Microbial modulation via cross-fostering prevents the effects of pervasive environmental stressors on microglia and social behavior, but not the dopamine system

Abstract

Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglial morphology and gene expression as well as decreased dopamine receptor expression and dopaminergic fiber input in the nucleus accumbens (NAc). Importantly, the gut-brain axis has been implicated in ASD, and both microglia and the dopamine system are sensitive to the composition of the gut microbiome. In line with this, we find that the composition of the gut microbiome and the structure of the intestinal epithelium are significantly shifted in DEP/MS-exposed males. Excitingly, both the DEP/MS-induced social deficits and microglial alterations in males are prevented by shifting the gut microbiome at birth via a cross-fostering procedure. However, while social deficits in DEP/MS males can be reversed by chemogenetic activation of dopamine neurons in the ventral tegmental area, modulation of the gut microbiome does not impact dopamine endpoints. These findings demonstrate male-specific changes in the gut-brain axis following DEP/MS and suggest that the gut microbiome is an important modulator of both social behavior and microglia.

Figure 1.. DEP/MS impairs social behavior in male but not female offspring.

a, Schematic of DEP/MS or CON procedure. b, Sociability assay. c, Social investigation time is significantly higher than object investigation time in all groups except DEP/MS males (N=6-8/group, un-paired t-tests (social vs. object): CON M: p<0.001, DEP/MS M: p=0.32, CON F: p<0.01, DEP/MS F: p<0.001). d, Social preference is significantly lower in DEP/MS males as compared to CON males. However, DEP/MS does not alter social preference in females (N=6-8/group; 2-way ANOVA [treatment x sex], treatment: p<0.001, sex: p=0.28, interaction: p<0.001, Bonferonni posthoc: CON male vs. DEP/MS male: p<0.001). e, Social Novelty Preference Test. f, CON males and females significantly prefer a novel social stimuli over a cage mate. DEP/MS males significantly prefer a familiar cage mate, while DEP/MS females show no preference (N=6-8/group, un-paired t-tests (novel vs. cage mate): CON M: p<0.001, DEP/MS M: p=0.04, CON F: p=0.02, DEP/MS F: p=0.84). g, Social novelty preference is significantly lower in DEP/MS males as compared to CON males. However, DEP/MS does not alter social novelty preference in females (N=6-7/group; 2-way ANOVA [treatment x sex], treatment: p<0.001, sex: p=0.23, interaction: p=0.04, Bonferonni posthoc: CON male vs. DEP/MS male: p<0.01). h, Schematic of all four groups design i, In male offspring, neither DEP or MS alone impairs sociability (N=8-10/group; one-way ANOVA [treatment], treatment p<0.001) j, Neither DEP alone, MS alone, nor DEP/MS alters sociability in females (N=7-10/group; one-way ANOVA [treatment], treatment p=0.71). k, Marble-burying. l, DEP/MS had no effect on marble burying behavior (N=8-10/group; 2-way ANOVA [treatment x sex], treatment: p=0.63, sex: p=0.004, interaction: p=0.71). m, Open Field. n, DEP/MS has no effect on center time in the open field (N=8-10/group; 2-way ANOVA [treatment x sex], treatment: p=0.90, sex: p=0.55, interaction: p=0.60). Data represent mean + SEM, *p<0.05. CON: vehicle/control,DEP/MS: diesel exhaust particles/maternal stress. M: males, F: females, E: Embryonic, P: postnatal.

Figure 2.. DEP/MS induces a hyper-ramified phenotype in male but not female microglia.

a, Male, but not female, microglia are hyper-ramified following DEP/MS, as assessed using 3DMorph (N=4-8 animals/group, avg. 11 microglia analyzed/mouse, 2-way ANOVA [treatment x sex], treatment: p=0.012, sex: p=0.55, interaction: p=0.03, Bonferonni posthoc: CON vs DEP/MS males: p=0.02). b, No differences were observed between CON and DEP/MS in cell density (N=7-9 animals/group, 2-way ANOVA [treatment x sex], treatment: p=0.12, sex: p=0.35, interaction: p=0.43) c, Imaris 3D reconstruction revealed larger microglial volume following DEP/MS in males, as well as more branchpoints (d) and more sholl intersections (e)(N=25-30 microglia from 7-8 animals, nested t-tests [CON vs DEP/MS], volume: p=0.02, branch endpoints: p=0.03, sholl intersections: p=0.02). In c and d, pale grey dots represent individual microglia while darker dots represent animal averages. f, Representative reconstructions of CON and DEP/MS microglia, scale=7μm. g, Volcano plots of microglia gene expression reveal numerous genes that are differentially expressed in males following DEP/MS at an adjusted p value cutoff of 0.1 (N=4/group, positive Log2 fold change indicates higher expression following DEP/MS. h, In contrast, very few genes are differentially expressed following DEP/MS in females at the same cutoff (N=4/group, positive Log2 fold change indicates higher expression following DEP/MS). i, GSEA analysis demonstrates up-regulation in pathways related to cell projection assembly and motility in males following DEP/MS (N=4/group, all pathways reached significance with FDR p<0.05). j, Genes related to cell motility and process extension/remodeling were up-regulated in males following DEP/MS. Data represent mean +/− SEM in a-e & j, *p<0.05. CON:vehicle/control, DEP/MS: diesel exhaust particles/maternal stress, N: number, NES: normalized enrichment score, p.m.b.: plasma membrane bound, GSEA: gene set enrichement analysis.

Figure 3.. Chemogenetic activation of VTA dopamine neurons rescues sociability in DEP/MS males.

a, Gene expression in the NAc at P45 for socially relevant receptors in males. mRNA for the dopamine D1 receptor (Drd1), D2 receptor (Drd2) and the kappa opioid receptor (Oprk1) are reduced following DEP/MS in males, while mu opioid receptor (Oprm1) and oxytocin receptor (Oxtr) mRNA are not (N=4-8/group, unpaired t-tests [CON vs DEP/MS], Drd1: p=0.036, Drd2: p=0.003, Oprm1: p=0.12, Oprk1: p=0.008, Oxtr: p=0.48). b, DEP/MS increases Oprk1 mRNA in females as compared to CON, but not mRNA for Drd1, Drd2, Oprm1, or Oxtr (N=4-7/group, unpaired t-tests [CON vs DEP/MS], Drd1: p=0.19, Drd2: p=0.43, Oprm1: p=0.32, Oprk1: p=0.017, Oxtr: p=0.66). c, Representative images of tyrosine hydroxylase (Th) fiber density in the NAc. d) Trend towards a significant interaction effect for Th mean grey value in the NAc with lower signal in DEP/MS males as compared to CON, but higher in females at P45 (N=4-9/group, 2-way ANOVA [treatment x sex], treatment: p=0.81, sex: p=0.016, interaction: p=0.053; CON vs DEP/MS males: p=0.12; females: p=0.22). e, Experimental timeline: Following prenatal DEP/MS, Dat-Cre+ male CON and DEP/MS males underwent stereotaxic microinjection of DREADD virus (Gq [excitatory] or mCherry [control] at PND23-24. At P33-35, social behavior was tested 30 min. after i.p. CNO injection. f, Mouse brain atlas image of location of microinjection. g, Representative 20x image of dopamine fibers (Th) co-labeled with mCherry in the VTA. h) Social preference is significantly increased in DEP/MS males following chemogenetic activation of dopaminergic cells in the VTA (N=7-12/group, one-way ANOVA [treatment], p=0.025). i, Chemogenetic activation of dopaminergic cells in the VTA restores social preference (N=7-12/group, unpaired t-test [social vs. object], p<0.001. Data are represented as mean + SEM, *p<0.05, #p=0.05. VTA: ventral tegmental area, NAc: nucleus accumbens, Th: tyrosine hydroxylase, P: postnatal, CON: vehicle/control, DEP/MS: diesel exhaust particles/maternal stress, i.p.: intraperitoneal, CNO: Clozapine-N-oxide, DREADD: Designer Receptors Exclusively Activated by Designer Drugs.

Figure 4.. DEP/MS shifts the composition of the gut microbiome and intestinal epithelial gene expression and anatomy.

a, Pielou’s evenness was significantly higher in DEP/MS males as compared to CON (N=6-13/group, p=0.02). b, CON and DEP/MS males clustered distinctly in PCoA analyses (N=6-13/group,weighted UniFrac dissimilarity, p=0.03). c-d, No changes in alpha or beta diversity were observed in females following DEP/MS exposure (N=11-13/group, Pielou’s evenness: p=0.66, weighted UniFrac dissimilarity: p=0.29). e, Representative image of section of the ileum assessed. f-g, DEP/MS exposure decreases tight-junction mRNA (Ocln, f; Zo1, g) in males, but increases expression in females (N=4-8/group, 2-way ANOVA [treatment x sex], Ocln: interaction: p<0.01, Zo1: interaction: p<0.01). h, DEP/MS decreases Oprm1 expression in both males and females as compared to CON (N=6-9/group, 2-way ANOVA [treatment x sex], treatment: p=0.02). i-k, DEP/MS has no effect on proinflammatory gene expression in the ileum (Tlr4, i; TNFα, j; Il-1β, N=4-8/group, 2-way ANOVA [treatment x sex], Tlr4: treatment: p=0.82, TNFα: treatment: p=0.38, Il-1β: treatment: p=0.36). l, Representative H&E staining of epithelium within the ileum. lines: orange=mucosal thickness, blue= villus length, grey=crypt length. Scale=100μm. m, DEP/MS increases villi length and mucosal thickness (n) in both males and females as compared to CON but has no effect on crypt length (o; N=4-7 animals/group, 2-way ANOVA [treatment x sex], villi length: treatment: p=0.01, mucosal thickness: treatment: p=0.01, crypt length: treatment: p=0.24). Data represent mean + SEM, *p<0.05. CON: control, DEP/MS: diesel exhaust particles/maternal stress.

Figure 5.. Cross-fostering at birth prevents DEP/MS-induced social deficits in male offspring.

a-c, Stratified Rank-Rank Hypergeometric Overlap (RRHO) analysis on RNA sequencing of isolated microglia from the NAc of male offspring following CON or DEP/MS (N=4/group). a, RRHO analysis revealed significant concordance between DEP/MS-regulated microglia genes as compared to gene differentials in germ-free adult male mice (Thion et al., 2018) b, Significant discordance was observed between DEP/MS-regulated microglia genes as compared to gene differentials in lipopolysaccharide (LPS) treated mice (Hanamsagar et al., 2017). c, Significant discordance was observed between DEP/MS-regulated microglia genes as compared to gene differentials across development (P4-P60; Hanamsagar et al., 2017). d, Schematic of cross-fostering procedure and subsequent behavioral testing and sample collection. e, Neither time spent on the nest, nor licking and grooming (f, L+G= licking and grooming) differed between CON and DEP/MS foster dams (N=6/group, un-paired t-tests [CON vs. DEP/MS]: time on nest: p=0.63, L+G: p=0.86). g, Pielou’s evenness differed significantly according to condition (N=7-9/group, p=0.01). D → fC males had significantly higher evenness within the cecal microbiome as compared to D → fD males (p=0.009) while evenness trended towards a difference between C → fC and D → fD males (p=0.067) h, The composition of the gut microbiome differed significantly according to condition on all four metrics of beta diversity (N=7-9/group, Bray-Curtis: p=0.003, Jaccard Index: p=0.001, unweighted UniFrac: p=0.007, weighted UniFrac: p=0.03, Bray-Curtis shown in PCoA). i-j, LEfSe revealed several genera of bacteria that were differentially abundant in DEP/MS-DEP/MS and DEP/MS-CON males including Helicobacter, Parabacteroides, and Bacteroides (N=7-9/group, 1-way ANOVA [treatment], p=0.002, posthoc: D→fD vs D→fC: p=0.007, D→fD vs C→fC: p=0.006, C→fC vs D→fC: p=0.99). k, Metabolomic analysis of cecal contents revealed a main effect of treatment such that, relative to control, SCFA concentrations differed signficantly in D→fD vs D→fC offspring (N=5/group, p<0.001). In D → fD males, all metabolites differed significantly from C→fC [baseline; single sample t-test] while none of the metabolites did so in D→fC males. l, Fostering to a CON dam on the day of birth restored a preference for the social stimulus (N=7-8/group, unpaired t-tests [Social vs. Object], D → fD: p=0.70, D → fC p=0.003) and significantly increased sociability in male DEP/MS offspring (m, N=7-8/group, unpaired t-test [D → fD vs. D → fC], p=0.005). Data represent Mean +/− SEM, *p<0.05. CON: vehicle and control, DEP/MS: diesel exhaust particles/maternal stress, p: postnatal, w.: weighted, unw.: unweighted, Rel. Ab.: relative abundance.

Figure 6.. Cross-fostering prevents changes in microglial hyper-ramification, but does not alter dopaminergic endpoints.

a, Imaris 3D reconstruction revealed that cross-fostering to a CON dam at birth reduced microglial volume following DEP/MS (a) as well as branchpoints (b) and sholl intersections (c; N=19-26 microglia from 6-8 animals/group, nested one-way ANOVA [C → fC vs. D → fD vs. D → fC], volume: p=0.014, branch endpoints: p=0.02, sholl intersections: p<0.001). In a and b, pale grey dots represent individual microglia while darker dots represent animal averages. d, Representative reconstructions of CON and DEP/MS microglia, scale=10μm. e, Volcano plot of microglia gene expression reveal numerous genes that are differentially expressed in D → fD vs D → fC males at an adjusted p value cutoff of 0.1 (N=4/group, positive Log2 fold change indicates higher expression in D → fC). f, Top most significantly altered genes in D → fD vs. D → fC males. g, Neither D1 nor D2 (h), mRNA expression differed in the nucleus accumbens between D → fD and D → fC males (N=7-8/group, un-paired t-test [D → fD vs. D → fC]; D1R: p=0.23, D2R: p=0.50). i, Representative image of ventral tegmental area (VTA) analyzed. j, Tyrosine hydroxylase + cell number did not differ in the VTA between D → fD and D → fC males (N=7/8/group, un-paired t-test [D → fD vs. D → fC], p=0.73). Data represent mean +/− SEM *p<0.05.