Fig 1.
GM-CSF mediates detrimental host responses to Cryptococcus gattii.
(A) Pulmonary GM-CSF cytokine levels in WT mice infected with C. gattii. Days 3, 7, and 10 data include n = 8-10 total mice per timepoint from N = 2 independent experiments; Days 0 (naive) and 5 data are from n = 5 total mice per timepoint from N = 1 experiment. (B) Kaplan-Meier survival curve of WT (white circles) and Csf2-/- (blue circles) mice, n = 14-15 total mice per group from N = 3 independent experiments. (C-E) Fungal burden was measured in lung (C), mediastinal lymph node (D), and brain (E). For the lung data, Day 5 includes n = 6-8 total mice per group from N = 2 independent experiments, and Day 10 includes n = 12-14 total mice per group from N = 3 independent experiments. For lymph node and brain data, Day 5 consists of n = 3-4 total mice per group from N = 1 experiment, and Day 10 consists of n = 8-10 total mice per group from N = 2 independent experiments. (F) Representative images of unaltered whole lungs surgically collected from WT and Csf2-/- mice at Days 5 and 10 p.i. Photo credit: Alison Ricafrente, First Author. (G-L) Representative lung sections from WT (G, I, K) and Csf2-/- (H, J, L) mice stained with Masson’s trichrome. For Panels G and H, scale bar = 100 μM at 10X magnification. The black boxes are magnified in Panels I and J to show alveolar spaces. The red boxes are magnified in Panels K and L to show the adventitial sheath. For Panels I-L, scale bar = 12 μM at 40X magnification. Fungal cells (yellow arrowheads); Airway (AW); Blood vessel (BV); Collagen fibers (black arrows). Histology images are representative of n = 4-5 total mice per group from N = 1 experiment; all fields of one lung slice from each mouse were evaluated. Data are presented as mean ± SEM and analyzed using one-way ANOVA (A), Mantel-Cox test (B), or two-way ANOVA (C-E). *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001.
Fig 2.
GM-CSF regulates the generation of monocyte-derived macrophages during C. gattii infection.
(A-B) Representative H&E-stained lung sections of infected WT (A) and Csf2-/- (B) mice at Day 10 p.i. showing pulmonary infiltrates, scale bar = 200 μM at 4X magnification. Black boxes are magnified in bottom panels, scale bar = 12 μM at 60X magnification. Fungal cells (yellow arrowhead); Foamy macrophages (white arrowhead). (C-G) Enumeration of lung cells from WT (white circles) and Csf2-/- (blue circles) mice at Days 5 and 10 p.i., including alveolar macrophages (C), interstitial macrophages (D), Ly6Chi monocytes (E), Ly6Clo monocytes (F), and monocyte-derived dendritic cells (moDCs) (G). (H-J) Pulmonary levels of cytokines MCP-1/CCL2 (H), IL-1⍺ (I), and IL-1β (J). Histology images are representative of n = 4-5 total mice per group from N = 1 experiment; all fields of one lung slice from each mouse were evaluated. For lung cell and cytokine data, Day 5 includes n = 4-5 total mice per group from N = 1 experiment; Day 10 includes n = 7-10 total mice per group from N = 2 independent experiments. Data are presented as mean ± SEM and analyzed using two-way ANOVA (C-J). *, P < 0.05. **, P < 0.01. *** P < 0.001. ****, P < 0.0001.
Fig 3.
Monocytes are detrimental to the host during C. gattii infection.
(A) Monocyte ablation strategy using CCR2-DTR+ and WT littermate mice, treated with 200 ng of diphtheria toxin by i.p. injection a day prior to infection with C. gattii and at Days 1 and 3 p.i. Organs were harvested at Day 3 or 7 p.i. for fungal burden measurements. (B) Kaplan-Meier survival curve of WT littermate (white circles) and CCR2-DTR+ (gray circles) mice, n = 28-31 total mice per group from N = 6 independent experiments. (C-E) Fungal burden was measured in lung (C), MLN (D), and brain (E). For lungs, Day 3 includes n = 26-28 total mice per group from N = 6 independent experiments; Day 7 includes n = 8-9 total mice per group from N = 2 independent experiments. For MLN and brains, Day 3 consists of n = 16-17 total mice per group from N = 3 independent experiments; Day 7 consists of n = 8-9 total mice per group from N = 2 independent experiments. (F) Representative images of unaltered whole lungs surgically collected from WT littermate and CCR2-DTR+ mice at Day 7 p.i. Photo credit: Alison Ricafrente, First Author. (G-J) Representative Masson’s trichrome stain of lung sections from WT littermate and CCR2-DTR+ mice comparing alveoli (G-H), scale bar = 100 μM at 10X magnification, and bronchovascular bundles (I-J), scale bar = 25 μM at 40X magnification. Airway (AW); Blood vessel (BV); Collagen fibers (black arrows); Fungal cells (yellow arrowheads). Histology images are representative of n = 4 total mice per group from N = 1 experiment; all fields of one lung slice from each mouse were evaluated. Data are presented as mean ± SEM and analyzed using Mantel-Cox test (B) and two-way ANOVA (C-E). *, P < 0.05. ***, P < 0.001. ****, P < 0.0001. Mouse illustration in (A) is from NIAID NIH BioArt Source (bioart.niaid.nih.gov/bioart/279).
Fig 4.
Monocyte-derived macrophages are permissive for C. gattii infection.
(A-D) BMDM were challenged with 2 × 104 C. neoformans H99-GFP or C. gattii R265-GFP at an MOI of 1:40 and analyzed for fungal uptake (A), fungal killing (B), expression of M1 and M2 polarization markers (C), and TNF cytokine secretion (D). Results are from n = 6-9 total replicates per group from N = 2-3 independent experiments. (E-I) WT mice were infected with 105 C. gattii, and on Day 7 p.i. samples from infected and naive lungs were collected for macrophage isolation. Alveolar macrophages were isolated from bronchoalveolar lavage (BAL) and interstitial macrophages were isolated from whole lung single cell suspensions. TNF expression by interstitial and alveolar macrophages from the lungs of naive (blue) or Day 7 p.i. (red) mice was measured by intracellular cytokine staining. Data are shown on a histogram (E), as compared to a fluorescence minus one (FMO) control (black dotted line) and macrophages from naive mice stimulated with lipopolysaccharide (LPS) ex vivo (black line), alongside quantitation of TNF+ macrophages per sample (F, G). The dotted line shows the percentage of macrophages from naive mice stimulated with LPS that expressed TNF (F, G). RNA from macrophages was also analyzed by qRT-PCR for expression of M1 and M2 polarization markers (H-I). Results are from n = 5 WT mice from N = 1 experiment. Data are presented as mean ± SEM and analyzed using Mann-Whitney test (A-D, F, G) and one-way ANOVA (H, I). **, P < 0.01. ****, P < 0.0001.
Fig 5.
GM-CSF induces the pulmonary influx of eosinophils over neutrophils during infection.
(A-B) Representative H&E-stained lung sections from WT (A) and Csf2-/- (B) mice at Day 10 p.i. showing perivascular cell infiltrates, scale bar = 25 μM at 20X magnification. Black boxes are magnified in the bottom panels, scale bar = 12 μM at 60X magnification. BV (Blood vessel). Histology images are representative of n = 4-5 total mice per group from N = 1 experiment; all fields of one lung slice from each mouse were evaluated. (C-E) Flow cytometry of lung cells from WT (white circles) and Csf2-/- (blue circles) mice, including eosinophils (C) neutrophils (D) and the calculated eosinophil-to-neutrophil (Eos:Neu) cellular ratio (E); a ratio of 1:1 is indicated by the dotted line. Results are from n = 7-9 total mice per group at Day 5 p.i. from N = 2 independent experiments; and n = 16 total mice per group at Day 10 p.i. from N = 4 independent experiments. (F-J) Pulmonary levels of cytokines IL-5 (F), G-CSF (G), IL-12p40 (H), IL-12p70 (I), and IL-17 (J). Naive results are from n = 5 total mice per group from N = 1 experiment; Day 5 results are from n = 3-4 total mice per group from N = 1 experiment; and Day 10 results are from n = 8-10 total mice per group from N = 2 independent experiments. Data are presented as mean ± SEM and analyzed using two-way ANOVA (C-J). *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001.
Fig 6.
Monocytes and monocyte-derived macrophages reinforce a high eosinophil-to-neutrophil ratio in the infected lung.
(A-B) Representative H&E-stained lung sections from WT littermates (A) and CCR2-DTR+ (B) mice at Day 7 p.i. showing perivascular cell infiltrates, scale bar = 25 μM at 20X magnification. Black boxes are magnified in bottom panels, scale bar = 12 μM at 60X magnification. BV (Blood vessel). Histology images are representative of n = 4 total mice per group from N = 1 independent experiment; all fields of one lung slice from each mouse were evaluated. (C-E) Flow cytometry of lung cells from naive WT (white triangles) and infected WT littermates (white circles) and infected Csf2-/- mice (gray circles), including eosinophils (C), neutrophils (D), and the calculated Eos:Neu cellular ratio (E); a ratio of 1:1 is indicated by the dotted line. Naive results are from n = 6 WT littermates from N = 2 independent experiments; Day 3 results are from and n = 15-19 total mice per group from N = 3 independent experiments; and Day 7 results are from n = 5 total mice per group from N = 1 experiment. (F-J) Pulmonary levels of cytokines IL-5 (F), G-CSF (G), IL-12p40 (H), IL-12p70 (I), and IL-17 (J) normalized to WT mean (black dotted line on left y-axis). Absolute WT mean cytokine levels shown by orange triangles and right y-axis. Naive results are from n = 3 total mice per group from N = 1 experiment; Day 3 results are from n = 8-9 total mice per group from N = 2 independent experiments; and Day 7 results are from n = 9 total mice per group from N = 2 independent experiments. Data are presented as mean ± SEM and analyzed using two-way ANOVA (C-E) or Mann-Whitney test (F-J). Absolute WT Mean cytokine values are not included in statistical analysis. *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001.
Fig 7.
Neutrophils are beneficial to the host after C. gattii infection.
(A) Neutrophil ablation strategy using Mrp8cretg iDTR+ mice and iDTR+ littermate control mice. All mice were treated with 200 ng of diphtheria toxin by i.p. injection daily, starting one day prior to i.t. infection with C. gattii. (B) At Day 3 and 7 p.i., tail vein blood was collected to measure blood neutrophils, and at Day 7 p.i., whole lungs were collected to measure total neutrophils (C) and fungal burden by CFU (D). Results are from n = 9-12 total mice per group from N = 3 independent experiments. Data are presented as mean ± SEM and analyzed using Mann-Whitney test. *, P < 0.05. **, P < 0.01. ****, P < 0.0001. Mouse illustration in (A) is from NIAID NIH BioArt Source (bioart.niaid.nih.gov/bioart/279).
Fig 8.
Schematic model of GM-CSF and monocyte signaling during Cryptococcus gattii infection.
After C. gattii enters the lungs, the fungus may induce various cells, such as Type I (AECI), Type II alveolar epithelial cells (AECII), endothelial cells, or fibroblasts to release GM-CSF. This GM-CSF induces recruited CCR2+Ly6Chi monocytes to differentiate into alveolar and interstitial macrophages. However, these macrophages cannot undergo M1 polarization, which then enables C. gattii to proliferate and invade host tissues unchecked. GM-CSF and monocyte-derived macrophages also facilitate the pulmonary influx of eosinophils while inhibiting the entry of neutrophils through the regulation of cytokines including IL-5, G-CSF, IL-12p40, and IL-17. The accumulation of eosinophils leads to harmful airway inflammation. The absence of anti-fungal neutrophils allows further fungal proliferation. Altogether, the secretion of GM-CSF and the generation of monocyte-derived macrophages fuel an imbalance of eosinophils and neutrophils that ultimately results in host mortality. Created in BioRender. Ricafrente, A. (2025) https://BioRender.com/lpowwec.