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Abstract：
BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation.
METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.
RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.
CONCLUSIONS: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.
METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.
RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.
CONCLUSIONS: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.
摘要：
背景：早产儿视网膜病变（ROP），常表现为支气管肺发育不良（BPD），是影响极早产儿的最常见疾病之一，并且是全球儿童严重视力障碍的主要原因。炎症小体级联和小胶质细胞的激活在ROP和BPD的发展中起作用。含有caspase募集结构域（ASC）的凋亡相关斑点样蛋白在炎症小体组装中至关重要。利用氧诱导的视网膜病变（OIR）和BPD的小鼠模型，这项研究旨在检验高氧诱导ASC斑点形成的假设，导致小胶质细胞活化和视网膜病变，以及人源化单克隆抗体对ASC斑点形成的抑制作用，IC100，针对ASC，将改善小胶质细胞活化和异常视网膜血管形成。
方法：我们首先使用BPD模型测试了表达ASC融合蛋白与C末端黄嘌呤（荧光GFP同种型）的ASC-黄嘌呤报告小鼠视网膜中的ASC斑点形成。通过将新生小鼠暴露于室内空气（RA）或85％O2，从出生后第1天到第14天，眼睛损伤。在P14上解剖视网膜，并使用视网膜平坦的支架来检测具有AF-594缀合的异凝集素B4（IB4）和黄嘌呤标记的ASC斑点的血管内皮。为了评估IC100对OIR模型的影响，新生ASC黄嘌呤报告小鼠和野生型小鼠（C57BL/6J）从P1到P6暴露于RA，然后从P7到P11暴露于75％O2，然后从P12到P18暴露于RA。在P12时，将小鼠随机分为以下组：RA与安慰剂PBS（RA-PBS），O2与PBS（O2-PBS），O2+IC100玻璃体内注射（O2-IC100-IVT），和O2+IC100腹腔注射(O2-IC100-IP)。视网膜血管形成通过用IB4平装染色来评价。通过免疫荧光染色检测同种异体移植物炎症因子1（AIF-1）和CD206的小胶质细胞活化。在H&E染色切片上分析视网膜结构,并通过模式视网膜电图（PERG）分析功能。进行视网膜的RNA测序(RNA-seq)以确定IC100处理在OIR中的转录作用。
结果：在BPD和OIR模型中，由于高氧暴露，视网膜中的ASC斑点显着增加，并与异常血管系统共定位，这与小胶质细胞活化增加有关。用IC100-IVT或IC100-IP治疗显著减少血管闭塞和玻璃体内新生血管形成。IC100-IVT治疗也减少了视网膜小胶质细胞活化，恢复了视网膜结构,改善视网膜功能.RNA-seq显示IC100处理纠正了与血管生成相关的基因的诱导,白细胞迁移,和由O2引起的VEGF信号。IC100还校正了与细胞连接组装相关的基因的抑制，神经元投影,和由O2引起的神经元识别。
结论：这些数据证明了ASC在OIR发病机理中的关键作用以及人源化治疗性抗ASC抗体在治疗OIR小鼠中的功效。因此，这种抗ASC抗体可能在与氧应激和视网膜病变相关的疾病中被考虑，比如ROP。
方法：我们首先使用BPD模型测试了表达ASC融合蛋白与C末端黄嘌呤（荧光GFP同种型）的ASC-黄嘌呤报告小鼠视网膜中的ASC斑点形成。通过将新生小鼠暴露于室内空气（RA）或85％O2，从出生后第1天到第14天，眼睛损伤。在P14上解剖视网膜，并使用视网膜平坦的支架来检测具有AF-594缀合的异凝集素B4（IB4）和黄嘌呤标记的ASC斑点的血管内皮。为了评估IC100对OIR模型的影响，新生ASC黄嘌呤报告小鼠和野生型小鼠（C57BL/6J）从P1到P6暴露于RA，然后从P7到P11暴露于75％O2，然后从P12到P18暴露于RA。在P12时，将小鼠随机分为以下组：RA与安慰剂PBS（RA-PBS），O2与PBS（O2-PBS），O2+IC100玻璃体内注射（O2-IC100-IVT），和O2+IC100腹腔注射(O2-IC100-IP)。视网膜血管形成通过用IB4平装染色来评价。通过免疫荧光染色检测同种异体移植物炎症因子1（AIF-1）和CD206的小胶质细胞活化。在H&E染色切片上分析视网膜结构,并通过模式视网膜电图（PERG）分析功能。进行视网膜的RNA测序(RNA-seq)以确定IC100处理在OIR中的转录作用。
结果：在BPD和OIR模型中，由于高氧暴露，视网膜中的ASC斑点显着增加，并与异常血管系统共定位，这与小胶质细胞活化增加有关。用IC100-IVT或IC100-IP治疗显著减少血管闭塞和玻璃体内新生血管形成。IC100-IVT治疗也减少了视网膜小胶质细胞活化，恢复了视网膜结构,改善视网膜功能.RNA-seq显示IC100处理纠正了与血管生成相关的基因的诱导,白细胞迁移,和由O2引起的VEGF信号。IC100还校正了与细胞连接组装相关的基因的抑制，神经元投影,和由O2引起的神经元识别。
结论：这些数据证明了ASC在OIR发病机理中的关键作用以及人源化治疗性抗ASC抗体在治疗OIR小鼠中的功效。因此，这种抗ASC抗体可能在与氧应激和视网膜病变相关的疾病中被考虑，比如ROP。
