Enforced Expression of Hoxa3 Inhibits Classical and Promotes Alternative

Enforced Expression of Hoxa3 Inhibits Classical and Promotes Alternative Activation of Macrophages In Vitro and In Vivo This information is current as of September 26, 2021. Hadeel Al Sadoun, Matthew Burgess, Kathryn E. Hentges and Kimberly A. Mace J Immunol 2016; 197:872-884; Prepublished online 24 June 2016; doi: 10.4049/jimmunol.1501944 Downloaded from http://www.jimmunol.org/content/197/3/872

Supplementary http://www.jimmunol.org/content/suppl/2016/06/23/jimmunol.150194 http://www.jimmunol.org/ Material 4.DCSupplemental References This article cites 65 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/197/3/872.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 © 2016 The Authors All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Enforced Expression of Hoxa3 Inhibits Classical and Promotes Alternative Activation of Macrophages In Vitro and In Vivo

Hadeel Al Sadoun, Matthew Burgess, Kathryn E. Hentges, and Kimberly A. Mace

The regulated differentiation of macrophages (mws) and their subsequent activation into proinflammatory or prohealing subtypes is critical for efficient wound healing. Chronic wounds such as diabetic (db) ulcers are associated with dysregulation of macrophage function. Whereas non-db mws polarize to an M2-like, prohealing phenotype during the late stages of healing, db-derived mws continue to display an M1-like, proinflammatory, or a mixed M1-like/M2-like phenotype. We have previously shown that sustained expression of Hoxa3 reduces the excessive number of leukocytes within the db wound; however, the effect of Hoxa3 on mw polarization was unknown. In this study, we show that Hoxa3 protein transduction of mws in vitro enhances macrophage Downloaded from maturation, inhibits M1 polarization, and promotes M2 polarization, in part via regulation of Pu.1/Spi1 and Stat6. Sustained expression of Hoxa3 in vivo in db wounds reduces the number of Nos2+ (M1-like) mws, increases the number of Arg1+ and VEGF+ (M2-like) mws, and accelerates healing in a DNA-binding independent manner. Our findings suggest a role for Hox protein activity in promoting M1-to-M2-like phenotypic switching via interactions with myeloid transcription factors and provide insight into mechanisms regulating this process in db wound healing. The Journal of Immunology, 2016, 197: 872–884. http://www.jimmunol.org/ he number of patients suffering from chronic wounds is are not sufficient to control chronic inflammation, and only a mild nearing epidemic proportions. Patients with both type 1 improvement in wound healing has been observed in the clinic. T and type 2 diabetes have impaired wound healing, and Therefore, a stronger focus on reprogramming cell behavior in the 15% of these patients go on to develop chronic, nonhealing wound, in particular the therapeutic manipulation of macrophages wounds, 84% of which result in lower limb amputation (reviewed (mws), could be an effective strategy. in Ref. 1). Current treatments are not effective because of a poor mws are involved in all phases of wound repair and regeneration understanding of the mechanisms underlying chronic wounds; (9, 10). During the early stages of healing, proinflammatory (M1- thus, they are the leading cause of nontraumatic amputations in the like) mws dominate the wound environment, where they are in- developed world today (2, 3). During the past two decades, the volved in killing pathogens and removing debris. As the wound by guest on September 26, 2021 role of dysregulated inflammation in impaired healing associated progresses to late stages of healing, most mws acquire the pro- with aging and diabetes has become apparent. This is due in part healing (M2-like) state that is critical for healing progression (11– to increased and prolonged expression of proinflammatory cyto- 13). In mice, the M2-like state is characterized by high levels of kines and chemokines, such as TNF and IL-1 (4) as well as a de- arginase production (11, 14). M2 mws can be generated in vitro by crease in growth factors such as fibroblast growth factors, TGF-b, the stimulation of the IL-4R and/or IL-13R, which signal via the platelet-derived growth factor, and vascular endothelial growth phosphorylation of Stat6 (15–17). This pathway is also important factor (VEGF), which normally function to promote new tissue to wound healing in vivo (18). However, the transition of proin- growth and may also act as anti-inflammatory factors (5–8). flammatory mws to prohealing mws is inhibited by pathological However, therapies targeted at manipulating these factors alone environments, such as aging (19) or diabetes. In addition, diabetes affects the process of how progenitor cells mature in the bone marrow (BM), how myeloid cells infiltrate the wound tissue, and Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United how mws respond to signals from the environment (13, 20, 21). Kingdom Mws from diabetic (db) mice continue to display these defects ORCIDs: 0000-0001-9666-6787 (M.B.); 0000-0001-8917-3765 (K.E.H.); 0000- 0002-3184-878X (K.A.M.). even when cultured outside the db environment, showing an ab- Received for publication September 2, 2015. Accepted for publication May 23, 2016. errant polarization response to both pro- and anti-inflammatory stimuli (12, 13, 22–24). Extrinsic signals coming from local This work was supported by the Saudi Arabian Cultural Bureau (to H.A.S.), the Medical Research Council (to M.B. and K.A.M.), and the Healing Foundation (to cells and Th2 lymphocytes, including IL-4, TGF-b, and IL-10 are K.A.M.). insufficient to induce the phenotypic switch from M1-like to M2- Address correspondence and reprint requests to Dr. Kimberly A. Mace, Healing like mws (13, 22). This deficit in reparative mws is associated with Foundation Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K. E-mail address: [email protected] decreased angiogenesis, extracellular matrix remodeling and wound contraction (reviewed in Ref. 25). Therefore, there is a The online version of this article contains supplemental material. need to identify factors that can stimulate the phenotypic switch of Abbreviations used in this article: AA, alternatively activated; BM, bone marrow; BMDM, BM-derived mw; CM, conditioned medium; db, diabetic; eGFP, enhanced mws within the wound from a proinflammatory to a prohealing GFP; mw, macrophage; MFI, mean fluorescence intensity; NA, nonactivated; qRT- phenotype to promote resolution of inflammation and wound PCR, quantitative RT-PCR; VEGF, vascular endothelial growth factor. healing. This is an open-access article distributed under the terms of the CC-BY 3.0 Unported Enforced expression of Hoxa3 has been shown to promote license. resolution of inflammation and wound healing in db wounds (21, Copyright Ó 2016 The Authors 0022-1767/16 26). Hoxa3 is a member of the homeobox gene family which www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501944 The Journal of Immunology 873 encode master regulator transcription factors that specify seg- Overexpression of Hoxa3 in mouse mws via protein mental identity along the anterio-posterior axis (27). In addition, transduction many Hox genes are expressed in early hematopoietic progenitors The HEK293T cell line (American Type Culture Collection) was maintained where they direct lineage commitment, cell proliferation, and in DMEM supplemented with 10% FBS and 13 penicillin/streptomycin. At self-renewal (28–30). Hox proteins can be delivered to cells di- 20% confluency, cells were transfected with 10 mg SP-Hoxa3-mCherry (SP- rectly via their capacity to cross-biological membranes in a re- Hoxa3mCh) or SP-mCherry expression plasmids using the calcium phosphate ceptor- and energy-independent manner (31, 32). Importantly, method (39), and live imaging was used to monitor transfection efficiency. Conditioned medium (CM) from the transfected cells was collected at 24, protein transduction of Hox proteins is a potentially safer method 48, and 72 h posttransfection, filtered via a 0.45-mm filter, and used to than gene transfer. For example, Hoxb4 protein transduction can supplement mouse mws in culture at a 1:1 volume with mw medium on days promote hemopoietic stem cell self-renewal without inducing 5 and 6 of differentiation. Hoxa3mCh in the medium entered mws via passive leukemia (33). translocation, as previously described in Refs. 31–34. Enforced Hoxa3 expression in hematopoietic progenitors was Detection of Hoxa3-mCherry protein in CM and mouse mws shown to promote their differentiation into Gr1+CD11b+ cells, a Lysates from mws treated with Hoxa3mCh or mCherry CM were prepared proangiogenic myeloid population, and rescue their diabetes- using IP lysis buffer (50 mM Tris-acetate [pH 7.5], 300 mM NaCl, 1 mg/ml induced defects (34). However, the specific effects of enforced BSA, 2% Igepal, 1 mM EDTA, 13 protease inhibitor mixture [Promega], Hoxa3onmws were unknown. and 13 sodium orthovanadate). Two micrograms of rabbit anti-mCherry We found that Hoxa3 protein transduction had an effect on both (BioVision) was labeled with biotin (DSB-X; Molecular Probes) and in- mw maturation and activation and attenuated the inflammatory cubated with 50 mg protein lysate, together with 0.4 mg prewashed Dynabeads (FlowComp Flexi system; Invitrogen). The mixture was left phenotype of mws, possibly via regulation of Spi-1/Pu.1, an Ets overnight at 4˚C on a rotator before washing and elution with immuno- Downloaded from family transcription factor that is required for the generation of precipitation wash buffer (0.1% SDS, 500 mM Tris-acetate [pH 7.5], mature mws (35–38). Our data show that Hoxa3 inhibits M1-like/ 300 mM NaCl, and 0.5% Igepal) using a magnet (EasySep; StemCell proinflammatory gene expression and induces expression of Technologies). CM from 293T cells transfected with SP-Hoxa3mCh or SP-mCherry and markers of M2-like/prohealing mws as well increasing Stat6 purified lysate from mws treated with CM were analyzed by Western phosphorylation, showing a link between Hoxa3 function and the blotting using 10% SDS-PAGE and polyvinylidene difluoride membrane,

IL-4 signaling pathway. Furthermore, sustained expression of using the Bio-Rad Turbo Transfer System. Blots were probed at 4˚C http://www.jimmunol.org/ Hoxa3 in vivo in db wounds also promotes the switch from an M1- overnight with 0.1 mg rabbit anti-mCherry Ab in a volume of 6 ml and like, proinflammatory mw phenotype to an M2-like, prohealing visualized using the Pierce ECL detection system (Thermo Fisher Scien- tific). Images were generated using the ChemiDoc MP System (Bio-Rad) mw phenotype. These findings have therapeutic relevance because and Image Lab Software. this switch in mw phenotype is a key event in efficient wound healing (13, 24). Flow cytometry analysis BM-derived mws (BMDMs) were trypsinized and resuspended in flow Materials and Methods cytometry buffer (PBS with 2% FBS). Following this, 1 3 106 cells were Animals blocked with 0.5 mg rat anti-mouse CD16/32 (2.4G2; BD Pharmingen) in a

volume of 100 ml for 30 min on ice. Cells were then stained with the fol- by guest on September 26, 2021 All animals were housed at the University of Manchester animal care fa- lowing Abs: 5 mg/ml eFluor450 anti-F4/80 (BM8; eBioscience), 1.25 mg/ml cility, and all procedures were approved by the local ethical review com- PE Cy7 anti-CD115 (25-1152; eBioscience), or 1.2 mg/ml eFluor450 anti- mittee and the Home Office and were performed in accord with the CD11b (M1/70; eBioscience). Flow cytometry was performed with a For- requirements of the Animal (Scientific Procedures) Act 1986. Leprdb/db, the tessa LSR II (BD Biosciences) and analyzed using FlowJo software. most well-characterized mouse model of type 2 diabetes, and heterozygous control Leprdb/+ mice were purchased from Harlan (Oxfordshire, U.K.) or RNA extraction and cDNA synthesis The Jackson Laboratory (Bar Harbor, ME). All animals used were 8–16 wk old and were age- and sex-matched to controls. A minimum of three bi- RNA was isolated from wound- mws and/or BMDMs at the specified time ological replicates per group were used for in vitro experiments. For points by cell or tissue homogenization in TRIzol (Life Technologies). in vivo wound healing studies, six mice per group were used. RNA was treated using the DNase I enzyme at room termperature for 10 min (Qiagen). One microgram of RNA was used for the reverse tran- Wound model scription reaction using Bioscript RT (Bioline) and random primers in a total volume of 25 ml. The resulting cDNA was diluted to 1:15 to be used db/db db/+ Lepr (type 2 diabetes model) and heterozygous control Lepr (non- as a template for quantitative RT-PCR (qRT-PCR). db) mice were anesthetized, and the dorsum was shaved and sterilized with antiseptic wipes. Eight millimeter in diameter full thickness wounds were Gene expression analysis excised including the panniculus carnosus layer. Plasmid DNA (either empty vector or containing wild-type or mutant Hoxa3) was mixed 1:1 qRT-PCR analysis was performed using the following TaqMan Gene with 1% methyl cellulose in water, and 25 mg in a volume of 50 ml was Expression Assays: Itgam (Mm00434455_m1), Emr1 (Mm00802529_m1), spotted and air-dried to form a pellet. Animals received buprenorphine at Csf1r (Mm01266652_m1), Ym1/Chi313 (Mm00675889_m1), Arg1 the time of surgery, and pellets were applied to open wounds. Animals (Mm00475988_m1), Tgfb (Mm01178820_m1), Mrc1 (Mm01329362_m1), were housed in separate cages until tissue was harvested at the described Nos2 (Mm01309897_m1), Tnf (Mm00443258-m1), Cd86 (Mm00444543-m1), time points by removing the entire wound area, including a 2-mm pe- Ccl2 (Mm00441242_m1), Spi1 (Mm00488142_m1), Stat6 (Mm01160477_m1), rimeter, or for wound healing studies, wounds were measured at the in- and Socs1 (Mm00782550-s1). Hsp90ab1 (Mm00833431_g1) and Hist2h2aa1 dicated time points using planimetry. Wound areas were calculated using (Mm00501974_s1) were used as reference genes. Reactions were performed on Adobe Photoshop, and statistics were performed using Microsoft Excel. a StepOnePlus cycler (Applied Biosystems) and analyzed using the d-d Ct method. All analyses were performed using Microsoft Excel 2013. Culturing of BM-derived mws Coimmunoprecipitation of Hoxa3 and Spi-1/Pu.1 protein BM from tibia and femurs of type 2 db and control non-db mice was flushed into DMEM (Sigma-Aldrich), passed through a 19-gauge needle and a 293T cells cotransfected with 5 mg pSport6-Pu.1 and pcDNA3.1- 70-mm filter (BD Falcon) to generate a single-cell suspension. A total of Hoxa3mCherry were harvested 48 h posttransfection. Cell lysates ob- 1 3 106 cells/ml were plated in mw medium (DMEM supplemented with tained using 1% Triton X-100 and 0.01% Igepal in PBS. Two micrograms 10% FBS [Sigma-Aldrich]), 13 penicillin/streptomycin (Sigma-Aldrich), of rabbit anti-mCherry (BioVision) and/or goat anti-Pu.1 (D-19; Santa 10% L929 cell (American Type Culture Collection)–conditioned medium Cruz Biotechnology) were labeled with biotin (DSB-X). Cell lysates (300 containing M-CSF on day 0. Cells were allowed to differentiate for 7 d at mg) were incubated with biotinylated anti-mCherry or anti-Pu.1 and then 37˚C under 5% CO2. Fresh medium was added on day 3; the medium was mixed with prewashed Dynabeads, according to the manufacturer’s in- changed completely on day 5. structions. The eluate was then analyzed by Western blot. 874 Hoxa3 PROMOTES M2 POLARIZATION

Luciferase reporter assay buffer when harvesting cell lysates for pStat6 detection. Cell lysate (10 mg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene RAW 264.7 mws (ATCC-LGC Standards) were treated with 20% M-CSF and mCh difluoride membrane. The blot was incubated with either goat polyclonal 50% CM from either mCherry or Hoxa3 cultures for 24 h. Cells were anti-Pu.1 (1:200, D-19, sc-5949; Santa Cruz Biotechnology), rabbit transfected with 1 mg pMAX enhanced GFP (eGFP), 1 mg pSport6-Pu.1, and monoclonal anti-Stat6 (1:2000, YE361, ab32520; Abcam), or rabbit 1 mg pGL2-7.2fms luciferase vector (40) with a 1:4 ratio of FuGENE HD polyclonal anti-pStat6 (1:1000, Tyr641, ab195647; Abcam). Protein was transfection reagent (Promega) and incubated for 24 h. Cell eGFP expres- detected using ECL (Thermo Fisher Scientific). sion was measured by fluorescence microscopy, and luciferase activity assayed using the Luciferase Assay System (E1500), according to the manufacturer’s protocol (Promega). The level of luciferase activity is shown as relative light units calculated as light units/mean eGFP fluorescence. Results Protein transduction of Hoxa3 in mouse mws promotes mw activation maturation mCh Hoxa3 - or mCherry-treated mws from db or non-db mice were split into We and others have shown that mws isolated from the db envi- the following three groups: 1, nonactivated mws; 2, mws that were clas- ronment show differentiation and maturation defects, which likely sically activated (CA) using serum-free mw medium supplemented with 100 ng/ml LPS (Sigma-Aldrich) and 100 ng/ml IFN-g (Sigma-Aldrich); contribute to their dysfunction in vivo (13, 22). Sustained ex- and 3, mws alternatively activated (AA) using serum-free medium suppression of Hoxa3 in wounds of type 2 db mice results in sig- plemented with 20 ng/ml IL-4 (PeproTech) and 50 mg/ml anti–IFN-g. nificant reduction in the total number of inflammatory cells within Twenty-four hours postactivation, RNA was harvested from each group the wound (21), but the mechanism underlying this effect on mws independently for gene expression. Forty-eight hours postactivation, cells were harvested or supernatants collected for arginase assays, ELISA, and was not identified. As protein transduction of Hoxa3 in hema- Greiss assays. topoietic stem cell/progenitors was shown to promote their Downloaded from differentiation into Gr1+CD11b+ cells, a proangiogenic granulocytic– NO release (Greiss assay) monocytic cell population (34), we aimed to test whether over- NO release was determined using supernatants from mws with Greiss expression of Hoxa3 by protein transduction on monocyte/mw reagents, as described previously (41). A colorimetric plate reader was precursors from type 2 db mice could rescue their differentiation used to measure absorbance at 570 nm and background absorbance at defects. To test this, we used a construct in which Hoxa3 is fused 630 nm.

to mCherry and to the Ig k-chain signal peptide (Fig. 1A) to allow http://www.jimmunol.org/ Arginase assay the secretion of Hoxa3 into the medium (43). Transfection efficiency of Hoxa3-mCherry (Hoxa3mCh) and mCherry control ex- Arginase activity in mw lysates was measured using the spectrophotometric method, as described previously (42). Briefly, 10 mM MnCl and 0.5 M pression plasmids in 293T cells was monitored by live imaging 2 mCh L-arginine were sequentially added to the mws and incubated for 1 h at 37˚C. (Fig. 1B). The presence of Hoxa3 and mCherry protein in the The reaction was stopped by the addition of an acid solution, and the for- CM and in the targeted mouse mws was confirmed via immuno- mation of urea by arginase was analyzed by adding isonitrosopropiophenone precipitation (Fig. 1C) and confocal imaging (Fig. 1D), respec- at 100˚C for 45 min. The colored product was measured at 570 nm and the mCh background absorbance at 450 nm. tively. Quantification of Hoxa3 and mCherry control protein localization within mws was performed to validate appropriate ELISA nuclear localization of the Hoxa3mCh protein (Fig. 1E). by guest on September 26, 2021 mCh Detection of mouse TNF-a and IL-12 was determined using a cytokine- Following Hoxa3 or mCherry control protein transduction specific ELISA kit (eBioscience) as described previously (13). For mouse for 24 h, total RNA was isolated from target mws and qRT-PCR TGF-b, a Ready-Set-Go mouse TGF-b ELISA kit (eBioscience) was used conducted to measure expression of Emr1, encoding F4/80 (a according to the manufacturer’s instructions. mw-specific marker), Itgam, encoding CD11b (common myeloid Wound processing for immunofluorescence staining marker), and Csf1r, encoding the M-CSF receptor (CD115) to assess mw maturation. Mws derived from non-db and db mice Wounds were processed from three type 2 db or control non-db mice showed significant increases in Emr (p , 0.05 and p = 0.07, re- treated with CMV-Hoxa3 or plasmid control as described in (19). Wounds mCh were stained as described previously, with some modifications (13). In spectively; Fig. 2A) and Csf1r (p = 0.05) in response to Hoxa3 . brief, 5 mm sections were generated from formalin-fixed wounds and A significant increase in Itgam (p , 0.05) in the non–db-derived stained with H&E using a Shandon Varistain 24-4 (Thermo Scientific) to population was observed, but there was no apparent change in the locate the granulation tissue and peri-wound dermis. Matching sections db-derived mws (Fig. 2A). We also analyzed changes in expres- were labeled with the following Abs diluted in PBS + 0.1% Tween 20 sion of these markers at the protein level by flow cytometry (PBT): rat anti-Mac3 (1:100, clone M3/84 (RUO); BD Pharmingen), mCh goat anti-Arg1 (1:200, sc-18354; Santa Cruz Biotechnology), rabbit anti- (Fig. 2B). Protein transduction of Hoxa3 showed a trend of Nos2 (1:100, sc- 651; Santa Cruz Biotechnology), rabbit anti-Vegf increased mean fluorescence intensity (MFI) of cell surface F4/80 (1:400, EP1176Y; Abcam), and rabbit anti-Tgfb (1:150, EPR12678(B); protein in non–db-derived mws (Hoxa3mCh MFI = 27725.5 6 Abcam). Next, the following secondary Abs were applied: donkey anti- 13624 versus mCherry MFI = 17380.8 6 13690, p = 0.2) and in rat Alexa Fluor 488 (1:500, A-21208; Invitrogen Life Technologies), mCh 6 donkey anti-goat Cy5 (1:500, ab6566; Abcam), and donkey anti-rabbit the db-derived population (Hoxa3 MFI = 11746 7681 versus Alexa Fluor 555 (1:500, A-31572; Invitrogen Life Technologies). Four mCherry MFI 8913 6 5499, p = 0.1). CD115 was significantly fields from each wound were imaged for statistical analysis of Mac3+ mCh 2 2 increased by Hoxa3 protein transduction in non–db-derived Nos2+Arg1 , Mac3+Nos2+Arg1+, or Mac3+Nos2 Arg1+. For VEGF and mCh 6 + + + + mws(Hoxa3 MFI 10188.5 2577 versus mCherry MFI TGF-b staining, the number of Mac3 ,VEGF,andMac3VEGF or 6 mCh Mac3+,TGFb+,andMac+TGFb+ cells were determined. All sample 6213 962, p = 0.05), and db-derived mws(Hoxa3 MFI = analyses were performed using Imaris 3D/4D image processing and 35915 6 11729 versus mCherry MFI = 21824 6 5971, p , 0.1). analysis software (Bitplane). The percentage of each phenotype was However, no detectable changes were noted in CD11b levels be- calculated as the mean 6 SEM of all images in each category (n = 12). tween Hoxa3mCh-treated and mCherry-treated mws. Altogether, Western blotting the Hoxa3-mediated regulation of mw maturation markers suggests that Hoxa3mCh protein transduction promotes the maturation Whole-cell lysates were obtained from mouse mws at the specified time of non–db- and db-derived mws. The discrepancies in transcript points by lysing the cells in complete radioimmunoprecipitation lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM and protein levels of Itgam may be due to posttranscriptional PMSF, 2 mM sodium orthovanadate, and 13 protease inhibitor mixture regulation of this protein, which has been shown in the db envi- [Fisher, BPE9706-1] in PBS). Phosphatase inhibitor was added to the lysis ronment (13). The Journal of Immunology 875

FIGURE 1. Protein transduction of Hoxa3mCh in mouse mws. (A) Schematic representation of signal peptide (SP)-mCherry control and SP-Hoxa3mCh constructs. Expression is directed by the CMV promoter (prom). (B) Fluorescent images of mCherry and Hoxa3mCh protein expression in transfected 293T cells. (C) Western blot detection of Hoxa3mCh and mCherry (mCh) in CM (left blot) and anti- mCherry immunoprecipitated lysate (lys) from 293T cells transfected with Hoxa3mCh (right blot,

left lane), mouse mws treated with CM containing Downloaded from mCherry (right blot, middle lane), or Hoxa3mCh (right blot, right lane), respectively. (D) Represen- tative confocal image of mws treated with mCherry CM (left panels) or Hoxa3mCh CM (right panels) showing anti-mCherry immunolocalization (top panels), DAPI staining (middle panels), and merged channels (bottom panels). (E) Quantiﬁcation of nu- http://www.jimmunol.org/ clear localization of mCherry and Hoxa3mCh following treatment with the respective CM, n =40 mws. Original magniﬁcation 3400. ***p , 0.001. by guest on September 26, 2021

Hoxa3 upregulates Spi1 levels in mouse mws in Spi1/Pu.1 protein (Fig. 3B). In the db-derived mws treated with mCh Spi1 (also known as Pu.1), Cepba, and Runx1 transcription factors Hoxa3 , Spi1/Pu.1 protein levels were also significantly in- ∼ ∼ are critical for monocyte and mw differentiation and are known to creased by 1.5-fold at 24 h and by 7.4-fold at 48 h compared upregulate expression of myeloid differentiation markers, in- with control-treated cells (Fig. 3C). Levels of Spi1/Pu.1 were quan- cluding Csf1r and Itgam (44). We hypothesized that Hoxa3 may tified relative to tubulin (Fig. 3D, 3E). These data suggest that promote maturation of murine mws via modulating the function of Hoxa3 may induce Spi1/Pu.1 at the transcriptional and posttran- Spi1, Cepba, and/or Runx1 transcription factors. We first assessed scriptional levels. This Hoxa3-mediated upregulation of Spi1/Pu.1 whether any differences were evident in the levels of expression of protein in db-derived mws may be one mechanism by which these transcription factors between non-db and type 2 db mws. The Hoxa3 promotes mw maturation. db-derived mws showed a significant reduction in Spi1 (p , 0.05; Supplemental Fig. 1A), whereas Cebpa and Runx1 levels did Hoxa3 and Pu.1 proteins physically interact not show significant differences among the two populations We next investigated whether Hoxa3 may physically interact with (Supplemental Fig. 1B, 1C). We then assayed whether Hoxa3mCh the Pu.1/Spi1 transcription factor and function as a cofactor on protein transduction for 24 or 48 h could affect Spi1, Cebpa,or myeloid differentiation target genes. Other Hox proteins, such as Runx1 mRNA levels. Interestingly, Hoxa3 upregulated Spi1 Hoxc13, have been shown to interact with ETS proteins, including mRNA levels in non–db-derived mws after 48 h (p , 0.05; Fig. Pu.1/Spi1, by physical and functional interactions that are impli- 3A) and in the db-derived mwsat24h(p , 0.05) and 48 h (p , cated in the differentiation of erythroid leukemia cell lines (27). 0.005; Fig. 3A) but did not affect the levels of Cebpa or Runx1 We examined biochemical interactions between Pu.1 and Hoxa3 (data not shown). We further investigated whether the changes by performing immunoprecipitation assays on cell lysates from observed in Spi1 mRNA levels were consistent with the Spi1/Pu.1 293T cells that were cotransfected with mCherry-tagged Hoxa3 protein levels in non–db- and db-derived mws. Surprisingly, Spi1/ and Pu.1. The anti-Pu.1 Ab coimmunoprecipitated Hoxa3 but not Pu.1 protein levels in non-db mws were significantly increased at the mCherry-only control (Fig. 3E), whereas the anti-mCherry Ab 24 h following Hoxa3mCh treatment, despite a lack of significant coimmunoprecipitated Pu.1/Spi1 in the Hoxa3mCh-Spi1 cotrans- increase in mRNA at this time point. However, as expected at 48 h fection only (Fig. 3F), suggesting that Hoxa3 and Pu.1/Spi1 pro- following Hoxa3mCh treatment, we detected a ∼14.8-fold increase teins physically interact. 876 Hoxa3 PROMOTES M2 POLARIZATION

FIGURE 2. Analysis of mw maturation following Hoxa3mCh protein transduction. (A) qRT-PCR analysis of mw maturation markers Emr1 (encoding F4.80), Csf1r (encoding CD115/M-CSFR), and Itgam (encoding CD11b) in non–db- and db-derived mws treated with either

Hoxa3mCh (n) or mCherry control (N) CM for 24 h (RE, Downloaded from relative expression to reference gene, n = 3, error bars = SEM; *p , 0.05). (B) Representative histogram plots from ﬂow cytometry analysis of same markers in (A) in non–db- (top panels) and db-derived (bottom panels) mws treated with either Hoxa3mCh (black shading) or mCherry control (white) CM for 24 h. http://www.jimmunol.org/ by guest on September 26, 2021

To test whether this interaction affects Spi1 DNA binding or or mCherry CM were either left in a naive state (nonactivated regulation of its target genes, we analyzed Spi1 activation of two [NA]) or CA using LPS and IFN-g. Production of NO as well as Csf1r enhancers using a luciferase reporter assay system. Spi1 the proinflammatory cytokines TNF and IL-12 were then assessed. has been shown to physically bind to two enhancer regions near We found that Hoxa3mCh significantly reduced NO produc- the Csfr1 promoter, one upstream of the transcriptional start site tion from CA mws (Fig. 4A). In addition, the level of IL-12 and one downstream, in the first intron, and abolition of this cytokine production was reduced in CA Hoxa3mCh-treated non– binding results in loss of enhancer activity (45, 46). We found db-derived mws by more than 4-fold compared with control- that protein transduction of Hoxa3 in combination with Spi1 treated cells (Fig. 4B). In db-derived mws, Hoxa3 also inhibited significantly promoted Csfr1 enhancer activity when both sites IL-12 production in CA mwsby∼2-fold compared with were present (pGL2-7.2fms construct) in response to M-CSF control-treated cells (Fig. 4B). In a similar manner, Hoxa3 compared with control protein transduction (Fig. 3G). However, markedly reduced TNF production by ∼53-fold in CA non– protein transduction of Hoxa3 did not affect the upstream-only db-derived mws (Fig. 4C) and showed a trend in reducing TNF reporter activation (pGL-0.5fms construct), suggesting it pro- production in CA db–derived mws compared with controls motes Spi1 activation through the downstream enhancer (data (p = 0.18; Fig. 4D). not shown). To determine whether protein transduction of Hoxa3 can limit the expression of M1 marker genes in CA mws, we as- Hoxa3 inhibits M1 proinflammatory polarization of BMDMs sessed the expression of M1 markers in NA and CA db and One of the reported functions of Hoxa3 in accelerated healing of non–db-derived mws (Fig. 4E–H). Hoxa3mCh-treated CA mws the type 2 db mouse model is reducing the total number of inflam- showed significantly reduced expression of all M1 marker matory cells recruited to or retained in the wound via downreg- genes tested including NO synthase (Nos2;Fig.4E),Tnf ulation of NF-ĸB pathway genes (24). The NF-ĸB pathway (Fig. 4F), chemokine C-C motif ligand 2 (Ccl2)(Fig.4G),and promotes proinflammatory gene expression and an M1-like Cd86 (Fig. 4H). These data, therefore, support the hypothesis phenotype. On the basis of these results, we wished to test the that enforced expression of Hoxa3 can limit M1 mw polari- ability of Hoxa3 to control the proinflammatory phenotype of zation in the presence of proinflammatory stimuli such as LPS mws. Non–db- and type 2 db–derived mws treated with Hoxa3mCh and IFN-g. The Journal of Immunology 877 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Hoxa3 protein transduction increases Spi1 mRNA and protein levels in mouse mws and physically interacts with Spi1 protein. (A) Spi1 expression after 24 or 48 h of control (mCherry, N)orHoxa3mCh (n) protein transduction in non–db (ndb)- or db-derived mws (RE, relative expression to reference gene, n = 3, error bars = SEM; *p , 0.05, **p , 0.01). Representative Western blot analysis of Spi1 levels in control-treated (mCherry) or Hoxa3mCh-treated ndb (B)ordb(C)mws. (D) Quantiﬁcation of Spi1 protein levels from Western blots as shown in (B)and(C). Spi1 levels were normalized to tubulin levels on same blot (n = 3, error bars = SEM; *p , 0.05, **p , 0.01). (E) Coimmunoprecipitation (Co-IP) of Spi1 and Hoxa3mCh proteins in transfected 293T cells. Left lane shows whole-cell lysate input from 293T cells transfected with Spi1 and Hoxa3mCh expression plasmids, lane 2 shows Co-IP using anti-Pu.1/Spi1 Ab from cells cotransfected with negative control (mCherry) and Spi1 expression plasmids, and lane 3 shows Co-IP using anti-Pu.1/Spi1 Ab from cells cotransfected with Hoxa3mCh and Spi1 expression plasmids. Blot was probed with anti-mCherry Ab. The predicted size of Hoxa3mCh is 73 kDa and mCherry is 26 kDa. (F) Co-IP of Spi1 and Hoxa3mCh proteins in transfected 293T cells. Left lane shows whole-cell lysate input from 293T cells transfected with Spi1 and Hoxa3mCh expression plasmids, lane 2 shows Co-IP using anti-mCherry Ab from cells transfected with Spi1 and control plasmids, and lane 3 shows Co-IP using anti-mCherry Ab from cells transfected with Spi1 and Hoxa3mCh expression plasmid. Blot was probed with anti-Pu.1/Spi1 Ab. The predicted size of Spi1/Pu.1 is 31 kDa. (G) Hoxa3 promotes Spi1/Pu.1 activation of the Csfr1 enhancer. Luciferase report activity in RAW 264.7 macrophages cotransfected with pGL2-7.2fms luciferase reporter plus Spi1 expression plasmid and treated with mCherry or Hoxa3mCh protein CM. Luciferase activity was normalized to ﬂuorescence from eGFP transfection control, and values represent mean 6 SEM of n = 4 experimental replicates. *p , 0.05. mCh, mCherry; Tnsfxn, transfection.

Hoxa3 promotes M2 prohealing polarization of BMDMs assay showed significantly higher levels of urea in AA non–- Prohealing mws (M2-like) are the major source of growth factors db-derived mws and a trend of increased urea levels in AA db– mCh such as TGF-b that contribute to tissue repair and wound healing derived mws treated with Hoxa3 compared with control-treated (8). Previous work from our laboratory has shown that enforced mws (Fig. 5A). Hoxa3 also increased the levels of anti-inflammatory expression of Hoxa3 upregulates Tgfb in myeloid cells (34), cytokine/growth factor TGF-b by ∼5-fold in AA non–db-derived suggesting it may promote M2-like polarization. Therefore, we mws (Fig. 5B) and by ∼2.9-fold in AA db–derived mws(p = 0.06; assessed the profile of Hoxa3mCh-treated mws for M2 markers Fig. 5B). Further analyses of the regulatory effect of Hoxa3 protein such as arginase and TGF-b production. Arginase, a hallmark of transduction on levels of these M2 markers were obtained by per- AA mws in mice, was assessed by measuring urea in cell lysates, forming qRT-PCR on Arg1, Mrc1 (mannose receptor CD206), Tgfb, which is a product from arginase degradation of L-arginine. This and chitinase 3-like 3 (Chi3l3, also known as Ym1)onRNA 878 Hoxa3 PROMOTES M2 POLARIZATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Analysis of proinflammatory (M1) mw markers in Hoxa3mCh- or mCherry-treated BMDMs from non-db (ndb) and db mice. (A) Production of NO (as measured by nitrate as the final product in the Griess assay) in NA or CA mws from ndb or db mice and treated with mCherry (N) or with Hoxa3mCh (n). (B) IL-12 release from NA or CA mws from ndb or db mice, treated with mCherry (N) or Hoxa3mCh (n). (C and D) TNF release from NA or CA mws from ndb and db mice, respectively, treated with mCherry (N) or with Hoxa3mCh (n). (E–H) Relative expression (RE) of Nos2, Tnf, Cd86, and Ccl2 to Hsp90 (reference gene, reference, n =5;*p , 0.05, ***p , 0.005). extracted from NA and AA mws treated with Hoxa3mCh or mCherry wounds with Hoxa3 could influence mw phenotype in vivo. Pre- control CM. Hoxa3mCh-treated AA non–db- and db-derived mws vious studies from our laboratory found that wounds of db mice showed significant upregulation of Tgfb, Mrc1,andChi3l3 mRNA show significant increases in M1-like (Nos2+) cells and significant in AA non–db- and db-derived mws (Fig. 5C–F). Altogether, the decreases in M2-like (Arg1+) cells when compared with non-db data suggest that Hoxa3 promotes M2 polarization by reducing M1 wounds at day 7 following wounding (13). Because day 7 in the markers and upregulating M2 markers, which may contribute to its healing process represents the phase where most mws in non-db capacity to promote wound resolution in vivo. wounds have transitioned from a proinflammatory phenotype to a prohealing phenotype, we therefore investigated the effect of Hoxa3 inhibits M1-like and promotes M2-like polarization of treating db mouse wounds with Hoxa3 at this time point. Sections macrophages in day 7 db wounds were taken from full thickness excisional wounds that were treated The data above suggested enforced expression of Hoxa3 suppress with Hoxa3 or control and subsequently labeled with Abs for the M1 phenotype and promote the M2 phenotype in cultured mws. detecting Mac3 (pan-macrophage marker), Nos2 (M1 marker), We therefore wished to investigate whether treating type 2 db and Arg1 (M2 marker). Analysis was performed on granulation The Journal of Immunology 879 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Analysis of prohealing (M2) mw markers in Hoxa3mCh- or mCherry-treated BMDMs from non-db (ndb) and db mice. (A) Arginase assay mCh (urea production from L-arginine) in NA or AA mws from ndb or db mice treated with mCherry (N) or with Hoxa3 (n). (B) TGF-b production from NA or AA mws from ndb or db mice treated with mCherry (N) or Hoxa3mCh (n). (C–F) Relative expression (RE) of Arg1, Tgfb1, Mrc1, and Chi3l3(Ym1) to Hsp90 (reference gene, n =5;*p , 0.05, ***p , 0.005). tissue and peri-wound dermis (Fig. 6A). In Hoxa3-treated wounds, Hoxa3 upregulates IL-4 signaling pathway effector Stat6 and 7 d postinjury, triple staining for Mac3, Nos2, and Arg1 showed a increases phospho-Stat6 levels + 2 mCh trend of reduced Nos2 Arg1 (M1-like) mws in the Hoxa3 -treated The IL-4 signaling pathway strongly promotes M2 macrophage wound as compared with control treated and significantly in- 2 + polarization via Stat6 activation. Upon IL-4 stimulation, Stat6 is creased numbers of Nos2 Arg1 (M2-like) mws (Fig. 6B, 6D). phosphorylated by the activated JAKs, and translocates through the There appeared to be no difference in the number of mixed phe- nucleus to function as a transcription factor that upregulates Arg1, notype Nos2+Arg1+ mws. Mrc1, and Chi3l3/Ym1 (M2 genes) (15, 16). To determine whether Additional markers for proreparative mws, TGF-b and VEGF, Hoxa3 promotes the M2-like mw phenotype by interacting with were also analyzed, as several previous studies have shown that the IL-4 signaling pathway, we investigated Stat6 activation fol- TGFb+ and VEGF+ macrophages are critical for effective healing lowing Hoxa3mCh protein transduction in vitro. We assessed the (12, 47). Sections from db wounds treated with Hoxa3 or control levels of Stat6 transcript, and total Stat6 protein as well phospho- were double labeled with Abs detecting Mac3 (mw marker) and mCh VEGF or TGF-b, and analysis was performed as above. In Hoxa3- Stat6 in Hoxa3 -treated mws. We found that Hoxa3 upregulated treated wounds, 7 d postinjury, double staining for Mac3 and Stat6 transcript and Stat6 protein levels in both non-db and db- VEGF showed a significant increase in the number of Mac3+ derived mws following IL-4 stimulation (Fig. 7A–C). Interestingly VEGF+ mw populations in diabetic wounds (p , 0.05; Fig. 6C, Hoxa3 increased total Stat6 protein accumulation in the unsti- 6E). However, although Hoxa3 significantly increased the number mulated cells (Fig 7C), although no detectable transcript differ- of total TGFb+ cells in the wound (p , 0.05), it did not increase ence was apparent (Fig. 7A, 7B), suggesting that Hoxa3 may also the Mac3+TGFb+ subpopulation (Supplemental Fig. 2A, 2B), mediate stabilization of Stat6 protein. In addition, phospho-Stat6 suggesting that these populations may be partially distinct. Neg- was also markedly increased in the presence of Hoxa3 in non–db- ative controls for all Abs are shown in supplementary material derived and db-derived mws (Fig. 7C). Analysis of Hoxa3-treated (Supplemental Fig. 3A–C). Altogether, the data in this paper whole wound samples from type 2 db mice also revealed up- provide evidence for Hoxa3 promoting an M2-like phenotype in regulation in Stat6 mRNA levels (Fig. 7D), suggesting Stat6 is a wound macrophages in vivo. downstream target of Hoxa3 in vivo as well. We did not observe 880 Hoxa3 PROMOTES M2 POLARIZATION

FIGURE 6. Enforced expression of Hoxa3 in vivo inhibits M1-like polarization and promotes M2-like polarization in wounds of db mice. (A) Representative section from day 7 wound of db mouse showing areas of analysis (indicated by boxes; scale bar, 1 mm). (B and C) Rep- resentative images of immunofluorescent Downloaded from (IF) detection of Mac3 (mw marker; green), Nos2 (proinflammatory marker; red), and Arg1 (prohealing marker; blue) in (B) and DAPI (blue), Mac3 (green), and VEGF (prohealing mw marker; red) in (C) from day 7 wound of db mouse treated with empty vector control or with http://www.jimmunol.org/ Hoxa3 expression plasmid (scale bar, 10 mm). (D) Quantification of Mac3+Nos2+, Mac3+Arg1+ and Mac3+Nos2+Arg1+ cells in IF sections from (B). (E) Quanti- fication of Mac3+, VEGF+, and Mac+ VEGF+ cells in IF sections from (C); *p , 0.05. FoV, field of view. by guest on September 26, 2021

any change in Il4 or Tgfb1 mRNA expression in vivo (data not current study. In addition, we wished to test whether DNA binding shown), suggesting that Hoxa3 modulation of Stat6 is independent is required for this function. To do this, we induced a mutation to of these genes. We also analyzed Socs1 expression in response to the reading frame encoding the homeodomain of Hoxa3 to change Hoxa3, as Socs1 is known to be activated in response to IL-4 asparagine 51 to glutamine (N51Q), abolishing DNA binding (49). signaling, and is crucial for IL-4–dependent M2 polarization in Interestingly, we found that the N51Q mutant was able to promote mws (48). Protein transduction of Hoxa3mCh significantly increased wound healing in db mice as effectively as the wild type Hoxa3 Socs1 expression in db-derived mws and showed a trend of in- (Fig. 7F). This finding is consistent with a model in which Hoxa3 creased expression in non–db-derived mws (Fig. 7E). Taken to- promotes macrophage maturation and polarization via interaction gether, our findings suggest that enforced expression of Hoxa3 can with other DNA-binding partners, such as Spi1/Pu.1. promote alternative activation of mws via increasing cellular pools of Stat6 and promoting Stat6 phosphorylation in murine mws. Discussion To assess the therapeutic potential of Hoxa3 on dysregulated mws, Hoxa3 accelerates wound healing in type 2 db mice in a we used an ex vivo culture model of BMDMs from type 2 db and DNA-binding independent manner non-db mice. Hox proteins possess the ability to translocate across We have previously shown that enforced expression of Hoxa3 in db biological membranes in an energy-free, endocytosis-free manner wounds accelerates wound healing (26). However, our prior work because of the penetratin peptide located in the third a helix of was performed using a larger excisional wound model than the homeodomain proteins (31, 32). Using this property, we delivered The Journal of Immunology 881 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Hoxa3 regulation of IL-4-Stat6/phospho-Stat6 signaling pathway during M2 activation. Relative expression (RE) of Stat6 in mCherry (N)or Hoxa3mCh-treated (n) non–db (ndb)-derived mws(A) or db-derived mws(B), stimulated with IL-4, normalized to mws without IL-4 stimulation (no IL-4). Hist2h2aa1 and Hsp90ab1 were used as reference genes, and values represent mean 6 SEM of n = 5 biological replicates. *p , 0.05. (C) Representative Western blots of total protein lysates from mws pretreated with Hoxa3mCh or mCherry CM that were left unstimulated (no IL-4) or stimulated with IL-4 and probed with the indicated Abs. Blots were repeated three times on mws from three different mice. (D) Stat6 expression in day 4 wounds of db mice treated with empty vector (N)orHoxa3 expression plasmid (n) relative to reference. (E) Socs1 expression in NA and AA ndb-derived mws (upper graph) and db- derived mws (lower graph) treated with control (N) or Hoxa3mCh (n). (F) Mean percent open wound following 0.8-cm excisional wounding on days indicated in ndb mice treated with control plasmid (blue line), db mice treated with control plasmid (orange line), db mice treated with wild-type Hoxa3 plasmid (green line), and db mice treated with mutant Hoxa3 plasmid encoding a change in homeodomain aa 51 from Asn to Gln (N51Q). Hoxa3, A3, n = 6/group, error bars indicate SEM, *p , 0.05 when comparing Hoxa3-treated db versus control-treated db. cntl, control.

Hoxa3 into cultured mws using protein transduction. This method mws are critical to wound healing as they secrete growth factors is potentially a safe and useful method to overexpress transcription and cytokines necessary for wound resident cell proliferation, factors ex vivo to reprogram cells for therapeutic use because it migration, and other behaviors (12, 50, 51). They also contribute does not involve gene manipulation or possible damage to the cell to resolution of the inflammatory phase via efferocytosis (removal that may be caused by other methods such as viral transduction. of dead/dying cells in the wound), particularly of neutrophils. The The passive translocation of Hox proteins was previously de- presence of neutrophils until late stages of healing in db wounds scribed with Hoxa3 and Hoxb4 using a coculture system to deliver can negatively impact the healing process by causing oxidative them into hemopoietic stem cells (33, 34). In this study, we damage and exacerbating tissue damage by secreting or leaking demonstrate for the first time, to our knowledge, the successful excessive proteases and elastases (52, 53). Khan et al. (54) found delivery of Hoxa3 protein into mws using a CM enriched with that successful efferocytosis can switch the mw phenotype in the Hoxa3 rather than the more complicated coculture system ap- db wound from proinflammatory to anti-inflammatory. Further- proach. This is superior to a coculture system as it is a step closer more, several studies have suggested that db-derived mws fail to to good manufacturing practice conditions, which would be re- perform this function effectively (55–57). However, until now it quired for potential therapeutic use. has not been clear whether delayed/failed maturation of db mws 882 Hoxa3 PROMOTES M2 POLARIZATION may underlie defective efferocytosis in db wounds, leading to hancing Spi1 function when overexpressed. Upregulation of Csf1r, excessive accumulation of leukocytes. The link between abnormal a known downstream target of Spi1, supports this hypothesis. In maturation of myeloid cells and diabetes was demonstrated in addition, luciferase assays indicate Hoxa3 collaborates with Spi1 non-obese db mice in which administration of GM-CSF promoted to activate the promoter/enhancer elements controlling Csf1r ex- the maturation of DCs and subsequently reduced the development pression. However, transcription of Itgam, which encodes CD11b of diabetes (58) as well as in obese db mice in which adminis- and is another known target of Spi1 (61), was not upregulated tration of G-CSF–promoted Gr-1+CD11b+ cell maturation and by Hoxa3 overexpression, suggesting only a subset of Spi1 targets wound healing (59); thus, the cause and effect relationship be- are modulated by Hoxa3 interactions. Interestingly, abolish- tween these two states may be intertwined. Our data suggest that ment of Hoxa3 DNA-binding did not affect its ability to promote this relationship can be extrapolated to macrophages in diabetic wound healing, providing further evidence that Hoxa3 modu- chronic wounds as well. lates macrophage behavior via direct interactions with myeloid Several studies have demonstrated that diabetes induces a transcription factors. Further studies to clarify the importance of dysfunctional mw polarization phenotype (13, 22, 60), even when Hoxa3-Spi1 interactions on mw maturation and polarization dur- cells are removed from the db environment and cultured ex vivo ing wound healing will address this question. (13). Although environmental stimuli play a large role in mw In addition to promoting mw maturation, Hoxa3 attenuated the phenotype, this last observation suggests a role for cell intrinsic proinflammatory response of mws to LPS and IFN-g and reduced factors in regulating mw maturity and phenotype as well. Tran- NO, IL-12, and TNF release in cell supernatants. Analysis of scription factors are likely candidates for such cell intrinsic factors M1 gene expression supported the hypothesis that Hoxa3 in- because they control cell differentiation and phenotype by regu- hibited M1 polarization. This is consistent with the previously Downloaded from lating hundreds of target genes that affect multiple processes described anti-inflammatory effect of Hoxa3 in vivo, where sus- within the cell. Hoxa3 has been shown to be aberrantly regulated tained expression of Hoxa3 reduced the recruitment and/or retention in db wounds, and enforced expression of this potent transcription of inflammatory cells and resulted in downregulation of genes in- factor can significantly inhibit inflammation, promote angiogen- volved in multiple inflammatory pathways, including NF-ĸB(21). esis, and accelerate healing (21, 26, 34). In this study, we inves- In addition to suppressing the M1 polarization phenotype,

tigated the effect of Hoxa3 protein transduction and in vivo gene enforced expression of Hoxa3 also promotes the M2 polariza- http://www.jimmunol.org/ transfer on mw maturation and phenotype both ex vivo and in vivo. tion phenotype. We found Hoxa3 upregulated Tgfb,amarker Hoxa3-treated mws showed upregulation of maturation markers of M2 mws, at the mRNA level and increased TGF-b growth F4/80 and CD115 as well a master regulator of mw development, factor release from non–db- as well db-derived mws. This is Spi1/Pu.1, at both transcriptional and protein levels, thus sup- consistent with a previous study showing upregulation of Tgfb porting the hypothesis that Hoxa3 rescues diabetes-induced mw in Gr-1+CD11b+ myeloid cells transfected with Hoxa3 (34). maturation defects. In addition, we found that Hoxa3 can directly TGF-b functions during the later stages of wound healing to interact with Spi1, suggesting Hoxa3 may act as a cofactor en- promote angiogenesis, stimulate myoﬁbroblast differentiation by guest on September 26, 2021

FIGURE 8. Model of multiple effects of Hoxa3 on db mws. db-derived mws have aberrant maturation and dysregulated polarization phenotypes. Enforced expression of Hoxa3 in ex vivo–differentiated mouse mws promotes differentiation of mws and increases expression of F4/80 and CD115 via upregulation of the Spi1/Pu.1 transcription factor. Hoxa3 also rescues the aberrant polarization phenotype in db mws, promoting the M2/prohealing polarization state in BM-derived as well in day 7 db wound mws. This occurs in part by Hoxa3-mediated increases in cellular pools of Stat6 protein as well as phosphorylation. In parallel, Hoxa3 also inhibits the heightened M1/proinflammatory mw phenotype in db mws, possibly by inhibiting NF-kB activation. The Journal of Immunology 883 and wound closure, and promote collagen deposition (reviewed 6. Bitar, M. S., and Z. N. Labbad. 1996. Transforming growth factor-b and insulin- like growth factor-I in relation to diabetes-induced impairment of wound heal- in Ref. 62). Hoxa3 treatment of wounds also increased the ing. J. Surg. Res. 61: 113–119. number of TGF-b–positive cells in the wound, although not 7. Beer, H. D., M. T. Longaker, and S. Werner. 1997. Reduced expression of PDGF specifically in macrophages. Hoxa3 treatment also increased and PDGF receptors during impaired wound healing. J. Invest. Dermatol. 109: 132–138. arginase production in mwsfrombothnon-dbandtype2db 8. Frank, S., G. Hubner,€ G. Breier, M. T. Longaker, D. G. Greenhalgh, and mice. Arginase is thought to promote wound healing by shifting S. Werner. 1995. Regulation of vascular endothelial growth factor expression in the production of NO to urea and ornithine. Ornithine is a cultured keratinocytes. Implications for normal and impaired wound healing. J. Biol. Chem. 270: 12607–12613. precursor of polyamines and collagen (reviewed in Ref. 63). 9. Brancato, S. K., and J. E. Albina. 2011. Wound macrophages as key regulators of Although arginase is not highly expressed in human monocytes repair: origin, phenotype, and function. Am. J. Pathol. 178: 19–25. or mws when stimulated in vitro (64), it has been shown to be 10. Koh, T. J., and L. A. DiPietro. 2011. Inflammation and wound healing: the role of the macrophage. Expert Rev. Mol. Med. 13: e23. expressed in human neutrophils (65), which may indicate that 11. Gordon, S., and F. O. Martinez. 2010. Alternative activation of macrophages: these cells play a similar role in human wounds with regard to mechanism and functions. Immunity 32: 593–604. € w 12. Lucas, T., A. Waisman, R. Ranjan, J. Roes, T. Krieg, W. Muller, A. Roers, and arginase function. Stat6 function in murine and human m sis S. A. Eming. 2010. Differential roles of macrophages in diverse phases of skin conserved, and thus, our finding that Hoxa3 treatment induces repair. J. Immunol. 184: 3964–3977. upregulation of Stat6 and increased phosphorylation of Stat6 13. Bannon, P., S. Wood, T. Restivo, L. Campbell, M. J. Hardman, and K. A. Mace. 2013. Diabetes induces stable intrinsic changes to myeloid cells that contribute will be relevant to human mw biology. Stat6-dependent gene to chronic inflammation during wound healing in mice. Dis. Model. Mech. 6: regulation is critical to M2 mw behavior (reviewed in Ref. 11). 1434–1447. Impairedwoundhealinginbothtype1andtype2dbmouse 14. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8: 958–969. models and human patients has been linked to a failure in mwsto 15. Hebenstreit, D., G. Wirnsberger, J. Horejs-Hoeck, and A. Duschl. 2006. Sig- Downloaded from switch from the M1 phenotype to the M2 phenotype (13, 22, naling mechanisms, interaction partners, and target genes of STAT6. Cytokine 60). In this study, we have shown that enforced expression of Growth Factor Rev. 17: 173–188. 16. Maier, E., A. Duschl, and J. Horejs-Hoeck. 2012. STAT6-dependent and Hoxa3 in type 2 db-derived mws can promote this switch during -independent mechanisms in Th2 polarization. Eur. J. Immunol. 42: 2827– wound healing via multiple mechanisms (Fig. 8). Hoxa3 treat- 2833. w 17. Stein, M., S. Keshav, N. Harris, and S. Gordon. 1992. Interleukin 4 potently ment promotes m maturation via upregulation of and direct enhances murine macrophage mannose receptor activity: a marker of alternative interaction with Spi1, which may lead to enhanced mw function. immunologic macrophage activation. J. Exp. Med. 176: 287–292. http://www.jimmunol.org/ This in turn may attenuate proinflammatory responses and 18. Knipper, J. A., S. Willenborg, J. Brinckmann, W. Bloch, T. Maaß, R. Wagener, T. Krieg, T. Sutherland, A. Munitz, M. E. Rothenberg, et al. 2015. Interleukin-4 augment anti-inflammatory behaviors. Hoxa3 also inhibits M1 receptor a signaling in myeloid cells controls collagen fibril assembly in skin polarization via suppression of proinflammatory gene expres- repair. Immunity 43: 803–816. sion and promotes M2 polarization by enhancing IL-4 signaling 19. Campbell, L., E. Emmerson, H. Williams, C. R. Saville, A. Krust, P. Chambon, K. A. Mace, and M. J. Hardman. 2014. Estrogen receptor- a promotes alternative through increased Stat6 activation. Altogether, these functions macrophage activation during cutaneous repair. J. Invest. Dermatol. 134: 2447– of Hoxa3 shift the balance from an M1-dominant wound envi- 2457. ronment to an M2-dominant wound environment, thus acceler- 20. Mangialardi, G., A. Oikawa, C. Reni, and P. Madeddu. 2012. Bone marrow microenvironment: a newly recognized target for diabetes-induced cellular ating healing. damage. Endocr. Metab. Immune Disord. Drug Targets 12: 159–167. We conclude that driving intrinsic changes in mws are key to 21. Mace, K. A., T. E. 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