Fig 1.
Monocyte phenotyping reveals tightly controlled distribution of subsets.
(A) Gating strategy for phenotyping human monocyte subsets, including (from left to right) size discrimination, doublet exclusion, selection of HLA-DR+lin− cells, and separation according to expression of CD14 and CD16. (B) Representative histograms of cell surface markers on monocyte subsets from one of 25 healthy subjects phenotyped (C = classical, I = intermediate, NC = non-classical). Classical monocytes (red), intermediate monocytes (yellow), and non-classical monocytes (blue) are shown overlaid on negative controls (dashed). (C) Mean expression (±SD) of surface markers on all subjects phenotyped. (D) Absolute numbers of total (black), classical (red), intermediate (yellow), and non-classical monocytes (blue) in circulation (bar = mean). (E) Frequency of each subset as a percentage of total circulating monocytes. (F) Change in the frequency of each subset in each of 10 subjects re-phenotyped 4–6 months following initial phenotyping. The average change in frequency (Δ) of each subset is shown.
Fig 2.
Morphology of sorted monocyte subsets.
(A) Low power magnification of monocyte subsets after magnetic enrichment and high-speed sorting from leukocyte concentrates (photomicrographs taken at 10X). (B) Differential interference contrast (DIC) image of monocyte subsets after two-day culture and adherence to substrate at low magnification (upper panel) and high magnification (lower panel). (C) Fluorescent image of the cells shown in lower panel B, with actin (orange), vinculin (green), β-tubulin (green) and nucleus (blue) stained to show morphological features.
Fig 3.
Cytokine secretion by sorted monocyte subsets stimulated with agonists to TLRs 1–9.
Secretion of TNFα (A), IL-1β (B) and IL-6 (C) by monocyte subsets (mean ± SD, n = 6) in response to extracellular TLR agonists pam3CSK4 (1μg/mL), HKLM (108 cells/mL), poly(I:C) (10 μg/mL), LPS (1 μg/mL), flagellin (10 μg/mL), FSL-1 (1μg/mL), imiquimod (10 μg/mL), ssRNA40 (10 μg/mL), and ODN2006 (5 μM) in culture for 18 hours is shown (*p<0.05).
Fig 4.
Classical monocytes differentiate to monocyte-derived DCs.
(A) DIC images of sorted monocyte subsets after seven days in DC differentiation medium, with classical monocytes displaying fully differentiated DC morphology, intermediate monocytes also displaying DC morphology, although the cells are fewer and smaller, and non-classical monocytes showing no differentiation to DC in the few living cells, but otherwise evidence of cell death (upper panel, photomicrographs taken at 20X). DIC images (bottom panels) were captured at 60X (bar = 10μm). (B) Fluorescent images of the cells pictured in lower panel of A, with actin (orange), vinculin (green), β-tubulin (green) and nucleus (blue) stained to show morphological features (bar = 10μm). (C) MFI (mean ±SD) of surface marker expression for all subjects (n = 5) (* p<0.05, ** p<0.01, **** p<0.0001, † p = 0.06). (D) Representative histograms from one subject (C = classical, I = intermediate, NC = non-classical). Plots from each subset–classical monocytes (red), intermediate monocytes (yellow), and non-classical (blue)–cultured in DC differentiation medium for seven days are shown overlaid on plots from control cultures for each subset cultured in unsupplemented complete medium for seven days (dashed). (E) T cell proliferation in allogeneic MLRs using mo-DCs derived from classical monocytes (right bracket), undifferentiated unfractionated monocytes (center bracket), or PBMC (left bracket) as stimulators are shown (mean ± SD, 2 technical replicates from one subject). (F) IFN-γ production in dividing T cells in allogeneic MLRs shown in E. (G) Phagocytosis assay using DC differentiated from classical, intermediate, and nonclassical monocytes (right bracket) or undifferentiated monocytes (center bracket) Controls include PBMCs incubated with beads at 0°C and 37°C, as well as monocyte-derived macrophages (left bracket). Bars represent mean ± SD of 2 technical replicates from one subject.
Fig 5.
Monocytes do not differentiate to pDCs, but do secrete type I IFN in response to intracellular TLR stimulation.
(A) Photomicrographs of sorted classical, intermediate, and non-classical monocytes after one week of culture in pDC differentiation medium. All cultures display features of both differentiated DCs and macrophages, with colony formation and reticular networking (bar = 50μm). (B) Representative histograms from one of two healthy subjects (C = classical, I = intermediate, NC = non-classical). Classical monocytes (red), intermediate monocytes (yellow), and non-classical monocytes (blue) cultured for seven days in pDC medium are shown overlaid on undifferentiated, uncultured monocytes (dashed). Plots from mature pDCs are shown in the bottom row (grey shaded). (C) IFN-α secretion by monocytes cultured in pDC differentiation medium for seven days and stimulated with agonists to intracellular TLR (mean +/- SD, n = 2) (D) IFN-α secretion by undifferentiated monocytes in response to stimulation with agonists to intracellular TLR. TLR agonists were added in the presence of lipid transfection reagents in both C and D.
Fig 6.
All monocyte subsets differentiate to macrophages with culture in GM-CSF- or M-CSF-containing medium.
(A) DIC images of macrophages differentiated from sorted classical, intermediate, or non-classical monocytes cultured for five days in medium with low-dose GM-CSF to induce M1 differentiation (bar = 10μm). (B) Fluorescent images of the cells in panel A, where actin (orange), vinculin (green), β-tubulin (green) and nucleus (blue) stains show morphological features. Of note, all subsets display rounded but firmly adherent appearance with actin-rich podosomes and extensions indicating attachment to the substrate (bar = 10μm). (C) DIC images of macrophages differentiated from sorted classical, intermediate, or non-classical monocytes cultured for five days in medium with M-CSF to induce M2 differentiation (bar = 10μm). (D) Fluorescent images of the cells in panel C, where actin (orange), vinculin (green), β-tubulin (green) and nucleus (blue) stains show morphological features. Note the presence of ventral membrane podosome formation in cells from all subsets (bar = 10μm). (E) Heatmap showing normalized expression of macrophage-associated surface markers for each subset (C = classical, I = intermediate, NC = non-classical) as undifferentiated, uncultured monocytes (left panel), cultured in M1 medium (center panel), or cultured in M2 medium (right panel). (F) Heatmap showing secretion of macrophage-associated cytokines for each subset (C = classical, I = intermediate, NC = non-classical) as undifferentiated, uncultured monocytes (top panel), cultured in M1 medium (middle panel), or cultured in M2 medium (bottom panel). Each box represents the mean of two technical replicates; data is shown separately for each subject tested. (G) Phagocytosis by undifferentiated monocytes and monocytes differentiated in either M1 or M2 macrophage-induction medium. Bars represent mean +/- SD of two technical replicates from one subject. Controls include PBMCs incubated with beads at 0°C and 37°C (left bracket).