IL-17 Stimulates Differentiation of Human Anti-Inflammatory and of Apoptotic Neutrophils in Response to IL-10 and This information is current as of October 2, 2021. Gaetano Zizzo and Philip L. Cohen J Immunol 2013; 190:5237-5246; Prepublished online 17 April 2013; doi: 10.4049/jimmunol.1203017

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-17 Stimulates Differentiation of Human Anti-Inflammatory Macrophages and Phagocytosis of Apoptotic Neutrophils in Response to IL-10 and Glucocorticoids

Gaetano Zizzo and Philip L. Cohen

Exposure of human /macrophages to anti-inflammatory agents, such as IL-10 or glucocorticoids, can lead to two separate fates: either Fas/CD95-mediated or differentiation into regulatory and efferocytic M2c (CD14brightCD16+CD163+Mer tyrosine kinase+) macrophages. We found that the prevalent effect depends on the type of Th environment and on the stage of -to- differentiation. In particular, the presence of IFN-g (Th1 inflammation) or the prolonged

exposure to IL-4 (chronic Th2 inflammation) promotes apoptosis of monocytes/macrophages and causes resistance to M2c Downloaded from differentiation, thus provoking impaired clearance of apoptotic neutrophils, uncontrolled accumulation of apoptotic cells, and persistent inflammation. In contrast, the presence of IL-17 (Th17 environment) prevents monocyte/macrophage apoptosis and elicits intense M2c differentiation, thus ensuring efficient clearance of apoptotic neutrophils and restoration of anti-inflammatory conditions. Additionally, the Th environment affects the expression of two distinct Mer tyrosine kinase isoforms: IL-4 down- regulates the membrane isoform but induces an intracellular and Gas6-dependent isoform, whereas IFN-g downregulates both

and IL-17 upregulates both. Our data support an unexpected role for IL-17 in orchestrating resolution of innate inflammation, http://www.jimmunol.org/ whereas IFN-g and IL-4 emerge as major determinants of IL-10 and resistance. The Journal of Immunology, 2013, 190: 5237–5246.

nnate inflammation is the first line of defense against micro- latory cells, another way to silence proinflammatory monocytes/ bes. Once the is removed or neutralized, a variety macrophages is to induce their apoptosis. Some studies have shown I of mechanisms act to downregulate innate inflammation that glucocorticoids and IL-10 also have proapoptotic effects on and protect tissues from uncontrolled chronic injury. Such mech- monocytes/macrophages (1–4). However, increased macrophage anisms include apoptosis of monocytes/macrophages (1–4), switch apoptosis may interfere with phagocytosis of ANs, and so con- by guest on October 2, 2021 of proinflammatory (classically activated) into regulatory (alter- tribute to further accumulation of apoptotic bodies. As such, natively activated) macrophages (5–7), and apoptosis of activated macrophage apoptosis can be detrimental for resolution of innate neutrophils with subsequent phagocytosis (8, 9). responses; indeed, it can stimulate abnormal adaptive responses Recent data highlight that a discrete subset of alternatively ac- and development of autoimmunity (15). tivated (M2) macrophages, induced by glucocorticoids or by M-CSF Optimal resolution of innate inflammation also depends on the plus IL-10 and called “M2c,” are importantly involved in reso- fate of dying neutrophils recruited into inflamed tissues. In par- lution of innate inflammation, thanks to their unique ability to ticular, early apoptosis of activated/aged neutrophils promotes phagocytose early apoptotic neutrophils (ANs) and to release anti- resolution by inhibiting further neutrophil oxidative burst and the inflammatory (5, 10–13). In particular, the upregulation release of proinflammatory mediators, and by eliciting regulatory of Mer receptor tyrosine kinase (MerTK) by these macrophages is pathways in efferocytic macrophages (8–10). Regulatory pathways crucial for intense efferocytosis and IL-10 production follow- can also be induced by neutrophil release of ectosomes, which ing apoptotic cell recognition and/or Gas6 stimulation (10–12, 14). stimulate macrophage production of anti-inflammatory cytokines Besides turning activated monocytes/macrophages into regu- following MerTK-mediated recognition of phosphatidylserine ex- posed on their surface (16). In contrast, some alternative fates of Section of Rheumatology, Department of Medicine, Temple University, Philadelphia, activated neutrophils can fuel ongoing inflammation, such as late PA 19140; and Temple Autoimmunity Center, Temple University, Philadelphia, PA apoptosis (secondary necrosis) and a newly described form of 19140 programmed cell death called NETosis (9, 17). Both secondary Received for publication October 31, 2012. Accepted for publication March 12, necrosis and NETosis generate extracellular release of chromatin 2013. and proteases, which may act as danger signals and supply anti- This work was supported by National Institute of Allergy and Infectious Diseases Grant 5U19AI082726 (Philadelphia Autoimmunity Center of Excellence), as well as genic material fostering chronic activation of immune system, a bequest from B. Wicks. uncontrolled tissue damage, and development of autoimmunity Address correspondence and reprint requests to Dr. Philip L. Cohen, Section of (18–25). Late apoptosis results from impaired clearance of early Rheumatology, Department of Medicine, Temple University, 3322 N. Broad Street, membrane-intact apoptotic cells (19, 26), as occurs in the presence Philadelphia, PA 19140-5185. E-mail address: [email protected] of macrophages with low or absent MerTK expression (10, 18). The online version of this article contains supplemental material. Whether a prompt clearance of early ANs can also have a role in Abbreviations used in this article: AC, apoptotic cell; AN, apoptotic neutrophil; MerTK, preventing NETosis remains to be determined. Altogether, multiple Mer tyrosine kinase; PI, propidium iodide. mechanisms aimed at resolving innate inflammation use MerTK to Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 enhance regulatory activity of anti-inflammatory macrophages www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203017 5238 IL-17 ORCHESTRATES RESOLUTION OF INNATE INFLAMMATION and to transmit regulatory signals from activated and dying neu- blots were developed and visualized by ECL using Amersham ECL re- trophils to surrounding macrophages. M2c polarization and MerTK agents (GE Healthcare). induction lead to noninflammatory clearance of activated neutro- Induction of apoptosis and phagocytosis assay phils and homeostatic control of innate immunity, whereas mac- rophage apoptosis and impaired clearance of early ANs exacerbate Human neutrophils were isolated from Ficoll-Hypaque pellets through dextran erythrocyte sedimentation and lysis of contaminating eryth- immune responses. rocytes by incubation with ice-cold ammonium chloride (0.15 M) and Activation of innate immune cells during disease occurs within potassium bicarbonate (0.01 M) solution. Neutrophils were resuspended a discrete immunopathological context, depending on polarization at 1 3 106 cell/ml in 10% FBS/RPMI 1640, labeled with 2.5 mM of adaptive . In the present study, we investigated CFSE (Sigma-Aldrich), and incubated for 20 h at 37˚C in 5% CO2. Allophycocyanin-conjugated annexin V (BD Biosciences) and propidium the specific influences of Th cytokines on macrophage respon- iodide (PI; Sigma-Aldrich) were used to measure apoptosis by flow cyto- siveness to M2c polarizing agents (IL-10 and glucocorticoids) in metry. The composition of neutrophils routinely obtained after incubation terms of monocyte/macrophage apoptosis, M2c macrophage dif- was 71.7 6 14.1% for early apoptotic cells (ACs) (annexin V+PI2), 4.3 6 2.0% for late ACs (annexinV+PI+), and 0.4 6 0.2% for necrotic cells ferentiation, MerTK induction, and phagocytosis of ANs. We found 2 + that IL-17 strongly amplifies M2c differentiation and MerTK- (annexinV PI ). ANs were added for 30 min to cultured macrophages at a 5:1 ratio. Flow cytometry was used to quantify percentages of dependent clearance of neutrophils, whereas IFN-g and chronic CD14-labeled macrophages that phagocytosed CFSE-labeled ACs. For exposure to IL-4 induce Fas/CD95-mediated apoptosis of monocytes/ inhibition studies, macrophages were preincubated with a goat polyclonal macrophages and uncontrolled accumulation of apoptotic neutro- anti-human MerTK Ab (5 mg/ml; R&D Systems) or a goat polyclonal phils. Furthermore, the Th environment affects differential expres- control IgG (5 mg/ml; SouthernBiotech) for 30 min before adding ANs. sion of surface and intracellular MerTK isoforms. Results suggest Fluorescence microscopy that the Th17 environment promotes successful resolution of innate Downloaded from Cultured macrophages were stained in 24-well plates using an FITC- inflammation by glucocorticoids and IL-10, whereas Th1 and conjugated anti-CD95 Ab (BioLegend) and a biotinylated mouse mono- chronic Th2 diseases may be resistant to therapies aimed at con- clonal anti-CD16 Ab (Ancell), followed by incubation with Cy3-conjugated trol of innate immunity. streptavidin (BioLegend). Cells were viewed under a Leica DMI6000B inverted fluorescence microscope, which was controlled by Slidebook Materials and Methods software. Cell cultures ELISA http://www.jimmunol.org/ Monocytes from buffy coats of healthy blood donors were isolated by Gas6 levels were measured in supernatants of 4-d cell cultures in serum-free Ficoll-Paque Plus gradient (GE Healthcare) and magnetic separation, conditions using sandwich ELISA according to standard procedures (27). using a kit for human monocyte enrichment by negative selection (EasySep; Standard curves were prepared with rhGas6 (R&D Systems). Purified goat StemCell Technologies), according to the manufacturers’ instructions. anti-human Gas6 Ab (R&D Systems) was used for capture. Biotinylated + + Purity of CD14 cells was .90%, as assessed by flow cytometry. CD14 goat polyclonal anti-human Gas6 Ab (R&D Systems), followed by HRP- 6 cells were cultured for 8 d in 24-well plates at 0.8 3 10 cells/ml at 37˚C in conjugated streptavidin (BioLegend), was used for detection. 5% CO2 in complete RPMI 1640 medium containing 10% human AB serum, L-glutamine, penicillin, and streptomycin. Cell treatments included: Statistical analysis IFN-g (10 ng/ml; R&D Systems), IL-4 (20 ng/ml; Novus Biologicals), IL-

6 by guest on October 2, 2021 17 (100 ng/ml; R&D Systems), GM-CSF (100 ng/ml; PeproTech), M-CSF Data are expressed as means SEM. Statistical significance among dif- (50 ng/ml; PeproTech), and LPS (from Escherichia coli 026:B6, 10 mg/ml; ferent cell treatments was assessed by a Student paired t test or by one-way repeated measures ANOVA when more than two treatment groups were Sigma-Aldrich). Dexamethasone (100 nM; Sigma-Aldrich) was given to , cultured monocytes on day 0, or to differentiated macrophages on day 5, as compared. Statistical significance was defined as p 0.05. Analysis and specified in the text. IL-10 (50 ng/ml; PeproTech) was added on day 5. To graphing were performed using GraphPad Prism software. measure the exact amounts of Gas6 released by cultured cells in super- natants, some experiments were performed in serum-free conditions. For Results 6 these experiments, cells were cultured at 0.8 3 10 cells/ml in X-VIVO 15 Macrophage response to IL-10: IL-17, but not IFN-g or IL-4, medium (Lonza) for 4 d, and incubated with one or more treatments from day 0. Prior to participation, all subjects gave informed consent to donate elicits M2c differentiation and MerTK-dependent efferocytosis their blood samples. The study was approved by the Institutional Review We examined the susceptibility of untreated (M0), IFN-g–conditioned Board of Temple University. (M1), IL-4–conditioned (M2a), and IL-17–conditioned (hereafter Flow cytometry called “M17”) macrophages to switch into M2c (CD14bright CD163+MerTK+) cells in response to IL-10. We found that the Phenotypic analysis was carried out in cultured monocytes/macrophages after washing in buffer containing 2% BSA. The following mouse IL-10 effects on macrophage polarization were significantly en- mAbs were used: anti-CD14 (PE-Cy7), anti-CD163 (allophycocyanin- hanced by IL-17. Although detectable, IL-10 effects were weak or PerCP-Cy5.5), anti-CD206 (allophycocyanin-Cy7), anti-CD209 (PerCP- and/or did not reach full statistical significance in the absence of Cy5.5), and anti-CD95 (allophycocyanin or FITC) (BioLegend); anti-CD16 IL-17. Along with a potent induction of CD163, the combination (allophycocyanin-Cy7) (BD Biosciences); and anti-CD204 (allophycocyanin) and anti-MerTK (clone 125518; PE) (R&D Systems). MerTK expres- of IL-17 plus IL-10 induced the highest levels of CD14 and was sion was evaluated using appropriate PE-labeled isotype control (Bio- able to upregulate MerTK (Fig. 1A–D). In contrast, both IFN-g Legend). Cells were analyzed using FACSCalibur (BD Biosciences) and and IL-4 inhibited M2c response, preventing IL-10 induction of FlowJo software. CD163, CD14, and MerTK on the cell surface (Fig. 1A–D). Western blot Analogous results were obtained whether IL-10 was added from day 5 to already differentiated M1, M2a, or M17 macrophages, as Cell lysates were obtained in buffer containing 50 mM HEPES, 150 mM a polarizing agent (Fig. 1A), or from day 0 to cultured monocytes NaCl, 10% glycerol, 1% Triton X-100, and freshly added cocktails of protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were resolved together with IFN-g, IL-4, or IL-17, as a differentiating agent (Fig. on a SDS-PAGE 8% polyacrylamide gel. Proteins, transferred to polyvi- 1B–D). In the presence of IL-17, IL-10 also induced greater se- nylidene difluoride membranes (Millipore), were probed with biotinylated cretion of the MerTK ligand Gas6 compared with IL-10 alone goat polyclonal anti-human MerTK (R&D Systems), mouse monoclonal (Fig. 1E). In accord with our previous results (10), IL-4 induced anti-CD95 (BD Biosciences), or rabbit anti–b-actin Abs (Santa Cruz Bio- technology). Membranes were then incubated, as appropriate, with HRP- high levels of Gas6, but in this case the addition of IL-10 exerted conjugated streptavidin (BioLegend), secondary donkey anti-mouse Abs no additive effect (Fig. 1E). Subsequently, we analyzed the ability (Pierce), or goat anti-rabbit Abs (Santa Cruz Biotechnology). Immuno- of M0, M1, M2a, and M17 macrophages to phagocytose ANs The Journal of Immunology 5239 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Macrophage response to IL-10: IL-17, but not IFN-g or IL-4, elicits M2c differentiation and MerTK-dependent efferocytosis. (A)Human monocytes were sorted from healthy PBMCs through negative selection magnetic beads and cultured in complete medium without cytokines (M0 differentiation) or in the presence of IFN-g (10 ng/ml; M1 differentiation), IL-4 (20 ng/ml; M2a differentiation), or IL-17 (100 ng/ml; M17 differ- entiation). On day 5, cells were treated with IL-10 (50 ng/ml) for an additional 3 d (M2c polarization). Gray histograms show cells cultured with IL-10; white histograms show cells cultured without IL-10. Expression of the M2c markers CD14, CD163, and MerTK was measured by flow cytometry. (B–E) To measure autocrine production of Gas6 along with surface expression of M2cmarkers,cellswereculturedfor4dinserum-freemediumwithIFN-g, IL-4, or IL-17 in the presence or absence of IL-10. Expression levels of CD14 (B), CD163 (C), and MerTK (D) were assessed by flow cytometry and depicted as percentages of CD14bright cells or mean fluorescence intensity (MFI) fold variation compared with levels obtained with culturing cells without IL-10. Gas6 levels (E) in culture supernatants were quantified by ELISA. Data are representative of four independent experiments. (F)Early ANs were obtained by incubating healthy human neutrophils in 10% FBS/RPMI 1640 for 20 h. CFSE-labeled ANs were added for 30 min, at a 5:1 ratio, to CD14-labeled macrophages differentiated for 7–8 d in complete medium without cytokines or in the presence of IFN-g, IL-4, or IL-17, with or without IL-10 in the last 3 d. For inhibition studies, macrophages were preincubated with a goat polyclonal anti-human MerTK Ab (5 mg/ml; R&D Systems) for 30 min before the addition of ANs. Data are representative of four independent experiments. Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. following exposure to IL-10. Consistent with the MerTK expres- IL-4 impedes IL-10 differentiation of M2c macrophages, but sion pattern, IL-10 significantly increased phagocytosis of ANs induces Gas6 and a 150-kDa intracellular MerTK isoform only in M17 cells, and this effect was abrogated in the presence of IL-4 and IL-10 are both considered major anti-inflammatory an anti-MerTK blocking Ab (Fig. 1F). cytokines. However, they induce different types of M2 macro- Detection of phenotype markers and quantification of phago- phages, namely M2a for IL-4 (CD206+CD209+) and M2c for IL-10 cytosis were not altered by macrophage autofluorescence, which (CD163+) (5, 10, 28). We examined the effects of combining IL-4 was absent or negligible in our experimental conditions (Supple- and IL-10 on macrophage phenotype. We found that IL-4 is mental Fig. 1). dominant over IL-10. The great majority of cells incubated with These data indicate that IL-17, but not IFN-g or IL-4, potentiates both cytokines were in fact CD209+, including a minor population the anti-inflammatory response of human macrophages to IL-10, of double-positive CD209+CD163+ cells, whereas very few cells resulting in more intense M2c polarization, greater secretion of were single positive for CD163 (Fig. 2A). In the co-presence of Gas6, and enhanced clearance of ANs via MerTK. IL-4 and IL-10, CD209 expression was even enhanced compared 5240 IL-17 ORCHESTRATES RESOLUTION OF INNATE INFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-4 impedes IL-10 differentiation of M2c macrophages, but it induces Gas6 and a 150-kDa intracellular MerTK isoform. (A–C) Macro- phages were differentiated for 8 d in complete medium plus IL-4 (20 ng/ml; M2a differentiation), IL-10 (50 ng/ml; M2c differentiation), or both cytokines C from day 3 for 5 d. Expression levels of CD163, CD209, CD206, CD14, and MerTK was assessed by flow cytometry and are depicted in ( ) as mean by guest on October 2, 2021 fluorescence intensity (MFI) fold variation compared with levels obtained with culturing cells without any cytokine. (D and E) To increase the number of terminally differentiated macrophages on day 8 and clearly detect a minor MerTK isoform, macrophages were differentiated in the presence of growth factors M-CSF (50 ng/ml) or GM-CSF (100 ng/ml) from day 0, adding IL-4 or IL-10 on day 3 for an additional 5 d. A 150-kDa (intracellular) MerTK isoform was observed by Western blot. The 200-kDa (surface) MerTK isoform was detected by both Western blot (D) and flow cytometry (E). Data are representative of three independent experiments. Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01. with cells treated with IL-4 alone, whereas CD206 was unchanged macrophages do not express MerTK on their surface, they still (Fig. 2B, 2C); conversely, membrane expression of the M2c express Gas6 and an intracellular 150-kDa MerTK glycoform. receptors CD163, CD14, and MerTK was significantly decreased compared with IL-10 alone (Fig. 2B, 2C). As we showed above Early response to glucocorticoids: IL-17 and IL-4, but not (Fig. 1E), both IL-10 and IL-4 stimulated Gas6 secretion. This is IFN-g, allow differentiation of M2c MerTK-dependent in apparent contradiction with IL-4 inhibition of the surface 200- efferocytic macrophages kDa MerTK isoform (11). Because prolonged Gas6 stimulation We examined the susceptibility of M0, M1, M2a, and M17 mac- was recently found to induce a poorly glycosylated intracellular rophages to switch into M2c cells in response to glucocorticoids. isoform of MerTK with lower molecular mass (150 kDa) (29), we For this purpose, we cultured monocytes in the presence or absence investigated whether IL-4, as well as IL-10, might still upregulate of IFN-g, IL-4, or IL-17 with or without dexamethasone from day the Gas6-dependent isoform. To facilitate the potential detection 0. We found that dexamethasone induced an M2c phenotype in all of this minority glycoform, we increased exposure time of Western cell populations except for M1 macrophages. IFN-g lowered ex- blots (up to 30 min); additionally, we cultured cells with macro- pression levels of CD14 (Fig. 3A) and significantly suppressed the phage growth factors (M-CSF or GM-CSF) to prolong times of M2c marker CD16/FcgRIII (Fig. 3B). Lower expression of other culture and therefore Gas6 exposure. In particular, because GM- M2c receptors (e.g., CD204, CD163) was also observed (not CSF downregulates MerTK (10), it was used to decrease MerTK shown). Additionally, IFN-g–treated cells were refractory to glu- background levels at baseline to increase the contrast for potential cocorticoid upregulation of MerTK (Fig. 3C) and Gas6 (Fig. 3D). new bands upon IL-4 or IL-10 treatment. Ultimately, we were able By Western blot, we observed dexamethasone-specific induction to clearly detect IL-4–specific induction of the 150-kDa MerTK of the 200-kDa MerTK isoform and confirmed IFN-g inhibition isoform; IL-10 was able to upregulate both 200- and 150-kDa iso- of MerTK (Fig. 3E). In line with higher levels of Gas6 in super- forms (Fig. 2D). The 150-kDa isoform was not detectable on the cell natants (Fig. 3F), cell treatments with dexamethasone in the surface, as assessed by flow cytometry (Fig. 2E). These studies show presence of IL-4 or IL-17 resulted in the upregulation of the in- that IL-4 exerts dominant effects on the M2 macrophage phe- tracellular 150-kDa MerTK isoform (Fig. 3E). Subsequently, we notype, impeding IL-10 induction of M2c cells. Although M2a tested dexamethasone effects on phagocytosis of ANs by M0, M1, The Journal of Immunology 5241 Downloaded from

FIGURE 3. Early response to glucocorticoids: IL-17 and IL-4, but not IFN-g, allow M2c differentiation and MerTK/Gas6 induction. Monocytes were cultured for 4 d in serum-free medium with or without dexamethasone from day 0 (100 nM; M2c differentiation) in the presence or absence of IFN-g (10 ng/ml), IL-4 (20 ng/ml), or IL-17 (100 ng/ml). Expression levels of CD14 (A), CD16 (B), and MerTK (C) were measured by flow cytometry and depicted as http://www.jimmunol.org/ mean fluorescence intensity (MFI) fold variation compared with untreated cells. Gas6 levels (D) in culture supernatants were measured by ELISA. Ex- pression of 200- and 150-kDa MerTK isoforms (E) was assessed by Western blot. Data are representative of four independent experiments. Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

M2a, and M17 macrophages. For inhibition studies, we used a goat a goat polyclonal control IgG (Fig. 4A, 4B). Consistent with IFN-g polyclonal anti-MerTK neutralizing Ab whose specificity was pre- resistance to glucocorticoid induction of MerTK (Fig. 3), cells liminarily verified by comparing its effects with those exerted by cultured with IFN-g plus dexamethasone showed significantly by guest on October 2, 2021

FIGURE 4. Early response to glucocorticoids: IL-17 and IL-4, but not IFN-g, allow differentiation of MerTK-dependent efferocytic macrophages. (A– D) CFSE-labeled ANs were added for 30 min, at a 5:1 ratio, to CD14-labeled macrophages differentiated for 7–8 d in complete medium with or without dexamethasone from day 0 (100 nM; M2c differentiation) in the presence of absence of IFN-g (10 ng/ml), IL-4 (20 ng/ml), or IL-17 (100 ng/ml). For inhibition studies, macrophages were preincubated with a goat polyclonal anti-human MerTK Ab (5 mg/ml; R&D Systems) for 30 min before the addition of ANs. (A and B) Specificity of anti-MerTK Ab was preliminarily assessed by comparing its effects with those exerted by a goat polyclonal control IgG (5 mg/ml; SouthernBiotech). All data shown are representative of four independent experiments. Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01. 5242 IL-17 ORCHESTRATES RESOLUTION OF INNATE INFLAMMATION lower clearance of ANs compared with macrophages differentiated stimulation of efferocytosis compared with dexamethasone treat- in the presence of dexamethasone alone or dexamethasone plus IL-4 ment from day 0; phagocytic activity progressively decreased as or IL-17 (Fig. 4C, 4D). Moreover, glucocorticoid induction of glucocorticoid treatment was delayed (Fig. 5A). Strikingly, in the efferocytosis by M1 cells was not dependent on MerTK. In contrast, presence of IL-17, the glucocorticoid promotion of efferocytosis dexamethasone effects on efferocytosis by M0, M2a, and M17 were was instead preserved over time. When dexamethasone was given significantly reduced by the addition of an anti-MerTK blocking Ab from day 5, IL-17 was able to rescue dexamethasone induction of (Fig. 4C, 4D). These data indicate that a Th1 environment promotes highly efferocytic (CFSEbright) cells, causing an effect analogous monocyte resistance to differentiation into anti-inflammatory and to what was seen with early glucocorticoid treatment (Fig. 5A, efferocytic macrophages in response to early glucocorticoid treat- 5B). In contrast, in the absence of IL-17, CFSEbright macrophages ment. In contrast, Th2 or Th17 conditions allow optimal gluco- were significantly fewer than with early glucocorticoid treatment corticoid differentiation of monocytes into MerTK+ M2c cells. (Fig. 5B). Addition of dexamethasone from day 5 did not result into a clearcut augmentation of total efferocytic (CFSE+) macro- Late response to glucocorticoids: IL-17, but not IFN-g or IL-4, phages (Fig. 5C, 5D). Similar results were obtained in the presence promotes MerTK-dependent efferocytosis by terminally of IFN-g (Fig. 5C, 5D). IL-4 facilitated glucocorticoid-induced differentiated macrophages efferocytosis, yet this was independent of MerTK activity (Fig. The first 24 h of culture are critical for glucocorticoid-mediated 5C, 5D). Remarkably, IL-17–differentiated macrophages exhibited augmentation of AC clearance by macrophages (30). We observed, the greatest response to dexamethasone, and M17 phagocytosis of in fact, that the addition of dexamethasone at later stages of ANs was significantly abrogated by blocking MerTK (Fig. 5C, 5D). macrophage differentiation (from day 3 or day 5) gave a weaker These results indicate that a Th17 environment uniquely disposes Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Late response to glucocorticoids: IL-17, but not IFN-g or IL-4, promotes MerTK-dependent efferocytosis by terminally differentiated mac- rophages. (A and B) Macrophages were differentiated for 8 d in complete medium in the presence or absence of IL-17 (100 ng/ml). Dexamethasone was added from day 0 (M2c differentiation) or during monocyte-to-macrophage differentiation from day 3 or from day 5. CFSE-labeled ANs were then coincubated with CD14-labeled macrophages, at a 5:1 ratio, for 30 min. Among macrophages, in (A) we could distinguish nonefferocytic CFSEnull macrophages (left peak), moderately efferocytic CFSElow macrophages (central peak), and highly efferocytic CFSEbright macrophages (right peak). Numerical values refer to per- centages of CFSEbright macrophages. Gray peaks refer to assays performed in the presence of a goat polyclonal anti-human MerTK blocking Ab (5 mg/ml; R&D Systems), added for 30 min prior to macrophage coincubation with ANs. CFSEtotal macrophages in (B) refers to total efferocytic (CFSEbright plus CFSElow) macrophages. (C and D) Cells were cultured in complete medium with no cytokines (M0 differentiation) or in the presence of IFN-g (10 ng/ml; M1 differentiation), IL-4 (20 ng/ml; M2a differentiation), or IL-17 (100 ng/ml; M17 differentiation); from day 5, cells were treated with dexamethasone (100 nM) for an additional 3 d (M2c polarization). CFSE-labeled ANs were then coincubated with CD14-labeled macrophages, at a 5:1 ratio, for 30 min. For inhibition studies, macrophages were preincubated with a goat polyclonal anti-human MerTK Ab (5 mg/ml; R&D Systems) for 30 min before the addition of ANs. Data are representative of four independent experiments. Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5243 terminally differentiated macrophages to highly efficient clearance and prevented MerTK expression, whereas the presence of IL-17 of ANs in response to glucocorticoid treatment by rescuing and allowed MerTK upregulation and prevented CD95 expression amplifying M2c differentiation and MerTK induction. (Fig. 6D–F). The presence of IL-4 variably influenced glucocor- To exclude that our results might have been conditioned by ticoid responses depending on the stage of macrophage differen- potential endotoxin contamination of reagents, we conducted tiation: early dexamethasone administration allowed MerTK control experiments looking at the effects of IL-10 and dexa- upregulation (Fig. 6D, 6E), whereas late administration increased methasone on macrophage phenotype and phagocytosis of ANs in CD95 expression and prevented MerTK induction (Fig. 6E). As the presence or absence of LPS. LPS did not significantly affect for MerTK, an inverse relationship with CD95 expression was macrophage response to IL-10 and dexamethasone, added either on found for the expression pattern of M2c markers such as CD16 day 0 or on day 5 (Supplemental Fig. 2). (Fig. 6F). These data indicate that M2c macrophage differentiation and macrophage apoptosis upon glucocorticoid or IL-10 stimu- Response to IL-10 and glucocorticoids: IL-17, but not IFN-g lation are mutually exclusive responses, and that the final effect is or IL-4, protects macrophages from CD95 upregulation and primarily influenced by the Th cytokine environment and by the apoptosis timing of cell stimulation. Th1 and protracted Th2 environments We explored the effect of IFN-g, IL-4, or IL-17 on monocyte program monocytes/macrophages to respond to anti-inflammatory susceptibility to apoptosis following exposure to glucocorticoids treatments by undergoing apoptosis, thereby preventing MerTK or IL-10. Resistance of IFN-g–cultured cells to glucocorticoid expression and phagocytosis of ANs. In contrast, the Th17 envi- induction of MerTK and M2c markers such as CD16 was asso- ronment constantly protects macrophages from apoptosis and elicits ciated with unchanged cell morphology upon treatment, as indi- active resolution of macrophage inflammation with noninflam- cated by unmodified forward and side scatter at flow cytometry matory clearance of ANs. Our principal findings regarding cy- Downloaded from (Fig. 6A). In the presence of IFN-g, indeed, either glucocorticoids tokine and glucocorticoid effects of macrophage differentiation or IL-10 significantly induced Fas/CD95 expression (Fig. 6B) and and phagocytosis are summarized in Table I. apoptosis, as assessed by increased proportions of CD95+annexin V+ cells (Fig. 6C). PI staining, indicative of cell death by necrosis, Discussion was instead negative (not shown). Dexamethasone upregulation of Macrophages are, at once, the main effectors and regulators of CD95 was inversely related to MerTK induction (Fig. 6D, 6E). innate inflammation. Owing to macrophage plasticity, intervention http://www.jimmunol.org/ Specifically, the presence of IFN-g allowed CD95 upregulation of anti-inflammatory agents in the microenvironment can promptly by guest on October 2, 2021

FIGURE 6. Response to IL-10 and glucocorticoids: IL-17, but not IFN-g or IL-4, constantly protects macrophages from CD95 upregulation and ap- optosis. (A) Cells were cultured for 4 d in serum-free medium with or without dexamethasone from day 0 (100 nM; M2c differentiation) in the presence or absence of IFN-g (10 ng/ml). Panels representing CD16 and MerTK expression, along with panels representing side scatter (SSC) and forward scattor (FSC) are shown. Data shown are representative of four independent experiments. (B–D) Cells were cultured for 3 d in complete medium in the presence or absence of IFN-g (10 ng/ml; M1 differentiation), IL-4 (20 ng/ml; M2a differentiation), or IL-17 (100 ng/ml; M17 differentiation) with or without IL-10 (50 ng/ml) or dexamethasone (100 nM) from day 0. Apoptosis of monocytes/macrophages was assessed by flow cytometry using CD95 (B–D), annexin V(C), and PI (not shown) stainings. Data are representative of four independent experiments. (E and F) Macrophages were differentiated for 8 d in complete medium in the presence or absence of IFN-g, IL-4, or IL-17; dexamethasone was added to some cell cultures from day 5. Expression of markers of macrophage apoptosis (CD95) and M2c polarization (MerTK, CD16) was assessed by Western blot (E) and fluorescence microscopy (original magnification 340) (F). Pooled data are represented as mean values 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001. 5244 IL-17 ORCHESTRATES RESOLUTION OF INNATE INFLAMMATION

Table I. Summary of findings regarding cytokines and glucocorticoid effects on macrophage differentiation and on apoptosis and phagocytosis of apoptotic neutrophils

Differentiation of M2c Regulatory Macrophages (CD14, CD16, CD163) and Phagocytosis of Apoptotic Neutrophils (↑MerTK)

IL-10 Early GCs (Day 0) Late GCs (Day 5) M0 1/2 111 1/- M1 22 2 M2a 2 111 2 M17 111 111 111 Apoptosis of Monocytes-Macrophages (CD95) and Accumulation of Apoptotic Neutrophils (↓MerTK)

IL-10 Early GCs (Day 0) Late GCs (Day 5) M0 1/21/2 111 M1 111 111 111 M2a 21/2 111 M17 22 2 Effects on M2c differentiation, MerTK expression and apoptosis exerted by IL-10 and early or late glucocorticoid administration on human macrophages differentiated in the absence of cytokines (M0), or in the presence of IFNg (M1), IL-4 (M2a) or IL-17 (M17). GCs, Glucocorticoids; 111, significant effects; 1/2, partial effects; 2, no effects; ↑, increase; ↓, decrease. Downloaded from shift macrophages from proinflammatory to regulatory suppressive ulation of MerTK and M2c receptors and increased expression cells (10, 31). In this study, we show that the response of human of CD95. monocytes and macrophages to anti-inflammatory mediators is By inducing monocyte/macrophage apoptosis and preventing dramatically influenced by polarization of the coexisting adaptive phagocytosis of ANs, IFN-g and IL-4 may promote autoimmune response. The presence in the microenvironment of the Th cyto- responses. In fact, coincubation of serum from lupus patients with http://www.jimmunol.org/ kines IFN-g, IL-4, or IL-17 has crucial effects on the overall healthy macrophages induces macrophage apoptosis (26), and elic- outcome of monocyte/macrophage exposure to IL-10 and gluco- iting macrophage apoptosis exacerbates autoimmunity in lupus corticoids in terms of regulatory M2c macrophage differentiation, models (15). Additionally, in lupus and other chronic inflammatory macrophage phagocytosis of ANs, or monocyte/macrophage ap- diseases, impaired clearance of early ANs accounts for late apoptosis, optosis. Owing to dynamic changes of immunological environment tissue damage, and aberrant immune activation (17–20, 24, 26). during in vivo inflammation, the study was conducted on well- Th1 and chronic Th2 inflammation may represent major deter- determined macrophage subsets obtained after in vitro polari- minants of macrophage resistance to the anti-inflammatory effects zation. The main findings are summarized in Table 1. of IL-10 and glucocorticoids. In the presence of IFN-g and IL-4,

We found that the IFN-g/Th1 environment makes monocytes/ IL-10 may have roles different from M2c polarization and im- by guest on October 2, 2021 macrophages highly resistant to M2c polarization and phagocytosis mune suppression: in Th1 diseases and lymphoproliferative dis- of ANs. In this regard, Heasman et al. (32) previously reported orders, as well as in organ rejection, IL-10 may cooperate with IFN-g inhibition of glucocorticoid augmentation of apoptotic cell IFN-g in promoting expansion of B and/or NK cells (7); in chronic clearance; however, these authors failed to elucidate the mecha- Th2 diseases and fibrotic disorders, IL-10 may enhance IL-4 in- nisms of such phenomenon. In this study, we determined that duction of remodeling M2a macrophages (35, 36). IFN-g potently inhibits glucocorticoid and IL-10 differentiation of Resistance to glucocorticoids is frequently observed in clinical human monocytes into regulatory M2c (CD14brightCD16+CD163+ practice (37). Several IFN-g–mediated pathologies, including ath- MerTK+Gas6+) macrophages; similarly, terminally IFN-g–differ- erosclerosis, acute respiratory distress syndrome, or many cases of entiated M1 macrophages do not switch into M2c cells upon inflammatory bowel disease, have poor or no response to gluco- treatment. M2c macrophages are, in fact, a subset of alternatively corticoids (38–40); similarly, refractory or relapsing forms of activated macrophages (5) that we recently characterized as highly vasculitis show persistent elevation of IFN-g levels in tissues specialized in the clearance of early (membrane-intact) apoptotic despite glucocorticoid treatment (41). IL-4 overexpression is in- cells due to intense MerTK upregulation (10). ANs maintain their volved in glucocorticoid resistance of several chronic Th2 dis- intact membranes longer than most other apoptotic cells (9) and, eases characterized by impaired apoptotic cell clearance and fibrotic consistently, MerTK has been shown to play a central role in their remodeling, such as chronic asthma, chronic obstructive pul- clearance (33). monary disease, and idiopathic pulmonary fibrosis (20, 21, 37, In the presence of IFN-g, monocytes/macrophages show ab- 42, 43); also, glucocorticoid-resistant asthma is associated with normal responses to IL-10 and glucocorticoids: instead of differ- local production of IFN-g (44). Our data provide new insights into entiating into M2c cells, they upregulate the proapoptotic receptor the mechanisms of IL-10 and glucocorticoid resistance in these Fas (CD95) and undergo apoptosis. Increased apoptosis may ex- diseases, showing that IFN-g and IL-4 strongly inhibit the ex- plain impaired M2c differentiation and efferocytosis; alternatively, pansion of M2c regulatory macrophages, thus preventing efficient because MerTK exerts an important antiapoptotic role in macro- efferocytosis and restoration of anti-inflammatory conditions. From phages (34), it is also possibile that defective MerTK expression this view, glucocorticoid-resistant airway fibrotic remodeling may promotes apoptosis. reflect refractoriness of fibrogenic M2a macrophages to M2c The IL-4/Th2 environment allows glucocorticoids to differen- polarization. tiate monocytes into M2c macrophages; however, terminally IL-4– Strikingly, the IL-17/Th17 environment significantly amplifies differentiated M2a macrophages are refractory to switching into the anti-inflammatory effects of IL-10 and sustains macrophage M2c cells in response to either glucocorticoids or IL-10. Similarly susceptibility to glucocorticoids. Facilitation of M2c polarization to IFN-g, chronic exposure to IL-4 results, instead, in downreg- by IL-17 is associated with macrophage protection from CD95 The Journal of Immunology 5245 upregulation and apoptosis; this is consistent with the previous In the course of this study, we observed that different Th cytokine demonstration in a mouse model of allergen-induced airway in- environments promote macrophage expression of different MerTK flammation that IL-17 inhibits CD95 expression and prolongs isoforms. Graham and colleagues (29) recently found that Gas6 survival of bronchoalveolar macrophages (45). To date, IL-17 induces an intracellular 150-KDa MerTK glycoform. In accord has been identified as a proinflammatory cytokine importantly with Gas6 upregulation by IL-4, we observed that M2a macro- involved in immune responses against (46), in chronic phages specifically express the 150-kDa MerTK isoform. This inflammation, and in autoimmunity (47, 48). Nonetheless, recent observation helps us to interpret our previous finding that IL-4 studies suggest that IL-17 effects are more complex than what was stimulates Gas6 production by M2a macrophages but does not initially thought. In fact, IL-17 not only stimulates granulopoiesis upregulate MerTK on their membrane (10). Consistent with the and neutrophil recruitment to sites of inflammation (48–50), but it higher levels of Gas6 produced by glucocorticoids in the presence also promotes neutrophil apoptosis (51–53) and macrophage of IL-17 compared with glucocorticoids alone, the 150-kDa phagocytosis of ANs (53). Thus, IL-17 could orchestrate multiple MerTK isoform was also detectable in the presence of IL-17. The steps in acute inflammation, including its resolution once the intracellular MerTK glycoform is not involved in membrane triggering agent has been neutralized. Silverpil et al. (53) recently recognition of apoptotic cells, but it might play a role in gene reported that IL-17 is able to stimulate phagocytosis of ANs by modulation, as suggested by its capability to translocate to nucleus murine bronchoalveolar macrophages. In humans, instead, we (29). Further investigation will be needed to better define its role. found that IL-17 is not responsible per se for phagocytosis, but it The cytokines tested in the present study were used at standard significantly amplifies and prolongs IL-10 and glucocorticoid in- doses, on the basis of previous similar in vitro studies (ranging from duction of phagocytosis. It is noteworthy, however, that Silverpil 10 to 100 ng/ml). For many of these cytokines, previous in vivo et al. (53) primed macrophages with LPS before IL-17 adminis- studies have determined their plasma levels, generally lower Downloaded from tration; therefore, autocrine production of IL-10 induced by LPS than the concentrations used in this study (in the range of picograms (54) could have allowed macrophage exposure to IL-17 and IL-10 per milliliter). However, plasma levels do not reflect the local tissue in combination. Intervention of IL-10 in the microenvironment is levels reached in sites of inflammation. We chose concentrations of probably required to convert IL-17 from a proinflammatory to a cytokines to reflect their peak bioactivity and to detect and compare regulatory molecule. The coexistence of IL-17 and IL-10, in fact, their full effects. disposes innate immune cells to restore anti-inflammatory con- Although macrophage autofluorescence might complicate flow http://www.jimmunol.org/ ditions. IL-10 alone is poorly effective on otherwise untreated M0 cytometry analysis, we did not clearly observe autofluorescent macrophages, whereas IL-17 enables M17 macrophages to re- cell populations. Accordingly, previous data reported that auto- spond to IL-10 efficiently, in terms of M2c polarization, MerTK fluorescence is low or negligible in monocytes/macrophages cul- expression, Gas6 production, and M2c-related phagocytosis of tured in vitro for a few days (59). Macrophage autofluorescence is ANs via MerTK. Our findings are consistent with previous data instead a characteristic of resident tissues macrophages (e.g., mu- reporting the efficient role of IL-10 in suppressing production of rine bone marrow and peritoneal macrophages, human alveolar macro- proinflammatory cytokines by macrophages exposed to IL-17 phages) and might be in part due to in vivo uptake of remnants and (55). The presence of both IL-17 and IL-10 in the milieu can be environmental particles (60). by guest on October 2, 2021 ascribed to multiple cell sources, including both T cells and In conclusion, different Th cytokine environments determine macrophages. In fact, Th17 cells differentiated in the presence different responses of innate immunity to anti-inflammatory treat- of high levels of TGF-b and/or low levels of IL-23 are able to ments. Our data highlight the unexpected role of IL-17 in orches- secrete both cytokines (56); moreover, IL-10 inhibits pro- trating resolution of innate inflammation, by amplifying and duction of IFN-g, but it does not suppress IL-17 production by protracting macrophage susceptibility to M2c differentiation and Th17 cells (57), thus favoring coexistence of IL-17. Finally, IL-17 phagocytosis of ANs. In contrast, IFN-g and IL-4 limit the ex- stimulates IL-10 production by macrophages themselves (55). pansion of regulatory macrophages and might elicit autoimmunity The facilitating role of IL-17 on the clearance of ANs may owing to induction of apoptotic macrophages and uncontrolled constitute an important feedback loop controlling the effects of accumulation of ANs. IL-17 on granulopoiesis and acute inflammation. Consistent with this view, the activation of liver X receptors by ANs was recently Disclosures reported in mice to elicit efficient clearance of senescent neu- The authors have no financial conflicts of interest. trophils via MerTK and to suppress the IL-23/IL-17 cytokine cascade (33). Additionally, we suggest that IL-17 could modulate innate immune responses by switching proinflammatory forms of References neutrophil programmed cell death into regulatory ones. It is 1. Estaquier, J., and J. C. Ameisen. 1997. 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