Maternal immune activation (MIA) and poor maternal nutritional habits are risk factors for the occurrence of neurodevelopmental disorders (NDD). Human studies show the deleterious impact of prenatal inflammation and low n-3 polyunsaturated fatty acid (PUFA) intake on neurodevelopment with long-lasting consequences on behavior. However, the mechanisms linking maternal nutritional status to MIA are still unclear, despite their relevance to the etiology of NDD. We demonstrate here that low maternal n-3 PUFA intake worsens MIA-induced early gut dysfunction, including modification of gut microbiota composition and higher local inflammatory reactivity. These deficits correlate with alterations of microglia-neuron crosstalk pathways and have long-lasting effects, both at transcriptional and behavioral levels. This work highlights the perinatal period as a critical time window, especially regarding the role of the gut-brain axis in neurodevelopment, elucidating the link between MIA, poor nutritional habits, and NDD. Fig. 1 EFFECT OF N-3 PUFA DEFICIENCY ON MIA-INDUCED BEHAVIORAL DEFICITS IN NEONATES AND IN ADULT OFFSPRING.: All graphs show Means ± SEM. a Experimental setup. b Average time spent by pups to achieve the Fox battery tests (negative geotaxis and righting reflex; 3 trials per day from PND4 to PND6). N = 14-19. Two-way ANOVA: MIA effect, F(1,62) = 11.67, p = 0.0011. c Average vocalization time (15-min sessions at PND7-8). N = 14-19. Kruskal-Wallis test followed by Mann-Whitney comparison; n-3 sufficient-Saline vs n-3 sufficient-LPS, **p < 0.01. d Neonate average locomotion measured as the distance traveled (in cm/min) during 1 min-session from PND5 to PND8. N = 14-19. Kruskal-Wallis test followed by Mann-Whitney comparison; n-3 sufficient-Saline vs n-3 deficient-saline, **p = 0.009, n-3 deficient-Saline vs n-3 deficient-LPS, ***p < 0.001. e Time course of locomotor activity of newborns from PND5 to PND8. N = 14-19. Two-way ANOVA on repeated measures followed by Bonferroni's multiple comparisons test: n-3 deficient-Saline vs n-3 deficient-LPS, *p = 0.02. f Time course of the distance traveled in the Morris Water Maze during the learning phase (in cm). N = 10. Two-way ANOVA on repeated measures: diet effect, F(1,36) = 9.22, p = 0.004. g Percentage of time spent in the target quadrant. N = 10. One-sample t test; n-3 sufficient-Saline, ***p < 0.001; n-3 sufficient-LPS, **p = 0.004; n-3 deficient-saline, ***p < 0.001; n-3 deficient-LPS, p = 0.11. h. Time course of the distance traveled in the Morris Water Maze during the reversal learning phase (in cm). N = 10. Two-way ANOVA on repeated measures: MIA effect, F(1,36) = 3.27, p = 0.008; time effect, F(1,36) = 19, p < 0.001. i Basal locomotor activity (in cm). N = 12. Kruskal-Wallis test followed by Mann-Whitney comparison; n-3 deficient-Saline vs n-3 deficient-LPS, **p = 0.008. j Time spent in the light box (anxiogenic area) of the light-dark test. N = 11. Kruskal-Wallis test followed by Mann-Whitney comparison. k Percentage of time spent in the center of the open-field arena (anxiogenic area). N = 8-11. Kruskal-Wallis test followed by Mann-Whitney comparison. Fig. 2 DIETARY N-3 PUFA DEFICIENCY EXACERBATES MIA-INDUCED ALTERATIONS OF THE HIPPOCAMPAL LIPID AND TRANSCRIPTIONAL PROFILES IN ADULTHOOD.: Quantification of the levels of total PUFAs (a), n-6 PUFAs (b), DTA n-6 (c) or DPAn-6 (d) in the hippocampus of adult mice, expressed as the percentage of total fatty acids. All graphs show Means ± SEM. N = 6. Two-way ANOVA followed by Bonferroni's multiple comparisons test: Total PUFAs: n-3 sufficient-Saline vs n-3 deficient-Saline, ***p = 0.0001; n-3 deficient-Saline vs n-3 deficient-LPS, *p = 0.0156; n-3 deficient-Saline vs n-3 sufficient-LPS, ***p = 0.0003. Total n-6 PUFAs: n-3 sufficient-Saline vs n-3 deficient-Saline, ***p < 0.0001; n-3 deficient-Saline vs n-3 deficient-LPS, **p = 0.0055; n-3 deficient-Saline vs n-3 sufficient-LPS, ***p < 0.0001; n-3 sufficient-Saline vs n-3 deficient LPS, ***p < 0.0001; n-3 deficient-LPS vs n-3 sufficient-LPS, ***p < 0.0001. DTA n-6: n-3 sufficient-Saline vs n-3 deficient-Saline, ***p < 0.0001; n-3 deficient-Saline vs n-3 deficient-LPS, **p = 0.0013; n-3 deficient-Saline vs n-3 sufficient-LPS, ***p < 0.0001; n-3 sufficient-Saline vs n-3 deficient LPS, ***p = 0.0004; n-3 deficient-LPS vs n-3 sufficient-LPS, *p = 0.0158. DPA n-6: n-3 sufficient-Saline vs n-3 deficient-Saline, ***p < 0.0001; n-3 deficient-Saline vs n-3 deficient-LPS, **p = 0.0045; n-3 deficient-Saline vs n-3 sufficient-LPS, ***p < 0.0001; n-3 sufficient-Saline vs n-3 deficient LPS, ***p < 0.0001; n-3 deficient-LPS vs n-3 sufficient-LPS, ***p < 0.0001. e Venn diagram highlighting the number of genes that were modulated by MIA in the hippocampi of adult n-3 sufficient (blue) or n-3 deficient (red) mice. Lower panel: Number of genes that were up- or down-regulated in n-3 sufficient and n-3 deficient mice. Representation of the 20 most significantly dysregulated genes in n-3 sufficient (f) and n-3 deficient (g) mice. Genes that appear in both n-3 sufficient and n-3 deficient mice are bold. h PCA analysis of MIA-induced differentially expressed genes (DEG) in both dietary groups. Confidence ellipses appear around each group. i, j Gene Ontology analysis of DEGs (light red and blue: up-regulated genes; dark red and blue: down-regulated genes). Fig. 3 EFFECT OF N-3 PUFA DEFICIENCY AND MIA ON MICROGLIA-NEURON CROSSTALK PATHWAYS, SPINE DENSITY, OLIGODENDROCYTE AND MYELIN PROTEIN EXPRESSION.: All graphs show Means ± SEM. a Colocalization of Iba-1 and PSD95 proteins immunoreactivity in the CA1 region of the hippocampus of PND14 pups. Representative confocal image of Iba-1 (green) PSD95 (red) costaining (Top panel: scale bar = 10 µm) and Imaris 3D reconstruction (Bottom panel, scale bar = 1 µm). N = 72-122. Kruskal-Wallis test followed by Mann-Whitney comparisons; n-3 deficient-Saline vs n-3 sufficient-Saline, ***p < 0.0001; n-3 deficient-LPS vs n-3 sufficient-LPS, ***p < 0.0001. b qRT-PCR quantification of microglia-neuron interaction mRNA markers in the hippocampus of PND14 mice (data normalized to the saline group, dotted line). N = 4-6. Kruskal-Wallis test followed by Mann-Whitney comparisons; *p < 0.05, **p < 0.01 (all comparisons in Table S2). c Quantification and representative images of Golgi staining spine density in the CA1 region of the hippocampus at PND28. N = 8-21. Kruskal-Wallis test followed by Mann-Whitney comparisons; n-3 deficient-Saline vs n-3 deficient-LPS, ***p < 0.0001; n-3 deficient-Saline vs n-3 sufficient-Saline, **p = 0.001; n-3 deficient-LPS vs n-3 sufficient-LPS, *p = 0.025. d Western blot-based quantification and representative images of PSD95 protein expression in the hippocampus of PND28 mice. N = 4-8. Kruskal-Wallis test followed by Mann-Whitney comparisons; n-3 deficient-Saline vs n-3 deficient-LPS, **p = 0.004; n-3 deficient-LPS vs n-3 sufficient-LPS, *p = 0.03. Quantification of Olig2 (e), PLP (f), APC (g), MAG (h) and MBP (i) immunoreactivity in the hippocampus of PND14 mice. N = 4-7. Two-way ANOVA. Olig2: diet effect, F(1,20) = 3.48, p = 0.08; MIA effect, F(1,20) = 4.78, p = 0.041. PLP: MIA effect, F(1,22) = 5.01, p = 0.036. APC: diet effect, F(1,17) = 4.96, p = 0.0397. Fig. 4 EFFECT OF N-3 PUFA DEFICIENCY AND MIA ON GUT MICROBIOTA COMPOSITION AT PND14 AND PND21.: All graphs show Means ± SEM. a 16S rRNA-sequencing-based alpha diversity analysis of the microbiota, measured by Shannon index in PND14 mice. N = 8-12. Two-way ANOVA: diet effect, F(1,39) = 12.76, p < 0.001; MIA effect, F(1,39) = 5.39, p = 0.026. Bacteria phyla (b) and family (c) observed in all experimental groups at PND14. d PCA of all subjects at PND14. Confidence ellipses appear around each group. e 16S rRNA-sequencing-based alpha diversity analysis, measured by Shannon index in PND21 mice. N=8-13. Two-way ANOVA: n-3 sufficient-Saline vs n-3 sufficient-LPS, ***p = 0.0005; n-3 sufficient-LPS vs n-3 deficient-LPS, *p = 0.019; n-3 sufficient-Saline vs n-3 deficient-Saline, **p = 0.0078. Bacteria phyla (f) and family (g) observed in all experimental groups at PND21. h PCA of all subjects at PND14. Confidence ellipses appear around each group. Quantification of MLN lymphocytes cytokine release measured by ELISA at PND14 (i) and PND21 (j). N = 6-15; Kruskal-Wallis test followed by Mann-Whitney comparisons; *p < 0.05, **p < 0.01, ***p < 0.001 (all comparisons in Table S2). Z-score of T cells inflammatory reactivity in PND14 (k) and PND21 (l) mice. N = 7-15. Kruskal-Wallis test followed by Mann-Whitney comparisons; PND14: n-3 deficient-Saline vs n-3 deficient-LPS, ***p < 0.001. PND21: n-3 sufficient-Saline vs n-3 sufficient-LPS, **p = 0.0011, n-3 sufficient-LPS vs n-3 deficient-LPS, ***p = <0.0004. Fig. 5 CORRELATIONS BETWEEN MICROBIAL MODIFICATIONS, GUT INFLAMMATION, AND NEUROBIOLOGICAL PARAMETERS.: a Spearman's correlation matrix between gut immune cells reactivity (e.g cytokine release after T-cells stimulation) and bacterial genera in PND14 mice (*p = 0.05). b Spearman's correlation matrix between neurobiological measurements (PLP, Olig2, Iba-1 and MAP2) and bacterial genera in PND14 mice (*p = 0.05). c Spearman's correlation matrix between gut immune cells reactivity (e.g cytokine release after T-cells stimulation) and bacterial genera in PND21 mice (*p = 0.05). d Spearman's correlation matrix between neurobiological measurements (PLP, Olig2, Iba-1, and MAP2) and bacterial genera in PND21 mice (*p = 0.05). e Spearman's correlation between gut immune cells reactivity (e.g released cytokines after stimulation) and neurobiological parameters in PND21 mice (*p = 0.05). N = 20-22. Escherichia-Shig: Escherichia-Shigella; Eubacterium copro: Eubacterium coprostanoligenes group; Lachno NK4A136: Lachnospiraceae NK4A136 group; Lachno UCG-008: Lachnospiraceae UCG-008; Prevo UCG-001: Prevotellaceae UCG-001; Rikenellaceae RC9: Rikenellaceae RC9 gut group; Ruminococcus gg: Ruminococcus gnavus group. f Schematic summarizing the main findings. Exposure of n-3 PUFA deficient dams to MIA alters the gut microbiota composition and increases the inflammatory reactivity of the gut T-lymphocytes in the offspring during the post-natal period. This is correlated with an impairment in microglia-neuron crosstalk during this phase, with consequences on hippocampus function and memory abilities later in life.

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