AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experime...AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.展开更多
Neuroinflammation has been recognized as a factor in the pathogenesis ofneurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disea...Neuroinflammation has been recognized as a factor in the pathogenesis ofneurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disease progression and neuropathology in several neurodegenerative diseases. However, detailed correlations among pe- ripheral inflammation, neuroinflammation and neurodegeneration remain unknown. In the present study, we pre- pared a peripheral inflammation model with lipopolysaccharides （LPS）-stimulated RAW264.7 macrophages to ex- plore its activation on BV2 microglia. We found that LPS induced the production of IL-1β, IL-6 and TNF-a in the culture medium of RAW264.7 macrophages. We further showed that LPS plus ATP activated inflammasome, evidenced by the upregulation of caspase-1 and IL-113, which was suppressed by ZYVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 micro- glia, stimulating the release of IL-1β, IL-6 and TNF-a from BV2 cells. ZYVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 cells conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation subsequently evokes neuroinflammation and may aggravate neural damage. Inflammasome and caspase- 1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.展开更多
The cysteine protease caspase-1(Casp-1)contributes to innate immunity through the assembly of NLRP3,NLRC4,AIM2,and NLRP6 inflammasomes.Here we ask whether caspase-1 activation plays a regulatory role in house dust mit...The cysteine protease caspase-1(Casp-1)contributes to innate immunity through the assembly of NLRP3,NLRC4,AIM2,and NLRP6 inflammasomes.Here we ask whether caspase-1 activation plays a regulatory role in house dust mite(HDM)-induced experimental allergic airway inflammation.We report enhanced airway inflammation in caspase-1-deficient mice exposed toHDMwith a marked eosinophil recruitment,increased expression of IL-4,IL-5,IL-13,aswell as full-length and bioactive IL-33.Furthermore,mice deficient for NLRP3 failed to control eosinophil influx in the airways and displayed augmented Th2 cytokine and chemokine levels,suggesting that the NLPR3 inflammasome complex controls HDM-induced inflammation.IL-33 neutralization by administration of soluble ST2 receptor inhibited the enhanced allergic inflammation,while administration of recombinant IL-33 during challenge phase enhanced allergic inflammation in caspase-1-deficient mice.Therefore,we show that caspase-1,NLRP3,and ASC,but not NLRC4,contribute to the upregulation of allergic lung inflammation.Moreover,we cannot exclude an effect of caspase-11,because caspase-1-deficient mice are deficient for both caspases.Mechanistically,absence of caspase-1 is associated with increased expression of IL-33,uric acid,and spleen tyrosine kinase(Syk)production.This study highlights acritical role of caspase-1 activation andNLPR3/ASCinflammasomecomplex in the down-modulation of IL-33 in vivo and in vitro,thereby regulating Th2 response in HDM-induced allergic lung inflammation.展开更多
目的探讨三黄连合剂通过NOD样受体蛋白3(NOD-like receptor protein domain associated protein 3,NLRP3)/凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)/半胱氨酸天冬氨酸蛋白酶1(cysteine aspa...目的探讨三黄连合剂通过NOD样受体蛋白3(NOD-like receptor protein domain associated protein 3,NLRP3)/凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)/半胱氨酸天冬氨酸蛋白酶1(cysteine aspartic acid specific protease-1,Caspase-1)通路对哮喘患儿气道炎症的作用及机制。方法选取2021年1月—2023年10月就诊的90例哮喘患儿,采用数字表法随机分为观察组与对照组,各45例。观察2组哮喘患儿不良反应、1 s用力呼气容积(forced expiratory volume in the first second,FEV_(1)%)、最大呼气流量(peak expiratory flow,PEF)、用力肺活量(forced vital capacity,FVC)、白细胞介素-1β(interleukin-1β,IL-1β)及TNF-α(tumor necrosis factor-α,TNF-α)表达水平差异。结果观察组不良反应发生率为6.66%,对照组为11.11%,差异无统计学意义(P>0.05)。观察组总有效率为95.56%,高于对照组的80.00%(P<0.05)。治疗后FEV_(1)%、PEF、FVC、哮喘控制测试表(asthma control test,ACT)评分高于对照组(P<0.05)。治疗后观察组血清IL-1β、IL-6、IL-8、IL-17、IL-18水平低于对照组(P<0.05)。治疗后观察组患儿血清NLRP3、ASC、Caspase-1、IL-1β蛋白表达水平低于对照组(P<0.05)。结论三黄连合剂治疗能减轻哮喘患儿气道炎症反应,降低炎症因子水平,改善肺功能,减轻病情,提高疗效,其可能通过NLRP3/ASC/Caspase-1通路发挥作用。展开更多
目的探讨自身免疫性甲状腺炎伴发抑郁症动物模型的制备与评价,并基于NOD样受体蛋白-3(NOD-like receptor protein 3,NLRP3)/含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)/消皮素D(gasdermin...目的探讨自身免疫性甲状腺炎伴发抑郁症动物模型的制备与评价,并基于NOD样受体蛋白-3(NOD-like receptor protein 3,NLRP3)/含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)/消皮素D(gasdermin D,GSDMD)通路加以验证。方法32只NOD.H-2H4小鼠随机分为正常组(N组)、抑郁组(DP组)、自身免疫性甲状腺炎伴抑郁症组(AIT+DP组)、自身免疫性甲状腺炎组(AIT组),每组8只。N组正常饲养,DP组采取5周慢性不可预知温和刺激(chronic unpredictable mild stress,CUMS),AIT组予0.05%碘化钠水溶液建立自身免疫性甲状腺炎模型,AIT+DP组在建立AIT动物模型基础上施加5周CUMS建立AIT+DP动物模型。通过观测小鼠甲状腺组织结构及淋巴细胞浸润情况和血清甲状腺过氧化物酶抗体(thyroid peroxidase antibody,TPOAb)和甲状腺球蛋白抗体(anti-thyroid autoantibodies,TGAb)水平评价小鼠自身免疫性甲状腺炎模型是否制备成功;通过测定体重、糖水偏好率、旷场行为学(中央象限时间、中央象限比例、站立次数、排便次数、毛发梳理时间),大脑皮质、海马病理变化及大脑皮质小胶质细胞焦亡相关蛋白水平评价小鼠抑郁状态。模型小鼠同时符合上述自身免疫性甲状腺炎与抑郁症相关指标检测,则表明AIT+DP动物模型制备成功。结果与N组比较,AIT组与AIT+DP组血清TGAb、TPOAb水平显著增加(P<0.01),甲状腺可见大量炎细胞浸润,DP组与AIT+DP组小鼠中央象限时间、中央象限比例、站立次数、排便次数、毛发梳理时间有不同程度降低,大脑皮质神经胶质细胞增多,神经元细胞减少,伴有部分细胞核萎缩,NLRP3、IL-1β、Caspase-1、GSDMD-N蛋白表达水平显著上调,AIT+DP组尤为明显(P<0.01)。结论0.05%碘化钠水溶液与CUMS可较好地模拟AIT+DP模型动物外在表现与内在指标变化,可为AIT+DP疾病的研究提供动物模型参考。展开更多
基金Supported by NIH R21 AI085416 (to Shi J)NIH NCRR P20-RR017686 (to PI:Daniel MarcusShi J)
文摘AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.
基金supported in part by Nanjing Medical Science and Technology Development Foundation(No.ZKX12037,No.YKX13129)in part by National Natural Science Foundation of China(No.81271418)
文摘Neuroinflammation has been recognized as a factor in the pathogenesis ofneurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disease progression and neuropathology in several neurodegenerative diseases. However, detailed correlations among pe- ripheral inflammation, neuroinflammation and neurodegeneration remain unknown. In the present study, we pre- pared a peripheral inflammation model with lipopolysaccharides （LPS）-stimulated RAW264.7 macrophages to ex- plore its activation on BV2 microglia. We found that LPS induced the production of IL-1β, IL-6 and TNF-a in the culture medium of RAW264.7 macrophages. We further showed that LPS plus ATP activated inflammasome, evidenced by the upregulation of caspase-1 and IL-113, which was suppressed by ZYVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 micro- glia, stimulating the release of IL-1β, IL-6 and TNF-a from BV2 cells. ZYVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 cells conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation subsequently evokes neuroinflammation and may aggravate neural damage. Inflammasome and caspase- 1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.
基金support by University of Orleans,la Region Centre(HabitAsthmeN8201200073535)and Conseil General45 to F.M.as PhD fellowship.
文摘The cysteine protease caspase-1(Casp-1)contributes to innate immunity through the assembly of NLRP3,NLRC4,AIM2,and NLRP6 inflammasomes.Here we ask whether caspase-1 activation plays a regulatory role in house dust mite(HDM)-induced experimental allergic airway inflammation.We report enhanced airway inflammation in caspase-1-deficient mice exposed toHDMwith a marked eosinophil recruitment,increased expression of IL-4,IL-5,IL-13,aswell as full-length and bioactive IL-33.Furthermore,mice deficient for NLRP3 failed to control eosinophil influx in the airways and displayed augmented Th2 cytokine and chemokine levels,suggesting that the NLPR3 inflammasome complex controls HDM-induced inflammation.IL-33 neutralization by administration of soluble ST2 receptor inhibited the enhanced allergic inflammation,while administration of recombinant IL-33 during challenge phase enhanced allergic inflammation in caspase-1-deficient mice.Therefore,we show that caspase-1,NLRP3,and ASC,but not NLRC4,contribute to the upregulation of allergic lung inflammation.Moreover,we cannot exclude an effect of caspase-11,because caspase-1-deficient mice are deficient for both caspases.Mechanistically,absence of caspase-1 is associated with increased expression of IL-33,uric acid,and spleen tyrosine kinase(Syk)production.This study highlights acritical role of caspase-1 activation andNLPR3/ASCinflammasomecomplex in the down-modulation of IL-33 in vivo and in vitro,thereby regulating Th2 response in HDM-induced allergic lung inflammation.
文摘目的探讨三黄连合剂通过NOD样受体蛋白3(NOD-like receptor protein domain associated protein 3,NLRP3)/凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)/半胱氨酸天冬氨酸蛋白酶1(cysteine aspartic acid specific protease-1,Caspase-1)通路对哮喘患儿气道炎症的作用及机制。方法选取2021年1月—2023年10月就诊的90例哮喘患儿,采用数字表法随机分为观察组与对照组,各45例。观察2组哮喘患儿不良反应、1 s用力呼气容积(forced expiratory volume in the first second,FEV_(1)%)、最大呼气流量(peak expiratory flow,PEF)、用力肺活量(forced vital capacity,FVC)、白细胞介素-1β(interleukin-1β,IL-1β)及TNF-α(tumor necrosis factor-α,TNF-α)表达水平差异。结果观察组不良反应发生率为6.66%,对照组为11.11%,差异无统计学意义(P>0.05)。观察组总有效率为95.56%,高于对照组的80.00%(P<0.05)。治疗后FEV_(1)%、PEF、FVC、哮喘控制测试表(asthma control test,ACT)评分高于对照组(P<0.05)。治疗后观察组血清IL-1β、IL-6、IL-8、IL-17、IL-18水平低于对照组(P<0.05)。治疗后观察组患儿血清NLRP3、ASC、Caspase-1、IL-1β蛋白表达水平低于对照组(P<0.05)。结论三黄连合剂治疗能减轻哮喘患儿气道炎症反应,降低炎症因子水平,改善肺功能,减轻病情,提高疗效,其可能通过NLRP3/ASC/Caspase-1通路发挥作用。
