Metabolic imprinting drives epithelial memory during mucosal fungal infection

Abstract

Epithelial cells at barrier sites are emerging as active participants in innate immune memory, yet the underlying metabolic and epigenetic mechanisms remain unclear. Here, we uncover a previously unrecognized form of trained immunity in oral epithelial cells that enhances protection against fungal infection. Using a mouse model, we show that mucosal exposure to Candida albicans confers sustained protective memory that is independent of adaptive immunity and myeloid cells. Mechanistically, mucosal memory is driven by proline catabolism via proline dehydrogenase (Prodh) in epithelial cells, which sustains mitochondrial function, epigenetic remodeling, and promotes cytokine production upon secondary challenge. Unlike classical trained immunity in immune cells, epithelial memory is independent of glycolysis but partially sustained by fatty acid oxidation via carnitine palmitoyltransferase-I (CPT1). These findings uncover a distinct metabolic-epigenetic axis that underlines long-term epithelial memory in the oral mucosa and reveal novel non-hematopoietic mechanisms of mucosal defense against fungal pathogens.

Keywords: Candida albicans; fatty acid oxidation; oral epithelium; proline catabolism.

Figure 1.. Mucosal priming enhances protection against oral reinfection.

A. Schematic of the C. albicans reinfection model and experimental timeline for assessment. Created with BioRender.com B. Oral fungal burden in immunocompetent wild-type mice at indicated time points during reinfection. N=6; Two-tailed Mann–Whitney Test. The y-axis represents the limit of detection (20 colony-forming units [CFUs] per gram of tissue). C. Heat map of chemokine and cytokine levels in tongue homogenates from immunocompetent wild-type mice after 11 days of primary infection and 8h post-reinfection. N=4-6. D. Recruitment of immune cells to the tongue at 8h of reinfection. N=6; Two-tailed Mann–Whitney Test. E. Oral fungal burden in Rag1^−/− and Ccr2^−/− mice at 2 days post-reinfection. N=6; Two-tailed Mann–Whitney Test. F. Oral fungal burden in neutrophil-depleted mice 1 day post-reinfection. N=6; Two-tailed Mann–Whitney Test.

Figure 2.. β-glucan priming induces epithelial memory.

A. Schematic of the in vitro β-glucan priming model in human oral epithelial cells. Created with BioRender.com B. Chemokine and cytokine levels in supernatants from naïve and BG-primed epithelial cells after 8h of C. albicans infection. N=6; One-way ANOVA with Tukey’s multiple comparisons test. UNINF – uninfected, INF – Infected. C. Chemokine and cytokine levels in supernatants from naïve, LPS-treated, and BG-primed epithelial cells after LPS restimulation for 24h. N=6; One-way ANOVA with Tukey’s multiple comparisons test. D. Chemokine and cytokine levels in supernatants from naïve, LPS-treated, and LPS-primed epithelial cells after LPS restimulation for 24h. N=6; One-way ANOVA with Tukey’s multiple comparisons test. E. Volcano plot showing pathways associated with differentially accessible regions (DARs) identified by ATAC-seq after, analyzed using g: profiler. F. Stacked bar plot depicting canonical pathways significantly enriched between 24 h after β-glucan-stimulated and control oral epithelial cells, as determined by Ingenuity Pathway Analysis (IPA). N=4. G. Representative immunoblot showing histone methylation in β-glucan primed epithelial cells after 48h rest. N – naïve, P – β-glucan primed H. Schematic of fungal infection model and timeline of epithelial-enriched tissue collection. I. Representative immunoblot showing histone methylation in epithelial-enriched tissue from wildtype mice after primary infection. N=3. J. Global DNA methylation levels in epithelial cells primed with β-glucan and mannan for 24h followed by 48h resting. N=6; One-way ANOVA with Dunnett’s multiple comparisons test. N – naïve, BG – β-glucan, M – mannan.

Figure 3.. Recognition of β-glucan induces proline catabolism in epithelial cells.

A. Schematic of key metabolites measured in epithelial cells involved in glycolysis, serine/glycine biosynthesis, the tricarboxylic acid (TCA) cycle, and proline metabolism following stimulation without or with β-glucan (BG). Created with BioRender.com B. Extracellular concentrations of glucose, proline, and glutamine in epithelial cells stimulated without or with β-glucan indicated time points. N=6; One-way ANOVA with Dunnett’s multiple comparisons test. C. Heat map showing the relative abundance of intracellular metabolites in epithelial cells stimulated without or with BG for the indicated time points. N=6. Abbreviations: Pyr – pyruvate, Lac – lactate; Cit – citrate, αKG – α-ketoglutarate, Suc – succinate, Fum – fumarate, Mal – malate, Ala – Alanine, Glu – glutamate, Gln – glutamine, Pro – proline, Asp – aspartate, Asn – asparagine, Ser – serine, Gly – glycine, Val – valine, Leu – leucine, IIe – isoleucine. D. Intracellular concentrations of selected metabolites in epithelial cells stimulated with BG for the indicated time points. N=5. One-way ANOVA with Dunnett’s multiple comparisons test. E-F. Mitochondrial oxidative function was quantified in epithelial cells stimulated without or with β-β-glucan for 24h, measured by Seahorse extracellular flux analysis. N=9; Unpaired student’s t-test. G. Schematic of the fungal infection and timeline of epithelial-enriched tissue metabolite measurement. Created with BioRender.com H. Concentrations of metabolites in epithelial-enriched tissues from immunocompetent wildtype mice after primary infection. N=8. Two-tailed Mann–Whitney Test.

Figure 4.. Epithelial memory is promoted by proline catabolism.

A-C. Representative immunoblot analysis of proline dehydrogenase (PRODH; proline catabolism) and pyrroline-5-carboxylate synthase (P5CS; proline biosynthesis) expression in epithelial cells stimulated without or with β-glucan for the indicated times (A), in β-glucan primed epithelial cells after 48h resting (B), and in epithelial-enriched tissue from immunocompetent wildtype mice after 5 days of primary infection (C). N – naïve, P – β-glucan primed, S – Sham, Ca – Candida albicans D. Schematic diagram of the mitochondrial proline catabolism pathway. E. Intracellular metabolite concentrations in epithelial cells treated without or with 10mM tetrahydrofurfuryl acid (THFA; a proline dehydrogenase inhibitor) and stimulated with BG for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. F. Levels of chemokines and cytokines in culture supernatants of epithelial cells primed without or with THFA and BG after 8h of infection with C. albicans. N=6. Unpaired Student t-test. G. Global DNA methylation levels in epithelial cells trained with β-glucan and THFA for 24h, followed by 48h of resting in culture media. N=6. Two-tailed Mann–Whitney Test. H. Concentrations of metabolites in epithelial-enriched tissues from Prodh^wt/wt and Prodh^−/− mice 5 days post-infection. I. Oral fungal burden in Prodh^wt/wt and Prodh^−/− mice during reinfection. N=6. One-way ANOVA with Dunnett’s multiple comparisons test. The y-axis represents the limit of detection (20 CFUs/g of tissue). J. Proinflammatory cytokine response in tongue homogenates in Prodh^wt/wt and Prodh^−/− mice upon reinfection. N=6; Two-tailed Mann–Whitney Test. K. Global DNA methylation levels in epithelial-enriched tissues from Prodh^wt/wt and Prodh^−/− mice 5 days after primary infection.

Figure 5.. Protective epithelial memory occurs independently of glycolysis.

A. Glucose concentrations in the media of epithelial cells treated without or with β-glucan (BG) and 10mM tetrahydrofurfuryl acid (THFA) for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. B. Intracellular pyruvate and lactate concentrations in epithelial cells treated without or with β-glucan and 10mM THFA for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. C. Intracellular reactive oxygen species levels in epithelial cells treated without or with THFA and BG for 2h. N=9; One-way ANOVA with Tukey’s multiple comparisons test. D. Representative immunoblot analysis of hypoxia-inducible factor 1-alpha (HIF-1α), glucose transporter 1-3 (GLUT1 and GLUT3) expression in epithelial cells treated without or with β-glucan and 10mM THFA for 24h. N=3. One-way ANOVA with Dunnett’s multiple comparisons test. E. Proposed schematic diagram of proline catabolism-mediated activation of glycolysis. Created with BioRender.com F. Schematic diagram of glycolysis and metabolite measurement approach. G. Glucose concentrations in media of epithelial cells treated without or with BG and 10mM 2-Deoxy-D-glucose (2DG; glycolysis inhibitor) for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. H. Intracellular metabolite concentrations in epithelial cells treated without or with BG and 10mM 2DG for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. I. Levels of chemokines and cytokines in culture supernatants of epithelial cells primed without or with 2DG/BG after 8h of infection with C. albicans. N=6. Unpaired Student t-test. J. Global DNA methylation levels in epithelial cells treated with β-glucan and 2DG for 24h followed by resting in culture media for 48h. N=6; Two-Tailed Mann–Whitney Test.

Figure 6.. β-glucan recognition induces fatty acid oxidation in epithelial cells.

A. Intracellular fatty acid concentrations in epithelial cells stimulated without or with β-glucan for 6 and 24h. N=5; One-way ANOVA with Dunnett’s multiple comparisons test. B. Intracellular fatty acid concentrations in BG-primed epithelial cells after 48h of rest. N=5; Two-tailed Mann–Whitney Test. C. Fatty acids concentrations in epithelial-enriched tissue from immunocompetent wild-type mice. N=6. Two-tailed Mann–Whitney Test. Abbreviations: PA:C16:0 – palmitic acid, POA:C16:1n7 – Palmitoleic acid, SA:C18:0 – Stearic acid, OA: C18:1n9 – oleic acid, α-LA:C18:3n3 – α-linoleic acid and LA:C18:2n6 – linoleic acid. D. Schematic of fatty acid oxidation and TCA cycle. E-G. Representative immunoblot analysis of carnitine palmitoyltransferase (CPT1/2) expression in epithelial cells stimulated without or with β-glucan for the indicated times (E), in β-glucan primed epithelial cells after 48h (F) and in epithelial-enriched tissue from immunocompetent wild-type mice after fungal clearance with primary infection(G).

Figure 7.. Epithelial memory is partially dependent on fatty acid oxidation.

A. Fatty acid concentrations in epithelial cells treated without or with 100μM etomoxir (ETO; carnitine palmitoyltransferase inhibitor) and stimulated with BG for 24h. N=5. One-way ANOVA with Tukey’s multiple comparisons test. Abbreviations: PA:C16:0 – palmitic acid, POA:C16:1n7 – Palmitoleic acid, SA:C18:0 – Stearic acid, OA: C18:1n9 – oleic acid, and α-LA:C18:3n3 – α-linoleic acid. B. Levels of chemokines and cytokines in culture supernatants of epithelial cells primed without or with ETO/BG after 8h of infection with C. albicans. N=6. Unpaired student t-test. C. Global DNA methylation in epithelial cells treated with β-glucan and ETO for 24h and rested in culture media for 48h. N=6. Two-tailed Mann–Whitney Test. D. Representative immunoblot of histone methylation in primed epithelial cells without or with ETO/BG for 24h, followed by resting in culture media for 48h. E. Levels of chemokines and cytokines in culture supernatants of epithelial cells primed without or with the histone methyltrasferase inhibitor MTA (5-deoxy-5-methylthio-adenosine)/BG, followed by 8h of infection with C. albicans. N=6. Unpaired student’s t-test. F. Levels of chemokines and cytokines in the media supernatants of OECs primed without or with BG/10μM 2-hydroxyglutarate (2-HG) after 8h of infection with C. albicans. N=6, Un-paired t-test.