Early-life weaning is a critical transition that can disrupt immune homeostasis and reshape the gut microbiota. In piglets, sudden separation from maternal nutrition and the introduction of solid feed often lead to immune dysregulation, gastrointestinal disturbance, and impaired growth. While immune activation after weaning is well known, how it affects protein translation in immune cells and whether gut microbes play a role have been less clear.
To investigate, a research team led by Dr. Xin Zong at Zhejiang University analyzed peripheral blood mononuclear cells (PBMCs) from healthy and weaning piglets. The team combined ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq) to capture both transcript levels and active translation. They also used 16S rRNA sequencing to profile gut microbiota composition.
The analysis showed that weaning stress caused broad transcriptional and translational remodeling. RNA-seq identified 94 upregulated and 219 downregulated genes, while Ribo-seq found 245 genes with increased ribosome occupancy and 60 with reduced occupancy. Translation efficiency analysis revealed 70 genes with increased efficiency and 7,312 with decreased efficiency. Immune genes as a group had higher ribosome occupancy in weaning piglets. Key inflammatory mediators, including CD82, CCL25, and TNFSF13, showed significantly increased translation efficiency.
The researchers next examined codon usage. The genetic code is redundant, and different synonymous codons can be used at different frequencies. This codon usage bias can influence how efficiently messenger RNAs are translated into proteins. In healthy piglets, codons ending in G or C at the third position (GC3 codons) already showed higher usage bias than A/U-ending codons. After weaning, this preference became stronger. Genes with enhanced translation were enriched for GC3 codons, including those encoding leucine, glutamate, and lysine. The weaning group had significantly greater GC3-biased codon usage than healthy controls, while the overall codon adaptation index did not differ between groups.
Weaning stress was also accompanied by gut microbiota dysbiosis. The microbial dysbiosis index was elevated, and alpha diversity was reduced in weaning piglets. Genera enriched in healthy piglets, such as Romboutsia, Lactobacillus, and Turicibacter, were associated with AT-rich codon usage patterns. In contrast, genera expanded under weaning stress, such as Fusobacterium and Streptococcus, were linked to GC-enriched codon profiles. Correlation analysis showed that specific microbial taxa, including Romboutsia sp. and Lachnoclostridium sp., were significantly correlated with the translation efficiency of immune transcripts such as CCL25, CD24, and TNFSF13.
Cross-species relative synonymous codon usage analysis further revealed coordinated codon preference between gut microbiota and host immune cells. Host immune cells and dominant bacterial taxa separated according to healthy versus weaning conditions. In synonymous families encoding glutamate, valine, and lysine, bacteria enriched in healthy piglets tended to favor AT-ending codons, while taxa enriched under weaning stress preferred the corresponding GC-ending codons, mirroring host codon usage profiles. In cell experiments, supplementation with valine, lysine, or glutamate modestly enhanced cell viability and partially mitigated lipopolysaccharide (LPS)-induced viability loss. Under inflammatory stimulation, these amino acids differentially modulated cytokine expression, including TNF-alpha and IL-1beta.
The authors conclude that weaning stress is associated with a coordinated shift toward GC-rich codon usage in both gut microbes and host immune genes, and that this shift may contribute to enhanced translation of key immune genes. They emphasize that the findings reveal an association, not a causal relationship, between microbiota composition and codon-usage-linked translational regulation. Future studies using antibiotic perturbation, fecal microbiota transplantation, and germ-free or gnotobiotic models will be needed to test causality.
The study was supported by the National Natural Science Foundation of China (32372890), the National Key Research and Development Program of China (2022YFD1301500), and the National Ten Thousand Talents Program for Young Top-notch Talents. Sequencing data have been deposited in the NCBI Sequence Read Archive under accession PRJNA1067344.
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