Gut-Evolved Candida albicans Induces Metabolic Changes in Neutrophils

Abstract

Serial passaging of the human fungal pathogen Candida albicans in the gastrointestinal tract of antibiotics-treated mice selects for virulence-attenuated strains. These gut-evolved strains protect the host from infection by a wide range of pathogens via trained immunity. Here, we further investigated the molecular and cellular mechanisms underlying this innate immune memory. Both Dectin-1 (the main receptor for β-glucan; a well-described immune training molecule in the fungal cell wall) and Nod2 (a receptor described to mediate BCG-induced trained immunity), were redundant for the protection induced by gut-evolved C. albicans against a virulent C. albicans strain, suggesting that gut-evolved C. albicans strains induce trained immunity via other pathways. Cytometry by time of flight (CyTOF) analysis of mouse splenocytes revealed that immunization with gut-evolved C. albicans resulted in an expansion of neutrophils and a reduction in natural killer (NK) cells, but no significant numeric changes in monocytes, macrophages or dendritic cell populations. Systemic depletion of phagocytes or neutrophils, but not of macrophages or NK cells, reduced protection mediated by gut-evolved C. albicans. Splenocytes and bone marrow cells of mice immunized with gut-evolved C. albicans demonstrated metabolic changes. In particular, splenic neutrophils displayed significantly elevated glycolytic and respiratory activity in comparison to those from mock-immunized mice. Although further investigation is required for fully deciphering the trained immunity mechanism induced by gut-evolved C. albicans strains, this data is consistent with the existence of several mechanisms of trained immunity, triggered by different training stimuli and involving different immune molecules and cell types.

Keywords: Candida albicans; immunometabolism; infectious diseases; live attenuated vaccines; neutrophils; trained immunity.

Figure 1

Protection against systemic candidiasis using the evolved strain is not dependent on Dectin-1, NOD2, TLR2 or TLR4. Survival of Dectin-1 (A), NOD2 (B), TLR2 (C) and TLR4 (D) KO mice immunized with 5x10⁵ CFUs of the gut-evolved strain R24 were challenged at day 14 post-immunization with a lethal dose of the WT C. albicans (SC5314) (5x10⁵ CFUs). n = 10 mice/group. Log-rank survival test was performed in Prism v9. ****P < 0.0001.

Figure 2

Cytometry by Time-Of-Flight (CyTOF) analysis of splenocytes from mice vaccinated with the evolved strains R24 and W2N identifies multiple changes in the innate immunity populations. (A) tSNE plots of CD45⁺CD90⁻CD3⁻CD19⁻ myeloid spleen cells from non-vaccinated mice and mice vaccinated with a low dose of the wild-type C. albicans strain SC5314 (10⁴ CFUs/mouse) or with the evolved C. albicans strains R24 and W2N (5x10⁵ CFUs/mouse) illustrating color-coded cell populations that clustered based on cell surface marker expression. (B) Bar graphs show the mean ± SEM of the Neutrophils and NK populations in the splenocytes from vaccinated and non-vaccinated mice. n=3 mice/group. One-way ANOVA with multiple comparisons (ANOVA p-value of 0.0002 for neutrophils and 0.0031 for NK cells), **P < 0.01, *P < 0.05.

Figure 3

Gut-evolved C. albicans R24-induced protection from systemic fungal infections is mediated by neutrophils. (A) WT mice systemically immunized with C. albicans R24 were injected at day 7 post-immunization with either clodronate liposomes or control liposomes (purchased from http://clodronateliposomes.com) i.v. at 200 µl liposome suspension/mouse every other day. Animals were challenged at day 14 post-immunization with the lethal dose of the WT C. albicans (SC5314) (5x10⁵ CFUs). 100% of mice immunized with the gut evolved strain (R24) succumbed after systemic challenge by WT SC5314 when treated with clodronate liposomes, but not with the control liposomes. Macrophages were specifically depleted using CSF1R neutralizing antibody (B) and the role of monocyte recruitment was studied in CCR2^−/− mice (C). NK cells and neutrophils were specifically depleted using NK1.1 neutralizing antibody (D) or Ly6G neutralizing antibody (E), respectively. None of the immunized mice succumbed after systemic challenge by WT SC5314 when treated with CSF1R or NK1.1 antibodies, while the immunized mice treated with the Ly6G neutralizing antibody succumbed to the challenge with a lethal dose of the WT C. albicans (E), or with a lethal dose of P. aeruginosa (F). n = 10 mice/group. Log-rank survival test was performed in Prism v9. ***P < 0.001, ****P < 0.0001.

Figure 4

R24 vaccination increase both mitochondrial respiration and glycolytic rate/potential. (A) Ex vivo Oxygen consumption rate (OCR) in Splenocytes and Bone Marrow cells from mice vaccinated 14 days with the gut evolved strain R24 (5x10⁵ CFUs) and non-vaccinated mice. Splenocytes and BM cells were purified, plated in 96-well plates and exposed to oligomycin, FCCP, and ROT plus AA (see “Materials and methods”). OCR was measured as pmol O₂/min/µg. (B) Proton Efflux Rate (PER) in Splenocytes and Bone Marrow cells from mice vaccinated 14 days with the gut evolved strain R24 (5x10⁵ CFUs) or non-vaccinated mice was determined by the glycolytic rate assay as mpH/min. (C) Basal Respiration, ATP production, maximal respiration and spare respiratory capacity were obtained by the mitochondrial stress analysis and are represented as mean ± SEM of n = 5 individual samples. (D) Basal glycolysis and compensatory glycolysis were obtained by the glycolytic rate assay and are represented as mean ± SEM of n = 5 individual samples. (E) Ex vivo Oxygen Consumption Rate (OCR) in neutrophils sorted from spleens from R24 vaccinated mice and non-vaccinated mice. (F) Basal Respiration, ATP production, maximal respiration and spare respiratory capacity are represented as mean ± SEM of n = 5 individual samples. Mann-Whitney tests between R24-vaccinated and mock-immunized mice were performed using Prism v9. *P < 0.05, **P < 0.01, ***P < 0.001.