ORIGINAL RESEARCH published: 03 April 2017 doi: 10.3389/fimmu.2017.00373 Activation of Adhesion GPCR EMR2/ ADGRE2 Induces Macrophage Differentiation and inflammatory Responses via Gα16/Akt/MAPK/ NF-κB Signaling Pathways Kuan-Yu I1, Yi-Shu Huang1†, Ching-Hsun Hu1, Wen-Yi Tseng2†, Chia-Hsin Cheng1, Martin Stacey3, Siamon Gordon1,4, Gin-Wen Chang1 and Hsi-Hsien Lin1,5,6* Edited by: 1 Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan, 2 Division of Céline Cougoule, Rheumatology, Allergy and Immunology, Chang Gung Memorial Hospital-Keelung, Keelung, Taiwan, 3 Faculty of Biological Centre national de la recherche Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK, 4 Sir William Dunn School of Pathology, scientifique (CNRS), France University of Oxford, Oxford, UK, 5 Department of Anatomic Pathology, Chang Gung Memorial Hospital-Linkou, Taoyuan, Reviewed by: Taiwan, 6 Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan Frédéric Velard, Université de Reims Champagne- Ardenne, France EMR2/ADGRE2 is a human myeloid-restricted adhesion G protein-coupled receptor crit- Junjie Zhang, ically implicated in vibratory urticaria, a rare type of allergy caused by vibration-induced University of Southern California, USA mast cell activation. In addition, EMR2 is also highly expressed by monocyte/macro- *Correspondence: phages and has been linked to neutrophil migration and activation. Despite these findings, Hsi-Hsien Lin [email protected] little is known of EMR2-mediated signaling and its role in myeloid biology. In this report, †Present address: we show that activation of EMR2 via a receptor-specific monoclonal antibody promotes Yi-Shu Huang and Wen-Yi Tseng, the differentiation of human THP-1 monocytic cell line and induces the expression of Kennedy Institute of Rheumatology, pro-inflammatory mediators, including IL-8, TNF- , and MMP-9. Using specific signaling University of Oxford, Oxford, UK α inhibitors and siRNA knockdowns, biochemical and functional analyses reveal that the Specialty section: EMR2-mediated signaling is initiated by Gα16, followed by the subsequent activation of This article was submitted Akt, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and nuclear factor to Inflammation, a section of the journal kappa-light-chain-enhancer of activated B cells. Our results demonstrate a functional Frontiers in Immunology role for EMR2 in the differentiation and inflammatory activation of human monocytic cells Received: 23 December 2016 and provide potential targets for myeloid cell-mediated inflammatory disorders. Accepted: 15 March 2017 Published: 03 April 2017 Keywords: cytokine, EMR2, GPCR, macrophage, inflammation, signaling Citation: I K-Y, Huang Y-S, Hu C-H, Abbreviations: aGPCR, adhesion GPCR; CM, conditioned medium; CTF, C-terminal fragment; DAG, diacylglycerol; DC, Tseng W-Y, Cheng C-H, Stacey M, dendritic cell; ECD, extracellular domain; EMR2, EGF-like module-containing mucin-like hormone receptor-like 2; ERK, Gordon S, Chang G-W and Lin H-H extracellular signal-regulated kinase; f-MLF, N-formyl-methionyl-leucyl-phenylalanine; GAIN, GPCR autoproteolysis-induc- (2017) Activation of Adhesion GPCR ing; GPCR, G protein-coupled receptor; GPS, GPCR proteolysis site; IP3, inositol triphosphate; JNK, c-Jun N-terminal kinase; EMR2/ADGRE2 Induces Macrophage LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPS, lipopolysaccharide; Mφ, macrophage; mAb, monoclo- Differentiation and Inflammatory nal antibody; MAPK, Mitogen-activated protein kinases; MMP, matrix metalloproteinases; Mo, monocyte; Nφ, neutrophil; Responses via Gα16 /Akt/MAPK/ NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NTF, N-terminal fragment; PLC, phospholipase C; NF-κB Signaling Pathways. PMA, phorbol 12-myristate 13-acetate; PI3K, Phosphoinositide 3-kinase; PIP2, phosphatidylinositol biphosphate; PIP3, phos- Front. Immunol. 8:373. phatidylinositol 3,4,5 trisphosphate; PTX, pertussis toxin; ROS, reactive oxygen species; SIRS, systemic inflammatory response doi: 10.3389/fimmu.2017.00373 syndrome; TNF, tumor necrosis factor; 7TM, seven transmembrane. Frontiers in Immunology | www.frontiersin.org 1 April 2017 | Volume 8 | Article 373 I et al. EMR2 Signaling in Monocytic Cell INTRODUCTION protein motifs and a GPCR autoproteolysis-inducing (GAIN) domain (20–22). During receptor biosynthesis, aGPCRs are nor- Professional phagocytes such as macrophages (Mφ), neutrophils mally bisected at a consensus GPCR proteolysis site via the GAIN (Nφ), and dendritic cells (DCs) are critical for the recognition domain-mediated autoproteolytic reaction into a N-terminal and elimination of invading pathogens (1, 2). The processes of ECD-fragment (NTF) and a C-terminal 7TM-fragment (CTF), phagocytosis as well as the subsequent microbial killing and which remain conjugated as a dual-subunit receptor (13, immune activation/resolution by these innate immune effector 21). Recent advances indicate that aGPCR activation is likely cells are largely mediated via a diverse array of receptors and their mediated by ligand-induced NTF displacement, followed by signaling reactions (1, 3). In this regard, one receptor of interest the unfolding and binding of an internal agonist peptide to is EMR2/ADGRE2, a human myeloid-restricted adhesion G the 7TM core of CTF (23, 24). The mechanistic insights of the protein-coupled receptor (aGPCR) highly homologous to F4/80, “tethered agonism” of aGPCRs are increasingly being unraveled, the widely acclaimed surface marker that defines murine tissue including the coupling of unique G proteins to distinct aGPCR Mφ (4–6). members (21, 25–27). However, an orderly depiction of aGPCR- As a human ortholog of F4/80, EMR2 similarly contains mediated signaling pathways is currently lacking. In the present multiple epidermal growth factor-like modules in its extracellular report, we investigated and identified the involvement of αG 16/ domain (ECD), which binds to its endogenous ligand dermatan Akt/mitogen-activated protein kinase (MAPK)/nuclear factor sulfate (4, 7, 8). Initially identified as a myeloid-restricted tran- kappa-light-chain-enhancer of activated B cells (NF-κB) in script expressed in monocytes (Mos)/Mφ, Nφ, and myeloid DC EMR2 receptor-mediated signaling. Our results indicate that (4), EMR2 protein expression was later shown to be upregulated EMR2 activation/signaling plays a functional role in the dif- during the in vitro differentiation of φM but downregulated ferentiation and inflammatory activation of human monocytic following DC maturation (9). On the other hand, the strongest cells. The EMR2-induced signaling cascades reported here may in vivo EMR2 protein signal was detected in CD16+ blood Mos help identify potential targets for the therapeutic management and BDCA-3+ myeloid DC (10). Foamy Mφ in atherosclerotic of inflammatory disorders, such as SIRS and vibratory urticaria. vessels and splenic Gaucher cells are highly EMR2-positive, whereas multiple sclerosis brain foam cells express little if any EMR2 (11). The differential expression patterns of EMR2 in MATERIALS AND METHODS distinct myeloid populations strongly suggest a regulatory role of EMR2 in myeloid cell function (12, 13). Reagents and Antibodies Indeed, binding and activation of EMR2 by a ECD-specific All chemicals and reagents were purchased from Sigma-Aldrich 2A1 monoclonal antibody (mAb) strongly enhanced the inflam- (St. Louis, MO, USA) unless otherwise specified. Anti-mAbs matory responses of Nφ to a panel of stimuli, while 2A1 treatment used for Western blotting against extracellular signal-regulated alone (without inflammatory stimuli) did not seem effective kinase (ERK)1/2, p-ERK1/2, p38, p-p38 (Thr180/Tyr182), (14). In addition, 2A1-induced EMR2 activation was shown to c-Jun N-terminal kinase (JNK), p-JNK (Thr183/Tyr185), κI B-α, modulate the production of multiple cytokines and survival of p-IκB-α (Ser32), p-Ikkα/β (Ser176/180), and p-Akt (Ser473) were lipopolysaccharide-stimulated Nφ (15). Hence, EMR2 activation obtained from Cell Signaling Technology (Beverly, MA, USA). seems to have a priming effect on φN activation. Furthermore, Anti-Gα16 mAb was from Abcam (Cambridge, UK). Anti-F(ab′)2 upregulated EMR2 expression was identified in φN of patients fragment goat anti-mouse (GAM) IgG (H + L) was from Jackson suffering from systemic inflammatory response syndrome (SIRS), ImmunoResearch (West Grove, PA, USA). Anti-CD11b-PE, anti- and a significant association was noted between the percentage CD62L-PE, anti-CD81-PE, anti-CD9-APC, and anti-CD4-FITC of EMR2-expressing Nφ and the extent of organ failure in SIRS for flow cytometry and anti-phosphotyrosine, anti-β-actin mAb patients. As a result, EMR2 was proposed recently as a novel Nφ for Western blotting were purchased from BD Biosciences. The biomarker for SIRS (14, 16). A more recent study demonstrated mAbs used for cell stimulation were 2A1 (EMR2-specific mAb) that Nφ of liver cirrhosis patients with infection have higher (AbD Serotec) and mouse monoclonal IgG1 (Clone 11711) (R&D EMR2 expression levels, which showed strong correlation with System) as described previously (18). disease severity and predicted overall mortality (17). Likewise, we previously showed that Mφ activated by 2A1-induced Cell Culture EMR2 ligation promoted secretion of several pro-inflammatory THP-1 (ATCC®TIB-202™), HL-60 (ATCC®CCL-240™), and cytokines (18). More recently,