Influence of Lipoxin-A4 Treatment on Cytokine, Chemokine Genes Expression, and Phenotypic Distribution of Lymphocyte Subsets During Experimental Liver Fibrosis

Abstract

Objective: Lipoxins are anti-inflammatory, pro-resolving molecules that are secreted by immune cells such as neutrophils and macrophages. Lipoxins are a metabolite of the arachidonic acid pathway that resolve inflammation in fibrotic liver by producing several anti-inflammatory molecules. In this study, phenotypic distribution activation markers of lymphocytes in the spleen and expression levels of chemokines (chemokine (C-X-C motif) receptor 3, chemokine (C-X-C motif) ligand 10) cytokines (interferon gamma, tumor necrosis factor alpha, interleukin-6, interleukin-10) in the liver of lipoxin A4-treated fibrotic mice were investigated.

Materials and methods: Liver fibrosis was induced in BALB/c mice by thioacetamide administration. Lipoxin A4 was administered during last 2 weeks of induction. Fibrosis level was determined by using Knodell scoring. Lymphocytes were identified by flow-cytometry. Expression levels of genes were measured by quantitative real time polymerase chain reaction in liver homogenates.

Results: Lipoxin A4 treatment caused an elevation of T-lymphocyte percentage in the spleen. Interestingly, administration of lipoxin A4 significantly reduced B-lymphocyte population in spleen of fibrotic group. CD8+ cytotoxic T cell frequency significantly reduced in thioacetamide-induced mice; however, lipoxin A4 administration increased that percentage significantly. Lipoxin A4 treatment significantly reduced frequency of activated (CD8+CD69+) cytotoxic T cells. Expression levels of chemokines significantly reduced in the liver after lipoxin A4 treatment. While expression levels of interferon gamma, tumor necrosis factor alpha, and interleukin-6 significantly reduced in the liver after lipoxin A4 treatment, an anti-inflammatory cytokine interleukin-10 expression was almost at similar levels in all experimental groups.

Conclusion: Lipoxin A4 performs its anti-inflammatory effect by reducing the frequency of activated T cells and expression levels of chemokines cytokines responsible from inflammatory immune response in the liver.

Figure 1.

Mononuclear cell infiltration level and the formation of connective and supporting tissues associated with the formation of fibrosis have been determined by H&E and trichrom staining, respectively. Figures show representative histologic views of liver samples for each group. Figures show H&E staining and trichrom staining views with 40× magnifications.

Figure 2.

Distribution of T-lymphocyte (CD3⁺) (•) and B-lymphocyte (CD19⁺) (▪) populations in spleen. Figure 2A presents data and mean values for each subject in all experimental groups. Figure 2B, histogram image of representative CD3 and CD19 populations after lymphocyte gate acquisition. The symbols * and # indicate statistically significant parameters of CD3 and CD19, respectively.

Figure 3.

Distribution of helper T-lymphocytes (Th) (CD4⁺) (•) and cytotoxic T-lymphocytes (Tc) (CD8⁺) (▪) cell populations in spleen. Figure 3A presents data and mean values for each subject in all experimental groups. Figure 3B, histogram image of representative CD4 and CD8 populations after CD3 T-lymphocyte gate acquisition. * indicates statistically significant CD8 values between TAA and TAA + LXA4.

Figure 4.

Distribution of CD4⁺CD69⁺ (•) and CD8⁺CD69⁺ (▪) cell populations in spleen. Figure 4A presents data and mean values for each subject in all experimental groups. Figure 4B presents histogram image of representative CD4⁺CD69⁺ and CD8⁺CD69⁺ populations after CD3 T-lymphocyte gate acquisition. * indicates significant difference on CD8⁺CD69⁺ values between TAA and TAA + LXA4 groups. # indicates significant difference on CD4⁺CD69⁺ and CD8⁺CD69⁺ values between Naïve and TAA groups.

Figure 5.

Distribution of CD4⁺CD127⁺ (•) and CD8⁺CD127⁺ (▪) cell populations in spleen. Figure 5A presents data and mean values for each subject in all experimental groups. Figure 4B presents histogram image of representative CD4⁺CD69⁺ and CD8⁺CD69⁺ populations after CD3 T-lymphocyte gate acquisition.

Figure 6.

DNA samples after qRT-PCR and visualization of those PCR products at gel electrophoresis (at 2% gel electrophoresis, 45 minutes, 90 V running conditions (A). First and ninth lines 100bp DNA marker (GelPilot, QiaGen); second line is for IFN-γ, third line is for TNF-α; fourth line is for IL-6; fifth line is for IL-10; sixth line is for CXCL10; seventh line is for CXCR3, and eighth line is for GAPDH. qRT-PCR gene expression levels of chemokine (B) and cytokine (C) genes in liver tissues. The data represent the mean ± SD of 2^{(Avg − Δ(ΔCt))}values in 2 separate experiments. In figure 6A, * and # indicate significant differences of CXCR3 and CXCL10 between TAA and TAA+LXA4 groups, respectively. In figure 6B, *, #, and ŧ indicate significant differences of IFN-γ , TNF-α, and IL-6 between TAA and TAA + LXA4 groups, respectively.