Indoleamine 2,3-dioxygenase 1 deficiency attenuates CCl4-induced fibrosis through Th17 cells down-regulation and tryptophan 2,3-dioxygenase compensation

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1) is an intracellular rate-limiting enzyme in the metabolism of tryptophan along the kynurenine pathway, subsequently mediating the immune response; however, the role of IDO1 in liver fibrosis and cirrhosis is still unclear. In this study, we investigated the role of IDO1 in the development of hepatic fibrosis and cirrhosis. Patients with hepatitis B virus-induced cirrhosis and healthy volunteers were enrolled. For animals, carbon tetrachloride (CCl4) was used to establish liver fibrosis in wild-type and IDO1 knockout mice. Additionally, an IDO1 inhibitor (1-methyl-D-tryptophan) was administered to WT fibrosis mice. Liver lesions were positively correlated with serum IDO1 levels in both the clinical subjects and hepatic fibrosis mice. A positive correlation between serum IDO1 levels and liver stiffness values was found in the cirrhosis patients. Notably, IDO1 knockout mice were protected from CCl4-induced liver fibrosis, as reflected by unchanged serum alanine transaminase and aspartate transaminase levels and lower collagen deposition, α-smooth muscle actin expression and apoptotic cell death rates. On the other hand, tryptophan 2,3-dioxygenase (TDO), another systemic tryptophan metabolism enzyme, exhibited a compensatory increase as a result of IDO1 deficiency. Moreover, hepatic interleukin-17a, a characteristic cytokine of T helper 17 (Th17) cells, and downstream cytokines' mRNA levels showed lower expression in the IDO1-/- model mice. IDO1 appears to be a potential hallmark of liver lesions, and its deficiency protects mice from CCl4-induced fibrosis mediated by Th17 cells down-regulation and TDO compensation.

Keywords: 1-methyl-D-tryptophan; T helper 17 cells; indoleamine 2,3-dioxygenase 1; liver fibrosis; tryptophan 2,3-dioxygenase.

Figure 1. The serum level of IDO1 was decreased in patients with HBV-induced cirrhosis and positively correlated with the degree of cirrhosis

(A) Participant flow diagram illustrating the two study groups. (B) ELISA evaluating the serum IDO1 level in participants. (C) The serum levels of ALT and AST in participants were detected with an automatic biochemical analyser. (D) Pearson's linear correlation tests for IDO1 and liver stiffness value in patients. (E) and (F) Pearson's linear correlation tests for IDO1, ALT, AST, GGT, ALB, DB, TBA, CHE and PTA levels in patients’ serum. r_p: Pearson's correlation coefficient. The data are presented as the means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 2. Serum IDO1 was time-dependently elevated in a CCl₄-induced fibrosis mouse model and positively correlated with liver lesions

(A) Experimental plan for mice. (B) Liver lesions were assessed by measuring serum ALT and AST levels in WT control and model mice. (C) ELISA for detecting serum IDO1 level in WT control and model mice. (D) Pearson's linear correlation tests for serum IDO1, ALT and AST levels in the WT model mice. (E) Western blot analysis of the expression of IDO1 and α-SMA in the livers of WT control and model mice (4-week). (F) Immunofluorescence analysis to detect the expression of IDO1 and α-SMA in the livers of WT control and model mice (4-week). (G) ELISA for detecting serum IFN –γ and IL-4 in mice and clinical subjects. The data are presented as the means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3. IDO1 deficiency attenuated the development of CCl₄-induced fibrosis in mice

(A) Changes in mean body weight in WT and IDO1^–/– mice during injection and the body weights of WT and IDO1^–/– mice at the end of the 4th week. (B) Liver lesions were assessed by measuring serum ALT and AST levels in WT and IDO1^–/–mice. (C) Western blot analysis of the expression of α-SMA, BAX and cleaved caspase 3 in WT and IDO1^–/– mice. (D) TUNEL assay to detect apoptotic cell death in liver tissues. (E) H&E, Sirius red and α-SMA immunofluorescence staining of liver sections. The data are presented as the means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0).

Figure 4. IDO1 deficiency elevated the expressions of TDO and GCN2 kinase during liver fibrosis

(A) LC-MS/MS analysis to detect the concentrations of TRP and KYN in liver tissues of WT and IDO1^–/– mice. (B) Quantitative real-time PCR analysis to detect TDO mRNA in the liver tissues of WT and IDO1^–/– mice. (C) and (D) Western blot analysis of the expression of TDO and GCN2 kinase in the livers of WT and IDO1^–/– mice. (E) Immunofluorescence analysis to detect the expression of CD8a in the livers of WT and IDO1^–/– mice. The data are presented as the means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5. IDO1 deficiency reduced IL-17a and downstream cytokines during Liver fibrosis

(A) Quantitative real-time PCR analysis to detect IL-17a mRNA in the liver tissues of WT and IDO1^–/– mice. (B) Western blot analysis of the expression of IL-17a in the livers of WT and IDO1^–/– mice. (C) Immunofluorescence analysis to detect the expression of IL-17a in the livers of WT and IDO1^–/– mice. (D) ELISA evaluating the serum IL-17a level in mice. (E) Western blot analysis of the expression of STAT3 in the livers of WT and IDO1^–/– mice. (F) Quantitative real-time PCR analysis to detect IL-6, TGF-β1, TNF-α and IL-1β mRNA in the liver tissues of WT and IDO1^–/– mice. The data are presented as the means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6. A model depicting the key role of IDO1 deficiency in the pathogenesis of CCl₄-induced liver fibrosis

After treatment with CCl₄, TDO in liver was increased under the condition of IDO1 deficiency. The compensation of TDO can degrade TRP, the depletion of which activates GCN2 kinase, subsequently suppressing the proliferation of both CD8+ cells and Th17 cells. Liver lesions are ameliorated due to less hepatocyte damage induced by CD8+ cells. HSCs can be activated by cytokines, including IL-6, IL-1β, TNF-α and TGF-β1, that are secreted by Kupffer cells under stimulation with Il-17a. Consequently, IDO1 deficiency protects mice from CCl₄-induced fibrosis mediated by Th17 cells and TDO compensation.