The Hypoxic Microenvironment of Candida albicans Biofilms Shapes Neutrophil Responses

Magdalena Juszczak ^1,2* Aleksandra Brankiewicz ^1,2 Marcin Zawrotniak ¹ Maria Rapala-Kozik ¹

• ¹ Department of Comparative Biochemistry and Bioanalysis, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
• ² Doctoral School of Exact and Natural Sciences, Jagiellonian University, Krakow, Poland

The microenvironment of Candida albicans biofilms exerts a profound influence on host immune responses. Here, we demonstrate that human neutrophils infiltrating C. albicans biofilms experience progressive hypoxia, which intensifies with biofilm maturation. This hypoxia results from high fungal metabolic activity and oxygen consumption during growth and extracellular matrix (ECM) production. Within the biofilm microenvironment, neutrophils exhibit increased stabilization of hypoxia-inducible factor 1-alpha (HIF-1α), upregulated expression of the anti-apoptotic protein Mcl-1, elevated secretion of MIP-1β, and reduced caspase 3/7 activity, collectively suggesting a biofilm-induced pro-survival phenotype. Simultaneously, biofilms markedly suppress neutrophil extracellular trap (NET) formation and reactive oxygen species (ROS) production while enhancing degranulation. Comparative analyses using mannan-deficient C. albicans mutants highlight the critical role of ECM composition in modulating hypoxia-driven immune responses. Pharmacological inhibition of HIF-1α with LW6 and PX478 partially restores NETosis and ROS production, underscoring its pivotal role in neutrophil function. These findings provide novel insights into the impact of biofilm-induced hypoxia on neutrophil responses, identifying HIF-1α as a key regulator of immune adaptation in fungal biofilms. Targeting hypoxia pathways may offer new therapeutic strategies to modulate neutrophil responses and enhance host defenses against fungal infections.