Fig 1.
The viability test of F. pedrosoi-spores, -hyphae and induced sclerotic cells.
The F. pedrosoi-spores, -hyphae and induced sclerotic cells were pre-stained with FUN1 Cell Stain (Cat: F7030, Invitrogen), a unique two-color fluorescent viability probe for yeast and fungi, and the viability was measured using flow cytometry assay according to the protocol by setting the 488 nm laser line as the excitation source, standard FITC channel (FITC-A) for detection of green emission (dead/metabolically inactive cells) and PE channel (PE-A) for red emission (live/metabolically active cells). The fungal cells with red emission or two-color emissions were considered viable (Percentage in Q2 and Q3 regions). In addition, the fungal cells without FUN1 treatment were set as self-fluorescence control. The fungal cells which were heat-killed by incubation in a water-bath at 70°C for 90 min and stained with FUN1 were set as the dead cell control.
Fig 2.
Intraperitoneal infection of BALB/c mice with F. pedrosoi-spores developed to be self-healing.
(A) Infectious foci in the abdomen at 10 days post-inoculation, as indicated by red arrows (top panel); PAS staining (×400) for the infected spleen was performed, and the majority of spores with budding were indicated by red circle (bottom panel). (B) Spleens from BALB/c mice at 30 days post-inoculation and normal control, scale bar = 1 mm (top panel); PAS staining (×400) for the spleen from BALB/c mice at 30 days post-inoculation (bottom panel).
Fig 3.
Recalcitrant infection of BALB/c mice was induced by intraperitoneal injection of F. pedrosoi hyphae with the formation of sclerotic cells in the nidus.
(A-C) Spleens respectively from BALB/c mice at 10 days (A), 30 days (B) and 50 days (C) post-inoculation as well as the normal control (left column). The infectious foci in the spleens and abdomen were indicated by red arrows. Scale bar = 1 mm. (A-C) HE staining (×400) for the infected spleens at 10 days (A), 30 days (B) and 50 days (C) post-inoculation (right column).
Fig 4.
In vitro induced transformation of saprophytic F. pedrosoi into sclerotic cells.
Optical microscope was used to characterize the morphology of saprophytic F. pedrosoi growing on PDA (A) and in-vitro transformed sclerotic cells cultured in ATCC830 medium plus PAF for 50 days (×400) (B). Scale bar = 20 μm (A and B).
Fig 5.
Recalcitrant infection of BALB/c mice was induced by intraperitoneal injection of in vitro-transformed F. pedrosoi sclerotic cells.
(A and B) Spleens respectively from BALB/c mice at 10 days (A) and 50 days (B) post-inoculation as well as the normal control (top row). The infectious foci in the spleens and abdomen were indicated by red arrows and red circle. Scale bar = 1 mm. (A and B) HE staining (×400) for infected spleens at 10 days (A) and 50 days (B) post-inoculation (bottom row).
Fig 6.
IFN-γ production was initially inhibited, followed by pan-inhibition of Th1/Th2/Th17 cytokines production in splenocytes of BALB/c mice intraperitoneally infected with F. pedrosoi sclerotic cells.
(A and B) BD Cytometric Beads Array (CBA) murine cytokine kit was introduced to measure the Th1/Th2/Th17 cytokine levels in culture supernatants of splenocytes from BALB/c mice at 10 days, 30 days and 50 days after intraperitoneally inoculation with F. pedrosoi-spores, -hyphae or transformed sclerotic cells respectively (n = 12 per group). The BALB/c mice of the same age intraperitoneally inoculated with normal saline were set as inoculation control (n = 12). Before measurement, the splenocytes from BALB/c mice were adjusted to 2×106 cells in a volume of 2 mL RPMI1640 plus 10% FCS, and were pretreated with 1×Cell Stimulation Solution at 37°C for 6h. (A) Seven bead populations with distinct APC-A fluorescence intensities had been coated with capture Abs specific for IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10 proteins, as indicated by A1-A7 in turns. The concentration of specific cytokine mentioned above can be revealed by the fluorescence intensity of PE-conjugated detection Ab, and be calculated according to the standard curve established by cytokine standards using FCAP Array software. The assay diluent incubated with beads and PE-conjugated Ab was set as blank control. (B) Graph showing the concentration (pg/mL) of Th1/Th2/Th17 cytokines produced by splenocytes from uninfected and F. pedrosoi-infected BALB/c mice mentioned above at the indicated days post-inoculation. Data represent the mean±SEM (n = 4 per group at each indicated time point) and statistical analysis was performed using Univariate ANOVA and LSD-t test. Significant: * P<0.05; Highly Significant: ** P<0.01.
Fig 7.
Intraperitoneal administration of IFN-γ effectively reduced fungal load in the infected mice spleens, and dampened peritoneal dissemination of F. pedrosoi-sclerotic cells.
Abbreviations: F. pedrosoi-SC (F. pedrosoi-sclerotic cells); F. pedrosoi-SC+IFN-γ (F. pedrosoi-sclerotic cells and exogeneous IFN-γ) (A) The fungal load in the infected spleens of BALB/c mice respectively at 10 days, 30 days and 50 days post-inoculation was compared between the group intraperitoneally injected with F. pedrosoi-SC and that injected with F. pedrosoi-SC+IFN-γ. Data represent the mean±SEM (n = 4 per group at each indicated time point) and statistical analysis was performed using Univariate ANOVA and LSD-t test. The Significant: * P<0.05; Highly Significant: ** P<0.01. (B) The peritoneal dissemination of F. pedrosoi-SC in BALB/c mice was compared between the group intraperitoneally injected with F. pedrosoi-SC and that with F. pedrosoi-SC+IFN-γ respectively at 10 days, 30 days and 50 days post-inoculation. The abdominal and retroperitoneal organs including the colon, kidneys, spleen, liver and bowels were respectively isolated from the mouse injected with F. pedrosoi-SC+IFN-γ at 50 days post-inoculation for fungal examination (from top to bottom, right panel). The infectious foci were indicated by red circles.
Fig 8.
Exogeneous IFN-γ contributed to the formation and maintenance of micro-abscess, and restored the decrease in neutrophil ROS production in the mouse spleen infected with F. pedrosoi-sclerotic cells.
Abbreviations: F. pedrosoi-SC (F. pedrosoi-sclerotic cells); F. pedrosoi-SC+IFN-γ (F. pedrosoi-sclerotic cells and exogeneous IFN-γ) (A) HE staining (×400) for the infected spleens from the BALB/c mice intraperitoneally inoculated with F. pedrosoi-SC or with F. pedrosoi-SC+IFN-γ respectively at 10 days, 30 days and 50 days post-inoculation. The sclerotic cells with transverse septation in the infectious foci were indicated by red arrows. (B and C) Neutrophil ROS was measured in the infected spleens from the BALB/c mice intraperitoneally injected with F. pedrosoi-spores, -hyphae, -SC, and -SC+IFN-γ at the indicated days post-inoculation. (B) Flow cytometry assay was introduced to measure ROS generation in neutrophils. Briefly, the infected nodules in the spleens were aseptically isolated, and the neutrophils were selected by using side scatter (SSA) and APC-conjugated anti-mouse Ly-6G (Gr-1) mAb. The cells incubated with APC-conjugated rat-derived IgG2a kappa were set as isotype control. ROS levels in neutrophils were measured using 2’, 7’-dichlorodihydrofluorescin diacetate (DCHF-DA) as fluorescence probe, and were represented as mean fluorescence intensity (MFI). The neutrophils without adding DCHF-DA were set as blank control. (C) Neutrophil ROS in the infected spleens was compared among the groups of BALB/c mice mentioned above at indicated days post-inoculation. Data represent the mean±SEM (n = 4 per group at each indicated time point) and statistical analysis was performed using Univariate ANOVA and LSD-t test. Highly Significant: ** P<0.01.
Fig 9.
Dectin-1, MR and Dectin-2 mainly bind to the surface of F. pedrosoi-hyphae, but not the transformed sclerotic cells.
(A-C) The binding of murine-derived Dectin-1, Mannose Receptor (MR), and Dectin-2 to F. pedrosoi cultured in ATCC 830 medium with 10−6 M PAF at 35°C for 30 days was detected respectively by PE-conjugated anti-murine Dectin-1 mAb (A), FITC-conjugated anti-murine MR mAb (B), and PE-conjugated anti-murine Dectin-2 mAb (C) using confocal microscope. Left column: fluorescence field; Middle column: bright field; Right column: merged images. Scale bar = 20 μm. The transformed sclerotic cells with cross-septation and swelling chlamydospores were indicated by red circle (A) or red arrows (B and C). The fungal cells incubated only with PE-conjugated anti-murine Dectin-1 or Dectin-2 mAb, or only with FITC-conjugated anti-murine MR mAb were set as blank control.
Fig 10.
Some chitin-like component exclusively accumulates on the surface of in-vitro transformed sclerotic cells and hyphal tip.
(A-C) FITC-conjugated Wheat Germ Agglutinin (WGA), a lectin that specifically binds to chitin, was introduced to detect chitin distribution on the surface of F. pedrosoi using confocal microscope. Left column: fluorescence field; Middle column: bright field; Right column: merged images. The binding of FITC-conjugated WGA to F. pedrosoi cultured in ATCC 830 medium with 10−6 M PAF for 40 days (A) or 50 days (B) was detected using confocal microscope. (C) F. pedrosoi cultured in ATCC 830 medium with 10−6 M PAF for 40 days with chitinase pretreatment was set as control. Scale bar = 20 μm. The majority of transformed sclerotic cells with cross-septation as well as swelling chlamydospores were indicated by red arrows (A) and circles (B and C).
Fig 11.
Three-dimensional conformation of chitin distribution re-constructs the figuration of in-vitro transformed sclerotic cells.
(A-D) Three-dimensional distribution of chitin on the surface of F. pedrosoi cultured in ATCC 830 medium with 10−6 M PAF for 40 days were analyzed by FITC-conjugated WGA using confocal tomoscanning. (A) F. pedrosoi in the bright field. In-vitro transformed sclerotic cells with cross-septation were indicated by red circles. (B) Three-dimensional conformation of chitin distribution on F. pedrosoi was represented by FITC-conjugated WGA in the fluorescence field. (C) The binding of FITC-conjugated WGA on the surface of F. pedrosoi was analyzed by confocal tomoscanning. Each cross-section thickness was set as 0.5μm. Scale bar = 20 μm. (D) Three-dimensional conformation of chitin distribution represented by FITC-conjugated WGA was reconstructed according to confocal tomoscanning.
Fig 12.
Chitin accumulation on the surface of F. pedrosoi-sclerotic cells compensatorily increased after chitinase treatment in a time-dependent manner.
(A-F) Abbreviations: F. pedrosoi-spore (F. p-spore); F. pedrosoi-hyphae (F. p-hyphae); In-vitro transformed F. pedrosoi-sclerotic cells in ATCC 830 medium with 10−6 M PAF for 50 days (FPSC); Heat-killed FPSC (HK-FPSC); Chitinase (CHIA). (A and B) The binding capacity of murine Dectin-1 to F. p-spores, F. p-hyphae, FPSC or HK-FPSC was respectively detected by PE-conjugated anti-murine Dectin-1 mAb using flow cytometry, and was represented as mean fluorescence intensity (MFI). (C and D) The binding capacity of murine Dectin-2 to F. p-spores, F. p-hyphae, FPSC or HK-FPSC was respectively detected by PE-conjugated anti-murine Dectin-2 mAb using flow cytometry, and was represented as mean fluorescence intensity (MFI). (A-D) The fungal cells incubated only with PE-conjugated isotypes were set as blank control. (E and F) The binding capacity of FITC-conjugated WGA to F. p-spore, F. p-hyphae, FPSC or HK-FPSC before and after chitinase treatment at indicated time points was detected by flow cytometry, and was represented as mean fluorescence intensity (MFI). (B, D and F) Data represent the mean±SEM from three individual experiments performed in triplicates, and statistical analysis was performed using Univariate ANOVA and LSD-t test. Significant: * P<0.05; Highly Significant: ** P<0.01.
Fig 13.
Exclusive accumulation of chitin was also detected on the surface of in-vivo transformed F. pedrosoi-sclerotic cells which were isolated from the nidus with chitinase expression.
(A and B) Expression of acidic mammalian chitinase (AMCase) in the footpads of nu/nu-BALB/c mice at 80 days (A, n = 3), or in the spleens of BALB/c mice at 50 days (B, n = 3) after inoculation with F. pedrosoi was detected by rabbit anti-AMCase polyAb using immunohistochemistry. In vivo-transformed sclerotic cells in the nidus at 50 days after inoculation with F. pedrosoi-hyphae were indicated by red arrows. (C) AMCase expression in the infected footpads of nu/nu-BALB/c mice or in the infected spleens of BALB/c mice was detected by rabbit anti-AMCase polyAb using western blotting at indicated days after inoculation with F. pedrosoi-hyphae (n = 5 per subgroup of BALB/c and nu/nu- BALB/c mice). The nu/nu-BALB/c or BALB/c mice subcutaneously or intraperitoneally inoculated with NS were set as normal controls (n = 15). β-actin in the infected footpads or spleens was set as loading control, and markers were shown. (D) Confocal microscope was introduced to detect the binding of FITC-conjugated WGA to F. pedrosoi isolated from the infected spleens of BALB/c mice at 50 days post-inoculation. (E) F. pedrosoi isolated from infected spleens of BALB/c mice at 50 days post-inoculation with chitinase pretreatment was set as control. In-vivo transformed sclerotic cells were indicated by red circles. Left column: fluorescence field; Middle column: bright field; Right column: merged images. Scale bar = 20 μm.
Fig 14.
Chitin contributes to an inhibited IFN-γ production in splenocytes of BALB/c mice after intraperitoneal stimulation of heat-killed F. pedrosoi sclerotic cells.
(A-C) The BALB/c mice were intraperitoneally injected for 3 times at 7-day intervals with in-vitro transformed F. pedrosoi-sclerotic cells which were heat-killed and treated with chitinase or not. (A) HE staining (×400) for the spleens isolated from BALB/c mice at 36 days after initial injection of heat-killed F. pedrosoi-sclerotic cells with chitinase treatment (left panel) or not (right panel). The sclerotic cells with cross -septation in the nidus were indicated by red circles. (B) BD Cytometric Beads Array (CBA) murine cytokine kit was introduced to measure the Th1/Th2/Th17 cytokine levels in culture supernatants of splenocytes from BALB/c mice at 7 days, 14 days, 21 days and 36 days after initial injection of heat-killed F. pedrosoi-sclerotic cells with chitinase treatment or not (n = 16 per group). The BALB/c mice intraperitoneally inoculated with normal saline were set as inoculation control (n = 16). Before measurement, the splenocytes from BALB/c mice were adjusted to 2×106 cells in a volume of 2 mL RPMI1640 plus 10% FCS, and were pretreated with 1×Cell Stimulation Solution at 37°C for 6h. Seven bead populations with distinct APC-A fluorescence intensities had been coated with capture Abs specific for IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10 proteins, as indicated by A1-A7 in turns. The concentration of specific cytokine mentioned above can be revealed by the fluorescence intensity of PE-conjugated detection Ab, and be calculated according to the standard curve established by cytokine standards using FCAP Array software. The assay diluent incubated with beads and PE-conjugated Ab was set as blank control. (C) Graph showing the concentration (pg/mL) of Th1/Th2/Th17 cytokines produced by splenocytes from BALB/c mice intraperitoneally stimulated with heat-killed F. pedrosoi-sclerotic cells with chitinase treatment or not as well as inoculation controls at the indicated days after initial inoculation. Data represent the mean±SEM (n = 4 per group at each indicated time point), and statistical analysis was performed using Univariate ANOVA and LSD-t test. Significant: * P<0.05; Highly Significant: ** P<0.01.