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CCL5 Promotes Resolution-Phase Macrophage Reprogramming in Concert with the Atypical Chemokine Receptor D6 and Apoptotic Polymorphonuclear Cells Miran Aswad, Simaan Assi, Sagie Schif-Zuck and Amiram Ariel

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http://www.jimmunol.org/content/suppl/2017/07/01/jimmunol.150254 2.DCSupplemental This article cites 54 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/199/4/1393.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2017; 199:1393-1404; Prepublished online 3 July 2017; doi: 10.4049/jimmunol.1502542 http://www.jimmunol.org/content/199/4/1393

The Journal of Immunology

CCL5 Promotes Resolution-Phase Macrophage Reprogramming in Concert with the Atypical Chemokine Receptor D6 and Apoptotic Polymorphonuclear Cells Miran Aswad,*,†,1 Simaan Assi,*,†,1 Sagie Schif-Zuck,*,† and Amiram Ariel*,†

D

uring the active resolution of inflammation (1–3) immune response elements are eliminated (4). This is hallmarked by leukocyte apoptosis and clearance by macrophages (5–7). Apoptotic cell engulfment by phagocytes is mediated by signals that are expressed on the surface of apoptotic cells and their corresponding receptors (reviewed in Ref. 7, 8), and leads to macrophage reprogramming and immune silencing (6, 9–11) in response to bacterial moieties through specific kinases, such as p38MAPK and JNK (12). Macrophage reprogramming is defined by a reduction in the release of proinflammatory cytokines and chemokines, concomitant with the production of TGF-b and IL-10 (13– 15), cytokines that can promote resolution and wound repair. In addition, the uptake of apoptotic cells promotes the expression of 15lipoxygenase-1, which is involved in the generation of proresolving lipid mediators (16, 17), by macrophages. It also induces the production of angiogenic growth factors (18) to promote wound healing. Macrophages differentiate to many functional phenotypes following the acquisition of immune-related and tissue-specific signals (19, 20). Recent reports indicate that macrophages acquire distinct phenotypes during the resolution of acute inflammation (21, 22). A new phenotype distinguishable from either M1 or M2

*Department of Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel; and †Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel 1

M.A. and S.A. contributed equally to this work.

ORCIDs: 0000-0002-5492-6074 (M.A.); 0000-0001-5415-9488 (S.S.-Z.). Received for publication December 8, 2015. Accepted for publication June 7, 2017. This work was supported by grants from the Israel Science Foundation (Grant 534/ 09), the Rosetrees Trust, and the Wolfson Foundation. Address correspondence and reprint requests to Dr. Amiram Ariel, Departments of Biology and Human Biology, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa 3498838, Israel. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: PMN, polymorphonuclear cell; PPI, post–peritonitis initiation; PS, phosphatidylserine; rM, resolution-phase macrophage; WT, wild type. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502542

was displayed by CD11blow, resolution-phase macrophages (rMs) generated from CD11bhigh ones upon the engulfment of threshold numbers of apoptotic polymorphonuclear cells (PMN) (22). This phenotypic conversion of macrophages results in significant immune silencing in addition to the reduction in surface expression of CD11b and F4/80 (22). Specifically, CD11blow macrophages stop producing TNF-a and IL-1b, but increase the production of TGF-b and the expression of 12/15- lipoxygenase, and emigrate to the lymphatics (22). Chemokines and their receptors have long been appreciated for their role in regulating leukocyte migration during inflammation (23, 24). However, atypical chemokine receptors, and under certain anti-inflammatory settings classic chemokine receptors, were reported to be involved in chemokine clearance (25–28). Notably, chemokines are reported to exert resolution-promoting activities in addition to their classic proinflammatory ones. For instance, CCL2 was found to promote the engulfment of apoptotic cells by macrophages (29), whereas CCL5 was reported to exert antiinflammatory actions under some settings (30–32). D6 is an atypical chemokine receptor that binds promiscuously to inflammatory chemokines, internalizes them, and directs them for lysosomal degradation without transmitting intracellular signaling events that characterize classical chemokine receptors (reviewed in Ref. 33, 34). Thus, it is considered an instrumental part of the resolution of inflammation. This phenomenon is due to constitutive recycling of D6 in a ligand-independent manner (33, 34). Interestingly, recent reports have indicated that most of the D6 protein is present in intracellular stores, whereas only a small amount reaches the cell surface; this surface expression can be modulated by various means (35, 36). Notably, D6 has been shown to be expressed on apoptotic PMN and promotes their reprogramming effect on rMs; hence D6+/+ senescent PMN, but not their D62/2 counterparts, promoted the secretion of IL-10 while inhibiting the release of proinflammatory cytokines and chemokines by macrophages that engulfed these cells (37). In this study, we report CCL5 is abundantly produced in resolving peritoneal exudates and by rMs. Moreover, treatment with CCL5 in vivo enhanced macrophage reprogramming during the

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The engulfment of apoptotic polymorphonuclear cells (PMN) during the resolution of inflammation leads to macrophage reprogramming culminating in reduced proinflammatory and increased anti-inflammatory mediator secretion. The atypical chemokine receptor D6/ACKR2 is expressed on apoptotic PMN and plays an important role in regulating macrophage properties during and after engulfment. In this study, we found that the inflammatory chemokine CCL5 is mostly retained (75%) during the resolution of zymosan A peritonitis in mice. Moreover, this chemokine is secreted by resolution-phase macrophages (2.5 ng/ml) and promotes their reprogramming in vivo in D6+/+ mice (2-fold increase in IL-10/IL-12 ratio) but not their D62/2 counterparts. In addition, CCL5 enhanced macrophage reprogramming ex vivo exclusively when bound to D6+/+ apoptotic PMN. Signaling through p38MAPK and JNK in reprogrammed macrophages was enhanced by CCL5-bound apoptotic PMN (3.6–4 fold) in a D6-dependent manner, and was essential for reprogramming. Thus, CCL5 exerts a novel proresolving role on macrophages when acting in concert with apoptotic PMN-expressed D6. The Journal of Immunology, 2017, 199: 1393–1404.

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resolution of inflammation, as did the exposure of rMs to CCL5bound apoptotic PMN. Finally, we show that the actions of apoptotic PMN-bound CCL5 are mediated through enhancement of p38MAPK and JNK activation.

Materials and Methods Reagents ELISA kits for mouse TNF-a, IL-10, IL-12, IL-6, CCL2, CCL3, and CCL5 were obtained from R&D Systems (Minneapolis, MN). Roscovitine and LPS (from Escherichia coli, clone 055:B5) from Sigma-Aldrich. CCL5 was from PeproTech. JNK and p38MAPK inhibitors (SP600125 and VX702, respectively) were from Selleck Chemicals. The CCR1 and CCR5 antagonists (BX471 and Maraviroc, respectively) were from Cayman Chemical and Sigma-Aldrich.

Murine peritonitis

Cytokine secretion ex vivo Macrophages were isolated from peritoneal exudates 66 h PPI by immunostaining with PE-conjugated rat anti-F4/80 Abs and PE selection magnetic beads (StemCell Technologies). Then, the cells were incubated alone or with senescent PMN from D6+/+ or D62/2 mice (24 h PPI, ex vivo culture for 16 h). After 24 h culture supernatants were collected and their TNF-a, IL-10, CCL2, CCL3, or CCL5 contents were determined by standard ELISA.

LPS responsiveness ex vivo Exudate D6+/+ macrophages were isolated 66 h PPI, incubated (1 3 106 cells in 0.5 ml of culture media) with senescent PMN from D6+/+ or D62/2 mice (24 h PPI, ex vivo culture for 16 h), and then incubated with LPS (1 mg/ml). In some experiments apoptotic PMN (treatment with roscovitine, 10 mM, 4–6 h) from D6+/+ or D62/2 mice were incubated (30 min) with CCL5 (1 mg/ml), washed twice (bound) or left with the chemokine (soluble), and incubated (1:3–1:5 macrophage to PMN ratio) with macrophages for 24 h. Then, macrophages were incubated with LPS as above. In some experiments macrophages were treated ex vivo with CCL5 (100 ng/ml) alone prior to LPS stimulation. In some experiments, JNK or p38MAPK inhibitors (SP600125 or VX702, respectively; 5–10 mm), or CCR1 or CCR5 antagonists (BX 471 (170 nM) or Maraviroc (5 mM), respectively) were added to the macrophages during the first 24 h of incubation to block the signaling induced by the apoptotic PMN. Alternatively, D6+/+ or D62/2 macrophages from vehicle- or CCL5 (100 ng per mouse, 18 h)-treated mice were isolated 48 or 66 h PPI, and immediately exposed to LPS. After incubation with LPS (16 h) the supernatants from all treatments were collected and their TNF-a, IL-12, IL-10, CCL2, CCL3, or CCL5 contents were determined by standard ELISA.

CCL5 binding to apoptotic PMNs Apoptotic peritoneal PMN were recovered from D6+/+ or D62/2 mice and incubated (30 min) with biotinylated CCL5 followed by incubation (30 min) with avidin-fluorescein (Fluorokine Biotinylated CCL5 #NFRN0; R&D Systems). Next, the cells were centrifuged and soluble CCL5 was collected. Alternatively, release of bound CCL5 was assessed by additional washing and incubation of CCL5-bound PMN for 60 min. Then, soluble CCL5 was collected following centrifugation. Surface-bound CCL5 levels were determined by flow cytometry of the PMN and soluble/released CCL5 was determined using an infinite 200 pro fluorometer (Tecan

Phosphorylation of p38MAPK and JNK D6+/+ macrophages were isolated from peritoneal exudates 66 h PPI, and were incubated with apoptotic D6+/+ or D62/2 PMNs alone (1:3 ratio), or supplemented with soluble or bound CCL5 as above for 1–5 min. Next, the cells were lysed in RIPA buffer, equal amounts of their protein content were run by 10% SDS-PAGE and evaluated for the phosphorylation of p38MAPK and JNK by Western blotting with phospho-p38MAPK, total p38MAPK, phospho-JNK, and total JNK specific primary Abs (Cell Signaling Technology, Danvers, MA) followed by the appropriate HRPconjugated secondary Ab (Jackson ImmunoResearch, West Grove, PA).

Quantitative RT-PCR Total RNA was extracted from F4/80+ peritoneal macrophages recovered from unchallenged mice or 66 h PPI using Tri Reagent (Sigma-Aldrich). First-strand cDNA was generated using a High Capacity cDNA RT kit (Applied Biosystems) and carried out by StepOnePlus Real-Time PCR Systems with the SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The following primers were used: mouse CCR1, forward 59-GTGGGCAATGTCCTAGTGATT-39, reverse 59-GGTAGATGCTGGTCATGCTTT-39; mouse CCR5, forward 59-TGCACAAAGAGACTTGAGGCA39, reverse 59-AGTGGTTCTTCCCTGTTGGCA-39; mouse D6, forward 59-TTCTCCCACTGCTGCTTCAC-39, reverse 59-TGCCATCTCAACATCACAGA-39, and mouse GAPDH 59 (as the endogenous control) 59-ACCACAGTCCATGCCATCAC-39, reverse 59-CACCACCCTGTTGCTGTAGCC-39. The relative mRNA levels of each receptor in each cell type were calculated with GAPDH as an internal control and with D6 expression in zymosan A–elicited macrophages given the value of 1.

Statistical analysis Ex vivo and in vivo experiments were performed at least three times with at least four replicates. Results were analyzed by one-way ANOVA with *p , 0.05, **p , 0.01, ***p , 0.005 indicated. Results are presented as averages 6 SE.

Results CCL5 is retained at high levels in resolving peritoneal exudates Chemokines are molecular cues that orchestrate leukocyte migration to sites of inflammation. Abrogation of neutrophil influx is a prerequisite for resolution of inflammation and mechanisms such as chemokine cleavage by proteolysis and chemokine sequestration are necessary to attain a resolving environment (3, 37, 38). However, previous studies (31, 37, 39, 40) indicate that D6 ligands might play a key role in promoting the resolution of inflammation and limiting unwarranted inflammatory consequences, particularly when acting on macrophages. Therefore, we examined the levels of select D6 ligands and the proinflammatory cytokines TNF-a and IL-6 in peritoneal exudates during the resolving phase of zymosan A–induced murine peritonitis. Our results indicate that although CCL5 levels at 72 h PPI remained at 75% of the levels at 24 h PPI (Fig. 1A), the levels of all other D6 ligands (CCL2 and CCL3), TNF-a, and IL-6 decreased by 9–10 fold at the same interval (Fig. 1B–E). Thus, our results suggest that CCL5 might be an effector chemokine in the resolution phase of inflammation. CCL5 is the predominant cytokine produced by rMs The engulfment of apoptotic leukocytes by macrophages results in their immune silencing and reprogramming, as reflected in reduced cytokine and chemokine production upon exposure to microbial agents, such as LPS (6, 10). However, macrophages treated with apoptotic cells alone upregulate the secretion of both pro- and anti-inflammatory cytokines (41, 42). Notably, Stables et al. (39) have shown that CCL5 is upregulated in rMs. Moreover, our results in Fig. 1 indicate a significant and selective retention of CCL5 in resolving peritoneal exudates. Therefore, we sought to determine whether rMs secrete CCL5, and whether this secretion is modulated by apoptotic PMN in a D6-dependent manner. Our results

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D62/2 mice were kindly obtained from Prof. Locati (Instituto Clinico Humanitas, Milan, Italy) and bred in a specific pathogen-free/viral Ab–free barrier facility. D6+/+ mice were obtained from Harlan Biotech, Israel. Briefly, D6+/+ male C57BL/6 mice (6–8 wk; protocol approved by the Committee of Ethics, The Technion, authorization number IL-065-04-2010) were injected i.p. with zymosan A (1 mg). After 24, 48, or 72 h, peritoneal contents were collected and the levels of TNF-a, IL-6, CCL2, CCL3, and CCL5 in cell-free exudates were determined by standard ELISA. Alternatively, D6+/+ or D62/2 mice were treated with CCL5 (100 ng/ml per mouse) at 48 h and exudate cells were collected at 66 h. Alternatively, D6+/+ mice were injected i.p. with anti-CCL5 neutralizing Ab (2 mg per mouse, clone 53405, R&D Systems), isotype control (clone RTK2758; BioLegend), or vehicle 18 h post–peritonitis initiation (PPI). Peritoneal cells were collected, enumerated, immunostained with FITC-conjugated rat anti-Gr-1 and PEconjugated rat anti-F4/80 (BioLegend), and analyzed by FACSCantoII (Becton Dickinson). Macrophages from each treatment were isolated using PE selection magnetic beads against F4/80 (StemCell Technologies, Vancouver, BC, Canada) and exposed to LPS ex vivo.

Group). Negative control supplied by the manufacturer were analyzed by flow cytometry, and the obtained values were subtracted from the CCL5 values to indicate specific binding.

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indicate CCL5 is the highest secreted cytokine/chemokine in comparison with TNF-a, IL-10, CCL2, and CCL3 (Fig. 2). Moreover, incubation with apoptotic PMN from either D6+/+ or D62/2 mice significantly increased the secretion of all cytokines. Importantly, incubation of apoptotic PMN from D62/2 mice with macrophages resulted in a higher secretion of CCL5 than incubation with their D6+/+ counterparts, thus suggesting that the reduced reprogramming of macrophages by D62/2 apoptotic PMN (37) is not due to reduced production of CCL5. Thus, CCL5 secretion from rMs might play an important role in regulating the resolution of inflammation. CCL5 blockade increases rM numbers and reduces their responses to LPS The engulfment of apoptotic leukocytes by macrophages results in their immune silencing and reprogramming, reflected by reduced proinflammatory cytokine and chemokine production in response to TLR ligands and conversion to the CD11blow phenotype (6, 10, 22). CD11blow macrophages, in turn, emigrate to remote sites to restore peritoneal homeostasis (22). Therefore, we examined whether neutralizing CCL5 during the resolution phase of peritonitis will affect macrophage numbers and LPS responsiveness ex vivo. Our results in

Fig. 3A indicate anti-CCL5 Abs significantly increased the percentage of F4/80+ mature macrophages in the peritoneum (24.3% increase from isotype), although it did not significantly change PMN or PMN-macrophage conjugate percentages or total leukocyte numbers (data not shown, n = 2) in comparison with isotype Ab treatment. Moreover, CCL5 neutralization reduced the LPS-induced TNF-a and IL-10 secretion (45.0 and 52.1% reduction from isotype) from rMs stimulated ex vivo with LPS (Fig. 3B, 3C), in comparison with isotype control. The secretion of IL-12, however, was not affected by CCL5 neutralization (Fig. 3D). Notably, the impact of CCL5 neutralization on macrophage numbers and LPS responsiveness was not significant at 48 h PPI (data not shown), suggesting that long-term exposure to CCL5 or high CCL5 concentrations are needed to affect macrophage properties. Moreover, no significant change in the percentage of CD11blow macrophages was observed with CCL5 neutralization (data not shown, n = 2). Thus, CCL5 seems to partially promote LPS responsiveness in rMs. CCL5 promotes macrophage reprogramming in vivo in a D6-dependent manner Our previous results indicate D6 deficiency in neutrophils hampers macrophage reprogramming during their interaction with

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FIGURE 1. CCL5 is selectively retained in resolving peritoneal exudates. Peritoneal fluids were collected from mice undergoing zymosan A–induced peritonitis at 24, 48, or 72 h PPI and their levels of CCL5 (A), TNF-a (B), IL-6 (C), CCL2 (D), or CCL3 (E) were determined by standard ELISA. The dilution factor was estimated at 1000-fold and final peritoneal concentrations were calculated accordingly. Results are means 6 SE of five replicates from a representative of three experiments. Significant differences by one-way ANOVA between peritonitis times are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

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FIGURE 2. rMs predominantly secrete CCL5. Peritoneal macrophages from D6+/+ mice were isolated at 66 h PPI, and incubated with D6+/+ or D62/2 senescent PMN. After 24 h, the culture supernatants were collected and evaluated for their content of CCL5, TNF-a, IL-10, CCL2, and CCL3 using standard ELISA. Results are representative of five experiments. Significant differences by one-way ANOVA between cytokines/chemokines or between treatments are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

secretion from D6+/+ macrophages (36, 27, and 36% reduction, respectively) upon LPS stimulation. As expected, TNF-a and IL-12 secretion from D62/2 macrophages were not reduced by CCL5 exposure, whereas IL-10 secretion by these macrophages was reduced and this reduction was enhanced by CCL5 (60% reduction, effectively eliminating the secretion stimulated by LPS). Because

FIGURE 3. CCL5 blockade increases rM numbers and limits their stimulation by LPS. Anti-CCL5 Abs, their isotype controls, or vehicle were injected to the peritoneum of mice 24 h PPI and peritoneal cells were collected and analyzed by flow cytometry for leukocyte subsets (A). In addition, macrophages were collected from each treatment and cultured with LPS (1 mg/ml) for 24 h. Then, their cell-free supernatants were collected and their content of TNF-a (B), IL-10 (C), and IL-12 (D) were determined using standard ELISA. Results are averages (A) 6 SE and representative (B–D) from three experiments. Significant differences by Student t test between mice treated with anti-CCL5, isotype Abs, or vehicle are indicated. *p , 0.05, ***p , 0.005.

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senescent PMN (37) and the results in Fig. 3 indicate CCL5 modulates rM properties. Therefore, we determined whether in vivo treatment with CCL5 affects D62/2 macrophage cytokine production and reprogramming. Our results (Fig. 4A–C) show that CCL5 did not change cytokine secretion by unstimulated macrophages. However, it significantly reduced TNF-a, IL-10, and IL-12

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IL-10 downregulates IL-12 secretion (43), we calculated the ratio of the LPS-stimulated secretion between IL-10 and 1L-12 as a reprogramming index. Our results indicated CCL5 significantly increased the reprogramming index of D6+/+ macrophages, but reduced it to negative values in their D62/2 counterparts (Fig. 4D). Notably, CCL5 did not change macrophage numbers in D6+/+ mice (Supplemental Fig. 1), but reduced their elevated numbers in D62/2 mice (37). Thus, D6 is essential for CCL5-enhanced macrophage reprogramming during the resolution of inflammation. CCL5 exposure ex vivo does not promote macrophage reprogramming Our results in Figs. 1, 3, and 4 suggest that high concentrations of CCL5 might directly promote reprogramming of rMs. To determine the direct actions of CCL5 on rMs, D6+/+ macrophages were exposed to vehicle or CCL5 and their cytokine secretion was determined. The results in Fig. 5 indicate that CCL5 exposure ex vivo increased LPS-stimulated secretion from macrophages of the anti-inflammatory cytokine IL-10 (178% increase; Fig. 5B). However, increases in TNF-a, IL-12, CCL2, and CCL3 were also found (128, 458, 897, and 492% increase, respectively; Fig. 5A, 5C–E), suggesting that exposure to soluble CCL5 ex vivo promoted LPS responsiveness of rMs in a nonselective manner. Thus, CCL5 is not promoting macrophage reprogramming directly. Binding of CCL5 to senescent PMN enhances their reprogramming of macrophages through D6 Our results in Fig. 5 suggested soluble CCL5 does not promote macrophage reprogramming. Apoptotic cells, however, serve as resolution and reprogramming cues for macrophages, as their recognition evokes distinct signaling events (12) that block the release of proinflammatory mediators from macrophages. Because D6 is expressed on the surface of apoptotic PMN (37), it can

present chemokine oligomers (44) to classic chemokine receptors (i.e., CCR5 and CCR1) expressed on macrophages, which will enhance the inhibitory signal evoked by apoptotic PMN. Therefore, we sought to determine whether CCL5 binds apoptotic PMN and whether D6 is essential for this binding. To this end we bound fluorescent CCL5 to apoptotic PMN from D6+/+ or D62/2 mice and determined the amount of bound and soluble chemokine directly after binding or after allowing the release of CCL5 during 1 h incubation. Our results (Fig. 6A) indicate D6 deficiency in PMN resulted in a significant reduction in CCL5 binding both prior and after the allowed release (49.6 and 55.4% of D6+/+ PMN, respectively). Importantly, the incubation with apoptotic PMN resulted in a significant decrease in the amount of soluble CCL5 (28.8–33.3% reduction) that did not depend on D6 (Fig. 6B). Moreover, no significant release of bound CCL5 from apoptotic PMN was observed. Thus, D6 is an important effector in the tight binding of CCL5 to apoptotic PMN. Next, we aimed to determine whether D6-mediated CCL5 binding to apoptotic PMN will promote their reprogramming of macrophages. To this end, D6+/+ macrophages were isolated from resolving exudates 66 h PPI and incubated with D6+/+ or D62/2 apoptotic PMN 6 soluble CCL5 or CCL5-bound apoptotic PMNs of either genotype (1:3 ratio) for 24 h. Then, the macrophages were stimulated with LPS, the cell-free supernatants were collected, and their content of cytokines and chemokines was determined. Our results (Fig. 6C, 6D) indicate in LPS-stimulated macrophages TNF-a and IL-12 secretion were reduced by senescent PMN alone or with bound CCL5 (21 and 60% reduction, respectively), and this reduction was significantly abrogated when the PMN were added with soluble CCL5 (7 and 40% reduction, respectively). In accordance with our previous report (37), apoptotic D6+/+ PMN increased IL-10 secretion from LPS-stimulated

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FIGURE 4. CCL5 promotes macrophage reprogramming in vivo in a D6-dependent manner. CCL5 (100 ng per mouse) or vehicle were injected to the peritoneum of D6+/+ or D62/2 mice 48 h PPI, and macrophages from peritoneal exudates were recovered 66 h PPI. Then, macrophages were cultured with LPS (1 mg/ml) for 24 h, their cellfree supernatants were collected, and their content of TNF-a (A), IL-10 (B), and IL-12 (C) was determined using standard ELISA. IL-10/1L-12 reprogramming index (D) was calculated as follows: (LPS-induced secretion of IL-10 2 vehicle-induced secretion of IL-10)/(LPS-induced secretion of IL-12 2 vehicle-induced secretion of IL-12). The averages of secretion from D6+/+ macrophages stimulated with LPS were 590 pg/ml for TNF-a, 1000 pg/ml for IL- 10, and 210 pg/ml for IL-12. Results were normalized to LPS-stimulated D6+/+ macrophages and averages 6 SE were calculated for three experiments. Significant differences by one-way ANOVA between D6+/+ and D62/2 macrophages and CCL5- or vehicle-treated mice are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

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p38 and JNK mediate the reprogramming of rMs

FIGURE 5. CCL5 exposure ex vivo does not promote macrophage reprogramming. Macrophages from D6+/+ mice were isolated 66 h PPI and incubated with CCL5 (100 ng/ml) for 24 h. Then, the cells were washed and incubated with LPS for an additional 24 h. The content of TNF-a (A), IL-10 (B), IL-12 (C), CCL2 (D), and CCL3 (E) in the culture media was determined using standard ELISA. Results are averages 6 SE representative of four experiments. Significant differences by one-way ANOVA between CCL5- and vehicle-treated macrophages are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

macrophages (2000% increase), which further increased (3368%) when CCL5 in its bound form was added to the PMN. As we reported previously (37), D62/2 senescent PMN were less effective than their wild-type (WT) counterparts in inhibiting TNF-a and promoting IL-10 secretion from macrophages. Importantly, the impact of bound CCL5 on cytokine secretion by LPS-stimulated macrophages was significantly abolished (IL-10 and IL-12) or even reversed (TNF-a) when it was applied to D62/2 PMN. Interestingly, D6 deficiency seems to reduce IL-12 inhibition by soluble CCL5 + senescent PMN, suggesting that even in the presence of excess soluble chemokine, the inhibitory signaling of D6-bound CCL5 dominates IL-12 regulation. Thus, we have identified a novel inhibitory/reprogramming mode of action for CCL5 upon binding to D6 on the surface of apoptotic PMN during efferocytosis. D6 deficiency hampers JNK and p38MAPK activation in macrophages by CCL5-bound apoptotic PMN A major signaling pathway involved in the regulation of cytokine secretion by activated immune cells involves the MAPKs, a family of serine/threonine kinases. The four well-characterized subfamilies of MAPKs are the ERKs (ERK1/2), c-Jun NH2-terminal

Our results indicate CCL5 bound to apoptotic PMN enhanced macrophage reprogramming as well as JNK and p38 phosphorylation following apoptotic PMN-macrophage encounter (Figs. 6, 7). Therefore, we sought to determine whether JNK or p38 signaling are essential for macrophage reprogramming by CCL5-bound apoptotic PMN. To this end, specific p38 and JNK inhibitors (VX702 and SP600125, respectively) were added to various forms of apoptotic PMN during the incubation with macrophages, which was followed by LPS stimulation of the macrophages for cytokine secretion. Our results indicate that the inhibitory effect of apoptotic PMN (at all forms) on TNF-a and IL-12 secretion was blocked or reversed to stimulation, respectively, by p38 inhibition (Fig. 8A, 8C). Notably, LPS-stimulated secretion of these cytokines was slightly reduced by p38 inhibition. In addition, JNK inhibition abrogated the inhibitory effect of apoptotic PMN on TNF-a and IL-12 secretion from macrophages when all forms of apoptotic PMN were used. The only exception to this rule was observed for TNF-a inhibition by CCL5-bound apoptotic PMN. With regards to IL-10 (Fig. 8B), p38 inhibition significantly reduced apoptotic cell–induced, but not LPS-induced, secretion and abolished the additive effect of bound CCL5. JNK inhibition did not reduce apoptotic cell–induced IL-10 secretion but did abolish the additive effect of bound CCL5. Thus, p38 and JNK signaling seems to be essential for macrophage reprogramming by apoptotic cells, and their action is enhanced when apoptotic PMN are bound with CCL5. CCR1 and CCR5 mediate macrophage reprogramming induced by apoptotic cell–bound CCL5 It was previously shown that the responses of rMs to apoptotic PMN (including cytokine secretion) are hampered due to D6 deficiency exclusively on these PMN (37). Therefore, we aimed to determine which CCL5-binding chemokine receptor expressed by rMs mediates their reprogramming by D6-bound CCL5 upon interaction with apoptotic PMN. Our results in Supplemental Fig. 2 indicate CCR1 and CCR5 are highly expressed in rMs (and resident peritoneal macrophages) whereas D6 is expressed at much

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kinases (JNK-1/2/3), p38 (p38a/b/g/d), and ERK5 (45). Moreover, apoptotic cells were found to stimulate JNK1/2 and p38 activation in macrophages (12). To determine whether CCL5 or D6 modulate JNK or p38 signaling evoked by apoptotic PMN, we treated rMs with D6+/+ or D62/2 apoptotic PMN that were untreated, added with soluble CCL5 or bound with CCL5, and evaluated their JNK and p38 phosphorylation. Our results indicate that JNK phosphorylation was induced (3-fold) by either apoptotic PMN or CCL5-bound apoptotic PMN from D6+/+ mice, with faster kinetics in the latter. Apoptotic PMN applied with soluble CCL5, in contrast, were unable to stimulate JNK phosphorylation in these macrophages. Notably, D62/2 PMN induced a small increase (2-fold) in JNK phosphorylation only when applied with soluble CCL5 for 5 min (Fig. 7A, 7B). Similarly, p38 phosphorylation (Fig. 7C, 7D) was stimulated dramatically in macrophages treated with D6+/+ apoptotic PMN or CCL5-bound apoptotic PMN (5- and 4-fold, respectively), but to a much lower extent by soluble CCL5-treated PMN (1.5-fold). Notably, p38 was phosphorylated faster, but to a lower extent upon treatment with CCL5-bound apoptotic PMN in comparison with apoptotic PMN alone. Importantly, p38 phosphorylation was induced by D62/2 apoptotic PMN after 5 min (although to a lower extent than their WT counterparts) but failed to do so when bound with CCL5. Thus, D6 is essential for the activation of JNK and p38 in macrophages treated with CCL5-bound apoptotic PMN.

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FIGURE 6. CCL5 bound to apoptotic PMN promotes macrophage reprogramming in a D6-dependent manner. (A and B) PMN from D6+/+ or D62/2 mice were isolated 24 h PPI and induced to undergo apoptosis with roscovitine (10 mM, 4 h). Then, the cells were washed and incubated with fluorescent CCL5 for 30 min. Next, the cellular and soluble components were separated by centrifugation and the supernatants were collected. The cells were further washed and incubated for 1 h at 37˚C to determine release of soluble CCL5 from PMN, and cellular and soluble components were separated again. The binding of CCL5 to apoptotic PMN and the retention or release of soluble CCL5 were determined by flow cytometry (A) and fluorometry (B), respectively, after each step. Results are averages 6 SE from three experiments. Significant differences by Student t test between D6+/+ and D62/2 PMN or different forms of soluble CCL5 are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. (C–E) Macrophages from D6+/+ mice were isolated 66 h PPI and incubated with apoptotic PMN. PMN were isolated 24 h PPI from D6+/+ or D62/2 mice and cultured for 4 h with roscovitine (10 mm) to promote apoptosis. Apoptotic cells were incubated (4˚C, 30 min) with CCL5 (100 ng/ml) and washed twice (bound form), or left unwashed (soluble form). Macrophages and CCL5-treated or untreated apoptotic neutrophils were cocultured at a 1:3 ratio for 24 h. Next, the supernatants were collected and the macrophages were treated with LPS (1 mg/ml) for an additional 24 h. Then, the cell-free supernatants were collected and their content of TNF-a, IL-10, and IL-12 was determined using standard ELISA. The averages of secretion in LPS-stimulated D6+/+ macrophages were 1.7 ng/ml for TNF-a, 2.9 ng/ ml for IL- 10, and 1.1 ng/ml for IL-12. Results were normalized to LPS-stimulated D6+/+ macrophages and the averages 6 SE were calculated for four experiments. Significant differences by oneway ANOVA between treatments are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

lower levels (over 600-fold) in these macrophages. Moreover, both CCR1 and CCR5 antagonists reduced IL-10 secretion by LPSstimulated rMs induced by apoptotic PMN, as well as CCL5bound apoptotic PMN (Fig. 8D). These antagonists also reversed

the inhibition of IL-12 secretion from these macrophages under the same settings (Fig. 8E). Notably, the CCR1 and 5 antagonists did not affect the inhibitory action of apoptotic PMN and CCL5 on TNF-a secretion (data not shown), hence suggesting additional

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FIGURE 7. Bound CCL5 enhances apoptotic PMN activation of JNK and p38MAPK in macrophages through D6. Macrophages from D6+/+ mice were incubated ex vivo (1:3 ratio) with apoptotic neutrophils (10 mM roscovitine, 4 h) from D6+/+ or D62/2 mice for 1–5 min. The apoptotic neutrophils were incubated (4˚C, 30 min) with vehicle or CCL5 (100 ng/ml) and washed twice (bound form), or left unwashed (soluble form) prior to incubation with the macrophages. Next, the cells were lysed and their protein content was run by SDS-PAGE (10%), and blotted for phospho-JNK, JNK, (A and B), phosphop38, and p38 (C and D). The bands corresponding to phosphoproteins were quantified by densitometry and normalized to the relevant total protein bands and to untreated controls (100%). Results are representative images (A and C) and averages 6 SE (B and D) from three independent experiments. Significant differences by one-way ANOVA between D6+/+ or D62/2 macrophages, or treatments are indicated. *p , 0.05, **p , 0.01, ***p , 0.005.

chemokine receptors might take part in the signal transduction induced by apoptotic PMN and CCL5. Thus, apoptotic PMNbound CCL5 promotes rM reprogramming through the activation of CCR1 and CCR5 (for illustration, please see Fig. 9).

Discussion CCL5 is considered to be a major effector chemokine in the attraction of leukocytes to inflamed tissues, in particular in peritoneal inflammation (23, 46). D6 was previously found to play a significant role in the resolution of inflammation, pre-

sumably through the binding, internalization, and lysosomal degradation of its ligands, which consist of most of the proinflammatory chemokines (26, 27, 47). In this study, we describe a novel role for CCL5 in regulating macrophage reprogramming during the resolution phase of zymosan A–induced peritonitis. CCL5 was found to be retained at bioactive concentrations (above 50 ng/ml, 25% reduction) during the resolution phase of inflammation (24–72 h), whereas all other proinflammatory cytokines and chemokines examined were reduced below 10 ng/ml (80–90% reduction; Fig. 1) at the same period. Moreover, rMs secreted

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CCL5 to the highest extent (2.5 ng/ml) in comparison with inflammatory cytokines and chemokines, and this secretion was enhanced by interactions with senescent PMN (Fig. 2). Importantly, CCL5 neutralization reduced IL-10, but not IL-12 production by these macrophages (Fig. 3). Moreover, CCL5 exposure in vivo resulted in significantly reduced secretion of the proinflammatory cytokines TNF-a and IL-12, but only slightly reduced the antiinflammatory cytokine IL-10 (Fig. 4). Hence, the reprogramming index, defined by the ratio of LPS-stimulated secretion of IL-10/IL12, was increased by CCL5 in WT mice but reduced in D62/2 mice. Of interest, CCL5 did not promote reprogramming ex vivo as it enhanced IL-10 secretion by rMs, but had a similar impact on inflammatory cytokines and chemokines (Fig. 5). Nevertheless, binding of CCL5 to apoptotic PMN from D6+/+ mice enhanced macrophage reprogramming (primarily through the increase in IL-10), but failed to do so in its soluble form, or when bound to D62/2 apoptotic PMN (Fig. 6). JNK and p38MAPK activation were enhanced by CCL5-bound apoptotic PMN from D6+/+ mice. Inhibition of these kinases by specific pharmacological inhibitors attenuated the reprogramming of macrophages by apoptotic PMN, and completely blocked the impact of bound CCL5. Although CCL5 has bona fide roles in attracting various immune cells to inflammation sites in multiple autoimmune and inflammatory settings (23, 24, 46, 48, 49), several studies have indicated

expression by rMs (39) and anti-inflammatory roles for this chemokine (30–32, 50). Notably, CCL5 was induced in an IL-10– dependent manner in anti-inflammatory B cell–trained macrophages (51). Most inflammatory chemokines, including CCL5, peak during early peritonitis and significantly decline by 24 h PPI (28, 37, 38). We now show that although CCL2 and CCL3, as well as other inflammatory cytokines, continue to decline during the following 48 h of spontaneously resolving inflammation, CCL5 levels remain at a bioactive concentration (Fig. 1). Moreover, our results indicate CCL5 is secreted to the highest extent (relative to both pro- and anti-inflammatory cytokines and chemokines) by rMs (Fig. 2). This secretion is promoted by senescent PMN from D6-deficient mice (as well as their WT counterparts) that were previously shown to exert reduced reprogramming of rMs. Hence, the decreased reprogramming of D6-deficient macrophages is not due to reduced CCL5 secretion. Importantly, these findings were supported by Stables et al. (39). Thus, CCL5 seems to be produced by rMs, in particular following their engulfment of apoptotic PMN, and thereby maintains consistent peritoneal levels during resolution. The expression of chemokine receptors on apoptotic leukocytes was previously reported (28, 37, 52), and the expression of D6 on apoptotic PMN, in particular, was found to have a significant impact on macrophages that engulfed these cells. Our findings now indicate in vivo exposure to CCL5 during the resolution of

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FIGURE 8. p38MAPK, JNK, CCR1, and CCR5 mediate the reprogramming of rMs by apoptotic neutrophils. Macrophages or neutrophils were isolated from D6+/+ mice at 66 or 24 h PPI, respectively. Neutrophils were cultured with roscovitine (10 mm, 4 h) for apoptosis induction, and apoptotic neutrophils were incubated with vehicle or CCL5 as above to generate soluble and bound forms. Macrophages and apoptotic neutrophils of all forms were cocultured with either p38MAPK (5 mM) or JNK (10 mM) inhibitors, or CCR1 (170 nM) or CCR5 (5 mM) antagonists for 24 h. Then, the cells were washed and incubated with LPS (1 mg/ml) for an additional 24–48 h. Next, the cellfree supernatants were collected and their content of TNF-a (A), IL-10 (B and D), and IL-12 (C and E) were determined using standard ELISA. The reversion of the effect of apoptotic PMN on IL-10 and IL-12 secretion by CCR antagonists was calculated for either apoptotic PMN or CCL5-bound apoptotic PMN as follows: [LPS stimulated secretion 2 (LPS + AC) stimulated secretion]/[LPS stimulated secretion 2 (LPS + AC + antagonist) stimulated secretion]. Results are averages 6 SE representative of three experiments. Significant differences by one-way ANOVA between D6+/+ or macrophages, different D62/2 treatments, or relative to LPS-treated macrophages (D and E) are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. AC, apoptotic cells.

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inflammation significantly abrogated TNF-a and IL-12 secretion by rMs exposed to LPS, whereas IL-10 secretion was slightly reduced. Therefore, the reprogramming index of these macrophages was significantly increased (Fig. 4). Importantly, D62/2 macrophages had a reduced reprogramming index under these settings. Moreover, exposure to CCL5 ex vivo leads to increased LPS responsiveness in terms of cytokine/chemokine secretion, regardless of the mediator’s properties (Fig. 5), whereas CCL5 neutralization in vivo reduced TNF-a and IL-10 secretion ex vivo (Fig. 3). These findings suggest that during the resolution of inflammation in vivo CCL5 acts in two functionally distinct forms. In concert with D6-expressing apoptotic PMN it promotes macrophage reprogramming. However, in its soluble form it partially promotes macrophage responsiveness to LPS and contributes to TNF-a production. Notably, both forms promote the production of the immunoregulatory cytokine IL-10 by rMs. CCL5-neutralizing Abs seem to block the activity of soluble CCL5 in vivo, but do not affect the activity of the apoptotic PMN-bound form, probably due to its localization to PMNmacrophage contact sites. Therefore, the impact of these Abs opposes the activity of soluble CCL5 ex vivo (Fig. 5), for the most part. Our results in Fig. 6 show that D6 is instrumental in the binding of CCL5 by apoptotic PMN and that binding of

CCL5 to D6+/+ apoptotic PMN, but not to their D62/2 counterparts, leads to increased reprogramming of macrophages that interacted with these PMN. This change was largely attributed to an increase in IL-10 secretion (Fig. 6D). Soluble CCL5 added to apoptotic PMN did not exert such actions. Notably, CCL5 neutralization also resulted in increased numbers of macrophages (Fig. 3), whereas CCL5 supplementation in D62/2 mice reduced the abnormal peritoneal macrophage numbers in these mice during resolution (Supplemental Fig. 1). These results indicate that soluble CCL5 also plays an important role in diminishing macrophage numbers during resolving inflammation, possibly by promoting their reprogramming and emigration from the peritoneum. Although D6 is not expressed on Ly-6Chi monocytes (53), which differentiate to form the vast majority of rMs in our model (data not shown), it has been shown to be involved in determining their fate in D6-deficient mice (53). Our results in Supplemental Fig. 2 and Fig. 8 confirm these findings and indicate that CCR1 and CCR5, rather than D6, are expressed by rMs and mediate IL-10 induction and IL-12 inhibition induced by apoptotic PMN alone or CCL5-bound apoptotic PMN. It remains to be determined which receptors expressed on these macrophages contribute to the inhibition of TNF-a secretion observed under these settings. Thus, the results presented in this report support a novel role for a CCL5–D6

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FIGURE 9. Illustration of the proposed role of a CCL5–D6 axis in the resolution of inflammation. This scheme illustrates the postulated role played by CCL5 and D6 in mediating the interactions between senescent/apoptotic PMN and macrophages during the resolution of inflammation. CCL5 is secreted by rMs and binds D6 on the surface of apoptotic PMN, possibly in the form of homo-oligomers. These CCL5 oligomers in turn bind and activate the classical chemokine receptors CCR1 and CCR5 expressed on the engulfing macrophage. We also postulate D6 patches with phosphatidylserine (PS) on the surface of apoptotic PMN and thus the signaling cascades from PS receptors and CCR1/CCR5 merge to promote macrophage reprogramming (resulting in a decreased TNF-a and IL-12 and increased IL-10 levels) through enhanced p38MAPK and JNK activation. This mode of action could explain the differences in macrophage reprogramming and signaling observed between cell-bound and soluble CCL5. In addition, we suggest these molecular pathways also explain D6’s involvement in promoting macrophage migration to the lymphatics (37).

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Disclosures The authors have no financial conflicts of interest.

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axis during the resolution of inflammation (Fig. 9). CCL5 is released by rMs and binds D6 on apoptotic PMN. As inflammatory chemokines are known to form homo-oligomers (54), our findings suggest that CCL5 forms such D6-bound complexes on the surface of apoptotic PMN and presents them to the classical chemokine receptors CCR1 and CCR5 expressed on macrophages, which in turn promote the IL-10–driven reprogramming cascade induced by apoptotic cell–displayed, phosphatidylserine (PS)containing membrane patches (55). Apoptotic cells were previously shown to promote macrophage reprogramming and survival through the activation of p38MAPK and JNK, and inhibition of ERK (12, 56). Our findings now show that CCL5 in its apoptotic PMN-bound form enhances the activating phosphorylation of p38MAPK and JNK in comparison with apoptotic PMN alone. This enhancement was evident during the early response (1 min) rather than the late one (5 min), when maximal phosphorylation was observed. Importantly, D6 deficiency or the presence of excess amounts of soluble CCL5 prevented the enhanced activation of p38MAPK and JNK, possibly due to the uncoupling of signaling from CCL5 and PS (Fig. 9). In this context it is important to note that soluble CCL5 was found to bind CCR5 on macrophages and promote their survival through activation of AKT and ERK. Taking into account these findings and that the increased LPS responsiveness of rMs following CCL5 exposure ex vivo (Fig. 5), it is plausible to suggest that soluble and apoptotic PMN-bound CCL5 exert different proresolving actions on macrophages through p38 MAPK/JNK (reprogramming) and AKT/ERK (survival). The present report uncovers novel roles for the chemokine CCL5 when acting in concert with the atypical chemokine receptor D6 upon its expression on apoptotic PMN. This newly found module functions to promote macrophage reprogramming during the resolution of inflammation. These findings are key to the understanding of chemokine biology during inflammation and its resolution, and might promote novel chemokine-based therapies to inflammatory disorders.

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