CCL5 Promotes Resolution-Phase Macrophage Reprogramming In

CCL5 Promotes Resolution-Phase Macrophage Reprogramming in Concert with the Atypical Chemokine Receptor D6 and Apoptotic Polymorphonuclear Cells This information is current as of September 28, 2021. Miran Aswad, Simaan Assi, Sagie Schif-Zuck and Amiram Ariel J Immunol 2017; 199:1393-1404; Prepublished online 3 July 2017; doi: 10.4049/jimmunol.1502542 Downloaded from http://www.jimmunol.org/content/199/4/1393

Supplementary http://www.jimmunol.org/content/suppl/2017/07/01/jimmunol.150254 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 54 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/199/4/1393.full#ref-list-1

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

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 Downloaded from 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.

low uring the active resolution of inflammation (1–3) immune was displayed by CD11b , resolution-phase macrophages (rMs) http://www.jimmunol.org/ response elements are eliminated (4). This is hallmarked generated from CD11bhigh ones upon the engulfment of threshold D by leukocyte apoptosis and clearance by macrophages numbers of apoptotic polymorphonuclear cells (PMN) (22). This (5–7). Apoptotic cell engulfment by phagocytes is mediated by phenotypic conversion of macrophages results in significant im- signals that are expressed on the surface of apoptotic cells and mune silencing in addition to the reduction in surface expression their corresponding receptors (reviewed in Ref. 7, 8), and leads to of CD11b and F4/80 (22). Specifically, CD11blow macrophages macrophage reprogramming and immune silencing (6, 9–11) in re- stop producing TNF-a and IL-1b, but increase the production of sponse to bacterial moieties through specific kinases, such as TGF-b and the expression of 12/15- lipoxygenase, and emigrate to p38MAPK and JNK (12). Macrophage reprogramming is defined by the lymphatics (22). a reduction in the release of proinflammatory cytokines and che- Chemokines and their receptors have long been appreciated for by guest on September 28, 2021 mokines, concomitant with the production of TGF-b and IL-10 (13– their role in regulating leukocyte migration during inflammation 15), cytokines that can promote resolution and wound repair. In (23, 24). However, atypical chemokine receptors, and under cer- addition, the uptake of apoptotic cells promotes the expression of 15- tain anti-inflammatory settings classic chemokine receptors, were lipoxygenase-1, which is involved in the generation of proresolving reported to be involved in chemokine clearance (25–28). Notably, lipid mediators (16, 17), by macrophages. It also induces the pro- chemokines are reported to exert resolution-promoting activities duction of angiogenic growth factors (18) to promote wound healing. in addition to their classic proinflammatory ones. For instance, Macrophages differentiate to many functional phenotypes fol- CCL2 was found to promote the engulfment of apoptotic cells lowing the acquisition of immune-related and tissue-specific sig- by macrophages (29), whereas CCL5 was reported to exert anti- nals (19, 20). Recent reports indicate that macrophages acquire inflammatory actions under some settings (30–32). D6 is an distinct phenotypes during the resolution of acute inflammation atypical chemokine receptor that binds promiscuously to inflam- (21, 22). A new phenotype distinguishable from either M1 or M2 matory chemokines, internalizes them, and directs them for lysosomal degradation without transmitting intracellular signaling events that characterize classical chemokine receptors (reviewed *Department of Biology, Faculty of Natural Sciences, University of Haifa, Haifa in Ref. 33, 34). Thus, it is considered an instrumental part of the 3498838, Israel; and †Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel resolution of inflammation. This phenomenon is due to constitu- 1M.A. and S.A. contributed equally to this work. tive recycling of D6 in a ligand-independent manner (33, 34). Interestingly, recent reports have indicated that most of the D6 ORCIDs: 0000-0002-5492-6074 (M.A.); 0000-0001-5415-9488 (S.S.-Z.). protein is present in intracellular stores, whereas only a small Received for publication December 8, 2015. Accepted for publication June 7, 2017. amount reaches the cell surface; this surface expression can be This work was supported by grants from the Israel Science Foundation (Grant 534/ 09), the Rosetrees Trust, and the Wolfson Foundation. modulated by various means (35, 36). Notably, D6 has been shown to be expressed on apoptotic PMN and promotes their Address correspondence and reprint requests to Dr. Amiram Ariel, Departments of +/+ Biology and Human Biology, Faculty of Natural Sciences, University of Haifa, Mt. reprogramming effect on rMs; hence D6 senescent PMN, but Carmel, Haifa 3498838, Israel. E-mail address: [email protected] not their D62/2 counterparts, promotedthesecretionofIL-10 The online version of this article contains supplemental material. while inhibiting the release of proinflammatory cytokines and Abbreviations used in this article: PMN, polymorphonuclear cell; PPI, post–perito- chemokines by macrophages that engulfed these cells (37). nitis initiation; PS, phosphatidylserine; rM, resolution-phase macrophage; WT, wild In this study, we report CCL5 is abundantly produced in re- type. solving peritoneal exudates and by rMs. Moreover, treatment with Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 CCL5 in vivo enhanced macrophage reprogramming during the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502542 1394 A CCL5–D6 AXIS PROMOTES THE RESOLUTION OF INFLAMMATION resolution of inflammation, as did the exposure of rMs to CCL5- Group). Negative control supplied by the manufacturer were analyzed by bound apoptotic PMN. Finally, we show that the actions of apo- flow cytometry, and the obtained values were subtracted from the CCL5 ptotic PMN-bound CCL5 are mediated through enhancement of values to indicate specific binding. p38MAPK and JNK activation. Phosphorylation of p38MAPK and JNK D6+/+ macrophages were isolated from peritoneal exudates 66 h PPI, and Materials and Methods were incubated with apoptotic D6+/+ or D62/2 PMNs alone (1:3 ratio), or Reagents 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 ELISA kits for mouse TNF-a, IL-10, IL-12, IL-6, CCL2, CCL3, and CCL5 were run by 10% SDS-PAGE and evaluated for the phosphorylation of were obtained from R&D Systems (Minneapolis, MN). Roscovitine and LPS p38MAPK and JNK by Western blotting with phospho-p38MAPK, total (from Escherichia coli, clone 055:B5) from Sigma-Aldrich. CCL5 was from p38MAPK, phospho-JNK, and total JNK specific primary Abs (Cell Sig- PeproTech. JNK and p38MAPK inhibitors (SP600125 and VX702, respectively) naling Technology, Danvers, MA) followed by the appropriate HRP- were from Selleck Chemicals. The CCR1 and CCR5 antagonists (BX471 and conjugated secondary Ab (Jackson ImmunoResearch, West Grove, PA). Maraviroc, respectively) were from Cayman Chemical and Sigma-Aldrich. Quantitative RT-PCR Murine peritonitis Total RNA was extracted from F4/80+ peritoneal macrophages recovered 2 2 D6 / mice were kindly obtained from Prof. Locati (Instituto Clinico from unchallenged mice or 66 h PPI using Tri Reagent (Sigma-Aldrich). Humanitas, Milan, Italy) and bred in a specific pathogen-free/viral Ab–free First-strand cDNA was generated using a High Capacity cDNA RT kit (Applied barrier facility. D6+/+ mice were obtained from Harlan Biotech, Israel. Biosystems) and carried out by StepOnePlus Real-Time PCR Systems with the Briefly, D6+/+ male C57BL/6 mice (6–8 wk; protocol approved by the SYBR Green Master Mix (Applied Biosystems) according to the manufac- Committee of Ethics, The Technion, authorization number IL-065-04-2010)

turer’s instructions. The following primers were used: mouse CCR1, forward Downloaded from were injected i.p. with zymosan A (1 mg). After 24, 48, or 72 h, peritoneal 59-GTGGGCAATGTCCTAGTGATT-39,reverse59-GGTAGATGCTGGTCA- contents were collected and the levels of TNF-a, IL-6, CCL2, CCL3, and TGCTTT-39; mouse CCR5, forward 59-TGCACAAAGAGACTTGAGGCA- CCL5 in cell-free exudates were determined by standard ELISA. Alterna- 39, reverse 59-AGTGGTTCTTCCCTGTTGGCA-39; mouse D6, forward 2 2 tively, D6+/+ or D6 / mice were treated with CCL5 (100 ng/ml per mouse) 59-TTCTCCCACTGCTGCTTCAC-39,reverse59-TGCCATCTCAACATCA- at 48 h and exudate cells were collected at 66 h. Alternatively, D6+/+ mice CAGA-39, and mouse GAPDH 59 (as the endogenous control) 59-ACCAC- were injected i.p. with anti-CCL5 neutralizing Ab (2 mg per mouse, clone AGTCCATGCCATCAC-39,reverse59-CACCACCCTGTTGCTGTAGCC-39. 53405, R&D Systems), isotype control (clone RTK2758; BioLegend), or The relative mRNA levels of each receptor in each cell type were calculated vehicle 18 h post–peritonitis initiation (PPI). Peritoneal cells were collected, with GAPDH as an internal control and with D6 expression in zymosan http://www.jimmunol.org/ enumerated, immunostained with FITC-conjugated rat anti-Gr-1 and PE- A–elicited macrophages given the value of 1. conjugated rat anti-F4/80 (BioLegend), and analyzed by FACSCantoII (Becton Dickinson). Macrophages from each treatment were isolated using Statistical analysis PE selection magnetic beads against F4/80 (StemCell Technologies, Van- Ex vivo and in vivo experiments were performed at least three times with at couver, BC, Canada) and exposed to LPS ex vivo. least four replicates. Results were analyzed by one-way ANOVAwith *p , , , Cytokine secretion ex vivo 0.05, **p 0.01, ***p 0.005 indicated. Results are presented as averages 6 SE. Macrophages were isolated from peritoneal exudates 66 h PPI by immunostaining with PE-conjugated rat anti-F4/80 Abs and PE selection Results magnetic beads (StemCell Technologies). Then, the cells were incubated CCL5 is retained at high levels in resolving peritoneal exudates by guest on September 28, 2021 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 Chemokines are molecular cues that orchestrate leukocyte migration TNF-a, IL-10, CCL2, CCL3, or CCL5 contents were determined by to sites of inflammation. Abrogation of neutrophil influx is a pre- standard ELISA. requisite for resolution of inflammation and mechanisms such as LPS responsiveness ex vivo chemokine cleavage by proteolysis and chemokine sequestration are necessary to attain a resolving environment (3, 37, 38). However, Exudate D6+/+ macrophages were isolated 66 h PPI, incubated (1 3 106 2 2 previous studies (31, 37, 39, 40) indicate that D6 ligands might play cells in 0.5 ml of culture media) with senescent PMN from D6+/+ or D6 / mice (24 h PPI, ex vivo culture for 16 h), and then incubated with LPS a key role in promoting the resolution of inflammation and limiting (1 mg/ml). In some experiments apoptotic PMN (treatment with roscovitine, unwarranted inflammatory consequences, particularly when acting 2 2 10 mM, 4–6 h) from D6+/+ or D6 / mice were incubated (30 min) with on macrophages. Therefore, we examined the levels of select D6 CCL5 (1 mg/ml), washed twice (bound) or left with the chemokine (sol- ligands and the proinflammatory cytokines TNF-a and IL-6 in uble), and incubated (1:3–1:5 macrophage to PMN ratio) with macrophages for 24 h. Then, macrophages were incubated with LPS as above. In peritoneal exudates during the resolving phase of zymosan A–in- some experiments macrophages were treated ex vivo with CCL5 (100 ng/ml) duced murine peritonitis. Our results indicate that although CCL5 alone prior to LPS stimulation. In some experiments, JNK or p38MAPK levels at 72 h PPI remained at 75% of the levels at 24 h PPI inhibitors (SP600125 or VX702, respectively; 5–10 mm),orCCR1orCCR5 (Fig. 1A), the levels of all other D6 ligands (CCL2 and CCL3), antagonists (BX 471 (170 nM) or Maraviroc (5 mM), respectively) were TNF-a, and IL-6 decreased by 9–10 fold at the same interval 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 (Fig. 1B–E). Thus, our results suggest that CCL5 might be an ef- macrophages from vehicle- or CCL5 (100 ng per mouse, 18 h)-treated mice fector chemokine in the resolution phase of inflammation. 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 CCL5 is the predominant cytokine produced by rMs and their TNF-a, IL-12, IL-10, CCL2, CCL3, or CCL5 contents were de- The engulfment of apoptotic leukocytes by macrophages results in termined by standard ELISA. their immune silencing and reprogramming, as reflected in reduced CCL5 binding to apoptotic PMNs cytokine and chemokine production upon exposure to microbial 2 2 agents, such as LPS (6, 10). However, macrophages treated with Apoptotic peritoneal PMN were recovered from D6+/+ or D6 / mice and incubated (30 min) with biotinylated CCL5 followed by incubation apoptotic cells alone upregulate the secretion of both pro- and (30 min) with avidin-fluorescein (Fluorokine Biotinylated CCL5 #NFRN0; anti-inflammatory cytokines (41, 42). Notably, Stables et al. (39) R&D Systems). Next, the cells were centrifuged and soluble CCL5 was have shown that CCL5 is upregulated in rMs. Moreover, our re- collected. Alternatively, release of bound CCL5 was assessed by additional sults in Fig. 1 indicate a significant and selective retention of CCL5 washing and incubation of CCL5-bound PMN for 60 min. Then, soluble CCL5 was collected following centrifugation. Surface-bound CCL5 levels in resolving peritoneal exudates. Therefore, we sought to determine were determined by flow cytometry of the PMN and soluble/released whether rMs secrete CCL5, and whether this secretion is modu- CCL5 was determined using an infinite 200 pro fluorometer (Tecan lated by apoptotic PMN in a D6-dependent manner. Our results The Journal of Immunology 1395

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), B C D

TNF-a ( ), IL-6 ( ), CCL2 ( ), or Downloaded from 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 representa-

tive of three experiments. Signiﬁcant http://www.jimmunol.org/ differences by one-way ANOVA between peritonitis times are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. by guest on September 28, 2021

indicate CCL5 is the highest secreted cytokine/chemokine in Fig. 3A indicate anti-CCL5 Abs significantly increased the per- comparison with TNF-a, IL-10, CCL2, and CCL3 (Fig. 2). centage of F4/80+ mature macrophages in the peritoneum (24.3% Moreover, incubation with apoptotic PMN from either D6+/+ or increase from isotype), although it did not significantly change PMN D62/2 mice significantly increased the secretion of all cytokines. or PMN-macrophage conjugate percentages or total leukocyte Importantly, incubation of apoptotic PMN from D62/2 mice with numbers (data not shown, n = 2) in comparison with isotype Ab macrophages resulted in a higher secretion of CCL5 than incu- treatment. Moreover, CCL5 neutralization reduced the LPS-induced bation with their D6+/+ counterparts, thus suggesting that the re- TNF-a and IL-10 secretion (45.0 and 52.1% reduction from isotype) duced reprogramming of macrophages by D62/2 apoptotic PMN from rMs stimulated ex vivo with LPS (Fig. 3B, 3C), in comparison (37) is not due to reduced production of CCL5. Thus, CCL5 se- with isotype control. The secretion of IL-12, however, was not af- cretion from rMs might play an important role in regulating the fected by CCL5 neutralization (Fig. 3D). Notably, the impact of resolution of inflammation. CCL5 neutralization on macrophage numbers and LPS responsiveness was not significant at 48 h PPI (data not shown), suggesting that CCL5 blockade increases rM numbers and reduces their long-term exposure to CCL5 or high CCL5 concentrations are responses to LPS needed to affect macrophage properties. Moreover, no significant The engulfment of apoptotic leukocytes by macrophages results in change in the percentage of CD11blow macrophages was observed their immune silencing and reprogramming, reflected by reduced with CCL5 neutralization (data not shown, n = 2). Thus, CCL5 proinflammatory cytokine and chemokine production in response to seems to partially promote LPS responsiveness in rMs. TLRligandsandconversiontotheCD11blow phenotype (6, 10, 22). CD11blow macrophages, in turn, emigrate to remote sites to restore CCL5 promotes macrophage reprogramming in vivo in a peritoneal homeostasis (22). Therefore, we examined whether neu- D6-dependent manner tralizing CCL5 during the resolution phase of peritonitis will affect Our previous results indicate D6 deficiency in neutrophils ham- macrophage numbers and LPS responsiveness ex vivo. Our results in pers macrophage reprogramming during their interaction with 1396 A CCL5–D6 AXIS PROMOTES THE RESOLUTION OF INFLAMMATION

FIGURE 2. rMs predominantly secrete CCL5. Perito- neal 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. Downloaded from

senescent PMN (37) and the results in Fig. 3 indicate CCL5 secretion from D6+/+ macrophages (36, 27, and 36% reduction,

modulates rM properties. Therefore, we determined whether in respectively) upon LPS stimulation. As expected, TNF-a and IL-12 http://www.jimmunol.org/ vivo treatment with CCL5 affects D62/2 macrophage cytokine secretion from D62/2 macrophages were not reduced by CCL5 production and reprogramming. Our results (Fig. 4A–C) show that exposure, whereas IL-10 secretion by these macrophages was re- CCL5 did not change cytokine secretion by unstimulated macro- duced and this reduction was enhanced by CCL5 (60% reduction, phages. However, it signiﬁcantly reduced TNF-a, IL-10, and IL-12 effectively eliminating the secretion stimulated by LPS). Because by guest on September 28, 2021

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. The Journal of Immunology 1397

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 cell- free 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 se- Downloaded from cretion 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 experi- http://www.jimmunol.org/ ments. 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.

IL-10 downregulates IL-12 secretion (43), we calculated the ratio of present chemokine oligomers (44) to classic chemokine receptors the LPS-stimulated secretion between IL-10 and 1L-12 as a re- (i.e., CCR5 and CCR1) expressed on macrophages, which will programming index. Our results indicated CCL5 significantly in- enhance the inhibitory signal evoked by apoptotic PMN. There- by guest on September 28, 2021 creased the reprogramming index of D6+/+ macrophages, but fore, we sought to determine whether CCL5 binds apoptotic PMN reduced it to negative values in their D62/2 counterparts (Fig. 4D). and whether D6 is essential for this binding. To this end we bound Notably, CCL5 did not change macrophage numbers in D6+/+ mice fluorescent CCL5 to apoptotic PMN from D6+/+ or D62/2 mice (Supplemental Fig. 1), but reduced their elevated numbers in D62/2 and determined the amount of bound and soluble chemokine di- mice (37). Thus, D6 is essential for CCL5-enhanced macrophage rectly after binding or after allowing the release of CCL5 during reprogramming during the resolution of inflammation. 1 h incubation. Our results (Fig. 6A) indicate D6 deficiency in PMN resulted in a significant reduction in CCL5 binding both CCL5 exposure ex vivo does not promote prior and after the allowed release (49.6 and 55.4% of D6+/+ PMN, macrophage reprogramming respectively). Importantly, the incubation with apoptotic PMN Our results in Figs. 1, 3, and 4 suggest that high concentrations of resulted in a significant decrease in the amount of soluble CCL5 CCL5 might directly promote reprogramming of rMs. To deter- (28.8–33.3% reduction) that did not depend on D6 (Fig. 6B). mine the direct actions of CCL5 on rMs, D6+/+ macrophages were Moreover, no significant release of bound CCL5 from apoptotic exposed to vehicle or CCL5 and their cytokine secretion was PMN was observed. Thus, D6 is an important effector in the tight determined. The results in Fig. 5 indicate that CCL5 exposure ex binding of CCL5 to apoptotic PMN. vivo increased LPS-stimulated secretion from macrophages of the Next, we aimed to determine whether D6-mediated CCL5 anti-inflammatory cytokine IL-10 (178% increase; Fig. 5B). binding to apoptotic PMN will promote their reprogramming of However, increases in TNF-a, IL-12, CCL2, and CCL3 were also macrophages. To this end, D6+/+ macrophages were isolated from found (128, 458, 897, and 492% increase, respectively; Fig. 5A, resolving exudates 66 h PPI and incubated with D6+/+ or D62/2 5C–E), suggesting that exposure to soluble CCL5 ex vivo pro- apoptotic PMN 6 soluble CCL5 or CCL5-bound apoptotic PMNs moted LPS responsiveness of rMs in a nonselective manner. Thus, of either genotype (1:3 ratio) for 24 h. Then, the macrophages CCL5 is not promoting macrophage reprogramming directly. were stimulated with LPS, the cell-free supernatants were collected, and their content of cytokines and chemokines was Binding of CCL5 to senescent PMN enhances their determined. reprogramming of macrophages through D6 Our results (Fig. 6C, 6D) indicate in LPS-stimulated macro- Our results in Fig. 5 suggested soluble CCL5 does not promote phages TNF-a and IL-12 secretion were reduced by senescent macrophage reprogramming. Apoptotic cells, however, serve as PMN alone or with bound CCL5 (21 and 60% reduction, re- resolution and reprogramming cues for macrophages, as their spectively), and this reduction was significantly abrogated when recognition evokes distinct signaling events (12) that block the the PMN were added with soluble CCL5 (7 and 40% reduction, release of proinflammatory mediators from macrophages. Because respectively). In accordance with our previous report (37), apo- D6 is expressed on the surface of apoptotic PMN (37), it can ptotic D6+/+ PMN increased IL-10 secretion from LPS-stimulated 1398 A CCL5–D6 AXIS PROMOTES THE RESOLUTION OF INFLAMMATION

kinases (JNK-1/2/3), p38 (p38a/b/g/d), and ERK5 (45). More- over, 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 Downloaded from apoptotic PMN in comparison with apoptotic PMN alone. Im- portantly, 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. http://www.jimmunol.org/ p38 and JNK mediate the reprogramming of rMs 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 FIGURE 5. CCL5 exposure ex vivo does not promote macrophage inhibitors (VX702 and SP600125, respectively) were added to by guest on September 28, 2021 reprogramming. Macrophages from D6+/+ mice were isolated 66 h PPI and various forms of apoptotic PMN during the incubation with incubated with CCL5 (100 ng/ml) for 24 h. Then, the cells were washed macrophages, which was followed by LPS stimulation of the and incubated with LPS for an additional 24 h. The content of TNF-a (A), macrophages for cytokine secretion. Our results indicate that the B C D E IL-10 ( ), IL-12 ( ), CCL2 ( ), and CCL3 ( ) in the culture media was inhibitory effect of apoptotic PMN (at all forms) on TNF-a and 6 determined using standard ELISA. Results are averages SE represen- IL-12 secretion was blocked or reversed to stimulation, respec- tative of four experiments. Significant differences by one-way ANOVA tively, by p38 inhibition (Fig. 8A, 8C). Notably, LPS-stimulated between CCL5- and vehicle-treated macrophages are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. 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 macro- macrophages (2000% increase), which further increased (3368%) phages when all forms of apoptotic PMN were used. The only when CCL5 in its bound form was added to the PMN. As we exception to this rule was observed for TNF-a inhibition by reported previously (37), D62/2 senescent PMN were less effective CCL5-bound apoptotic PMN. With regards to IL-10 (Fig. 8B), than their wild-type (WT) counterparts in inhibiting TNF-a and p38 inhibition significantly reduced apoptotic cell–induced, but promoting IL-10 secretion from macrophages. Importantly, the im- not LPS-induced, secretion and abolished the additive effect of pact of bound CCL5 on cytokine secretion by LPS-stimulated bound CCL5. JNK inhibition did not reduce apoptotic cell–in- macrophages was significantly abolished (IL-10 and IL-12) or duced IL-10 secretion but did abolish the additive effect of even reversed (TNF-a)whenitwasappliedtoD62/2 PMN. Inter- bound CCL5. Thus, p38 and JNK signaling seems to be essential estingly, D6 deficiency seems to reduce IL-12 inhibition by soluble for macrophage reprogramming by apoptotic cells, and their CCL5 + senescent PMN, suggesting that even in the presence of action is enhanced when apoptotic PMN are bound with CCL5. excess soluble chemokine, the inhibitory signaling of D6-bound CCR1 and CCR5 mediate macrophage reprogramming induced CCL5 dominates IL-12 regulation. Thus, we have identified a by apoptotic cell–bound CCL5 novel inhibitory/reprogramming mode of action for CCL5 upon binding to D6 on the surface of apoptotic PMN during efferocytosis. It was previously shown that the responses of rMs to apoptotic PMN (including cytokine secretion) are hampered due to D6 de- D6 deficiency hampers JNK and p38MAPK activation in ficiency exclusively on these PMN (37). Therefore, we aimed to macrophages by CCL5-bound apoptotic PMN determine which CCL5-binding chemokine receptor expressed by A major signaling pathway involved in the regulation of cytokine rMs mediates their reprogramming by D6-bound CCL5 upon in- secretion by activated immune cells involves the MAPKs, a family teraction with apoptotic PMN. Our results in Supplemental Fig. 2 of serine/threonine kinases. The four well-characterized subfam- indicate CCR1 and CCR5 are highly expressed in rMs (and resi- ilies of MAPKs are the ERKs (ERK1/2), c-Jun NH2-terminal dent peritoneal macrophages) whereas D6 is expressed at much The Journal of Immunology 1399 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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 one- way ANOVA between treatments are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. lower levels (over 600-fold) in these macrophages. Moreover, both the inhibition of IL-12 secretion from these macrophages under CCR1 and CCR5 antagonists reduced IL-10 secretion by LPS- the same settings (Fig. 8E). Notably, the CCR1 and 5 antagonists stimulated rMs induced by apoptotic PMN, as well as CCL5- did not affect the inhibitory action of apoptotic PMN and CCL5 on bound apoptotic PMN (Fig. 8D). These antagonists also reversed TNF-a secretion (data not shown), hence suggesting additional 1400 A CCL5–D6 AXIS PROMOTES THE RESOLUTION OF INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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), phospho- p38, 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 sumably through the binding, internalization, and lysosomal induced by apoptotic PMN and CCL5. Thus, apoptotic PMN- degradation of its ligands, which consist of most of the proin- bound CCL5 promotes rM reprogramming through the activa- flammatory chemokines (26, 27, 47). In this study, we describe a tion of CCR1 and CCR5 (for illustration, please see Fig. 9). novel role for CCL5 in regulating macrophage reprogramming during the resolution phase of zymosan A–induced peritonitis. Discussion CCL5 was found to be retained at bioactive concentrations (above CCL5 is considered to be a major effector chemokine in the 50 ng/ml, 25% reduction) during the resolution phase of inflam- attraction of leukocytes to inflamed tissues, in particular in mation (24–72 h), whereas all other proinflammatory cytokines peritoneal inflammation (23, 46). D6 was previously found to and chemokines examined were reduced below 10 ng/ml (80–90% play a significant role in the resolution of inflammation, pre- reduction; Fig. 1) at the same period. Moreover, rMs secreted The Journal of Immunology 1401

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 cell- free supernatants were collected and Downloaded from 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 http://www.jimmunol.org/ 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 by guest on September 28, 2021 D62/2 macrophages, different treatments, or relative to LPS-treated macrophages (D and E) are indicated. *p , 0.05, **p , 0.01, ***p , 0.005. AC, apoptotic cells.

CCL5 to the highest extent (2.5 ng/ml) in comparison with in- expression by rMs (39) and anti-inflammatory roles for this che- flammatory cytokines and chemokines, and this secretion was en- mokine (30–32, 50). Notably, CCL5 was induced in an IL-10– hanced by interactions with senescent PMN (Fig. 2). Importantly, dependent manner in anti-inflammatory B cell–trained macrophages CCL5 neutralization reduced IL-10, but not IL-12 production by (51). Most inflammatory chemokines, including CCL5, peak during these macrophages (Fig. 3). Moreover, CCL5 exposure in vivo early peritonitis and significantly decline by 24 h PPI (28, 37, 38). resulted in significantly reduced secretion of the proinflammatory We now show that although CCL2 and CCL3, as well as other cytokines TNF-a and IL-12, but only slightly reduced the anti- inflammatory cytokines, continue to decline during the following inflammatory cytokine IL-10 (Fig. 4). Hence, the reprogramming 48 h of spontaneously resolving inflammation, CCL5 levels remain index, defined by the ratio of LPS-stimulated secretion of IL-10/IL- at a bioactive concentration (Fig. 1). Moreover, our results indicate 12, was increased by CCL5 in WT mice but reduced in D62/2 CCL5 is secreted to the highest extent (relative to both pro- and mice. Of interest, CCL5 did not promote reprogramming ex vivo as anti-inflammatory cytokines and chemokines) by rMs (Fig. 2). This it enhanced IL-10 secretion by rMs, but had a similar impact on secretion is promoted by senescent PMN from D6-deficient mice inflammatory cytokines and chemokines (Fig. 5). Nevertheless, (as well as their WT counterparts) that were previously shown to binding of CCL5 to apoptotic PMN from D6+/+ mice enhanced exert reduced reprogramming of rMs. Hence, the decreased macrophage reprogramming (primarily through the increase in reprogramming of D6-deficient macrophages is not due to re- IL-10), but failed to do so in its soluble form, or when bound to duced CCL5 secretion. Importantly, these findings were supported D62/2 apoptotic PMN (Fig. 6). JNK and p38MAPK activation were by Stables et al. (39). Thus, CCL5 seems to be produced by rMs, enhanced by CCL5-bound apoptotic PMN from D6+/+ mice. Inhi- in particular following their engulfment of apoptotic PMN, and bition of these kinases by specific pharmacological inhibitors at- thereby maintains consistent peritoneal levels during resolution. tenuated the reprogramming of macrophages by apoptotic PMN, The expression of chemokine receptors on apoptotic leukocytes and completely blocked the impact of bound CCL5. was previously reported (28, 37, 52), and the expression of D6 on Although CCL5 has bona fide roles in attracting various immune apoptotic PMN, in particular, was found to have a significant cells to inflammation sites in multiple autoimmune and inflam- impact on macrophages that engulfed these cells. Our findings matory settings (23, 24, 46, 48, 49), several studies have indicated now indicate in vivo exposure to CCL5 during the resolution of 1402 A CCL5–D6 AXIS PROMOTES THE RESOLUTION OF INFLAMMATION Downloaded from http://www.jimmunol.org/

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 by guest on September 28, 2021 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). inflammation significantly abrogated TNF-a and IL-12 secretion CCL5 to D6+/+ apoptotic PMN, but not to their D62/2 coun- by rMs exposed to LPS, whereas IL-10 secretion was slightly terparts, leads to increased reprogramming of macrophages that reduced. Therefore, the reprogramming index of these macro- interacted with these PMN. This change was largely attributed to an phages was significantly increased (Fig. 4). Importantly, D62/2 increase in IL-10 secretion (Fig. 6D). Soluble CCL5 added to ap- macrophages had a reduced reprogramming index under these optotic PMN did not exert such actions. Notably, CCL5 neutrali- settings. Moreover, exposure to CCL5 ex vivo leads to increased zation also resulted in increased numbers of macrophages (Fig. 3), LPS responsiveness in terms of cytokine/chemokine secretion, whereas CCL5 supplementation in D62/2 mice reduced the ab- regardless of the mediator’s properties (Fig. 5), whereas CCL5 normal peritoneal macrophage numbers in these mice during res- neutralization in vivo reduced TNF-a and IL-10 secretion ex vivo olution (Supplemental Fig. 1). These results indicate that soluble (Fig. 3). These findings suggest that during the resolution of CCL5 also plays an important role in diminishing macrophage inflammation in vivo CCL5 acts in two functionally distinct numbers during resolving inflammation, possibly by promoting forms. In concert with D6-expressing apoptotic PMN it promotes their reprogramming and emigration from the peritoneum. macrophage reprogramming. However, in its soluble form it Although D6 is not expressed on Ly-6Chi monocytes (53), which partially promotes macrophage responsiveness to LPS and con- differentiate to form the vast majority of rMs in our model (data tributes to TNF-a production. Notably, both forms promote the not shown), it has been shown to be involved in determining their production of the immunoregulatory cytokine IL-10 by rMs. fate in D6-deficient mice (53). Our results in Supplemental Fig. 2 CCL5-neutralizing Abs seem to block the activity of soluble and Fig. 8 confirm these findings and indicate that CCR1 and CCL5 in vivo, but do not affect the activity of the apoptotic CCR5, rather than D6, are expressed by rMs and mediate IL-10 PMN-bound form, probably due to its localization to PMN- induction and IL-12 inhibition induced by apoptotic PMN alone or macrophage contact sites. Therefore, the impact of these Abs CCL5-bound apoptotic PMN. It remains to be determined which opposes the activity of soluble CCL5 ex vivo (Fig. 5), for the receptors expressed on these macrophages contribute to the inhi- most part. Our results in Fig. 6 show that D6 is instrumental in bition of TNF-a secretion observed under these settings. Thus, the the binding of CCL5 by apoptotic PMN and that binding of results presented in this report support a novel role for a CCL5–D6 The Journal of Immunology 1403 axis during the resolution of inflammation (Fig. 9). CCL5 is re- 12. Patel, V. A., A. Longacre, K. Hsiao, H. Fan, F. Meng, J. E. Mitchell, J. Rauch, leased by rMs and binds D6 on apoptotic PMN. As inflammatory D. S. Ucker, and J. S. Levine. 2006. 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