PD-1阻断对伯氏疟原虫感染小鼠免疫应答的影响

doi:10.12140/j.issn.1000-7423.2020.04.001

摘要/Abstract

摘要：

目的探讨程序性死亡受体-1（PD-1）阻断对小鼠抗伯氏疟原虫ANKA（Plasmodium berghei ANKA,PbA）感染免疫应答的影响。方法将BALB/c小鼠按照随机数字表法分为健康组、感染组和PD-1组,每组8只。感染组和PD-1组小鼠经腹腔注射1 × 10⁶个PbA感染红细胞,健康组小鼠不作任何处理。感染当天和感染后3、5、7 d,PD-1组每鼠腹腔注射200 μg PD-1单抗,感染组注射等剂量PD-1同型对照单抗。感染后4 d起隔天检测小鼠红细胞感染情况并记录小鼠生存情况。感染后5 d每组各处死4只小鼠,取脾组织,制备脾细胞悬液,流式细胞术检测脾细胞中髓样树突状细胞（mDCs）及PD-1配体（PD-L1）的表达水平、骨髓来源抑制性细胞（MDSCs）和Th1细胞的百分率和绝对数,ELISA检测脾细胞培养上清中细胞因子IFN-γ、IL-10和IL-6分泌水平。结果感染组小鼠在感染后5~6 d外周血开始出现疟原虫感染红细胞,随后红细胞感染率快速升高,在感染后16 d达到40%左右,PD-1组小鼠红细胞感染率与感染组的相比差异无统计学意义（P > 0.05）。感染组小鼠在感染后15 d出现死亡,23 d全部死亡;PD-1组小鼠在13 d出现死亡,18 d全部死亡,差异有统计学意义（P < 0.05）。流式细胞术检测结果显示,PD-1组脾细胞中CD11c⁺CD11b⁺ mDCs百分率为（2.21 ± 0.10）%,明显低于感染组的（4.51 ± 0.21）%（P < 0.05）;PD-1组CD4⁺T-bet⁺IFN-γ⁺ Th1百分率为（3.38 ± 0.54）%,低于对照组的（5.85 ± 0.42）%（P < 0.05）;mDCs上PD-L1的表达水平,PD-1组为（55.67 ± 6.35）%,高于感染组的（21.35 ± 4.45）%（P < 0.05）;Gr-1⁺CD11b⁺MDSCs的百分率,PD-1组为（9.03 ± 0.62）%,高于感染组的（3.58 ± 0.16）%（P < 0.05）。脾细胞培养上清中,感染组IFN-γ、IL-6和IL-10的分泌水平分别为（901.69 ± 73.37）、（200.94 ± 4.97）和（551.95 ± 121.71）pg/ml,PD-1组分别为（231.17 ± 57.69）、（86.16 ± 3.93）和（101.72 ± 20.73）pg/ml,PD-1组均低于感染组（P < 0.05）。结论 PD-1阻断可削弱小鼠对PbA感染的保护性免疫应答。

关键词: 伯氏疟原虫, 程序性死亡受体-1, 固有免疫, 细胞免疫

Abstract:

Objective To investigate the effect of programmed cell death-1(PD-1) blockade on immune responses to the infection of Plasmodium berghei ANKA (PbA) in mice.Methods BALB/c mice were divided by random number table method into 3 groups: the healthy group, infection group and PD-1 group, 8 mice each. Mice in the infection group and PD-1 group were injected intraperitoneally with 1 × 10⁶ PbA-infected red blood cells, while those in the healthy group did not receive any treatment. On the day of infection and on days 3, 5, and 7 after infection, mice in the PD-1 group were injected intraperitoneally with 200 μg of PD-1 monoclonal antibody, while the infection group was injected with equal dose of PD-1 homotype control monoclonal antibody. The erythrocyte infection rate and mice survival rate were monitored every other day from day 4 after infection. On day 5 after infection, 4 mice in each group were sacrificed. Spleen tissues were collected to prepare spleen cell suspensions. Myeloid dendritic cells (mDCs) and the expression of PD-1 ligand (PD-L1) in spleen cells, as well as the percentages and absolute numbers of bone marrow-derived inhibitory cells (myeloid-derived suppressor cells, MDSCs) and Th1 cells were detected by flow cytometry. IFN-γ, IL-6 and IL-10 levels in spleen cell culture supernatant were determined by ELISA.Results In the infection group, Plasmodium-infected red blood cells began to appear in peripheral blood 5-6 days after infection, and then the infection rate of red blood cells increased rapidly, reaching about 40% on day 16 after infection, but there was no difference between the PD-1 group and the infection group. In the infection group, deaths occurred from day 15 after infection and all died by day 23 after infection. In the PD-1 group, deaths occurred from day 13 and all died by day 18. Flow cytometry showed that the percentage of CD11c⁺CD11b⁺ mDCs in PD-1 group was (2.21 ± 0.10)%, which was significantly lower than that in the infection group [(4.51 ± 0.21)%, P < 0.05]; the percentage of CD4⁺T-bet⁺IFN-γ⁺ Th1 cells [(3.38 ± 0.54)%] was also lower than that in the infection group [(5.85 ± 0.42)%, P < 0.05]. The expression of PD-L1 on mDCs was higher in PD-1 group [(55.6 ± 6.35)%] than that in the infection group [(21.35 ± 4.45)%, P < 0.05]. The percentage of Gr-1⁺CD11b⁺ MDSCs in the PD-1 group [(9.03 ± 0.62)%] was higher than that in the infection group [(3.58 ± 0.16%), P < 0.05]. The levels of IFN-γ, IL-6 and IL-10 in the infection group were (901.69 ± 73.37) pg/ml, (200.94 ± 4.97) pg/ml and (551.95 ± 121.71) pg/ml, respectively, while those in the PD-1 group were (231.17 ± 57.69) pg/ml, (86.1 ± 3.93) pg/ml and (101.72 ± 20.73) pg/ml, respectively.Conclusion PD-1 blockade weakened the protective immune response to PbA infection in mice.

Key words: Plasmodium berghei, Programmed cell death-1, Innate immunity, Cellular immunity

中图分类号:

R531.3

王作玲, 潘艳艳, 孙晓丹, 曹雅明. PD-1阻断对伯氏疟原虫感染小鼠免疫应答的影响[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(4): 405-411.

WANG Zuo-ling, PAN Yan-yan, SUN Xiao-dan, CAO Ya-ming. Effect of PD-1 blockade on immune responses in mice infected with Plasmodium berghei[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(4): 405-411.

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