{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T16:30:42Z","timestamp":1792168242880,"version":"4.4.0"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,22]],"date-time":"2025-04-22T00:00:00Z","timestamp":1745280000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The microenvironment of <jats:italic>Candida albicans<\/jats:italic> biofilms create a hypoxic microenvironment, which exerts a profound influence on host immune responses during infection. Neutrophils are key defenders against <jats:italic>C.\u00a0albicans<\/jats:italic>; however, the impact of biofilm-induced hypoxia on neutrophil function remains unclear.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We co-cultured human neutrophils in vitro with <jats:italic>C. albicans<\/jats:italic> biofilms at various stages of maturation, using both wild-type strains and extracellular matrix (ECM)-deficient mutants. Intracellular hypoxia was assessed using a fluorescent oxygen-sensitive probe. Neutrophil effector functions were evaluated by measuring caspase-3\/7 activity, stabilization of hypoxia-inducible factor 1-alpha (HIF-1\u03b1), and accumulation of the anti-apoptotic Mcl-1 protein. Analyses included also quantification of reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) formation, chemokine secretion (IL-8 and MIP-1\u03b2), and neutrophil elastase release. To assess the role of hypoxia signaling in neutrophil responses, cells were treated with the selective HIF-1\u03b1 inhibitors LW6 and PX478.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Neutrophils infiltrating <jats:italic>C. albicans<\/jats:italic> wild-type biofilms experience progressive hypoxia, which intensifies with biofilm maturation. This hypoxia results from high fungal metabolic activity and extracellular matrix (ECM) production. Within the biofilm microenvironment, neutrophils exhibit increased stabilization of HIF-1\u03b1 and Mcl-1, elevated secretion of MIP-1\u03b2, IL-8, and reduced caspase 3\/7 activity, collectively suggesting a biofilm-induced pro-survival phenotype. Simultaneously, mature biofilms markedly suppress NET formation and ROS production while enhancing degranulation. Comparative analyses using mannan-deficient <jats:italic>C. albicans<\/jats:italic> mutants highlight the critical role of ECM composition in modulating hypoxia-driven immune responses. Pharmacological inhibition of HIF-1\u03b1 with LW6 and PX478 partially restores NETosis and ROS production, underscoring the pivotal role of this protein in regulation of neutrophil function.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>These findings provide novel insights into the impact of biofilm-induced hypoxia on neutrophil responses, identifying HIF-1\u03b1 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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1547559","type":"journal-article","created":{"date-parts":[[2025,4,22]],"date-time":"2025-04-22T05:26:42Z","timestamp":1745299602000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["The hypoxic microenvironment of Candida albicans biofilms shapes neutrophil responses"],"prefix":"10.3389","volume":"16","author":[{"given":"Magdalena","family":"Juszczak","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aleksandra","family":"Brankiewicz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcin","family":"Zawrotniak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Rapala-Kozik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,22]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3389\/fmed.2018.00028","article-title":"Candida biofilms: Threats, challenges, and promising strategies","volume":"5","author":"Cavalheiro","year":"2018","journal-title":"Front Med"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1126\/science.284.5418.1318","article-title":"Bacterial biofilms: A common cause of persistent infections","volume":"284","author":"Costerton","year":"1999","journal-title":"Science"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/S0966-842X(02)00002-1","article-title":"Candida biofilms and their role in infection","volume":"11","author":"Douglas","year":"2003","journal-title":"Trends Microbiol"},{"key":"B4","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.mib.2019.04.001","article-title":"Candida albicans biofilm growth and dispersal: Contributions to pathogenesis","volume":"52","author":"Wall","year":"2019","journal-title":"Curr Opin Microbiol"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1007\/s40588-018-0098-6","article-title":"The role of neutrophils in host defense against invasive fungal infections","volume":"5","author":"Desai","year":"2018","journal-title":"Curr Clin Microbiol Rep"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.3389\/fphys.2018.00113","article-title":"Neutrophil: A cell with many roles in inflammation or several cell types","volume":"9","author":"Rosales","year":"2018","journal-title":"Front Physiol"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1126\/science.1092385","article-title":"Neutrophil extracellular traps kill bacteria","volume":"303","author":"Brinkmann","year":"2004","journal-title":"Science"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2005.00659.x","article-title":"Neutrophil extracellular traps capture and kill Candida albicans and hyphal forms","volume":"8","author":"Urban","year":"2006","journal-title":"Cell Microbiol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.3390\/biom5020702","article-title":"The role of reactive oxygen species (ROS) in the formation of extracellular traps (ETs) in humans","volume":"5","author":"Stoiber","year":"2015","journal-title":"Biomolecules"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1000675","article-title":"A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus","volume":"185","author":"Pilsczek","year":"2010","journal-title":"J Immunol"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1038\/srep18302","article-title":"Classical ROS-dependent and early\/rapid ROS-independent release of neutrophil extracellular traps triggered by Leishmania parasites","volume":"5","author":"Rochael","year":"2016","journal-title":"Sci Rep"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2017.00414","article-title":"Aspartic proteases and major cell wall components in Candida albicans trigger the release of neutrophil extracellular traps","volume":"7","author":"Zawrotniak","year":"2017","journal-title":"Front Cell Infect Microbiol"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.3390\/cells8121611","article-title":"Farnesol, a quorum-sensing molecule of Candida albicans, triggers the release of neutrophil extracellular traps","volume":"8","author":"Zawrotniak","year":"2019","journal-title":"Cells"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2021.681030","article-title":"Extracellular nucleic acids present in the Candida albicans biofilm trigger the release of neutrophil extracellular traps","volume":"11","author":"Smolarz","year":"2021","journal-title":"Front Cell Infect Microbiol"},{"key":"B15","doi-asserted-by":"publisher","first-page":"e57571","DOI":"10.15252\/embr.202357571","article-title":"Candida albicans induces neutrophil extracellular traps and leucotoxic hypercitrullination via candidalysin","volume":"24","author":"Unger","year":"2023","journal-title":"EMBO Rep"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1086\/652783","article-title":"Interactions between human phagocytes and Candida albicans biofilms alone and in combination with antifungal agents","volume":"201","author":"Katragkou","year":"2010","journal-title":"J Infect Dis"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jis607","article-title":"Candida albicans biofilms do not trigger reactive oxygen species and evade neutrophil killing","volume":"206","author":"Xie","year":"2012","journal-title":"J Infect Dis"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcsw.2023.100111","article-title":"The role of the Candida biofilm matrix in drug and immune protection","volume":"10","author":"Roy","year":"2023","journal-title":"Cell Surf"},{"key":"B19","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1371\/journal.ppat.1005884","article-title":"The extracellular matrix of Candida albicans biofilms impairs formation of neutrophil extracellular traps","volume":"12","author":"Johnson","year":"2016","journal-title":"PloS Pathog"},{"key":"B20","doi-asserted-by":"publisher","first-page":"995","DOI":"10.1111\/j.1365-2958.2011.07626.x","article-title":"Contribution of the glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans","volume":"80","author":"Bonhomme","year":"2011","journal-title":"Mol Microbiol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2003700117","article-title":"Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance","volume":"117","author":"Kowalski","year":"2020","journal-title":"Proc Natl Acad Sci U.S.A"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00098-09","article-title":"Hypoxic adaptation by Efg1 regulates biofilm formation by Candida albicans","volume":"75","author":"Stichternoth","year":"2009","journal-title":"Appl Environ Microbiol"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21061984","article-title":"Candida albicans shields the periodontal killer Porphyromonas gingivalis from recognition by the host immune system and supports bacterial infection of gingival tissue","volume":"21","author":"Bartnicka","year":"2020","journal-title":"Int J Mol Sci"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2014.08.057","article-title":"Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures","volume":"24","author":"Fox","year":"2014","journal-title":"Curr Biol"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20040624","article-title":"Hypoxia-induced neutrophil survival is mediated by HIF-1\u03b1\u2013dependent NF-\u03baB activity","volume":"201","author":"Walmsley","year":"2005","journal-title":"J Exp Med"},{"key":"B26","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1016\/j.molcel.2008.04.009","article-title":"Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway","volume":"30","author":"Kaelin","year":"2008","journal-title":"Mol Cell"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1038\/s41581-022-00587-8","article-title":"The effect of HIF on metabolism and immunity","volume":"18","author":"Taylor","year":"2022","journal-title":"Nat Rev Nephrol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"B29","doi-asserted-by":"publisher","first-page":"fty043","DOI":"10.1093\/femspd\/fty043","article-title":"Hypoxia arising from concerted oxygen consumption by neutrophils and microorganisms in biofilms","volume":"76","author":"Wu","year":"2018","journal-title":"Pathog Dis"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2006.00792.x","article-title":"How do microbes evade neutrophil killing","volume":"8","author":"Urban","year":"2006","journal-title":"Cell Microbiol"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2006-03-013771","article-title":"The antiapoptotic protein Mcl-1 is essential for the survival of neutrophils but not macrophages","volume":"109","author":"Dzhagalov","year":"2007","journal-title":"Blood"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27113426","article-title":"Action sites and clinical application of HIF-1\u03b1 inhibitors","volume":"27","author":"Xu","year":"2022","journal-title":"Molecules"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1021\/jm0610292","article-title":"(Aryloxyacetylamino)benzoic acid analogues: A new class of hypoxia-inducible factor-1 inhibitors","volume":"50","author":"Lee","year":"2007","journal-title":"J Med Chem"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.233.3.3","article-title":"Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1\u03b1","volume":"3","author":"Welsh","year":"2004","journal-title":"Mol Cancer Ther"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.638609","article-title":"Mechanistic understanding of Candida albicans biofilm formation and approaches for its inhibition","volume":"12","author":"Atriwal","year":"2021","journal-title":"Front Microbiol"},{"key":"B36","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1038\/s41579-022-00692-2","article-title":"Gradients and consequences of heterogeneity in biofilms","volume":"20","author":"Jo","year":"2022","journal-title":"Nat Rev Microbiol"},{"key":"B37","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.1421437112","article-title":"Community participation in biofilm matrix assembly and function","volume-title":"Proc Natl Acad Sci USA","author":"Mitchell","year":"2015"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.3390\/jof3010014","article-title":"The Candida albicans biofilm matrix: Composition, structure, and function","volume":"3","author":"Pierce","year":"2017","journal-title":"J\u00a0Fungi"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01333-14","article-title":"Novel entries in a fungal biofilm matrix encyclopedia","volume":"5","author":"Zarnowski","year":"2014","journal-title":"mBio"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1093\/mmy\/myt007","article-title":"Paradoxical antifungal activity and structural observations in biofilms formed by echinocandin-resistant Candida albicans clinical isolates","volume":"52","author":"Walraven","year":"2014","journal-title":"Med Mycol."},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2020.08.002","article-title":"The role of HIF in immunity and inflammation","volume":"32","author":"McGettrick","year":"2020","journal-title":"Cell Metab"},{"key":"B42","doi-asserted-by":"publisher","first-page":"e0244366","DOI":"10.1371\/journal.pone.0244366","article-title":"Hypoxia-inducible factor 1 alpha limits dendritic cell stimulation of CD8 T cell immunity","volume":"15","author":"Tran","year":"2020","journal-title":"PloS One"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.633586","article-title":"Oxygen-mediated suppression of CD8+ T cell proliferation by macrophages: Role of pharmacological inhibitors of HIF degradation","volume":"12","author":"Gojkovic","year":"2021","journal-title":"Front Immunol"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1111\/imr.12453","article-title":"Clearance of apoptotic neutrophils and resolution of inflammation","volume":"273","author":"Greenlee-Wacker","year":"2016","journal-title":"Immunol Rev"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2010-12-324186","article-title":"Activation of hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model","volume":"118","author":"Elks","year":"2011","journal-title":"Blood"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1100\/2011\/131539","article-title":"Regulation of neutrophil survival\/apoptosis by Mcl-1","volume":"11","author":"Milot","year":"2011","journal-title":"Sci World J"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M411858200","article-title":"Hypoxia-inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis","volume":"280","author":"Piret","year":"2005","journal-title":"J Biol Chem"},{"key":"B48","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/j.chom.2024.11.012","article-title":"Intestinal E. coli-produced yersiniabactin promotes profibrotic macrophages in Crohn\u2019s disease","volume":"33","author":"Ahn","year":"2025","journal-title":"Cell Host Microbe"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2016.00518","article-title":"Formation of neutrophil extracellular traps under low oxygen levels","volume":"7","author":"Branitzki-Heinemann","year":"2016","journal-title":"Front Immunol"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1002213","article-title":"Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils","volume":"186","author":"McGovern","year":"2011","journal-title":"J Immunol"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21041183","article-title":"The impact of hypoxia on neutrophil degranulation and consequences for the host","volume":"21","author":"Lodge","year":"2020","journal-title":"Int J Mol Sci"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1136\/thoraxjnl-2015-207604","article-title":"Hypoxia upregulates neutrophil degranulation and potential for tissue injury","volume":"71","author":"Hoenderdos","year":"2016","journal-title":"Thorax"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.3390\/cells10102736","article-title":"Proteinous Components of Neutrophil Extracellular Traps Are Arrested by the Cell Wall Proteins of Candida albicans during Fungal Infection, and Can Be Used in the Host Invasion","volume":"10","author":"Karkowska-Kuleta","year":"2021","journal-title":"Cells"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.3892\/mmr.2015.3862","article-title":"LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells","volume":"12","author":"Sato","year":"2015","journal-title":"Mol Med Rep"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1547559\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,22]],"date-time":"2025-04-22T05:26:43Z","timestamp":1745299603000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1547559\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,22]]},"references-count":54,"alternative-id":["10.3389\/fimmu.2025.1547559"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1547559","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,22]]},"article-number":"1547559"}}