收稿日期: 2019-12-26
网络出版日期: 2020-07-07
基金资助
湖南省教育厅科学研究项目(15C1257);湖南省教育厅优秀青年项目(17B240)
IL-18 enhances the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection
Received date: 2019-12-26
Online published: 2020-07-07
Supported by
Scientific Research Project of Education Department of Hunan Province(15C1257);Outstanding Youth Project of Education Department of Hunan Province(17B240)
目的 探讨白细胞介素18（IL-18）是否能增强DNA疫苗候选抗原pcDNA3.1/SjOST48对日本血吸虫病感染的免疫保护效果。 方法 构建pcDNA3.1/SjOST48和pcDNA3.1/IL-18真核载体,表达重组蛋白。Western blotting检测HeLa细胞内重组质粒pcDNA3.1/SjOST48和pcDNA3.1/IL-18表达情况,ELISA检测细胞培养基中IL-18的表达情况。100只雌性BALB/c小鼠随机均分为PBS组（空白对照组）、pcDNA3.1组（空质粒组）、pcDNA3.1/IL-18组、pcDNA3.1/SjOST48组和pcDNA3.1/SjOST48+pcDNA3.1/IL-18组等5组,各组小鼠左后腿股四头肌肌内注射相应的重组质粒（30 μg）,共免疫3次,每次间隔14 d。末次免疫后2周,各组取5只小鼠采集血样,ELISA检测各组小鼠血清IgG抗体及其亚类（IgG1和IgG2a）水平。末次免疫后3周,各组取5只小鼠脾组织,无菌分离小鼠脾淋巴细胞,ELISA检测小鼠脾淋巴细胞上清中TNF-α、INF-γ、IL-2、IL-4、IL-6和IL-17含量及脾淋巴细胞增殖水平。末次免疫后2周,各组取15只免疫小鼠感染血吸虫尾蚴（20 ± 1）条/鼠。感染后6周处死小鼠,无菌分离肝,计算减卵率;门静脉灌注法收集成虫,计算减虫率;另取肝组织,切片后行HE染色,显微镜下观察肝虫卵肉芽肿数量及炎症浸润情况。 结果 末次免疫后2周,ELISA结果显示,pcDNA3.1/SjOST48+pcDNA3.1/IL-18组、pcDNA3.1/SjOST48组、pcDNA3.1/IL-18组小鼠的IgG抗体水平分别为0.82 ± 0.07、0.41 ± 0.06、0.16 ± 0.05,均高于pcDNA3.1组的0.12 ± 0.03（P < 0.05）;pcDNA3.1/SjOST48+pcDNA3.1/IL-18组较pcDNA3.1/SjOST48组和pcDNA3.1/IL-18组均增高（P < 0.05）;pcDNA3.1/SjOST48+pcDNA3.1/IL-18组和pcDNA3.1/SjOST48组的IgG2a/IgG1比值分别为4.02 ± 0.01、2.51 ± 0.01（P < 0.05）,均>1,其他3组均<1。末次免疫后3周,pcDNA3.1/SjOST48+pcDNA3.1/IL-18组小鼠脾淋巴细胞上清中的IL-2、TNF-α、INF-γ、IL-6和IL-17含量分别为（101.82 ± 8.90）、（738.02 ± 146.22）、（593.41 ± 51.07）、（685.64 ± 171.2）、（261.32 ± 48.19）pg/ml,高于pcDNA3.1/SjOST48组[（55.82 ± 9.69）、（538.21 ± 85.26）、（393.41 ± 51.07）、（335.64 ± 62.63）、（118.32 ± 8.91）pg/ml]（P < 0.05）和pcDNA3.1/IL-18组[（35.16 ± 6.43）、（284.40 ± 69.96）（141.91 ± 24.48）、（198.44 ± 38.15）、（47.66 ± 14.33）pg/ml]（P < 0.05）,三者均高于pcDNA3.1组[（12.91 ± 8.01）、（74.86 ± 6.64）（23.75 ± 6.06）、（82.75 ± 10.96）、（22.91 ± 13.80）pg/ml]（P < 0.05）;IL-4含量为（12.28 ± 7.08）pg/ml,与pcDNA3.1/SjOST48组[（15.03 ± 10.12）pg/ml]、pcDNA3.1/IL-18组[（13.59 ± 4.42）pg/ml]、pcDNA3.1组[（16.13 ± 10.08）pg/ml]比较差异无统计学意义（P > 0.05）。pcDNA3.1/SjOST48+pcDNA3.1/IL-18组脾淋巴细胞增殖水平为11.84 ± 0.16,高于pcDNA3.1/SjOST48组（5.93 ± 0.25）（P < 0.05）和pcDNA3.1/IL-18组（3.19 ± 0.36）（P < 0.05）,三者均高于pcDNA3.1组（2.08 ± 0.16）（P < 0.05）。末次免疫后2周,经日本血吸虫感染后6周,pcDNA3.1/SjOST48+pcDNA3.1/IL-18组小鼠的平均检获成虫数为（14.33 ± 1.08）;与pcDNA3.1组比较,减虫率和减卵率分别为46.19%和44.68%,高于pcDNA3.1/SjOST48免疫组（32.18%和35.78%）和pcDNA3.1/IL-18组（13.22%和16.68%）（P < 0.05）。各组小鼠肝组织切片HE染色结果显示,与其他4组比较,pcDNA3.1/SjOST48+pcDNA3.1/IL-18组小鼠肝组织虫卵结节较少,虫卵肉芽组织数量明显减少,炎症浸润不明显。 结论 IL-18能使pcDNA3.1/SjOST48免疫小鼠诱导较高水平的Th1型和Th17型免疫应答,并且增强其抗血吸虫感染的免疫保护效果。
关键词： 日本血吸虫; 白细胞介素18; pcDNA3.1/SjOST48; 候选抗原; 免疫保护性
谭潇 , 肖楚丽 , 肖非 , 王硕 , 卿蕊 , 黄泽智 . IL-18增强pcDNA3.1/SjOST48抗血吸虫感染的免疫保护性研究[J]. 中国寄生虫学与寄生虫病杂志, 2020 , 38(3) : 279 -285 . DOI: 10.12140/j.issn.1000-7423.2020.03.004
Objective To investigate if IL-18 can enhance the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection. Methods pcDNA3.1/SjOST48 and pcDNA3.1/IL-18 eukaryotic vectors were constructed to express recombinant proteins. Western blotting was performed to detect the expression of pcDNA3.1/SjOST48 and pcDNA3.1/IL-18 in HeLa cells, and ELISA was performed to examine IL-18 levels in culture medium. One hundred BALB/c female mice were randomly divided into 5 groups: PBS group (blank control), pcDNA3.1 group (empty vector), pcDNA3.1/IL-18 group, pcDNA3.1/SjOST48 group, and pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group, and were immunized with corresponding recombinant plasmid (30 μg) for three times at intervals of 14 days by intramuscular injection (100 μg/ml) at the quadriceps of the left hind leg femur. Two weeks after the last immunization, five mice in each group were selected for blood collection, and serum levels of IgG and its subclasses (IgG1 and IgG2a) were detected by ELISA. Three weeks after the last immunization, 5 mice of each group were selected for collection of spleen, to isolate spleen lymphocytes under sterile conditions; and the contents of TNF-α, INF-γ, IL-4, IL-6 and IL-8 in the lymphocytes culture supernatant and the proliferation of spleen lymphocytes were assessed by ELISA. Two weeks after the last immunization, 15 mice of each group were infected with 20 ± 1 cercariae of Schistosoma japonicum and sacrificed 6 weeks later. The liver tissue was obtained aseptically to calculate egg reduction rate; adult worms were collected by portal vein perfusion to calculate worm reduction rate. Another portion of liver tissue was used for hematoxylin & eosin (HE) staining to observe the amount of S. japonicum egg granuloma and inflammatory infiltration. Results ELISA results showed that at two weeks after the last immunization, the IgG antibody levels in mice of the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group, the pcDNA3.1/SjOST48 group, and the pcDNA3.1/IL-18 group were 0.82 ± 0.07, 0.41 ± 0.06, and 0.16 ± 0.05, respectively, all significantly higher than that in the pcDNA3.1 group (0.12 ± 0.03, P < 0.05). The IgG antibody level of the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group was significantly higher than those of the pcDNA3.1/SjOST48 group and the pcDNA3.1/IL-18 group (P < 0.05). The IgG2a/IgG1 ratio in the pcDNA3.1/SjOST48+pcDNA3.1/SjOST48 and pcDNA3.1/SjOST48 groups were 4.02 ± 0.01 and 2.51 ± 0.01 (P < 0.05), respectively, both >1, while those of the other 3 groups were all <1. At 3 weeks after the last immunization, the contents of IL-2, TNF-α, INF-γ, IL-6 and IL-17 in the supernatant of spleen lymphocytes in the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group were (101.82 ± 8.90) pg/ml, (738.02 ± 146.22) pg/ml, (593.41 ± 51.07) pg/ml, (685.64 ± 171.2) pg/ml and (261.32 ± 48.19) pg/ml, respectively, all significantly higher than those in the pcDNA3.1/SjOST48 group [(55.82 ± 9.69) pg/ml, (538.21 ± 85.26) pg/ml, (393.41 ± 51.07) pg/ml, (335.64 ± 62.63) pg/ml, (118.32 ± 8.91) pg/ml] (P < 0.05) and the pcDNA3.1/IL-18 group [(35.16 ± 6.43) pg/ml, (284.40 ± 69.96) pg/ml, (141.91 ± 24.48) pg/ml, (198.44 ± 38.15) pg/ml, and (47.66 ± 14.33) pg/ml] (P < 0.05). All the three groups had significantly higher contents of IL-2, TNF-α, INF-γ, IL-6 and IL-17 than the pcDNA3.1 group [(12.91 ± 8.01) pg/ml, (74.86 ± 6.64) pg/ml (23.75 ± 6.06) pg/ml, (82.75 ± 10.96) pg/ml, and (22.91 ± 13.80) pg/ml, respectively] (P < 0.05). The content of IL-4 in the supernatant of spleen lymphocytes in the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group was (12.28 ± 7.08) pg/ml, which showed no significant difference from those of the pcDNA3.1/SjOST48 [(15.03 ± 10.12) pg/ml], pcDNA3.1/IL-18 [(13.59 ± 4.42) pg/ml] and pcDNA3.1 groups [(16.13 ± 10.08) pg/ml] (P > 0.05). The proliferation level of spleen lymphocytes in the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group was 11.84 ± 0.16, higher than that in the pcDNA3.1/SjOST48 group (5.93 ± 0.25) (P < 0.05) and the pcDNA3.1/IL-18 group (3.19 ± 0.36) (P < 0.05), and all three were higher than that in the pcDNA3.1 group (2.08 ± 0.16) (P < 0.05). Six weeks after S. japonicum infection, the average number of adult worms detected from mice in the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group was (14.33 ± 1.08), and the worm reduction rate and egg reduction rate were 46.19% and 44.68%, respectively, significantly higher than those of the pcDNA3.1/SjOST48 immunized group (32.18% and 35.78%) and the pcDNA3.1 /IL-18 group (13.22% and 16.68%) (P < 0.05). HE staining of liver tissue showed that, in comparison to other groups, mice in the pcDNA3.1/SjOST48+pcDNA3.1/IL-18 group had fewer nodules of worm eggs on the liver surface and significantly decreased S. japonicum egg granulomas, and the inflammatory infiltration was unapparent. Conclusion IL-18 can induce a higher level of Th1 and Th17 immune response in mice immunized with pcDNA3.1/SjOST48, and enhance the immuno-protective effects of pcDNA3.1/SjOST48 against S. japonicum infection.
Key words： Schistosoma japonicum; IL-18; pcDNA3.1/SjOST48; Candicate antigen; Immune protection
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