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High Glucose Induces Pyroptosis of Retinal Microglia Through NLPR3

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High Glucose Induces Pyroptosis of Retinal Microglia Through NLPR3 A RQUIVOS B RASILEIROS DE ORIGINAL ARTICLE High glucose induces pyroptosis of retinal microglia through NLPR3 inflammasome signaling Altos níveis de glicose induzem piroptose da micróglia da retina por sinalização de inflamassomas NLPR3 Libin Huang1 , Junmei You1, Yao Yao1, Maosong Xie1 1. The First Affiliated Hospital of Fujian Medical University, China. ABSTRACT | Purpose: Diabetic retinopathy is currently (25 mM)-induced cytotoxicity, NOD-like receptor family pyrin considered a chronic inflammatory disease involving NOD-like domain containing 3 inflammasome activation, and pyroptosis receptor family pyrin domain containing 3 inflammasome of retinal microglia. Conclusions: NOD-like receptor family activation and retinal microglial pyroptosis. In this study, pyrin domain containing 3 inflammasome signaling may mo- we aimed to investigate whether NOD-like receptor family dulate retinal microglia-related inflammation and pyroptosis pyrin domain containing 3 inflammasome signaling induces under high-glucose conditions. pyroptotic death of retinal microglia under high-glucose con- ditions. Methods: Retinal microglia were stimulated by high Keywords: Diabetic retinopathy; Microglia; NLRP3 inflamma- glucose levels for 24 h. Cell viability, lactate dehydrogenase some; Pyroptosis; Gasdermin D release, and caspase-1 activity were detected in vitro. The expression of pro-inflammatory cytokine (interleukin-1β, RESUMO | Objetivo: Atualmente, a retinopatia diabética é activated microglia marker ionized calcium-binding adapter considerada uma doença inflamatória crônica envolvendo a molecule-1), NOD-like receptor family pyrin domain con- ativação de inflamassomas NLRP3 e piroptose da micróglia da taining 3, cleaved caspase-1, and cleaved gasdermin D were retina. Neste estudo, objetivamos investigar se a sinalização examined. Subsequently, retinal microglia were pretreated de inflamassomas NLRP3 induz a morte da micróglia da retina with the inhibitors of NOD-like receptor family pyrin domain sob condições de alta glicose. Métodos: A micróglia da retina containing 3 inflammasome signaling prior to stimulation with foi estimulada por altos níveis de glicose durante 24 horas. A high glucose, and their molecular and functional changes were viabilidade celular, a liberação de LDH e a atividade da caspase1 evaluated. Results: High-glucose (25, 50, or 100 mM) stimulation foram analisadas in vitro. Avaliou-se a expressão de citocina decreased cell viability, but enhanced lactate dehydrogenase pró-inflamatória (IL1β), de marcador de micróglia ativado (Iba1), release and caspase-1 activity in a dose-dependent manner. de NLRP3, de caspase1 clivada e de GSDMD clivada. Subsequen- Moreover, high glucose upregulated the protein expression of temente, a micróglia da retina foi pré-tratada com inibidores interleukin-1β, ionized calcium-binding adapter molecule-1, da sinalização de inflamassomas NLRP3 antes da estimulação NOD-like receptor family pyrin domain containing 3, cleaved com altos níveis de glicose e suas alterações moleculares e caspase-1, and cleaved gasdermin D. However, pretreatment funcionais foram avaliadas. Resultados: A estimulação com with the inhibitors of NOD-like receptor family pyrin domain altos níveis de glicose (25 mM, 50 mM ou 100 mM) diminuiu containing 3 inflammasome signaling inhibited high glucose a viabilidade celular, mas aumentou a liberação de LDH e a atividade da caspase1 de forma dependente da dose. Além disso, os altos níveis de glicose aumentaram a expressão das proteínas IL1β, Iba1, NLRP3, caspase1 clivada e GSDMD clivada. No entanto, o pré-tratamento com inibidores da sinalização de inflamassomas NLRP3 e a posterior estimulação com altos níveis de glicose (25 mM) induziu citotoxicidade, a ativação de inflamassomas NLRP3 e a piroptose da micróglia da retina. Submitted for publication: July 22, 2019 Accepted for publication: December 22, 2019 Conclusão: A sinalização de inflamassomas NLRP3 pode modular Disclosure of potential conflicts of interest: None of the authors have any potential a inflamação e a piroptose da micróglia da retina na presença conflicts of interest to disclose. de altos níveis de glicose. Corresponding author: Maosong Xie. E-mail: [email protected] Descritores: Retinopatia diabética; Microglia; NLRP3 Inflam- Approved by the following research ethics committee: The First Affiliated Hospital of Fujian Medical University (#2016-YK-163). massomos; Piroptose; Gasdermin D This content is licensed under a Creative Commons Attributions 4.0 International License. http://dx.doi.org/10.5935/0004-2749.20210010 Arq Bras Oftalmol. 2021;84(1):67-73 ■ 67 High glucose induces pyroptosis of retinal microglia through NLPR3 inflammasome signaling INTRODUCTION understood whether the activation of the NLRP3 inflam- Diabetic retinopathy (DR), the major ocular com- masome causes pyroptosis of retinal microglia in DR. plication of diabetes mellitus, is the leading cause of In the present study, we constructed an in vitro model blindness in working-age populations(1). Numerous stu- to determine the role of NLRP3 inflammasome signa- dies have documented the effectiveness of routine DR ling in modulating retinal microglial pyroptosis under screening and early treatment(2-3), while current thera- high-glucose conditions. Additionally, we intended to pies are unable to reverse the vision loss in the advanced interpret the pathogenesis of DR in terms of microglia background or proliferative stage of DR. pyroptosis and related inflammation. The exact mechanisms of DR remain unclear. Although DR has been traditionally described as a microvascular METHODS disorder, recent evidence demonstrated that early DR is Primary retinal microglia culture linked to retinal inflammation associated with microglia All animal procedures were approved by the Animal activation(4-5). Over-activated microglia (M1 phenotype) Care and Use Committee of the First Affiliated Hospital induce the release of inflammatory factors, contribute of Fujian Medical University (Fuzhou, China) (Approval to the development of neurovascular unit lesions, and No. 2016-YK-163), and conformed to the Association eventually lead to irreversible retinal dysfunction(6-7). for Research in Vision and Ophthalmology Statement on The NOD-like receptor family pyrin domain contai- the Use of Animals in Ophthalmic and Vision Research. ning 3 (NLRP3) inflammasome, one of the key sensors The primary microglial culture was performed as in microglial plasma, plays a critical role in inflamma- previously described(21). In brief, retinas of healthy new- tion mediated by inflammatory cytokines, such as in- born C57BL/6 mice (Shanghai SLAC Laboratory Animal terleukin-1β (IL-1β), from microglia(8). NLRP3 recognizes Co. Ltd., Shanghai, China) were digested with 0.125% different endogenous and exogenous stimuli, and com- bines with the adaptor apoptosis-associated speck-like trypsin for 30 min at 37°C to generate a single-cell protein containing a caspase activation and recruitment suspension. Subsequently, the cells were resuspended domain (ASC) and pro-caspase-1. Accordingly, they are in Dulbecco’s modified Eagle’s medium/F-12 culture assembled to a multiprotein complex(9). Activation of the medium containing 10% fetal bovine serum, 1% micro- NLPR3 inflammasome activates pro-caspase-1 cleavage, glia growth supplement (Sciencell, USA), 100 U/mL pe- μ 2 facilitates IL-1β secretion, and subsequently triggers nicillin, and 100 g/mL streptomycin, plated onto 75 cm a highly inflammatory type of programmed cell death culture flasks, and incubated at 37°C in a humidified atmosphere containing 5% CO . The culture medium termed pyroptosis(10). Gasdermin D (GSDMD) is a membra- 2 ne pore-forming protein involved in pyroptosis(11). The was changed at 24 h. After 2 weeks, mixed glial cells NLPR3 inflammasome could cleave GSDMD to generate were purified by shaking at 200 rpm for 1 h. The super- the N-terminal fragment of GSDMD, cause cell rupture, natant containing microglia were harvested and used in and increase the release of IL-1β(12). the following experiments. The purity of microglia was It has been reported that NLRP3 inflammasome determined through flow cytometry using fluorescein signaling is involved in the pathogenesis of various eye isothiocyanate-conjugated rabbit anti-CD11b and iso- diseases(13-15). Some studies have demonstrated that cas- type immunoglobulin G2b control antibodies (Abcam, pase-1 activity and IL-1β production were significantly Cambridge, UK). increased in microglia in vitro following exposure to hyperglycemic conditions(16-17), suggesting that micro- Cell treatment glial pyroptosis is a crucial factor in the pathogenesis of Microglia were incubated with D-glucose 5.5 (control), DR. A recent study found that NLRP3 gene knockout 25, 50, and 100 mM (Sigma-Aldrich, St. Louis, MO, downregulated the expression of caspase-1 and proin- USA) for 24 h. The cells were pretreated with the NLRP3 flammatory cytokines, and alleviated retinal ganglion cell inhibitor MCC950 (10 μM) or caspase-1 inhibitor Z-Tyr- death following optic nerve crush injury(18). Moreover, Val-Ala-Asp(OMe) fluoromethyl ketone (Z-YVAD-FMK; high glucose could trigger pyroptosis of retinal micro- 10 μM) (all from Sigma-Aldrich) for 30 min prior to vascular endothelial cells via the NLRP3 inflammasome treatment with high glucose to suppress NLRP3 inflam- signaling pathway(19-20). However, it remains poorly masome signaling. 68 Arq Bras Oftalmol. 2021;84(1):67-73 Huang H, et al. Cell viability anti-rabbit immunoglobulin
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