The bacterial peptidoglycan-sensing molecule Pglyrp2 modulates brain development and behavior

Abstract

Recent studies have revealed that the gut microbiota modulates brain development and behavior, but the underlying mechanisms are still poorly understood. Here, we show that bacterial peptidoglycan (PGN) derived from the commensal gut microbiota can be translocated into the brain and sensed by specific pattern-recognition receptors (PRRs) of the innate immune system. Using expression-profiling techniques, we demonstrate that two families of PRRs that specifically detect PGN (that is, PGN-recognition proteins and NOD-like receptors), and the PGN transporter PepT1 are highly expressed in the developing brain during specific windows of postnatal development in both males and females. Moreover, we show that the expression of several PGN-sensing molecules and PepT1 in the developing striatum is sensitive to manipulations of the gut microbiota (that is, germ-free conditions and antibiotic treatment). Finally, we used the PGN-recognition protein 2 (Pglyrp2) knockout mice to examine the potential influence of PGN-sensing molecules on brain development and behavior. We demonstrate that the absence of Pglyrp2 leads to alterations in the expression of the autism risk gene c-Met, and sex-dependent changes in social behavior, similar to mice with manipulated microbiota. These findings suggest that the central activation of PRRs by microbial products could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain.

Figure 1

Bacterial peptidoglycan from commensal gut microbiota and its sensing molecules are present in the developing brain. Peptidoglycan (PGN) levels were determined in serum (a) and the cerebellum (b) of specific pathogen free (SPF) and germ-free (GF) juvenile (that is, P25) male mice, as determined using the silkworm larvae plasma assay (serum: n=5 for SPF and n=7 for GF, and cerebellum: n=8 per group; **P<0.01 when compared with SPF control group). (c) The bar graphs show PGN levels in the developing cerebellum of C57BL/6 male mice (n=6 per group; ***P<0.001 when compared with the adult group). (d) PGN levels were measured during cerebellar development in both male (black bars) and female (red bars) mice at P3, P14, and P21 (n=5 for females and n=6 for males). Quantitative RT-PCR was used to examine the mRNA expression levels of Pglyrp1 (e), Pglyrp2 (f), Pglyrp3 (g), Pglyrp4 (h), Nod1 (i), Nod2 (j), Tlr2 (k) and PepT1 (l), which were examined in the striatum of male (black bars) and female (red bars) C57BL/6 mice during postnatal development (n=4 for P1 females and n=5 for the other postnatal age groups). Graphical visualization of PGN-sensing molecules and PepT1 expression levels during striatal development (m). The expression level of each gene examined was normalized to Hsp90ab1 levels and expressed relative to adult male levels (that is, P60). All data (a–l) are presented as means (±s.e.m.). (e–l) *P<0.05, **P<0.01, and ***P<0.001 when compared with their respective adult male group. Differences between male and female mice are indicated as follows: §P<0.05, §§P<0.01. The label is as follows: postnatal day (P).

Figure 2

Expression and cellular localization of PGRPs in the developing brain. Changes in striatal Pglyrp2 (a) and Pglyrp3 (b) protein levels during postnatal development. Protein levels were normalized to the Hsp90 protein levels and expressed relative to the adult group (that is, P60; n=4 per group). All data (a and b) are presented as means (±s.e.m.). *P<0.05, **P<0.01 and ***P<0.001 when compared with their respective adult groups (that is, P60). Distribution of Pglyrp2 in the cerebellum (c), frontal cortex (d–f) and hippocampus (g–i) at P3. Double immunofluorescence staining shows staining of Pglyrp2 (green) combined with either NeuN (a neuronal marker; red), Iba1 (a microglial marker; red), GFAP (an astrocyte marker; red) or calbindin (a marker for purkinje cells; red). The merged images show the colocalization of Pglyrp2 (green) and NeuN (red) in the frontal cortex (d) and hippocampus (g) and with calbindin (red) in the cerebellum (c). Note that Pglyrp2 is moderately colocalization with astrocytes (f and i), and with microglia to a lesser extend (e and h). Arrows indicate Pglyrp2-positive Purkinje cells (c), Pglyrp2-positive microglia (e and h) and Pglyrp2-positive astrocytes (f and i). The scale bars represent 50 μm. Hsp, heat shock protein; P, postnatal day; PGRP, peptidoglycan-recognition protein.

Figure 3

Manipulation of the gut microbiota alters the brain-specific expression of peptidoglycan-sensing molecules early in life. Gene expression levels of Pglyrp1 (a), Pglyrp2 (b), Pglyrp3 (c), Pglyrp4 (d), Nod1 (e), Nod2 (f), Tlr2 (g) and PepT1 (h) in the striatum of three-day-old germ-free (GF) and perinatal antibiotic-treated male and female mice. All data (a–h) are presented as means (±s.e.m.). Significant differences between GF (n=6 for both males and females) and specific pathogen free (SPF; n=5 for both males and females) mice and between perinatal antibiotic-treated (n=5 for males, and n=6 for females) and control (n=5 for males, and n=6 for females) mice are indicated as *P<0.05, **P<0.01 and ***P<0.001.

Figure 4

Genetic disruption of the PGN recognition protein 2 alters the expression of synapse-related genes and social behavior. Gene expression levels of c-Met (a), Bdnf (b) and Syp (c) in the prefrontal cortex and striatum of 3-day-old Pglyrp2 knockout (KO) and wild-type (WT) male and female mice (n=5 for male Pglyrp2 KO mice, n=6 for female Pglyrp2 KO mice and n=6 for WT mice). Bars show time (seconds) spent in the different chambers during the social approach session by Pglyrp2 KO and WT male (d) and female (g) mice (n=8 per group). Bars show time (seconds) spent interacting with the stimulus mouse or in close proximity to the novel object by male (e) and female (h) mice. Representative traces of movement patterns of Pglyrp2 KO (dark green) and WT (light green) male (f) and female (i) mice during the 10-min sociability test. All data (a–e, g and h) are presented as means (±s.e.m.). *P<0.05, **P<0.01 compared with their respective WT mice. PGN, peptidoglycan.