Abstract

   Diet-related maternal obesity has been implicated in neurodevelopmental
   disorders in progeny. Although the precise mechanisms and effective
   interventions remain uncertain, our research elucidates some of these
   complexities. We established that a prenatal high-fat diet triggered
   maternal immune activation (MIA), marked by elevated serum
   lipopolysaccharide levels and inflammatory-cytokine overproduction,
   which dysregulated the maternal tryptophan metabolism promoting the
   accumulation of neurotoxic kynurenine metabolites in the embryonic
   brain. Interventions aimed at mitigating MIA or blocking the kynurenine
   pathway effectively rescued the male mice social performance.
   Furthermore, excessive kynurenine metabolites initiated oxidative
   stress response causing neuronal migration deficits in the fetal
   neocortex, an effect that was mitigated by administering the
   glutathione synthesis precursor N-Acetylcysteine, underscoring the
   central role of maternal immune-metabolic homeostasis in male mice
   behavioral outcomes. Collectively, our study accentuated the profound
   influence of maternal diet-induced immuno-metabolic dysregulation on
   fetal brain development and provided the preventive strategies for
   addressing neurodevelopmental disorders.

   Subject terms: Obesity, Autism spectrum disorders
     __________________________________________________________________

   Diet-related maternal obesity is linked to neurodevelopmental disorders
   in offspring, yet underlying mechanisms are unclear. Here, authors show
   that maternal high-fat diets trigger immune-metabolic dysregulation,
   elevating neurotoxic kynurenine metabolites in fetal brains, and
   blocking these pathways rescues social deficits in male mice,
   highlighting preventive strategies for neurodevelopmental disorders.

Introduction

   Diet is a crucial factor in human health and plays a significant role
   in the prevalence of noncommunicable chronic diseases, now at epidemic
   levels^[40]1. Research in the United States indicates that over half of
   all females are either overweight or living with obesity at the time of
   pregnancy^[41]2. Excessive consumption of saturated fats is a primary
   factor contributing to this health concern^[42]3. Recent studies
   suggest that maternal obesity during pregnancy may elevate the risk of
   neurodevelopmental disorders, such as autism spectrum disorder (ASD) –
   characterized by repetitive behaviors and challenges in social
   interaction – in offspring^[43]4,[44]5. Notably, neurodevelopmental
   disorders are two to four times more frequently diagnosed in males than
   in females^[45]6. Despite this, effective treatments for
   neurodevelopmental disorders remain limited^[46]7. Consequently,
   understanding how maternal diet impacts fetal development and health is
   imperative for devising preventive strategies and therapies for
   neurodevelopmental disorders resulting from maternal HFD in the
   progeny.

   Escalating evidence suggests an essential mechanism by which a high-fat
   diet (HFD) influences physiology and health is via the induction of a
   state of chronic metabolic inflammation, termed metaflammation^[47]8.
   Maternal health during pregnancy significantly influences the health
   and disease risk of offspring^[48]9. Rodent model studies show that a
   gestational HFD intensifies the production of inflammatory cytokines
   such as IL-1β, IL-6, and TNF-α in the plasma, liver, and brain tissue
   of both the mother and the fetus^[49]10,[50]11. The maternal immune
   activation (MIA) hypothesis posits that inflammatory disturbances
   during gestation can influence fetal neurodevelopment^[51]12–[52]14,
   and human epidemiological studies substantiate a correlation between
   maternal inflammation during pregnancy and the incidence of
   neurodevelopmental disorders in offspring^[53]15. Meanwhile,
   MIA-induced behavioral changes in offspring exhibit a strong male
   bias^[54]16. Research in animals in which the innate immune system is
   engaged in directing brain masculinization has proposed the hypothesis
   that the male brain experiences a more inflammatory environment than
   the female brain during development, leading to male susceptibility to
   MIA^[55]17. However, little is known about whether maternal
   dietary-immune interactions are involved in disrupting fetal brain
   development and postnatal offspring behavioral phenotypes. Furthermore,
   as each proinflammatory state may involve multiple mechanisms^[56]18,
   more rigorous evidence is required to ascertain the mechanisms linking
   diet-induced MIA with heightened susceptibility to neurodevelopmental
   disorders in male offspring.

   Presently, it is widely acknowledged that tissue-specific and systemic
   immune responses and metabolic regulation are intricately connected,
   with one’s proper function dependent on the other^[57]19. Disturbing
   this immune-metabolic homeostasis can lead to various chronic metabolic
   disorders, particularly neurodevelopmental and autoimmune
   diseases^[58]20. Our recent study indicates that a sustained HFD
   triggers an inflammatory response mediated by gut microbes, which
   disrupts peripheral tryptophan (Trp)-kynurenine (Kyn)
   metabolism^[59]21. Various pathologies of the central nervous system
   (CNS) are accompanied by dysfunction in Trp metabolism^[60]22. The Kyn
   pathway, tightly controlled by the immune system, serves as the prime
   metabolic route for Trp degradation^[61]23. The dysregulation of this
   pathway often results in excess production of neuroactive metabolites
   that regulate glutamate receptor-mediated neurotoxicity and free
   radical production^[62]24. This phenomenon is implicated in
   neurodevelopmental and psychiatric disorders^[63]24,[64]25. However,
   whether disrupted interactions between maternal immune activities and
   metabolic responses help create a mechanistic understanding of maternal
   HFD-induced behavioral deficiencies in male offspring remains the
   subject of study.

   Here we report that prenatal HFD induced inflammatory response in mice
   during pregnancy that precipitated behavioral impairments in male
   offspring, a condition ameliorated by the administration of
   (+)-Naloxone, a toll-like receptor 4 (TLR4) antagonist. Furthermore,
   maternal HFD-induced MIA upregulated the Trp-Kyn pathway in both
   maternal circulation and fetal forebrain, causing the accumulation of
   neuroexcitatory Kyn metabolites in the embryonic brain. Administrating
   HFD-fed mice with 1-methyltryptophan (1MT) effectively inhibited the
   Kyn pathway to rescue social behavioral deficits of male offspring,
   reinforcing the central role of the deregulated Kyn pathway in maternal
   HFD-induced male offspring behavioral deficits. The built-up Kyn
   metabolites initiated the oxidative stress response and lipid
   peroxidation in the fetal brain, causing neuronal migration deficits in
   the cerebral cortex. Supplementing HFD-fed mice with N-Acetylcysteine
   (NAC), a precursor for glutathione synthesis, further confirmed these
   observations during gestation. Collectively, our results provide the
   mechanistic insight into maternal HFD-associated social behavioral
   deficits in male offspring as a consequence of up-regulated MIA
   interfering with the Kyn pathway to cause neurodevelopmental
   abnormalities in the fetal brain.

Results

Differential impact of prenatal and postnatal maternal HFD on male offspring
social behavior

   Current epidemiological research suggests a link between maternal HFD
   and the onset of neurodevelopmental disorders^[65]26. Diverse prenatal
   and postnatal factors can modulate offspring susceptibility to such
   disorders in myriad ways^[66]12. To disentangle the effects of maternal
   pre-pregnancy and postnatal HFD on offspring behavioral outcomes,
   cross-fostering experiments were conducted, involving the exchange of
   newborns between mothers on a standard diet and those on HFD
   (Fig. [67]1a)^[68]27. At six weeks of age, female mice were assigned to
   either a control diet or HFD for 6 weeks, before being paired with
   males to reproduce. The resulting offspring were then switched at birth
   to new mothers, with litters between 1 and 5 days old (which were
   removed) (Fig. [69]1a). Persistent HFD resulted in a significant
   increase in maternal weight (Fig. [70]S1a) and a reduction in litter
   size (Fig. [71]S1b). Despite these maternal effects, no significant
   difference in body weight was observed between male offspring from
   mothers on a control diet (mCD) and those exposed to prenatal HFD
   (mHFD) at postnatal day 35 (P35, Fig. [72]S1c), when behavioral tests
   were conducted (Fig. [73]1a). Notably, pups fostered by mothers on the
   HFD (mC-H) exhibited higher weight gain (Fig. [74]S1c). We evaluated
   social behavior, including sociability and social preference, using an
   adaptation of the three-chamber social interaction paradigm
   (Fig. [75]1b). In testing sociability, we measured the time in which
   the subject mice interacted with either a stranger mouse (Mouse 1) or
   an empty wired cup (Empty, Fig. [76]1b). Male offspring from mHFD
   (oHFD) displayed compromised sociability, as reflected by no preference
   for stranger mice over empty cups (Fig. [77]1c). Alternatively, in
   comparison to oCD male offspring, a reduced interaction time with a
   mouse was evident in oC-H male offspring (Fig. [78]S1d), although they
   demonstrated significant sociability (Fig. [79]1c). Social preference
   was determined by comparing the time spent by mice interacting with a
   familiar (Mouse 1) versus a stranger mouse (Mouse 2). Both oCD and oC-H
   male offspring spent significantly more time interacting with a new
   mouse than a familiar one, indicating normal novelty preference
   (Figs. [80]1d, [81]S1e). In contrast, oHFD male offspring did not
   prefer interaction with stranger mice (Figs. [82]1d, [83]S1e).
   Additionally, sustained maternal HFD both pre-pregnancy and post-partum
   led to increased marble-burying behavior in male offspring, indicating
   an enhanced inclination towards restrictive and repetitive activities
   (Fig. [84]1e). This behavioral deficit was found to be particularly
   pronounced in oHFD male offspring as compared to oC-H male offspring
   (Fig. [85]1e). In conclusion, our results suggest that continuous
   maternal HFD, particularly during the prenatal stage, significantly
   impaired the social behavior in male offspring.

Fig. 1. Maternal high-fat diet impairs male offspring behavioral outcomes.

   [86]Fig. 1
   [87]Open in a new tab

   a Schematic of the maternal diet regimen and breeding. b Schematic of
   the three-chamber social interaction task. c In the sociability test,
   oCD and oC-H male offspring spent more time interacting with a mouse
   than with an empty wire cage, whereas oHFD male offspring showed no
   preference for the mouse (n = 10 mice, 10 litters/group; oCD:
   t = 9.835, p = 1.195e-07; oHFD: t = 0.43, p = 0.966; oC-H: t = 4.202,
   p = 0.0007226). d In the social novelty test, unlike oCD and oC-H, oHFD
   male offspring exhibited no preference for interacting with a novel
   versus a familiar mouse (n = 10 mice, 10 litters/group; oCD: t = 8.153,
   p = 1.931e-07; oHFD: t = 0.244, p = 0.810; oC-H: t = 6.290,
   p = 4.835e-05). e Images (left) and quantification (right) of the
   number of buried marbles (n = 10 mice, 10 litters/group; F
   [2,27] = 9.434, oCD vs oHFD: p = 4.3e-07, oHFD vs oC-H: p = 0.00053,
   oCD vs oC-H: p = 0.036). Data are represented as mean ± SD. In c and d
   p-values were determined by the two-sided unpaired Student’s t-test. In
   e statistical significance was assessed by one-way analysis of variance
   (ANOVA) with Bonferroni’s multiple comparison test. Source data are
   provided as a Source Data file. NS not significant, *p-value ≤ 0.05,
   **p-value ≤ 0.01, ***p-value ≤ 0.001.

Maternal HFD induces immune activation resulting in male offspring behavioral
deficits

   Nutrient and resource absorption by the developing fetus is solely
   reliant on maternal circulation, underlining the importance of maternal
   health during pregnancy in influencing offspring development^[88]28. To
   uncover the effects of dietary insults on systemic homeostasis in
   pregnant mice, we assessed concentrations of 92 proteins in sera of the
   gestational day (GD) 18.5 mice employing the Olink Proteomics method.
   Principal component analysis (PCA) of these proteins revealed a
   distinct separation between mCD and mHFD mice (p < 0.001, Fig. [89]2a).
   Differentially expressed proteins (DEPs) analysis revealed 58 proteins
   significantly disturbed by maternal diet (p < 0.05, Fig. [90]2b).
   Pathway enrichment analysis based on DEPs informed that prenatal HFD
   triggered maternal immune activation (MIA) (Figs. [91]2c, [92]S2a).
   Notably, the interleukin-17 (IL-17) pathway was significantly
   upregulated in mHFD mice (Fig. [93]2c), as evidenced by enhanced
   expression of IL17a and IL17f (Figs. [94]2b, Fig. [95]S2c), which has
   been shown to promote neurodevelopmental disorders in offspring^[96]29.
   Activation of the IL-17 signaling pathway is commonly recognized as a
   response to infectious agents (e.g. Escherichia coli)^[97]30. We
   confirmed, in recent studies, that sustained HFD encourages the
   proliferation of lipopolysaccharide (LPS)-producing bacteria, thus
   increasing circulating endotoxin concentrations, subsequently evoking
   TLR4-mediated systemic inflammatory response^[98]21,[99]31. Similarly,
   prenatal HFD increased serum LPS levels in pregnant mice (Fig. [100]2d)
   and enhanced the LPS-mediated signaling pathway and toll-like receptor
   signaling pathway (Figs. [101]2c, [102]S2a).

Fig. 2. HFD triggers maternal inflammation linked to behavioral deficits in
male offspring.

   [103]Fig. 2
   [104]Open in a new tab

   a PCA score plot of serum proteomic data (n = 6 mice/group). b Volcano
   plots showing serum protein changes in HFD versus normal diet-fed mice
   at GD18.5. c Metascape enrichment network for HFD-altered serum protein
   (p < 0.05). d HFD increased lipopolysaccharide in maternal serum
   (normalized to mCD; n = 6 mice/group; t = 5.462, p = 0.002). e
   Schematic of (+)-Naloxone administration (mH-Nalo). f Volcano plot of
   (+)-Naloxone-altered serum proteins in HFD-fed mice. g (+)-Naloxone
   inhibited IL-17 signaling pathway. h (+)-Naloxone rescued social
   behavior deficits in male mice (n = 10 mice, 10 litters/group;
   sociability: oHFD: t = 0.43, p = 0.966; oH-Nalo: t = 7.562,
   p = 4.143e-06; social preference: oHFD: t = 0.244, p = 0.810; oH-Nalo:
   t = 6.620, p = 8.988e-06). i (+)-Naloxone reduced marble-burying number
   (n = 10 mice, 10 litters/group; t = 7.387, p = 7.513e-07). j IL-17
   cytokine blockade experimental design. IL-17A blocking antibody rescued
   the social performance (k, n = 10 mice, 10 litters/group; sociability:
   t = 5.53, p = 4.43e-05; social preference: t = 3.116, p = 0.006; l,
   n = 10 mice, 10 litters/group; t = 3.729, p = 0.002). Data are
   represented as mean ± SD. In a, permutational multivariate analysis of
   variance (PERMANOVA) by Adonis was used to determine statistical
   significance (F = 3.885, p = 8.412e-06). In b and f statistical
   significance was assessed by a two-sided unpaired t-test, adjusted
   using the false discovery rate (FDR) method. In g the enrichment score
   was calculated, normalized to obtain the Normalized Enrichment Score
   (NES). Nominal P-values were computed using an empirical
   phenotype-based permutation test (999 permutations), with multiple
   comparisons corrected by FDR. In d, h, i, and k–l p-values were
   determined by the two-sided unpaired Student’s t-test. Source data are
   provided as a Source Data file. LPS Lipopolysaccharide.

   Chronic inflammation during pregnancy has been associated with the
   onset of neurodevelopmental and psychiatric complications in
   offspring^[105]12. To determine the role of MIA in HFD-induced
   offspring behavioral deficits, (+)-Naloxone, an antagonist of
   TLR4^[106]32, was administered to HFD-fed mice during pregnancy
   (mH-Nalo, Fig. [107]2e). mH-Nalo mice, compared to mCD mice, exhibited
   prominently different serum protein profiles at GD 18.5 (p = 0.0027,
   Fig. [108]2a). Essential observation was the ability of prenatal
   (+)-Naloxone to inhibit HFD-induced MIA, downregulate the IL-17
   signaling pathway, and suppress the expression of proinflammatory
   cytokines IL-17a and IL-17f (Figs. [109]2f–g, [110]S2b). The inhibitory
   effect of (+)-Naloxone on HFD-induced maternal immune responses was
   confirmed by ELISA assay results (Fig. [111]S2c). This effect
   safeguarded the social behavior traits in the three-chamber test for
   oH-Nalo male offspring (Fig. [112]2h). Moreover, oH-Nalo male offspring
   illustrated lower levels of stereotyped marble-burying behavior
   (Fig. [113]2i). To further explore the role of upregulated IL-17
   expression in maternal HFD-induced social behavior deficits in
   offspring, pregnant mice were systemically administered an IL-17a
   antibody (mH-Anti-IL17a) to block IL-17 signaling or an IgG1 isotype as
   a control (mH-IgG1) beginning at GD0.5 (100 µg/mouse/2 days,
   Fig. [114]2j). Compared to the IgG1 injection, the IL-17a-blocking
   antibody administration effectively rescued social behavioral deficits
   in the offspring (Fig. [115]2k–l). These findings propose the maternal
   HFD-induced MIA as a key player in modulating male offspring behavioral
   deficits.

Association between MIA and upregulation of the maternal Kyn pathway

   While MIA has been previously identified as a critical factor affecting
   the disease-related phenotypes of the offspring^[116]33, there is still
   limited understanding of how MIA contributes to brain development.
   Empirical studies provide convincing evidence of obesity and dietary
   factors, despite their proinflammatory nature, on neurodevelopment
   through metabolic stress, oxidative stress, and neuroendocrine
   mechanisms^[117]34. To uncover the response of serum metabolites to the
   maternal diet, we conducted untargeted metabolomics on the serum of
   mice at gestational day 18.5 (GD18.5). PCA results demonstrated a
   significant variation in serum metabolic profiles between mice scores
   in the mCD group and those in the mHFD group along dimension 2
   (Fig. [118]3a). The variation in serum metabolites was found to be
   mitigated by (+)-Naloxone administration (Fig. [119]3a), alluding to a
   potential association between diet-induced MIA and maternal metabolic
   transitions. Co-expression analysis is a common tool to investigate
   molecular pathways that underlie disease phenotypes^[120]35. To
   elaborate on the metabolic response connected with maternal HFD-induced
   immune response, a weighted correlation network analysis (WGCNA) was
   performed on the 8950 serum metabolites from the 18 samples (n = 6 for
   mCD, mHFD, and mH-Nalo). This analysis clustered the serum metabolites
   into 16 co-expression modules, each normatively designated by a
   specific color (Fig. [121]3b). Subsequent analyzes of these modules
   were used to estimate eigenmetabolite, as a summary metric signifying
   the aggregate metabolite expression of each module. Correlation
   analysis between eigenmetabolite and serum proteins, impacted by
   maternal diet (n = 58), demonstrated that the MEorange and MEblack
   modules were positively co-varied with the serum levels of
   proinflammatory cytokines such as IL1b, IL6, Tnfrsf11b, IL17a, and
   IL17f (Fig. [122]3c). Notably, (+)-Naloxone administration effectively
   reversed the maternal HFD-influenced patterns of eigenmetabolite in the
   MEblack module but not in the MEorange module (Fig. [123]3d),
   suggesting that the metabolites in the MEblack module were more
   susceptible to inflammation related to maternal HFD.

Fig. 3. HFD-induced MIA promotes the kynurenine pathway in maternal
circulation.

   [124]Fig. 3
   [125]Open in a new tab

   a PCA of serum metabolic profiles (n = 6 mice/group). b WGCNA cluster
   dendrogram grouped serum metabolites into modules M1-16. c Correlation
   heatmap between metabolite modules and HFD-altered serum proteins. d
   Module eigengene expression across groups (n = 6 mice/group; MEblack: F
   [2,15] = 79.902, mCD vs mHFD: p = 2.41e-08, mHFD vs mH-Nalo:
   p = 3.62e-08; MEorange: F [2,15] = 5.429, mCD vs mHFD: p = 0.012, mHFD
   vs mH-Nalo: p = 0.058). e qMSEA identified metabolic pathways of
   MEblack module. f Summary of perturbed tryptophan metabolism pathways
   in serum (n = 6 mice/group; mCD vs mHFD: Trp, t = 5.002,
   p = 0.0002847077; Kyn, t = 5.569, p = 0.0001679119; 3-HK, t = 5.004,
   p = 0.0003999991; Kyna, t = 3.884, p = 0.003; Quin, t = 2.594,
   p = 0.03; Xanthurenic Acid, t = 2.994, p = 0.01; 3-HAA, t = 6.103,
   p = 7.704395e-05; Serotonin, t = 4.228, p = 0.002; Melatonin,
   t = 3.216, p = 0.009; ILA, t = 2.591, p = 0.03; Tryptophol, t = 2.611,
   p = 0.03; mHFD vs mH-Nalo: Trp, t = 4.511, p = 0.001; Kyn, t = 4.383,
   p = 0.001; 3-HK, t = 4.994, p = 0.0004063576; Quin, t = 4.517,
   p = 0.001; Xanthurenic Acid, t = 3.629, p = 0.005; 3-HAA, t = 4.738,
   p = 0.0006114107; Melatonin, t = 2.443, p = 0.03; Tryptophol,
   t = 4.005, p = 0.002). In d data are represented as mean ± SD. In a
   Adonis was used to determine statistical significance (F = 7.704,
   p = 5.374e-07). In c statistical significance was determined using a
   two-tailed unpaired Student’s t-test, with multiple comparisons
   corrected using the FDR method. Statistical significance in (d) was
   determined using a ANOVA followed by Dunnett’s multiple comparison
   test, and in (f) a two-tailed unpaired Student’s t-test was used. In e
   p-value was determined using permutation testing (999 permutations)
   with adjustment using FDR. Enrichment ratio = Hits / Expected. Source
   data are provided as a Source Data file.

   To extract more insight into MIA-induced metabolic stress, quantitative
   metabolite sets enrichment analysis (qMSEA) was performed on the
   metabolites of the MEblack module, leading to the revelation that
   pathways relating to Trp metabolism and glutathione metabolism were
   significantly overrepresented (Fig. [126]3e). Our previous study
   exhibited a strong association between disrupted Trp metabolism and
   neurological disorders in mice with obesity^[127]31. Consequently, this
   work further scrutinized the levels of Trp metabolites in the maternal
   serum and found an evident upregulation of the Kyn pathway in mHFD
   mice, as marked by depleted Trp reserves (0.44-fold) and increased
   levels of Kyn (2.59-fold), 3-hydroxykynurenine (3-HK, 2.42-fold),
   3-hydroxyanthranilic acid (3-HAA, 2.23-fold), xanthurenic acid
   (1.83-fold), and quinolinic acid (Quin, 1.55-fold) (Fig. [128]3f).
   Notably, (+)-Naloxone administration significantly reduced maternal
   serum levels of Kyn metabolites, while exerting minimal effects on
   serotonin and other metabolites in the indole pathway (Fig. [129]3f).
   Concomitantly, consistent with the beneficial effects on offspring
   social behavioral phenotypes, blockade of the IL-17 pathway effectively
   inhibited the accumulation of Kyn and 3-HK in maternal serum
   (Fig. [130]S3f). These findings suggest that the HFD-induced maternal
   immune response, particularly the activation of the IL-17 pathway,
   predominantly triggered the Kyn pathway of Trp metabolism in the
   maternal circulation.

Maternal HFD-induced MIA promotes the Kyn-Quin pathway in the fetal forebrain

   Kyn and its associated metabolites, cumulatively termed “kynurenines,”
   are renowned for their effects on the CNS and have been implicated in
   numerous mental and psychiatric disorders^[131]36. Like Trp, Kyn and
   3HK readily cross the blood–brain barrier^[132]37. A distinctive
   metabolic pattern (Fig. [133]S3a) coupled with elevated levels of Kyn
   and 3HK (Fig. [134]S3b), consistent with findings in the maternal serum
   (Fig. [135]S3c–e), was observed in the mHFD fetal forebrain at
   embryonic day 18.5 (E18.5). Microglia, the resident innate immune cells
   in the CNS, control the Kyn-Quin metabolism(Fig. [136]4a)^[137]38. We
   noticed that microglia (Iba1-positive cells) were primarily located in
   the ventricular zone (VZ) of the E18.5 fetal neocortex (Fig. [138]4b).
   Consistent with the enhanced maternal inflammatory responses
   (Fig. [139]2b–d), gene set enrichment analysis (GSEA) based on RNA-seq
   data indicated significant upregulation of the “inflammatory response
   LPS” pathway in mHFD fetal brain at E18.5 (Fig. [140]4d). Extant
   studies have highlighted that LPS exposure accelerates microglia
   development in mice (Fig. [141]4e)^[142]39. As microglia function in
   response to environmental cues, alterations in microglia morphology can
   point to changes in CNS immune activities and potential
   dysfunction^[143]40. Examining the density and morphological features
   of microglia, we found an uptick in microglia density (Fig. [144]4f)
   and complexity of traits, such as cellular cytosol area, process
   number, and average process length (Figs. [145]4c, [146]S4a), in mHFD
   fetal brain, indicating an increased inflammatory response. Besides,
   there was a significant rise in the kynurenine 3-monooxygenase (KMO)
   mRNA expression in the mHFD fetal brain (Fig. [147]4g), driving the
   transformation of Kyn to neuroactive and neurotoxic metabolites
   (Fig. [148]4a)^[149]41. This metabolic shift was further validated by
   the upregulation of the “Trp metabolic process” pathway, characterized
   by higher kynureninase (KYNU) mRNA expression, in the mHFD fetal brain
   (Fig. [150]S4c). Furthermore, metabolomics analysis distinguished 1932
   significantly modified metabolites between mCD and mHFD E18.5 fetal
   forebrain (p < 0.05), interplay with Trp metabolism and
   anti-inflammatory pathway (Fig. [151]4j). Consequently, Quin level in
   the fetal brain of mHFD mice was substantially higher compared to that
   of control diet mice (Fig. [152]4i).

Fig. 4. Maternal HFD enhances Quin production in the fetal forebrain.

   [153]Fig. 4
   [154]Open in a new tab

   a Kyn metabolism in microglia. b Confocal images and skeleton analysis
   of microglia labeled with Iba-1 in fetal brain. c Microglia
   morphologies in mCD and mHFD fetal brain (n = 30 cells from 3 mice).
   GSEA showing enrichment of the inflammatory response LPS pathway (d)
   and main fetal microglia pathway (e) (n = 3 for each group).
   (+)-Naloxone administration decreased the microglial density (f) n = 9
   slices from 3 mice, 3 litters/group; F [2,24] = 43.474, mCD vs mHFD:
   p = 1.80e-08, mHFD vs mH-Nalo: p = 2.08e-07) and mRNA expression of
   Kynurenine-3-monooxidase (KMO) (g) n = 6 mice, 6 litters/group; F
   [2,15] = 59.674, mCD vs mHFD: p = 8.09e-08, mHFD vs mH-Nalo:
   p = 6.98e-07) in the fetal brain. The concentrations of Kyn (h) F
   [2,15] = 5.516, mCD vs mHFD: p = 0.187, mHFD vs mH-Nalo: p = 0.011) and
   Quin (i) F [2,15] = 9.394, mCD vs mHFD: p = 0.002, mHFD vs mH-Nalo:
   p = 0.009) in the fetal brain tissue among three groups (n = 6 mice, 6
   litters/group). j qMSEA identified the metabolic pathways in the fetal
   brain significantly perturbed by maternal HFD (p < 0.05). Data are
   represented as mean ± SD. In d,e NES was calculated as described above.
   Statistical significance was determined using permutation testing (999
   permutations), with FDR correction. In g–i data are normalized to mCD.
   In f–i p-values were determined by ANOVA with Dunnett’s multiple
   comparison test. In j, enrichment ratio was calculated as described
   above, and statistical significance was assessed through permutation
   testing (999 permutations) with FDR correction. Source data are
   provided as a Source Data file. KYNU Kynureninase, KMO Kynurenine
   3-monooxygenase, Iba-1 Ionized calcium binding adapter molecule 1, DAPI
   4’,6-Diamidino-2-phenylindole, NMDAR N-methyl-D-aspartic acid receptor,
   α7nAChR α7 nicotinic acetylcholine receptor, AMPAR
   α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

   The Kyn pathway is known to be activated by proinflammatory cytokines
   and TLR ligands^[155]42. Interestingly, (+)-Naloxone administration in
   HFD-fed mice during pregnancy inhibited the peripheral Trp-Kyn pathway
   (Fig. [156]3f). Concurrently, the hyperactivity of microglia in the
   fetal forebrain of mH-Nalo was mitigated (Fig. [157]S4d), resulting in
   decreased microglia complexity and density (Figs. [158]4b, [159]4f,
   [160]S4a). The transcriptional profile of the mH-Nalo fetal brain was
   analogous to that of control diet mice (Fig. [161]S4b), indicating that
   MIA predominantly determined the maternal HFD-triggered disruption in
   fetal brain development. Furthermore, with (+)-Naloxone administration,
   the decrease in KMO and KYNU mRNA expressions (Figs. [162]4g, [163]S4d)
   led to the restraining of the Trp metabolism pathway (Fig. [164]S4d),
   which subsequently inhibited the production of Kyn metabolites (e.g.
   3HK and Quin) in the fetal forebrain (Figs. S3b, [165]4i). Similarly,
   inhibition of the Trp-Kyn metabolic shift in the embryonic brain was
   observed in mH-Anti-IL17 (Fig. [166]S3g). Lastly, the enhancement in
   kynurenic acid (Kyna) concentration in mH-Nalo fetal forebrain was
   noticed (Fig. [167]4h), a compound linked with neuronal
   protection^[168]43. In summary, our findings suggest a predominant role
   of maternal diet-induced immune activation in Kyn pathway activation
   and neurotoxic Quin accumulation in the fetal brain.

Maternal Trp modulation affects behavioral outcomes in the male offspring of
HFD-fed mice

   It was observed that maternal HFD-induced MIA brought about Trp
   metabolism dysfunction. Hence, we sought to understand if this
   metabolic response contributed to social behavioral deficits in
   progeny. Prior research indicated that serum Trp metabolites increase
   dose-dependently with dietary Trp intake^[169]44. Subsequently, Trp
   addition to the drinking water (0.8%)^[170]45 of gestational mice on a
   control diet (mC-Trp) or HFD (mH-Trp) was carried out (Fig. [171]5a).
   As envisaged, this led to a boost in Trp level in maternal circulation
   and fetal forebrain (Fig. [172]5b). Interestingly, the oC-Trp male
   offspring showed no significant difference in their social performance
   when compared with oCD male offspring (Figs. [173]5c, [174]S5a–c). On
   the other hand, extra Trp supplementation in HFD-fed mice led to
   enhancements in Kyn pathway metabolites, especially marked in Quin
   production within the fetal brain (Fig. [175]5b). Additionally, more
   severe stereotypic behavior was observed in the marble-burying test as
   per the oH-Trp male offspring, though the three-compartment test showed
   no difference when compared to oHFD male offspring, a potential floor
   effect due to low social performance (Figs. [176]5c, [177]S5a–c). To
   reaffirm the influence of maternal Kyn metabolites concerning social
   behavioral deficits in offspring, gestational mice under HFD were
   treated with 1-methyltryptophan (1MT), an indoleamine-pyrrole
   2,3-dioxygenase 1 (IDO1) antagonist to inhibit Kyn production (mH-1MT,
   Fig. [178]5a). By inhibiting the Kyn pathway (Fig. [179]5b), 1MT
   effectively counteracted maternal HFD-induced behavioral deficits,
   evident through reduced marble-burying and improved three-chamber test
   performance in the oH-1MT male offspring (Fig. [180]5c, [181]S5a-c).

Fig. 5. Maternal Trp modulation impacts embryonic brain development and
offspring behavioral phenotypes.

   [182]Fig. 5
   [183]Open in a new tab

   a Schematic of dietary Trp modulation. b Heatmap of Trp metabolite
   levels in maternal circulation and fetal brain (n = 6 mice/group).
   T-values were indicated (right). c Trp modulation affected male
   offspring’s marble burying test (n = 10 mice, 10 litters/group; oCD vs
   oC-Trp, t = 0.677, p = 0.507; oHFD vs oH-Trp, t = 2.717, p = 0.014;
   oHFD vs oH-1MT, t = 4.096, p = 0.000615341). d Fuzzy c-means clustering
   of gene expression patterns in fetal forebrain (n = 3 mice/group). e
   Coronal brain section showing somatosensory and motor cortex
   assessment. f Density of BrdU-positive cells located in each laminar
   bin (n = 9 slices from 3 mice; bin1: t = 4.694, p = 0.0002088483; bin2:
   t = 2.998, p = 0.009; bin5: t = 2.859, p = 0.011; bin6: t = 2.469,
   p = 0.025; bin7: t = 3.615, p = 0.002; bin8: t = 7.465,
   p = 9.243799e-07; bin9: t = 5.881, p = 1.814603e-05; bin10: t = 3.159,
   p = 0.006). g, h Cortical cell density evaluation (n = 9 slices from 3
   mice; t = 3.653, p = 0.002). Cortical columns were divided into ten
   equal-sized bins (i) and the number of Brn2-positive cells counted in
   each (j) n = 9 slices from 3 mice; bin1: t = 2.389, p = 0.030; bin2:
   t = 2.408, p = 0.028; bin3: t = 3.244, p = 0.005; bin5: t = 2.314,
   p = 0.034; bin6: t = 2.168, p = 0.046; bin7: t = 3.146, p = 0.006;
   bin10: t = 3.999, p = 0.001). Bin 10 represents the upper margin of the
   SVZ. k The density of Brn2-positive cells (n = 9 slices from 3 mice;
   t = 1.079, p = 0.297). Data are represented as mean ± SD. Statistical
   significance was assessed by the two-sided unpaired Student’s t-test.
   Source data are provided as a Source Data file. Brdu bromodeoxyuridine,
   BLBP Brain lipid-binding protein, PI Propidium Iodide.

   To unravel the mechanisms responsible for generating behavioral
   deficits in offspring as a result of maternal Kyn pathway dysfunction,
   the fuzzy C-means algorithm^[184]46 was employed on RNA-seq data
   obtained from the E18.5 fetal forebrain (Fig. [185]5d). We recognized a
   total of ten distinct gene clusters that responded to various
   regulatory factors (Figs. S6, [186]5d). Notably, the relative
   expression of clusters 1 and 6 correlated significantly with Kyn
   concentration in the fetal forebrain (Fig. [187]5c). Pertinently, GSEA
   based on these two clusters revealed that the processes related to
   microtubule organization and movement, crucial for neuronal migration,
   were down-regulated, indicating compromised development and
   localization of neurons in mHFD fetal forebrain (Figs. [188]S7a, b).
   Thus, these findings suggest that the dysregulated Kyn pathway in the
   fetal brain may detrimentally affect the male offspring social behavior
   by impacting the migration and localization of newborn neurons.

   The brains of patients diagnosed with ASD, a condition notable for
   impaired communication and reciprocal social interaction, have been
   found to possess a disorganized cortex with irregular laminar
   cytoarchitecture^[189]47. To study the proliferative activity of fetal
   neural precursor and the migration of newborn neurons, pregnant mice
   received a single injection of bromodeoxyuridine (BrdU, 50 mg/kg), a
   thymidine analog that incorporates into the DNA of dividing cells
   during the S-phase of the cellular cycle, at GD14.5. These mice were
   then sacrificed at GD18.5 to collect embryonic brains for subsequent
   analyzes. Cortical development ordinarily progresses in an inside-out
   manner, with the neurons of the inner layers born first^[190]48. As
   gestation continues, newborn neurons migrate past these initial layers
   to sequentially inhabit the exterior layers of the cortex. Our results
   showed that maternal HFD had no significant impact on the proliferative
   activity of neural stem cells located in the ventricular and
   subventricular zones (VZ/SVZ) (Figs. [191]S8a, b). Both the relative
   abundance of neurons and the distribution of cells within the cortical
   laminae are crucial to preserving the functional integrity of the
   neocortex^[192]49. Therefore, we counted cortical tissues to ascertain
   the distribution of BrdU-positive cells across 10 equally distributed
   laminar areas or “bins” (laminar distribution) (Fig. [193]5e). We
   observed a significant disruption in the laminar distribution of cells
   detected in the brains of mHFD fetuses, detailed in a depletion of
   cells born on E14.5 from the outer layers (bins 1 and 2) and an
   overrepresentation in the inner layers (bins 5-10) (Fig. [194]5f). We
   also observed a significant upsurge in BrdU-positive cell density
   across the entirety of the cortex in mHFD fetal forebrains
   (Fig. [195]5g–h). To confirm the arrest of neuronal migration in the
   deeper neocortical layers, cortical sections were immunostained for
   Brn2, a marker predominantly expressed in neurons situated in
   superficial layers (Fig. [196]5i). Consistent with the disrupted
   distribution of BrdU-positive cells in the fetal neocortex, the deeper
   layers revealed a substantial delay in Brn2-labeled cells
   (Fig. [197]5j). Notably, the maternal HFD did not show any discernible
   effects on the density of Brn2-positive neurons (Fig. [198]5k),
   suggesting that this dietary regimen did not influence the process of
   Brn2-positive neuron differentiation and maturation. Consistent with
   the improved social behaviors observed in male offspring
   (Fig. [199]2h–i), (+)-Naloxone administration effectively ameliorated
   the disruption of neuronal migration within the fetal neocortex
   (Fig. [200]S8c–f). These results highlight the critical influence of
   MIA in mHFD fetal neurodevelopmental disorders. Mechanistic insights
   gleaned from RNA-seq analysis (Figs. [201]5d, [202]S7a, b) informed our
   subsequent investigations toward the potential role of Kyn pathway
   dysregulation in fetal cortical abnormalities caused by maternal
   HFD-induced MIA. Examination of cortical development in mH-1MT fetuses
   revealed that the inhibition of the maternal Kyn pathway significantly
   counteracted the impaired neuronal migration in the embryonic neocortex
   (Fig. [203]S8c–f), establishing causal links between MIA-dysregulated
   Kyn metabolism and consequential deficits in male offspring social
   behavior resulting from cortical disorganization.

Inhibition of the maternal Kyn pathway attenuates oxidative stress in the
fetal brain

   Most metabolites of the Kyn pathway have been observed to stimulate the
   release of synaptosomal glutamate (Fig. [204]S9a), exerting detrimental
   effects via various mechanisms^[205]50. These include the production of
   reactive oxygen intermediates and the consumption of endogenous
   antioxidants^[206]51. Metabolomics carried out on both maternal serum
   and fetal forebrain revealed that the maternal HFD significantly
   perturbed the glutathione metabolism pathway (Figs. [207]3e, [208]4j),
   which is integral to antioxidant defense and cellular event
   regulation^[209]52. Moreover, an accumulation of reactive oxygen
   species (ROS) impacts various phases of cortical neuron development,
   including precursor cell proliferation, neuroblast migration and
   positioning, and neuronal maturation^[210]53. Corroborating the
   hyperactive Kyn pathway in the embryonic brain, GSEA results
   demonstrated a marked induction of oxidative stress response in the
   mHFD fetal forebrain relative to that of mCD (Fig. [211]6a). To
   conclusively ascertain ROS production levels in the brain,
   dihydroethidium (DHE) staining was implemented on the E18.5 fetal
   forebrain sections (Fig. [212]6b). Consistently, higher ROS levels were
   detected in the brain of the mHFD fetus compared to that of the mCD
   mice (Fig. [213]6c). Notably, the detrimental impact was amplified when
   Trp supplementation was introduced, causing an increase in ROS
   production in the mH-Trp fetal forebrain (Fig. [214]6b–c). A key
   mechanism by which ROS induces cellular disorders involves lipid
   peroxidation. The Brain tissues are susceptible to lipid peroxidation
   induced by ROS due to their high concentration of polyunsaturated
   lipids^[215]54. Untargeted lipidomics conducted on the E18.5 embryonic
   brain revealed a significant alteration in the lipid profiles resulting
   from maternal diet (Fig. [216]6d). Echoing the transcriptomic results
   (Fig. [217]S9a), the chemical similarity enrichment analysis (ChemRICH)
   exposed a significant increase in oxidized lipid production,
   particularly oxidized fatty acid (OxFA) and oxidized
   phosphatidylethanolamine (OxPE), in the mHFD fetal forebrain
   (Fig. [218]6e). Notably, 1-MT administration effectively inhibited the
   oxidative stress response (Fig. [219]S9b) and ROS accumulation
   (Fig. [220]6b–c) in the fetal brain. This protective effect was further
   confirmed by the reduced production of OxFA and OxPE in the mH-1MT
   fetal forebrain (Fig. [221]6f, [222]S9b–d). Collectively, these
   findings suggest a potential connection between Kyn metabolism
   dysfunction-induced oxidative stress response and the impairment of
   embryonic brain development.

Fig. 6. The dysregulated Kyn pathway causes oxidative stress in the fetal
brain.

   [223]Fig. 6
   [224]Open in a new tab

   a GSEA of activated oxidative stress pathway. b DHE staining of E18.5
   fetal brain sections. c Statistical analysis of relative arbitrary unit
   of DHE staining (n = 9 slices from 3 mice; F [3,32] = 91.625, mCD vs
   mHFD: p = 2.2e-08, mHFD vs mH-Trp: p = 2.1e-06; mHFD vs mH-1MT:
   p = 2.3e-07). Maternal HFD significantly altered lipid profiles in the
   fetal forebrain (d) and resulted in the overproduction of oxidized
   lipids (e) (n = 6 mice/group). f 1-MT treatment alleviated the
   accumulation of oxidized lipids in the embryonic brain (n = 6 for each
   group). Data are represented as mean ± SD. In c p-values were
   determined by ANOVA with Bonferroni’s multiple comparison test. In a
   NES was calculated as described above. Statistical significance was
   determined using permutation testing (999 permutations), with FDR
   correction. In c, p-values were determined using one-way ANOVA,
   followed by Bonferroni’s correction for multiple comparisons. In d
   PERMANOVA by Adonis was used to determine statistical significance
   (F = 6.113, p = 0.007). In e a one-sample Kolmogorov-Smirnov test was
   used to assess the null hypothesis that the p-values for lipids in a
   set follow a reference uniform distribution. FDR correction was
   performed for the set-level p-values. The nomenclature of lipid classes
   in (e) is available at [225]http://prime.psc.riken.jp. Source data are
   provided as a Source Data file.

N-Acetylcysteine supplementation attenuates cortical neuronal migration
deficits and social behavioral dysfunction in male offspring

   Given the co-occurrence between redox dysregulation and cortical
   developmental shortfalls observed in the fetal brain, we aimed to
   ascertain whether reducing Kyn metabolites-induced oxidative stress
   might remediate embryonic brain development anomalies and associated
   behavioral disorders in the progeny. Corroborated the perturbs in the
   glutathione metabolism pathway in both maternal circulation and fetal
   brain revealed by metabolite enrichment analyzes (Figs. [226]3e,
   [227]4j), maternal HFD significantly decreased the ratio of reduced to
   oxidized glutathione and total antioxidant capacity in the fetal brain
   (Fig. [228]7b–c). As such, we supplemented the drinking water of
   HFD-fed mice with N-Acetylcysteine (NAC), a precursor of tissue
   glutathione synthesis, during pregnancy (mH-NAC, Fig. [229]7a)^[230]55.
   This intervention markedly improved the reduced glutathione ratio and
   enhanced total antioxidant capability (Fig. [231]7b-c), contributing to
   rectifying the redox imbalance present in the fetal brain of mothers on
   HFD. Additionally, this beneficial effect was further validated by the
   downregulated oxidative stress response pathway and minimized ROS
   production in the mH-NAC fetal forebrain (Figs. [232]7d–e, [233]S10a).
   Subsequently, we noted significant shifts in lipid profiles in the
   mH-NAC fetal forebrain, marked by decreased oxidized lipid accumulation
   following maternal NAC administration (Fig. [234]S10b–d). An inspection
   of fetal cortical development revealed that NAC supplementation
   moderated the arrest of neuronal migration in the cortical deeper
   layers and the anomalous distribution of functional neurons
   (Fig. [235]7f, h). Interestingly, a minor drop in neuronal density was
   observed in the mH-NAC embryonic brain (Fig. [236]7g, i). These
   findings underscore the significant contribution of cellular redox
   equilibrium in preserving the structural and functional integrity of
   the neocortex. Advancing in line with these positive outcomes, social
   behavioral tests exhibited oH-NAC male offspring with superior
   sociability and social preference, and fewer buried marbles when
   compared to oHFD male offspring (Fig. [237]7j, k). In summary, our
   results validated the hypothesis that counteracting oxidative stress
   incited by Kyn metabolites is a viable strategy for correcting
   behavioral disorders in male offspring induced by a maternal HFD.

Fig. 7. N-Acetylcysteine supplementation rescues oxidative stress-induced
neuronal migration deficits and offspring behavioral dysfunction.

   [238]Fig. 7
   [239]Open in a new tab

   a NAC supplementation schematic. b Measurement of the ratio of reduced
   to oxidized glutathione in the fetal brain (n = 6 mice/group; F
   [2,15] = 22.823, mCD vs mHFD: p = 0.045, mHFD vs mH-NAC: p = 1.41e-05).
   c The total antioxidant capacity of the mH-NAC fetal brain was
   significantly improved (n = 6 mice/group; F [2,15] = 86.955, mCD vs
   mHFD: p = 0.000507, mHFD vs mH-NAC: p = 2.74e-09). d GSEA of oxidative
   stress response (n = 3 for each group). e NAC inhibited superoxide
   production in the fetal brain (n = 9 slices from 3 mice; t = 11.028,
   p = 1.064145e-09). f Neuronal migration in fetal cortex (n = 9 slices
   from 3 mice; bin1: t = 3.893, p = 0.001; bin7: t = 2.256, p = 0.038;
   bin7: t = 2.256, p = 0.038; bin8: t = 3.714, p = 0.002; bin9:
   t = 3.324, p = 0.004). h NAC supplementation partially restored the
   distribution of Brn2-positive cells in the cerebral cortex (n = 9
   slices from 3 mice; bin3: t = 2.903, p = 0.010; bin6: t = 2.786,
   p = 0.013; bin7: t = 3.548, p = 0.003; bin10: t = 4.388,
   p = 0.0004013904). A reduction in the total number of BrdU-positive (g)
   t = 2.694, p = 0.016) and Brn2-positive (i) t = 2.782, p = 0.013)
   neurons was observed in the mH-NAC fetal brain (n = 9 slices from 3
   mice). j Offspring social behavior in three-chamber test (n = 10 mice,
   10 litters/group; sociability: t = 3.318, p = 0.004; social preference:
   t = 5.259, p = 4.47265e-05). k Marble-burying test (n = 10 mice, 10
   litters/group; t = 6.332, p = 4.469088e-06). Data are represented as
   mean ± SD. In b-c, p-values were determined by ANOVA with Dunnett’s
   multiple comparison test. The rest of the statistical significance was
   assessed by the two-sided unpaired Student’s t-test. Source data are
   provided as a Source Data file.

Discussion

   Substantial research suggests that both genetic and environmental
   factors, along with their interplay, are instrumental in the etiology
   of neurodevelopmental disorders^[240]56. One notable observation
   alluded to repeatedly in epidemiological studies is that maternal
   obesity during pregnancy escalates the risk of neuropsychiatric
   disorders in offspring^[241]57. However, the fundamental mechanisms
   underlying this link, despite its clinical significance, are mostly not
   understood comprehensively. Our results provide perspectives into the
   mechanisms by which maternal consumption of HFD disrupts male offspring
   behavioral outcomes, highlighting the importance of maternal immune
   responses and metabolic regulation in modulating fetal brain
   developmental processes.

   The human diet has drastically altered over the past century,
   transitioning from agriculture-based meals to high-calorie processed
   foods laden with saturated fatty acids^[242]1. This change is widely
   identified by researchers as a key factor in the rise of obesity and
   related metabolic disorders^[243]58. Furthermore, recent research has
   illuminated the role of diet in the regulation of the immune system and
   correlating inflammatory diseases^[244]59. Our latest report stipulates
   that a consistent HFD impairs mitochondria in the colonocytes, thus
   enhancing access for host-derived respiratory substrates to intestinal
   pathogens, subsequently leading to an increase in Escherichia coli
   (E.coli)-derived LPS concentrations in circulation, initiating systemic
   inflammation^[245]21. When confronted with environmental hazards, an
   inflammatory response is triggered to provide protection against
   pathogens and facilitate tissue regeneration, thereby preserving
   cellular homeostasis^[246]60. However, excessive or erroneously
   regulated inflammation can result in detrimental imprinting of the
   immune system, thereby increasing susceptibility to chronic
   diseases^[247]61. Rodent studies further suggest that heightened
   inflammation during pregnancy due to bacterial infections or other
   inflammatory triggers has been correlated with neurodevelopmental and
   psychiatric disorders in the offspring^[248]12–[249]14. While the
   mechanisms of MIA-elicited impairments are largely unknown, prenatal
   administration of LPS has been linked to inflammatory cytokine
   production in maternal circulation and the fetal brain^[250]62. Recent
   mouse studies indicate a key role for IL-17a in inducing LPS-based,
   autism-like behaviors^[251]63. Similarly, our study has observed that
   (+)-Naloxone treatment can curb HFD-induced maternal inflammation,
   characterized by the down-regulation of the IL-17 signaling pathway,
   along with the production of IL-17a and IL-17f, thereby rectifying
   social behavioral deficiencies in male offspring (Fig. [252]2f–i).
   IL-17a has drawn attention due to its direct or indirect effects on
   brain cells and neurological and neuromodulatory phenotypes^[253]64.
   For instance, a high-salt diet increased IL-17a production resulting in
   brain endothelial damage and cognitive dysfunction, as seen through
   altered behaviors^[254]65. IL-17a has also been suspected to cause
   anxiety- and depression-like behaviors in mice^[255]66. Although
   certain maternal cytokines have been pinpointed as essential mediators
   of MIA on disease-related phenotypes in offspring, the underlying
   mechanisms of brain development alterations caused by these maternal
   cytokines remain unclear.

   The correlation between metabolism and immunity has been documented
   since the 1960s^[256]67. Recent advancements in immunometabolism
   research have further elucidated the intricate relationship between
   host metabolic processes and immune responses^[257]68. Notably, the
   presence of infection-induced proinflammatory cytokines has the
   capacity to influence both amino acid and lipid metabolism, emphasizing
   their pivotal role in the metabolic reprogramming of the host^[258]69.
   Therefore, we proposed a hypothesis that MIA-induced metabolic stress
   might play a role in offspring behavioral deficits associated with
   maternal HFD (Fig. [259]8). Comprehensive analysis of proinflammatory
   cytokines and metabolites revealed that maternal HFD significantly
   disrupted Trp-Kyn metabolism in the maternal circulation
   (Fig. [260]3c–f). A noteworthy fact is that over 95% of free Trp
   undergoes degradation via the Kyn pathway, forming metabolites with
   various biological activities in immune response and
   neurotransmission^[261]36. IDO1, the main rate-limiting enzyme of the
   Kyn pathway, is expressed in numerous cell types, such as microglia,
   and its activity is regulated by proinflammatory cytokines (e.g. IL-17)
   and TLR ligands^[262]36,[263]70,[264]71. Activation of the Kyn pathway
   triggers a negative feedback mechanism that mitigates the inflammatory
   response^[265]72. However, given that many Kyn metabolites are
   neuroactive, this protective effect may be counteracted by increased
   production of neurotoxic metabolites of the Kyn pathway (e.g., 3-HK and
   Quin)^[266]73. This fact provides a plausible explanation as to why
   extra supplementation of Trp to HFD-fed pregnant mice intensifies the
   severity of stereotypical behaviors in their offspring (Fig. [267]5c).
   Notably, the CNS receives ~60% of Kyn from the periphery, facilitated
   by transport across the blood-brain barrier^[268]74. Moreover, akin to
   Trp and Kyn, 3-HK can cross the blood-brain barrier, contributing to
   Quin production in microglia, while also having the potential to cause
   more direct harmful effects linked to neurodevelopmental
   abnormalities^[269]75. Consequently, abnormal accumulation of Kyn
   metabolites was identified in the fetal brain of mHFD mice, showing a
   strong correlation with maternal serum levels (Figs. [270]5b,
   [271]S3b–e). The metabolic process of Kyn in the brain is largely
   directed by the activity of microglia, which begin to populate the
   brain at E8.5^[272]76. Microglia in the developing brain are
   morphologically and functionally unique compared to those in the adult
   brain^[273]76. Inflammatory insults are shown to hasten the maturation
   and functional differentiation of microglia^[274]77. Morphological
   analysis revealed that microglia in the cerebral cortex of the mHFD
   fetus reveal a more complex profile (Fig. [275]4b–c), indicative of a
   hyperactivated phenotype which aids in converting Kyn to Quin.
   Inhibition of MIA via (+)-Naloxone or direct suppression of IDO1
   activities by 1-MT significantly mitigated the Kyn pathway in fetal
   brain and social performance in adolescent offspring (Figs. [276]2h–i,
   [277]5b–c; [278]S3b, [279]S5a–c), demonstrating the critical role of
   MIA-induced metabolic stress in offspring behavioral dysfunction.

Fig. 8. Schematic diagram of the mechanism underlying maternal HFD-induced
offspring behavioral dysfunction.

   [280]Fig. 8
   [281]Open in a new tab

   Prenatal HFD enhanced maternal inflammatory activities, promoting the
   accumulation of Kyn metabolites in the maternal bloodstream. Most Kyn
   metabolites readily cross the blood-brain barrier. Concurrently,
   maternal HFD-associated MIA stimulated the overactivity of microglia
   within the fetal brain, culminating in the transformation of Kyn into
   the neurotoxic compound Quin. An overabundance of Quin subsequently
   induced an oxidative stress response, which impairs neuronal migration
   within the fetal neocortex and postnatal social behavioral phenotypes.

   The mammalian neocortex, a highly organized and complex structure,
   plays significant roles in numerous higher cognitive, emotional, and
   sensorimotor functions^[282]78. It is vital to maintain proper
   regulation during neuronal migration from the deeper part of the
   developing brain towards the pial surface, as impairments in this
   process could lead to disorders such as brain malformation or
   psychiatric illnesses^[283]79. The published data from studies
   involving children with ASD exhibit abnormal laminar cytoarchitecture
   and cortical disorganization of neurons, especially in the prefrontal
   and temporal cortical tissue^[284]80,[285]81. Moreover, a mouse model
   of MIA exposed to viral infection suggests that a distinctive cortical
   phenotype may drive observable behavioral anomalies in
   offspring^[286]82. The gestation period, a critical time for the rapid
   establishment of foundational structures and neural networks in the
   fetal brain, also represents a period of particular
   vulnerability^[287]83. However, the effects of prenatal maternal HFD on
   the development of the fetal brain in utero have been sparingly
   reported. Conventional wisdom affirms that MIA incited by bacterial or
   viral infection inhibits axon guidance, neurogenesis, and cytoskeleton
   genes while promoting genes involved in translation, cell cycle, and
   DNA damage processes^[288]84. Transcriptomic analysis in our study
   revealed a strong correlation between excessive Kyn production and
   hampered neuronal development and migration in the fetal brain
   (Fig. [289]5d), suggesting that dysfunctional Kyn metabolism may be the
   critical factor in causing MIA-induced cerebral developmental deficits
   among mHFD progeny. Neurotoxic Kyn metabolites, such as Quin and 3-HK,
   have been demonstrated to interfere with neurodevelopment in several
   animal models by elevating extracellular levels of glutamate,
   obstructing glutamate uptake by astrocytes, depleting endogenous
   antioxidants, and triggering ROS and lipid peroxidation^[290]75.
   Simultaneously, recent research affirms that oxidative stress
   contributes to ASD pathogenesis^[291]85. Our findings suggest that
   maternal Trp modulation regulates the oxidative stress response and
   neurodevelopment within the fetal brain (Figs. [292]6a–c, [293]7f–i),
   which offers a mechanistic understanding of maternal HFD-induced
   cortical disorganization. Moreover, case-control studies have observed
   that children with ASD show aberrant plasma metabolite levels in the
   glutathione redox pathway, typically showcasing a decrease in reduced
   glutathione and an increase in oxidized glutathione disulfide^[294]86.
   Metabolomics analysis results indicate that HFD disrupts both maternal
   and embryonic glutathione metabolism (Figs. [295]3e, [296]4j),
   potentially aggravating the oxidative stress impairment caused by an
   overabundance of Kyn metabolites in the fetal brain. Notably,
   introducing NAC to HFD-fed pregnant mice has proven effective in
   restoring the balance in glutathione redox and mitigating ROS
   production in the fetal brain (Fig. [297]7b–e), thereby ameliorating
   social behavioral deficits in the offspring. These results further
   corroborate the hypothesis that the dysregulated Kyn pathway-induced
   oxidative stress response in the fetal brain is vital for the abnormal
   behavioral phenotype of HFD offspring.

   The current study bears several limitations that warrant
   acknowledgment. Previous research demonstrates that male offspring of