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. 2020 Oct 7:11:570524.
doi: 10.3389/fimmu.2020.570524. eCollection 2020.

The Differential and Dynamic Progression of Hepatic Inflammation and Immune Responses During Liver Fibrosis Induced by Schistosoma japonicum or Carbon Tetrachloride in Mice

Li-Jun Song  1   2   3 , Xu-Ren Yin  2 , Sha-Sha Mu  2 , Jia-Huang Li  1   4   5 , Hong Gao  6 , Ying Zhang  2 , Pan-Pan Dong  2 , Cong-Jin Mei  2 , Zi-Chun Hua  1   4   5
Affiliations

The Differential and Dynamic Progression of Hepatic Inflammation and Immune Responses During Liver Fibrosis Induced by Schistosoma japonicum or Carbon Tetrachloride in Mice

Li-Jun Song et al. Front Immunol. .

Abstract

Liver fibrosis can result from various causes and could progress to cirrhosis and cancer; however, there are no effective treatments due to that its molecular mechanism is unclear. liver fibrosis model made by Schistosoma japonicum (S. japonicum) infection or Carbon tetrachloride (CCl4) intraperitoneal injection is a conventional model used in liver fibrosis-related studies for mechanism or pharmaceutical research purposes. But the differences in the pathological progression, immune responses and the underlying mechanism between the two liver fibrosis model have not been carefully compared and characterized, which hinders us from correctly understanding and making better use of the two models. In the present study, the pathological changes to the liver, and the cytokines, inflammatory factors, macrophages, and lymphocytes subsets involved were analyzed in the liver fibrosis model of S. japonicum infection or CCl4 intraperitoneal injection. Additionally, the pathological progression, immune responses and the underlying injury mechanism in these two models were compared and characterized. The results showed that the changing trend of interleukin-13 (IL-13), transforming growth factor beta (TGF-β), inflammatory factors, and M1, M2 macrophages, were consistent with the development trend of fibrosis regardless of whether liver fibrosis was caused by S. japonicum or CCl4. For lymphocyte subsets, the proportions of CD3+ T cells and CD4+ T cells decreased gradually, while proportion of CD8+ T cells peaked at 6 weeks in mice infected with S. japonicum and at 12 weeks in mice injected with CCl4. With prolonged S. japonicum infection time, Th1 (CD4+IFN-γ+) immunity converted to Th2 (CD4+IL-4+)/Th17 (CD4+IL-17+) with weaker regulatory T cell (Treg) (CD4+CD25+FOXP3+) immunity. However, in liver fibrosis caused by CCl4, Th1 cells occupied the dominant position, while proportions of Th2, Th17, and Treg cells decreased gradually. In conclusion, liver fibrosis was a complex pathological process that was regulated by a series of cytokines and immune cells. The pathological progressions and immune responses to S. japonicum or CCl4 induced liver fibrosis were different, possibly because of their different injury mechanisms. The appropriate animal model should be selected according to the needs of different experiments and the pathogenic factors of liver fibrosis in the study.

Keywords: Schistosoma japonicum; carbon tetrachloride; differential progression; dynamic progression; hepatic inflammation; immune response; liver fibrosis.

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Figures

Figure 1
Figure 1
The schematic diagram, weight gain and serum ALT of mice infected with S. japonicum or injected with CCl4 intraperitoneally. (A) The schematic diagram of mice infected with S. japonicum. (B) The schematic diagram of mice injected with CCl4 intraperitoneally. (C) The weight gain of mice infected with S. japonicum (% of week 0). (D) Bodyweight gain of mice injected with CCl4 intraperitoneally (% of week 0). (E) Image of the morphology of liver in mice infected with S. japonicum and injected with CCl4 intraperitoneally. (F) The number of adult worm in mice infected with S. japonicum challenged with the cercariae (12 ± 1). (G) The number of eggs per gram of liver in mice infected with S. japonicum challenged with the cercariae (12 ± 1). (H) The serum ALT levels in mice infected with S. japonicum or injected with CCl4 intraperitoneally. ALT, alanine aminotransferase; SJ, S. japonicum. Vehicle, olive oil. Data represent the mean ± SE from three independent experiments. ** P < 0.01, *** P < 0.001.
Figure 2
Figure 2
The intensity of liver fibrosis and cytokine levels in the liver of mice infected with S. japonicum or injected with CCl4 intraperitoneally. (A) Masson staining of the liver in mice. Original magnification 100×. (B)The fibrosis areas of the liver of mice infected with S. japonicum or injected with CCl4 intraperitoneally. (C) The hydroxyproline level in the mice liver. (D–J) The relative mRNA expression levels of collagen I, collagen III, α-SMA, MMP-9, TIMP-1, IL-13, and TGF-β in the liver of mice infected with S. japonicum. (K–Q) The relative mRNA expression levels of collagen I, collagen III, α-SMA, MMP-9, TIMP-1, IL-13, and TGF-β in the liver of mice injected with CCl4 intraperitoneally. SJ, S. japonicum Vehicle, olive oil. Data represent the mean ± SE from three independent experiments. *** P < 0.001.
Figure 3
Figure 3
The mRNA levels of inflammatory factors in the liver of mice infected with S. japonicum or injected with CCl4 intraperitoneally. (A–D) The relative mRNA expression levels of F4/80, TNF-α, IL-1β and IL-6 in the liver of mice infected with S. japonicum. (E–H) The relative mRNA expression levels of F4/80, TNF-α, IL-1β, and IL-6 in the liver of mice injected with CCl4 intraperitoneally. SJ, S. japonicum. Vehicle, olive oil. Data represent the mean ± SE from three independent experiments. ** P < 0.01, *** P < 0.001.
Figure 4
Figure 4
Flow cytometric analysis of macrophages in the liver of mice infected with S. japonicum or injected with CCl4 intraperitoneally. Scatter diagram of (A) M1 macrophages or (C) M2 macrophages in the mice liver. Statistical data analysis of (B) M1 macrophages or (D) M2 macrophages in the mice liver. SJ, S. japonicum. Vehicle, olive oil. Data represent the mean ± SE from three independent experiments. ** P < 0.01, *** P < 0.001.
Figure 5
Figure 5
Flow cytometric analysis of CD3/CD4/CD8+ lymphocytes in the liver of mice infected with S. japonicum or intraperitoneally injected with CCl4. Scatter diagram of (A) CD3+ lymphocytes, (C) CD4+ lymphocytes or (E) CD8+ lymphocytes in the mice liver. Statistical data analysis of (B) CD3+ lymphocytes, (D) CD4+ lymphocytes or (F) CD8+ lymphocytes in the mice liver. SJ, S. japonicum. Vehicle, olive oil. Data represent the mean ± SE from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.
Figure 6
Figure 6
Flow cytometric for the detection of Th1/Th2/Th17/Treg cells in mice infected with S. japonicum or intraperitoneally injected with CCl4. Scatter diagram of (A) Th1 cells (CD4+IFN-γ+), (C) Th2 cells (CD4+IL-4+), (E) Th17 cells (CD4+IL-17+) or (G) Treg cells (CD4+CD25+Foxp3+) in total CD4+ T cells from mice liver. Statistical data analysis of (B) Th1 cells (CD4+IFN-γ+), (D) Th2 cells (CD4+IL-4+), (F) Th17 cells (CD4+IL-17+), or (H) Treg cells (CD4+CD25+Foxp3+) in total CD4+ T cells from mice liver. SJ, S. japonicum; Vehicle, olive oil; Treg, regulatory T cell. Data represent the mean ± SE from three independent experiments. Cells were gated on the CD4+ population. ** P < 0.01, *** P < 0.001.
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