NFAT5-Regulated Macrophage Polarization Supports the Proinflammatory Function of Macrophages and T Lymphocytes

This information is current as Mónica Tellechea, Maria Buxadé, Sonia Tejedor, Jose of September 24, 2021. Aramburu and Cristina López-Rodríguez J Immunol published online 17 November 2017 http://www.jimmunol.org/content/early/2017/11/17/jimmun ol.1601942 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 17, 2017, doi:10.4049/jimmunol.1601942 The Journal of Immunology

NFAT5-Regulated Macrophage Polarization Supports the Proinﬂammatory Function of Macrophages and T Lymphocytes

Mo´nica Tellechea, Maria Buxade´, Sonia Tejedor, Jose Aramburu, and Cristina Lo´pez-Rodrı´guez

Macrophages are exquisite sensors of tissue homeostasis that can rapidly switch between pro- and anti-inﬂammatory or regulatory modes to respond to perturbations in their microenvironment. This functional plasticity involves a precise orchestration of gene expression patterns whose transcriptional regulators have not been fully characterized. We had previously identiﬁed the transcription factor NFAT5 as an activator of TLR-induced responses, and in this study we explore its contribution to macrophage

functions in different polarization settings. We found that both in classically and alternatively polarized macrophages, NFAT5 Downloaded from enhanced functions associated with a proinflammatory profile such as bactericidal capacity and the ability to promote Th1 polarization over Th2 responses. In this regard, NFAT5 upregulated the Th1-stimulatory cytokine IL-12 in classically activated macrophages, whereas in alternatively polarized ones it enhanced the expression of the pro-Th1 mediators Fizz-1 and arginase 1, indicating that it could promote proinflammatory readiness by regulating independent genes in differently polarized macrophages. Finally, adoptive transfer assays in vivo revealed a reduced antitumor capacity in NFAT5-deficient macrophages against

syngeneic Lewis lung carcinoma and ID8 ovarian carcinoma cells, a defect that in the ID8 model was associated with a reduced http://www.jimmunol.org/ accumulation of effector CD8 T cells at the tumor site. Altogether, detailed analysis of the effect of NFAT5 in pro- and anti- inﬂammatory macrophages uncovered its ability to regulate distinct genes under both polarization modes and revealed its predominant role in promoting proinﬂammatory macrophage functions. The Journal of Immunology, 2018, 200: 000–000.

acrophages can react to multiple signals generated pressive plasticity to reversibly acquire pro- or anti-inﬂammatory during development or upon disruption of tissue ho- functions in response to microenvironment cues (2–4). Macro- meostasis to engage in processes such as organogen- phages can adopt different functional phenotypes that are gener-

M by guest on September 24, 2021 esis, antipathogen responses, tissue repair, or antitumor defense (1). ally classified with respect to two main types: those conditioned Their ability to perform these functions efficiently and to coor- by proinflammatory stimuli such as IFN-g, also referred as clas- dinate with other immune and nonimmune cells requires an im- sically activated or M1, and those polarized by anti-inflammatory cytokines such as IL-4 and IL-13, also known as alternatively Immunology Unit, Department of Experimental and Health Sciences, Pompeu Fabra activated or M2 macrophages (4–6). The capacity of macrophages to University, 08003 Barcelona, Spain function in pro- or anti-inflammatory modes plays a key role in both ORCIDs: 0000-0002-3020-8393 (M.B.); 0000-0001-9279-9523 (J.A.). the triggering and resolution of immune responses, but can also con- Received for publication November 15, 2016. Accepted for publication October 17, stitute a potential vulnerability for the immune system as imbalances in 2017. macrophage function can lead to pathological immune reactivity or This work was supported by grants from the Spanish Ministry of Economy and tolerance. In this regard, the tumor microenvironment provides an in- Competitiveness and Fondo Europeo de Desarrollo Regional/European Fund for Re- teresting scenario, where despite proinflammatory and potentially an- gional Development (SAF2012-36535, and SAF2015-71363-R) and Fundacio´ la Marato´ TV3 (1225-30 and 201619-30). We also acknowledge funding support from General- titumor macrophages being found (7), anti-inflammatory macrophages itat de Catalunya (Grant 2014SGR1153) and the Spanish Ministry of Economy and are the dominant type and control tumor progression by performing Competitiveness through the Marıá de Maeztu Program for Units of Excellence in key protumoral trophic and immunotolerant functions (8–12). Research and Development (Grant MDM-2014-0370). M.T. was supported by fel- lowships from Fundacio´ Catalunya-La Pedrera (2011) and Generalitat de Catalunya Specialization into distinct macrophage subtypes relies on dif- (Formacio´ Investigadors-Direccio´ General de Recerca program 2013). S.T. was sup- ferent transcription regulators that induce specific gene expression ported by a predoctoral fellowship of the Spanish Ministry of Economy and Com- petitiveness (Grant BES-2013-062670). C.L.-R. is a recipient of an Institucio´ programs (13, 14). The transcription factors STAT1 and IRF5 are Catalana de Recerca i Estudis Avançats (Generalitat de Catalunya) Acade`mia Award. central players in the induction of the proinflammatory polariza- Address correspondence and reprint requests to Dr. Cristina Lo´pez-Rodrı´guez, Im- tion of macrophages (15, 16), and activation of STAT6 is a central munology Unit, Department of Experimental and Health Sciences, Pompeu Fabra mechanism that induces their alternative polarization (5, 13). University, Carrer Doctor Aiguader 88, Barcelona 08003, Spain. E-mail address: [email protected] Together with these master regulators, other transcription factors are known to facilitate macrophage polarization. In this regard, The online version of this article contains supplemental material. alternative polarization was also found to be regulated in different Abbreviations used in this article: BMDM, bone marrow–derived macrophage; IKKb, inhibitor of kB kinase b; iNOS, inducible NO synthase; LLC, Lewis lung contexts by p50/NF-kB dimers (17, 18), IFN regulatory factor 4 carcinoma; MRC1, mannose receptor C type 1; PPAR, peroxisome proliferator acti- (19), Kruppel-like€ factor 4 (20), the CREB/C/EBPb axis (21), vated receptor; TAM, tumor-associated macrophage. c-Myc (22), and the nuclear receptors peroxisome proliferator This article is distributed under The American Association of Immunologists, Inc., activated receptor (PPAR) g and PPARd (23–25). The diversity of Reuse Terms and Conditions for Author Choice articles. transcription regulators capable of modulating the acquisition of Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 pro- and anti-inflammatory profiles by macrophages likely un-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601942 2 NFAT5-REGULATED MACROPHAGE POLARIZATION derlies their ability to register multiple types of cues for switching (from Escherichia coli 055:B5, catalog L2880; Sigma) for the indicated time their function accordingly. points. Despite the knowledge accumulated in transcription regulators RNA expression analysis of macrophage polarization, our understanding of this process is 3 6 still incomplete. For instance, it is intriguing that, apart from the Total RNA from BMDMs (1 10 ) was isolated using the High Pure RNA Isolation System (catalog 11828665001; Roche) and quantified in a core regulators of inflammatory polarization STAT1, IRF5, and NanoDrop (ND-1000) spectrophotometer. Then 100–600 ng of total RNA p65/NF-kB (13), other pathways involved in this process could was retro-transcribed to cDNA using the First Strand cDNA synthesis also conserve the ability to support alternative polarization. This system with random primers (catalog 04897030001; Roche). For real-time was observed for the Notch pathway, which supports inflamma- quantitative PCR, LightCycler 480 SYBR Green I Master Mix (catalog 04887352001; Roche), LightCycler 480 Multiwell Plates (catalog tory gene expression and antitumor responses (26, 27), but can 4729749001; Roche), and the LightCycler 480 Real-Time PCR System also control immunoregulatory or alternatively activated macro- (Roche) were used according to the manufacturer’s instructions. For phages (28, 29). Mouse models deficient for the transcription samples with low cell numbers (103–105 cells), the RNeasy Microkit factor NFAT5 have become instrumental for the identification of (catalog 74004; Qiagen) was used and the RNA was retro-transcribed using specific roles of this factor in different immune cells. In this the SuperScript III First-Strand Synthesis system (catalog 18080-051; Thermo Fisher Scientific). In all cases, samples were normalized to L32 regard, although its function in B and T lymphocytes has been (L32 ribosomal protein gene) mRNA levels using the LightCycler Soft- mainly studied in hypertonicity-induced transcription responses ware, version 1.5. Primer sequences are listed in Supplemental Table I. (30–32), in innate immune cells such as macrophages NFAT5 has Immunoblotting assays been shown to be required for TLR-induced responses to patho- gens (33), a role that NFAT5 performs in isotonic conditions (33) For Western blotting, BMDMs were lysed in Triton X-100 lysis buffer (1 3 Downloaded from 6 but that can be enhanced under hypernatremia (34). As we had 10 cells in 100 ml; 1% Triton X-100, 40 mM Hepes pH 7.4, 120 mM previously found that many of the TLR-induced NFAT5-regulated NaCl, 1 mM EDTA, 1 mM PMSF, 5 mg/ml leupeptin, 5 mg/ml aprotinin, and 1 mg/ml pepstatin A, 1 mM sodium fluoride, 1 mM sodium orthova- genes, such as inducible NO synthase (iNOS) or TNF-a, have a nadate, 10 mM sodium pyrophosphate, and 10 mM b-glycerophosphate). central role in inflammatory responses (33), we were interested in Protein concentration was quantified using the BCA assay (catalog 23227; understanding the contribution of NFAT5 to the balance between Thermo Fisher Scientific) and lysates were boiled in reducing 13 Laemmli buffer. Next, 10–50 mg of total protein were subjected to SDS-PAGE and classically or alternatively activated macrophage functions. http://www.jimmunol.org/ transferred to PROTRAN membranes (BA83; Schleicher & Schuell). Membranes were blocked with 5% dry milk in TBS and immunoblotted Materials and Methods with the specific Abs indicated below in TBST 0.05%. Membranes were Mice then washed with TBST, incubated with a HRP-conjugated anti-rabbit secondary Ab, washed with TBST, and developed with enhanced chemo- Mice heterozygous for Nfat5 were described previously (35, 36) and used luminescence detection (catalog RPN2106; ECL Western Blotting Detec- for generating NFAT5-deficient and littermate control mice in a 129/sv tion Reagents, Amersham). The primary Abs were rabbit polyclonal anti- background. NFAT5-floxed mice in a pure C57BL/6 background were NFAT5 Ab (PAI-123; Affinity BioReagents), rabbit anti-iNOS (catalog sc- described previously (36, 37) and used to generate different conditional 651; Santa Cruz Biotechnology), rabbit anti-arginase 1 (catalog 9819; Cell knockouts for NFAT5. By crossing them with mice carrying the Cre Signaling Technology), and rabbit anti–Fizz-1 (catalog ab39626; Abcam).

recombinase under the control of the Vav1 gene promoter [Vav-Cre (38)] by guest on September 24, 2021 or the LysM promoter [LysM-Cre (33)], we generated mice that deleted ELISA NFAT5 in hematopoietic and myeloid cells respectively. In addition, the Mx1-Cre transgene [Mx-Cre (39)] was used to generate an inducible de- Cell-free supernatants from BMDM cultures were analyzed for IL-12 and letion of Nfat5 in adult NFAT5-floxed mice by poly(I:C) (catalog tlrl-pic; IL-6 production using commercially available ELISA kits (R&D Systems), InvivoGen) injection. For this, Mx-Cre NFAT5-floxed and littermate Mx- mouse IL-12/IL-23p40 DuoSet ELISA (catalog DY2398-05), and mouse Cre control mice were injected three times every other day with poly(I:C) IL-6 DuoSet ELISA (catalog DY406-05), following the manufacturer’s (15 mg/kg) and bone marrow was extracted 12 d later to generate bone instructions. marrow–derived macrophages (BMDM). All mice were analyzed between Flow cytometry 6 and 10 wk of age. Mice were bred and maintained in specific pathogen- free conditions, and animal handling and experiments were in accordance Briefly, 2 3 105 cells were blocked for 20 min in 100 mlof13 PBS with protocols approved by the ethics committee of the Barcelona Bio- containing 10% FBS, 0.1% sodium azide (staining buffer), and 0.2 mgof medical Research Park/ Pompeu Fabra University Animal Care and Use an Ab against the Fc receptor CD16/32 (catalog 101302; BioLegend). Committee, and carried out in accordance with the Declaration of Helsinki Cells were then incubated with surface marker-specific Abs in the same and the European Communities Council Directive (86/609/EEC). solution (1 mg of Ab per 106 cells) for 30 min in the dark at 4˚C. After washing them twice with staining buffer, cells were analyzed with Production and stimulation of BMDM FACSCalibur or LSR II flow cytometers. Data analysis was carried out To obtain BMDM, mice were sacrificed and the femoral and tibial marrow using FlowJo software (Tree Star). Abs for flow cytometry were from flushed with DMEM supplemented with 2 mM glutamine, 50 mM eBioscience: CD4 PE-Cy5.5 (RM4-5 clone), CD8 APC (53-6.7 clone), b-mercaptoethanol, 1 mM sodium pyruvate, and penicillin/streptomycin CD45.2 PerCP-Cy5.5 (104 clone), Ly6G PE-Cy7 (1A8 clone), CD11b (incomplete medium) (all from Thermo Fisher Scientific) using 25G sy- FITC (M1/70 clone), Ly6C PE (HK1.4 clone), MHC-II (IA/IE) APC (M5/ ringes. Then 10 3 106 BM cells were incubated in complete medium 114.15.2 clone); from BD Pharmingen: CD44 FITC (IMT clone), and (incomplete plus 10% FBS) with 25% (v/v) of L929-conditioned medium CD62L PE (MEL-14 clone). (as a source of M-CSF) in 150 mm-diameter sterile Petri dishes (catalog Macrophage and CD4+ T cell cocultures 82.1184.500; Sarstedt) for 6–7 d. Differentiated macrophages were collected by gentle pipetting after incubating 10 min with ice cold 13 PBS, 5 mM Wild-type and NFAT5-deficient BMDMs were seeded in 12-well plates in EDTA. For Western blot, ELISA and mRNA analysis, six-well plates (cat- triplicate (0.7 3 106 cells per well), polarized to M1 and M2 phenotypes alog 140675; Nunc, Thermo Fisher Scientific) were used and macrophages for 18 h, and then treated with 0.3 ng/ml of LPS for 12 h before adding were plated at 1 3 106 cells per well. For CD4+ T lymphocyte cocultures, CD4+ T cells. CD4+ T cells were isolated from the spleen and peripheral 12-well plates (Labclinics, catalog PLC30012) were used with 0.7 3 106 lymph nodes of wild-type C57BL/6 mice by negative selection using the cells per well. For cocultures with tumor cells, 4 3 106 BMDMs were plated MagniSort Mouse CD4 T cell Enrichment Kit (catalog 8804-6821-74; in 100 mm-diameter plates (catalog 172958; Thermo Fisher Scientific) and eBioscience) according to the manufacturer’s instructions. Upon isolation, incubated with Lewis lung carcinoma (LLC) tumor cells. For macrophage cells were incubated with 1 mg of anti-CD3 per million cells (catalog polarization analysis, replated macrophages were treated with 100 U/ml of 553058; BD Pharmingen) for 1 h at 4˚C, washed with DMEM medium, recombinant murine IFN-g or 10 ng/ml of recombinant murine IL-4 (catalogs and incubated with the BMDM cultures in a 1:1 ratio for 48 h. CD4+ cells 12343536 and 12340043; ImmunoTools) for 24 h to induce M1 and M2 were harvested from the coculture supernatants by gentle pipetting, polarization, respectively. Cells were then activated with LPS at 0.3 ng/ml resuspended in 13 PBS plus 10% of FBS, isolated by positive selection The Journal of Immunology 3

(Dynabeads CD4 Positive Isolation Kit, catalog 11331D; Thermo Fisher peritoneal lavage using ice-cold 13 PBS and analyzed by flow cytometry Scientific), and lysed in RNA lysis buffer (Roche) for total RNA extrac- to determine the distribution of lymphocyte subsets. LLC cells derived tion. from C57BL/6 mice were kindly provided by Dr. I. Melero (Center for Applied Medical Research, Pamplona, Spain). LLC cells were grown in Phagocytosis and bacteria killing assays complete medium and maintained at subconfluency by passing them with 3 5 Escherichia coli (AmpR) saturated cultures were opsonized with mouse gentle pipetting. For solid tumor development, a 1:1 mixture of 1 10 serum before being incubated for 30 min at 37˚C with wild-type and LLC cells and wild-type or NFAT5-deficient macrophages were s.c. injected in the right back flank of 6–8 wk old C57BL/6 female mice. Al- NFAT5-deficient M1 or M2 polarized macrophages using a multiplicity of 3 5 infection of 1 (calculated for bacterial cell cultures as: OD 1.0 = 8 3 108 ternatively, LLC cells (2 10 cells per mouse) were injected in LysM- 600 Cre Nfat5fl/fl or littermate control mice. Tumor growth was periodically cells per ml). As a negative control of phagocytosis, a parallel assay was measured using a caliper and the tumor volume was calculated using the maintained at 4˚C. For the killing assay, infected macrophages were 3 3 treated for 1 h with a high dose of gentamicin (50 mg/ml) to kill extra- formula L W2 0.52, where L = maximal length and W = maximal cellular bacteria, then washed and maintained in a low dose of gentamicin width. Mice were sacrificed at day 12 or 15. (6 mg/ml) for 0, 3, and 6 h. To determine phagocytosis and killing abilities, BMDMs were lysed at the indicated time points with 1% deoxycholate in Isolation of peritoneal and tumor-infiltrating macrophages 13 PBS and the lysates were seeded in ampicillin-containing agar plates to Peritoneal macrophages from mice bearing ID8-Luc tumors were isolated by quantify bacterial colonies. peritoneal lavage and positive selection using magnetic beads (Dynabeads sheep anti-rat IgG, catalog 11035; Life Technologies) coated with rat anti- In vivo tumor models CD11b M1/70.15 mAb supernatant. For tumor-associated macrophage Two different syngeneic tumor models were used. ID8 cells derived from a (TAM) analysis in LLC tumor samples, tumors were minced using a scalpel C57BL/6 ovarian epithelial carcinoma were used as a syngeneic serous and digested with 0.5 mg/ml of collagenase A (catalog 10103578001; Roche) tumor model (40). ID8 cells stably expressing luciferase (ID8-luc) were a plus 0.01% of DNaseI (catalog D4263-5VL; Sigma) in complete DMEM Downloaded from kind gift of Dr. K.F. Roby (University of Kansas), Dr. J.R. Conejo-Garcia medium without b-mercaptoethanol during 1 h at 37˚C in rotation. Samples (The Wistar Institute), and Dr. L. Zitvogel and Dr. P. Roberti (Institut were then filtered through a 70 mm cell strainer to remove undigested Gustave Roussy). Cells were grown in DMEM supplemented with 5% FBS fragments and the filter washed by adding 20 ml of DMEM. Filtered cells and 13 insulin-transferrin-sodium selenite media supplement (1003, were then centrifuged for 8 min at 1200 rpm and pellets resuspended in 500 catalog I1884; Sigma). For in vivo tumor development assays, 5 3 106 ml of staining buffer. Macrophages were then isolated by positive selection subconfluent ID8-Luc cells in 200 ml of 1 x PBS were injected i.p. in 6–8 with rat anti-CD11b M1/70.15 mAb and sheep anti-rat IgG magnetic beads. wk old C57BL/6 female mice, either wild-type (LysM-Cre Nfat5 +/+)or LysM-Cre Nfat5fl/fl. Tumor growth was assessed between days 3 and 35 Reagents http://www.jimmunol.org/ m postinoculation by i.p. injection of 200 l of luciferin at 12.5 mg/ml Sodium chloride, Trizma base, glycine, EDTA, sodium orthovanadate, (catalog LUCK-500; GoldBio) and bioluminescent image detection and b-glycerophosphate, PMSF, leupeptin, pepstatin A, aprotinin, SDS, sodium analysis was carried out using the IVIS200 (PerkinElmer). Analysis of pyrophosphate, methanol, BSA, Triton X-100, Tween 20, and lysogeny broth peritoneal macrophages and T cell subsets was carried out 1 d after lu- were purchased from Sigma-Aldrich. Sodium fluoride was from Merck. minescence measurement, at days 4, 7, 14, and 36. Additional experiments HEPES was from Lonza. GM-CSF and M-CSF were from ImmunoTools. were carried out in wild-type C57BL/6 mice first inoculated with ID8-Luc 6 cells, and 30 d later injected i.p. with 9 3 10 wild-type or NFAT5- Statistical analysis deficient BMDM. Tumor growth was assessed by bioluminescence measurement 4 and 10 d after BMDM injection. On the last day of the Statistical analyses were carried out using the GraphPad Prism 5 software.

experiment mice were sacriﬁced and peritoneal cells were harvested by Normality (Gaussian distribution) of samples was determined by a by guest on September 24, 2021

FIGURE 1. Expression of NFAT5 in mouse and human macrophages. (A) Schematic diagram of macrophage (BMDM) conditioning with IFN-g (100 U/ ml) and IL-4 (10 ng/ml), with or without further stimulation with LPS (0.3 ng/ml). Macrophages were designated as M1 when pretreated with IFN-g for 24 h, and M2 when treated with IL-4 for 24 h. (B) mRNA expression of NFAT5 in wild-type macrophages treated with IFN-g or IL-4 for 24 h (left panel), or in wild-type (Mx-Cre Nfat5+/+, WT) and NFAT5-deficient (Mx-Cre Nfat5fl/fl, KO) macrophages treated with each respective cytokine followed by LPS stimulation (right panel). Values in the left panel are shown relative to NFAT5 expression in the absence of cytokines (which was given a value of 1), andin the right panel they are referred to wild-type macrophages activated with IFN-g plus LPS (100%). Values shown are the mean 6 SEM (n = 5 independently performed experiments). (C) Western blot of NFAT5 in wild-type macrophages stimulated as in (B). Tubulin Western blot is included as loading control. Two representative experiments are shown. (D) Expression of NFAT5 in human blood monocytes (two independent donors) treated for 6 d with 100 U/ml GM-CSF (GM) or 10 ng/ml M-CSF (M). 4 NFAT5-REGULATED MACROPHAGE POLARIZATION

D’Agostino–Pearson normality test before determining statistical signi- IL-4, as these cytokines are prototypical inducers of responses at ficance with an unpaired t test (for sets of samples with a Gaussian dis- both ends of the functional spectrum of macrophages. We also tribution) or Mann–Whitney U test (samples with a non-Gaussian analyzed the response of IFN-g and IL-4–polarized macrophages distribution). A two-way ANOVA test was used when comparing various groups of samples, and a one-sample t test was used when samples were to subsequent stimulation with LPS. LPS was used to boost the compared with a reference control sample (set as 100%). The statistical acquisition of proinflammatory features in IFN-g–polarized analyses used are indicated in each respective figure legend. macrophages, and in the case of IL-4–polarized ones, to induce a switch from an anti-inflammatory to a proinflammatory state Results (Fig. 1A). For simplicity, we have used the nomenclature M1 to NFAT5 regulates different genes in macrophages polarized refer to macrophages preconditioned with IFN-g, and M2 for with IFN-g or IL-4 macrophages conditioned with IL-4 alone. First, we observed that As a first approximation to explore new roles of NFAT5 in pro- and IFN-g and IL-4 alone did not change the basal expression of anti-inflammatory macrophage functions, we analyzed how the NFAT5 in mouse BMDM, but found that IFN-g pretreatment lack of NFAT5 affected macrophages conditioned with IFN-g or enhanced the induction of NFAT5 by LPS both at the mRNA and Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Expression of pro- and anti-inflammatory genes in wild-type and NFAT5-deficient macrophages. (A) Expression of the indicated genes in wild-type (Mx-Cre Nfat5+/+, WT) and NFAT5-deficient (Mx-Cre Nfat5fl/fl, KO) macrophages activated with IFN-g and LPS or with IL-4 alone. Values represented are relative to wild-type macrophages (100%), and show the mean 6 SEM of five independently performed experiments (*p , 0.05, **p , 0.01, ***p , 0.001). (B) Expression of the indicated genes in wild-type (Mx-Cre Nfat5+/+) and NFAT5-deficient (Mx-Cre Nfat5fl/fl) macrophages, either without pretreatment or pretreated 24 h with IFN-g or IL-4, and then stimulated with LPS as indicated. Data correspond to the mean 6 SEM of three to five experiments performed independently. Statistical significance was determined with an unpaired t test, with a one-sample t test when samples were compared with a reference control sample (set as 100%), or with a two-way ANOVA test when comparing various groups of samples. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5 protein levels, whereas IL-4 did not, or even reduced it mildly products iNOS, IL-12p40, and IL-6 in response to LPS stimulation (Fig. 1B, 1C). Similarly, we found that human macrophages de- (Figs. 2, 3, Supplemental Fig. 1E). rived from blood monocytes expressed more NFAT5 when treated We also found that lack of NFAT5 affected the expression of several with the proinflammatory cytokine GM-CSF, compared with those genes induced by IL-4. Arginase 1, CD163, YM1 (Chi3l3), and Fizz-1 treated with the anti-inflammatory polarizing factor M-CSF (Retnla) expression was decreased in IL-4–polarized NFAT5- (Fig. 1D). These results showed that expression of NFAT5 was deficient cells (Fig. 2, Supplemental Fig. 1E). Other gene products higher in proinflammatory or M1 macrophages than in alternative such as mannose receptor C type 1 (MRC1 or CD206) and Gas6 were or M2 ones. We then analyzed the contribution of NFAT5 to gene NFAT5 independent in IL-4 polarized macrophages, although we also expression in macrophages polarized with IFN-g or IL-4, using noticed that expression of MRC1 mRNA was more resistant to different mouse models of NFAT5 deletion in the whole body (35, downregulation by LPS in NFAT5-deficient macrophages. Attenuated 36) or in myeloid and hematopoietic cells (Supplemental Fig. 1A– induction of the prototypical IL-4–inducible products arginase 1 and D). IFN-g conditioning strongly enhanced the induction by LPS of Fizz-1 in NFAT5-deficient macrophages was also observed at the main proinflammatory gene products iNOS, IL-1b, IL-6, and IL- protein level (Fig. 3D). Regulation of IFN-g– and IL-4–responsive 12b compared with macrophages stimulated only with LPS or genes by NFAT5 was consistently observed in macrophages from two conditioned with IL-4 before LPS (Fig. 2, Supplemental Fig. 1E). different mouse models of conditional deletion of Nfat5 (induced by Parallel analysis of NFAT5-deficient macrophages revealed re- Mx-Cre and Vav-Cre), as well as macrophages differentiated from duced mRNA and protein expression of inflammatory gene NFAT5-deficient mice (Figs. 2, 3, Supplemental Fig. 1E). Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Expression of protein products of NFAT5-target genes in polarized NFAT5-deficient and control macrophages. (A) Western blot of iNOS in wild- type (Nfat5 +/+)andNFAT5-deficient(Nfat52/2) macrophages stimulated as indicated. Two representative experiments showing a long and a short exposure are shown. Tubulin and lamin B are included as loading controls. (B) Quantification of long (n =4)andshort(n = 3) exposures from independent iNOS Western blot analyses. iNOS quantification was normalized to the respective loading controls tubulin and lamin B. Statistical significance was determined with a one- sample t test. (C)ExpressionofIL-12b and IL-6 in wild-type and NFAT5-deficient (Mx-Cre or Vav-Cre Nfat5fl/fl mice) macrophages stimulated as indicated. Results show the mean 6 SEM, n = 3. Statistical significance was determined with a one-sample t test. (D) Expression of arginase 1 (Arg1) and Fizz-1 was analyzed by Western blot in wild-type and NFAT5-deficient (Vav-Cre Nfat5fl/fl mice) macrophages stimulated with IL-4 plus subsequent LPS, or with IFN-g. One representative out of two (Arg1) and four (Fizz-1) independently performed experiments is shown. *p , 0.05, **p , 0.01, ***p , 0.001. 6 NFAT5-REGULATED MACROPHAGE POLARIZATION

Altogether, our results showed that in addition to regulating gene expression in macrophages activated by different TLR ligands (33), NFAT5 also modulated their response to the polarizing cytokines IFN-g and IL-4. Our findings showing that NFAT5 could promote the expression of various IL-4–responsive genes in primary macrophages differed from those in a recent report using the cell line RAW264.7, in which NFAT5 repressed the expression of arginase 1, IL-10, and MRC1/CD206 induced by IL-4 (41). As experiments by Choi et al. only achieved partial downregulation of NFAT5 with small interfering RNA in the cell line RAW264.7, it is possible that they might have missed relevant differences that we have identified in this study using primary macrophages with a complete deletion of NFAT5. Altogether, this block of results indicates that, rather than promoting macrophage gene expression in a fixed proinflammatory direction, NFAT5 could modulate different outcomes depending on the stimulatory environment. Lack of NFAT5 affects IFN-g– and IL-4–regulated macrophage functions Downloaded from Our analysis of diverse genes characteristic of IFN-g or IL-4–stimulated macrophages suggested that NFAT5-deficient cells could exhibit functional defects or biases in response to these cytokines. IFN- g and IL-4 influence the intrinsic antimicrobial capacity of macrophages, for instance, IFN-g enhances their bactericidal activity to

allow a tighter control of pathogen load upon infection (42). We FIGURE 4. Phagocytic and bactericidal activity in wild-type and NFAT5- http://www.jimmunol.org/ compared the phagocytosis and bacteria-killing capacity of wild-type deficient macrophages treated with IFN-g or IL-4. (A)PhagocytosisofliveE. and NFAT5-deficient macrophages primed with IFN-g or IL-4. coli by IFN-g or IL-4–pretreated (24 h) wild-type (Mx-Cre Nfat5+/+)and fl/fl Macrophages pretreated with IFN-g or IL-4 were comparably capa- NFAT5-deficient (Mx-Cre Nfat5 ) macrophages. Values represented are ble of phagocytosing live E. coli, and lack of NFAT5 only caused relative to IL-4–pretreated wild-type macrophages (100%), and show the 6 B them a mild decrease in their phagocytic capacity (Fig. 4A). By mean SEM of three independently performed experiments. ( ) Bactericidal activity of IFN-g– or IL-4–pretreated (24 h) wild-type (Mx-Cre Nfat5+/+)and contrast, NFAT5-deficient macrophages exhibited a marked delay in fl/fl NFAT5-deficient (Mx-Cre Nfat5 ) macrophages on ingested E. coli was their capacity to kill intracellular bacteria, both under IFN-g and IL-4 analyzedat3and6hafterphagocytosis.Resultsarerepresentedasper- conditioning (Fig. 4B). centage of live intracellular bacteria relative to a sample of freshly infected Macrophages conditioned by IFN-g or IL-4 have the ability to macrophages (100%, indicated by the dotted line). Results show the mean 6 by guest on September 24, 2021 promote T lymphocyte polarization toward Th1 or Th2 respec- SEM of five independently performed experiments. Statistical significance tively, which in turn reinforces the feedback between macrophages was determined with an unpaired t test, or with a one-sample t test for and CD4 and CD8 T cells. NFAT5 enhanced the expression of the samples compared with a reference control sample (set as 100%). *p , 0.05. pro-Th1 cytokine IL-12 in IFN-g–polarized macrophages stimulated with LPS, but also induced Fizz-1 and arginase 1 in response to IL-4 (Figs. 2, 3, Supplemental Fig. 1E). Although Fizz-1 and NFAT5 influences macrophage-mediated antitumor responses arginase 1 are induced by IL-4 and are characteristic markers of in adoptive transfer models M2 macrophages, they have a pro-Th1 effect as they attenuate Th2 Collectively, our diagnostic experiments with IFN-g and IL-4 polarization in favor of Th1 (43, 44). These results led us to ask revealed that NFAT5 could regulate macrophage responses to about the ability of NFAT5-deficient macrophages to influence pro- and anti-inflammatory polarizing stimuli, which could influ- T cell polarization. Activation of CD4 T lymphocytes was elicited ence the specialization of T lymphocytes. These findings led us to by stimulation with a soluble anti-CD3 agonistic Ab in the pres- explore how NFAT5 would influence macrophage function in ence of macrophages preconditioned with IFN-g or IL-4 that were more complex settings where macrophage activity in one or other subsequently treated with LPS. We observed that LPS enhanced direction may affect the outcome of the overall immune response. the capacity of IFN-g– or IL-4–polarized macrophages to re- In this regard, the interaction between macrophages and cancer spectively skew Th1 or Th2 responses (Fig. 5). Despite some cells in the tumor microenvironment is shaped by a balance be- differences in Th polarization markers induced in CD4 cells by tween pro- and anti-inflammatory inputs that ultimately deter- macrophages derived from the two types of NFAT5-conditional mines tumor progression. Tumors can evade immune attack by knockout mice, we could observe that NFAT5-deficient macro- instructing local immune cells to become tumor tolerant. Although phages were less capable of promoting IL-2 and IFN-g expression tumors can harbor M1 inflammatory macrophages with antitumor in T cells, but were better inducers of IL-4 (Fig. 5). This result was capacity, the overall balance is shifted toward a dominant M2-like, consistent with the positive role of NFAT5 in inducing the pro-Th1 tolerance-promoting environment that attenuates proinflammatory mediators IL-12, arginase 1 and Fizz-1 (Figs. 2, 3, Supplemental responses and favor tumor progression (7, 8). Fig. 1E), and suggested that although NFAT5 could regulate di- We first assessed the response of peritoneal macrophages to a verse macrophage genes in response to IL-4 or IFN-g condition- syngeneic tumor in vivo, using the model of the ovarian epithelial ing, its net effect in macrophage-mediated T cell stimulation was carcinoma cell line ID8-Luc (C57BL/6 background). These cells to facilitate Th1 polarization at the expense of Th2. Therefore, induce tumors in the peritoneal cavity that resemble those seen in NFAT5 function in macrophages could contribute to promoting advanced stages of ovarian carcinoma (40), and have been used for type 1 proinflammatory responses and limit type 2 immune re- the analysis of tumor-infiltrating macrophages (45). Initial setting- sponses in different contexts. up experiments showed that peritoneal inoculation of ID8-Luc The Journal of Immunology 7 Downloaded from

FIGURE 5. T cell–costimulatory capacity of wild-type and NFAT5-deficient macrophages. (A) Expression of the indicated cytokine mRNA in CD4 T cells either left unstimulated or stimulated for 24 h with soluble anti-CD3 Ab in the presence of wild-type (Mx-Cre Nfat5+/+) and NFAT5-deficient (Mx- Cre Nfat5fl/fl) macrophages that had been pretreated with IFN-g or IL-4 for 24 h, with or without further stimulation with LPS as indicated. (B) Same +/+ fl/fl analysis in an independent model of wild-type (Vav-Cre Nfat5 ) and NFAT5-deficient (Vav-Cre Nfat5 ) macrophages. Results show the mean 6 SEM of http://www.jimmunol.org/ three to five independently performed experiments. Statistical significance was determined with an unpaired t test, or with a one-sample t test for samples compared with a reference control sample (set as 100%). *p , 0.05, ***p , 0.001. cells elicited the accumulation of different TAM subsets, as well showed an increased proportion of TAM-A cells (Ly6Chi MHC as an increase in the local proportion of effector-memory class IIneg) and a mild decrease in IL-12b mRNA expression, (CD62Lneg, CD44hi) CD4 and CD8 T cells (Supplemental Fig. although they did not present significant defects in the expression 2A, 2B). In this experimental setting, peritoneal CD11b+ macro- of other pro- and anti-inflammatory markers (Supplemental Fig. phages of ID8-Luc–bearing mice expressed diverse genes indic- 3A, 3B). We also did not find differences in the proportions of ative of both M1 and M2 activation profiles (Supplemental Fig. naive and effector CD4 and CD8 peritoneal T cells between both by guest on September 24, 2021 2C). We also observed that ID8-Luc tumor burden progressed mouse genotypes (data not shown). At later time points from 2 to slowly and in some mice decreased gradually over time 5 wk, we did not detect differences between both mouse genotypes (Supplemental Fig. 2D). Next, we compared the tumor burden and in tumor progression rates (Fig. 6A). Also, comparison of the macrophage activation parameters between myeloid-specific percentages of tumor-infiltrating macrophage subsets and gene NFAT5-deficient (LysM-Cre Nfat5fl/fl) and littermate control expression patterns at 5 wk did not show differences between mice at days 7 and 35 after inoculating ID8-Luc cells. We chose wild-type and LysM-Cre Nfat5fl/fl mice, although we observed that day 7 as it corresponded to an early peak of macrophage activation macrophages at 5 wk differed from 1 wk in expressing lower soon after tumor inoculation (Supplemental Fig. 2C), and 5 wk as levels of arginase 1 and CCL2, as well as having a lower TAM-A it could be informative of a longer-term interaction between the percentage (Supplemental Fig. 3A, 3B). We then tested a different tumor and the immune response. We found that tumor burden at syngeneic (C57BL/6) tumor model of LLC cells. LLC cells in- day 7 was moderately reduced in myeloid-specific NFAT5- jected s.c. gave rise to solid tumors whose growth progression deficient mice (Fig. 6A), whose peritoneal macrophages also and size were comparable in wild-type and myeloid-specific

FIGURE 6. Analysis of tumor burden in myeloid-specific NFAT5-deficient mice. (A) ID8-Luc peritoneal tumor burden assessed by intravital luminescence at different time points after inoculation in wild-type (LysM-Cre Nfat5+/+, WT) and myeloid-specific NFAT5-deficient mice (LysM-Cre Nfat5fl/fl, KO). Results of day 6 comprise two independent consecutive experiments, the first with seven mice of each genotype and the second with six; and results at days 17, 29, and 35 correspond to another independent experiment with seven mice of each genotype. Graphs show individual values for each mouse, plus the mean 6 SEM. Statistical significance was determined with an unpaired Mann–Whitney U test (*p , 0.05). (B) LLC tumor size at different days after s.c. inoculation in wild-type and myeloid-specific NFAT5-deficient mice. Results show the mean 6 SEM of n = 7 mice of each genotype. 8 NFAT5-REGULATED MACROPHAGE POLARIZATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Analysis of antitumor responses mediated by wild-type and NFAT5-deficient macrophages. (A) Left panel, Mice bearing ID8-Luc ovarian cancer cells (30 d tumors) were either left untreated or injected i.p. with wild-type or NFAT5-deficient macrophages. Tumor burden was assessed 5 and 10d later in each individual mouse by intravital luciferase luminescence. Each circle corresponds to one individual mouse and bars within each group of mice are the mean 6 SEM of luminescence readings. Right panel shows a similarly performed independent experiment analyzing tumor burden 4 d after macrophage injection in mice with 31 d tumors. Statistical significance was determined with an unpaired t test. (B) Percentages of peritoneal naive and memory CD8 (upper panels) and CD4 (lower panels) T cells, and their naive to effector ratio were analyzed by flow cytometry 4 d after injecting wild-type or NFAT5-deficient macrophages in mice that had developed peritoneal ID8-Luc tumors for 31 d. One control mouse without ID8-Luc and another with ID8-Luc but without exogenously injected macrophages are shown for reference. Each symbol corresponds to one individual mouse and bars within each group of mice are the mean 6 SEM. (C) Growth curves of LLC tumors implanted s.c. together with (Vav-Cre Nfat5+/+) or NFAT5-deficient (Vav-Cre Nfat5fl/fl) macrophages. Two independent experiments are shown, with 10 mice per experimental group (Exp #1), and six mice per group (Exp #2). Statistical significance was determined with an unpaired t test (*p , 0.05). (D) Expression of IL-12b, iNOS and arginase 1 in wild-type (Vav-Cre Nfat5+/+) and NFAT5-deficient (Vav- Cre Nfat5fl/fl) macrophages first activated as M1 and then cultured for 24 h in the presence or absence of LLC tumor cells (LLC/macrophage ratio 1:1). Panels below show an enlarged view of the relative expression of IL-12b, iNOS, and arginase 1 in wild-type and NFAT5-deficient (Figure legend continues) The Journal of Immunology 9

NFAT5-deficient (LysM-Cre Nfat5fl/fl) mice throughout the 14 d of which already expressed less iNOS and IL-12b than wild-type examination (Fig. 6B) and showed no obvious differences between ones upon IFN-g and LPS stimulation, suffered a more pro- both mouse genotypes in macrophage polarization markers and nounced downregulation of these genes and enhanced arginase 1 TAM subsets (Supplemental Fig. 3C, 3D). Altogether, results with induction by LLC cells (Fig. 7D). These results indicated that lack these tumor models suggested that lack of NFAT5 in endogenous of NFAT5 exacerbated the tumor-induced switch of macrophage myeloid cells in vivo could cause a modest impact in the dynamics toward an anti-inflammatory phenotype, and were consistent with of macrophage activation in one of the tumor types tested, but they the gene expression profile regulated by NFAT5 in polarized also suggested that its depletion from myeloid cells did not affect macrophages, as well as with our finding that NFAT5-deficient overall tumor progression. Interpreting the precise contribution of BMDM were poorer stimulators of Th1 responses and effector NFAT5 to the function of endogenous TAMs in these models was CD8 T cells (Figs. 5, 7B). difficult seeing that infiltrating macrophages in both types of tumors exhibited a considerable dispersion in subset distribution and Discussion magnitude of gene expression between individual mice. Also, Our detailed analysis of the role of NFAT5 in classical and al- LysM-Cre conditional knockout mice would not only lack NFAT5 ternative macrophage polarization indicated its prevalent proin- in different macrophage subsets but also in other myeloid cells flammatory role in different macrophage functions that range from such as neutrophils and dendritic cells, whose pro- and anti- the direct control of pathogen load and T cell polarization to anti- inflammatory activity at different stages of tumor development tumor activity in vivo. The overall effect of NFAT5 in facilitating may contribute with variable outcomes to tumor progression. In proinflammatory outcomes was paralleled by its high expression this regard, it has been shown that macrophages at different levels in macrophages stimulated with GM-CSF or IFN-g plus Downloaded from intratumoral maturation stages can differ considerably in expres- LPS, in comparison with its low expression in alternative mac- sion patterns of pro- and anti-inflammatory genes (12). rophages induced with M-CSF- or IL-4. In view of these results we decided to use different approaches, Whereas the contribution of NFAT5 to macrophage functions consisting of the adoptive transfer of homogeneous populations of associated with a proinflammatory profile was consistent with its in vitro–differentiated BMDM into tumor-bearing mice, or coin- capacity to enhance the expression of genes characteristic of

oculation of BMDM with tumor cells (45, 46). By challenging the classically or M1-polarized macrophages, we also identified http://www.jimmunol.org/ tumors with a relatively large number of exogenous, homoge- NFAT5 as a regulator of a group of markers of alternatively po- nously differentiated macrophages we expected to circumvent the larized macrophages. To a different extent, lack of NFAT5 impaired potential variability contributed by the deletion of NFAT5 in dif- the expression of markers such as Arg1, Cd163, Chi3l3 (YM1), or ferent myeloid cell lineages in LysM-Cre conditional knockout Retnla (Fizz-1). Intriguingly, arginase 1 and Fizz-1, which are mice, and the heterogeneity in the activity of these populations at induced in macrophages by Th2 cytokines, have been shown to different moments of tumor progression. Our results showed that function in a negative feedback loop as attenuators of Th2 cells to NFAT5-deficient BMDM injected in the peritoneum of mice with favor Th1 inflammatory responses (43, 44). Therefore, it can be ID8-Luc tumors were less effective than wild-type BMDM at proposed that the contribution of NFAT5 to the expression of reducing tumor burden (Fig. 7A) and also reduced the accumu- particular alternative macrophage markers also aligns with its by guest on September 24, 2021 lation of peritoneal CD8 T effector cells (Fig. 7B). We also found capacity to enhance pro-Th1 mediators like IL-12 in M1 macro- that NFAT5-deficient BMDM were less effective than wild-type phages, so that altogether NFAT5 would increase the effectiveness ones at restraining the growth of s.c. LLC carcinomas when tumor of macrophages to activate Th1 cells when switching from M2 to M1 cells were coinjected simultaneously with BMDM (Fig. 7C). These functions in response to inflammatory signals. Nonetheless, although results suggested that NFAT5 could confer antitumor advantage this interpretation agrees with our characterization of NFAT5 as a when a population of homogeneous macrophages was adoptively proinflammatory factor, its capacity to regulate diverse macrophage transferred in a sufficiently high proportion relative to tumor cells. features under both M1 and M2 inducers suggests that it could tune By contrast, in experiments where tumor cells were inoculated different modes of response in macrophages placed in complex alone in wild-type or myeloid-specific LysM-Cre Nfat5fl/fl mice, the stimulatory environments, without necessarily always driving them potential capacity of NFAT5 to modulate antitumor function in toward a fixed proinflammatory outcome. endogenous macrophages was possibly masked by the heteroge- The gene expression profile regulated by NFAT5 in polarized neity of myeloid cell subsets, whose contribution to tumor expan- macrophages suggested that it could enhance the capacity of sion and sensitivity to lacking NFAT5 could be heterogeneous. macrophages to promote type 1 versus type 2 immune responses Our results also suggested that tumor cells might be able to (47, 48). Direct coculture of CD4 T lymphocytes with polarized rapidly instruct recruited macrophages to suppress antitumor NFAT5-deficient macrophages showed that they were weaker in- functions so that tumor cells could persist and expand. This pos- ducers of Th1 and stronger inducers of Th2 responses than wild- sibility led us to analyze the response of NFAT5-deficient mac- type ones, and NFAT5-deficient macrophages inoculated at the rophages to tumor cells in direct coculture experiments. We found tumor site in mice bearing ovarian carcinoma cell tumors were that ID8-luc and LLC cells did not induce inflammatory markers less effective at inhibiting tumor growth and supporting the local iNOS and IL-12b in unpolarized wild-type macrophages (data not accumulation of effector CD8 T cells, altogether suggesting shown), and so we asked whether tumor cells could influence the an impaired capacity to elicit type 1 immune responses. In inflammatory phenotype of macrophages previously polarized as this regard, CD8 T lymphocytes are central players in antitumor M1. These experiments showed that LLC cells downregulated responses induced spontaneously or upon therapy (49–51), and iNOS and IL-12b and induced the expression of arginase 1 in studies in ovarian carcinoma patients have shown that the presence BMDM previously activated as M1 with IFN-g plus LPS of dense infiltrates of activated CD8 T cells associates with a good (Fig. 7D). We also observed that NFAT5-deficient macrophages, prognosis to antitumor treatment (52, 53).

macrophages after coculture with LLC cells. Results show the mean 6 SEM of four independently performed experiments. Statistical signiﬁcance was determined with a one-sample t test for samples compared with a reference control sample (set as 100%). *p , 0.05. n.s, not signiﬁcant. 10 NFAT5-REGULATED MACROPHAGE POLARIZATION

We have used in vivo models of tumor progression as one readout macrophages in a p65/NF-kB– and IKKb-dependent manner (33). to test how NFAT5 could contribute to macrophage function in a These observations suggest the possibility that macrophages might complex in vivo microenvironment, where the activity spectrum of use NFAT5 in concert with Notch and IKKb-regulated pathways to local macrophages would encompass both pro- and anti- tune their pro- and anti-inflammatory capabilities under different inflammatory functions (10, 45). Although we could detect dif- polarizing stimuli. It is intriguing that macrophages experiencing anti- ferences in some responses of tumor-infiltrating macrophages inflammatory or M2-promoting conditions do not entirely shut down between wild-type and LysM-Cre Nfat5fl/fl mice, our overall re- proinflammatory pathways, but maintain active a set of transcription sults suggested that NFAT5 played a limited role in how endog- factors and signaling pathways such as NFAT5, Notch/RBP-J and enous macrophages influenced tumor expansion. In contrast, we IKKb capable of mobilizing rapid inflammatory responses. As found that adoptively transferred NFAT5-deficient macrophages macrophages exhibit a considerable degree of plasticity in reacting had impaired antitumor capacity, which could be attributed in part to a broad diversity of potential inputs from their microenvironment, to their reduced ability to promote a proinflammatory environment having dually responsive transcription regulators could allow them to and engage CD8 effector T cells. This interpretation is consistent stay poised and maintain a state of readiness to rapidly react to danger with our finding that NFAT5 enhanced the capacity of macro- signals and disruptors of tissue homeostasis. phages to stimulate IFN-g and IL-2 production in CD4 T lymphocytes and promoted macrophage expression of iNOS and Acknowledgments IL-12, all of which can activate antitumor CD8 cytolytic responses We are grateful to Dr. Marc Schmidt-Supprian and Dr. Thomas Graf for (11, 51). These results, and our coculture experiments showing a providing the Mx1-Cre and the Vav1-Cre transgenic mouse models, respec- Downloaded from greater resistance of NFAT5 wild-type macrophages to the tumor- tively; Dr. Ignacio Melero for providing the LLC cells; and Dr. Katherine F. induced M1 to M2 switch, suggested that NFAT5 could facilitate Roby, Dr. Jose´ R. Conejo-Garcia, Dr. Laurence Zitvogel, and Dr. Paula macrophage-mediated mechanisms that ultimately support Roberti for making available the ovarian carcinoma ID8-Luc cell line, as immunosurveillance against tumors. Regarding the different re- well as for providing valuable advice with culture. We thank Dr. Miguel sults obtained with adoptively transferred and endogenous mac- Lo´pez-Botet, Dr. A´ ngel L. Corbı´, and Hector Huerga for insightful com- rophages, these could be discussed as several mutually ments with this work; Dr. Jordi Pou for advice with polarization of human fl/fl nonexclusive interpretations. Myeloid-specific LysM-Cre Nfat5 macrophages; and Dr. Oscar Fornas and team for guidance and expertise http://www.jimmunol.org/ mice would delete NFAT5 not only in different macrophage with flow cytometry assays. We thank Marıá Garcıá Belando for technical subsets, but also in other myeloid cells such as dendritic cells, support with mouse genotyping and members of our group for stimulating and helpful discussions. neutrophils, and eosinophils, all populations that could exhibit heterogeneous NFAT5-dependent effects with regards to tumor expansion. By contrast, in the adoptive transfer experiments we Disclosures inoculated a large number of a homogeneous type of macrophage, The authors have no financial conflicts of interest. likely tilting the equilibrium toward an immune response domi- nated by the transferred BMDM and not by endogenous myeloid References by guest on September 24, 2021 cells. This dominant role of adoptively transferred macrophages 1. Wynn, T. A., A. Chawla, and J. W. Pollard. 2013. Macrophage biology in de- has been reported in other systems such as the correction of lung velopment, homeostasis and disease. 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