Fig 1.
Neutrophils with DC characteristics (PMN-DCs) emerge during pulmonary blastomycosis.
(A) Neutrophil gating strategy showing representative lung sample 7 dpi with Blastomyces. (B) Canonical neutrophils (CD11b+, Ly6G+, Ly6Cint, CD11c-), PMN-DCs (CD11b+, Ly6G+, Ly6Cint, CD11c+), and MoDCs (CD11b+, Ly6G-, Ly6C+, CD11c+) were FACS sorted from Blastomyces-infected lungs at day 7 then stained with a Hema3 kit for microscopy. (C-D) Kinetic analysis of neutrophil differentiation after intratracheal challenge with Blastomyces showing relative proportion (C) and absolute numbers (D) of differentiated neutrophils (parent CD11b+, Ly6G+, Ly6Cint); the combined population is the sum of all neutrophils expressing either CD11c or MHC class II. (E-F) Surface expression of neutrophil marker (CXCR2) and antigen-presenting cell markers (MHC class I, CD80, CD86) on neutrophil populations and moDCs (S1C Fig) indicated by histograms (E) and relative expression (F) as indicated by mean fluorescence over fluorescence minus one (FMO) control (gray histogram). (G) Expression of CD64 on neutrophil and MoDC populations. Representative experiments shown; mean±SEM indicated; N = 3–5 mice/group; C-D show representative experiment from 3 kinetic experiments; E-G show representative results from at least two independent experiments at 7 dpi with Blastomyces.
Fig 2.
PMN-DCs associate with yeast and kill yeast at higher frequencies than canonical neutrophils.
Mice were challenged with DsRed B. dermatitidis, and lungs were harvested at 7 dpi. (A) Representative plots showing canonical neutrophils, PMN-DCs and moDCs indicating the cells of each population associated with yeast (% Uvitex+). Inset histograms show Uvitex+ events indicating live (DsRed+) and dead (DsRed-, % noted) yeast associated with each population. (B) The proportion of each population associated with yeast as indicated by Uvitex staining. (C) The killing rate–proportion of Uvitex+ yeast that are DsRed-. (D) The proportion of total yeast (all Uvitex+ events) associated with indicated leukocytes. (E) The contribution to yeast killing is indicated by the proportion of total killed yeast in the lung (all DsRed-Uvitex+ events) associated with each population. Panel A shows a single representative experiment of 4 independent experiments; N = 5 mice; means ± SEM indicated.
Fig 3.
Phagocyte functions of PMN-DCs.
(A-B) Mice were challenged with GFP B. dermatitidis, and lungs were harvested 7 dpi and stained with calcofluor white to mark extracellular yeast. (A) Representative flow plots showing association of gated leukocyte populations with extracellular (Calcfluor+, blue) and phagocytosed (Calcofluor-, green) yeast (GFP+). (B) Absolute number (histogram bars) of phagocytosed yeast by moDCs, canonical neutrophils or PMN-DCs with inset pie chart indicating the proportion of all yeast phagocytosis by leukocyte populations in the lung. (C) Surface expression of fungal-recognizing pattern recognition receptors on neutrophil populations and moDCs at 7 dpi with B. dermatitidis. Histograms show FMO controls (gray) and stained populations (red); relative expression of each receptor on each population. (E–F) Ex vivo staining of neutrophil populations and moDCs with ROS-indicator DHR-123 (C) or NO-indicator DAF-FM (D). Representative histograms shown on top; proportions of ROS+ and NO+ on the bottom left and ROS/NO production indicate by MFI on the right. All experiments are representative of at least two independent experiments. N = 5 mice. Means ± SEM indicated.
Fig 4.
Appearance of PMN-DCs and fungal killing by the cells during pulmonary aspergillosis and systemic candidiasis.
(A–C) Mice were infected IT with Uvitex-stained DsRed A. fumigatus spores, and lungs were harvest 48 hours later. (A) Proportions and absolute numbers of canonical and MHCII+ only neutrophils, PMN-DCs (MHCII+ CD11c+) and moDCs in the lung. (B) Representative plots showing association of leukocyte populations with live (DsRed+) and killed (DsRed-) Aspergillus spores (Uvitex+). (C) Proportion of leukocytes associated with live and killed spores (top) and killing rate (% DsRed-/Uvitex+) (bottom) of spores by leukocytes in the lungs. (D–E) Mice were challenged IV with C. albicans yeast; kidneys, spleens, and peripheral blood were harvested at day 1 or 3 or from naïve mice (day 0). (D) Representative plots showing neutrophils (CD11b+, Ly6G+, Ly6Gint, Siglec F-) with inset plots indicating the proportion of CD11c+ neutrophils expressing MHC class II. (E) Time course showing absolute numbers of canonical (MHCII- CD11c-), MHCII+ CD11c-, CD11c+ and MHCII+ CD11c+ neutrophils in tissues during systemic candidiasis. All data are representative of at least three independent experiments. N = 3–5 mice. Means±SEM indicated. For C, statistical comparisons were among leukocyte populations; for D, statistical comparisons were with Day 0 control.
Fig 5.
Differentiation of granulocyte/macrophage progenitor (GMP) cell line into PMN-DCs in vitro.
(A) ER-HoxB8 GMP cells (GFP+) were cultured for 4–5 days in the presence or absence of estrogen and then cultured for an additional 5 days with or without GM-CSF or IL-4 in the presence of bone marrow feeder cells; differentiation of GFP+ cells into PMN-DCs was tracked by CD11c and MHC class II expression. (B-C) GMP cells were matured into neutrophils (Day 0) or further differentiated into PMN-DCs (after 5 days with GM-CSF and IL-4 and feeder cells). (B) Undifferentiated (Day 0) or differentiated (Day 5) neutrophils (upper panel) were incubated overnight with DsRed A. fumigatus spores stained with Uvitex and then analyzed by flow cytometry; the association rate with live (DsRed+) or dead (DsRed-) A. fumigatus spores with each population from above plots is shown in the lower panel. (C) Undifferentiated neutrophils or differentiated PMN-DCs were incubated for 1 hour with β-glucan-coated AlexaFluor647 beads and analyzed by flow cytometry for association with cells. (D) Expression of CD11c and MHC class II on starting neutrophils (Day 0) vs. neutrophils differentiated for 5 days with or without feeder cells (MFI indicated). (E) GMP cells were matured into neutrophils (Day 0) and differentiated 5–7 days without feeder cells; representative flow plots and images of cells at day 0, 5 or 7. Mean ± SEM shown.
Fig 6.
Direct fungal killing by PMN-DCs and protection by adoptive transfer of PMN-DCs.
Canonical neutrophils or PMN-DCs were generated in vitro from the GMP cell line and co-cultured in vitro with fungi (A-D) or transferred IV into mice (E). (A) Fungal killing by canonical neutrophils or PMN-DCs during in vitro culture with C. albicans yeast (top) or B. dermatitidis yeast (bottom). (B) Scanning electron microscopy (SEM) of canonical neutrophils or PMN-DCs alone, with C. albicans or B. dermatitidis, or stimulated with PMA. Higher magnification images of highlighted boxes are shown to the right of wider image. For interactions with fungi, top images show interactions between cells and fungi highlighting phagocytosis and attempted phagocytosis; bottom images show NETs. (C) SEM comparison of NET structure and thickness released by canonical neutrophils or PMN-DCs in response to C. albicans or B. dermatitidis. (D) C. albicans was incubated for 2 hours to become filamentous before neutrophils or PMN-DCs were added in killing assays in the presence or absence of 50 μg/ml DNase I. (E) WT mice were infected IV with 105 C. albicans yeast and received 2 x 106 canonical neutrophils or PMN-DCs (or PBS vehicle) IV 24 hours later. Burden in kidneys shown at 3 dpi. (A, D) C. albicans viability was determined by XTT assay and compared with neutrophil-absent control to calculate percent killing. (A) B. dermatitidis was plated on BHI agar after to determine number of remaining viable yeast. Means ± SEM are shown. Data are representative of at least two independent experiments.
Fig 7.
Presentation of fungal antigen (calnexin) to transgenic Tg1807 T cells by PMN-DCs generated in vitro from ER-HoxB8 GMP cells.
(A–D) PMN-DCs or bone-marrow DCs (BMDCs) were incubated overnight with or without recombinant fungal calnexin, and the next day 5 x 104 cells were injected subcutaneously into CD90.2 mice that had received adoptive transfer of Tg1807 cells (CD90.1+, calnexin-specific CD4+ T cells). One week later skin draining lymph nodes were harvested. (A) IL-6 in supernatants from overnight culture of PMN-DCs or BMDCs with or without calnexin, n.d,:not detected. (B) Experimental design of delivering calnexin-loaded APCs into mice that had received congenic Tg1807 cells. (C) Proportion of Tg1807 cells activated in lymph nodes (indicated by CD44+ and CD62L-). (D) The absolute number of Tg1807 cells and activated Tg1807 cells for each treatment group (N = 5–6 mice). (E) Lymph node cells (as shown in C-D) were stimulated ex vivo for 3 days with calnexin, and IFN-γ and IL-17 assayed in culture supernatants, Ag: antigen pulsed. (F-G) PMN-DCs or BMDCs were incubated overnight with fungal calnexin or heat-killed Blastomyces yeast before enriched CD4+ Tg1807 cells were added. (F) Presence of IFN-γ and IL-17 in supernatants collected after 3 days was determined by ELISA. (G) Expression of MHC class II from PMN-DCs at the end of the assay (Day 11) shown with comparison of MHC class II expression before mixing with T cells (Day 7) and before PMN-DC differentiation (Day 0), MFI indicated (±SEM). Both in vivo and in vitro experiments were completed independently twice. Statistics: for C-E, statistical significance between calnexin-loaded PMN-DCs and no antigen controls; for F-G, statistical significance between samples with fungal antigen and unstimulated.