Spatial transcriptomics and N-glycoproteomics reveal breed-dependent immune and structural responses in Mycoplasma hyopneumoniae-infected pig lungs

Mycoplasma hyopneumoniae (Mhp) is a major cause of enzootic pneumonia in pigs, yet the molecular and spatial determinants underlying breed-specific susceptibility remain incompletely understood. Here, we integrated spatial transcriptomics, quantitative proteomics and N-glycoproteomics to compare lung responses to experimental Mhp infection in Chenghua pigs with higher susceptibility and Yorkshire pigs with relative resilience. Under an identical inoculation dose, Chenghua pigs exhibited more severe clinical and pathological manifestations than Yorkshire pigs, despite comparable pulmonary Mhp loads at this late stage of infection. This observation suggests that disease severity is associated with differences in host response organization rather than with overall pathogen burden. Spatial analyses revealed a shared spatial and cellular infection framework across breeds, with marked differences in regulatory balance. Across spatial clusters, Chenghua pigs exhibited coordinated activation of TNF-α-NF-κB and IL-6-JAK-STAT3 signaling, accompanied by relative attenuation of IFN-I responses. These inflammatory features co-occurred with hypoxia- and mTORC1-related metabolic reprogramming, activation of stress-response pathways, and engagement of tissue remodeling programmes. Transcription factor analyses further indicated increased activity of HIF1A, RELA and STAT3, together with reduced IRF9/STAT2-associated regulatory activity. Immune deconvolution and multiplex immunofluorescence revealed enrichment of M0/M2 macrophages, plasma cells and activated mast cells, alongside reduced M1 macrophage and CD8+ T-cell signatures. These changes delineated inflammatory niches characterized by relatively high IL1B expression and enrichment of mast cells. Ligand–receptor analysis revealed enhanced BAFF/APRIL–B-cell signaling features and amplification of MHC-II, RESISTIN and CXCL networks in Chenghua pigs. Proteomic and N-glycoproteomic profiling further revealed disruption of junctional and extracellular-matrix components, together with extensive breed-specific remodeling of barrier and antigen-presentation related glycoproteins in Chenghua pigs. Together, these findings provide a spatially resolved description of breed-dependent differences in immune, metabolic and structural responses observed during chronic Mhp infection.