Impaired NLRP3 inflammasome activation by chitin underlies refractory chromoblastomycosis caused by Fonsecaea pedrosoi muriform cells.

INTRODUCTION: Macrophage NLRP3 activation is crucial for antifungal immunity, yet its role in chromoblastomycosis-a chronic subcutaneous mycosis typically caused by Fonsecaea pedrosoi-remains unclear. Unlike prior self-resolving models using conidia and hyphae, clinical disease features muriform cells, a parasitic form resistant to macrophages and linked to chronicity. Notably, chitin, a weak NLRP3 agonist, accumulates on the surface of muriform cells. This study investigates whether chitin accumulation modulates NLRP3 activation and promotes fungal persistence.

METHODS: Human chromoblastomycosis lesions were analyzed by IHC (MPO, CD86, NLRP3) and RT-PCR for cytokine transcripts. WT and NLRP3-/- mice were footpad-inoculated with F. pedrosoi muriform cells for in vivo assessment. Flow cytometric bead array quantified IL-1β, IL-6, IL-10, TNF-α, and IL-17A in footpad homogenates, and the same panel (excluding IL-17A) in stimulated BMDM supernatants. NLRP3 pathway activation in BMDMs was confirmed by western blotting and immunofluorescence.

RESULTS: In human lesions, marked MPO+ neutrophil recruitment encapsulated muriform cells, yet multinucleated giant cells sequestered some fungal elements from neutrophils. Despite robust NLRP3 expression and elevated IL-1β, IL-6, TNF-α, and IL-10 versus normal skin (all p < 0.01), muriform cells persisted in tissue. In WT mice, lesions transitioned from purulent (day 7) to granulomatous (day 90), concurrent with declining IL-1β, IL-17A, IL-6, and TNF-α. MPO+ neutrophils abundantly surrounded muriform cells at day 7; at day 90, F4/80+ macrophages enveloped most fungi without elimination, forming multinucleated giant cells-recapitulating human pathology. In NLRP3-/- mice, IL-1β and IL-17A were produced in an NLRP3-independent manner and showed no decline over time. Muriform cells progressively budded and transitioned to hyphae both inside and outside giant cells; MPO+ neutrophils preferentially localized to hyphal areas, while macrophages remained around muriform cells, suggesting compartmentalized inflammation. In vitro, chitinase-treated muriform cells induced higher NLRP3-dependent IL-1β, IL-6, TNF-α, and IL-10 in WT BMDMs versus intact cells (all p < 0.01). Mechanistically, chitin accumulation partially suppressed NF-κB phosphorylation, reducing NLRP3 expression, caspase-1 cleavage, and ASC speck formation.

CONCLUSION: NLRP3 mediates host defense against muriform cells yet fails to resolve chronic infection. Chitin accumulation on muriform cells attenuates NLRP3 activation, thereby promoting chromoblastomycosis chronicity.