Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models
Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M. Penninger, Koh-Hei Sonoda, Takehiko Yokomizo
Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M. Penninger, Koh-Hei Sonoda, Takehiko Yokomizo
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Research Article Angiogenesis Inflammation

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

Abstract

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.

Authors

Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M. Penninger, Koh-Hei Sonoda, Takehiko Yokomizo

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Figure 5

M2-type macrophages express BLT1 and infiltrate into the injured eyes of aged mice.

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M2-type macrophages express BLT1 and infiltrate into the injured eyes of...
BMDMs from aged BLT1-WT (filled circles) and BLT1-KO (open circles) mice (>20 weeks old) were polarized to M1 and M2 macrophages, and BLT1 expression was examined by quantitative PCR (A) and Flow cytometry analysis (B). M0 denotes a sample of nonpolarized BMDMs. RAW264.7 (C) and THP-1 (D) cells were polarized to M0 (cross plots), M1 (open squares), and M2 (filled circles) macrophages, and expression of BLT1, PD-L2 (an M2 marker), and CD80 (an M1 marker) was examined by flow cytometry. The y axis shows the MFI relative to that of an isotype control (mouse IgG1). MFI, mean fluorescence intensity. n = 3 per group. (E–H) FACS analysis of M2 macrophages isolated from the laser-injured eyes of aged WT mice (>20 weeks old) as described in Figure 3, C and D. CD206+ M2 macrophages, CD11bhiF4/80hiCD206+; CD206+BLT1+M2 macrophages, CD11bhiF4/80hiCD206+BLT1+. n = 3–4 per group. (I) FACS analysis of M2 markers on the ocular-infiltrating BLT1+ and BLT1– macrophages on day 5 after laser injury from aged WT mice. CD206, CD301, and PD-L2 are specific surface markers of M2 macrophages. Cells were stained with anti-CD206, anti-CD301, and anti–PD-L2 mAbs (black outlines) or isotype controls (gray filled histograms). Overlays show the expression level of M2 markers on BLT1+ (black outlines) and BLT1– (black dotted lines) macrophages. These macrophages were gated as the CD45+F4/80hiCD11bhi population. (J and K) FACS analysis of BLT1 expression on the ocular-infiltrating CD206+ M2 macrophages on day 3 after laser injury from young or aged WT mice. n = 3 per group. (A–D) **P < 0.01; ***P < 0.005; ****P < 0.001 versus 0 hours (2-way ANOVA with Bonferroni’s post hoc test). (E–H) *P < 0.05; **P < 0.01 versus day 0 (1-way ANOVA with Dunnett’s post hoc test). (K) **P < 0.01 (Student’s t test). Results are representative of at least 2 independent experiments (A–I).