The effect of trained innate immunity on macrophage efferocytosis of melanoma cells.

(A) Schematic diagram of experimental layout.

(B-D) Trained or control macrophages were pre-stained with CFSE and were then co-cultured with pHrodo-labelled apoptotic B16F10 melanoma cells for 4 h. (B) Efferocytosis, (C) Representative flow cytometric plot; Numbers in outlined areas indicate the percentage of macrophages that is pHrodo+ red), (D) Ratio of mean fluorescence intensity (MFI) are shown and were calculated as the percentage of pHrodo+ macrophages and pHrodo MFI in macrophages, respectively. (B, D) Each paired line represents average values from nine different experiments (n = 44 separate cell isolations per group).

(E) Live cell imaging was performed in co-cultures as shown in (A). Levels of efferocytosis are shown over time (left, n = 8-10 separate cell isolations per group). Representative images in selected time points are shown (right). Arrows denote phagocytic macrophages.

*P<0.05, **P<0.01, ****P<0.0001. Paired (B, D) two-tailed Student’s t-test, Two-way ANOVA test (E). Data are presented as mean and mean ± s.e.m.

The impact of trained macrophages in efferocytosis of breast cancer cells.

Trained or control macrophages were pre-stained with CFSE and were then co-cultured with pHrodo-labelled apoptotic PY230 (A-C) and PY8119 (D-F) breast cancer cells for 4 h.

(A, D) Relative efferocytosis and (B, E) relative ratio of mean fluorescence intensity (MFI) are shown and were calculated as the percentage of pHrodo+ macrophages and pHrodo MFI in macrophages, respectively. Data are expressed relative to the control group, set as 1 (n=8-9 (A-C) and n = 9-10 (D-F) separate cell isolations per group).

(C, F) Representative flow cytometric plots are shown. Numbers in outlined areas indicate the percentage of macrophages that is pHrodo+ red.

*P<0.05, **P<0.01, two-tailed Student’s t-test (A-F). Data are presented as mean ± s.e.m. and are pooled from two experiments (A-F).

Trained innate immunity does not impact macrophage ADCP of melanoma cells.

(A) Schematic diagram of experimental layout.

(B) Macrophages derived from WT or FcγR-/- mice were pre-stained with CFSE and were then co-cultured with pHrodo-labelled B16F10 cells in the presence of 0.5 μg/ml TA99 Ab that recognises the tumour antigen tyrosine related protein-1 (TYRP1 or gp75) or isotype control for 4 h. Relative ADCP is shown and is calculated as the percentage of pHrodo+ macrophages. Data are expressed relative to the untreated control group, set as 1 (n = 3 separate cell isolations per group).

(C, D) Trained or control macrophages were pre-stained with CFSE and were then co-cultured with pHrodo-labelled B16F10 cells as in (B). (C) ADCP and (D) relative ADCP are shown and calculated as the percentage of pHrodo+ macrophages. Data are expressed relative to the control group, set as 1 (n = 6 separate cell isolations per group).

*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., non-significant. One-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± s.e.m. and are pooled from three independent experiments (B-D).

The role of MerTK pathway on the phenotype of trained macrophages.

(A) Trained or control macrophages were pre-stained with CFSE, were treated with 0.1μM of the MerTK inhibitor UNC2025 or control vehicle for 1h and were then co-cultured with pHrodo-labelled apoptotic melanoma cells for 4 h. Relative efferocytosis using flow cytometry is shown and was calculated as the percentage of pHrodo+ macrophages. Data are expressed relative to the control group, set as 1 (n=7-8 separate cell isolations per group).

(B, C) Relative mRNA expression of Gas6, Lxra, Abca1, Axl from control or trained macrophages. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in the control macrophages (n=15 separate cell isolations per group).

(C) Each paired line represents average values from different experiments shown in (B).

(D) The relative MerTK MFI in control or trained macrophages is shown. Relative MFI ratio was set as 1 in the control macrophages (n=17-18 separate cell isolations per group).

*P<0.05, **P<0.01, ***P<0.001, n.s., non-significant. One-way ANOVA with Tukey’s multiple comparisons test (A), two-tailed Student’s t-test (B, D). Data are presented as mean and mean ± s.e.m. and are pooled from two (A), three (B, C), four experiments (D).

The levels of immune mediators and active caspase-1 during macrophage efferocytosis of melanoma cells.

Trained or control macrophages were co-cultured with apoptotic melanoma cells for 4 or 20h. Protein concentrations of (Α) IL-1β and (D) IL-10 were measured in efferocytosis co-culture supernatants. Monocultures of trained or control macrophages and apoptotic melanoma cells were used as controls. N=9-10 separate cell isolations per group for macrophages and n=2 for B16F10 cells. (B, C) Trained or control macrophages, pre-stained with the DiD, were co-cultured with apoptotic melanoma cells for 4 h. The probe FAM-YVAD-FMK FLICA (10 μM) was added 1 h prior to the end of the co-culture. Relative caspase-1 activity is shown and was calculated as the percentage of positive macrophages that are labelled with the FLICA (n=8-9 separate cell isolations per group). Data are expressed relative to the control group, set as 1.

(C) Representative flow cytometric plots are shown. Numbers in outlined areas indicate the percentage of macrophages that is FAM-FLICA+.

*P<0.05, ***P<0.001, ****P<0.0001, n.s., non-significant. One-way ANOVA with Šídák’s multiple comparisons test (A, D), two-tailed Student’s t-test (B). Data are presented as mean ± s.e.m. and are pooled from two experiments (A-D).

The inhibitory effect of cytochalasin D on macrophage efferocytosis.

(A) Schematic diagram of experimental layout.

(B) CFSE-labelled macrophages were treated with the actin polymerization inhibitor cytochalasin D for 30 min prior to their co-culture with pHrodo-labelled apoptotic melanoma cells for 4 h (n=3 separate cell isolations).

Samples in which apoptotic cells were added to the macrophage cultures just before the flow cytometric analysis, without any incubation, served as negative control. Efferocytosis is shown as the percentage of pHrodo+ macrophages.

(C) Representative flow cytometric plots are shown. Numbers in outlined areas indicate percentage of macrophages that is pHrodo+ red. *P<0.05, **P<0.01, One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean ± s.e.m. and are derived from one experiment.

The effect of LPS on Tnf mRNA expression levels in trained macrophages.

Relative mRNA expression of Tnf in trained and control macrophages that were cultured in the presence or absence of 10 ng/ml LPS for 16-18 h. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in macrophages that were not treated with LPS (n=10 separate cell isolations for control and 9 for trained macrophages). ***P<0.001, n.s., non-significant. Mann-Whitney test. Data are presented as mean ± s.e.m. and are pooled from three experiments.

The role of trained innate immunity on macrophage phagocytosis of microbes and apoptotic neutrophils.

(A-D) Trained or control macrophages were treated with 50 μg/ml of pHrodo green E. coli particles for 1 h and 3 h. Percentage of macrophages that have engulfed E. coli particles is shown (A, C: n = 8-10 separate cell isolations per group).

(E, F) Trained or control macrophages, pre-stained with CFSE, were co-cultured with pHrodo red – labelled apoptotic neutrophils for 30 min. Percentage of macrophages that have engulfed apoptotic neutrophils is shown (n=8 separate cell isolations per group).

(B, D, F) Representative fluorescence-activated cell sorting plots for E. coli particle phagocytosis (B, D) and neutrophil efferocytosis (F) are shown. Numbers in outlined areas indicate the percentage of macrophages that is pHrodo+ green (B, D) and pHrodo+ red (F).

*P<0.05, **P<0.01. Two-tailed Student’s t-test (A, C, E). Data are presented as mean ± s.e.m. and are pooled from two experiments (A, C) or derived from one experiment (E).

The effect of MerTK blockade on macrophage gene expression.

Relative mRNA expression of Lxra and Abca1 in macrophages that were treated or not with 0.1 μΜ of the MerTK inhibitor UNC2025 for 60 minutes. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in the untreated macrophages (n=9-10 separate cell isolations per group). **P<0.01, ***P<0.001. Two-tailed unpaired t-test test. Data are presented as mean ± s.e.m. and are pooled from two experiments.