Maternal gut microbiota mediate intergenerational effects of high-fat diet on descendant social behavior

Abstract

Dysbiosis of the maternal gut microbiome during pregnancy is associated with adverse neurodevelopmental outcomes. We previously showed that maternal high-fat diet (MHFD) in mice induces gut dysbiosis, social dysfunction, and underlying synaptic plasticity deficits in male offspring (F₁). Here, we reason that, if HFD-mediated changes in maternal gut microbiota drive offspring social deficits, then MHFD-induced dysbiosis in F₁ female MHFD offspring would likewise impair F₂ social behavior. Metataxonomic sequencing reveals reduced microbial richness among female F₁ MHFD offspring. Despite recovery of microbial richness among MHFD-descendant F₂ mice, they display social dysfunction. Post-weaning Limosilactobacillus reuteri treatment increases the abundance of short-chain fatty acid-producing taxa and rescues MHFD-descendant F₂ social deficits. L. reuteri exerts a sexually dimorphic impact on gut microbiota configuration, increasing discriminant taxa between female cohorts. Collectively, these results show multigenerational impacts of HFD-induced dysbiosis in the maternal lineage and highlight the potential of maternal microbiome-targeted interventions for neurodevelopmental disorders.

Keywords: CP: Microbiology; CP: Neuroscience; DOHaD; Limosilactobacillus reuteri; intergenerational; maternal diet; microbiome; neurodevelopment; probiotics; social behavior.

Figure 1.. MHFD-descendant F₂ offspring display social dysfunction

(A) Schematic of breeding scheme to produce MRD- and MHFD-descendant F₂ generations with HFD exposure indicated. (B) Timeline of mating and behavior. Eppendorf tubes represent time of stool sample collection. (C) Schematic of Crawley’s 3 chamber (3C) test for sociability and preference for social novelty. (D and E) (D) MHFD F₂ male offspring show impaired sociability (MRD: t(86) = 4.517, p < 0.0001; MHFD: t(86) = 1.735, p = 0.1651) and (E) preference for social novelty (MRD: t(43) = 4.208, p = 0.0003; MHFD: t(43) = 0.1159, p = 0.9916) compared with MRD F₂ males. (F) MRD F₂ females show a strong preference in 3C sociability (MRD: t(28) = 5.406, p < 0.0001), with MHFD F₂ female offspring showing mild deficits in sociability (MHFD: t(28) = 2.181, p = 0.0741). (G–I) (G) Neither MRD nor MHFD F₂ female offspring showed a statistically significant preference for social novelty (MRD: t(28) = 1.934, p = 0.1224; MHFD: t(28) = 1.613, p = 0.222). Representative MRD-descendant (blue) or MHFD-descendant (red) F₂ offspring 3C track plots for (H) males and (I) females. (J) Reciprocal social interaction schematic. (K) Comparison of reciprocal social interaction times revealed decreased interaction among MHFD-descendant F₂ male, but not female, stranger pairs (male MHFD versus MRD: t(39) = 3.103, p = 0.0036; female MHFD versus MRD: t(28) = 1.918, p = 0.0653). Bar graphs show mean ± SEM with individual data points representing biological replicates. (D–G) N = 13–25 subjects per group; (K) N = 14–22 pairs per group.

Figure 2.. F₂ MHFD descendants harbor a distinct gut microbiome from F₂ MRD descendants but show a partial recovery of richness

(A) Principal-coordinate analysis (PCoA) of unweighted UniFrac distances from the averaged rarefied 16S rRNA gene amplicon sequencing dataset (8,310 reads/sample; n = 1,000 rarefactions) revealed statistically significant clusters based on diet and generation (p = 0.001, R² = 0.259). (B–D) Alpha diversity metrics of F₁ females used to generate F₂ offspring revealed a statistically significant loss of microbial diversity in MHFD compared with MRD dams, as measured by observed OTUs (MHFD F₁ versus MRD F₁: t(10) = 3.272, p = 0.0084) and Chao1 index (MHFD F₁ versus MRD F₁: t(10) = 2.494, p = 0.0317). The Shannon diversity index did not differ between cohorts (MHFD F₁ versus MRD F₁: t(10) = 1.204, p = 0.2564). (E–G) No significant differences in alpha diversity between F₂ groups were observed, as measured by observed OTUs (MHFD F₂ versus MRD F₂: t(45) = 1.192, p = 0.2395), Chao1 index (MHFD F₂ versus MRD F₂: t(45) = 0.8097, p = 0.4224), and Shannon index (MHFD F₂ versus MRD F₂: t(45) = 1.274, p = 0.2093). Bar graphs show mean ± SEM with individual data points representing biological replicates. (A–G) N = 5–15 subjects per group.

Figure 3.. LEfSe analysis indicates differentially abundant genus-level taxa between diet cohorts in F₁ and F₂ generations

(A) Histogram of the LDA scores (log10) computed for genus-level taxa with differential abundance in MHFD and MRD F₁ dams. (B) Histogram of the LDA scores (log10) computed for genus-level taxa with differential abundance in MHFD and MRD F₂ offspring. LDA, linear discriminant analysis; LEfSe, LDA effect size. (A and B) N = 5–15 subjects per group.

Figure 4.. Fecal microbiota transplants from both F₂ MRD and MHFD descendants rescue germ-free social deficits

(A) Timeline of germ-free (GF) fecal microbiota transplant (FMT), stool collection for 16S rRNA gene sequencing, and behavioral analysis. (B–D) (B) Representative 3C track plots for GF-Control (black), GF MRD-FMT (blue), or GF MHFD-FMT (red) males. 3C interaction times for (C) sociability (GF-Control: t(20) = 0.5364, p = 0.9349; MRD-FMT: t(20) = 4.688, p = 0.0004; MHFD-FMT: t(20) = 4.429, p = 0.0008) and (D) preference for social novelty (GF-Control: t(20) = 1.355, p = 0.4696; MRD-FMT: t(20) = 3.155, p = 0.0149; MHFD-FMT: t(20) = 4.558, p = 0.0006) showing social dysfunction in GF males is rescued by FMT from either MRD- or MHFD-descendant F₂ donors. Bar graphs show mean ± SEM with individual data points. (E) PCoA of unweighted UniFrac distances from the averaged rarefied 16S rRNA gene amplicon sequencing dataset (3,370 reads/sample; n = 1,000 rarefactions) revealed statistically significant clusters based on diet (p = 0.002, R² = 0.552). (F–H) Alpha diversity metrics did not differ between GF MRD-FMT or GF MHFD-FMT males, as measured by observed OTUs (GF MRD-FMT versus GF MHFD-FMT: t(12) = 1.391, p = 0.1895), Chao1 index (GF MRD-FMT versus GF MHFD-FMT: Mann-Whitney U = 22, p = 0.8518), and Shannon diversity index (GF MRD-FMT versus GF MHFD-FMT: Mann-Whitney U = 12, p = 0.1419). Bar graphs show mean ± SEM with individual data points representing biological replicates. (C–H) N = 7–8 subjects per group.

Figure 5.. L. reuteri rescues social deficits in male F₂ MHFD-descendant offspring and exerts differential effects on F₂ MRD versus MHFD offspring gut microbiota composition

(A) Experimental schematic. Three monogamous MRD lineage or MHFD lineage F₁ breeder cages were used to establish MRD- and MHFD-descendant F₂ generations, respectively. Upon weaning at 3 weeks old, all mice were given drinking water containing 10⁸ CFU/mL L. reuteri daily through behavior assessment. (B) Analysis of the reciprocal social interaction times between pairs revealed no differences between males (male MHFD + L reuteri versus MRD + L. reuteri: t(22) = 0.01855, p = 0.9854), with increased interaction times between F₂ MHFD + L. reuteri females compared with F₂ MRD + L. reuteri females (female MHFD + L. reuteri versus MRD + L. reuteri: t(25) = 6.340, p < 0.0001). (C and D) (C) F₂ male MHFD + L. reuteri offspring show typical preference during sociability (MRD: t(56) = 4.515, p < 0.0001; MHFD: t(56) = 4.373, p = 0.0001) and (D) social novelty (MRD: t(28) = 5.472, p < 0.0001; MHFD: t(28) = 4.28, p = 0.0004). (E–H) (E) Likewise, MHFD + L. reuteri F₂ female offspring show a statistically significant preference during sociability (MRD: t(36) = 2.633, p = 0.0246; MHFD: t(36) = 4.253, p = 0.0003) and (F) preference for social novelty (MRD: t(18) = 0.7226, p = 0.7228; MHFD: t(18) = 3.763, p = 0.0028). Representative MRD + L. reuteri (light blue) or MHFD + L. reuteri (light orange) track plots for (G) males and (H) females, respectively. Bar graphs show mean ± SEM with individual data points. (I) PCoA of unweighted UniFrac distances from the averaged rarefied 16S rRNA gene amplicon sequencing dataset (5,508 reads/sample; n = 1,000 rarefactions) revealed statistically significant clusters based on diet (p = 0.001, R² = 0.148). (J–L) L. reuteri administration differentially affects MRD and MHFD F₂ gut microbiome alpha diversity, as measured by observed OTUs (MHFD F₂ + L. reuteri versus MRD F₂ + L. reuteri: t(50) = 5.285, p < 0.0001), Chao1 index (MHFD F₂ + L. reuteri versus MRD F₂ + L. reuteri: t(52) = 4.143, p = 0.0001), and Shannon index (MHFD F₂ + L. reuteri versus MRD F₂ + L. reuteri: t(50) = 4.436, p < 0.0001). Bar graphs show mean ± SEM with individual data points representing biological replicates. B, N = 10–16 pairs per group; (C–F) N = 10–15 subjects per group; (I–L) N = 24–28 subjects per group.

Figure 6.. LEfSe analysis indicates differentially abundant genus-level taxa between diet lineage in L. reuteri-treated F₂ groups

Histogram of the LDA scores (log10) computed for genus-level taxa with differential abundance in MHFD and MRD F₂ offspring treated with L. reuteri. LDA, linear discriminant analysis; LEfSe, LDA effect size. N = 24–28 subjects per group.

Figure 7.. Neurotypical social behavior is observed in the MHFD-descendant F₃ generation

(A) Breeding schematic. MRD control and MHFD-descendant F₂ mice were bred to produce the MRD- and MHFD-descendant F₃ generations, respectively. (B–H) (B) Comparison of reciprocal social interaction times revealed no differences between male (F₃ MRD versus MHFD: t(22) = 1.824, p = 0.0817) or female pairs (F₃ MRD versus MHFD: t(24) = 0.9925, p = 0.3309). Both MRD- and MHFD-descendant F₃ males demonstrate normal sociability (C) (MRD: t(33) = 2.934, p = 0.0120; MHFD: t(33) = 4.322, p = 0.0003) and preference for social novelty (D) (MRD: t(33) = 5.493, p < 0.0001; MHFD: t(33) = 6.271, p < 0.0001) in the 3C task. F₃ MRD and MHFD female offspring show a statistically significant preference for mouse 1 during the sociability phase of the 3C task (E) (MRD: t(48) = 6.979, p < 0.0001; MHFD: t(48) = 4.663, p < 0.0001), while only MHFD-descendant F₃ females show a preference for social novelty (F) (MRD: t(48) = 1.722, p = 0.4632; MHFD: t(48) = 2.879, p = 0.0118). Representative MRD and MHFD track plots for (G) males and (H) females, respectively. Bar graphs show mean ± SEM with individual data points representing biological replicates. (B) N = 11–15 pairs per group; (C–F) N = 12–20 subjects per group.