Deficiency of the G Protein Gαq Ameliorates Experimental

Cellular & Molecular Immunology (2017) 14, 557–567 & 2017 CSI and USTC All rights reserved 2042-0226/17 $32.00 www.nature.com/cmi RESEARCH ARTICLE Deficiency of the G protein Gαq ameliorates experimental autoimmune encephalomyelitis with impaired DC-derived IL-6 production and Th17 differentiation Weiming Lai1,5, Yingying Cai1,5, Jinfeng Zhou1, Shuai Chen1, Chaoyan Qin1, Cuixia Yang1, Junling Liu2, Xin Xie3 and Changsheng Du1,4 Many G protein-coupled receptors (GPCRs) are reported to be involved in the pathogenesis of multiple sclerosis (MS), and ~ 40% of all identified GPCRs rely on the Gαq/11 G protein family to stimulate inositol lipid signaling. However, the function of Gα subunits in MS pathogenesis is still unknown. In this study, we attempted to determine the role of Gαq in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a well-known mouse model of MS. We discovered that compared with wild-type mice, Gαq-knockout mice exhibited less severe EAE symptoms, with lower clinical scores, reduced leukocyte infiltration and less extensive demyelination. Moreover, a significantly lower percentage of Th17 cells, one of the key players in MS pathogenesis, was observed in Gαq-knockout EAE mice. Studies in vitro demonstrated that deficiency of Gαq in CD4+ T cells directly impaired Th17 differentiation. In addition, deficiency of Gαq significantly impaired DC-derived IL-6 production, thus inhibiting Th17 differentiation and the Gαq-PLCβ-PKC and Gαq-MAPKs signaling pathways involved in the reduced IL-6 production by DCs. In summary, our data highlighted the critical role of Gαq in regulating Th17 differentiation and MS pathogenesis. Cellular & Molecular Immunology (2017) 14, 557–567; doi:10.1038/cmi.2016.65; published online 20 February 2017 Keywords: Dendritic cells; EAE; Gαq; IL-6; Th17 differentiation; MS INTRODUCTION MS.3 T cell polarization, leukocyte migration and infiltration Multiple sclerosis (MS) is a common autoimmune inflamma- into the CNS are very important steps involved in EAE tory disease of the central nervous system (CNS) characterized pathogenesis.4 However, how various environmental stimuli by immune-mediated demyelination, axonal loss and tissue participate in MS and EAE pathogenesis is unclear. G protein- destruction. The animal model, experimental autoimmune coupled receptors (GPCRs) are known to mediate most of the encephalomyelitis (EAE), has many pathological and histolo- physiological responses to environmental stimuli, suggesting gical similarities with MS. The pathogenesis of MS is still that GPCRs may play an important role in MS pathogenesis by unclear. Many environmental risk factors have been reported mediating signal transduction. to be involved in MS pathogenesis, such as viral infection, GPCRs, which are important drug targets, mediate a number sunlight exposure and cigarette smoking.1,2 Recently, many of biological processes by transmitting signals across the cell studies have shown that both Th1 and Th17 cells play a role in membrane.5 The binding of agonist and receptor leads to the 1Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China; 2Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 3National Center for Drug Screening, CAS Key Laboratory of Receptor Research, Chinese Academy of Sciences, Shanghai Institute of Materia Medica, Shanghai 201203, China and 4Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China 5These authors contributed equally to this work. Correspondence: Dr C Du, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, 1239 Si Ping Road, Shanghai 200092, China. E-mail: [email protected] Received: 23 August 2016; Revised: 3 November 2016; Accepted: 3 November 2016 Deficiency of the G protein Gαq ameliorates experimental autoimmune encephalomyelitis WLaiet al 558 activation of the G protein Gα subunit and the Gβγ dimer, paraformaldehyde ⩾ 12 h. Paraffin-embedded tissue sections which can regulate a set of independent effectors.6 Gα subunits (5 μm) were stained with H&E and Luxol fast blue to analyze are divided into five different families: Gαq, Gαs, Gαi, Gαvand inflammatory infiltration and demyelination. Image-Pro soft- Gα12, according to sequence similarity.7,8 The Gαq/11 family ware was used to calculate the number of infiltrating cells and has four family members; Gαq and Gα11 are ubiquitously the percentage of myelin loss in inflammatory foci per section α expressed, G 14 is expressed in the kidney, liver and lung, and for quantization of inflammation and demyelination levels, α 9 G 15/16 is only found in hematopoietic cells. Many cellular respectively. Paraffin-embedded sections of the spinal cords α proteins have been reported to interact with G q, such as were rehydrated and put in antigen retrieval solution at 95 °C β β 10 phospholipase C (PLC ), Akt and MAPK members. for 20 min before proceeding to immunohistochemistry. Sec- Many GPCRs have been reported to participate in MS or tions were incubated with rabbit polyclonal anti-NFH antibody EAE pathogenesis.11 In MS patients, β-adrenergic receptors (N4142, 1:500) and mouse anti-GFAP antibody (MAB360, have been reported to be increased in PBMCs and correlated 1:500) at 4 °C overnight. The secondary antibody was con- with disease activity;12 asignificant decrease in kappa opioid jugated to Alexa Fluor 546 (Thermo Fisher, A-11010, 1:1 000) receptors has been found in the spinal cords of MS patients and or 488 (Thermo Fisher, A-11001, 1:1 000), and nuclei were are thought to be related to neuropathic pain.13 CVT-6883, an fl A2BAR-specific antagonist, alleviates EAE pathogenesis via stained with DAPI. An Olympus IX51 inverted uorescence fl reducing IL-6 secretion from dendritic cells (DCs);14 CXCR3 microscope was used for uorescence detection. signaling reduces the severity of EAE by controlling the distribution of effector and regulatory T cells in the CNS.15 Reverse transcription and quantitative real-time PCR However, the function of G proteins in MS or EAE is still not Total RNA was extracted from mouse tissues (lymph nodes, clear. It has been estimated that almost 40% of all identified spleen, cerebrum and lumbar spinal cord) using TRI reagent GPCRs rely on the Gαq/11 family to stimulate signaling. In this (Molecular Research Center, Inc.). Reverse transcription was study, we found that Gαq-KO mice showed less severe clinical performed with random hexamer primers and murine leuke- symptoms of EAE and reduced CNS infiltration of inflamma- mia virus reverse transcriptase (Promega, Madison, WI, USA). tory cells. Further research demonstrated that Gαq-KO mice Quantitative real-time PCR was assayed in a LightCycler showed reduced Th17 differentiation by inhibiting IL-6 pro- quantitative PCR apparatus with SYBR Green 2 × qPCR α β duction by DCs, which was mediated by G q-PLC and Master Mix (Bioneer, Seoul, South Korea). Expression was α MAPK signaling. Our results revealed a critical role of G q normalized relative to β-actin and then to the expression of the fl in regulating EAE pathogenesis by in uencing Th17 differen- control. The primer sequences are listed in Supplementary tiation and IL-6 production in DCs. Table 1. MATERIALS AND METHODS Mice Flow cytometry + C57BL/6 mice were purchased from Nanjing Biomedical Research Lymphocytes from tissues or CD4 T cells from in vitro differen- Institute of Nanjing University (Nanjing, China). Gαq-KO mice tiation assays were incubated with PMA (50 ng/ml; Sigma, St were derived from the C57BL/6 strain, and genotype identification Louis, MO, USA), ionomycin (750 ng/ml; Sigma) and brefeldin A was based on PCR.16 All mice were maintained at the Tongji (10 μg/ml; Sigma) for 5 h at 37 °C. Surface markers were University animal care facility in pathogen-free conditions. The incubated with relevant antibodies for 30 min at 4 °C in the dark. experiments were carried out in accordance with the Tongji Then, the cells were subjected to fixation and permeabilization, University Animal Care Committee guidelines. followed by intracellular cytokine (IL-17 and IFN-γ) staining with relevant antibodies for 30min at 4°C in the dark. A Foxp3 EAE induction staining buffer set (eBioscience, San Diego, CA, USA) was used to – Female mice (8 10 weeks of age) were immunized with detect Treg cells after fixation, permeabilization and staining with μ MOG35-55 (200 g) in CFA, which contained heat-killed antibody for 30 min at 4 °C in the dark. Flow cytometric analysis Mycobacterium tuberculosis (5 mg/ml). Pertussis toxin (200 ng/ was carried out with a Guava EasyCyte 8HT system and mouse) was IP injected on day 0 and day 2. Clinical appearance GuavaSoft software. was assessed daily and scored as follows: 0, normal; 1, paralyzed tail; 2, mildly paralyzed hind legs; 3, totally paralyzed hind legs; 3.5, paraplegia with mildly paralyzed forelimbs; 4, paraplegia ELISA with paralyzed forelimbs; 4.5, moribund and 5, death. Serum was collected from the orbital venous blood. To collect culture supernatants, lymphocytes were isolated from the Histopathological and immunohistochemical analysis draining lymph nodes, seeded into 96-well plates (2 × 105/ μ μ For the histological analysis, mice were anesthetized with well/100 l and restimulated with MOG35–55(20 g/ml) for chloral hydrate and

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