CCL17 in Inflammation and Pain Kevin M.-C

CCL17 in Inflammation and Pain Kevin M.-C. Lee, Andrew Jarnicki, Adrian Achuthan, Andrew J. Fleetwood, Gary P. Anderson, Christian Ellson, Maria Feeney, Louise K. Modis, Julia E. Smith, John A. This information is current as Hamilton and Andrew Cook of October 2, 2021. J Immunol published online 27 May 2020 http://www.jimmunol.org/content/early/2020/05/26/jimmun ol.2000315 Downloaded from

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 27, 2020, doi:10.4049/jimmunol.2000315 The Journal of Immunology

CCL17 in Inﬂammation and Pain

Kevin M.-C. Lee,* Andrew Jarnicki,† Adrian Achuthan,* Andrew J. Fleetwood,* Gary P. Anderson,† Christian Ellson,‡ Maria Feeney,‡ Louise K. Modis,‡ Julia E. Smith,‡ John A. Hamilton,*,x and Andrew Cook*

It has been reported that a GM-CSF→CCL17 pathway, originally identified in vitro in macrophage lineage populations, is implicated in the control of inflammatory pain, as well as arthritic pain and disease. We explore, in this study and in various inflammation models, the cellular CCL17 expression and its GM-CSF dependence as well as the function of CCL17 in inflammation and pain. This study used models allowing the convenient cell isolation from Ccl17E/+ reporter mice; it also exploited both CCL17-dependent and unique CCL17-driven inflammatory pain and arthritis models, the latter permitting a radiation chimera approach to help identify the CCL17 responding cell type(s) and the mediators downstream of CCL17 in the control of inflammation and pain. We present evidence that 1) in the particular inflammation models studied, CCL17 expression is predominantly

in macrophage lineage populations and is GM-CSF dependent, 2) for its action in arthritic pain and disease development, CCL17 Downloaded from acts on CCR4+ non–bone marrow–derived cells, and 3) for inﬂammatory pain development in which a GM-CSF→CCL17 pathway appears critical, nerve growth factor, CGRP, and substance P all appear to be required. The Journal of Immunology, 2020, 205: 000–000.

ranulocyte-macrophage CSF, originally defined by its We have recently shown that GM-CSF- and IFN-regulatory ability to generate in vitro colonies of granulocytes and factor 4 (IRF4)–dependent signaling can regulate myeloid cell http://www.jimmunol.org/ G macrophages from precursor cells (1), can also act as a numbers and the macrophage phenotype during inflammation proinflammatory cytokine (2–6). In this capacity it can act on (19). Furthermore, we identified the chemokine, CCL17 (TARC), myeloid populations to control their numbers at a site of inflam- as a downstream mediator of GM-CSF in monocytes/macrophages mation, possibly as a prosurvival factor, and/or activate them to and in vivo models, with GM-CSF inducing CCL17, via Jmjd3- modulate function (4, 7). There is a lot of supporting evidence regulated IRF4, to mediate inflammation (19–22). Thus GM-CSF from animal models that GM-CSF is a likely inflammatory ther- joins the list of potential stimuli, for example, IL-4, capable of apeutic target for many conditions (4, 8–16); companies are cur- enhancing CCL17 expression (20). How broadly significant this rently targeting GM-CSF or its receptor, with success being GM-CSF control over CCL17 expression and function is un- by guest on October 2, 2021 recently reported in phase II trials in rheumatoid arthritis (17) and known. Blockade of CCL17 can ameliorate GM-CSF–dependent hand osteoarthritis (OA) (18). inflammatory pain and arthritic pain and disease (20–22). GM-CSF, however, is not downstream of CCL17 (20). Although CCL17 is implicated in the attraction of T cells such as Th2 cells (23) via its *Department of Medicine, The Royal Melbourne Hospital, The University of Mel- receptor, CCR4, we have shown it to be required for T cell–inde- † bourne, Parkville, Victoria 3050, Australia; Department of Pharmacology, The pendent inflammatory arthritis and OA models (20–22), in which University of Melbourne, Parkville, Victoria 3050, Australia; ‡Adaptive Immunity Research Unit, GlaxoSmithKline, Stevenage, Hertfordshire SG1 2NY, United King- it did not appear to be acting as a chemotactic agent, but rather dom; and xAustralian Institute for Musculoskeletal Science, The University of involved in a broader biology. Much more needs to be done to Melbourne and Western Health, St. Albans, Victoria 3021, Australia understand this new GM-CSF–linked CCL17 biology and assess its ORCIDs: 0000-0002-6609-3524 (K.M.-C.L.); 0000-0003-3640-2338 (A.A.); 0000- significance. 0001-6590-2927 (M.F.); 0000-0002-9493-9224 (J.A.H.). In the current study, we therefore sought more information on Received for publication March 23, 2020. Accepted for publication April 28, 2020. GM-CSF–linked CCL17 biology in the context of inflammation This work was supported by a grant from the National Health and Medical Research and pain. More specifically, we sought 1) to define better which Council of Australia (1085240) to J.A.H. and A.C. infiltrating myeloid cell types express CCL17 during inflamma- K.M.-C.L., G.P.A., C.E., M.F., L.K.M., J.E.S., J.A.H., and A.C. conceived the study and designed the studies; K.M.-C.L., A.J., A.A., and A.J.F. conducted the experi- tory reactions and assess the GM-CSF dependence or not of such ments; K.M.-C.L., A.J., and A.C. analyzed the data; K.M.-C.L., J.A.H., and A.C. expression, 2) to identify the CCL17R and the source of the wrote the original draft of the manuscript; and all authors reviewed and edited the CCL17 responsive cells in CCL17-dependent inflammatory pain manuscript. and/or arthritis models, and 3) to identify downstream mediators Address correspondence and reprint requests to Dr. Kevin M.-C. Lee, The University → of Melbourne, Department of Medicine (RMH), The Royal Melbourne Hospital, governing pain regulation by the GM-CSF CCL17 pathway. Parkville, VIC 3050, Australia. E-mail address: [email protected] We provide evidence that in Ag-induced peritonitis (AIP) and The online version of this article contains supplemental material. LPS-induced lung inflammation, CCL17 is expressed predominantly Abbreviations used in this article: AIP, Ag-induced peritonitis; BALF, bronchoalveo- in macrophage lineage populations (macrophages and monocyte- lar lavage fluid; BM, bone marrow; cDC, conventional dendritic cell; COX, cyclo- derived dendritic cells [MoDCs]) in a GM-CSF–dependent manner oxygenase; DC, dendritic cell; GSK, GlaxoSmithKline; i.n., intranasal(ly); i.pl., + intraplantar(ly); IRF4, INF-regulatory factor 4; mBSA, methylated BSA; MHCII, and that CCL17 acts on CCR4 non–bone marrow (BM)–derived cells MHC class II; MoDC, monocyte-derived dendritic cell; NGF, nerve growth factor; for its actions in arthritic pain and disease development; we also report OA, osteoarthritis; RSV, respiratory syncytial virus; WT, wild-type; ZIA, zymosan- that, downstream of the GM-CSF→CCL17 pathway, nerve growth induced arthritis. factor (NGF), CGRP, and substance P all appear to be required for Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 inflammatory pain development.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000315 2 CCL17 IN INFLAMMATION AND PAIN

Materials and Methods incapacitance meter on at least three occasions prior to the com- Mice mencement of the experiment. Three measurements were taken for each time point and averaged. The following mice were used: Ccl17E/+ (a single copy of Ccl17 has been replaced by EGFP) (24), Ccr42/2 (25), and Il62/2 mice (The Walter and Histology Eliza Hall Institute, Parkville, VIC, Australia). All gene-deficient mice At termination, the knee joints were removed, fixed, decalcified, and were backcrossed onto the C57BL/6 background for more than 10 gen- paraffin embedded (20, 28, 32). Frontal sections (5 mm) were stained with erations (The Walter and Eliza Hall Institute). For lung experiments, H&E. For the mBSA/CCL17 model, cellular infiltration, synovitis (syno- C57BL/6 mice were from Animal Resource Centre (Perth, WA, Australia). vial hyperplasia), pannus formation, cartilage damage, and bone erosions Mice were fed standard rodent chow and water ad libitum. Mice of both were scored separately from 0 (normal) to 5 (severe), as described previ- sexes (8–12 wk) were used. ously (20, 28). For ZIA, cell infiltration, proteoglycan loss (Safranin O/fast AIP green stain) and bone erosions were scored separately from 0 (normal) to 3 (severe) as before (20, 31). AIP was as described previously (20, 26, 27). Mice were immunized (day 221) with methylated BSA (mBSA), emulsified in CFA, intradermally in BM chimeras 2 2 2 the base of the tail. A booster injection was given at day 7. On day 0, C57BL/6 wild-type (WT) or Ccr4 / recipient mice received total body mice were challenged i.p. with 200 mg of mBSA, and 4 d later, peritoneal irradiation (two exposures 3 5.5 Gy, 3 h apart). BM cells were harvested exudate cells were harvested. For Ab blockade experiments, mice received from the femurs and tibiae of WT and Ccr42/2 donor mice and injected i.v. 150 mg of anti–GM-CSF mAb (22E9) or isotype control (IgG2a) on days 1 into the irradiated recipient mice (1 3 106 cells). Effective BM reconsti- and 2 after i.p. challenge. tution was determined 6 wk later (by flow cytometry using Abs against the different congenic CD45 allotypes [Ly5.1 and Ly5.2]) where possible; only LPS-induced lung inflammation 2 2 Ly5.2 Ccr4 / mice were available. Downloaded from Mice were lightly anesthetized with isoflurane (3% in O2), challenged intranasally (i.n.) with 10 mg of LPS (Escherichia coli serotype 026:B6; Inflammatory pain models Sigma-Aldrich) in 40 ml of PBS (26), and 3 d later, bronchoalveolar lavage Inflammatory pain was induced by intraplantar (i.pl.) injection (10 ml) of fluid (BALF) cells were harvested. For Ab blockade experiments, mice either mouse CCL17 (50 or 100 ng; BioLegend) (20, 21), mouse CCL22 received 100 mg of either anti–GM-CSF mAb (22E9) or isotype control (50, 100, and 200 ng; PeproTech), zymosan (100 mg; Sigma-Aldrich), (IgG2a) i.n. 30 min prior to LPS challenge. mouse GM-CSF (50 ng; PeproTech), or saline into the left hind Cell population analysis footpad. NGF activity was blockedusingeitheranti-NGFmAb(from GlaxoSmithKline [GSK]) or anti-NGF mAb (clone Ig 30, Absolute http://www.jimmunol.org/ AIP cells were collected by lavage with 5 ml of cold PBS, followed by Antibody) (10 mg per paw) given i.pl. at the same time as the stimulus. As washing and cell counting as previously descried (27), then analyzed by isotype controls, anti–respiratory syncytial virus (anti-RSV) mAb (GSK) and flow cytometry. Fc receptors on cells were blocked with normal mouse IgG2a, respectively, were used. CGRP and substance P activities were serum (1/4 dilution) and stained with fluorochrome-conjugated mAbs blocked using the CGRP antagonist, CGRP8-37 (0.1 mM in 10 ml given i.pl. specific for mouse allophycocyanin-conjugated CD115 (AFS98), allo- 10 min prior to the stimulus; Tocris Bioscience) and the neurokinin 1 re- phycocyanin-Cy7–conjugated CD11b (M1/70), PE-Cy7–conjugated ceptor antagonist SR140333 (25 mg per paw in 10 ml given i.pl. at the same Ly6G (1A8V500-conjugated I-A/I-E (M5/114.15.2), PE-Cy7–conjugated time as the stimulus; Tocris), respectively. CD11c (N418), and the corresponding isotype controls, all either from BD Biosciences or BioLegend. Cells were analyzed using CytoFlex Flow Statistics Cytometer (Beckman Coulter). For cell counts and proportions, an unpaired Student t test was used; for by guest on October 2, 2021 For BALF cells, mice were killed with a s.c. injection of pentobarbitone pain readings, a two-way ANOVA was used; and for histologic scores, (200 mg/kg) and bronchoalveolar lavage was performed by tracheotomy the Mann–Whitney two-sample rank test or two-way ANOVA was used proximal to the larynx. Mice were lavaged in situ with a 400-ml aliquot of (Prism, version 8.0.2; GraphPad, San Diego, CA). A Bonferroni post hoc test PBS, followed by three 300-ml aliquots of PBS. BALF cells were washed, was used when appropriate. Data were plotted as mean 6 SEM with signi- Fc nonspecific binding–blocked with anti-mouse CD16/CD32, and stained ficance p values as indicated. A p value ,0.05 was considered significant. with fluorochrome-conjugated mAbs specific for mouse allophycocyanin/ Cy7–conjugated Ly6G, allophycocyanin-conjugated CD11b, PE/Cy7– Study approval conjugated CD11c, Pacific Blue–conjugated MHC class II (MHCII), PE-conjugated Siglec F, and the corresponding isotype controls either All experiments were approved by The University of Melbourne Animal from BD Biosciences or BioLegend. Cells were detected using a BD Ethics Committee and the GSK Policy on the Care, Welfare and Treatment LSRFortessa FACS machine and analyzed using FlowJo v5.0 software of Animals. (BD Biosciences). Results mBSA-induced arthritis models GM-CSF is important for myeloid cell CCL17 expression Monoarticular arthritis was induced as before (20, 28, 29) by intra-articular during an inflammatory response injection of 100 mg of mBSA in 10 ml of saline into the right knee on day We have previously shown that a blockade of GM-CSF leads to 0, the left knee being injected with saline, followed by a s.c. injection in the scruff of the neck on days 0–2 of mouse CCL17 (600 ng; R&D fewer myeloid cells in the peritoneal cavity during AIP (26, 33) and Systems) or saline. Mice were sacrificed (day 7), and knee joints were in the bronchoalveolar lavage fluid (BALF) after i.n. LPS ad- collected for histology. ministration (26). We have also shown that in AIP, Ccl17 ex- Zymosan-induced arthritis model pression is lower in total peritoneal cells following GM-CSF blockade and, using CCL17/EGFP reporter (Ccl17E/+) mice, that For the induction of the zymosan-induced arthritis (ZIA) model (20, 21, 30, CCL17/EGFP expression is detected predominantly in the CD115 m 31), mice were injected with 300 g of sonicated zymosan (Sigma- (CSF-1R)+ inflammatory macrophage lineage populations (20). Aldrich) in a 10-ml volume into the left knee joint, whereas the contra- lateral knee received saline as a control. On day 7, arthritic joints were From these studies, it was unclear whether the lower CCL17 ex- collected for histologic analysis. pression in total exudate cells seen following GM-CSF blockade is due simply to there being fewer cells that express CCL17 or Pain reading whether GM-CSF actually regulates cellular CCL17 expression As an indicator of pain-related behavior (in this study referred to as (20). We therefore examined the effect of neutralizing anti–GM- pain), the differential distribution of weight over a 3 s period between CSF mAb administration in Ccl17E/+ mice, once again using the the inflamed limb or paw relative to the noninflamed limb or paw was measured using an incapacitance meter (IITC Life Science). This AIP and LPS-induced lung inflammation models; these are convenient technique has been validated for measurement of both arthritic knee models in which it is possible to lavage large numbers of exudate cells and footpad pain (20, 22, 27, 30). Mice were acclimatized to the on which to perform FACS analysis. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. GM-CSF is important for CCL17 expression during AIP. AIP was induced in Ccl17E/+ miceonday0,followedbyi.p.treatmentwith isotypecontroloranti–GM-CSFmAbondays1and2(seeMaterials and Methods). Peritoneal exudate cells were harvested on day 4 for analysis. (A) Representative FACS plots showing the gating strategy to deﬁne monocytes/macrophages, MoDCs, cDCs, and neutrophils. (B)NumberofAIP cells. (C) Representative FACS plots showing CCL17/EGFP+ macrophages and MoDCs. Numbers indicate the frequency of cells within the indicated gates. (D) The frequencies (percentage) of CCL17/EGFP+ cells in AIP. (E) Number of CCL17/EGFP+ cells in AIP. Data are mean 6 SEM (n = 4–5 mice per group). p values were obtained using a Student t test. *p , 0.05, **p , 0.01, ***p , 0.001, anti-GM-CSF mAb versus isotype. N.D., not detected.

AIP. Notwithstanding the challenges faced in defining populations MHCII+ macrophages (R2) and MoDCs (R3) (Fig. 1B). Fig. 1C categorically in the mononuclear phagocyte system (26), peritoneal shows representative FACS plots of CCL17/EGFP expression exudate myeloid cell populations in the AIP model in Ccl17E/+ in MHCII+ macrophages and MoDCs in AIP (day 4) following mice were identified at day 4, once again using the following markers isotype or anti–GM-CSF mAb treatment. In the isotype control (27): CD115+CD11b+ mononuclear phagocyte system populations group, MoDCs (R3) and cDCs (R4) showed the greatest per- (CD11c2MHCII2 monocytes [R1], CD11c2MHCII+ macrophages centage of CCL17/EGFP+ cells, whereas MHCII2 monocytes [R2], CD11c+MHCII+ MoDCs [R3]), CD11b2CD11c+MHCII+ (R1) did not express CCL17/EGFP (Fig. 1D); following anti– conventional dendritic cells (cDCs) (R4), and CD1152CD11b+Ly6G+ GM-CSF mAb treatment, MHCII+ macrophages, MoDCs, and neutrophils (R5) (Fig. 1A). Note that, as previously (26), the CD11c2 neutrophils all showed a reduction in the percentage of CCL17/ MHCII2 (R1) population in the inflamed peritoneal cavity is referred EGFP+ cells, with cDCs showing a trend (Fig. 1D). Thus, to as monocytes based on their morphology and their being pre- even when there was no decrease in the cell number for an dominantly Ly6C+. individual cell type, a decrease in the proportion of CCL17 As before (26, 33), anti–GM-CSF mAb treatment led to expressing cells was evident. This resulted in there being fewer total peritoneal exudate cells on day 4 of AIP compared fewer CCL17/EGFP+ cells in each positive population fol- with isotype treatment (Fig. 1B); in particular, there were fewer lowing anti–GM-CSF mAb treatment (Fig. 1E), indicating that 4 CCL17 IN INFLAMMATION AND PAIN

GM-CSF is an important stimulus for CCL17 expression in this then 3 d post-i.n. LPS. BALF cell populations were identiﬁed using model. the following markers (34): Ly6G+ neutrophils, SiglecF+CD11chi al- 2 lo hi 2/lo LPS-induced lung inﬂammation. We have previously shown that veolar macrophages, SiglecF CD11c CD11b MHCII monocytes, 2 GM-CSF blockade reduced the number of BALF exudate macro- and SiglecF CD11cloCD11bhiMHCIIhi DCs (Supplemental Fig. 1A). phages, monocytes, and neutrophils appearing after i.n. LPS ad- At baseline, there was no CCL17/EGFP detected in BALF cells ministration (26). In order to determine, in this model, which cells (data not shown). As found before (35), 3 d post-i.n. LPS, there was an express CCL17 and, again, whether their expression was GM-CSF increase in the total number of BALF cells (2.2 6 0.1 versus 43.2 6 dependent, we analyzed BALF cells initially from naive lungs, and 1.7 3 105 cells per milliliter, saline versus LPS, respectively) with Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. CCR4 is required for the regulation by CCL17 of arthritic pain and optimal disease development. (A and B) mBSA/CCL17 arthritis (intra- articular mBSA and s.c. CCL17 or saline [days 0–2]) was induced in WT and Ccr42/2 mice. (C and D) ZIA was induced in WT and Ccr42/2 mice. (A and C) Change in weight distribution (pain) over time. (B and D) Representative histologic pictures of knee joints [H&E (B and D) and Safranin O/fast green stain (D), original magnification 3100] and quantification of arthritis and proteoglycan loss at day 7. Arrows indicate the following features, respectively: solid arrows, cellular infiltration; dotted arrows, proteoglycan loss. Data are mean 6 SEM (n = 10 mice per group). p values were obtained using a two-way ANOVA test for pain (weight distribution) and histology. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, mBSA/saline versus mBSA/CCL17 for WT mice. #p , 0.05, ###p , 0.001, ####p , 0.0001, WT versus Ccr42/2 mice. F, femur; P, patella; T, tibia. The Journal of Immunology 5

7.0 6 0.5% of neutrophils, 50.5 6 14.2% of alveolar macrophages, requirement of CCR4, a high affinity receptor for CCL17, for 3.9 6 0.4% of monocytes, and 35.2 6 8.8% of DCs now expressing inflammatory arthritic pain and disease development. To determine CCL17/EGFP. As before (26), blockade of GM-CSF 30 min prior to this, we used Ccr42/2 mice in the CCL17-driven (mBSA/CCL17) i.n. LPS led to a reduction in the total number of BALF cells com- arthritis model and in the CCL17-dependent ZIA model, both pared with isotype-treated mice at day 3, with a significant reduction being lymphocyte independent (20, 21). in the number of alveolar macrophages (R2) and a trend toward fewer mBSA/CCL17 arthritis. To explore whether CCL17 might be acting neutrophils (R1) (Supplemental Fig. 1B). Interestingly, for monocytes via CCR4 in its control of arthritic pain and disease, we again used and DCs (R3 and R4, respectively) there was no change in cell our novel mBSA/CCL17 arthritis model, which is driven by ex- number. Alveolar macrophages, monocytes, and neutrophils, how- ogenous CCL17 (20). Although administration of exogenous + ever, had a significant reduction in their number of CCL17/EGFP systemic cytokine may not necessarily inform about the role of cells following GM-CSF blockade (Supplemental Fig. 1C). As seen the endogenous molecule (4, 7, 36), it is a convenient approach for the AIP model above, these findings equated to a decrease in the to explore potential pathways downstream of a particular cyto- percentage of cells in each population expressing CCL17/EGFP kine, for example, CCL17 (20, 21). Following the induction of following GM-CSF blockade ([shown for alveolar macro- mBSA/CCL17 in WT mice, pain (incapacitance meter) devel- phages (Supplemental Fig. 1D)]. Once again, these data suggest oped by day 5 (Fig. 2A), as before (20, 21); however, arthritic that GM-CSF can be an important stimulus for CCL17 expression pain was not seen in Ccr42/2 mice (Fig. 2A). In addition, Ccr42/2 in inflammatory myeloid cells during an inflammatory reaction. mice were protected from mBSA/CCL17–driven arthritis development (Fig. 2B). CCR4 is required for the regulation by CCL17 of arthritic pain

ZIA. We previously showed that ZIA pain and optimal disease Downloaded from and optimal disease development development require both endogenous CCL17 and GM-CSF (20). Having identiﬁed which cells are capable of producing CCL17 during We now report that whereas WT mice developed ZIA pain (day 1) the inﬂammatory reactions studied above, we next investigated the and disease (day 7) as before (20, 21, 30), Ccr42/2 mice did not http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. CCR4+ non-BM-derived cells are required for CCL17-driven arthritic pain and disease. (A and B)WT(A)orCcr42/2 (B) BM cells were adoptively transferred into irradiated WT or Ccr42/2 mice, followed by the induction of mBSA/CCL17 arthritis model. (A and B) Change in weight distribution (pain) over time. Representative histologic pictures of knee joints (H&E, original magnification 3100) and quantification of arthritis at day 7. Solid arrows indicate cellular infiltration. Data are mean 6 SEM (n = 8–10 mice per group). p values were obtained using a two-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, mBSA/saline versus mBSA/CCL17 for WT mice, WT→WT mice and Ccr42/2→WT mice. ##p , 0.01, ###p , 0.001, ####p , 0.0001, mBSA/CCL17 for WT versus Ccr42/2 mice, WT→WT versus WT→Ccr42/2 mice and Ccr42/2→WT versus Ccr42/2→Ccr42/2 mice. F, femur; P, patella. 6 CCL17 IN INFLAMMATION AND PAIN develop pain (Fig. 2C); this readout is a highly relevant measure of arthritic pain in a single knee joint in arthritis models (20, 22, 28, 32, 37). Ccr42/2 mice also had less synovial hyperplasia and proteoglycan loss (Fig. 2D). Thus, CCR4 is required for arthritic pain and optimal disease development when CCL17 is given exogenously or acting endogenously. CCR4+ non-BM-derived cells are required for CCL17-driven arthritic pain and disease development CCR4 is expressed in various hemopoietic and nonhemopoietic cell types (38, 39). Having shown above that CCL17-driven arthritic pain and disease require CCR4, we next determined, using a BM chimera approach, whether CCR4+ BM or non-BM-derived cells were required in this model. As expected, WT chimeras (WT→WT) developed mBSA/CCL17 arthritic pain and disease (Fig. 3A), whereas transfer of WT BM cells to Ccr42/2 mice (WT→Ccr42/2) resulted in no mBSA/CCL17 arthritic pain or disease (Fig. 3A). By contrast, Ccr42/2 chimeras (Ccr42/2→Ccr42/2) did not develop mBSA/CCL17 arthritic pain or disease (Fig. 3B), whereas transfer of Ccr42/2 BM to WT mice Downloaded from (Ccr42/2→WT) led to the development of arthritic pain and disease (Fig. 3B). These data suggest that non-BM-derived CCR4+ cells are required for mBSA/CCL17 arthritic pain and disease development. CCL17, but not CCL22, induces inflammatory pain via CCR4

As for the mBSA/CCL17 model, we have previously established a http://www.jimmunol.org/ cyclooxygenase (COX)-dependent CCL17-driven inflammatory pain model in the mouse paw to explore potential pathways FIGURE 4. CCL17, but not CCL22, induces inflammatory pain via downstream of CCL17 (20, 21). We therefore used this model to 2 2 CCR4. (A) i.pl. injection of CCL17 (50 ng) or saline in WT and Ccr4 / determine the requirement for CCR4 for CCL17-driven inflam- mice. (B) i.pl. injection of saline, CCL17 (50 ng) and different doses of matory pain. As before (20, 21), i.pl. injection of CCL17 led to CCL22 (50, 100, and 200 ng) in WT mice. (A and B) Change in weight 2/2 pain at 6 h in WT mice (Fig. 4A); however, Ccr4 mice did not distribution (pain) over time. Data are mean 6 SEM [n = 10 mice per develop pain. Because CCL22 is another ligand for CCR4, albeit group (A), n = 5 mice per group (B)]. p values were obtained using a two- with different responses (23), we tested whether it was also able to way ANOVA. *p , 0.05, ***p , 0.001, saline versus CCL17. ##p , 0.01, induce inflammatory pain. Interestingly, i.pl. CCL22 did not in- WT CCL17 versus Ccr42/2 CCL17. by guest on October 2, 2021 duce inflammatory pain at the doses tested (50–200 ng) (Fig. 4B).

NGF, CGRP, and substance P are required for the regulation by by an incapacitance meter at 4 h in the isotype control-treated CCL17 of inflammatory pain mice (Fig. 5B). Even with this higher CCL17 dose, mice dis- We have previously reported that the components of the GM- played no detectable pain in the presence of this anti-NGF mAb CSF→CCL17 pathway are required for zymosan-induced in- (Fig. 5B). Following zymosan or GM-CSF administration, anti- flammatory pain (20). This widely used i.pl. model is also COX NGF mAb–treated mice again had no detectable pain similar to dependent (20). We have developed, in the mouse, a GM-CSF- saline-treated mice (Fig. 5C, 5D). driven inflammation model that, as for CCL17-driven inflam- CGRP dependence. Mice were treated with CGRP8–37 [CGRP matory pain model discussed above, allows the exploration of antagonist (41), 0.1 mM in 10 ml per paw] or vehicle 10 min prior potential pathways downstream of GM-CSF in this case (20). Like to i.pl. injection of CCL17, zymosan, or GM-CSF (Fig. 6A–C). CCL17-driven inflammatory pain, the GM-CSF–driven pain in the CGRP8–37–treated mice, in comparison with vehicle-treated mice, mouse paw is also COX dependent (20). The neurotrophin, NGF, also prevented the pain induced by each of the stimuli (Fig. 6A–C, and the neuropeptides CGRP and substance P, are important me- respectively). diators in pain development, including in human subjects (40). We Substance P dependence. Mice were treated with the neurokinin 1 therefore explored whether CCL17-driven inflammatory pain was receptor antagonist SR140333 [i.e., substance P receptor antagonist dependent on NGF, CGRP, and substance P; likewise, we explored (42), 25 mg per paw], or vehicle at the same time as i.pl. CCL17, whether the same mediators were required for zymosan- and GM- zymosan, or GM-CSF was administered. Blockade of substance CSF–driven inflammatory pain. P action also prevented the pain induced by the three stimuli NGF dependence. Mice were treated with anti-NGF mAb (10 mg (Fig. 6D–F, respectively). per paw) or isotype control at the same time as i.pl. CCL17, These data suggest that all three pain mediators are required for zymosan, or GM-CSF was administered. Upon control (anti-RSV) the induction of the inflammatory pain dependent on the GM- mAb treatment, mice injected with each of the stimuli developed CSF→CCL17 pathway (see Discussion). pain by 4–6 h, as expected (20) (Fig. 5A–C, respectively). Fol- lowing CCL17 injection, anti-NGF mAb–treated mice showed a Discussion reduction in the degree of pain in this experiment (Fig. 5A). Given We have previously demonstrated that a GM-CSF→CCL17 pathway, this last result, CCL17-driven pain was also examined in the originally discovered in vitro in monocytes/macrophages, is required presence of a different anti-NGF mAb (clone Ig 30). A higher dose for the development of inflammatory and arthritic pain (20). To of CCL17 (100 ng) was also used so pain was clearly measurable address the main questions outlined in the Introduction around 1) The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. NGF is required for CCL17-, zymosan-, and GM-CSF–driven inﬂammatory pain. i.pl. injection of (A) CCL17 (50 ng), (B) CCL17 (100 ng), (C) zymosan (100 mg), or (D) GM-CSF (50 ng) in WT mice. (A, C, and D) Anti-RSV mAb or anti-NGF mAb (GSK) was injected i.pl. at the same time as the various stimuli; saline was used as a control stimulus. (B) Isotype or anti-NGF mAb (clone Ig 30) was injected i.pl. at the same time as the CCL17 stimulus (100 ng). Change in weight distribution (pain) over time. Data are mean 6 SEM [n = 10 mice per group (A–C), n = 5 mice per group (D)]. p values were obtained using a two-way ANOVA. *p , 0.05, **p , 0.01, ****p , 0.0001, anti-RSV versus anti-NGF; isotype versus anti-NGF mAb (clone Ig 30). #p , 0.05, ####p , 0.0001, saline versus anti-RSV mAb. $$$p , 0.001, saline versus anti-NGF mAb. by guest on October 2, 2021

GM-CSF–dependent CCL17 cellular expression, 2) the identity of study, following induction of an inflammatory reaction in both the the CCL17R, and 3) downstream mediators governing CCL17 peritoneal cavity and lung, CCL17 was expressed predominantly action in its regulation of inflammation and pain, a number of in macrophage lineage populations and cDCs. Blockade of convenient inflammation models were used. This range allowed GM-CSF led to fewer macrophage lineage cells present in the us to begin to assess the generality of our findings by studying inflammatory exudates, as previously reported (20, 26, 33, 35). different sites of inflammation, as well as to continue to explore Interestingly, the reduction in the proportion of CCL17/EGFP+ the role of the GM-CSF→CCL17 pathway in inflammation and cells in a particular population was greater than any reduction in pain (20–22). In the current study, we have found CCL17 to be its number and was even reduced in cell populations in which the expressed predominantly in macrophage lineage populations cell number was not altered by GM-CSF blockade. These data sug- during inflammatory responses in the peritoneum and lung, and gest that during anti–GM-CSF mAb therapy for autoimmune/ we found this expression to be GM-CSF regulated. By using inflammatory reactions, it is easier to reverse the GM-CSF ef- CCL17-driven pain and arthritis models and a CCL17-dependent fects on cell function (e.g., CCL17 expression) than on their arthritis model (ZIA), we found that CCR4 was required for pain numbers with potential implications for the dosing and type of and disease development; for CCL17-driven arthritic pain and responses to such therapy. These results confirm and extend our disease progression, the required CCR4+ cells were non-BM- previous findings showing GM-CSF is an upstream regulator of derived. Finally, using the CCL17-, GM-CSF–, and zymosan- CCL17 in several inflammatory models (19, 20). driven inflammatory pain models, we provided evidence that In both our CCL17-driven pain and arthritis models and in a common pain mediators, namely NGF, CGRP, and substance P, CCL17-dependent arthritis model (ZIA), we found that CCR4 was were all needed for pain induction. It should be noted that al- required, and thus supporting the evidence that CCL17 can signal though the measurement of changes in weight distribution of the via CCR4 (43) and in this sense validating our overall approach hind limbs as an indicator of arthritic pain is a highly relevant and with the various models. Using a BM chimera strategy, we further well-established method for measuring pain in single arthritic showed that non-BM-derived CCR4+ cells are required for the knee joint (20, 22, 28, 32, 37), we acknowledge this does not fully development of CCL17-driven arthritic pain and disease. CCL17 capture the pain phenotype. was originally defined as a T cell chemokine (24, 44), but these We have earlier shown in vitro that GM-CSF–stimulated data argue against migrating T cells being the responding pop- monocytes and macrophages secrete CCL17, a response that is ulation, consistent with our prior observation that the mBSA/ IRF4-dependent (20), and during an inflammatory response CCL17 arthritis model does not require lymphocytes (20). The in vivo, that macrophages and MoDCs are the main cell types, chimera data pointing to a non-BM-derived–responding pop- along with cDCs, that express CCL17 (19, 20). In the current ulation(s) are also consistent with our earlier observation that 8 CCL17 IN INFLAMMATION AND PAIN Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. CGRP and substance P are required for CCL17-, zymosan-, and GM-CSF–driven inflammatory pain. i.pl. injection of (A and D) CCL17 (50 ng), (B and E) zymosan (100 mg), or (C and F) GM-CSF (50 ng) in WT mice. (A–C) Vehicle or CGRP8–37 was injected i.pl. 10 min prior to stimulus. (D–F) Vehicle or SR140333 was injected i.pl. at the same time. Change in weight distribution (pain) over time. Data are mean 6 SEM [n = 10 mice per group (A), n = 5 mice per group (B–F)]. p values were obtained using a two-way ANOVA. *p , 0.05, ***p , 0.001, ****p , 0.0001, vehicle versus CGRP8–37 or SR140333. cellular infiltration (synovitis) in the collagenase-induced OA peritoneal inflammation model that macrophages switch to a pro- model was not affected by an absence of CCL17 (22); in contrast, fibrotic phenotype and secrete CCL17, thereby activating fibroblasts joint pain and late stage joint damage were reduced. Also, its (46). In support of a role for CCL17 in fibrosis, neutralization of absence did not modulate zymosan-induced peritonitis (19). CCL17, but not CCL22, led to a reduction in bleomycin-induced In addition to T cells, hemopoietic cells such as platelets, NK pulmonary fibrosis (47). CCR4 has also been found to be cells, monocytes, macrophages, and DCs have been reported to expressed in keratinocytes (48). Both CCR4 (49) and the GM- express CCR4 (45). The non-BM source of the CCR4+ responsive CSFR (50, 51) have been reported to be expressed in nocicep- cells suggests that it is of nonhemopoietic origin. In line with our tive neurons, although recent RNA sequencing data for their chimera result, we previously found CCR4 mRNA to be widely expression in purified murine dorsal root ganglion neurons suggest expressed in synovial cells in the collagenase-induced OA model, otherwise (52–54). Nonsteroidal anti-inflammatory drug sensitiv- with a predominance in non-BM-derived fibroblasts and endothelial ity of both GM-CSF– and CCL17-driven arthritic pain suggests cells (22). Interestingly, it was recently shown in an injury-driven an indirect action of both CCL17 and GM-CSF on neurons (20). The Journal of Immunology 9

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Supplementary Figure 1. GM-CSF is important for CCL17 expression during LPS-induced lung inflammation. LPS-induced lung inflammation was induced in Ccl17E/+ mice treated with isotype or anti-GM-CSF mAb (t= -30 mins) and BALF cells were analysed on day 3 (see Materials and Methods). (A) Representative FACS plots showing the gating strategy to define BALF neutrophils, alveolar macrophages, monocytes and DCs. (B) Number of BALF cells. (C) Number of CCL17/EGFP+ cells. (D) Representative FACS plots showing CCL17/EGFP+ alveolar macrophages. Numbers indicate the frequency of cells within the indicated gates. Data are mean ± SEM; n=3-5 mice/group. P values were obtained using a Student’s t-test. *p<0.05, isotype vs. anti-GM-CSF mAb