# Cumulative environmental exposures adversely impact social behaviour and are associated with dysregulation of genes and proteins involved in epigenetic, ribosomal, and immune regulation in male mice

## TL;DR

Cumulative environmental stressors in mice lead to social behavior issues and changes in genes and proteins linked to immune and epigenetic functions, especially in males.

## Contribution

A novel 'triple-hit' mouse model was developed to study cumulative environmental effects on neurodevelopmental disorder risk and immune-related molecular changes.

## Key findings

- Male mice exposed to combined stressors showed elevated NDD-related behavioral risk and social deficits.
- Altered inflammatory and ribosomal pathways were observed in brain glia and peripheral immune cells.
- Proteomic analysis revealed decreased proteins involved in inflammation, translation, and synaptic function in both brain and blood.

## Abstract

This study investigated how cumulative environmental exposures influence offspring behaviour and inflammation-related molecular signatures in the brain and peripheral immune system.

A novel "triple-hit" mouse model was developed using C57Bl/6JAusB mice (N = 70), combining preconceptual social stress, antenatal high-fat diet, and a postnatal immune challenge (poly(I:C), 10 mg/kg). At 12 weeks, offspring underwent behavioural tests relevant to neurodevelopmental disorders (NDDs), including the Elevated Plus Maze, 3-Chamber Social Preference, Self-Grooming, and Marble Burying. A composite NDD-risk index was calculated. Single-cell RNA sequencing (scRNA-seq) and bulk proteomics were performed on male triple-hit offspring to identify differentially expressed genes and proteins associated with inflammatory pathways.

Male triple-hit offspring showed elevated NDD-related behavioural risk and social deficits, not observed in females. scRNA-seq revealed altered inflammatory and ribosomal pathways in brain glia and peripheral immune cells. Proteomic analysis showed decreased abundance of proteins involved in inflammation, translation, chromatin remodelling, and synaptic function in both brain and blood.

Combined environmental stressors may drive male-specific behavioural and inflammatory changes relevant to NDDs. The identification of overlapping inflammatory signatures in brain and peripheral immune cells supports a role for shared immune mechanisms in brain–immune axis dysfunction. However, these pathway-level findings should be interpreted as preliminary hypotheses and warrant independent validation to confirm their mechanistic significance.

The online version contains supplementary material available at 10.1007/s00011-025-02152-y.