γδ T cells-secreted IL-17A aggravates liver fibrosis in mice infected with Schistosoma japonicum via activating hepatic stellate cells

doi:10.12140/j.issn.1000-7423.2020.03.007

Abstract

Abstract:

Objective To investigate the effect of IL-17A-producing γδ T cells on the development of early liver fibrosis induced by Schistosoma japonicum infection. Methods Ten wild-type (WT) female C57BL/6 mice were randomly divided into the healthy control group and the WT infection group, five mice each. Another group of five C57BL/6-background T cell antigen receptor δ chain gene knockout (TCR δ KO) mice served as the TCR δ KO infection group. Mice in the two infection groups were infected with S. japonicum cercariae (20 ± 2 each) by using abdominal patches, while the healthy control group was not infected. Six weeks after infection, the mice were anaesthesized and sacrificed, and liver tissues were collected, homogenized, and lysed. The pellets after lysis underwent density gradient centrifugation to obtain liver nonparenchymal cells. The content of IL-17A expressed by γδ T cells among the liver nonparenchymal cells was determined by flow cytometry. The IL-17A concentration in the lysate supernatant was examined by ELISA. Another part of the liver tissue was fixed in 4% paraformaldehyde, followed by hematoxylin-eosin (HE) and Masson staining to observe the deposition of collagen fibers. Meanwhile, peripheral blood was collected the mice of all groups to determine the serum concentration of IL-17A. A human hepatic stellate cell line (LX-2 cells) was used to examine the relative transcription level of mRNA of α-smooth muscle actin (α-SMA) and collagen type I (Col 1) in the LX-2 cells by fluorescent quantitative PCR. The assay was carried out by assigning the cell culture into 3 groups, of which the test group was assayed by adding IL-17A to a final concentration of 10 ng/ml, while the positive group and negative control group was assayed by adding IL-13 to a final concentration of 10 ng/ml and equal volume of PBS. Results The results of flow cytometry showed that the percentage of IL-17A-secreting Vγ2 subtype γδ T cells in the WT infection group was (48.0 ± 1.0)%, which was significantly higher than that in the control group [(17.0 ± 1.6)%, P < 0.01]. Masson staining revealed that the collagen deposition area in the mice of the TCR δ KO group was (3.5 ± 0.2) × 10⁴ μm², which was smaller than that in the WT infection group [(6.4 ± 0.5) × 10⁴ μm², P < 0.01]. ELISA assay showed that the IL-17A concentration in the sera and liver lysate supernatant of the infected TCR δ KO mice was (34.0 ± 22.3) pg/ml and (101.6 ± 18.6) pg/ml, respectively, both lower than those in the WT infection group [(143.9 ± 33.8) pg/ml, P < 0.05; (217.2 ± 19.2) pg/ml, P < 0.01, respectively]. Fluorescent quantitative PCR showed that the mRNA expression of α-SMA and Col 1 in LX2 cells after IL-17A stimulation was up-regulated by (4.0 ± 0.7) and (8.7 ± 1.2) folds, respectively, compared to the control group (P < 0.05, P < 0.01). Conclusion The IL-17A secreted from γδ T cells may involve in the development of hepatic fibrosis induced by S. japonicum infection in mice probably via activating hepatic stellate cells.

Key words: Schistosoma japonicum, γδ T cell, IL-17A, Liver fibrosis

CLC Number:

R383.24

SUN Lei, HU Yuan, SHEN Yu-juan, CAO Jian-ping. γδ T cells-secreted IL-17A aggravates liver fibrosis in mice infected with Schistosoma japonicum via activating hepatic stellate cells[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 299-303.

Figures/Tables 3

References 27

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基因名称 Gene name	引物序列（5′→3′） Primer sequence（5′→3′）
GAPDH	F: AAGGTCGGAGTCAACGGATTTGGT
	R: AGCCTTGACGGTGCCATGGAATTT
α-SMA	F: TCTGGAGATGGTGTCACCCACAAT
	R: AATAGCCACGCTCAGTCAGG
Col 1	F: GGACACAGAGGTTTCAGTGG
	R: CCAGTAGCACCATCATTTCC