Aspergillus fumigatus modulates mast cell activation and reshapes mast cell–fibroblast cross-talk

Fungal infections such as those caused by Aspergillus fumigatus represent a growing global health challenge. Meanwhile, antifungal immunity studies do not consider the role of tissue-resident mast cells (MCs) and recruited MC progenitors undergoing in situ maturation in these conditions. This study evaluated the modulation of MC responses by A. fumigatus using myeloid progenitors immortalized via 4-hydroxytamoxifen (4-OHT)-inducible HoxB8, and characterized MC-fibroblast cross-talk and tissue remodeling using 2D and 3D co-cultures. Three MC models were evaluated: bone marrow-derived MCs (BMMCs; 4–8 weeks culture with rmIL-3 (2 ng/mL)), immortalized MC progenitors (iMCps; maintained with rmIL-3 and 4-OHT), and differentiated MCs (iMCds; obtained after 6–8 days of 4-OHT withdrawal). Differentiation was validated by toluidine blue staining and flow cytometry for c-KIT, FcεRI, and integrin alpha4beta7. Functional assays evaluated acute degranulation (β-hexosaminidase activity and CD107a expression) and prostaglandin (PG)D₂ secretion upon fungal stimulation compared to compound 48/80. IL-6, CCL3, IL-10, and IL-13 were measured after overnight PMA stimulation. MC–fibroblast (NIH-3T3) cross-talk was assessed in flat-bottom plates (2D) or agarose-coated U-bottom plates (3D) with heat-inactivated A. fumigatus (1:10), measuring cell growth, morphometry, and PGD₂; IL-6; TNF-alpha; IL-33; CCL3 for 3-7 days. A. fumigatus acted as a suppressive immunomodulator, dampening compound 48/80-induced degranulation, PGD₂ generation, and PMA-induced IL-6, IL-10, and CCL3, while sparing IL-13. In co-cultures, the MCs and the fungus altered fibroblast density and secretomes, while fungal interaction with iMCps remodeled 3D spheroid morphology. These findings suggest that MCs modulate stromal architecture and mediator release, an effect that is further altered by fungal exposure.