IL-18 enhances the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection

doi:10.12140/j.issn.1000-7423.2020.03.004

Abstract

Abstract:

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

CLC Number:

R383.24

TAN Xiao, XIAO Chu-li, XIAO Fei, WANG Shuo, QING Rui, HUANG Ze-zhi. IL-18 enhances the immunoprotective effect of pcDNA3.1/SjOST48 against Schistosoma japonicum infection[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 279-285.

Figures/Tables 6

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分组 Group	成虫数 Worm burden	减虫率 Worm reduction rate/%	虫卵数/×10⁴ Average number of eggs per gram liver tissue/×10⁴	减卵率 Egg reduction rate/%
PBS	26.63 ± 3.41		36.86 ± 6.25
pcDNA3.1	26.32 ± 2.34		35.25 ± 4.21
pcDNA3.1/IL-18	23.11 ± 1.76	13.22^a	30.71 ± 3.91	16.68^a
pcDNA3.1/SjOST48	18.06 ± 1.21	32.18^a	23.67 ± 2.54	35.78^a
pcDNA3.1/IL-18+pcDNA3.1/SjOST48	14.33 ± 1.08	46.19	20.39 ± 2.31	44.68