Profibrogenic role of IL-15 through IL-15 receptor alpha-mediated trans-presentation in the carbon tetrachloride-induced liver fibrosis model

Abstract

Background: Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis.

Methods: We induced liver fibrosis in Il15^-/- , Il15ra^-/- and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl₄). Liver fibrosis was evaluated by Sirius red and Mason's trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry.

Results: Both Il15^-/- and Il15ra^-/- mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. Il15ra^-/- mice showed further reduction in collagen deposition compared to Il15^-/- mice. However, Col1a1 and Col1a3 genes were similarly induced in the fibrotic livers of wildtype, Il15^-/- and Il15ra^-/- mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, Il15^-/- and Il15ra^-/- mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45⁺ immune cells and CD68⁺ macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice.

Conclusion: Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα.

Keywords: IL-15; IL-15Rα; carbon tetra chloride (CCl) 4; liver fibrosis; pre-clinical study.

Figure 1

IL-15 or IL-15Rα deficiency reduces CCl₄-induced liver fibrosis. (A) Il15 gene expression was determined by RT-qPCR in the livers of wildtype (WT), Il15^–/– and Il15ra^–/– mice treated with vehicle (oil) or CCL₄. (B) Serum ALT levels in WT, Il15^–/– and Il15ra^–/– mice following vehicle or CCl₄ treatment at the endpoint. Values from individual mice are shown. (C) Sirius Red/Fast green staining of WT, Il15^–/– and Il15ra^–/– liver tissues from oil- or CCl₄-treated mice. Data shown are representative of at least 8 mice per group from 3 to 4 separate experiments. (D) Quantification of Sirius Red stained area was performed on seven to ten were randomly selected fields in the different regions of liver sections from 3 to 4 mice per group. (E) Masson’s trichrome staining of WT, Il15^–/– and Il15ra^–/– liver tissues from oil and CCl₄-treated mice. Data shown are representative of 4-5 mice per group from three independent experiments. (A, B, D) Mean + standard error of the mean (SEM). Statistical significance was calculated using Mann-Whitney test (A) or ordinary one-way ANOVA with Tukey’s post-hoc test: ***p<0.001; ****p<0.0001.

Figure 2

Impact of IL15 or IL15Rα deficiency on the expression of fibrogenic genes. Indicated genes associated with hepatic fibrogenesis were assessed in the livers of oil- or CCl₄- treated mice from the three genotypes. Mean + SEM. Statistical significance was calculated using Mann-Whitney’s test: *p<0.5; **p<0.1; ***p<0.001.

Figure 3

IL15 or IL15Rα deficiency reduces mononuclear cell infiltration in livers of CCl₄-treated mice. (A) Hematoxylin and eosin-stained sections of livers from oil- and CCl₄-treated WT, Il15^–/– and Il15ra^–/– mice. Lower (bar = 100 μm) and higher (bar = 250 μm) magnification images are shown for CCl₄-treated to indicate mononuclear cell infiltration in the latter (white arrowheads). Data are representative of 4-5 mice per group from two independent experiments. (B) Representative immunofluorescence staining of liver sections of from oil- and CCl₄-treated WT, Il15^–/– and Il15ra^–/– mice for COL1A1 in red and CD45 in green color. Nuclei were counter stained with Hoechst 33342. Images are representative of 2-3 mice per group from two independent experiments. Quantification of (C) COL1A1 staining and (D) CD45 infiltration areas was performed on at least five randomly selected fields from different liver sections collected from 2 to 3 mice per group. Mean + SEM are shown. One-way ANOVA with Tukey’s post-hoc test: *p<0.05, ****p<0.0001.

Figure 4

Loss of IL15 or IL15Rα reduces CD68⁺ macrophage infiltration in livers of CCl₄-treated mice. (A) Cd68, Ccl2, Ccl5 and Cx3cl1 gene expression was assessed in the livers of oil- and CCl₄- treated WT, Il15^–/– and Il15ra^–/– mice. Mean + SEM. Mann-Whitney’s test: *p<0.1; ***p<0.001; ****p<0.0001, ns, not significant. N=3-10 mice per group. (B) Tissues were stained with alpha-SMA and CD68 antibodies. Nuclei were counter stained with Hoechst. Images are representative of 2-3 mice per group from two independent experiments. Quantification of (C) αSMA staining and (D) CD68 infiltration areas was performed on at least five randomly selected fields from different liver sections collected from 2 to 3 mice per group. Mean + SEM. One-way ANOVA with Tukey’s post-hoc test: *p<0.05, **p<0.1, ****p<0.0001.

Figure 5

Phenotype of lymphoid cells in the livers of oil- and CCl₄- treated mice. (A) Flow cytometry analyzes of representative mice from each group for the different lymphoid cell subsets in the liver are shown. (B) Numbers of total and the indicated IHLs were calculated from the proportion of cells determined by flowcytometry and the cell yield per gram of liver tissue. Two-way ANOVA with Tukey’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6

Phenotype of myeloid cells in the livers of oil- and CCI₄- treated mice. (A, B) Flow cytometry analyses of representative mice from each group for the different myeloid cell subsets in the liver are shown. (C–E) Total numbers of neutrophils (C), eosinophils (D) and monocytes (E) within the CD45⁺CD11b⁺Ly6G^-CD11c^-Eo^- gate. Representative data from at least 3 mice in the oil-treated group and 5 mice in the CCl4-treated group are shown. Mean + SEM from at least 3 mice in the oil-treated group and 5 mice in the CCl4-treated group are shown. One-way ANOVA with Tukey’s post-hoc test: * p<0.05, ** p<0.01.

Figure 7

IL-15 signaling deficiency reduces the infiltration of monocytes during CCl₄-induced liver fibrosis. (A) Flow cytometry analyzes of representative mice from each group for the different monocyte cell subsets in the liver are shown. (B–D) the numbers of monocyte subsets were calculated from the proportion of cells determined by flow cytometry and the cell yield per gram of liver tissue. As most of the Ly6C high cells are also positive for CCR2 and CX3CR1 (A, bottom panel), the same subsets are included in the pro-inflammatory monocytes (B) and in the pro-/anti-inflammatory monocytes (D). Mean + SEM from at least 3 mice in the oil-treated group and 5 mice in the CCl₄-treated group are shown. One-way ANOVA with Tukey’s post-hoc test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.