Mast cell activation in response to fungi-associated molecular patterns and its influence on fibroblasts in 2D and 3D culture models.

Mast cells (MCs) are granular cells that reside at barrier sites. They express a variety of receptors, including pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), which are conserved microbial products. Recent studies have shown that MCs are capable of orchestrating immune responses in different contexts. However, the response of MCs to pathogens, such as fungi and their structural components, including chitin and β-glucans (e.g., zymosan), remains poorly characterized.

We used immortalized mast cell precursors (iMCps) from C57BL/6 mouse bone marrow, carrying a tamoxifen (4-OHT) inducible HoxB8 cassette that blocks differentiation and sustains self-renewal. Toluidine blue staining assessed granulation; flow cytometry measured c-KIT/FcεRI expression. To exam fungal-component effects on MC activation, cells received zymosan or chitin overnight for cytokine assays (±PMA 20 ng/mL) or 30 min for CD107a degranulation assays (±48/80 1ug/mL). A SYTOX Green assay assessed DNA extracellular trap (DET) release. MCs (500 cells) were co-cultured with NIH/3T3 fibroblasts (2000 cells) ± zymosan/chitin in 2D (crystal violet biomass quantification) and 3D agarose-coated wells (morphology characterization). Supernatants were collected for cytokine quantification. 

Removal of 4-OHT from the mIL-3 (2 ng/mL)-supplemented culture medium generated differentiated MCs (iMCds) within 6 days, confirmed by increased metachromasia and c-KIT/FcεRI. Neither zymosan nor chitin induced degranulation or cytokine release, though zymosan triggered DET release. In 2D, MCs modulated fibroblast growth. At 72 h, iMCd-containing supernatants had lower IL-6, IL-33, CCL3, and TNF-α than other groups. Both iMCps and iMCds altered spheroid morphology into two distinct populations, and iMCd spheroid supernatants showed reduced IL-33. 

Our data suggest that fungal components are able to modify MCs activation profile and its capacity to modulate the microenvironment.