mice (Fig. 2D,E). MBP expression at four weeks of age was not different among these groups (Fig. 2F,G). These results were further demonstrated by immunostaining as shown in Fig. 2H,I. These data demonstrate that microbiota from a poor growth preterm infant is associated with delayed oligodendrocyte development and myelination in the early (two weeks of age) postnatal brain.

Microbiota influences neurotransmission pathways

Glutamate and γ-aminobutyric acid (GABA) are the main excitatory and inhibitory neurotransmitters in the central nervous system, respectively. Together they are involved in over 85% of the synapses that underlie learning and memory, motor activity, sensory and many other functions in the mammalian cortex54,55,56. Serotonergic and dopamine signaling control mood, sleep, concentration, and motivation. Dysfunction and imbalance of these pathways can contribute to cognitive deficits57,58. It has been suggested that changes in serotonergic signaling may contribute to the altered anxiety phenotype in GF mice59. Since previous molecular and behavioral studies have implicated the gut microbiota in the development of neuronal circuits9,60, we studied the expression of synaptic transmission plasticity-related genes by quantitative RT-PCR (Fig. 3). Microbiota indeed impacted Glutamatergic, GABAergic, serotonergic, dopaminergic and ion channel pathways. Furthermore M PI -L and M PI -H had differential impacts on different neurotransmission pathways. For example, at four weeks of age, M PI -L was generally associated with increased glutamatergic pathway activity demonstrated by increased expression levels (red) and decreased serotonin and dopamine pathway expression levels (blue) in Fig. 3 when compared to M PI -H.

Figure 3 Effect of microbiota on gene expression related to neurotransmission pathways. Expression of genes related to neurotransmission pathways. aRepresents ionotropic receptors and brepresents metabotropic receptors. Transcript values measured by RT-PCR were first normalized to Gapdh (n = 3–9 for two weeks and n = 3–7 for four weeks). Comparison to M PI -H values as fold changes are presented in heatmap format to highlight differential effects on gene expression in GF, M PI -L and M PI -H mice. *Indicates a statistical difference when compared to M PI -H mice. In this pseudo-colored heat map, increasing red intensities indicate genes with higher expression levels compared to M PI -H mice, and increasing blue intensities indicate genes with lower expression levels compared to M PI -H mice. Full size image

Colonization of GF mice with human fecal samples from a preterm infant with poor growth is associated with neuroinflammation in the brain

IL-1β and TNF are major regulators of neuroinflammation associated with an inflammatory/cytotoxic phenotype in the brain61,62. Neuronal nitric oxide synthase (NOS1) is a key enzyme implicated in neurotoxicity in the perinatal cortex63,64. As a potential mechanism by which different microbiota can affect brain development, we examined the overt inflammatory status in the developing brain. Our data showed that at two weeks of age, GF mice had significantly higher Il-1β and Tnf mRNA expression in the brain when compared to M PI -L and M PI -H mice (Fig. 4A,B). M PI -L mice exhibited significantly higher Nos1 expression when compared to GF and M PI -H mice in the cortex (Fig. 4C). These data demonstrate that microbiota can affect the neuroinflammation status of the developing brain.

Figure 4 Effects of microbiota on neuroinflammation. Relative brain transcripts of Il-1β (A), n = 3–9 at two weeks and n = 2–8 at four weeks of age), Tnf (B), n = 3–9 at two weeks and n = 2–6 at four weeks of age), and Nos1 (C), n = 3–8 at two weeks and n = 2–7 at four weeks of age) were measured by RT-PCR. Two-way ANOVA was used to detect the effects of age and microbiota among the groups. Post-hoc test was used to identify differences when a main effect was noted. Bars with indicates a significant difference between the two bars (at least p < 0.05). Full size image

Colonization of GF mice with human fecal samples from preterm infants did not change the brain fatty acids profile

Preterm infants are subjected to brain fatty acid deficiency because fatty acid accumulation in the brain is at the highest rate from the intrauterine and neonatal period up to two years of age37. To evaluate if there were effects of growth phenotype–related preterm infant microbiota on brain fatty acid composition, we measured several PUFAs and myelin-related fatty acid levels in the brain. The levels of C20:4 (AA) (Fig. 5A), C22:6 (DHA) (Fig. 5B) and C18:1 (Oleic acid) (Fig. 5C) at both two weeks and four weeks of age were not different among GF, M PI -L and M PI -H mice, demonstrating the colonization of microbiota from either a poor growth preterm infant or a good growth preterm infant did not influence these fatty acids in the brain.

Figure 5 Effects of microbiota on brain fatty acid profile. Brain AA (A), DHA (B), and oleic acid (C) contents were measured by GC in the brain tissues collected both at two (n = 3–8) and four (n = 3–6) weeks of age. Two-way ANOVA was used to detect the effects of age and microbiota among the groups. Data are presented as mol% ± s.e.m. Full size image

Effects of different microbiota colonization to GF mice on fecal SCFA concentration

SCFAs are among the major metabolites produced by anaerobic bacterial fermentation in the gut65. We found that fecal contents of acetic acid (Fig. 6A), propionic acid (Fig. 6B), butyric acid (Fig. 6C), isovaleric acid (Fig. 6D), hexanoic acid (Fig. 6E), and isobutyric acid (Fig. 6F) were not different among the three groups at four weeks of age, demonstrating the colonization of microbiota from either a poor growth preterm infant or a good growth preterm infant did not influence the SCFA production in the fecal samples in our experimental setting.

Figure 6 SCFA analysis of fecal samples. Effects of microbiota on fecal acetic acid, propionic acid, and butyric acid at four weeks of age were measured by GC. Fatty acid composition is expressed as a percent of total identified fatty acids and concentrations as µg/mg sample. One-way ANOVA was used to detect differences among groups. Full size image

Colonization of germ free mice with human fecal samples from a preterm infant with poor growth resulted in decreased circulating IGF-1, IGFBP3 levels and brain IGF-1 levels and increased Igfbp3 brain transcript levels

To understand the mechanisms by which different growth phenotype related-microbiota colonization resulted in different brain development phenotypes, we tested whether the effect of microbiota is mediated through circulating levels of IGF-1. Serum levels of IGF-1 measured by ELISA were significantly lower in GF and M PI -L mice compared with the M PI -H mice at two weeks of age (Fig. 7A, p < 0.05). At four weeks of age, M PI -L mice still had significantly lower circulating IGF-1 compared to M PI -H mice (Fig. 7B, p < 0.05). In serum, the majority of the IGFs exist in a 150-kDa complex including the IGF molecule, IGF binding protein 3 (IGFBP-3), and the acid labile subunit (ALS). This complex prolongs the half-life of serum IGFs and facilitates their endocrine actions. We found that there was no difference in serum IGFBP3 levels among the mice at two weeks of age (Fig. 7C), but at four weeks of age, IGFBP3 serum level in M PI -L mice was significantly lower than that of the M PI -H mice (Fig. 7D, p < 0.05). These data suggest that poor growth-phenotype related microbiota is associated with reduced circulating IGF-1 and IGFBP3 levels in mouse pups.

Figure 7 Effects of microbiota on the levels of circulating IGF-1 and IGFBP3. Circulating IGF-1 (A,B) and IGFBP3 levels (C,D) were measured by ELISA at both two weeks (n = 3–6) and four weeks (n = 3–8) of age. One-way ANOVA was used to detect the difference among the groups. Bars with indicate a significant difference between the two bars (at least p < 0.05). Full size image

To assess contribution of circulating IGF-1 to brain IGF-1 status, we measured local production of IGF-1 in the brain at both the protein and mRNA level. The brain IGF-1 levels of GF mice were significantly higher than those of M PI -L and M PI -H pups (at least p < 0.05) at two weeks of age (Fig. 8A). At four weeks old, the levels of IGF1 in the brains of both GF and M PI -L mice were significantly lower than those of M PI -H mice (Fig. 8B). However, there were no differences in brain Igf1 (Fig. 8C,D) or Igf1r (Fig. 8E,F) mRNA levels among the three experimental groups at either two weeks or four weeks old of age.

Figure 8 Effects of microbiota on the brain levels of IGF-1, IGF1R and IGFBP3. Brain IGF-1 (A,B) were measured by ELISA at both two weeks (n = 3–7) and four weeks (n = 2–6) of age. Relative brain transcripts of Igf1 (C, n = 2–9 for two weeks and D, n = 2–7 for four weeks), Igf1r (E, 2 weeks) and (F, four weeks), and Igfbp3 (G, two weeks) and (H, 4 weeks) (all n = 3–9 for two weeks and n = 2–8 for four weeks) were measured by RT-PCR. One-way ANOVA was used to detect the difference among the groups. Bars with indicates a significant difference between the two bars (at least p < 0.05). Full size image

IGFBP3 antagonizes the local biologic effects of IGF-1 by having higher affinity for IGF-1 than the IGF-1 receptor66,67. At two weeks of age, there were no differences in Igfbp3 transcript levels among the three experimental groups (Fig. 8G). At four weeks old of age, M PI -L mice had significantly higher Igfbp3 mRNA levels than M PI -H mice (Fig. 8H, p < 0.05). These data demonstrate that poor growth phenotype-associated microbiota resulted in decreased circulating and brain IGF-1, decreased circulating IGFBP3 and increased IGFBP3 brain levels when compared to good growth phenotype-associated microbiota.

Colonization of GF mice with human fecal samples from a preterm infant with poor growth decreased liver IGF-1 levels

Liver synthesis and secretion of IGF-1 is responsible for 80% of the circulating IGF-1. To test whether the effect of different microbiota on circulating IGF-1 level is due to liver production of IGF-1, we examined both the protein and transcript levels of IGF-1 in the liver. IGF-1 tissue levels measured from liver homogenates by ELISA were significantly lower in M PI -L mice compared to GF mice (Fig. 9A, p < 0.05) at four weeks of age. Liver Igf1 transcript levels were not affected by the different microbiota (Fig. 9B). Our data demonstrate that microbiota from a preterm infant with poor growth negatively regulated liver levels of IGF-1.