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 Supplementary http://www.jimmunol.org/content/suppl/2017/11/17/jimmunol.160194 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 tran- scription 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 macro- phages. 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 Marı´a 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 Avanc¸ats (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).