Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction

Abstract

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-β are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.

Figure 1. MerTK is up-regulated during monocyte-to-macrophage differentiation, and is further enhanced by M-CSF

(A–D) Monocytes were sorted from human healthy PBMCs through negative selection magnetic beads, and analyzed by flow cytometry at day 0, and after 1 and 3 days of culture. Monocytes were gated using an anti-CD14 antibody, and stained with anti-MerTK antibody. An anti-CD42b antibody was also used, to distinguish platelet-monocyte conjugates from isolated monocytes. (E–G) Monocytes were cultured in complete medium in the presence of M-CSF (50 ng/ml) or GM-CSF (100 ng/ml), or in the absence of colony stimulating factors (CSFs), and analyzed for MerTK expression on day 3. Data shown are representative of three independent experiments.

Figure 2. Macrophage MerTK expression promptly adapts to changes of immunological environment

(A–C) CD14+ cells were cultured in complete medium in the presence of M-CSF (50 ng/ml) or GM-CSF (100 ng/ml). On day 5, cells were treated with GM-CSF (100 ng/ml), M-CSF (50 ng/ml), IFNγ (10 ng/ml) + LPS (1 μg/ml), IL-4 (20 ng/ml) or dexamethasone (Dex; 100 nM), for an additional 3 days. MerTK expression was analyzed by Western blot (A), or measured by flow cytometry as MFI fold variation compared to levels obtained with culturing cells with M-CSF alone (B) or GM-CSF alone (C) for 8 days. Data shown are representative of three independent experiments. (D–E) Cells were incubated with dexamethasone (Dex; 1–1000 nM), in the presence or absence of M-CSF (50 ng/ml), for 3 days in serum-free medium. MerTK up-regulation was measured by flow cytometry as MFI fold increase compared to expression levels in untreated cells. Data shown are representative of four independent experiments. (F–G) Cells were cultured in complete medium in the presence of M-CSF (50 ng/ml) or GM-CSF (100 ng/ml), or in the absence of colony stimulating factors. On day 5, cells were treated with IL-4 (20 ng/ml) or IL-10 (50 ng/ml), for an additional 3 days. Dendritic cells (DCs) were differentiated in the presence of GM-CSF and IL-4 from day 0 for 8 days. MerTK expression was analyzed by Western blot (F). Densitometry of Western blots was performed to quantify MerTK expression following cell treatment with IL-10, M-CSF, or both (G). Densitometry values were normalized to β-actin, and are reported as fold variation compared to MerTK expression levels in untreated cells. Data are representative of three independent experiments. (H) Cells were incubated with GM-CSF or GM-CSF + IL-4 for 8 days in complete medium, to differentiate M1 macrophages or DCs, respectively. Cells were stained for CD209 (DC-SIGN), CD1a and MerTK. MerTK+ cells were quantified as percentages of total cells by flow cytometry. *P <0.05; **P <0.01; ***P <0.001.

Figure 3. MerTK expression is restricted to (CD163+CD16+CD206+) M2 macrophages

Macrophages were differentiated from peripheral monocytes for 7–8 days in complete medium, in the presence of GM-CSF (100 ng/ml), IFNγ (10 ng/ml), IL-4 (20 ng/ml), M-CSF (50 ng/ml), dexamethasone (100 nM), or IL-10 (50 ng/ml). Cells were stained for MerTK, CD14, CD163, CD204/SR-A1, CD16, CD206, CD209, and analyzed by flow cytometry. Histograms show MFI fold variations compared to levels in untreated cells (basal). Data shown are representative of eight to twelve independent experiments. *P <0.05; **P <0.01; ***P <0.001.

Figure 4. IL-10 is required for M-CSF to induce M2c differentiation and up-regulate MerTK

(A) CD14+ cells were cultured in the presence or absence of M-CSF (50 ng/ml), in either serum-containing or serum-free medium, for 3 days. (B) Cells were incubated with IL-10 (1 ng/ml) and/or increasing doses of M-CSF (0.05 to 50 ng/ml). MerTK up-regulation was measured by flow cytometry as MFI fold increase compared to expression levels in untreated cells. Data shown are representative of three independent experiments. (C) Cells were cultured in serum-containing medium with or without M-CSF (50 ng/ml), in the presence or absence of a neutralizing mouse monoclonal anti-human IL-10 antibody (5 μg/ml; Biolegend, clone JES3-9D7), for 3 days. Cells were stained for CD163 and MerTK, and quantified by flow cytometry as percentages of total cells. (D) Cells were cultured in serum-free medium in the presence of M-CSF (50 ng/ml), IL-10 (50 ng/ml), M-CSF + IL-10, TGFβ (20 ng/ml), dexamethasone (Dex; 100 nM), for 4 days. Cells were stained for MerTK, CD163, CD16 and CD163. MerTK expression is shown as MFI fold variation compared to levels in untreated cells, as well as MerTK+ cell percentages; CD163, CD16 and CD14 MFI fold variations are also reported. Data shown are representative of four independent experiments. The expression of IL-10 receptor (IL-10R, or CD210) was measured after 3-day cytokine stimulations. Data shown are representative of three independent experiments. *P <0.05; **P <0.01; ***P <0.001.

Figure 5. MerTK confers to M2c (M-CSF+IL-10) macrophages enhanced ability to clear early ACs

(A) Early apoptotic cells (ACs) were obtained incubating human neutrophils, isolated from peripheral blood healthy donors, in 10% FCS-RPMI for 20 hours. According to annexin V and propidium iodide (PI) staining, early ACs were around 65–70% of total neutrophils. (B and C) CFSE-labeled apoptotic neutrophils were added for 60 minutes to 7-day differentiated M0 (untreated), M1 (IFNγ, 2.5 ng/ml), M2a (IL-4, 20 ng/ml) and M2c (M-CSF, 50 ng/ml + IL-10, 50 ng/ml) macrophages, labeled with a fluorochrome-conjugated anti-CD14 antibody, at a 5:1 ratio. M2c macrophages showed significantly enhanced ability to clear early ACs, expressed as higher percentages of phagocytic (CSFE+) macrophages. Pre-incubation of M2c macrophages with a goat polyclonal anti-human MerTK antibody (5 μg/ml; R&D Systems) for 30 minutes before addition of apoptotic neutrophils abolished such superiority of M2c cells to phagocytose ACs compared to other macrophage subsets. Blocking MerTK diminished not only the number of CFSE+ macrophages, but also the mean phagocytosis activity per single cell, depicted as CFSE MFI. Altogether, it resulted in a significant decrease of the phagocytosis index, determined by multiplying the percentage of CFSE+ macrophages by the CFSE MFI of phagocytic macrophages. Data shown are representative of three independent experiments. *P <0.05; **P <0.01. (D) By fluorescence microscopy (Leica TCS SP5 confocal laser scanning microscope, 40X/1.25 NA oil objective), M2c macrophages stained for CD14 (red) and CD163 (green) were shown to engulf Hoechst 33342-labeled apoptotic neutrophils (blue) (left panel, yellow arrows). Pre-incubation of M2c macrophages with an anti-MerTK blocking antibody inhibited engulfment, but not the physical interaction between macrophages and apoptotic neutrophils (right panel, white arrows).

Figure 6. M2c markers universally define MerTK+ macrophages prone to AC clearance

(A–C) CD14+ cells were cultured in serum-free medium in the presence of IFNγ (10 ng/ml; M1), IL-4 (20 ng/ml; M2a), M-CSF (50 ng/ml) + IL-10 (50 ng/ml; M2c), or in the absence of cytokines (M0), for 4 days. Cells were stained for MerTK, CD163, CD14, CD16 and CD204. Co-expression of MerTK and M2c surface markers was studied by flow cytometry (A). For each M2c receptor (CD14, CD16, CD163, CD204), an Expression Ratio was obtained by dividing “percentage of macrophages expressing a given M2c receptor among MerTK+ macrophages” by “percentage of macrophages expressing a given M2c receptor among total macrophages in culture”, after differentiation in M0, M1, M2a or M2c conditions. Frequencies of each M2c receptor (percentages of positive cells) among MerTK+ macrophages and among total macrophages were analyzed for potential significant differences between the two sets of data (B). IL-4-treated cells were also stained for CD209 and CD206 (C). Data shown are representative of four independent experiments. (D–E) CD14+ cells treated with IFNγ or IL-4 for 3 days in serum-free medium were co-incubated with CFSE-labeled apoptotic neutrophils for 1 hour, and then stained for CD14 and CD163. For inhibition studies, cells were pre-incubated with a goat polyclonal anti-human MerTK antibody (2 μg/ml; R&D Systems) or a goat control IgG (2 μg/ml; SouthernBiotech) for 30 minutes before addition of apoptotic neutrophils. Percentages of CFSE+ phagocytic macrophages were determined among the major populations of M1 or M2a cells (CD14^dimCD163−) and the minor populations of M2c-like cells (CD14^brightCD163+). Data from one representative experiment (D) and three independent experiments (E) are reported. *P <0.05; **P <0.01.

Figure 7. M2c-like CD14^brightCD16+ circulating monocytes utilize MerTK to phagocitose ACs

(A–E) Freshly isolated monocytes were analyzed by flow cytometry directly from PBMCs, without magnetic sorting, in order to include also CD16+ (HLA-DR+) monocytes. On the basis of CD14 and CD16 expression levels, monocytes were divided into 3 categories: CD14^brightCD16− (red histograms and peaks), CD14^brightCD16+ (blue) and CD14^dimCD16+ (green). Platelet-monocyte conjugates were depicted by flow cytometry as events also positive for the platelet marker CD42b in each monocyte subset (D). Percentages in (E) refer to positivity of CD14^brightCD16+ cells for the receptors indicated. Data shown are representative of four independent experiments. (F–H) freshly isolated PBMCs were co-incubated with CFSE-labeled apoptotic neutrophils at a 1:1 ratio for 4 hours. For inhibition studies, cells were pre-incubated with a goat polyclonal anti-human MerTK antibody (2 μg/ml; R&D Systems) or a goat control IgG (2 μg/ml; SouthernBiotech) for 30 minutes before addition of apoptotic neutrophils. Percentages of CFSE+ phagocytic monocytes were determined within each monocyte subset. Data from three independent experiments (F–G) and one representative experiment (H) are reported. *P <0.05; **P <0.01; ***P <0.001.

Figure 8. Gas6 is released by M2c macrophages, and amplifies IL-10 production via MerTK

(A) Gas6 was measured by ELISA in supernatants of CD14+ cells cultured in serum-free medium, in the presence of different treatments (M-CSF, 50 ng/ml; IL-10, 50 ng/ml; dexamethasone, 100 nM; TGFβ, 20 ng/ml; GM-CSF, 100 ng/ml; IFNγ, 10 ng/ml; IL-4, 20 ng/ml), for 4 days. Data shown are representative of four independent experiments. (B–C) CD14+ cells were cultured in serum-free conditions for 3 days in the absence of colony stimulating factors, and stimulated from day 1 with LPS (50 ng/ml) ± rhGas6 (1 μg/ml) for 48 hours; TNFα and IL-10 levels were measured in supernatants by ELISA. Data shown are representative of three independent experiments. (D) Cells were cultured in serum-free conditions for 3 days in the presence of M-CSF (50 ng/ml), and stimulated from day 1 with LPS ± rhGas6 for 48 hours; rhGas6 significantly increased LPS-induced IL-10 release in culture supernatants, as assessed by ELISA. IL-10 increase was prevented by blocking MerTK with a goat polyclonal anti-human MerTK antibody (5 μg/ml; R&D Systems) during LPS + rhGas6 stimulation. Data shown are representative of four independent experiments. (E) Cells were cultured in serum-free conditions for 4 days in the presence or absence of M-CSF (50 ng/ml) ± IL-10 (50 ng/ml). On day 1, a recombinant MerFc (5 μg/ml; R&D Systems) or a goat polyclonal anti-human Gas6 blocking antibody (5 μg/ml; R&D Systems) was added to precipitate Gas6 endogenously produced by cultured cells. From day 2, cells were stimulated with LPS for 48 hours. Neither MerFc or anti-Gas6 antibody were able to restore TNFα secretion, inhibited in M2c (M-CSF+IL-10) cells. Data shown are representative of three independent experiments. *P <0.05; **P <0.01; ***P <0.001.