Fig 1.
Differential expression of uptake receptors during GM-CSF driven differentiation.
Bone marrow was harvested from C57BL/6 mice and was cultured in GM-CSF and stained with antibodies to CD36, CD206, or Dectin-1. (A) Representative histograms showing the expression of CD36, CD206, and Dectin-1 from day 3 to 7. Data were compiled from at least three independent experiments showing mean fluorescence intensity (MFI) of (B) CD36 and (C) MMR expression in the five cell populations. Statistical analysis was conducted using one way ANOVA Tukey multiple comparison test. Letters over bars indicate statistically significant differences in means (p<0.05).
Fig 2.
The uptake capacity of GM-CSF driven myeloid cells increased as cell progress in development and modestly decreased in more differentiated cell types.
Sorted cell populations were fed 0.5 mg/mL Dextran Alexa Flour 488 (10,000 MW) or S. aureus BioParticle Alexa flour 594 conjugate for 1 h at 37°C (red), or at 4°C (blue) as negative control. The uptake of the fluorescent tracers was measured by flow cytometry. (A) Representative histograms of Dextran uptake (top panels) and BioParticle uptake (bottom panels) by the five isolated myeloid cell populations. Uptake of dextran (B) and bioparticles (C). Uptake values were calculated by subtracting the MFI of the ice control for each population from the MFI of that population incubated at 37°C. These data are compiled from at least four independent experiments. Statistical analysis was performed using SAS, one-way ANOVA Tukey’s multiple comparison tests. Letters over bars indicate statistically significant differences in means (p<0.05).
Fig 3.
Phagosomal pH of each developmental stage.
Sorted populations were pulsed with 10 μL pHrodo E. coli BioParticles for 15 min, washed and chased for 90 min. The uptake of the pHrodo E. coli BioParticles and the phagosomal acidification was measured by flow cytometry. (A) A representative histograms of pHrodo E. coli BioParticle uptake at 15 min (top) and phagosomal acidification at 90 min (bottom) in all the five isolated populations. (B) The MFI of pHrodo E. coli BioParticle uptake at 15 min and 90 min, in all the five isolated myeloid cell populations. Data are compiled from at least four independent experiments. (C) The phagosomal pH of each myeloid cell population was calculated vs a standard curve at varying pHs maintained in Hepes buffer containing nigericin and monensin. The statistical analysis was performed using SAS, one way ANOVA Tukey’s multiple comparison test. Letters over bars indicate statistically significant differences in means (p<0.05).
Fig 4.
Proteolytic activity increases as myeloid cells become more differentiated.
Sorted populations were pulsed with 10 μg/mL DQ™ ovalbumin (DQ-OVA) for 15 min, washed and chased for 30 min, 1hr or 2hr. Proteolysis (indicated by increased fluorescence) of DQ-OVA over time was measured by flow cytometry. (A) A representative histogram of DQ-OVA uptake at 15 min (blue) and degradation at 30 min (red). (B) The MFI of DQ-OVA at 15 min and degradation at 30 min, 1hr and 2hr in all the five isolated myeloid cell populations. (C) The MFI of DQ-OVA degradation only within OVA-Alexa positive cells measured at 1 hr post incubation. Compiled data come from a minimum of three independent experiments. Statistical analysis was conducted using one way ANOVA Tukey multiple comparison test. Asterisk indicate a p<0.05 when compared with the 15 min pulse.
Fig 5.
moMac/moDP and moDC exhibited both antigen processing and presentation functions.
The sorted cell populations were incubated with different concentrations of OVA peptide or OVA protein and co-cultured with CFSE labeled T cells. (A) The dot plots illustrate CD4+ OT-II T cells stimulated with 1 μM of OVA323-339 (top) or 100 μg/mL of OVA protein (bottom). T cell proliferation and activation was measured using CFSE fluorescence dilution at day 5 by flow cytometry. (B) and (C) Compiled data of CD25 expression by OT-II T cells in the presence of OVA peptide (1 μM) or OVA protein (100 μg/mL), respectively. (D) The dot plots of CD8+ OT-I T cells stimulated with 10 nM of OVA257-264 (top) or 300 μg/mL of OVA protein (bottom). T cell proliferation and activation was measured at day 4 by flow cytometry. (E) and (F) CD25 expression by OT-I T cells in the presence of OVA peptide (10 nM) or OVA protein (300 μg/mL), respectively. Compiled data are derived from a minimum of 3 independent experiments. Statistical analysis was conducted using one-way ANOVA Tukey multiple comparison test. Letters over bars indicate statistically significant differences in means (p<0.05).
Fig 6.
The MHCIIlo and MHCIIint cell populations are similar in uptake capacity and phagosomal acidification when compared with MHCIIhi cells.
Cells were sorted into three populations on day 5 of culture: MHCIIlo, MHCIIint and MHCIIhi after first gating on CD11c+, and CD11b+ cells. (A) Sorted cells were fed S. aureus BioParticle-Alexa flour 594 conjugate for 1 h. Representative histograms of BioParticle uptake at 4°C (blue) or 37°C (red). (B) Compiled MFI of BioParticle uptake from three experiments. (C) Representative histograms of cells pulsed with pHrodo E. coli BioParticles at 15 min (red) and chased for 90 min (brown) or at 4°C for 90 min (blue). (D) The compiled MFI of pHrodo E. coli BioParticle at 15 min and 90 min. (E) Calculated phagosomal pH of each cell population based on standard curves. (F) Representative histograms of cells pulsed with 10 μg/mL DQ-OVA for 15 min (red) and chased for 30 min (brown) or at 4°C for 1 hr (blue). (G) Compiled MFI of DQ-OVA at 15 min and degradation at 30 min and 1hr. (H) The compiled MFI of DQ-OVA degradation within OVA-Alexa positive cells measured at 1 hr post incubation. Statistical analysis was performed using SAS, one-way ANOVA Tukey’s multiple comparison test. Letters over bars indicate statistically significant differences in means (p<0.05).
Fig 7.
CMP, GMP and monocyte derived ex-vivo are similar in functionality to in vitro GM-CSF derived counterpart.
Freshly harvested ex-vivo bone marrow cells were sorted into 3 populations has previously described. (A) Sorted cells were fed E. coli BioParticle-Alexa flour 594 conjugate for 1 h. Representative histograms of BioParticle uptake at 4°C (blue) or 37°C (red). (B) Compiled MFI of BioParticle uptake from three experiments. (C) Representative histograms of cells pulsed with pHrodo E. coli BioParticles at 15 min (red) and chased for 90 min (brown) or at 4°C for 90 min (blue). (D) The compiled MFI of pHrodo E. coli BioParticle at 15 min and 90 min. (E) Calculated phagosomal pH of each cell population based on standard curves. (F) The compiled MFI of DQ-OVA degradation within OVA-Alexa positive cells measured at 1 hr post incubation.