Gadolinium Chloride Inhibits the Production of Liver Interleukin-27 and Mitigates Liver Injury in the CLP Mouse Model

Abstract

Background: Liver macrophages play an important regulatory role in the inflammatory response of liver injury after severe infection. Interleukin- (IL-) 27 is an inflammatory cytokine that plays an important role in diseases caused by bacterial infection. However, the relationship between IL-27 and liver macrophages in liver injury after severe infection is not yet clear.

Methods: A cecal ligation puncture (CLP) model was established in wild-type (WT) and IL-27 receptor- (WSX-1-) deficient (IL-27r^-/-) mice, and recombinant IL-27 and gadolinium chloride (GdCl3) were injected into WT mice in the designated groups. The serum and liver IL-27, IL-6, tumor necrosis factor alpha (TNF-α), and IL-1β expression levels were evaluated by ELISA, quantitative PCR, or Western blotting; serum ALT and AST were detected by detection kits; and the severity of liver damage was evaluated by hematoxylin and eosin staining and the TUNEL assay of the liver tissue from the different groups. Liver macrophage polarization was evaluated by immunofluorescence. In addition, the polarization of peritoneal macrophage was evaluated by flow cytometry.

Results: The serum and liver IL-27 expression levels were elevated in WT mice after CLP-induced severe infection, which were consistent with the changes in HE scores in the liver tissue. The levels of serum ALT, AST, liver IL-6, TNF-α, and IL-1β mRNA and liver pathological injury scores were further increased when pretreated with recombinant IL-27 in WT mice, but these levels were decreased in IL-27r^-/- mice after CLP-induced severe infection compared to WT mice. In WT mice pretreated with GdCl3, liver pathological scores, serum ALT and AST, TUNEL-positive cell proportion from liver tissues, liver IL-27 expression, and the liver macrophages M1 polarization proportion decreased after CLP; however, the serum IL-27, IL-6, TNF-α, and IL-1β levels and the pathological lung and kidney scores were not significantly changed. When supplemented with exogenous IL-27, the liver pathological scores, serum ALT, AST, TUNEL-positive cell proportion of liver tissues, liver IL-27 expression, and the liver macrophage M1 polarization proportion increased. The in vitro, IL-27 expression increased in peritoneal macrophages when stimulated with LPS. Recombinant IL-27 together with LPS promoted the elevations in IL-6, TNF-α, and IL-1β levels in supernatant and the M1 polarization of peritoneal macrophages.

Conclusion: IL-27 is an important cytokine in the inflammatory response to liver injury after severe infection. The reduction of liver injury by gadolinium chloride in severe infection mice models may relate to the inhibition of liver IL-27 production. These changes may be mainly related to the decrease of liver macrophages M1 polarization. IL-27 may have a positive feedback on these macrophages.

Figure 1

(a) Serum IL-27 levels of sham WT mice (Sham) and WT mice after CLP (CLP) (by ELISA), ^∗∗∗p < 0.001. (b) EBI3 and P28 mRNA levels in the liver tissue of sham WT mice (Sham) and WT mice after CLP (CLP) (by qPCR), ^∗∗∗p < 0.001. (c) EBI3 and P28 protein levels in the liver tissue of sham WT mice (Sham) and WT mice after CLP (CLP) (by Western blot), ^∗∗∗p < 0.001. (d) Hematoxylin and eosin- (H&E-) stained liver tissues and histological scores for the liver from WT, WT+recombinant IL-27 (R IL-27) pretreatment, and IL-27r^−/− mice (n = 5 per group) (sham and after CLP) (×200, ×400 magnifications), ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs sham group; NS: p > 0.05, #p < 0.05, ##p < 0.01. (e) IL-6, TNF-α, and IL-1β mRNA levels in the liver tissue (by qPCR) of WT, WT+recombinant IL-27 (R IL-27) pretreatment, and IL-27r^−/− mice (sham and after CLP), ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs sham group; NS: p > 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001. (f) Serum ALT and AST levels of WT, WT+recombinant IL-27 (R IL-27) pretreatment, and IL-27r^−/− mice (sham and after CLP). ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs sham group; NS: p > 0.05, ###p < 0.001.

Figure 2

(a) Hematoxylin and eosin- (H&E-) stained liver tissues and histological scores for liver from WT mice (n = 5 per group) with or without GdCl3 and recombinant IL-27 (R IL-27) pretreatment (sham and after CLP), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, NS: p > 0.05. (b) Serum ALT and AST levels of WT mice after CLP with or without GdCl3 and recombinant IL-27 (R IL-27) pretreatment, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (c) DNA fragmentation analysis (TUNEL) and TUNEL-positive cell proportion of the liver from WT mice (n = 5 per group) after CLP with or without GdCl3 and recombinant IL-27 (R IL-27) pretreatment (×200, ×400 magnifications), ^∗p < 0.05, ^∗∗p < 0.01. (d) EBI3 and P28 protein levels in the liver tissue of WT mice after CLP with or without GdCl3 pretreatment. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

(a) Serum IL-27, IL-6, TNF-α, and IL-1β levels (by ELISA) of WT mice after CLP with or without GdCl3 pretreatment, NS:p > 0.05. (b) Hematoxylin and eosin- (H&E-) stained lung and kidney tissues and histological scores from sham WT mice (Sham) and WT mice after CLP (n = 5 per group) with or without GdCl3 pretreatment (×200, ×400 magnifications), ^∗∗p < 0.01, NS: p > 0.05. (c) M1 and M2 polarization numbers of liver macrophages in sham WT mice (sham) and WT mice after CLP (n = 5 per group) with or without GdCl3 and recombinant IL-27 (R IL-27) pretreatment (by immunofluorescence) (×200, ×800 magnifications). Blue fluorescence stands for DAPI, red fluorescence stands for F4/80, green fluorescence stands for CD206, and pink fluorescence stands for iNOS. The F4/80 + iNOS image showed only the M1 type macrophages (cells with red and pink color), while the F4/80 + CD206 image showed only the M2 type macrophages (cells with red and green color), and the merged image showed both the M1 type and M2 type of macrophage. ① stands for M1 type macrophages, ② stands for M2 type macrophages, and ③ stands for macrophages that are not polarized to M1 or M2 type. ^∗p < 0.05, ^∗∗p < 0.01, NS: p > 0.05. (d) The IL-27p28 expression in liver macrophages after CLP (n = 5 per group) with or without GdCl3 pretreatment (by immunofluorescence) (confocal microscopy ×200, ×600 magnifications). Blue fluorescence stands for DAPI, red fluorescence stands for F4/80, and green fluorescence stands for IL-27p28. ^∗p < 0.05. (e) EBI3 and P28 mRNA levels of liver macrophages in WT-CLP and WT-CLP + GdCl3 mice groups (by qPCR), ^∗p < 0.05.

Figure 4

(a) EBI3 and P28 protein and mRNA levels of peritoneal macrophages stimulated by LPS(100 ng/ml) (by western-blot and qPCR), ^∗∗∗p < 0.001 vs control. (b) IL-6, TNF-α, and IL-1β levels in cell supernatant after LPS stimulated, with or without recombinant IL-27 (R IL-27) (by ELISA), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (c) M1 and M2 polarization of peritoneal macrophages after LPS stimulated, with or without recombinant IL-27 (R IL-27) (by flow cytometry). ^∗∗p < 0.01, ^∗∗∗p < 0.001.