Plasticity of antimicrobial and phagocytic programs in human macrophages

Summary

Macrophage (MΦ) polarization is triggered during the innate immune response to defend against microbial pathogens, but can also contribute to disease pathogenesis. In a previous study, we found that interleukin‐15 (IL‐15) ‐derived classically activated macrophages (M1 MΦ) have enhanced antimicrobial activity, whereas IL‐10‐derived alternatively activated macrophages (M2 MΦ) were highly phagocytic but lacked antimicrobial activity. Given that the ability to modulate MΦ polarization from M2 MΦ to M1 MΦ may promote a more effective immune response to infection, we investigated the plasticity of these MΦ programs. Addition of IL‐10 to M1 MΦ induced M2‐like MΦ, but IL‐15 had little effect on M2 MΦ. We determined the set of immune receptors that are present on M2 MΦ, elucidating two candidates for inducing plasticity of M2 MΦ, Toll‐like receptor 1 (TLR1) and interferonγ (IFN‐γ) receptor 1. Stimulation of M2 MΦ with TLR2/1 ligand (TLR2/1L) or IFN‐γ alone was not sufficient to alter M2 MΦ phenotype or function. However, co‐addition of TLR2/1L and IFN‐γ re‐educated M2 MΦ towards the M1 MΦ phenotype, with a decrease in the phagocytosis of lipids and mycobacteria, as well as recovery of the vitamin‐D‐dependent antimicrobial pathway compared with M2 MΦ maintained in polarizing conditions. Similarly, treatment of M2 MΦ with both TLR2/1L and anti‐IL‐10 neutralizing antibodies led to polarization to the M1‐like MΦ phenotype and function. Together, our data demonstrate an approach to induce MΦ plasticity that provides the potential for re‐educating MΦ function in human mycobacterial disease to promote host defense and limit pathogenesis.

Abbreviations

CYP27b1

cytochrome P450 family 27 subfamily B member 1

IFN

interferon

M1

classically activated macrophage

M2

alternatively activated macrophage

MΦ

macrophage

TLR

Toll‐like receptor

Introduction

The differentiation of monocytes into macrophages (MΦ) is triggered by exposure to microbes as well as host signals, including activated lymphocytes and damaged cells.1 Differentiation results in two different MΦ polarization states: (i) classically activated M1 MΦ induced by interferon‐γ (IFN‐γ) + lipopolysaccharide stimulation2 and (ii) alternatively activated M2 MΦ, which are further subcategorized into M2a, M2b, and M2c MΦ induced by stimulation of interleukin‐4 (IL‐4) or IL‐13, immune complexes, and IL‐10 or transforming growth factor‐β, respectively.3, 4 Functionally, M1 MΦ contribute to host defense against intracellular pathogens by mediating a direct antimicrobial activity. However, the inflammatory responses mediated by M1 MΦ can lead to tissue injury. By contrast, M2 MΦ are phagocytic, contributing to the clearance of helminths and nematodes.5 The phagocytic function of M2 MΦ in combination with their ability to dampen inflammation promotes wound healing. However, the induction of M2 MΦ is deleterious in some instances, for example in contributing to the persistence of intracellular bacterial infection.6

Since the discovery of phagocytes in 1884,7 immunologists have generally linked two key functions of the innate immune response, phagocytosis and antimicrobial responses, as being co‐regulated for optimal host defense. Previously, however, we discovered by studying human leprosy as a model, that MΦ phagocytic and antimicrobial responses are differentially programmed by distinct cytokines, which are expressed at the site of infection.8 Interleukin‐15, expressed in lesions from the resistant form of the disease, tuberculoid leprosy (T‐lep), induced monocytes to differentiate into M1‐like MΦ, expressing CD209 and key components of the vitamin‐D‐dependent antimicrobial pathway. By contrast, IL‐10, expressed in lesions from the progressive form of the disease, lepromatous leprosy (L‐lep), induced monocytes to differentiate into M2 MΦ, that expressed CD209, CD163, and scavenger receptors that contributed to phagocytic activity.

The phenotypic and functional programs of IL‐15‐ and IL‐10‐derived MΦ reflect the MΦ gene programs at the site of disease in leprosy, evidenced by the divergence of antimicrobial versus phagocytic pathways in the different clinical forms.8 M1 MΦ express the 25‐hydroxylase enzyme, CYP27b1, which is differentially expressed in T‐lep versus L‐lep lesions. CYP27b1 converts inactive vitamin D [25‐hydroxy vitamin D (25D)] into its active form [1,25‐dihydroxy vitamin D (1,25D)], leading to the production of antimicrobial peptides cathelicidin and β‐defensin 2 that contribute to killing of mycobacteria.9, 10 The M2 MΦ phagocytic gene program was differentially expressed in L‐lep lesions. These M2 MΦ are highly phagocytic, but are unable to initiate the vitamin‐D‐dependent antimicrobial response against mycobacteria.

The disease spectrum of leprosy is not static, but dynamic, as patients can develop a ‘reversal reaction’, which typically leads to a shift from the progressive to the self‐limiting form of the disease, associated with a switch from M2 MΦ to M1 MΦ in lesions.8 This suggested plasticity in the MΦ programs is associated with changes in the immune response to Mycobacterium leprae, the intracellular bacterium that causes leprosy. we investigate the mechanisms by which IL‐15‐polarized MΦ versus IL‐10‐polarized MΦ are re‐educated in an effort to understand MΦ plasticity in mycobacterial infection and diseases in which MΦ contribute to pathogenesis.

Materials and methods

Reagents

Interleukin‐10 (R&D Systems, Minneapolis, MN, USA) and IL‐15 (R&D Systems) were used for MΦ differentiation. Antibodies for cell surface staining are as follows: CD163 (BD Biosciences, Franklin Lakes, NJ, USA), CD209 (BD Biosciences), CD36 (BD Biosciences), Toll‐like receptor 1 (TLR1) (EBioscience, Waltham, MA, USA), IFN‐γ receptor 1 (IFNGR1; R&D Systems), IFNGR2 (R&D Systems), and isotypes (Thermofisher, Waltham, MA, USA). Synthetic 19 000 molecular weight lipoprotein derived from mycobacteria (EMC Microcollections, Tübingen, Germany) and recombinant human IFN‐γ (BD Biosciences) were used for MΦ re‐education. Anti‐IL‐10 (Invitrogen, Carlsbad, CA, USA) and IgG1 isotype control (BioLegend, Hercules, CA, USA) were used for neutralization studies.

Macrophage differentiation and reeducation

Peripheral blood was acquired from healthy donors with informed consent (UCLA Institutional Review Board #125.15.0‐f). As previously described, adherent monocytes were isolated and differentiated8 into M1 or M2 MΦ using 300 ng/ml IL‐15 or 10 ng/ml IL‐10, respectively. After 3 days of differentiation, cells were detached using 5 mm phosphate‐buffered saline–ethylenediamine tetraacetic acid solution and viable cells were counted by Trypan blue counterstain. CD209⁺ macrophages were then enriched by CD209⁺ beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. CD209⁺ macrophages were then replated and stimulated for an additional 3 days with the following ligands alone or in combination: 300 ng/ml IL‐15, 10 ng/ml IL‐10, TLR2/1L (10 μg/ml), IFN‐γ (1·25 ng/ml), and assessed for re‐education by cell surface labeling of CD209 and CD163 as previously described.8

Microarray analysis

Microarray data from IL‐10‐ and IL‐15‐stimulated adherent human peripheral blood mononuclear cells (PBMCs) were downloaded from Gene Omnibus accession GSE59184.8 In brief, adherent PBMCs from four healthy donors were stimulated with IL‐10 or IL‐15 (R&D Systems) in RPMI‐1640 supplemented with 10% fetal calf serum. Cells were harvested at 6 and 24 hr after stimulation, and monocytes were purified by CD14 MicroBeads (Miltenyi Biotec). Total RNA was isolated and Affymetrix Human U133 Plus 2.0 array was used. All probes for each gene are shown in the Figure 2(a) dendrogram.

IL‐10 secretion and neutralization studies

Interleukin‐10 MΦ were treated with TLR2/1L alone or in combination with IFN‐γ for 24 hr and supernatants were assayed for IL‐10 protein levels by enzyme‐linked immunosorbent assay as previously described.11 For IL‐10 neutralization studies, IL‐10 MΦ were incubated with anti‐IL‐10 blocking antibody or isotype control before TLR2/1L treatment for 24 hr. Cells were then labeled for CD163 cell surface expression as previously described.8

Endocytosis assays

Re‐educated IL‐10 MΦ were incubated for 4 hr at 37° with Dil(1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate)‐labeled CuSO₄‐oxidized low‐density lipoprotein (oxLDL) (Intracel Resources, Frederick, MD, USA) and assayed for uptake as previously described.8 Some macrophages were also labeled with CD36 antibody and mean fluorescence intensity was acquired by flow cytometry according to established methods.8

Real‐time quantitative polymerase chain reaction

Interleukin‐10 MΦ were stimulated with IL‐10, TLR2/1L + IFN‐γ, or TLR2/1L for 24 hr and mRNA expression of CYP27b1, CAMP, or DEFB4 was assayed using primers as previously described.8

Results

IL‐10 is sufficient to reprogram M1 MΦ, but IL‐15 is not sufficient to reprogram M2 MΦ

We investigated pathways of MΦ plasticity in vitro using IL‐15‐differentiated M1 MΦ and IL‐10‐differentiated M2 MΦ. Adherent PBMCs were first differentiated with IL‐15 or IL‐10 for 3 days to generate CD209⁺ CD163⁻ M1 MΦ or CD209⁺ CD163⁺ M2 MΦ as previously described.8 At day 3 for the IL‐10‐derived M2 MΦ, approximately 60% of CD209⁺ cells expressed the M2 MΦ marker CD163 (Fig. 1a). In contrast, for IL‐15‐derived M1 MΦ, approximately 5% of CD209⁺ cells expressed CD163. Differentiated MΦ were immunomagnetically sorted for CD209⁺ to achieve ~80% CD209⁺ cells (see Supplementary material, Fig. S1) and then treated with IL‐10 or IL‐15 for an additional 3 days to determine if their phenotype can be reprogrammed.

Figure 1.

Interleukin‐10 (IL‐10) is sufficient to reprogram classically activated macrophages (M1 MΦ), but IL‐15 is not sufficient to reprogram alternatively activated macrophages (M2 MΦ). (a) Adherent peripheral blood mononuclear cells (PBMC) were stimulated with IL‐15 or IL‐10 for 3 days and labeled with antibodies to cell surface markers CD163 and CD209, representative of n = 4. (b) IL‐15‐derived M1 MΦ and IL‐10‐derived M2 MΦ were washed and treated with either IL‐15 or IL‐10 for an additional 3 days and labeled with antibodies to cell surface markers CD163 and CD209 at day 6, representative plot of n = 4. (c) Quantification of average percentage ± SEM of CD163⁺ cells from (b). **P < 0·01.

Interleukin‐15‐derived M1 MΦ, when further cultured in the presence of IL‐15, retained their CD209⁺ CD163⁻ phenotype (Fig. 1b). However, treatment of the IL‐15‐derived M1 MΦ with the M2‐polarizing cytokine IL‐10 significantly increased the percentage of CD163⁺ cells from 0% to > 60% at day 6 (Fig. 1c), indicating that IL‐10 is sufficient to reprogram M1 MΦ. Interleukin‐10‐derived M2 MΦ, when further cultured in the presence of IL‐10, retained their CD209⁺ CD163⁺ phenotype. Treatment with IL‐15, however, was not sufficient to reprogram M2 MΦ, as treatment of IL‐10‐derived M2 MΦ with the M1‐polarizing cytokine IL‐15 resulted in a partial decrease in the percentage of CD163‐positive cells from 91% to 67% at day 6 (Fig. 1c, P = 0·005, n = 4).

Differential expression of MΦ receptors suggests ligands to alter M2 MΦ phenotype

The functional response of cells is determined in part by the expression of specific receptors that can be triggered by distinct ligands. Hence, we hypothesized that the MΦ receptor repertoire can be exploited to identify ligands that can alter the MΦ phenotype. Using previously published transcriptional signatures of adherent human PBMCs stimulated with IL‐10 or IL‐15, or time zero unstimulated cells,8 we compiled expression data for receptors known to be involved in driving MΦ polarization (Fig. 2a). Concordant with Fig. 1, M1 MΦ express IL‐10 receptor genes IL10RA and IL10RB, thereby making them susceptible to IL‐10‐induced reprogramming into M2 MΦ. On the other hand, IL‐10‐derived M2 MΦ express decreased levels of IL‐15 receptor complex genes IL15RA, IL2RB, and IL2RG compared with M1 MΦ, which may explain why IL‐15 treatment was only partially able to reprogram M2 MΦ. Assessment of cell surface markers on the IL‐10‐derived M2 MΦ revealed heightened expression of other receptors known to drive M1 MΦ polarization, including the IFNGR1 and members of the TLR family. TLR2 (although not up‐regulated in IL‐10‐treated monocytes), TLR1 and IFNGR1 are known to be associated with host defense against mycobacterial infection.9, 12 In addition, treatment of monocytes with IFN‐γ and a mycobacterial lipopeptide activated TLR2/1‐enhanced expression of the antimicrobial protein cathelicidin,13 a characteristic of M1 MΦ.8 Cell surface expression of inflammatory receptors TLR1 and IFNGR1, but not IFNGR2, was confirmed by flow cytometry to be more highly expressed on M2 MΦ at day 3 compared with M1 MΦ or freshly isolated monocytes (Fig. 2b).

Figure 2.

Differential macrophage (MΦ) receptors suggest ligands to change MΦ phenotype. (a) Heat map displaying receptor expression data from transcriptional signatures of adherent human peripheral blood mononuclear cells (PBMC) (n = 4) stimulated with interleukin‐10 (IL‐10) or IL‐15 for the indicated time‐points. Each row represents one probe and each column represents a sample taken at the time‐point indicated. Red indicates higher expression and green depicts lower expression. (b) Adherent PBMC stimulated with IL‐10 or IL‐15 for 3 days or freshly isolated monocytes were labeled with specific antibodies to Toll‐like receptor 1 (TLR1), interferon‐γ receptor 1 (IFNGR1), or IFNGR2. Receptor expression is quantified as average ΔMFI ± SEM (n = 3) for each surface marker. (c) IL‐10‐derived alternatively activated MΦ (M2 MΦ) were treated with the ligands indicated for an additional 3 days and cell surface markers were assessed at day 6 by flow cytometry. Representative flow density plots and average percentage of CD163⁺ ± SEM (n = 4) are shown. **P < 0·01, *P < 0·05.

TLR2/1L + IFN‐γ stimulation is sufficient to reprogram M2 MΦ

Due to the prominent expression of TLR1 and IFNGR1 on M2 MΦ, we speculated that activation of IFN‐γ receptor and/or TLR1 may be sufficient to fully reprogram M2 to M1‐like MΦ. To test whether stimulation of these receptors would lead to a change in the IL‐10 phenotype, CD209⁺ IL‐10 MΦ were purified and stimulated with either TLR2/1L, IFN‐γ, or the combination of TLR2/1L plus IFN‐γ for three additional days. As a control, CD209⁺ IL‐10 MΦ were purified and cultured with either IL‐15 or IL‐10 for three additional days. At day 6, only treatment of the IL‐10‐derived M2 MΦ with the combination of TLR2/1L and IFN‐γ led to a substantial decrease in CD163 surface expression. Although M2 MΦ that were further cultured in IL‐10 were > 80% positive for CD163, M2 MΦ that were further treated with TLR2/1L and IFN‐γ were < 20% positive for CD163 (Fig. 2c). Treatment with TLR2/1L, IFN‐γ, or IL‐15 alone resulted in a moderate decrease in the percentage of CD163⁺ cells to 46·3%, 61·2%, and 66·9%, respectively. Increasing the concentration of IFN‐γ did not alter the percentage of CD163⁺ cells (see Supplementary material, Fig. S2).

TLR2/1L‐induced secretion of IL‐10 counteracts suppression of CD163 expression

Toll‐like receptor activation is reported to be a key driver of M1 MΦ polarization; however, stimulation with TLR2/1L failed to markedly decrease CD163 expression. This incomplete repolarization may be due to an increase in IL‐10 secretion, as TLR2/1L treatment of human monocytes is reported to induce high levels of IL‐10,14 which may counteract its ability to decrease CD163 expression. To identify factors that may antagonize the IL‐10‐induced program, an upstream analysis was performed by Ingenuity Pathways Analysis on the gene expression data derived from IL‐10‐treated monocytes (Fig. 3a). Interferon‐γ was identified as the most significant repressor of the IL‐10‐induced program with P‐value 3·9 × 10⁻³⁴, suggesting that the ability of TLR2/1L plus IFN‐γ to reverse M2 MΦ polarization is at least in part due to IFN‐γ‐mediated suppression of TLR2/1L‐induced IL‐10 secretion.

Figure 3.

Toll‐like receptor 2/1 ligand (TLR2/1L) ‐induced secretion of interleukin‐10 (IL‐10) prevents change in macrophage (MΦ) phenotype by TLR2/1L. (a) Top potential upstream regulators of the IL‐10‐induced gene program as determined by Ingenuity Pathways Analysis. (b) Quantification of average IL‐10 secretion ± SEM (n = 2) after stimulation of IL‐10‐derived alternatively activated MΦ (M2 MΦ) with TLR2/1L, interferon‐γ (IFN‐γ, or combination of TLR2/1L + IFN‐γ for 24 hr. (c) IL‐10‐derived M2 MΦ were incubated with αIL‐10‐blocking antibody or isotype control and stimulated with TLR2/1L for 3 days. Cells were then labeled with antibody to CD163 and percentage of CD163⁺ cells was assessed by flow cytometry. Average percentage of CD163⁺ ± SEM (n = 3) is shown. *P < 0·05, **P < 0·01.

To investigate whether IFN‐γ can suppress the levels of IL‐10 protein induction, M2 MΦ were stimulated with TLR2/1L or TLR2/1L in combination with IFN‐γ for 24 hr, and the secretion of IL‐10 protein was measured by enzyme‐linked immunosorbent assay. As in previously published data,15 stimulation with TLR2/1L alone induced secretion of IL‐10 protein (Fig. 3b). Co‐stimulation with IFN‐γ repressed TLR2/1L‐induced secretion of IL‐10 by approximately 50%. M2 MΦ were also stimulated with TLR2/1L for 3 days in the presence of αIL‐10 neutralizing antibody or isotype control to determine if TLR2/1L‐induced secretion of IL‐10 prevented a complete decrease of CD163 expression. Treatment of IL‐10‐derived M2 MΦ with TLR2/1L in the presence of neutralizing antibody led to a substantial down‐regulation of CD163 expression to approximately 15% positive cells versus 40% positive cells in the isotype control. These data indicate that TLR2/1L‐induced IL‐10 secretion prevents a marked decrease in CD163 expression and complete MΦ reprogramming.

Re‐education of M2 MΦ to M1‐like MΦ leads to a loss of phagocytic function

In addition to CD163 surface expression, M2 MΦ are characterized by enhanced phagocytic function and an increase in uptake of oxidized lipoprotein and mycobacteria compared with M1 MΦ, which in leprosy is associated with the presence of foamy MΦ at the site of disease.8 To determine whether the change in MΦ phenotype also led to a change in MΦ function, MΦ were cultured with stimuli as in Fig. 2(c) and assayed for DiI‐labeled oxLDL uptake via flow cytometry. Co‐treatment of IL‐10‐derived M2 MΦ with TLR2/1L and IFN‐γ led to a marked decrease in oxLDL uptake, decreasing from a mean florescence intensity (MFI) of 319 ± 77 (SEM) with IL‐10 treatment to 121 ± 13 SEM (P = 0·038) (Fig. 4b). To determine whether this effect on oxLDL uptake was due to an alteration in the receptors that mediate the uptake of oxLDL, we assessed the surface expression of CD36, a scavenger receptor highly induced by IL‐10 and the major receptor for oxLDL in these MΦ. The surface expression of CD36 was significantly decreased upon treatment of IL‐10‐derived MΦ with both TLR2/1L and IFN‐γ (Fig. 4b,c). To test the phagocytosis of bacteria, live Mycobacterium leprae fluorescently labeled with PKH were added at a multiplicity of infection of ten to one to IL‐10‐derived M2 MΦ treated with IL‐10 or TLR2/1L and IFN‐γ for an additional 3 days. Results were analyzed by flow cytometry and showed that the proportion of cells positive for M. leprae decreased from 30% to 12% comparing IL‐10 with TLR2/1L and IFN‐γ‐treated MΦ (P = 0·049, n = 2).

Figure 4.

Reprogramming of alternatively activated macrophages (M2 MΦ) to classically activated MΦ (M1‐like MΦ) correlates with a loss of phagocytic function. (a) Interleukin‐10 (IL‐10) ‐derived M2 MΦ were treated with the ligand indicated for an additional 3 three days and then assayed for DiI‐labeled oxLDL uptake using flow cytometry. Average uptake determined by DiI MFI ± SEM (n = 5). (b) IL‐10‐derived M2 MΦ were treated with the ligand indicated for an additional 3  days and average CD36 surface expression ± SEM (n = 3) was determined by flow cytometry, representative histogram of CD36 expression is pictured in (c). (d) IL‐10‐derived M2 MΦ were treated with IL‐10 or TLR2/1L + interferon‐γ for an additional 3 days and then assayed for PKH26‐labeled Mycobacterium leprae uptake by flow cytometry. The M. leprae association was determined by average PKH26 per cent positive cells ± SEM (n = 2). *P < 0·05.

Reprogramming of M2 MΦ to M1‐like MΦ correlates with an induction of the vitamin D antimicrobial pathway

Toll‐like receptor 2/1L or IFN‐γ treatment of adherent monocytes can trigger activation of the vitamin D pathway, involving up‐regulation of the 25‐hydroxylase enzyme CYP27b1 which converts 25D to bioactive 1,25D, leading to the production of antimicrobial peptides cathelicidin and β‐defensin‐2 (DEFB4).9, 13, 16 Given that IL‐10‐derived M2 MΦ failed to up‐regulate CYP27b1, cathelicidin and DEFB4,8 we investigated whether induction of plasticity to repolarize these cells to M1 MΦ would allow induction of this pathway. The IL‐10‐derived M2 MΦ were treated with TLR2/1L plus IFN‐γ, IL‐10 or medium alone for 24 hr and CYP27b1 was measured by polymerase chain reaction. The TLR2/1L plus IFN‐γ‐treated M2 MΦ showed an induction of CYP27b1 of 11‐fold over medium alone, but further IL‐10 stimulation did not have a significant difference, albeit threefold (Fig. 5a). In addition, treatment of reprogrammed M2 MΦ with 25D3 for 24 hr led to induction of cathelicidin and DEFB4 antimicrobial peptide gene expression compared with without 25D3 supplementation. Addition of 25D3 to IL‐10‐derived M2 MΦ treated with IL‐10 did not significantly induce antimicrobial peptides compared with unstimulated. These results show that M2 MΦ stimulated with TLR2/1L and IFN‐γ were able to induce expression of cathelicidin and DEFB4 (Fig. 5b). In summary, TLR2/1L plus IFN‐γ treatment is able to re‐educate M2 MΦ into M1‐like MΦ, down‐regulating expression of CD163 and scavenger receptors, diminishing phagocytic function, and inducing CYP27b1 expression to enable induction of the genes encoding antimicrobial peptides.

Figure 5.

Reprogramming of alternatively activated macrophages (M2 MΦ) to classically activated‐like MΦ (M1‐like MΦ)correlates with an induction of antimicrobial activity. (a) Interleukin‐10 (IL‐10) ‐derived M2 MΦ were stimulated with the indicated stimulants for 24 hr and assayed for average CYP27b1 mRNA fold change ± SEM (n = 3 to n = 5) compared with unstimulated M2 MΦ by quantitative polymerase chain reaction. (b) IL‐10‐derived M2 MΦ were stimulated by indicated ligands in the presence of 25D3 vitamin D (black bars) or without vitamin D (white bars) and CAMP or DEFB4 expression was determined by quantitative polymerase chain reaction. Average log₂ fold‐change ± SEM (n = 3) of condition with 25D3 versus without 25D3 is shown. *P < 0·05.

Discussion

As part of the innate immune response, monocytes respond to distinct signals that induce their differentiation into polarized MΦ characterized by distinct functional programs. The plasticity of MΦ programming allows for optimization of the immune response to respond rapidly to threats such as microbial infection. To understand the signals that drive MΦ plasticity in humans, we studied IL‐15‐differentiated M1 MΦ that express the vitamin D‐dependent antimicrobial pathway and IL‐10‐differentiated M2 MΦ that are highly phagocytic, both of which are relevant to the host response to intracellular mycobacteria.8 We demonstrate that IL‐15‐derived MΦ could be repolarized into M2‐like MΦ by treatment with IL‐10, leading to down‐regulation of the vitamin D antimicrobial pathway and up‐regulation of phagocytic activity. The re‐education of M2 MΦ into M1‐like MΦ by co‐addition of TLR2/1L and IFN‐γ led to up‐regulation of key components of the vitamin D antimicrobial pathway and down‐regulation of phagocytic activity. The regulation of MΦ plasticity allows flexibility in the innate immune response to defend the host against intracellular bacteria.

The M2 MΦ play a critical role in tissue homeostasis, in particular, IL‐10‐derived MΦ have been implicated in the uptake of lipids,17 lipoproteins,18 apoptotic cells,19 and hemoglobin,20 key functions of MΦ in maintaining tissue homeostasis. In response to tissue damage, the phagocytic capacity of M2 MΦ results in the clearance of cellular debris and apoptotic cells, allowing for tissue repair and regeneration.21 In contrast, M1 MΦ produce pro‐inflammatory mediators that not only result in antimicrobial responses but also contribute to tissue injury. Hence, the re‐education of M1 MΦ into M2‐like MΦ would likely be beneficial in cases of bystander tissue damage to maintain tissue homeostasis. We show that IL‐10 not only maintains M2 MΦ polarization, but can also re‐educate M1 MΦ to become M2‐like MΦ, including enhancement of phagocytic function. Hence, treatment with IL‐10 has been shown to lessen the harmful effects of MΦ‐induced inflammation.21 Other compounds that trigger M2 MΦ differentiation, such as pomegranate juice polyphenols, have been reported to have anti‐inflammatory properties in immune‐mediated diseases.22

In the context of intracellular bacterial infection, M2 MΦ promote disease pathogenesis, as these cells are highly phagocytic but weakly express antimicrobial genes, such that bacteria are taken up but not killed. In contrast, M1 MΦ induce antimicrobial responses against the foreign invader. Several studies have highlighted the ability of intracellular bacterial pathogens to promote M2 MΦ polarization and re‐educate M1 MΦ into M2‐like MΦ to enhance bacterial survival.23, 24, 25 Therefore, in the context of intracellular bacterial infection, it is critical to understand the factors that induce plasticity of M2 MΦ into M1 MΦ. In contrast to the ability of IL‐10 to induce plasticity of M1 MΦ into M2‐like MΦ, IL‐15 was not sufficient to induce plasticity of M2 MΦ into M1‐like MΦ. Analysis of M1 and M2 MΦ receptor expression led to the discovery that co‐addition of TLR2/1L with IFN‐γ reprogrammed M2 MΦ to M1‐like MΦ, resulting in the decrease of phagocytic function, and the induction of antimicrobial genes such as CYP27b1, CAMP, and DEFB4. This is concordant with the synergy between TLR2/1L and IFN‐γ to induce the genes encoding these antimicrobial peptides in monocytes.14 Hence, re‐polarization of M2 MΦ into M1 MΦ could be a powerful tool in augmenting host defense against intracellular pathogens, as well as to generate antitumor effects.26, 27

The conversion of M2 MΦ into M1‐like MΦ was not only triggered by the addition of TLR2/1L plus IFN‐γ, but also by the addition of TLR2/1L plus anti‐IL‐10 neutralizing antibodies. Interleukin‐10 has long been known to inhibit MΦ function including cytokine production28 and antimicrobial function.29 In human mycobacterial infection, IL‐10 inhibited M. leprae‐induced cytokine responses in PBMCs,30 TLR2/1L cytokine release from monocytes,31 and NOD2 induction of IL‐32.32 Interleukin‐10 also blocks the antimicrobial function of macrophages against mycobacteria, including phagosome maturation required to kill the intracellular bacteria,33, 34 and the vitamin‐D‐dependent antimicrobial pathway against M. leprae.12 In addition, IL‐10 has been shown to contribute to susceptibility to Mycobacterium tuberculosis in vivo,35 although in mice a major source are T cells. Our data suggest that the production of IL‐10 by human M2 MΦ locks MΦ plasticity, creating a barrier for reprogramming of M2 MΦ into M1‐like MΦ.

Research into potential targets for host‐directed therapy against mycobacterial infection has provided insight into strategies to alter MΦ function. These studies have identified specific targets of MΦ function including phagosome maturation,36, 37 autophagy,38 and induction of antimicrobial peptides.39, 40, 41, 42 These targeted approaches modulate specific MΦ functions but may not be sufficient to trigger conversion of M2 phagocytic into M1 antimicrobial MΦs. Given our data that IFN‐γ is not sufficient to induce MΦ plasticity, it is not surprising that IFN‐γ alone has not been fully effective as a therapy against leprosy43 or tuberculosis.44, 45 Instead, our data suggest that IFN‐γ must be combined with an innate modulator or an inhibitor of the immunosuppressive cytokine IL‐10 to fully effect conversion of M2‐like into M1‐like MΦs. Further studies are needed to identify and refine strategies to awaken the antimicrobial MΦ to combat intracellular bacteria.

Author contribution

The following contributions were made to this study: conceptualization: D.M. and R.L.M; methodology: D.M., D.C., S.R.K.; investigation: D.M., B.G.F, M.M., R.M.B.T; formal analysis: D.M., B.G.F, M.M.; writing – original draft: D.M., M.M.; writing – review and editing: D.M. R.L.M, M.P., S.R.K.; supervision: R.L.M., M.P.; and funding acquisition: R.L.M, M.P.

Disclosures

None.

Funding

NIAMS NIH3P50AR06302, NIAMS AR40312, NIAID AI22553.

Supporting information

Figure S1. (A) Adherent peripheral blood mononuclear cells were stimulated with interleukin‐15 (IL‐15) or IL‐10 for 3 days enriched for CD209⁺ cells by magnetic bead enrichment. Cell were then labeled with antibodies to cell surface markers CD163 and CD209. Numbers indicate the per cent positive in each quadrant (n = 2).

Figure S2. Increasing concentrations of interferon‐γ did not further decrease percentage of CD163⁺ cells.