Inflammatory reprogramming of alveolar macrophages by the bacterial peptide iE-DAP via PEPT2 transport.

Alveolar macrophages serve as key sentinels of pulmonary immunity, yet the mechanisms by which they recognize and respond to bacterial-derived peptides remain poorly understood. Here, we demonstrate for the first time that the peptide transporter PEPT2 is functionally expressed in NR8383 cells, a rat alveolar macrophage-derived cell line, and mediates the uptake of the bacterial-derived dipeptide iE-DAP, triggering pro-inflammatory responses. iE-DAP treatment significantly increased TNF-α and IL-6 expression in a PEPT2-dependent manner. Using SWATH-based quantitative proteomics, we identified 1,198 upregulated and 1404 downregulated proteins following iE-DAP exposure, among which proteasome components and NF-κB subunits were markedly induced, while lysosome-related proteins were downregulated. Inhibitor studies confirmed that iE-DAP activates the MAPK/NF-κB pathway, with a unique involvement of the JNK cascade in macrophages. Notably, the upregulation of cathepsin B (CTSB) and its potential role in inflammasome activation via NLRP3 resemble key elements of pyroptotic cell death, suggesting a link between lysosomal dysfunction and exaggerated inflammatory signaling. These findings indicate that PEPT2 facilitates the uptake of iE-DAP in alveolar macrophages, leading to enhanced proteasome activity, lysosomal impairment, and M1 polarization, ultimately amplifying inflammatory signaling. Our study uncovers a novel innate mechanism by which bacterial peptides can induce alveolar macrophage activation and suggests that dysregulated PEPT2-NOD1-proteasome signaling may contribute to the pathogenesis of pulmonary inflammatory disorders. These insights provide a foundation for targeting peptide transporters in the regulation of macrophage-driven lung inflammation. The online version contains supplementary material available at 10.1007/s43188-025-00335-7.