The respiratory sensitizer methylene diphenyl diisocyanate (MDI) reprograms human cells of the alveolar tract

Abstract

This study applied a suite of human-relevant, non-animal cell models to investigate early events associated with respiratory sensitization induced by 4,4'-methylene diphenyl diisocyanate (MDI), a representative low-molecular-weight respiratory sensitizer. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage-like cells (THP-1M) and primary human monocyte-derived macrophages were exposed to MDI (3-300 µM, 4 h), resulting in a restricted cytokine response characterized primarily by enhanced IL-1α and IL-1β production. In contrast, lipopolysaccharide (LPS, 1 ng/mL-10 µg/mL) induced a pronounced pro-inflammatory response consistent with classical M1-like activation. Flow cytometric analysis of THP-1M indicated a partial M2a-like immunomodulatory phenotype following MDI exposure, characterized by increased CD1a and reduced CD14 expression. This response was accompanied by changes in selected central carbon metabolites, whereas LPS induced changes more consistent with M1-like activation. In human alveolar epithelial type I-like cells (hAELVi), MDI induced changes in selected central carbon metabolites, indicating epithelial metabolic responsiveness. In epithelial-macrophage coculture (hAELVi/THP-1M), metabolic responses to MDI were attenuated while chemokine production (CCL2, CCL5, CXCL8) was enhanced, suggesting macrophage-mediated modulation of epithelial responses and immune cell recruitment potential. In an air-liquid interface coculture model, these early responses were associated with dendritic cell-like activation of THP-1 cells, marked by OX40L upregulation, indicating Th2-skewing potential. Overall, the findings support interacting epithelial-immune key events relevant to respiratory sensitization and highlight the value of incorporating epithelial-immune interactions into new approach methods (NAMs) based hazard assessment. SHORT SUMMARY: 4,4'-Methylene diphenyl diisocyanate (MDI) induced epithelial metabolic changes in vitro and promoted a partial M2a-like immunomodulatory macrophage response associated with alterations in central carbon metabolites. In epithelial-macrophage co-culture, macrophages attenuated metabolic responses while enhancing chemokine production, suggesting modulation of epithelial responses and immune cell recruitment potential. In an air-liquid interface co-culture model, these early events were associated with dendritic cell-like activation of THP-1 cells, marked by OX40L upregulation and indicative of Th2-skewing potential. Together, the findings support interacting key events relevant to proposed adverse outcome pathway concepts for respiratory sensitization and highlight candidate biomarkers and mechanistically relevant cellular responses that may support human-relevant NAM development for low-molecular-weight respiratory sensitizers.

Keywords: 4,4’-Methylene diphenyl diisocyanate (MDI); Epithelial-immune interactions; Immunometabolism; Macrophage polarization; New approach methods (NAMs); Respiratory sensitization; Th2-skewing.