Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We culture...Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We cultured bone marrow-derived macrophages (BMDMs) to investigate the immune response to challenge byC. albicans. The p62 gene was knocked down by transfection with p62 small interfering RNA (siRNA) in the p62 siRNA group. BMDMs transfected with nonsense siRNA served as the negative control (NC) group. These two groups of BMDMs were challenged withC. albicans in vitro. We detected p62 expression through quantitative reverse transcription PCR and western blotting. The phagocytosis ability of BMDMs was evaluated by flow cytometry and microscopic examination using an Olympus FV1000 laser scanning confocal microscope. Moreover, we determined the level of reactive oxygen species (ROS) in BMDMs. The mRNA levels of proinflammatory cytokines were determined by quantitative reverse transcription PCR.Results: After stimulation byC. albicans, the relative expression of p62 mRNA was increased in a dose-dependent manner, the relative expression of p62 and the ratio of BMDMs toC. albicans is 1.893 ± 0.2156 (1:1,P < 0.05), 2.873 ± 0.4787 (1:3,P < 0.05) and 3.556 ± 0.2892 (1:5,P < 0.01). The p62 protein level was also increased. After transfection with p62 siRNA, the mRNA and protein levels of p62 were significantly decreased in BMDMs (P < 0.05). After 0.5, 1 and 2 hours of co-culture of BMDMs withC. albicans, flow cytometry showed that the phagocytosis rates ofC. albicans by BMDMs were significantly lower in the p62 siRNA group than in the NC group (39.70 ± 1.69%vs. 55.23 ± 0.72%, 46.70 ± 0.89%vs. 60.80 ± 1.78%, 51.90 ± 0.98%vs. 64.43 ± 2.0%, respectively, allP < 0.05). Consistent results were seen in the production of ROS (4269 ± 392.6vs. 13426 ± 1859.7, 4967 ± 721.2vs. 13687 ± 2611.2, 7647 ± 1950.0vs. 17719 ± 1814.2, respectively, allP < 0.05). The ROS levels were higher in BMDMs of the NC group than in BMDMs transfected with p62 siRNA at 0.5, 1, and 2 hours after treatment withC. albicans. BMDMs was co-cultured withC. albicans for 4 and 12 hours, the mRNA levels of interleukin-1β and interleukin-18 in NCs were also higher than p62 siRNA group, interleukin-1β: (6.14 ± 1.63vs. 12.12 ± 0.54, 8.81 ± 0.86vs. 26.2 ± 4.67, respectively, allP < 0.05), IL-18: (0.38 ± 0.02vs. 0.97 ± 0.06, 0.44 ± 0.02vs. 2.23 ± 0.46, respectively, allP < 0.05).Conclusion: p62 plays an important role in the process of phagocytosis in BMDMs challenged byC. albicans through ROS production and expression of proinflammatory cytokines.展开更多
基金The study was supported by the National Natural Science Foundation of China(Nos.82103749,81773338,and 82173432)ChineseAcademy ofMedicalSciencesMedicine andHealthTechnology InnovationProject(No.2017-I2M-1-017)+1 种基金Natural Science Foundation of Jiangsu Province(No.BK20190144)the Nanjing Incubation Program for National Clinical Research Center(No.2019060001).
文摘Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We cultured bone marrow-derived macrophages (BMDMs) to investigate the immune response to challenge byC. albicans. The p62 gene was knocked down by transfection with p62 small interfering RNA (siRNA) in the p62 siRNA group. BMDMs transfected with nonsense siRNA served as the negative control (NC) group. These two groups of BMDMs were challenged withC. albicans in vitro. We detected p62 expression through quantitative reverse transcription PCR and western blotting. The phagocytosis ability of BMDMs was evaluated by flow cytometry and microscopic examination using an Olympus FV1000 laser scanning confocal microscope. Moreover, we determined the level of reactive oxygen species (ROS) in BMDMs. The mRNA levels of proinflammatory cytokines were determined by quantitative reverse transcription PCR.Results: After stimulation byC. albicans, the relative expression of p62 mRNA was increased in a dose-dependent manner, the relative expression of p62 and the ratio of BMDMs toC. albicans is 1.893 ± 0.2156 (1:1,P < 0.05), 2.873 ± 0.4787 (1:3,P < 0.05) and 3.556 ± 0.2892 (1:5,P < 0.01). The p62 protein level was also increased. After transfection with p62 siRNA, the mRNA and protein levels of p62 were significantly decreased in BMDMs (P < 0.05). After 0.5, 1 and 2 hours of co-culture of BMDMs withC. albicans, flow cytometry showed that the phagocytosis rates ofC. albicans by BMDMs were significantly lower in the p62 siRNA group than in the NC group (39.70 ± 1.69%vs. 55.23 ± 0.72%, 46.70 ± 0.89%vs. 60.80 ± 1.78%, 51.90 ± 0.98%vs. 64.43 ± 2.0%, respectively, allP < 0.05). Consistent results were seen in the production of ROS (4269 ± 392.6vs. 13426 ± 1859.7, 4967 ± 721.2vs. 13687 ± 2611.2, 7647 ± 1950.0vs. 17719 ± 1814.2, respectively, allP < 0.05). The ROS levels were higher in BMDMs of the NC group than in BMDMs transfected with p62 siRNA at 0.5, 1, and 2 hours after treatment withC. albicans. BMDMs was co-cultured withC. albicans for 4 and 12 hours, the mRNA levels of interleukin-1β and interleukin-18 in NCs were also higher than p62 siRNA group, interleukin-1β: (6.14 ± 1.63vs. 12.12 ± 0.54, 8.81 ± 0.86vs. 26.2 ± 4.67, respectively, allP < 0.05), IL-18: (0.38 ± 0.02vs. 0.97 ± 0.06, 0.44 ± 0.02vs. 2.23 ± 0.46, respectively, allP < 0.05).Conclusion: p62 plays an important role in the process of phagocytosis in BMDMs challenged byC. albicans through ROS production and expression of proinflammatory cytokines.
