Inflammation ( # 2014) DOI: 10.1007/s10753-014-9990-2
Aurora Kinase A Regulates M1 Macrophage Polarization and Plays a Role in Experimental Autoimmune Encephalomyelitis Lixia Ding,1 Haijuan Gu,1 Xiaoming Gao,1 Sidong Xiong,1,4 and Biao Zheng2,3,4
Abstract—Macrophage polarization is a dynamic and integral process of tissue inflammation and remodeling. Here we demonstrate an important role of Aurora kinase A in the regulation of inflammatory M1 macrophage polarization. We found that there was an elevated expression of Aurora-A in M1 macrophages and inhibition of Aurora-A by small molecules or specific siRNA selectively led to the suppression of M1 polarization, sparing over the M2 macrophage differentiation. At the molecular level, we found that the effects of Aurora-A in M1 macrophages were mediated through the down-regulation of NF-κB pathway and subsequent IRF5 expression. In an autoimmune disease model, experimental autoimmune encephalitis (EAE), treatment with Aurora kinase inhibitor blocked the disease development and shifted the macrophage phenotype from inflammatory M1 to anti-inflammatory M2. Thus, this study reveals a novel function of Aurora-A in controlling the polarization of macrophages, and modification of Aurora-A activity may lead to a new therapeutic approach for chronic inflammatory diseases. KEY WORDS: Aurora kinase; M1 macrophage polarization; inflammation; autoimmune diseases.
INTRODUCTION Macrophages are heterogeneous and comprised of phenotypically and functionally distinct subsets that have different roles in inflammatory processes and require specific tissue milieu for their differentiation and maintenance [1–6]. Macrophage polarization is highly susceptible to cytokine milieu, environmental stimuli through Toll-like Lixia Ding and Haijuan Gu contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10753-014-9990-2) contains supplementary material, which is available to authorized users. 1
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, China 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, East China Normal University, Shanghai, 200241, China 3 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA 4 To whom correspondence should be addressed to Sidong Xiong at Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, China. E-mail: [email protected]; and Biao Zheng at Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA. E-mail: [email protected]
receptors (TLRs) and cytokine receptors. For example, lipopolysaccharide (LPS) stimulation can generate classically activated macrophages (M1) that are programmed to produce pro-inflammatory cytokines, such as interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α and play a crucial role in the initiation and perpetuation of inflammatory responses. Alternatively activated macrophages (M2) primed by IL-4 can exhibit anti-inflammatory properties characterized by the production of IL-10 and a prominent phagocytic function [2, 4, 5]. It has been shown that macrophages can be induced to polarize into opposing M1 and M2 phenotypes and functions when tissue cytokine milieu changes. A balanced M1 to M2 ratio is believed to be essential for the homeostasis of the immune system, and there is evidence that this balance is shifted towards M1 polarization, displayed by both resident macrophages and recruited macrophages from peripheral blood, representing one of the hallmarks in histopathology in different types of diseases including cardiovascular, metabolic, and muscular–skeletal and autoimmune diseases, such as atherosclerosis, rheumatoid arthritis (RA) and multiple sclerosis (MS) [7–9]. In MS, activation of resident microglia and CNS infiltration of activated M1 macrophages is thought to
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Ding, Gu, Gao, Xiong, and Zheng contribute to the amplification of inflammatory responses and tissue injury at the site of pathology [10, 11]. In accordance with this, accumulation of polarized M1 macrophages was considered as a predominant mechanism of demyelination in brain lesions of MS. Therefore, one of the therapeutic approaches for these diseases is to restore the balance of M1 and M2 macrophages and identification of novel targets involved in this process that could be aimed for the effective treatment of diseases such as atherosclerosis and MS. However, it remains elusive as to how an altered M1 and M2 polarization is triggered by various inflammatory insults and what key players and underlying mechanisms are involved. The Aurora kinases belong to the serine threonine kinase superfamilies that play an important role in mitosis by regulating various steps in centrosome formation and chromosome segregation [12, 13]. There are three members of the Aurora kinase family in mammalian cells, Aurora-A, Aurora-B, and Aurora-C, each with different locations and cellular functions. In contrast to normal tissues, aberrant amplification and/or over-expression of Aurora-A are frequent findings in wide, various types of tumors and chronic inflammation [14–17]. There is a close association of Aurora-A over-expression with inflammation in gastric tumorigenesis using different in vivo and in vitro models [16]. In addition, phosphorylation status is the main regulatory mechanism of Aurora-A as its activity largely depends on the phosphorylation of the specific residue Thr288 [18]. Aurora-A is susceptible to small molecule inhibitors, which target the ATP-binding pocket to compete with ATP substrates. A growing number of inhibitors have been developed and shown promising therapeutic efficacy in preclinical studies [17, 19–21]. In this study, we have identified the selectively elevated expression of Aurora-A during the polarization of M1 macrophages under both in vitro and ex vivo conditions. We further investigated the role of AuroraA in the process of M1 macrophage polarization using the Aurora kinase inhibitor VX-680 and specific siRNAs through the regulation of interferon responsive factor (IRF)-5 expression and NF-κB signaling pathway. More importantly, treatment of VX-680 in an autoimmune animal model inhibited the activation and function of M1 macrophages, leading to amelioration of the disease. This study reveals a novel function of Aurora-A in controlling the pathogenic polarization of M1 macrophages in autoimmune diseases and has important implications for the study of Aurora-A as a potential therapeutic target for inflammatory diseases.
MATERIALS AND METHODS Induction and Treatment of EAE C57BL/6 mice were purchased from Shanghai Laboratory Animal Center. All experiments were performed with mice at 6–10 weeks old, with protocols approved by the Institutional Animal Care and Use Committee by Soochow University. The encephalitogenic peptide (residues 35–55, Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-SerArg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) of myelin oligodendrocyte glycoprotein (MOG) was purchased from BioAsia Biotechnology. To induce acute EAE, we injected mice s.c. (in the back region) with 300 ug of the MOG35–55 peptide in CFA containing 5 mg/ml heat-killed Mycobacterium tuberculosis (H37Ra strain; BD Diagnostics). On the day of immunization and 48 h later, the mice were also injected i.v. with pertussis toxin (200 ng/mouse; List Biological Laboratories) in PBS. VX-680 (Santa Cruz) and vehicle control were administered intraperitoneally into mice every day, starting from the day of immunization at a dose of 40 mg/kg. Clinical signs of EAE were assessed daily using the EAE scoring scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of 1 or 2 hind limbs); 3, paraplegia (complete paralysis of 2 hind limbs); 4, paraplegia with fore limb weakness or paralysis; 5, moribund state or death [22]. Histology Tissues for histological analysis were removed from mice at 15 days after immunization and immediately fixed in 4 % paraformaldehyde. Paraffin-embedded 5- to 10-μm sections of spinal cord were stained with Luxol fast blue or H&E and then examined by light microscopy. The degree of demyelination and inflammatory infiltrates was quantified on an average of three spinal cord transverse sections per mouse for a total of six mice per group using the following scoring scale of severity of inflammation: 0, no inflammation; 1, cellular infiltrate only in the perivascular areas and meninges; 2, mild cellular infiltrate in parenchyma; 3, moderate cellular infiltrate in parenchyma; 4, severe cellular infiltrate in parenchyma. Spinal cord demyelination was scored as follows: 1, traces of subpial demyelination; 2, marked subpial and perivascular demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the CNS parenchyma; 5, extensive perivascular and subpial demyelination involving the whole cord
Aurora Kinase A Regulates M1 Macrophage Polarization section with presence of cellular infiltrates in the CNS parenchyma [23, 24].
[3H]-thymidine. Levels of cytokine production in culture supernatants were measured by BioPlex according to the manufacturer’s instructions.
Cell Culture Bone marrow cells were cultured in RPMI-1640 (Gibco), 10 % FBS supplemented with 100 ng/ml macrophage colony-stimulating factor (M-CSF, eBiosicence). After 6 days of culture, the adherent cells were collected and then stimulated with LPS (10 ng/ml) for M1 macrophages and IL-4 (20 ng/ml) for M2 macrophages. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, and the protocol was approved by the Institutional Review Board at Soochow University. Enriched populations of CD14+ monocytes were further purified by CD14 Microbeads (Miltenyi Biotec). M1 macrophages were in vitro differentiated after 6 days of culture of human monocytes in RPMI-1640 medium (Invitrogen) supplemented with GM-CSF (50 ng/ml), followed by LPS stimulation (10 ng/ml) for 24 h. M2 macrophages were in vitro differentiated with M-CSF (100 ng/ ml; R&D Systems) for 6 days, followed by IL-4 stimulation (20 ng/ml) for 24 h.
Flow Cytometry For cell surface staining, cells were stained with antibodies to CD11b (eBioscience). For the cell proliferation assay, cells were labeled with CFSE (eBioscience). For the cell apoptosis assay, cells were stained by Annexin V and analyzed by BD LSRII (BD Biosciences). siRNA Transfection Bone marrow-derived macrophages were transfected with Aurora-A specific siRNA (Dharmacon) or nontargeting control siRNAs using a Mouse Macrophage Nucleofector® Kit (LONZA) according to the manufacturer’s instructions prior to M1 macrophage polarization. Statistical Analysis Statistical significance was determined by performing a two-tail Student’s t test or one-way ANOVA. p values of
Aurora Kinase A Regulates M1 Macrophage Polarization and Plays a Role in Experimental Autoimmune Encephalomyelitis Lixia Ding,1 Haijuan Gu,1 Xiaoming Gao,1 Sidong Xiong,1,4 and Biao Zheng2,3,4
Abstract—Macrophage polarization is a dynamic and integral process of tissue inflammation and remodeling. Here we demonstrate an important role of Aurora kinase A in the regulation of inflammatory M1 macrophage polarization. We found that there was an elevated expression of Aurora-A in M1 macrophages and inhibition of Aurora-A by small molecules or specific siRNA selectively led to the suppression of M1 polarization, sparing over the M2 macrophage differentiation. At the molecular level, we found that the effects of Aurora-A in M1 macrophages were mediated through the down-regulation of NF-κB pathway and subsequent IRF5 expression. In an autoimmune disease model, experimental autoimmune encephalitis (EAE), treatment with Aurora kinase inhibitor blocked the disease development and shifted the macrophage phenotype from inflammatory M1 to anti-inflammatory M2. Thus, this study reveals a novel function of Aurora-A in controlling the polarization of macrophages, and modification of Aurora-A activity may lead to a new therapeutic approach for chronic inflammatory diseases. KEY WORDS: Aurora kinase; M1 macrophage polarization; inflammation; autoimmune diseases.
INTRODUCTION Macrophages are heterogeneous and comprised of phenotypically and functionally distinct subsets that have different roles in inflammatory processes and require specific tissue milieu for their differentiation and maintenance [1–6]. Macrophage polarization is highly susceptible to cytokine milieu, environmental stimuli through Toll-like Lixia Ding and Haijuan Gu contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10753-014-9990-2) contains supplementary material, which is available to authorized users. 1
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, China 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, East China Normal University, Shanghai, 200241, China 3 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA 4 To whom correspondence should be addressed to Sidong Xiong at Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, China. E-mail: [email protected]; and Biao Zheng at Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA. E-mail: [email protected]
receptors (TLRs) and cytokine receptors. For example, lipopolysaccharide (LPS) stimulation can generate classically activated macrophages (M1) that are programmed to produce pro-inflammatory cytokines, such as interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α and play a crucial role in the initiation and perpetuation of inflammatory responses. Alternatively activated macrophages (M2) primed by IL-4 can exhibit anti-inflammatory properties characterized by the production of IL-10 and a prominent phagocytic function [2, 4, 5]. It has been shown that macrophages can be induced to polarize into opposing M1 and M2 phenotypes and functions when tissue cytokine milieu changes. A balanced M1 to M2 ratio is believed to be essential for the homeostasis of the immune system, and there is evidence that this balance is shifted towards M1 polarization, displayed by both resident macrophages and recruited macrophages from peripheral blood, representing one of the hallmarks in histopathology in different types of diseases including cardiovascular, metabolic, and muscular–skeletal and autoimmune diseases, such as atherosclerosis, rheumatoid arthritis (RA) and multiple sclerosis (MS) [7–9]. In MS, activation of resident microglia and CNS infiltration of activated M1 macrophages is thought to
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Ding, Gu, Gao, Xiong, and Zheng contribute to the amplification of inflammatory responses and tissue injury at the site of pathology [10, 11]. In accordance with this, accumulation of polarized M1 macrophages was considered as a predominant mechanism of demyelination in brain lesions of MS. Therefore, one of the therapeutic approaches for these diseases is to restore the balance of M1 and M2 macrophages and identification of novel targets involved in this process that could be aimed for the effective treatment of diseases such as atherosclerosis and MS. However, it remains elusive as to how an altered M1 and M2 polarization is triggered by various inflammatory insults and what key players and underlying mechanisms are involved. The Aurora kinases belong to the serine threonine kinase superfamilies that play an important role in mitosis by regulating various steps in centrosome formation and chromosome segregation [12, 13]. There are three members of the Aurora kinase family in mammalian cells, Aurora-A, Aurora-B, and Aurora-C, each with different locations and cellular functions. In contrast to normal tissues, aberrant amplification and/or over-expression of Aurora-A are frequent findings in wide, various types of tumors and chronic inflammation [14–17]. There is a close association of Aurora-A over-expression with inflammation in gastric tumorigenesis using different in vivo and in vitro models [16]. In addition, phosphorylation status is the main regulatory mechanism of Aurora-A as its activity largely depends on the phosphorylation of the specific residue Thr288 [18]. Aurora-A is susceptible to small molecule inhibitors, which target the ATP-binding pocket to compete with ATP substrates. A growing number of inhibitors have been developed and shown promising therapeutic efficacy in preclinical studies [17, 19–21]. In this study, we have identified the selectively elevated expression of Aurora-A during the polarization of M1 macrophages under both in vitro and ex vivo conditions. We further investigated the role of AuroraA in the process of M1 macrophage polarization using the Aurora kinase inhibitor VX-680 and specific siRNAs through the regulation of interferon responsive factor (IRF)-5 expression and NF-κB signaling pathway. More importantly, treatment of VX-680 in an autoimmune animal model inhibited the activation and function of M1 macrophages, leading to amelioration of the disease. This study reveals a novel function of Aurora-A in controlling the pathogenic polarization of M1 macrophages in autoimmune diseases and has important implications for the study of Aurora-A as a potential therapeutic target for inflammatory diseases.
MATERIALS AND METHODS Induction and Treatment of EAE C57BL/6 mice were purchased from Shanghai Laboratory Animal Center. All experiments were performed with mice at 6–10 weeks old, with protocols approved by the Institutional Animal Care and Use Committee by Soochow University. The encephalitogenic peptide (residues 35–55, Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-SerArg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys) of myelin oligodendrocyte glycoprotein (MOG) was purchased from BioAsia Biotechnology. To induce acute EAE, we injected mice s.c. (in the back region) with 300 ug of the MOG35–55 peptide in CFA containing 5 mg/ml heat-killed Mycobacterium tuberculosis (H37Ra strain; BD Diagnostics). On the day of immunization and 48 h later, the mice were also injected i.v. with pertussis toxin (200 ng/mouse; List Biological Laboratories) in PBS. VX-680 (Santa Cruz) and vehicle control were administered intraperitoneally into mice every day, starting from the day of immunization at a dose of 40 mg/kg. Clinical signs of EAE were assessed daily using the EAE scoring scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of 1 or 2 hind limbs); 3, paraplegia (complete paralysis of 2 hind limbs); 4, paraplegia with fore limb weakness or paralysis; 5, moribund state or death [22]. Histology Tissues for histological analysis were removed from mice at 15 days after immunization and immediately fixed in 4 % paraformaldehyde. Paraffin-embedded 5- to 10-μm sections of spinal cord were stained with Luxol fast blue or H&E and then examined by light microscopy. The degree of demyelination and inflammatory infiltrates was quantified on an average of three spinal cord transverse sections per mouse for a total of six mice per group using the following scoring scale of severity of inflammation: 0, no inflammation; 1, cellular infiltrate only in the perivascular areas and meninges; 2, mild cellular infiltrate in parenchyma; 3, moderate cellular infiltrate in parenchyma; 4, severe cellular infiltrate in parenchyma. Spinal cord demyelination was scored as follows: 1, traces of subpial demyelination; 2, marked subpial and perivascular demyelination; 3, confluent perivascular or subpial demyelination; 4, massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the CNS parenchyma; 5, extensive perivascular and subpial demyelination involving the whole cord
Aurora Kinase A Regulates M1 Macrophage Polarization section with presence of cellular infiltrates in the CNS parenchyma [23, 24].
[3H]-thymidine. Levels of cytokine production in culture supernatants were measured by BioPlex according to the manufacturer’s instructions.
Cell Culture Bone marrow cells were cultured in RPMI-1640 (Gibco), 10 % FBS supplemented with 100 ng/ml macrophage colony-stimulating factor (M-CSF, eBiosicence). After 6 days of culture, the adherent cells were collected and then stimulated with LPS (10 ng/ml) for M1 macrophages and IL-4 (20 ng/ml) for M2 macrophages. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, and the protocol was approved by the Institutional Review Board at Soochow University. Enriched populations of CD14+ monocytes were further purified by CD14 Microbeads (Miltenyi Biotec). M1 macrophages were in vitro differentiated after 6 days of culture of human monocytes in RPMI-1640 medium (Invitrogen) supplemented with GM-CSF (50 ng/ml), followed by LPS stimulation (10 ng/ml) for 24 h. M2 macrophages were in vitro differentiated with M-CSF (100 ng/ ml; R&D Systems) for 6 days, followed by IL-4 stimulation (20 ng/ml) for 24 h.
Flow Cytometry For cell surface staining, cells were stained with antibodies to CD11b (eBioscience). For the cell proliferation assay, cells were labeled with CFSE (eBioscience). For the cell apoptosis assay, cells were stained by Annexin V and analyzed by BD LSRII (BD Biosciences). siRNA Transfection Bone marrow-derived macrophages were transfected with Aurora-A specific siRNA (Dharmacon) or nontargeting control siRNAs using a Mouse Macrophage Nucleofector® Kit (LONZA) according to the manufacturer’s instructions prior to M1 macrophage polarization. Statistical Analysis Statistical significance was determined by performing a two-tail Student’s t test or one-way ANOVA. p values of
