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The Regulation of Macrophage Polarization by Hypoxia-PADI4 Coordination in Rheumatoid Arthritis

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The Regulation of Macrophage Polarization by Hypoxia-PADI4 Coordination in Rheumatoid Arthritis The Regulation of Macrophage Polarization by Hypoxia-PADI4 Coordination in Rheumatoid Arthritis Yu Cheng Shanghai East Hospital https://orcid.org/0000-0002-1380-3780 Yuying Si Shanghai East Hospital Lan Wang Shanghai East Hospital Menglei Ding Shanghai East Hospital Shanshan Yu Shanghai East Hospital Cuncun Chen Shanghai East Hospital Liu Lu Shanghai East Hospital Yide Guo Shanghai East Hospital Ming Zong shanghai east hospital Lieying Fan ( [email protected] ) Shanghai East Hospital Research article Keywords: Rheumatoid arthritis, Macrophage polarization, Hypoxia, PADI4 Posted Date: November 2nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-98320/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Version of Record: A version of this preprint was published at International Immunopharmacology on October 1st, 2021. See the published version at https://doi.org/10.1016/j.intimp.2021.107988. Page 2/23 Abstract Background: Hypoxia, a common feature of rheumatoid arthritis (RA), induces the overexpression of peptidyl arginine deiminase 4 (PADI4) in broblast-like synoviocytes (FLSs) and macrophages. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the role of hypoxia-PADI4 for macrophage polarization in RA patients. Methods: synovial tissue(ST) and synovial uid(SF) were collected from 3 OA patients and 6 RA patients. The distribution of M1 and M2 in ST and cytokines in SF were examined by immunohistochemical analysis and BioPlex immunoassays. THP-1 macrophages and BMDM polarization were determined under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions. The effects of PADI4 on macrophages were determined by transfection of adenovirus vector-coated PADI4 (AdPADI4) and the use of PADI4 inhibitor. To mimic the environment of RA joints, THP-1 macrophages polarization was examined after coculturing with RA-FLSs. Finally, the roles of PADI4 in joint synovial lesions on macrophage polarization were investigated in collagen-induced arthritis(CIA) rats. Results: We found increased macrophage polarization of M1 and M2 in the RA ST, compared with OA ST. The ratio of M1/M2 for RA and OA was 1.633 ± 0.1443 and 2.544 ± 0.4429, respectively. The concentration of M1- and M2-type cytokines was higher in RA than that in OA patients. Hypoxia contributed to the increase of the gene and protein expression of M1 and M2 markers. M1- but not M2- type gene expression showed a positive relationship with PADI4 expression while the level of expression of M2-type genes showed no signicant difference. RA-FLSs could promote the copolarization of M1 and M2, and PADI4 inhibitor reversed M1 activation. The degree of joint swelling and destruction was effectively alleviated, and the number of macrophages especially M1 decreased in CIA rats after downregulating PADI4 expression. Conclusion: Hypoxia is responsible for the copolarization of M1 and M2. Hypoxia-associated PADI4 is responsible for M1 macrophage activation, implying that inammatory environment can be eased by decreasing PADI4 expression and improving the hypoxic environment. Background Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by abnormal activation of the immune system, resulting in synovitis and destruction of joint function (1). Synovitis is characterized by an overabundance of RA broblast-like synoviocytes (RA-FLSs), monocytes, macrophages, and other immune cells within the RA synovial tissue (2). Among these cells, it has been well recognized that macrophages play an essential role in the initiation and perpetuation of RA by secreting multiple inammatory and anti-inammatory factors (3). Mature macrophages have two polarization states: classical activation (M1) and the alternative activation (M2) phenotype (4). M1 macrophages, induced by Th-1 cytokine interferon-γ (IFN-γ) or lipopolysaccharide (LPS), contribute to the development of inammation and joint destruction by producing numerous pro-inammatory factors, Page 3/23 such as IL-12 and TNF-α, and specically expressing NOS-2 and CCR7 as biomarkers. The Th-2 cytokines IL-4, IL-10, and IL-13, however, cause macrophages to polarize into the M2 phenotype. They can then secrete anti-inammatory cytokine IL-10, increasing the specic expression of the mannose receptor (CD206) (5). The high concentration of TNF-α in the RA joint synovium can promote M1 polarization (6), and the broblast factor secreted by RA-FLSs can promote M2 polarization (7). However, the role and molecular mechanism of M1/M2 in RA synovial lesions remain largely unknown. An early step in the development of RA is the activation of RA-FLSs. This is followed by mass production of inammatory mediators, including cytokines and chemokines, which mediate the recruitment and interaction with immune cells (8). This process enhances the oxygen consumption caused by the proliferation of cells in RA synovium, which leads to the oxygen tension in RA, indicated by the 2–4% values, which in diseased synovial tissues can be even lower than 1% of O2 (hypoxia condition); healthy controls show levels around 8% (9). Macrophages, as well as other leukocytes, are sensitive to tissue hypoxia, which can increase inammatory cytokine production (10), yet how hypoxia affects macrophage polarization is not well understood in vitro. Peptidyl arginine deiminase IV (PADI4) is involved in the post-translational modication of arginine residues (11). A study revealed that a large number of activated macrophages and FLSs in RA synovial tissues are involved in the overexpression of PADI4 (12). The contribution of PADI4 to the apoptosis of RA-FLSs has been revealed (13). Considering the multiple roles of PADI4 in the regulation of gene expression and immunological functions, PADI4 may be a potential target for the therapy of autoimmune diseases. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the polarization of macrophages in RA synovial tissue and elucidate the role of hypoxia-PADI4 in macrophage polarization. A collagen-induced arthritis (CIA) mouse model (13) was used to explore the PADI4-mediated development of inammation. Materials And Methods Patients and controls Clinical samples were the resident synovial tissue collected from patients undergoing knee arthroscopic or routine examination at Shanghai East Hospital, including six patients with RA and three patients with osteoarthritis (OA). All of the patients provided consent and met the diagnostic criteria of the American College of Rheumatology (ACR) criteria for RA (14) and OA (15). The Ethics Committee approved the study protocol of Shanghai East Hospital. Animals Page 4/23 Twelve SD rats were administered intradermally at the base of the tail with a dose of bovine type II collagen (100 μg) emulsied in complete Freund's adjuvant on day 0. Then a booster injection was provided on day 21 with bovine type II collagen (100 μg) emulsied with incomplete Freund's adjuvant. The rats were then randomly divided into two groups: the CIA model group and the PADI4 inhibition group. Normal non-immunized rats were selected as the normal control group. The rats were sacriced on the nal day; joints were collected from all of the groups. Immunohistochemical and immunouorescence analyses Synovial tissues from humans and joints from rats were xed in 10% neutral buffered formalin and then embedded in paran. Paran sections were deparanized and rehydrated. After blocking with 3%H2O2, the sections were incubated with a goat polyclonal antibody against human CCR7 (1:500, Abcam, Cambridge, MA, US) and rabbit polyclonal antibody against human mannose receptor (CD206, 1:500, Abcam, Cambridge, MA, US) at 4°C overnight. Next, the sections were incubated with secondary antibody for 30 min at room temperature. Immunoreactive signals were visualized using DAB (diaminobenzidine 3). For double immunouorescence staining, the sections were incubated with anti-CCR7 antibody or anti- CD206 antibody at 4°C overnight, then incubated with Donkey anti-Goat IgG Alexa Fluor 488 (1:200, Thermo Fisher, US) and Donkey anti-Rabbit IgG Alexa Fluor 594 (1:200, Thermo Fisher, US) for 30 min at room temperature. The cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and visualized under a uorescence microscope. Cytokine immunoassay Synovial uid samples were collected, three RA, and three OA samples as controls. Undiluted media samples were plated, and analytes (IL-1, IL-6, IL-8, IL-10, IL-12 IL-13, IL-17, IFN-γ, and TNF-α) were assessed according to the protocol included with the ProcartaPlex™ Platinum Human Multiplex Assay. Each plate was read on the BioPlex 200 (BioRad). Cell culture Human monocytic THP-1 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Roswell Park Memorial Institute medium (RPMI) 1640 culture medium containing 10% heat-inactivated fetal bovine serum (FBS), 20 mg/mL penicillin, and 20 mg/mL streptomycin. Cells were treated with 100 nM PMA (Sigma-Aldrich, #P8195) for 24 h and differentiated into M0 macrophages as previously reported (16), then the cells were further cultured in a normoxic (21% O2) and a hypoxia (3% O2) incubator. M1 was obtained from M0 by being treated with 20 μg/mL IFN-γ (PeproTech) and 100 ng/mL LPS (#L8630, Sigma), while M2 was treated with 20 μg/mL IL-4 (PeproTech) for another 48 h. Page 5/23 Bone marrow (BM) cells were harvested from the femurs and tibias of 6- to 10-week-old C57BL/6 mice (SJA Laboratory Animal Co., Ltd). The cells were cultured in Dulbecco's modied Eagle's medium (DMEM) (Gibco) supplemented with 10% FBS (Wisent Biomart) and recombinant mouse M-CSF (40 ng/ml; PeproTech). After one week, BMDMs were replated and untreated (M0) macrophages were then stimulated with Escherichia coli LPS O111: B4 (100 ng/ml; Sigma) and IFN-γ (20 ng/ml; PeproTech) for 24 h (M1) or with IL-4 (20 ng/ml; PeproTech) for 24 h (M2).
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