Introduction

The identification of novel mechanisms to harness immune responses is essential for bolstering tumour suppression. There is an increasing need to better understand the regulation of macrophage responses to cancer to inform the development of novel therapeutic approaches and to increase the efficacy of current immunotherapies.

Macrophages represent a major orchestrator of immune responses in a plethora of pathologies^1–3. While residing in the tumour microenvironment, macrophages display remarkable heterogeneity and plasticity turning into essential components that can either promote or suppress tumour growth^4–6. The tightly coordinated recognition and uptake of ‘foreign’ material through the process of phagocytosis is a major macrophage effector function that shapes host immune responses to both microbial and sterile stimuli^7,8. In the tumour setting, macrophages can engulf cancer cells, that are opsonised with antibodies recognising surface tumour antigens, through antibody dependent-cellular phagocytosis (ADCP)⁹ and mediate the clearance of apoptotic cancer cells through the specialised type of phagocytosis called efferocytosis¹⁰. These two different types of phagocytosis, that represent a cardinal function of macrophages, have been linked to distinct effects on tumour growth¹¹. While ADCP promotes anti-tumour effects⁹, efferocytosis promotes a tumour-tolerant condition by inducing the release of immunosuppressive mediators such as interleukin 10^10,12. Inhibition of signaling pathways or phagocyte receptors that upregulate efferocytosis, such as Mer proto-oncogene tyrosine kinase (MerTK), has therefore been effective in reducing tumour growth in several experimental cancer models^13,14.

Trained innate immunity (TII or trained immunity) defines long-lasting adaptations of innate immune cells based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors^15–17. Specifically, TII represents a state of enhanced immune responsiveness of the myeloid compartment to secondary challenges. We have previously shown that TII contributes to tumour suppression using experimental models of melanoma and lung cancer¹⁸. The anti-tumour effects of TII were attributed at least in part to epigenetic and transcriptomic reprograming in neutrophils and enhanced tumour cell killing by release of reactive oxygen species¹⁸. The tumour suppressive phenotype of TII has been corroborated by several studies employing different tumour models and utilising different agents to induce TII^19–22. While the role of TII in promoting anti-tumour immunity is established, the impact of TII in efferocytosis or ADCP to shape anti-tumour immunity remains poorly explored.

The application of immunotherapies including checkpoint inhibitors that target the adaptive immune compartment for the treatment of melanoma has significantly improved the clinical management of this malignancy²³. However, current drugs have significant limitations as some patients suffer from side effects or do not benefit from treatment^24–27.

Macrophages are abundant in the tumour microenvironment of melanoma and their therapeutic targeting may be a promising strategy to complement existing therapies and enhance treatment success^28,29. Of note, macrophages have been shown to contribute to ADCP³⁰ of melanoma cells. In addition, targeted inhibition of efferocytosis incites anti-tumour immunity in melanoma³¹.

Here, we studied the role of TII on macrophage phagocytosis of melanoma cells and the cytokine profile of phagocytic macrophages. Our data demonstrate that ‘trained’ macrophages show decreased efferocytosis of tumour cells accompanied by reduced secretion of IL-1β. These findings unravel a hitherto unrecognized role of TII in the control of macrophage anti-tumour functions. The decreased macrophage efferocytosis of tumour cells has the potential to be harnessed therapeutically.

Materials and methods

Mice and mouse experiments Mice

C57BL/6 wild type (WT) mice and Fcer1gtm1Rav (FcγR^-/-; provided from Dr. James Hewitson, University of York) mice^32,33 were bred in-house under specific pathogen-free conditions on a standard 12/12 h light/dark cycle according to the institutional guidelines at the Biological Services Facility (BSF), University of York. Male and female mice that were 8-12 weeks old were used in experimental procedures. Food and water were provided ad libitum.

Mouse experiments

To induce trained innate immunity, mice were pre-treated with a single dose of 1 mg of β-glucan from Trametes versicolor or Saccharomyces cerevisiae (Invivogen) in PBS or with PBS vehicle alone (control) intraperitoneally (i.p.). Seven days after injection, bones were harvested and bone marrow was used for bone marrow-derived macrophage (BMDM) differentiation. All animals were visually inspected daily and were within accepted humane endpoints.

Ethics statement

All animal experiments were carried out under the authority of a UK Home Office Licence (project licence number PPL PP7424874) that obtained approval by the University of York Animal Welfare and Ethics Review Board. All procedures were performed in compliance with ARRIVE guidelines. No unexpected adverse events were recorded during this study.

Generation of mouse bone marrow-derived macrophages (BMDMs) and neutrophils

The isolation of bone marrow and differentiation into BMDMs (hereafter mentioned as macrophages) was performed as previously described³⁴. Briefly, bone marrow was flushed from femurs and tibias of mice and cells were plated and cultured in the presence of recombinant macrophage colony-stimulating factor (M-CSF; 20 ng/ml, PeproTech or Proteintech). Culture medium was replaced every two days, and after seven days differentiated BMDMs were used for further experiments. To isolate neutrophils, bone marrow cells, after erythrocyte lysis, were centrifuged in 62% Percoll gradient (Cytiva) as previously described³⁴.

Mouse cell lines

The B16F10 melanoma cell line (Creative Biogene) and the breast cancer cell lines PY8119 and PY230 (provided by Will Brackenbury, University of York) were utilised. B16F10 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco) and 2 mM L-Glutamine (Gibco). PY8119 cells were cultured in F-12K Nutrient Mix medium (Gibco) supplemented with 5% FBS and 100 U/ml penicillin, 100 μg/ml streptomycin. Same medium was used for the culture of PY230 cells supplemented with 0.1% MITO+ serum extender (Corning).

Flow cytometric analysis

Cell surface staining of MerTK on macrophages was performed in 5% FBS/PBS using 6 μg/ml of anti-MerTK (PE-conjugated, clone: DSSMMER, Invitrogen) or the IgG2a λ isotype control (PE-conjugated, clone: G013C12, BioLegend) for 25 minutes.

Levels of apoptosis were assessed using the annexin V apoptosis detection kit APC (Invitrogen) in combination with propidium iodide as previously described³⁴.

Flow cytometric analysis of stained cells was performed on a CytoFLEX (Beckman Coulter) and data were analysed with CytExpert software.

Cell treatments

Trained or non-trained macrophages were stimulated with 10 ng / ml lipopolysaccharide (LPS, Invivogen) for 16–18 h followed by RNA extraction and assessment of Tnf mRNA levels.

To induce apoptosis, cancer cells were treated with 1 μM of the protein kinase C inhibitor staurosporine³⁵ for 16–18 h (Cell Signalling Technology). To induce apoptosis in bone marrow neutrophils, cells were cultured in HBSS containing 1% FBS for 16 – 18 h³⁴.

To inhibit phagocytosis, macrophages were treated with the actin polymerization inhibitor Cytochalasin D (10 - 40 μΜ; Merck Group or Cayman Chemical Company) for 30 minutes, culture medium was changed and then phagocytic cargo was added.

To block signaling through the MerTK receptor, macrophages were treated with the inhibitor UNC2025 (0.1 μΜ, purity ≥ 98%; Cayman Chemical Company) for 1 hour prior to their co-culture with apoptotic cells or prior to RNA isolation.

Activity of caspase-1 was measured in macrophages after co-culture by incubating with FAM-YVAD-FMK FLICA (10 μM, Bio-Rad, as per manufacturer instructions) for 60 minutes with flow cytometry.

Phagocytosis assays

Unstained macrophages or macrophages that were stained with either carboxyfluorescein succinimidyl ester (CFSE, 0.75 μM; Biosciences) or with DiD (3 μM; Thermo Fisher Scientific) were co-cultured with (i) apoptotic cells pre-labelled with 5 μM pHrodo Red SE (Invitrogen) in a 1:5 ratio of macrophages to apoptotic cells, (ii) pHrodo-labelled melanoma cells in the presence of 0.5 μg/ml anti-mouse/human Ab recognising TYRP1/TRP1 (gp75) on melanoma cells (clone: TA99, Bio X Cell) or the mouse IgG2a isotype control (clone: C1.18.4, Bio X Cell) in a 1:10 ratio of macrophages to tumour cells or (iii) pHrodo green E. coli Bioparticles (50 μg/ml, Invitrogen).

Flat bottom 96-well plates that were either non - TC-treated or TC-treated were used for flow cytometry and live cell imaging, respectively. Co-culture plates were spun for 1 minute at 250g prior to the initiation of the phagocytosis assay. Three technical replicates were used for each sample. For live cell imaging, the Livecyte instrument (Phasefocus) with a 10x magnification and a frame size of 1 mm x 1mm was used. At least 50 macrophages per region of interest were evaluated.

Quantitative RT-PCR

Total RNA was extracted from cultured cells using TRIzol (Ambion Inc) according to manufacturer’s instructions and was quantified by spectrometry at 260 and 280 nm using a Nanodrop instrument (ThermoFischer). Complementary DNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems) and gene-specific primers in a QuantStudio 3 Real time PCR detection system (Thermo Fisher Scientific). 18S was used as an internal control for normalization. Data were analyzed using the comparative (ΔΔCt) method. The following primers were used: 18S-F (GTT CCG ACC ATA AAC GAT GCC), 18S-R (TGG TGC CCT TCC GTC AAT), Tnf-F (AAG CCT GTA GCC CAC GTC GTA), Tnf-R (GGC ACC ACT AGT TGG TTG TCT TTG), Lxra-F (CTC AAT GCC TGA TGT TTC TCC T), Lxra-R (TCC AAC CCT ATC CCT AAA GCA A), Abca1-F (GGT TTG GAG ATG GTT ATA CAA TAG TTG T), Abca1-R (CCC GGA AAC GCA AGT CC), Axl-F (ATG GCC GAC ATT GCC AGT G), Axl-R (CGG TAG TAA TCC CCG TTG TAG A), Gas6-F (TGC TGG CTT CCG AGT CTT C), Gas6-R (CGG GGT CGT TCT CGA ACA C).

Enzyme-linked immunosorbent assays (ELISAs)

Mouse IL-10 and IL-1β proteins were measured in cell culture supernatants using kits from Biolegend and Invitrogen, respectively, according to the instructions of the manufacturer.

Statistical analysis

Statistical analyses were carried out with GraphPad Prism 10 software. For infection experiments, researchers were blinded to mouse genotype. This also applies to histology and flow cytometry analyses. Results are presented as mean plus standard error of the mean. Two tailed parametric and non-parametric tests were used as appropriate after testing for normality. Multiple-group comparisons were performed using analysis of variance (ANOVA) and the Tukey’s, Dunnett’s or Šídák’s multiple comparison tests. A P value of < 0.05 was considered to be statistically significant. Statistical information is provided in each figure legend.

Results

Efferocytosis of apoptotic tumour cells is decreased by trained macrophages

Having previously identified a tumour suppressive role for TII that is mediated by myeloid cells¹⁸, we sought to determine the impact of TII as a regulator of macrophage uptake of apoptotic tumour cells. To mimic efferocytosis conditions, macrophages pre-stained with CFSE were co-cultured with pHrodo red-labelled apoptotic melanoma cells (supplementary Fig. 1A). Pre-treatment of macrophages with the actin polymerization inhibitor cytochalasin D prior to their co-culture with tumour cells resulted in dose-dependent blockade of efferocytosis confirming the specificity of the assay (supplementary Fig. 1B, C). The effect of TII on macrophage efferocytosis of tumour cells was investigated using bone marrow derived macrophages (BMDMs) from the bone marrow of mice treated with β-glucan (hereafter described as trained macrophages). The increased levels of Tnf in trained macrophages upon their stimulation with lipopolysaccharide (LPS) confirmed the induction of TII³⁶ in our experimental setting (supplementary Fig. 2). Flow cytometric analysis of the co-cultures revealed that efferocytosis was attenuated in trained macrophages (Fig. 1B-D). The dynamic behaviour of trained macrophages during efferocytosis of melanoma cells was mapped using real time imaging verifying their decreased capacity for tumour cell uptake (Fig. 1E).

Figure 1.

To determine whether the effect of TII on macrophage efferocytosis applies to other types of cancer, the two breast cancer cell lines PY230 and PY8119 were used in our co-culture experiments. Consistently with our results on decreased efferocytosis of melanoma cells, we demonstrated that trained macrophages exerted attenuated efferocytic activity upon their co-culture with apoptotic breast cancer cells (Fig. 2) suggesting a broad role of TII on shaping efferocytosis of cancer cells.

Figure 2.

TII does not have any effect on macrophage ADCP of melanoma cells

We next investigated the role of TII on the macrophage ADCP. To induce ADCP, the co-culture was performed in the presence of the well-characterised anti-mouse/human TA99 Ab that recognises the tumour antigen Tyrp1³⁷ (Fig. 3A). Specificity of ADCP induction was demonstrated using macrophages from mice deficient in Fcγ receptors^32,33 (Fig. 3B). Following co-culture of TA99-opsonised melanoma cells with either trained or non-trained macrophages, levels of ADCP were comparable between the two groups (Fig. 3C, D), suggesting that TII does not play any role in macrophage ADCP of melanoma cells under these conditions.

Figure 3.

Given that macrophage phagocytosis can be differentially modulated by different types of phagocytic cargo^38,39, we sought to investigate the behaviour of trained macrophages towards different key phagocytic cargos. To this end, trained or non-trained macrophages were cultured together with microbial bioparticles or apoptotic neutrophils to mimic phagocytosis during infection or resolution phase of sterile inflammatory responses, respectively. The uptake of both E. coli particles (supplementary Fig. 3A-D) and apoptotic neutrophils (supplementary Fig. 3E, F) was elevated in trained macrophages compared to the phagocytic activity shown in control macrophages. These findings underline the ability of trained macrophages to shape differential responses to distinct phagocytic cues.

Induction of TII combined with MerTK inhibition further downregulate efferocytosis of tumour cells

MerTK functions as an efferocytic receptor recognising the ‘eat-me’ signal PS on the outer surface membrane of apoptotic cells with the help of bridging molecules such as Gas6. As MerTK blockade has already been shown to promote tumour suppression³¹, we investigated the consequences of MerTK inhibition on trained macrophage efferocytosis.

To this end, trained or control macrophages were cultured with apoptotic melanoma cells in the presence of the small-molecule MerTK inhibitor UNC2025⁴⁰. In trained macrophages, the presence of UNC2025 resulted in decreased efferocytosis compared to that one in control macrophages treated with the inhibitor (Fig. 4A). Gene expression analysis of molecules downstream of MerTK signaling¹⁴ in macrophages treated with the UNC2025 verified the downregulation of Liver X receptor alpha (Lxra) and ATP-binding cassette transporter (Abca1) (supplementary Fig. 4). We then assessed the impact of TII in the expression levels of molecules implicated in the MerTK pathway and found that the components of MerTK-dependent signaling Gas6, Lxra and Abca1 were downregulated in trained macrophages (Fig. 4B, C). MerTK protein levels were downregulated in trained macrophages (Fig. 4D). Therefore, decreased efferocytosis of tumour cells by trained macrophages may be attributed at least in part to the downregulated molecular machinery orchestrating MerTK-dependent efferocytosis.

Figure 4.

Secretion of IL-1β by trained macrophages upon efferocytosis of tumour cells is blunted

Macrophage efferocytosis not only paves the way towards the clearance of apoptotic cells, but also promotes metabolic alterations in macrophages^41–43. At the same time, metabolic alterations in efferocytic macrophages induced by digested apoptotic material substantially regulates the cytokine secretion by efferocytic macrophages⁴⁴. Since upregulation of interleukin-1β (IL-1β) has been linked to both enhanced efferocytosis⁴⁵ and tumour growth^46,47, we determined the levels of IL-1β production in our co-culture setting. Production of IL-1β was induced as a result of efferocytosis, as IL-1β was minimally detected in either macrophage or apoptotic melanoma cell monocultures (Fig. 5A). Trained macrophages displayed lower levels of IL-1β production as shown in co-culture supernatants compared to those from control macrophages (Fig. 5A), further underscoring the tumour suppressive effect of TII through inhibition of efferocytosis. Consistently, we demonstrated that levels of active caspase-1 were decreased in trained macrophages after efferocytosis of apoptotic tumour cells (Fig. 5B, C) thereby explaining at least in part the decreased levels of active IL-1β in co-culture supernatants.

Figure 5.

We also determined the levels of immunosuppressive cytokine interleukin 10 (IL-10), previously described to be upregulated after efferocytosis¹², in the supernatants of efferocytosis co-cultures. Levels of IL-10 in the co-culture supernatant were comparable between the trained and control macrophages (Fig. 5D).

Together, these data unveil a novel role for TII in shaping macrophage-cancer cell interactions, providing a better understanding on mechanisms that may be targeted to block efferocytosis-dependent tumour growth.

Discussion

The orchestration of host immune responses against cancer involves several immune cell types and events including monocyte infiltration, macrophage activation, cytokine release and phagocytosis of cancer cells, all of which aim to contribute to tumour suppression⁴⁸. Although immune cell interactions play important roles in the immunopathology of cancer, the network of molecules and mechanisms that coordinate this complex process warrants further elucidation. Therefore, identification of immune regulatory networks that aim to harness cancer-associated immunosuppression are required.

In an experimental model of melanoma, induction of TII has been shown to promote myeloid cell – dependent tumour suppression through reprograming the phenotype of neutrophils to inhibit tumour growth¹⁸. Here, we investigated whether TII regulates macrophage phagocytosis of tumour cells. We show for first time that trained macrophages have decreased phagocytic activity of apoptotic tumour cells and that is accompanied by lower levels of secreted IL-1β. We also found that Gas6, Lxra, Abca1 and MerTK, all previously linked to enhanced efferocytosis^34,49, were downregulated by trained macrophages. Under the tested conditions, levels of ADCP were comparable between trained and control macrophages. Use of antibodies recognising alternative melanoma surface antigens may promote ADCP by trained macrophages.

Further studies would be useful to address why trained macrophages demonstrate a cargo-dependent phagocytic behaviour, i.e. decreased efferocytosis of tumour cells, unaltered ADCP and enhanced phagocytosis of apoptotic neutrophils and E. coli particles. In agreement with the literature, although using a different approach to induce TII, enhanced phagocytosis of pathogen particles³⁶ and apoptotic neutrophils⁵⁰ by trained phagocytes has been demonstrated. Our findings on trained macrophage efferocytosis of different apoptotic cargos further support previous reports suggesting that the cellular identity of the engulfed apoptotic cell instruct distinct macrophage phenotypes⁵¹ that may be potentially linked to enhanced effectiveness of macrophage-based cell therapies.

Targeting efferocytosis to bolster anti-tumour immunity has recently attracted attention^10,13. Of note, efferocytosis has been linked to cancer therapeutic resistance among several types of cancer^52,53. The role of TII on attenuating macrophage efferocytosis in the tumour setting was corroborated by showing that inhibition of the efferocytosis receptor MerTK further decreased the uptake of apoptotic tumour cells in co-cultures with trained macrophages. As blockade of MerTK may have a direct impact in cancer cells⁵⁴, further studies are required to address whether MerTK inhibition affects solely efferocytosis in our experimental setting.

The decreased caspase-1 activity in trained efferocytic macrophages was reflected by lower levels of IL-1β levels. Efferocytosis has been shown to induce the activation of NLRP3 inflammasome signaling in myeloid cells thereby promoting IL-1β secretion and tumour growth⁴⁵. Further focus on the identification of specific inflammasomes that contribute to the attenuated efferocytosis by trained macrophages may support the development of novel cancer immunotherapeutic targets. In addition, future studies will provide further insight into the potential role of MerTK inhibition on macrophage IL-1β secretion during efferocytosis.

Given the macrophage heterogeneity⁵⁵ and the different types of central and peripheral trained innate immunity⁵⁶, the use of BMDMs as the only source of macrophages may represent a limitation of this study. However, BMDMs have been utilised extensively for the elucidation of mechanisms orchestrating cargo-dependent macrophage phagocytosis⁵¹. Future validation experiments using different types of macrophages will strengthen the impact of this study.

Lack of in vivo data on macrophage efferocytosis is another limitation of our study. Further analyses are required to address the physiological relevance of trained macrophages in efferocytosis of tumour cells in vivo.

Cancer combination therapies, including the administration of immunotherapy, have contributed to the improvement of the clinical management of cancer particularly in aggressive types such as melanoma^57,58 and also breast cancer^59,60.

The anti-tumour effect of β-glucan and the vaccine Bacillus Calmette-Guerin, both inducers of TII, is being tested in clinicals trials^61–63. TII - dependent decrease of efferocytosis may be a promising adjuvant immunotherapeutic strategy to complement existing cancer therapies and enhance treatment success.

In summary, the present study pinpoints a novel role for trained innate immunity in attenuating macrophage efferocytosis of tumour cells and demonstrates that the decreased efferocytosis by trained macrophages is associated with lower levels of active caspase-1 and IL-1β secretion. This novel role of trained innate immunity could be therapeutically exploited in pre-clinical models to expand our current mechanistic and translational understanding of the anti-tumour potential of trained immunity.

Supplementary figures

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Figure 3.

Supplementary Figure 4.

Acknowledgements

We would like to thank staff at the Imaging and Cytometry Lab in the University of York Bioscience Technology. We thank Prof. Will Brackenbury (University of York) for providing the PY8119 and PY230 cell lines and Dr. James Hewitson (University of York) for providing the Fcer1gtm1Rav mice.

Additional information

Author Declarations

We confirm all relevant ethical guidelines have been followed, and any necessary ethics committee approvals have been obtained.

Author contributions

AC contributed to experimental design, performed experiments, analysed and interpreted data; JL performed experiments; DL contributed to experimental design and interpreted data; DB contributed to experimental design, interpreted data and supervised research; IK conceived and designed the study, performed experiments, supervised research, interpreted data, and wrote the paper.

All authors critiqued and edited the manuscript.

Funding Statement

This work was funded by the Hull York Medical School (PhD studentship to AC) and was funded by the Academy of Medical Sciences Springboard grant (SBF007\100172), the Royal Society grant (RGS\R2\202032) and the Rosetrees Trust grant (Seedcorn2021 100043) awarded to I.K.. D.B. is funded by the Academy of Medical Sciences Springboard Award (SBF006\1025) and a Medical Research Council New Investigator grant (MR/Z504221/1).