Immunomodulatory effects of natural killer cells on the CD4+ T cell subset in mice with cerebral malaria

doi:10.12140/j.issn.1000-7423.2020.03.012

Abstract

Abstract:

Objective To explore the immunomodulatory effects of natural killer (NK) cells on the CD4⁺T cell subset in mice with cerebral malaria. Methods Female C57BL/6 mice were randomly divided into the health control group, infection group and NK elimination group, 14 mice in each. The mice in the infection group and NK elimination group were injected intraperitoneally with 1 × 10⁶ Plasmodium berghei ANKA (PbA)-infected red cells. The mice in NK elimination group were intraperitoneally injected with anti-Asialo GM-1(15 μl), a blocking antibody against NK cells, on day 1 before and day 2 after infection, while the mice in the health control and infection groups were injected with equal volumes of PBS. From day 3 on post-infection, blood was collected from the tail vein to make blood smears for examining parasitemia, and death of mine was recorded. On days 3 and 5 after infection, 3 mice were sacrificed in each group. The spleens were taken to make single cell suspensions for assaying Th1 type cells and regulatory T cells (Tregs) by flow cytometry. The spleen cells were cultured in vitro for 48 h, then the culture supernatant was collected to assay the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10) by ELISA. On day 6 after infection, 3 mice each group received Evans blue (EB) intravenously, brain tissues were incubated in vitro for 48 h, supernatant was collected and the EB content passing through the blood-brain barrier of the mice was detected by microplate reader. Results In the infection group, mice deaths began to occur on day 6 after infection, accompanied by neurological symptoms, and all died on day 8. In the NK elimination group, deaths began to occur on day 7 after infection, and all mice died on day 12. On day 7 after infection, mice in the NK elimination group had significantly higher parasitemia (5.2%-10.5%) than those in the infection group (2.4%-5.0%) (P < 0.05). On day 5 after infection, the percentage and absolute counts of Th1 type cells were (2.65 ± 0.06)% and (2.09-2.25) × 10⁶in the infection group, which were significantly higher than those in the health control group (1.37 ± 0.02)% and (0.41-0.47) × 10⁶ (P < 0.01). and those in the NK elimination group [(1.82 ± 0.07)% and (1.48-1.86) × 10⁶] were significantly lower than those in the infection group (P < 0.01 or P < 0.05). The percentages of Tregs on days 3 and 5 in the infection group were(1.21 ± 0.06)% and (1.70 ± 0.13)%, respectively, which were significantly higher than those in the health control group[(0.90 ± 0.01)% and (0.91 ± 0.02)%] (P < 0.01). The absolute counts of Tregs in the infection group on day 3 and 5 post-infection were (0.63-0.73) × 10⁶ and (1.39-1.62) × 10⁶, respectively, which were higher than those in the health control group[(0.27-0.31) × 10⁶ and (0.47-0.56) × 10⁶] (P < 0.01); the percentages of Tregs in the NK elimination group were (1.86 ± 0.07)% and (2.15 ± 0.04)%, and the absolute counts were (0.77-0.90) × 10⁶ and (1.68-2.15) × 10⁶, respectively, all significantly higher than those in the infection group (P < 0.01 or P < 0.05). On days 3 and 5 after infection, the concentrations of IFN-γ in the cultural supernatant of splenocytes in the infection group were (790.75 ± 84.80) and (989.58 ± 199.59) pg/ml, respectively, and the TNF-α concentrations were (2 637.47 ± 283.50) and (3 124.58 ± 964.70) pg/ml, respectively, which were higher than those in the health control group [(73.69 ± 16.67) and (75.19 ± 15.97) pg/ml, (290.01 ± 187.46) and (290.51 ± 186.76) pg/ml respectively] (P < 0.01 or P < 0.05). In the NK elimination group, the IFN-γ concentrations were (15.83 ± 4.27) and (266.63 ± 108.62) pg/ml, and the TNF-α concentrations were (165.89 ± 71.02) and (842.77 ± 311.94) pg/ml, respectively, which were lower than those in the infection group (P < 0.01 or P < 0.05). On day 3 after infection, the IL-10 concentration in the NK elimination group was (3 588.20 ± 1 436.38) pg/ml, which was higher than that in the infection group [(1 255.77 ± 190.01) pg/ml] (P < 0.05). On day 5 after infection, the IL-10 concentration in the infection group was (4 991.36 ± 1 030.89) pg/ml, which was higher than that in the health control group as (848.50 ± 501.79) pg/ml (P < 0.05). In the NK elimination group, the IL-10 concentration was (9 317.95 ± 1 077.89) pg/ml, which was higher than that in the infection group (P < 0.05). On day 6 after infection, the EB content in the brain tissue of the infection group was (4.02 ± 0.10) μg/ml, which was higher than that in the health control group [(2.09 ± 0.06) μg/ml] (P < 0.05), and the EB content in the NK elimination group was (3.14 ± 0.02) μg/ml, which was lower than that in the infection group (P < 0.05). Conclusion The elimination of NK cells weakens the Th1 immune response, enhances the Tregs immune response, reduces the blood-brain barrier permeability and prolongs the survival time of mice with cerebral malaria.

Key words: Cerebral malaria, Natural killer cells, Th1 immune response, Regulatory T cells

CLC Number:

R531.33

YAN Jin, LI Dan-ni, FU Wei-xin. Immunomodulatory effects of natural killer cells on the CD4⁺ T cell subset in mice with cerebral malaria[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 332-338.

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References 21

[1]	World Health Organization. World malaria report 2019[R]. Geneva: WHO, 2019.
[2]	Ashley EA, Pyae Phyo A, Woodrow CJ. Malaria[J]. Lancet, 2018,391(10130):1608-1621.
[3]	Nacer A, Movila A, Sohet F, et al. Experimental cerebral malaria pathogenesis: hemodynamics at the blood brain barrier[J]. PLoS Pathog, 2014,10(12):e1004528.
[4]	Chen HM, Fu YZ, Chen JP, et al. The roles of vascular endothelial growth factor in cerebral malaria[J]. J Trop Med, 2017,17(2):269-273. (in Chinese)
[4]	( 陈宏敏, 傅毅振, 陈金平, 等. 血管内皮生长因子在脑型疟疾中的作用[J]. 热带医学杂志, 2017,17(2):269-273.)
[5]	Cariaco Y, Lima WR, Sousa R, et al. Ethanolic extract of the fungus Trichoderma stromaticum decreases inflammation and ameliorates experimental cerebral malaria in C57BL/6 mice[J]. Sci Rep, 2018,8(1):1547.
[6]	Kho S, Marfurt J, Handayuni I, et al. Characterization of blood dendritic and regulatory T cells in asymptomatic adults with sub-microscopic Plasmodium falciparum or Plasmodium vivax infection[J]. Malar J, 2016,15:328.
[7]	Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. Ⅰ. Distribution of reactivity and specificity[J]. Int J Cancer, 1975,16(2):216-229.
[8]	Campbell KS, Hasegawa J. Natural killer cell biology: an update and future directions[J]. J Allergy Clin Immunol, 2013,132(3):536-544.
[9]	Artavanis-Tsakonas K, Riley EM. Innate immune response to malaria: rapid induction of IFN-Gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes[J]. J Immunol, 2002,169(6):2956-2963.
[10]	Crouse J, Xu HC, Lang PA, et al. NK cells regulating T cell responses: mechanisms and outcome[J]. Trends Immunol, 2015,36(1):49-58.
[11]	Jiao L, Gao XL, Joyee AG, et al. NK cells promote type 1 T cell immunity through modulating the function of dendritic cells during intracellular bacterial infection[J]. J Immunol, 2011,187(1):401-411.
[12]	de Souza JB, Hafalla JC, Riley EM, et al. Cerebral malaria: why exprimental murine are required to undersand the pathogenesis of disease[J]. Parasitology, 2010,137(5):755-772.
[13]	Hora R, Kapoor P, Thind KK, et al. Cerebral malaria--clinical manifestations and pathogenesis[J]. Metab Brain Dis, 2016,31(2):225-237.
[14]	Storm J, Craig AG. Pathogenesis of cerebral malaria: inflammation and cytoadherence[J]. Front Cell Infect Microbiol, 2014,4:100.
[15]	Du YT, Zhao W, Xu L, et al. Effect of artesunate combined with erythropoietin on the expression of cerebral malaria-associated factors in mice[J]. Chin J Parasitol Parasit Dis, 2019,37(5):525-531. (in Chinese)
[15]	( 杜云婷, 赵薇, 徐兰, 等. 青蒿琥酯与促红细胞生成素联合使用对小鼠脑型疟相关因子表达的影响[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(5):525-531.)
[16]	Amani V, Vigário AM, Belnoue E, et al. Involvement of IFN-gamma receptor-medicated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection[J]. Eur J Immunol, 2000,30(6):1646-1655.
[17]	Berretta F, Piccirillo CA, Stevenson MM. Plasmodium chabaudi AS infection induces CD4⁺ Th1 cells and Foxp3⁺ T-bet⁺ regulatory T cells that express CXCR3 and migrate to CXCR3 ligands[J]. Front Immunol, 2019,10:425.
[18]	Berretta F, St-Pierre J, Piccirillo CA, et al. IL-2 contributes to maintaining a balance between CD4⁺Foxp3⁺ regulatory T cells and effector CD4⁺ T cells required for immune control of blood-stage malaria infection[J]. J Immunol, 2011,186(8):4862-4871.
[19]	Kurup SP, Obeng-Adjei N, Anthony SM, et al. Regulatory T cells impede acute and long-term immunity to blood-stage malaria through CTLA-4[J]. Nat Med, 2017,23(10):1220-1225.
[20]	Wilson KD, Ochoa LF, Solomon OD, et al. Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria[J]. J Neuroinflammation, 2018,15(1):173.
[21]	Ng SS, Souza-Fonseca-Guimaraes F, Rivera FL, et al. Rapid loss of group 1 innate lymphoid cells during blood stage Plasmodium infection[J]. Clin Transl Immunol, 2018,7(1):e1003.