Integrative multi-omics analysis reveals systemic and intestinal responses to heat stress in finishing pigs

Heat stress (HS) is a major environmental threat to swine production that impairs growth performance and health. Because of their limited thermoregulatory capacity, pigs are highly vulnerable to HS, which results in compromised intestinal integrity, systemic inflammation, and metabolic inefficiency. To elucidate the mechanisms underlying HS acclimation in pigs, we conducted a longitudinal multi-omics analysis integrating fecal microbiome, whole-blood transcriptome, and immune cell deconvolution in finishing pigs under thermoneutral (TN) or HS conditions. HS markedly reduced the average daily gain and feed intake. Microbiome profiling revealed condition-specific shifts: TN pigs showed enrichment of short-chain fatty acid (SCFA)-producing genera, such as <italic>Prevotella</italic> and <italic>Streptococcus</italic>, whereas HS pigs exhibited increased <italic>Clostridium sensu stricto 1</italic>. Functional predictions indicated preservation of the antioxidant and immunomodulatory pathways (glutathione, retinol, and aminoacyl-tRNA biosynthesis) in TN pigs, whereas HS pigs displayed branched-chain amino acid catabolism, reflecting metabolic acclimation under stress. Transcriptomic analysis revealed acute changes at week 1, with 516 differentially expressed genes enriched in hematopoiesis, focal adhesion, cytoskeletal remodeling, and thyroid hormone signaling. By Week 2, these gene responses had declined, suggesting partial acclimation. Network analysis identified cytoskeletal genes (<italic>ACTB</italic>, <italic>MYL9</italic>, <italic>ACTN4</italic>, and <italic>COL4A4</italic>) as the central regulators. Immune deconvolution further showed the HS-driven elevation of cytotoxic T and myeloid subsets, in contrast to B cell populations which were maintained under TN, highlighting divergent immune trajectories. Integration of the microbiome, transcriptome, and immune data revealed two axes: (1) cytotoxic T cells positively associated with <italic>Clostridium sensu stricto 1,</italic> but negatively associated with cytoskeletal genes, and (2) B cells positively linked to <italic>Prevotella</italic>, <italic>Lactobacillus</italic>, and structural genes. Only the B-cell structural axis formed a coherent cross-layer module, indicatin a recovery-oriented response. These findings demonstrate that resilience to HS requires the coordination of humoral immunity, cytoskeletal reinforcement, and SCFA-producing microbiota. The identified biomarker axis (<italic>Prevotella</italic>, B cells, and cytoskeletal genes) provides a mechanistic basis for developing precise strategies to enhance thermal tolerance in swine.