Fumiyuki Sasaki

ROCK-Isoform-Specific Polarization of Macrophages Associated with Age-Related Macular Degeneration

Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia

Leukotriene B4 (LTB4) has been implicated in the pathogenesis of allergic diseases. BLT2, a low‐affinity LTB4 receptor, is activated by LTB4 and 12(S)‐hydroxyheptadeca‐5Z,8E,10E ‐trienoic acid (12‐HHT). Although the high‐affinity LTB4 receptor BLT1 has been shown to exert proinflammatory roles, the role of BLT2 in allergic inflammation has not been clarified. To study the function of BLT2 in development of asthma, we used mice model of ovalbumin (OVA)‐induced allergic airway disease. The 12‐HHT levels were elevated in bronchoalveolar lavage (BAL) fluids of OVA‐sensitized/challenged wild‐type mice. BLT2‐deficient mice exhibited enhanced eosinophilia in BAL fluids after OVA exposure. Interleukin (IL)‐13 levels in BAL fluids and IL‐13‐producing CD4+ T cells in the lungs were elevated in BLT2‐deficient mice compared to wild‐type mice, whereas the levels of IL‐4, IL‐5, and interferon (IFN)‐γ in BAL fluids and serum OVA‐specific IgE were comparable. Transfection of BLT2‐specific small interfering RNA enhanced IL‐13 production in CD4+ T cells in vitro. Expression of BLT2 mRNA in CD4+ T cells was significantly reduced in patients with asthma compared to healthy control subjects. These findings indicate that BLT2 has a protective role in allergic airway inflammation and that diminished BLT2 expression in CD4+ T cells may contribute to the pathophysiology of asthma.—Matsunaga, Y., Fukuyama, S., Okuno, T., Sasaki, F., Matsunobu, T., Asai, Y., Matsumoto, K., Saeki, K., Oike, M., Sadamura, Y., Machida, K., Nakanishi, Y., Kubo, M., Yokomizo, T., Inoue, H., Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia. FASEBJ. 27, 3306‐3314 (2013). www.fasebj.org

Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)‐hydroxyheptadeca‐5Z,8E,10E‐trienoic acid (12‐HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2‐overexpressing Madin‐Darby canine kidney (MDCK) II cells and in the small intestine of BLT2‐transgenic mice. BLT2‐deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK‐BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK‐Mock cells, and 12‐HHT stimulation accelerated TER recovery only in MDCK‐BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12‐HHT/BLT2 axis up‐regulated claudin‐4 (CLDN4) expression in MDCK‐BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12‐HHT‐dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12‐HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12‐HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein‐p38 MAPK pathway.—Ishii, Y., Saeki, K., Liu, M., Sasaki, F., Koga, T., Kitajima, K., Meno, C., Okuno, T., Yokomizo, T. Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins. FASEB J. 30, 933–947 (2016). www.fasebj.org

A high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled receptor study

The FLAG sequence (DYKDDDDK) is an artificial sequence widely used to detect, quantify, and purify proteins expressed as FLAG-fusion proteins. Several highly specific monoclonal antibodies for FLAG are commercially available; however, they are not always sensitive enough to detect proteins expressed at low levels and can give rise to unacceptable levels of background signal when used for immunostaining in vitro and in vivo. The current study reports the successful establishment of hybridoma cells that produce an extremely high-affinity antibody to FLAG, namely 2H8 Ab. 2H8 Ab stained FLAG-tagged G-protein-coupled receptors more strongly than commercially available antibodies in both flow cytometry and immunostaining experiments with no background staining. 2H8 was sensitive enough to detect FLAG-tagged G-protein-coupled receptors and soluble proteins in crude preparations, which could not be achieved using commercially available antibodies. Only 10 ng of 2H8 Ab was required to immunoprecipitate FLAG-tagged G-protein-coupled receptors from cell lysates. Of note, 2H8 stained FLAG-tagged BLT2, a low-affinity leukotriene B4 receptor, expressed in vivo in the small intestine of mice under control of the villin promoter. Thus, 2H8 Ab is a promising tool for analyzing various FLAG-fusion proteins, particularly G-protein-coupled receptors, both in vitro and in vivo.

Absence of LTB4/BLT1 axis facilitates generation of mouse GM-CSF-induced long-lasting antitumor immunologic memory by enhancing innate and adaptive immune systems.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.

Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.

Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.

A Novel Anti-FLAG Monoclonal Antibody Is Useful to Study GPCRs

Epitope tagging is a technique widely used in molecular and cellular biology. FLAG (DYKDDDDK), influenza virus hemagglutinin (YPYDVPDYA), and c-myc (EQKLISEEDL) tags are famous and are frequently used because high-affinity antibodies against these tags are commercially available. This technique is extremely useful for G protein-coupled receptor (GPCR) research because it is generally difficult to establish specific monoclonal antibodies (mAb) against GPCRs. We unexpectedly established a novel anti-FLAG mAb (2H8) during an attempt to generate an anti-mouse leukotriene B4 receptor 1 mAb. This mAb is a powerful tool to analyze various FLAG-fusion proteins, particularly GPCRs, both in vitro and in vivo. In this chapter, we describe experimental protocols to utilize the 2H8 mAb for flow cytometric, immunofluorescence staining, and immunoprecipitation analyses of various FLAG-tagged GPCRs.