Expression and function of arginase in livers of mice infected with Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2020.03.008

Abstract

Abstract:

Objective To investigate the expression and function of arginase (ARG) in livers of mice infected with Echinococcus granulosus. Methods Eighteen female BALB/c mice were randomly divided into the infection and control group, 9 mice each. Mice in the infection group were each given 2 000 live protoscoleces of E. granulosus by intraperitoneal injection, while the control group was injected with the same volume of saline. Liver leukocytes were isolated 270 days after infection. The expression levels of ARG and nitric oxide synthase (NOS) were examined by Western blotting, and the protein abundance was analyzed with the Image J software. The ARG activity in liver leukocytes, and urea and nitric oxide contents in liver tissues were assessed with the ARG activity detection kit, urea detection kit and nitric oxide (NO) detection kit, respectively. The expression of ARG-1 in various myeloid cells and CD3ζ in T cells in the liver were analyzed by flow cytometry. Data were analyzed using the SPSS 20.0 software. Results Western blotting showed that the gray value of ARG-1 expression in liver leukocytes in the infection group was 1.03 ± 0.01, which was significantly higher than that in the control group (0.12 ± 0.01, P < 0.01); the expression gray value of ARG-2 and inducible nitric oxide synthase(iNOS) in the infection group was 0.54 ± 0.01 and 0.64 ± 0.02, while that of control group was 0.55 ± 0.02 and 0.59 ± 0.02, respectively; no significant differences were found between the groups (P > 0.05). In the infection group, the ARG activity in liver leukocytes, and urea and NO contents in liver tissues was (0.03 ± 0.00) U/ml, (0.66 ± 0.02) mmol/L and (14.63 ± 1.55) μmol/L, respectively, being significantly different from those in the control group [(0.02 ± 0.00) U/L, (0.04 ± 0.01) mmol/L, and (29.22 ± 0.36) μmol/L, respectively] (P < 0.05). Flow cytometry showed that the relative fluorescence density of ARG-1 expression in CD11b⁺CD11c⁺, CD11b⁺F4/80⁺, CD11b⁺Gr-1⁺, CD11b⁺Gr-1⁺Ly-6C-Ly-6G⁺, CD11b⁺Gr-1⁺Ly-6C⁺-Ly-6G^- and CD11b⁺Ly-6G⁺ liver cells in the infection group were 1.41 ± 0.12, 1.21 ± 0.06, 1.52 ± 0.16, 1.30 ± 0.03, 1.58 ± 0.12 and 1.21 ± 0.04, respectively, all items being significantly higher than those in the control group (1.00 ± 0.14, 1.00 ± 0.05, 1.00 ± 0.01, 1.00 ± 0.07, 1.00 ± 0.03 and 1.00 ± 0.02; P < 0.05). The relative mean fluorescence density of CD3ζ in liver CD4⁺ and CD8⁺ T cells in the infection group were 0.82 ± 0.04 and 0.78 ± 0.05, respectively, both significantly lower than those in the control group (1.00 ± 0.05, 1.00 ± 0.07; P < 0.05). Conclusion Higher expression of ARG-1 in liver leukocytes was found in mice infected with E. granulosus, which may antagonize the NOS to some extent; meanwhile, the expression of CD3ζ in the CD4⁺ and the CD8⁺ T cells was down-regulated.

Key words: Echinococcus granulosus, Arginase, Nitric oxide synthetase, Flow cytometry, CD3ζ

CLC Number:

R383.33

CAO Sheng-kui, ZHANG Xiao-fan, WEI Yu-huan, PAN Jia-ming, CAO Jian-ping, SHEN Yu-juan, CHEN Jia-xu. Expression and function of arginase in livers of mice infected with Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 304-309.

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References 22

[1]	Budke CM, Carabin H, Ndimubanzi PC, et al. A systematic review of the literature on cystic echinococcosis frequency worldwide and its associated clinical manifestations[J]. Am J Trop Med Hyg, 2013,88(6):1011-1027. pmid: 23546806
[2]	Wu WP, Wang H, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parasit Dis, 2018,36(1):1-14. (in Chinese)
	( 伍卫平, 王虎, 王谦, 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(1):1-14.)
[3]	Vuitton DA. Echinococcosis and allergy[J]. Clinic Rev Allerg Immunol, 2004,26(2):93-104.
[4]	Zheng YD. Strategies of Echinococcus species responses to immune attacks: implications for therapeutic tool development[J]. Int Immunopharmacol, 2013,17(3):495-501. pmid: 23973651
[5]	Grubor NM, Jovanova-Nesic KD, Shoenfeld Y. Liver cystic echinococcosis and human host immune and autoimmune follow-up: a review[J]. World J Hepatol, 2017,9(30):1176-1189. pmid: 29109850
[6]	Caldwell RW, Rodriguez PC, Toque HA, et al. Arginase: amultifaceted enzyme important in health and disease[J]. Physiol Rev, 2018,98(2):641-665.
[7]	Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition[J]. Biochem J, 2001,357(3):593-615.
[8]	Amri M, Touil-Boukoffa C. A protective effect of the laminated layer on Echinococcus granulosus survival dependent on upregulation of host arginase[J]. Acta Trop, 2015,149:186-194. pmid: 26048557
[9]	Zea AH, Rodriguez PC, Culotta KS, et al. L-arginine modulates CD3ζ expression and T cell function in activated human T lymphocytes[J]. Cell Immunol, 2004,232(1/2):21-31.
[10]	Xiao N, Qiu JM, Nakao M, et al. Short report: identification of Echinococcus species from a yak in the Qinghai-Tibet plateau region of China[J]. Am J Trop Med Hyg, 2003,69(4):445-446.
[11]	Gong WC, Huang FJ, Sun L, et al. Toll-like receptor-2 regulates macrophage polarization induced by excretory-secretory antigens from Schistosoma japonicum eggs and promotes liver pathology in murine schistosomiasis[J]. PLoS Negl Trop Dis, 2018,12(12):e0007000.
[12]	Díaz A, Casaravilla C, Allen JE, et al. Understanding the laminated layer of larval Echinococcus Ⅱ: immunology[J]. Trends Parasitol, 2011,27(6):264-273. doi: 10.1016/j.pt.2011.01.008 pmid: 21376669
[13]	Paredes R, Godoy P, Rodríguez B, et al. Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility[J]. J Cell Biochem, 2011,112(1):189-199.
[14]	El Kasmi KC, Qualls JE, Pesce JT, et al. Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens[J]. Nat Immunol, 2008,9(12):1399-1406.
[15]	Rodriguez PC, Quiceno DG, Zabaleta J, et al. Arginase Ⅰ production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses[J]. Cancer Res, 2004,64(16):5839-5849. pmid: 15313928
[16]	Bowcutt R, Bell LV, Little M, et al. Arginase-1-expressing macrophages are dispensable for resistance to infection with the gastrointestinal helminth Trichuris muris[J]. Parasite Immunol, 2011,33(7):411-420.
[17]	Paduch K, Debus A, Rai B, et al. Resolution of cutaneous leishmaniasis and persistence of Leishmania major in the absence of arginase 1[J]. J Immunol, 2019,202(5):1453-1464.
[18]	Cao SK, Pan W, Liu H, et al. Expression and activity of arginase from monocytic-type myeloid-derived suppressor cells in rats infected with Echinococcus granulosus[J]. Chin J Parasitol Parasit Dis, 2016,34(1):27-31. (in Chinese)
	( 曹胜魁, 潘伟, 刘华, 等. 细粒棘球蚴感染小鼠单核髓源抑制性细胞精氨酸酶的表达和活性研究[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(1):27-31.)
[19]	Peng SS, Yu T, Wang L, et al. Influence of type 2 macrophages (M2) in echinococcosis[J]. Int J Clin Exp Pathol, 2016,9(3):4110-4116.
[20]	Munder M, Eichmann K, Morán JM, et al. Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells[J]. J Immunol, 1999,163(7):3771-3777.
[21]	Oberlies J, Watzl C, Giese T, et al. Regulation of NK cell function by human granulocyte arginase[J]. J Immunol, 2009,182(9):5259-5267.
[22]	Bronte V, Serafini P, Mazzoni A, et al. L-arginine metabolism in myeloid cells controls T-lymphocyte functions[J]. Trends Immunol, 2003,24(6):301-305.

细胞类型Cell type	ARG-1表达的细胞比例/% Percentages of cells expressing ARG-1/%		ARG-1表达的相对平均荧光密度 Relative MFI of ARG-1 expression
细胞类型Cell type	感染组Infection group	对照组Control group	感染组Infection group	对照组Control group
CD11b⁺CD11c⁺	1.85 ± 0.24	1.69 ± 0.16	1.41 ± 0.12	1.00 ± 0.14
CD11b⁺F4/80⁺	5.39 ± 1.17	6.19 ± 0.40	1.21 ± 0.06	1.00 ± 0.05
CD11b⁺Gr-1⁺	2.11 ± 0.55	1.27 ± 0.52	1.52 ± 0.16	1.00 ± 0.01
CD11b⁺Gr-1⁺Ly-6C^--Ly-6G⁺	1.63 ± 0.46	0.73 ± 0.18	1.30 ± 0.03	1.00 ± 0.07
CD11b⁺Gr-1⁺Ly-6C⁺-Ly-6G^-	1.85 ± 0.40	0.89 ± 0.43	1.58 ± 0.12	1.00 ± 0.03
CD11b⁺Ly-6G⁺	1.86 ± 0.45	1.15 ± 0.09	1.21 ± 0.04	1.00 ± 0.02