Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner This information is current as of October 2, 2021. Blanca Soler Palacios, Concha Nieto, Pilar Fajardo, Arturo González de la Aleja, Nuria Andrés, Ángeles Dominguez-Soto, Pilar Lucas, Ana Cuenda, José Miguel Rodríguez-Frade, Carlos Martínez-A, Ricardo Villares, Ángel L. Corbí and Mario Mellado Downloaded from J Immunol published online 26 June 2020 http://www.jimmunol.org/content/early/2020/06/25/jimmun ol.1901330 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/06/25/jimmunol.190133 Material 0.DCSupplemental

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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 June 26, 2020, doi:10.4049/jimmunol.1901330 The Journal of Immunology

Growth Hormone Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner

Blanca Soler Palacios,* Concha Nieto,† Pilar Fajardo,* Arturo Gonza´lez de la Aleja,† Nuria Andre´s,* A´ ngeles Dominguez-Soto,† Pilar Lucas,* Ana Cuenda,* Jose´ Miguel Rodrı´guez-Frade,* Carlos Martı´nez-A,* Ricardo Villares,* A´ ngel L. Corbı´,† and Mario Mellado*

Growth hormone (GH), a pleiotropic hormone secreted by the , regulates immune and inflammatory responses. In this study, we show that GH regulates the phenotypic and functional plasticity of macrophages both in vitro and in vivo.

Specifically, GH treatment of GM-CSF–primed monocyte–derived macrophages promotes a significant enrichment of anti- Downloaded from inflammatory and dampens the proinflammatory cytokine profile through PI3K-mediated downregulation of activin A and upregulation of MAFB, a critical transcription factor for anti-inflammatory polarization of human macrophages. These in vitro data correlate with improved remission of inflammation and mucosal repair during recovery in the acute dextran sodium sulfate–induced colitis model in GH-overexpressing mice. In this model, in addition to the GH-mediated effects on other immune cells, we observed that macrophages from inflamed gut acquire an anti-inflammatory/reparative profile. Overall, these data

indicate that GH reprograms inflammatory macrophages to an anti-inflammatory phenotype and improves resolution during http://www.jimmunol.org/ pathologic inflammatory responses. The Journal of Immunology, 2020, 205: 000–000.

rowth hormone (GH) is produced and secreted by control, GH is a pleiotropic hormone with myriad functions, in- somatotropic cells of the anterior pituitary gland in cluding anabolic actions on muscle and bone and catabolic effects G and is a main regulator of postnatal growth on adipose tissue (1). It also promotes fluid retention in the kidney and development. Although initially implicated in somatic growth (2), exerts metabolic effects on the liver (3), triggers sexual mat- uration (4), and induces resistance (5). Many of these ef- fects are mediated through insulin-like growth factor (IGF)-1, by guest on October 2, 2021 *Departamento de Inmunologı´a y Oncologı´a, Centro Nacional de Biotecnologı´a/Consejo which is produced in response to GH in liver and other tissues (6). Superior de Investigaciones Cientı´ficas, 28049 Madrid, Spain; and †Departamento de Biologı´a Molecular y Celular, Centro de Investigaciones Biolo´gicas/Consejo Studies both in vitro and in vivo have also demonstrated a role for Superior de Investigaciones Cientı´ficas, 28040 Madrid, Spain GH in immune regulation. GH (GHR) is expressed by ORCIDs: 0000-0001-6031-1896 (B.S.P.); 0000-0003-0790-3440 (C.N.); 0000-0001- various leukocyte subsets (7), including B and T cells (8) and 8398-7354 (P.F.); 0000-0002-9013-5077 (A.C.); 0000-0001-7562-6700 (R.V.); 0000- macrophages (9), and GH has been shown to mediate thymic 0001-6325-1630 (M.M.). development (8), promote T cell engraftment in SCID mice (10), Received for publication November 6, 2019. Accepted for publication May 24, 2020. improve B cell responses and Ab production (11, 12), and mod- This work was supported in part by grants from the Spanish Ministry of Science, Inno- vation and Universities (SAF2017-82940-R Agencia Estatal de Investigacio´n/Fondo ulate the activity of NK cells (13) and macrophages (9). In myeloid Europeo de Desarrollo Regional (AEI/FEDER), Unio´n Europea [UE] [to M.M.], cells, GH stimulates the proliferation of RAW 264.7 macrophages SAF2017-83785-R AEI/FEDER, UE [to A´ .L.C.] and FJCI-2016-29990 AEI/FEDER, (14) and functions as a human macrophage–activating factor (15). UE [to B.S.P.]), from the Redes Tema´ticas de Investigacio´n Cooperativa en Salud Program of Instituto de Salud Carlos III (RD12/0012/0006 and RD12/0012/0007, Beneficial effects of GH administration have been reported in Red de Investigacio´n en Inflamacio´n y Enfermedades Reuma´ticas), and the Regional autoimmunity, as it alters tolerization mechanisms through acti- Government of Madrid (B2017/BMD-3804 [to C.M.-A.]). vation of regulatory T cells and modulation of Th17 cell plasticity, The sequence presented in this article has been submitted to the Sequence Read reduces type I diabetes development (16), and contributes to im- Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA555143. prove collagen-induced arthritis symptoms (17). In inflammatory Address correspondence and reprint requests to Dr. A´ ngel L. Corbı´ or Dr. Mario Mellado, Department of Molecular and Cellular Biology, Centro de Investigaciones diseases, GH limits mucosal inflammation in experimental colitis Biolo´gicas/Consejo Superior de Investigaciones Cientı´ficas, Calle Raminor de Maeztu (18) and has protective effects in patients with active Crohn’s 9, 28040 Madrid, Spain (A´ .L.C.) or Centro Nacional de Biotecnologı´a/Consejo disease (19, 20). Superior de Investigaciones Cientı´ficas, Calle Darwin 3, Campus Cantoblanco U.A.M., 28049 Madrid, Spain (M.M.). E-mail addresses: [email protected] (A´ .L.C.) Tissue-resident macrophages are a heterogeneous and highly or [email protected] (M.M.) plastic cell population whose effector functions are dependent on The online version of this article contains supplemental material. their ontogeny and the surrounding tissue microenvironment. Their Abbreviations used in this article: Ct, cycle threshold; CTV, Cell Trace Violet; functional versatility enables them to play critical roles in both the DSS, dextran sodium sulfate; GH, growth hormone; GHR, GH receptor; GHTg, initiation and resolution of inflammatory and immune responses. GH-overexpressing transgenic; GM-Mf, macrophage primed by GM-CSF; GSEA; -set enrichment analysis; IGF, insulin-like growth factor; iNOS, inducible NO Reflecting this functional versatility, macrophages primed by GM- synthase; Listeria-GFP, Listeria monocytogenes coupled to GFP; M-Mf, macrophage CSF (GM-Mf) or macrophages primed by M-CSF (M-Mf) exert primed by M-CSF; rhGH, recombinant human GH; RNAseq, RNA sequencing; opposite functions in terms of T cell stimulation, inflammatory RT-qPCR, real-time quantitative PCR; WT, wild-type. cytokine production, tumor cell growth, and methotrexate sensi- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 tivity (21–25). Indeed, GM-Mf and M-Mf are considered as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901330 2 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES pro- and anti-inflammatory macrophages, respectively, as they dis- with ProLong Gold reagent with DAPI (Invitrogen, San Diego, CA) and play specific transcriptional profiles resembling those of mac- visualized on a TCS SP5 Confocal Microscope (Leica Microsystems, rophages from the synovia of rheumatoid arthritis patients or Wetzlar, Germany). tumor-associated macrophages, respectively (26, 27). Cell culture We report in this study that GH downmodulates the transcrip- Human PBMCs were isolated from buffy coats of healthy donors by density tional and cytokine profile of proinflammatory GM-Mf by re- gradient centrifugation using endotoxin-tested (,0.12 endotoxin units/ml) ducing, in a PI3K-dependent manner, the secretion of activin A, a Ficoll Paque Plus (GE Healthcare, Uppsala, Sweden), according to stan- involved in tilting the balance of macrophages toward dard procedures. Monocytes were purified from PBMCs by magnetic cell a proinflammatory phenotype (28), and upregulating the expres- sorting using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes (.94% CD14+ cells) were cultured at 0.5 3 106 sion of MAFB, a transcription factor that promotes the anti- cells per milliliter for 7 d (37˚C, 5% CO2) in RPMI 1640 medium sup- inflammatory polarization of macrophages (29, 30). In line with plemented with 10% FCS, sodium pyruvate and L-glutamine, and con- these in vitro results, we demonstrate that GH-overexpressing taining GM-CSF (1000 U/ml) or M-CSF (10 ng/ml) (both from transgenic (GHTg)–mice show improved remission of inflamma- ImmunoTools, Friesoythe, Germany) to generate GM-Mf and M-Mf, tion and mucosal repair during recovery from dextran sodium respectively. Cytokines were added every 2 d. To evaluate the direct effect of GH on monocytes, cells were incubated with recombinant human GH sulfate (DSS)–induced acute colitis. Indeed, together with possible (rhGH) (1 mg/ml Genotonorm; Pfizer, New York, NY; for 7 d, 37˚C, 5% effects on other immune cells, the polarization of gut macrophages CO2). To determine the effect of GH on differentiated GM-Mf and from GHTg mice was shifted toward an anti-inflammatory/reparative M-Mf, cells were treated with rhGH (1 mg/ml) in fresh medium for 24 h. profile during the recovery phase. Overall, our results point to GH as To evaluate the effect of IGF-1 on differentiated GM-Mf, cells were treated with rhIGF-1 (100 ng/ml) for 24 h, or it was added at the same time a regulatory factor for macrophage polarization under physiological

as GM-CSF. For signaling analysis, GM-Mf and M-Mf were stimulated Downloaded from and pathological settings. with rhGH (1 mg/ml) for the indicated times. Where appropriate, GM-Mf and M-Mf were treated with LY294002 (10 mM, 60 min) prior to rhGH addition. Functional polarization of GM-Mf and M-Mf was determined Materials and Methods by quantification of cytokine (TNF-a, IL-10, IL-6) production. Murine Mice GM-Mf and M-Mf bone marrow–derived macrophages were generated using human M-CSF (10 ng/ml; ImmunoTools) or murine GM-CSF Mice transgenic for bovine GH under the control of the phosphoenolpyr- (20 ng/ml; PreproTech), respectively, as previously described (21, 33). uvate carboxykinase promoter were on a C57BL/6J background (31) and http://www.jimmunol.org/ were maintained by repeated backcrosses to C57BL/6J females. A total of Global analysis 40 GHTg mice and 40 control littermates (10–14 wk old) were subjected to the DSS-induced experimental model of colitis, using matched sex ratios RNA sequencing (RNAseq) was performed on RNA obtained from three in each experiment. Mice were handled according to national and Euro- independent human samples of untreated or GH-treated (1 mg/ml, 24 h) pean Union guidelines, and experiments were approved by the Animal GM-Mf, using the BGISEQ-500 platform (BGI, Hong Kong, China), with Experimentation Ethics Committee of the Madrid regional government 20 million reads and 100 paired-end sequencing reads. Statistical analysis (PROEX 316- 15). of RNAseq data were performed following previously described proce- dures (29). Differential gene expression analysis by DSeq2 revealed no Acute experimental colitis induced by DSS statistically significant differences between the experimental conditions. RNAseq data have been deposited in the Sequence Read Archive (https://

Acute colitis was induced with 3% DSS (w/v, molecular mass 36,000– www.ncbi.nlm.nih.gov/sra) under accession no. PRJNA555143. Gene-set by guest on October 2, 2021 50,000 Da; MP Biomedicals, Solon, OH) in drinking water for 5 d, fol- enrichment analysis (GSEA) was performed using data sets available at the lowed by a recovery period of an additional 9 d as indicated. Mice were Web site (http://software.broadinstitute.org/gsea/index.jsp) (34), as well as monitored for body weight and signs of distress, diarrhea, and rectal data sets containing the genes differentially expressed by either human bleeding and (when required) were killed. The colon was removed, washed M-Mf (anti-inflammatory gene set) or GM-Mf (proinflammatory gene with PBS, and opened longitudinally for analysis. set) (GSEA68061) (28, 35), and the genes specifically downregulated in When needed, infiltrating immune cells were obtained by enzymatic MAFB-silenced human M-Mf (GSE84622) (29). digestion of the intestinal tissues. Briefly, tissue samples were incubated (30 min, 37˚C) in 10 ml HBSS with 0.4 mg/ml Dispase (Life Technologies) Quantitative real-time PCR and 10,000 U/ml penicillin/streptomycin. FBS was added to 5% (v/v), and tissue debris was removed by sequential filtering through 100, 70, and Mouse samples were collected in RNAlater, and total RNA was extracted 40 mm cell strainers. Cells were collected by centrifugation (150 3 g,10 with TriReagent (both from Sigma-Aldrich, St Louis, MO). Total RNA min, 4˚C). Colon cells were stained with combinations of fluorescence- from cultured cells was extracted using the Nucleospin RNA/Protein Kit labeled Abs to the cell surface markers CD45, CD11b, F4/80, Gr1, CD11c (Macherey-Nagel, Du¨ren, Germany). RNA was retrotranscribed using and CD86 and analyzed in a Gallios Flow Cytometer (Beckman Coulter, SuperScript II Reverse Transcriptase (Invitrogen), and real-time quantitative Brea, CA). The profiles obtained were analyzed with FlowJo software (BD PCR (RT-qPCR) analysis was performed with Power SYBR Green PCR Biosciences); leukocytes were gated as CD45+ cells. F4/80+ cells from Master Mix (Applied Biosystems, Foster City, CA). Triplicate samples mouse colon samples were isolated using anti-F4/80 MicroBeads Ultra- were quantified using the ABI Prism HT7900 sequence detection system Pure (Miltenyi Biotech) (∼80% purity). (Applied Biosystems). Oligonucleotides for selected genes were designed employing the Roche Universal ProbeLibrary Assay Design Center Histologic analysis and scoring (Table I). Assays were performed in triplicates and results normalized according to the expression levels of TBP and GAPDH; for murine sam- Full-length colon samples were longitudinally divided into two halves for ples, results were normalized using Actb expression levels. Results were histological and transcriptional analysis. Inflammation and crypt damage obtained using the 22ΔCt comparative threshold method for quantification. were scored on formalin-fixed, paraffin-embedded colon sections stained with H&E. The scoring scheme (32) included inflammation severity ELISA (0, none; 1, mild; 2, moderate; and 3, severe), inflammation extent (0, none; 1, mucosa; 2, submucosa; and 3, transmural), crypt damage (0, none; Supernatants from untreated and GH-treated GM-Mf or M-Mf were 1, basal one-third damage; 2, basal two-thirds damage; 3, crypt loss, sur- evaluated for the presence of cytokines and growth factors using com- face epithelium present; and 4, crypt and surface epithelium loss), and mercially available ELISA kits for activin A and CCL2 (Quantikine im- percentage of colon involvement (0, 0%; 1, ,25%; 2, 25–50%; 3, 50–75%; munoassay; R&D Systems, Minneapolis, MN) and for IL-6, IL-10, and and 4, .75%). Histological evaluation was performed by two independent TNF-a (Human ELISA Max Standard; BioLegend, San Diego, CA) fol- investigators in a double-blinded fashion. lowing the protocols supplied by the manufacturers. For analysis, formalin-fixed, paraffin-embedded colon sections were Flow cytometry stained with anti–arginase-1 (BD Biosciences, San Jose, CA) and K13-a anti–inducible NO synthase (iNOS) (Novus Biologicals, Littleton, CO) GHR expression was determined in human samples using an anti-GHR Abs and immunodetected with Alexa 488–labeled goat anti-rabbit IgG mAb (36). The incubations were done in the presence of 20 mg/ml hu- (Molecular Probes, Eugene, OR). Slides were mounted for fluorescence man IgG to prevent biding through the Fc portion of the Abs. Infiltrating The Journal of Immunology 3

Table I. Human and mouse oligonucleotides used for RT-qPCR analysis

Gene Forward Reverse hCCR2 59-CCCATCATCTATGCCTTCGT-39 59-GGCAATCCTACAGCCAAGAG-39 hEGLN3 59-ATCGACAGGCTGGTCCTCTA-39 59-GATAGCAAGCCACCATTGC-39 hFOLR2 59-GAGAGAGGCCAACTCAGACAC-39 59-CCAGACCATGTCTTTCTGTCC-39 hIGF1 59-TGTGGAGACAGGGGCTTTTA-39 59-ATCCACGATGCCTGTCTGA-39 hINHBA 59-CTCGGAGATCATCACGTTTG-39 59-CCTTGGAAATCTCGAAGTGC-39 hMMP12 59-TGTCACTACCGTGGGAAATAAG-39 59-AACACTGGTCTTTGGTCTCTCAG-39 hCCL2 59-AGTCTCTGCCGCCCTTCT-39 59-GTGACTGGGGCATTGATTG-39 hHMOX1 59-GCCATGAACTTTGTCCGGTG-39 59-TTTCGTTGGGGAAGATGCCA-39 hSTAB1 59-CGCCTGAAGCCCCACC-39 59-TCTTGCCACGGTGAGAAGTC-39 HIL-10 59-TCACTCATGGCTTTGTAGATGC-39 59-GTGGAGCAGGTGAAGAATGC-39 hCLEC5A 59-ACGGCTTCATTACCACAAGG-39 59-CTTGCTTGATAAAATTCCCAGTC-39 hTBP 59-CGGCTGTTTAACTTCGCTTC-39 59-CACACGCCAAGAAACAGTGA-39 hGAPDH 59-GAAGATGGTGATGGGATTTC-39 59-GAAGGTGAAGGTCGGAGTC-39 mArg1 59-AGCCGCTGGAACCCAGAGAGA-39 59-AACAAAGGCCAGGTCCCCGTG-39 mNos2 59-GGCAGCCTGTGAGACCTTTG-39 59-GCATTGGAAGTGAAGCGTTTC-39 mCd206 59-CCACAGCATTGAGGAGTTTG-39 59-ACAGCTCATCATTTGGCTCA-39 mYm1 59-TCTGGGTACAAGATCCCTGAA-39 59-TCATATGGAGATTTATAGAGGGGACT-39 mIl10 59-CAGAGCCACATGCTCCTA-39 59-GTCCAGCTGGTCCTTTGT-39 mInhba 59-ATCATCACCTTTGCCGAGTC-39 59-TCACTGCCTTCCTTGGAAAT-39 mTnf 59-TCTTCTCATTCCTGCTTGTGG-39 59-GGTCTGGGCCATAGAACTGA-39 Downloaded from mActb 59-TACCCAGGCATTGCTGACAG-39 59-ACTTGCGGTGCACGATGGA-39 immune cells from mouse intestinal tissues were phenotypically charac- [pH 8] and 1.5 M NaCl), membranes were incubated with anti-MAFB terized using anti-CD45, anti-CD19, anti-CD11b, anti-Gr1, anti-F4/80, (BioLegend), anti-pGSK3a/b, -AKT, -pAKT, ( Technol- anti-CD11c and anti-CD86 mAb (BioLegend), as described previously ogy, Danvers, MA), anti-ERK1/2 or anti-pERK1/2 (Santa Cruz Biotech- (37). Analysis was performed on a Gallios Flow Cytometer (Beckman nology, Santa Cruz, CA), or anti-vinculin (Sigma-Aldrich) Abs, followed http://www.jimmunol.org/ Coulter). by the corresponding HRP-conjugated secondary Ab. Allogeneic CD4+ T lymphocyte activation assay Statistical analysis PBMCs were isolated from healthy donors as described and depleted of For comparison of means, and unless otherwise indicated, statistical CD14+ cells using specific MicroBeads (Miltenyi Biotec) and magnetic significance of the data were evaluated using paired Student t test and cell sorting. Allogenic cells were then labeled with 5 mM Cell Trace Violet two-way ANOVA (Sidak multiple comparations test). The p value , 0.05 (CTV; Invitrogen) for 20 min at 37˚C and washed twice with RPMI 1640 was considered significant (*p , 0.05, **p , 0.01, ***p , 0.001, medium containing 10% FCS. CTV-labeled cells were seeded onto 96-well ****p , 0.0001). Statistical parameters used in the GSEA analysis plates containing GM-Mf (1 or 2 3 105 cells per well) or M-Mf (1 3 104 were as previously described (34). cells per well) in the presence or absence of rhGH (1 mg/ml, 24 h, 37˚C). by guest on October 2, 2021 Cells were cocultured in RPMI 1640 medium with 10% FCS (10 d) and Results supplemented with rhGH (1 mg/ml) 24 h before cell proliferation eval- uation. As negative controls, we used isolated CTV-labeled cells in Improved mucosal repair from DSS-induced colitis in GHTg the presence or absence of rhGH. CTV-labeled cells exposed to PMA mice correlates with altered macrophage polarization (100 ng/ml, 24 h) were used as positive control. Cell viability and pro- Previous reports have demonstrated that GH modulates immune liferation (dye dilution) were determined on CD4+ cells using a Gallios Flow Cytometer (Beckman Coulter). The percentage of dividing cells (16, 17) and inflammatory (17–20) responses. Because GH has also was calculated using FlowJo (Tree Star, Ashland, OR). been shown to modulate adipocyte differentiation in a macrophage- dependent manner (9), we sought to determine whether GH affects Phagocytosis assay macrophage polarization during inflammatory responses. To this Listeria monocytogenes coupled to GFP (Listeria-GFP), kindly provided end, we initially made use of a murine model of DSS-induced by Dr. E. Veiga (Centro Nacional de Biotecnologı´a/Consejo Superior de acute colitis in which GH limits pathological conditions (18). Investigaciones Cientı´ficas, Madrid, Spain), was grown in brain-heart in- fusion medium (overnight, 37˚C), diluted, recovered at midlogarithmic We observed that both GHTg and wild-type (WT) mice survived growth phase (OD600nm = 0.5), and washed in PBS before use. 5 d of treatment with 3% DSS. We did not conduct survival Cells were infected with Listeria-GFP as described (38). Briefly, 30 min analysis during the recovery phase as euthanasia was performed at (37˚C, 5% CO2) after inoculation, gentamicin (100 mg/ml) was added the designed time points to cover the kinetics of immune cell (60 min; 37˚C) to cultures to eliminate extracellular bacteria. Cells were infiltration, but we did not observe differences in mortality in any then extensively washed, fixed with paraformaldehyde (4% w/v, 20 min, room temperature) and seeded (30 min, 37˚C) onto poly-L-lysine–coated of the groups of mice maintained until the end of the experiment plates (20 mg/ml, 10 min, room temperature; Sigma-Aldrich) prior to their (9 d). Along the 5-d DSS treatment, both GHTg and WT mice incubation with wheat germ agglutinin (Thermo Fisher Scientific, Wal- were positive for occult blood (Hemoccult test) with no significant tham, MA) and DAPI (Sigma-Aldrich) for 5 min at 4˚C. Cell staining was differences between the two groups of mice (Fig. 1A). Likewise, evaluated by confocal microscopy (Zeiss Axiovert LSM 510-META) and images were analyzed and quantified using ImageJ software (National weight loss (Fig. 1B) and histological scores for severity of in- Institutes of Health). flammation were similar in both groups (Fig. 1C, upper panel). Lastly, we also detected comparable levels of destruction of the Western blotting crypt structure, a disturbed epithelial layer and massive infiltration Cell lysates were prepared in RIPA buffer (50 mM HEPES, 150 mM NaCl, of inflammatory cells in mucosal and submucosal layers in both 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM NaF, mouse strains (Fig. 1C, middle [WT] and bottom [GHTg]; for 5 mM EDTA, 0.1 mM NaPO4, 5% ethylene glycol) supplemented with higher resolution images of these micrographs, see Supplemental 10 mg/ml of aprotinin and , 10 mM sodium orthovanadate, and 1 mM PMSF. Lysates (50 mg) were subjected to electrophoresis and Fig. 1A [WT] and Supplemental Fig. 1B [GHTg]), confirming the transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Ger- lack of significant differences between the two groups of mice many). After blocking with 5% BSA in TBS buffer (100 mM Tris 1 HCl during disease onset. 4 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Improved mucosal repair after DSS-induced colitis in GHTg mice correlates with altered expression of macrophage polarization–specific markers. (A) Percentage of GHTg and WT mice that were Hemoccult positive as determined by daily evaluation during DSS treatment and recovery. (B) Percentage of body weight change during DSS treatment and recovery in GHTg mice and WT littermates. Mean 6 SD of 44 mice are shown. Two-way ANOVA. **p , 0.01. (C) Histologic inflammation score and the representative photomicrographs of H&E-stained sections from longitudinally opened and spirally arranged colon obtained from GHTg mice and WT littermates at day DSS + 1. Original magnification 310; scale bar, 1000 mm. (D) Representative photomicrographs of H&E-stained colon sections from GHTg mice and WT littermates at day DSS + 7. Scale bar, 1000 mm (black arrows: area with crypt damage; white arrows: area showing infiltrating foci). Zoom images (original magnification 340): regions of the colon with immune infiltration (insets) that were obtained from consecutive sections stained with anti-arginase and anti-iNOS mAbs. Nuclei were counterstained with DAPI. Scale bar, 50 mm. Black arrow depict area with crypt damage; white arrow depict area showing infiltrating foci. (E) Histologic scores for severity, extent of inflammation, crypt damage, and percentage of colon involvement. Mean 6 SD of four mice at DSS + 7 are shown. Paired Student t test. ***p , 0.001, ****p , 0.0001.

However, although both groups of mice showed similar relative group showing an increase in the number of iNOS+ cells com- weight loss during disease onset, the recovery phase was sig- pared with GHTg mice (Fig. 1D, bottom). Of note, histological nificantly quicker in GHTg mice in terms of changes in body scores were also significantly different between GHTg and WT weight (Fig. 1B). Histological analysis of samples harvested 7 d littermates at this stage, with a lower grade of mucosal de- after the DSS treatment (DSS + 7) (Fig. 1D, upper, Fig. 1E; struction and inflammation in the colon of GHTg mice (Fig. 1E). Supplemental Fig. 1C [WT] and Supplemental Fig. 1D [GHTg] Indeed, flow cytometry analysis of the infiltrating macrophages for higher resolution images), further supported the role of GH in of the intestinal tissues revealed a higher percentage of anti- enhancing tissue repair after DSS-induced colitis based on the inflammatory macrophages (CD45+F4/80+Gr1+CD11b+ CD86high) extent and severity of inflammation, crypt damage, and per- in GHTg mice (0.72 6 0.13) than in controls (0.28 6 0.09) centage of affected colon. To assess the state of macrophage (Table II). We also found a higher CD4+/CD8+ ratio in GHTg than polarization at the recovery phase, we determined the expression in control animals (0.74 versus 0.38) in the recovery phase (DSS + of conventional proinflammatory (iNOS) and anti-inflammatory/ 7), whereas the opposite occurred during inflammation (DSS + 1 reparative (arginase 1) macrophage markers in the recovering d, GHTg 0.24 versus controls 0.68) (Table II). Notwithstanding tissue. Immunohistological analysis of DSS + 7 mice revealed the possibility of a GH-mediated effect on other immune cells, that the proportion of arginase-1+ cells infiltrating the colon was which might also contribute to improved recovery from DSS- higher in GHTg mice than in WT littermates, with the latter induced colitis in GHTg mice, our results indicate a GH-triggered The Journal of Immunology 5

Table II. Immune cell infiltration (gated for CD45+) of affected intestinal tissue at DSS + 1 and DSS + 7 in WT and GHTg mice subjected to the DSS-induced model of colitis

DSS + 1 DSS + 7

WT GHTg WT GHTg CD3+ CD4+ CD82 8.20 6 2.46 4.52 6 0.84 7.14 6 0.92 9.32 6 1.60 CD42 CD8+ 12.04 6 5.57 18.41 6 4.63 18.49 6 7.90 12.56 6 3.07 CD3+ CD69+ CD4+ CD82 2.01 6 0.45 1.61 6 0.33 2.40 6 0.26 2.39 6 0.61 CD42 CD8+ 8.14 6 3.20 16.38 6 4.81 14.71 6 6.41 8.41 6 2.65 F4/80+ Gr1+ CD11chighCD86low 3.86 6 1.52 5.42 6 2.27 0.44 6 0.01 0.19 6 0.09 CD11clowCD86high 1.82 6 0.36 2.05 6 0.39 0.28 6 0.09 0.72 6 0.13 change in the polarization state of macrophages within the inflamed revealed a significant positive enrichment of M-Mf-specific tissue. genes, but not GM-Mf-specific genes (28, 35), following GH exposure (Fig. 2D). We validated these findings by RT-qPCR GH treatment modulates phenotypic and functional GM- analysis, which showed that GH treatment of GM-Mf signifi- macrophage polarization cantly upregulated the expression of genes associated with the The above results led us to hypothesize that GH might directly M-Mf anti-inflammatory profile (HMOX1, STAB1, IGF1, and modulate macrophage polarization. To address this issue, we FOLR2), whereas the GM-Mf–specific genes CCR2, MMP12, Downloaded from switched to the analysis of human monocyte–derived proin- and EGLN3 were significantly downregulated (Fig. 3A). The ef- flammatory (GM-Mf) and anti-inflammatory (M-Mf) macro- fect of GH was specific for GM-Mf, as it failed to modify the phages. Human GHR was found on monocytes, proinflammatory transcriptome of M-Mf (Fig. 3B) or monocytes (Supplemental GM-Mf, and anti-inflammatory M-Mf, as demonstrated by flow Fig. 2B). Overall, these data revealed that GH modulates the hu- cytometry using a GHR-specific mAb (36) (Supplemental Fig. man macrophage gene signature, favoring the expression of genes

2A). Because the expression of the major GH effector molecule specifically linked to anti-inflammatory M-Mf. To correlate these http://www.jimmunol.org/ IGF-1 (39) is restricted to anti-inflammatory M-Mf (28), we observations with data from GHTg mice, we derived murine GM- evaluated the influence of GH on macrophage polarization by Mf and M-Mf from bone marrow progenitors using GM-CSF or comparing the transcriptome of GM-Mf untreated or exposed to 1 M-CSF, respectively, followed by GH exposure. RT-qPCR anal- mg/ml of rhGH for 24 h (Fig. 2A–C). GSEA of the RNAseq data ysis showed that GH treatment of murine GM-Mf significantly by guest on October 2, 2021

FIGURE 2. GH modulates the global transcriptomal signature of GM-Mf.(A) Relative mRNA expression of M-Mf–specific genes (HMOX1, IGF1, FOLR2) in untreated or rhGH-treated (1 ng/ml–2 mg/ml) GM-Mf. Results are shown as 22ΔCt relative to the mean of internal GAPDH and TBP expression. Determinations were done in triplicates; box plots represent the mean and the minimum to maximum values (n = 3). Paired Student t test. *p , 0.05. (B) Experimental design of the gene-profiling experiment. (C) MA plot representing the comparative analysis of the transcriptome from untreated versus rhGH- treated GM-Mf.(D) GSEA on the “log fold change–ranked” list of genes obtained from untreated versus rhGH-treated GM-Mf according to limma analysis, using the previously defined M-Mf (left panel) and GM-Mf (right panel) gene sets (GSE68061). Vertical black lines indicate the position of each of the genes comprising the M-Mf and GM-Mf gene sets. 6 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. GH drives an M-Mf–specific gene signature in human and murine GM-Mf, but does not alter the expression profile of M-Mf.(A) Relative mRNA expression of marker genes corresponding to GM-Mf (CCR2, MMP12, EGLN3) and M-Mf (HMOX1, STAB1, IGF1, FOLR2) polarization in untreated (white bars) or rhGH-treated (gray bars) GM-Mf.(B) Relative mRNA expression of marker genes as in (A) in untreated (white bars) or rhGH- treated (gray bars) M-Mf. In both cases, results are shown as 22ΔCt relative to the mean of internal GAPDH and TBP expression. Determinations were done in triplicates; box and whisker plots represent the mean, lower and upper deciles, and the minimum to maximum values (n = 8). Paired Student t test. (C) Relative mRNA expression of mouse GM-Mf–specific (Nos2, Inhba, Tnf) and M-Mf–specific (Arg1, Cd206, Ym1) polarization genes in untreated or rhGH-treated GM-Mf and M-Mf. Results are shown as mean 6 SD values of 22DCt relative to internal Actb expression. Determinations were done in triplicates (n = 4). Paired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. upregulated the expression of genes associated with the M-Mf GM-Mf or M-Mf and found that GH also induced a significant anti-inflammatory profile (Arg1, Cd206, and Ym1), whereas it increase in the levels of CCL2 in GM-Mf supernatants (Fig. 4A). downregulated the expression of GM-Mf–associated inflamma- Altogether, these results indicate that GH treatment reprograms tory markers (Nos2, Inhba, and Tnf) (Fig. 3C). Thus, these data human macrophages, as it impairs the phenotypic and functional confirmed that GH also modulates the mouse macrophage gene polarization of GM-Mf and causes a shift toward an anti- signature, favoring the acquisition of anti-inflammatory markers. inflammatory profile. As macrophage polarization is essentially defined by the pre- f vailing cytokine secretion profile, we assessed the ability of GH to GH treatment of GM-M reduces activin secretion influence the cytokine signature of GM-Mf and M-Mf. To do this, Activins are pluripotent growth and differentiation factors of the we cultured both macrophage populations in the absence or TGF-b superfamily (40). Like TGF-b, activins exert immunosti- presence of rhGH for 24 h and then measured the levels of TNF-a, mulatory and immunosuppressive functions (41). Activin A is IL-6, and IL-10 in the corresponding culture supernatants. Results considered a crucial modulator of inflammatory responses (42) showed that, compared with control cultures, GH exposure for and, in fact, activin A skews macrophage polarization by pro- 24 h triggered a significant decrease of TNF-a and a significant moting the acquisition of a proinflammatory phenotype (28). We increase of IL-10 in GM-Mf supernatants, whereas the levels of thus measured the levels of activin A in the culture supernatants of IL-6 were unaffected (Fig. 4A). By contrast, and in accordance untreated or rhGH-treated GM-Mf and M-Mf and observed that with the gene profile of M-Mf, GH treatment failed to modify the GH treatment led to a significant decrease in activin A in GM-Mf cytokine signature of anti-inflammatory macrophages (Fig. 4A). cultures (Fig. 4B), whereas it did not modify activin A secretion Because M-Mf secrete CCL2 (28), we also evaluated CCL2 from M-Mf (Fig. 4B). These data agree with the decrease in the levels in the culture supernatant of untreated or rhGH-treated mRNA levels of the activin A-encoding gene (INHBA) (43) The Journal of Immunology 7

detected in rhGH-treated GM-Mf (Fig. 4C). Thus, our results suggest an active role of GH in downregulating activin A ex- pression and secretion by GM-Mf, which would ultimately con- tribute to shape the transcriptional and functional profile of these macrophages. The physiological effects of GH are partially mediated by IGF-1 produced in the liver (44). IGF-1 is nonetheless produced by several cell types (45), including M-Mf (46). We thus evaluated whether GH-induced GM-Mf repolarization might be mediated by IGF-1. We found that treatment of fully differentiated proin- flammatory GM-Mf with IGF-1 had no effect on their charac- teristic gene expression profile (Supplemental Fig. 3A, 3B), or on activin A secretion (Supplemental Fig. 3C), suggesting that the polarizing action of GH is independent of IGF-1 production. GH treatment of GM-Mf macrophages induces MAFB expression Recent reports indicate that MAFB plays an essential role in human macrophage anti-inflammatory polarization (29, 30). MAFB is known to constrain the ability of M-CSF to instruct myeloid cell Downloaded from proliferation, promotes macrophage differentiation (47), and controls the acquisition of an anti-inflammatory transcriptional profile of M-Mf (29). We thus performed GSEA on the GH- treated GM-Mf RNAseq data and observed a significant pos- itive enrichment of MAFB-regulated genes (Fig. 5A). Indeed,

Western blot analysis showed that GH treatment increased the http://www.jimmunol.org/ level of MAFB protein in GM-Mf, whereas no changes were observed in the already elevated expression of MAFB in M- Mf (29) (Fig. 5B, left). In line with these results, and with the previous demonstration that MAFB protein stability is con- trolled by GSK3b-mediated phosphorylation and subsequent proteasomal degradation (48), we found a significant increase in the inactivating serine 9 phosphorylation of GSK3b (49) in rhGH-treated GM-Mf but not in rhGH-treated M-Mf (Fig. 5B, right). The relevance of these results was supported by guest on October 2, 2021 by the increase in the expression of MAFB-dependent genes (IL-10, CCL2) and the decreased levels of the proinflammatory gene CLEC5A, whose expression is inhibited by MAFB (29), in GH-treated GM-Mf (Fig. 5C). These observations indi- cate that GH modifies the polarization state of GM-Mf likely through the inhibition of GSK3b, which would block MAFB degradation and result in the upregulation of MAFB- dependent genes and the subsequent acquisition of an anti- inflammatory profile. Kinetic analysis indicated that MAFB expression in GM-Mf was evident ∼1 h after exposure to GH and that GH-induced serine 9 phosphorylation of GSK3b remained elevated 4 and 24 h after treatment (48, 49) (Fig. 6A, Supplemental Fig. 4). To dissect the molecular mechanisms underlying these events, and based on previous data on the regulation of GSK3b activity (50, 51), we tested the initial signaling events triggered by GH in GM- Mf. We detected GH-mediated phosphorylation of ERK1/2 and AKT after 30 min and 1 h, respectively (Fig. 6A, Supplemental Fig. 4). Of note, blocking PI3K activity in GM-Mf with LY294002 (52) inhibited the GH-mediated phosphorylation of GSK3b and the increase in MAFB expression (Fig. 6A) and FIGURE 4. GH treatment modifies the secreted cytokine profile of GM- activin A secretion (Fig. 6B). These data are in accord with a Mf and decreases the secretion of activin A. (A). TNF-a, IL-6, IL-10, and previous report describing a role for AKT in GSK3b inactivation CCL2 abundance in culture supernatants of untreated (white bars) or rhGH-treated (gray bars) GM-Mf and M-Mf by ELISA. Shown are the mean 6 SD of triplicate determinations (n = 8). Paired Student t test. (B) Abundance of activin A in culture supernatants of untreated (white bars) or mean of internal GAPDH and TBP expression. Results in (B) and (C) rhGH-treated (gray bars) GM-Mf and M-Mf by ELISA. (C) INHBA ex- correspond to triplicate determinations (n = 8); box and whisker plots pression determined by RT-qPCR in untreated (white bars) or rhGH-treated represent the mean, lower and upper deciles, and the minimum to maxi- 2Δ (gray bars) GM-Mf and M-Mf. Results are shown as 2 Ct relative to the mum values. Paired Student t test. *p , 0.05, **p , 0.01. 8 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES Downloaded from http://www.jimmunol.org/

FIGURE 5. GH-mediated upregulation of MAFB in GM-Mf correlates with M-Mf marker expression. (A) GSEA analysis on the “log fold change– ranked” list of genes obtained from untreated versus rhGH-treated GM-Mf according to limma analysis, using the previously defined list of genes downregulated in MAFB-silenced cells (GSE84622). (B) Representative Western blot of MAFB (left panel) and pGSK3b (right panel) expression in lysates of untreated or rhGH-treated GM-Mf and M-Mf. Vinculin was used as a loading control. Densitometric analysis of MAFB and pGSK3b levels normalized to that of vinculin. Mean 6 SD (n = 8). Paired Student t test. **p , 0.01, ****p , 0.0001. (C) CLEC5A, CCL2, and IL-10 mRNA expression levels by guest on October 2, 2021 determined by RT-qPCR in untreated or rhGH-treated GM-Mf and M-Mf. Results are shown as 22ΔCt relative to the mean of internal GAPDH and TBP expression and correspond to triplicate determinations (n = 8); box and whisker plots represent the mean, lower and upper deciles, and the minimum to maximum values. Paired Student t test. *p , 0.05.

(49). Correspondingly, LY294002 pretreatment of GM-Mf abro- rhGH-treated GM-Mf with allogeneic T cells for 10 d and ex- gated the GH-mediated upregulation of CCL2, FOLR2, IGF1, amined T cell proliferation by evaluation of dye dilution using and IL-10 and the downregulation of CLEC5A and IHNBA (Fig. flow cytometry (Fig. 7B). Results showed that rhGH-treated 6C). Overall, these results demonstrate the involvement of a GM-Mf promoted a significantly lower proliferation of allo- PI3K-GSK3b-MAFB axis in the GH-mediated polarization of genic T cells than untreated GM-Mf (Fig. 7C). These results GM-Mf. indicate that GH-mediated repolarization of GM-Mf not only affects phenotypic and transcriptional parameters, but also nega- GH treatment alters the effector functions of tively affects effector functions that characterize proinflammatory GM-Mf macrophages GM-Mf (T cell activation) while favoring the acquisition of We next examined whether the phenotypic, transcriptional, and functions preferentially associated with anti-inflammatory M-Mf signaling effects of GH on GM-Mf influenced the effector (bacterial capture and uptake). functions normally mediated by this macrophage subtype. As innate immune cells, macrophages efficiently phagocytose Colon-infiltrating recovery-phase macrophages from pathogens and stress-inducing agents (53), with M-Mf dis- DSS-induced colitis exhibit an enhanced anti-inflammatory playing a higher phagocytic activity than GM- Mf (22). We thus MAFB-dependent signature in GHTg mice tested whether GH treatment altered the phagocytic properties of To investigate the pathophysiological relevance of the GH-induced GM-Mf by coculturing untreated and rhGH-treated GM-Mf or MAFB-mediated switch in macrophage polarization, we evaluated M-Mf with Listeria-GFP for 30 min, and determining bacterial the polarization state of colon-infiltrating recovery-phase macro- uptake by confocal microscopy. Results showed that bacterial phages from mice subjected to DSS-induced colitis. In agreement uptake was significantly higher in rhGH-treated GM-Mf than with immunohistochemistry results, analysis of colon samples from in untreated cells, whereas this effect was absent in M-Mf DSS + 7 mice revealed a higher expression of Arg1 and signifi- (Fig. 7A). cantly diminished levels of Nos2 in GHTg mice (Fig. 8A). We Macrophages are also Ag-presenting and T cell–activating cells then isolated the infiltrated F4/80+ cells at DSS + 7 d (Fig. 8B) and (54), and GM-Mf display a considerably higher T cell–activating observed a significant increase of Arg1, Cd206, and Ym1, three capacity than M-Mf (55). To determine whether GH modifies the specific markers of anti-inflammatory macrophages (56, 57), in T cell–activating ability of GM-Mf, we cocultured untreated and cells from GHTg mice compared with control littermates, and a The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. GH-mediated MAFB upregulation in GM-Mf is dependent on PI3K activity. (A) Western blot analysis of MAFB, pGSK3a/b, pAKT, and pERK1/2 expression in lysates of untreated or rhGH-treated GM-Mf pretreated or not with the PI3K inhibitor LY294002. Anti-AKT and anti-ERK1/2 mAbs were used as loading controls. One representative experiment from five is shown. (B). ELISA determination of activin A in culture supernatants of untreated (solid bars) or rhGH-treated (1 mg/ml, 24 h, striped bars) cells. GM-Mf were pretreated with LY294002 (gray background bars) or untreated (white background bars). Mean 6 SD from three independent experiments are shown. Paired Student t test. *p , 0.05, ****p , 0.0001. (C) FOLR2, IL-10, CCL2, IGF1, CLEC5A, and INHBA mRNA expression levels were determined by RT-qPCR in untreated (white background bars) and pretreated with LY294002 (gray background bars) GM-Mf treated with GH (striped bars) or untreated (solid bars). Data show mean 6 SD of triplicate determinations, relative to the mean of GAPDH and TBP expression (n = 3). Paired Student t test. *p , 0.05, **p , 0.01. reduced expression of Nos2, Tnf, and Inhba (Fig. 8C). These data GHR-knockout mice, the loss of GH action exacerbates inflam- corroborate the switch in macrophage polarization toward an anti- mation in adipose tissue (58). We previously showed that trans- inflammatory phenotype observed in GHTg mice. Accordingly, genic expression of GH ameliorates inflammation in diabetic although a direct effect of GH on other immune cells cannot be NOD mice (16) and reduces the incidence of arthritis in a discarded, the presence of macrophages with a more reparative collagen-induced murine model (17). Similarly, GH attenuates the and less proinflammatory phenotype correlates with the improved inflammatory burden in inflammatory bowel disease (59) and re- recovery from DSS-induced colitis in GHTg mice. Overall, these duces NF-kB activation in colitis (18). A preliminary study in results support the in vivo relevance of the macrophage-repolarizing humans revealed that GH might be a beneficial treatment for ability of GH, and further illustrate the capacity of GH to drive Crohn’s disease (20). Although some of these observations have macrophage polarization to a more anti-inflammatory/reparative been associated with GH resistance processes, they might also be phenotype. related to a potential role of GH modulating the immune system and favoring anti-inflammatory responses. Discussion The phenotypic and functional heterogeneity of macrophages is GH modulates immune function through direct effects on immune essential to create the appropriate extracellular environment for cells that express the GHR. For example, in macrophage-specific immune responses to occur. Macrophages are highly plastic and 10 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES

anti-inflammatory genes (Arg1, Cd206, and Ym1) and reduction of GM-Mf-specific inflammatory genes (Nos2, Inhba, and Tnf). Functional in vitro analysis showed that GH reduces TNF-a re- lease by GM-Mf and concomitantly increases their ability to secrete IL-10. Likewise, we also detected CCL2 secretion by rhGH-treated GM-Mf. By contrast, GH failed to alter TNF-a, IL-10, or CCL2 production in M-Mf. Further studies are needed to determine whether these findings are a consequence of a partial effect of GH on M-Mf, a dosage-mediated effect, or other unknown cofactors required to complete the repolarization process. Initially characterized as inducers of follicle-stimulating hor- mone production, activins are now recognized to regulate the growth of numerous cell types, for example, by contributing to the maintenance of pluripotency in embryonic stem cells, and exerting antitumorigenic effects (41). Moreover, activin A is a crucial modulator of inflammatory responses (42), and its expression is upregulated in response to inflammatory medi- ators (62, 63). Activin A, which is encoded by the INHBA gene, induces arginase A expression in murine peritoneal Downloaded from macrophages and is important for the differential gene ex- pression profiles and effector functions exhibited by proin- flammatory macrophages (28). We observed that, in vitro, rhGH treatment of GM-Mf reduces the expression of INHBA and activin A, pointing to a possible role for GH in triggering GM-Mf repolarization. http://www.jimmunol.org/ We also found that GH triggers MAFB upregulation via a FIGURE 7. GH treatment increases bacterial uptake by GM-Mf and PI3K-dependent pathway. MAFB is a member of the MAF diminishes the ability of macrophages to trigger allogenic T cell prolif- family of transcription factors and is involved in many cellular eration. (A) GM-Mf treated or not with rhGH and M-Mf were infected functions, such as control of lymphangiogenesis (64), or dif- with Listeria-GFP, fixed, and added to poly-L-lysine–coated plates. Left ferentiation of pancreatic a and b cells (65, 66). Within the panel shows representative confocal images of GFP fluorescence (green), murine myeloid lineage, MAFB restricts the ability of M-CSF membrane staining (wheat germ agglutinin, red) and nuclei (DAPI, cyan). to trigger myeloid cell proliferation (67), and also critically Scale bar, 10 mm. Right panel shows GFP fluorescence quantitation using determines the acquisition of the anti-inflammatory tran- ImageJ. Equatorial-plane images from 200 to 250 cells were collected in by guest on October 2, 2021 scriptional and functional profiles in human macrophages (29, random fields. Results are mean 6 SD of background-subtracted GFP intensities (arbitrary units [a.u.]) (n = 3) Paired Student t test. (B)Rep- 30). Simultaneously, it negatively regulates genes associated resentative flow cytometry panels to illustrate the procedure for CD4+ with proinflammatory polarization (29, 30). Our GSEA of proliferation determination. In the coculture, viable CD4+ cells were RNAseq data indicated a significant enrichment of MAFB- identified using anti–CD4-PerCpCy5.5 mAb and then CTV dilution specific genes in rhGH-treated GM-Mf. In the current study, (gated) was evaluated. (C) Percentage of CD4+ cell proliferation, measured GH reduced activin A secretion and promoted MAFB upreg- by CTV dilution in cells cocultured with GM-Mf or M-Mf in the absence ulation, and thus supporting the role of GH in GM-Mf reprog- + (2) or presence (+) of rhGH. As controls proliferation of CD4 cells ramming. In accord with these results, activin A was shown to cultured in the absence or presence of rhGH without macrophages and of + suppress MAFB expression in a pancreatic islet cell line (68). CD4 cells exposed to PMA for 24 h are shown. Results are expressed as a MAFB stabilization is controlled by GSK3b (48), an inhibi- percentage of the maximum CD4+ cell proliferation (CD4+ cell prolifer- tory serine/threonine kinase that is inactivated by phosphory- ation in the presence of GM-Mf without GH stimulation, 100%). Mean 6 SD (n = 5). Paired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001, lation at serine-9 (49). We found that these GH-mediated ****p , 0.0001. processes were blocked in cells pretreated with LY294002, a classical PI3K inhibitor, suggesting a role for p-AKT in this signaling event. Activated AKT has been associated with can adopt proinflammatory or anti-inflammatory/resolving prop- phosphorylation of GSK3b and inhibition of its activity (69), erties depending on microenvironmental signals, and the deregu- indicating that GSK3b is a substrate for AKT, as was described lation of this balance leads to chronic inflammatory diseases (60). several years ago (70). Nonetheless, the effect triggered by GH Modulation of macrophage polarization is thus a potential target was not completely abolished by LY294002 treatment. Al- for therapeutic intervention in these chronic pathologies (61). The though several mechanisms might be behind this effect, our results of the current study reveal that GH does not promote results indicate that GH also triggered ERK1/2 activation, a monocyte polarization, but repolarizes the GM-Mf phenotype to pathway that has also been implicated in GSK3b inactivation an anti-inflammatory phenotype. Consistent with this, GSEA of (71, 72). RNAseq data demonstrated a significant enrichment of M-Mf– The polarization state of macrophages not only conditions specific genes in rhGH-treated GM-Mf, some of them also con- the secreted cytokine profile, but also affects other cellular firmed by RT-qPCR analysis (HMOX1, IL-10, and IGF1), whereas processes such as phagocytosis, production of reactive oxygen the expression of others associated with GM-Mf were reduced species (22), and capacity to trigger allogenic T cell prolifer- (INHBA, MMP12, and EGLN3). The reprogramming effect of GH ation. As expected, bacterial capture ability was higher in on human macrophages was also seen in murine GM-Mf, whose M-Mf than in GM-Mf (22), and we found that GH treatment treatment with GH resulted in upregulation of M-Mf–associated increased the bacterial capture and microbial killing properties The Journal of Immunology 11

of GM-Mf. In addition, GH treatment significantly limited the ability of GM-Mf to promote allogeneic T cell proliferation. All of these data confirm the role of this hormone in repola- rizing GM-Mf. Available evidence supports the use of GH to treat inflam- matory bowel diseases, including Crohn disease and ulcerative colitis (73). Although in most of the cases the observed effects have been associated with metabolic changes triggered by GH (3), a role for GH in enhancing survival, remission of inflam- mation, and mucosal repair during recovery has been linked to a local decrease of IL-1b production in a murine model of DSS- induced colitis in GHTg mice (74). This in vivo data suggest that GH reduces inflammation by altering the cytokine profile se- creted by immune cells. We thus analyzed the in vivo relevance of GH-mediated GM-Mf reprogramming by comparing the phenotype of macrophages infiltrating the intestinal tissue of GHTg and WT littermates after DSS-induced colitis. As reported (74), GH did not influence the susceptibility of mice to acute DSS-induced colitis, but instead improved the remission of in- flammation and mucosal repair during recovery. Importantly, Downloaded from GHTg mice presented a high proportion of infiltrating M-Mf (arginase 1+) when compared with WT littermates, which showed GM-Mf (iNOS+) infiltration. Flow cyometry analysis of these isolated macrophages confirmed the data. In GHTg mice, we found higher numbers of anti-inflammatory macrophages (CD45+F4/80+Gr1+CD11b+CD86high)thanincontrols.Inaddi- http://www.jimmunol.org/ tion, the infiltrating F4/80+ cells in GHTg mice showed higher expression of Arg1, Cd206,andYm1, three genes whose ex- pression marks murine anti-inflammatory macrophages (56, 57), whereas the expression of inflammatory genes (Nos2, Inhba,and Tnf)wasreduced. It has been previously described that IGF-1 secreted by satellite cells, myofibers, fibroblasts, endothelial cells, and in- flammatory cells, plays an important role in muscle regeneration by guest on October 2, 2021 (75), with the authors of this study proposing an autocrine role for IGF-1 in modulating murine macrophage polarization to an anti-inflammatory phenotype. This observation correlates with in vitro analyses showing that both murine and human mac- rophages express significant quantities of IGF-1 propeptides, which are upregulated by stimulation with so-called M2- polarizing stimuli, including IL-4, IL-13, or M-CSF (76, 77). Our in vitro studies clearly showed an increase of IGF-1 pro- ductionbyGM-Mf treated with GH, but we excluded an effect ofIGF-1onGM-Mf reprogramming, per se. It is nonetheless known that, in vivo, IGF-1 influences the magnitude of tissue inflammation by redirecting epithelial cells to a phagocytic phenotype (78). In conclusion, our findings provide strong support for a role of GH in reprograming inflammatory macrophages to an anti- inflammatory phenotype, which contributes to dampening the in- flammatory microenvironment where the immune response takes place.

shown. Student t test. *p , 0.05, **p , 0.01, ****p , 0.0001. (B) After enzymatic digestion of the intestinal tissue of both GHTg and control littermates at DSS + 7 d (left panel, Before), F4/80+ cells were isolated using magnetic beads, and the sample purity analyzed by flow cytometry FIGURE 8. Colon-infiltrating recovery-phase macrophages from DSS- using an anti-F4/80 mAb (gated for CD45+) (right panel, After). (C) Ex- induced colitis exhibit an enhanced anti-inflammatory and MAFB-depen- pression levels of Arg1, Nos2, and MAFB-dependent genes were deter- dent signature in GHTg mice. (A) Expression levels of Arg1 and Nos2 mined by RT-qPCR using isolated F4/80+ cells from GHTg mice and WT 2Δ were determined by RT-qPCR using full-length colon samples from littermates’ colon at day DSS + 7 (n = 3). Mean 6 SD values of 2 Ct, GHTg mice and WT littermates at day DSS + 7 (n = 4). Mean 6 SD values relative to Actb expression, from triplicate determinations are shown. of 22ΔCt, relative to Actb expression, from triplicate determinations are Student t test. **p , 0.01. 12 GH TRIGGERS REPOLARIZATION OF HUMAN INFLAMMATORY MACROPHAGES

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+ % of Max 100 20 40 60 80 0 relative to the mean of internal 89 10 10 10 Relative expression 0.1 1.0 GH -4 -3 -2 10 Monocytes 0 INHBA - 10 Mo 1 + Ø 10 11 2 ( CCR2 Relative expression 10 10 10 10 10 10 10 10 1.0 GH 3 100 20 40 60 80 0 -7 -6 -5 -4 -3 -2 -1 0.37 MMP12 , - INHBA 10 Mo 0 B. Relative mRNA expression of marker genes mRNAmarker Relative of B. expression GHR + GM-M 10 1 Ø , GAPDH 99.6 Relative expression 10 10 10 10 10 10 10 10 MMP12 1.0 GH 2 -7 -6 -5 -4 -3 -2 -1 10 FOLR2 - 3 100 20 40 60 80 0 Mo and o M-M or ) 0.82 + 10 TBP 0

Relative expression M-M GH 10 10 10 10 10 1.0 10 10 -5 -4 -3 -2 -1 1 Ø expression. Determina Ø HMOX1 - 10 A. ( 2 Mo 99.2 FOLR2 + Expression levels 10 3 10 10 10 10 10 10 10 10 Relative expression10 1.0 GH 10 , -9 -8 -7 -6 -5 -4 -3 -2 -1 HMOX1 - IGF1 Mo + 32 - ,

A CCR2 CLEC5A EGLN3 10 INHBA MMP12 1.0 100 1.0 1.0

0.1 1.0 Relative expression Relative expression Relative expression Relative expression Relative expression 0.1 10 10-2 0.1 0.1 rhIFG1 - + rhIFG1 - + rhIFG1 - + rhIFG1 - + rhIFG1 - + GM-MØ GM-MØ GM-MØ GM-MØ GM-MØ B C CCR2 CLEC5A EGLN3 FOLR2 MMP12 20 1.0 10 1.0 0.1 1.0

15 ng/ml) 10 0.1 1.0 0.1 10-2 0.1

5 Activin A ( A Activin

Relative expression Relative expression Relative expression Relative expression Relative expression 0 10-2 0.1 10-2 0.1 10-2 rhIFG1 - + rhIFG1 - + rhIFG1 - + rhIFG1 - + rhIFG1 - + rhIFG1 - + GM-MØ GM-MØ GM-MØ GM-MØ GM-MØ GM-MØ

Supplemental Figure 3. Characterization of polarized GM-MØ exposed to recombi- nant IGF-1. A) Relative mRNA expression of marker genes corresponding to GM-MØ in untreated (white bars) or rhIGF-1-treated (grey bars) GM-MØ (24h, 37ºC). Results are shown as 2 relative to the mean of internal GAPDH and TBP expression. B) Relative mRNA expression of marker genes corresponding to GM-MØ in untreated (white bars) or rhIGF-1-treated (grey bars) GM-MØ. rhIGF-1 was added at the same time as the GM-CSF. Results are expressed as in A). Determinations were done in triplicate (n=3); box and whis- ker plots represent the mean, lower and upper deciles and the minimum to maximum values. Paired Student’s t-test. C) ELISA determination of activin A in culture supernatants of untreated (white bars) or rhIGF-1-treated (24h, 37ºC; grey bars) GM-MØ. Determinations were done in triplicate (n=3); box and whisker plots represent the mean, lower and upper deciles and the minimum to maximum values MAFB pGSK3 40 40

- LY .) - LY .) .u .u +LY **** +LY 30 30 ** * 20 **** 20 * 10 10 Normalized D.O. (a Normalized D.O. (a 0 0 GH (1M) - 5’ 15’ 30’ 1h 4h 24h GH (1M) - 5’ 15’ 30’ 1h 4h 24h pAKT pERK 6 - LY 80 - LY .) .)

.u +LY *** *** .u +LY ** 60 4 ** 40 2 20

Normalized D.O. (a 0 Normalized D.O. (a 0 GH (1M) - 5’ 15’ 30’ 1h 4h 24h GH (1M) - 5’ 15’ 30’ 1h 4h 24h

Supplemental Figure 4. GH-mediated MAFB upregulation in GM-MØ is dependent on PI3K activity. Densitometric