Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression

Abstract

Background: Biliary atresia is characterized by extrahepatic bile duct obliteration along with persistent intrahepatic portal inflammation. Steroids are standard in the treatment of cholangitis following the Kasai portoenterostomy, and were advocated for continued suppression of the ongoing immunologic attack against intrahepatic ducts. Recent reports, however, have failed to demonstrate an improved patient outcome or difference in the need for liver transplant in postoperative patients treated with a variety of steroid regimes compared with historic controls. In the wake of progressive liver disease despite biliary decompression, steroids are hypothesized to suppress inflammation and promote bile flow without any supporting data regarding their effect on the emerging cellular and molecular mechanisms of liver repair. We have previously shown in a reversible model of cholestatic injury that repair is mediated by macrophages, neutrophils, and specific matrix metalloproteinase activity (MMP8); we questioned whether steroids would alter these intrinsic mechanisms.

Methods: Rats underwent biliary ductal suspension for 7 d, followed by decompression. Rats were treated with IV dexamethasone or saline at the time of decompression. Liver tissue obtained at the time of decompression or after 2 d of repair was processed for morphometric analysis, immunohistochemistry, and quantitative RT-PCR.

Results: There was a dramatic effect of dexamethasone on the inflammatory component with the initiation of repair. Immunohistochemistry revealed a reduction of both ED1+ hepatic macrophages and ED2+Kupffer cells in repair compared with saline controls. Dexamethasone treatment also reduced infiltrating neutrophils by day 2. TNF-alpha expression, increased during injury in both saline and dexamethasone groups, was markedly reduced by dexamethasone during repair (day 2) whereas IL-6, IL-10, and CINC-1 remained unchanged compared with saline controls. Dexamethasone reduced both MMP8 and TIMP1 expression by day 2, whereas MMP9, 13, and 14 were unchanged compared with sham controls. Despite substantial cellular and molecular changes during repair, collagen resorption was the same in both groups

Conclusion: Dexamethasone has clear effects on both the hepatic macrophage populations and infiltrating neutrophils following biliary decompression. Altered MMP and TIMP gene expression might suggest that steroids have the potential to modify matrix metabolism during repair. Nevertheless, successful resorption of collagen fibrosis proceeded presumably through other MMP activating mechanisms. We conclude that steroids do not impede the rapid intrinsic repair mechanisms of matrix degradation required for successful repair.

Figure 1. Experimental design

Biliary suspension surgery was performed and cholestatic injury occurred over the following 7 days. Saline (n=12) or Dexamethasone (n=16) (7.2μg/100g per rat) was administered IV on day 7 of injury at bile duct decompression and on day 2 of repair prior to necropsy. The day of decompression is defined here as day 0. Rats were sacrificed 6 hours following injections at day 0 and at 2 days of repair for each group

Figure 2. Inflammatory cells of the liver

ED1+ (A), ED2+ (B) and PMN (C) stained liver sections derived from sham, injured (day 0) and repairing (day 2) animals treated with saline or Dexamethasone were digitally imaged and counted. The number of ED1+, ED2+ cells and PMN following cholestatic injury are significantly elevated when compared to saline sham controls. 2A and 2B * indicates significant (p≤0.05) increase in cell number over sham controls and Dexamethasone treatment at day 0 compared to saline controls. 2C * indicates significant (p≤0.05) increase in cell number over sham controls. 2A, 2B and 2C ** indicates significant decrease in cell number in Dexamethasone-treated animals compared to saline controls (p≤0.05). Declines in ED1+ and ED2+ cells following decompression are in contrast with PMN cell counts that remain elevated in saline controls suggesting a role for the persistence of neutrophils during repair.

Figure 3. Quantitative real time RT-PCR

TNF-α (A), IL-6 (B), IL-10 (C), CINC-1 (D) mRNA expression was quantified in liver homogenates derived from sham, injured and repairing animals following saline or Dexamethasone treatment. Data are relative to 18S expression and are given as fold increase over saline sham. 3A * indicates a significant (p≤0.05) decrease in TNF-α expression in Dexamethasone-treated animals at day 2 compared to saline controls 3D * indicates a significant (p≤0.05) increase in CINC-1 expression in injured animals at day 0 compared to sham controls.

Figure 4. Quantitative real time RT-PCR

MMP-8 (A), MMP-2 (B), MMP-9 (C), MMP-13 (D) mRNA expression was quantified in liver homogenates derived from sham, injured and repairing animals following saline or Dexamethasone treatment. Data are relative to 18S expression and are given as fold increase over saline sham. 3A * indicates a significant (p≤0.05) increase in MMP-8 expression in injured animals at day 0 compared to sham controls whereas ** indicates significant (p≤0.05) decrease in expression in Dexamethasone-treated animals at day 2 of repair compared to saline controls.

Figure 5. Quantitative real time RT-PCR

TIMP-1 (A), TIMP-2 (B) mRNA expression was quantified in liver homogenates derived from sham, injured and repairing animals following saline or Dexamethasone treatment. Data are relative to 18S expression and are given as fold increase over saline sham. 5A and 5B * indicates significant increase in TIMP gene expression (p≤0.05) in the injured animals at day 0 compared to sham controls. 5A ** indicates a significant (p≤0.05) decrease in TIMP-1 expression in the Dexamethasone-treated animals compared to saline controls.

Figure 6. Fibrotic Repair

Collagen is expressed as a percentage of total area of Sirius red stained liver sections derived from injured and repairing animals treated with either saline or Dexamethasone. Biliary obstruction * significantly (p≤0.05) increased the deposition of collagen following 7 days of injury compared to sham operated controls. Collagen content, as measured by Sirius red, was significantly (p≤0.05) decreased in the saline-treated animals ** following decompression (day 2). Dexamethasone-treatment did not block the decompression related decline in collagen despite changes in the inflammatory cellular response. Measures of collagen content were not different from injured animals and significantly (p≤0.05) elevated when compared to saline treated matched controls.