A bioartificial liver device secreting interleukin-1 receptor antagonist for the treatment of hepatic failure in rats

Abstract

Background: Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial liver (BAL) device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL device for the treatment of FHF.

Materials and methods: We injected an adenoviral vector encoding human IL-1 receptor antagonist (AdIL-1Ra) into the liver of D-galactosamine (GalN) intoxicated rats via the portal vein. We also transfected primary rat hepatocytes and reversibly immortalized human hepatocytes (TTNT cells) with AdIL-1Ra, and incorporated these transfected hepatocytes into our flat-plate BAL device and evaluated their efficacy in our GalN-induced FHF rat model after 10 h of extracorporeal perfusion.

Results: Rats injected with AdIL-1Ra showed significant reductions in the plasma levels of hepatic enzymes. Primary rat hepatocytes transfected with AdIL-1Ra secreted IL-1Ra without losing their original synthetic function. Incorporating these cells into the BAL device and testing in a GalN-induced FHF rat model resulted in significant reductions in plasma IL-6 levels and significantly improved animal survival. Incorporating the AdIL-1Ra transfected TTNT cells in the BAL device and testing in the GalN-induced FHF rat model resulted in significantly reduced plasma IL-6 levels, and a trend toward improved survival was seen.

Conclusion: Hepatocytes producing IL-1Ra are a promising cell source for BAL devices in the treatment of GalN-induced FHF.

FIG. 1

Schematic diagram of the extracorporeal perfusion system, including the flat-plate bio-artificial liver device with an internal membrane oxygenator. (Color version of figure is available online.)

FIG. 2

Effect of human IL-1Ra gene delivery on blood parameters in the fulminant hepatic failure rat model. The levels of AST (A), ALT (B), human IL-1Ra (C), and rat IL-6 (D) 24 h after hepatic failure induction in rats receiving human AdIL-1Ra delivered gene or AdLacZ delivered gene (*P < 0.05). AST, aspartate aminotransferase; ALT, alanine aminotransferase; IL-1Ra, interleukin-1 receptor antagonist; IL-6, interleukin-6; AdIL-1Ra, adenoviral vector encoding human IL-1Ra gene; AdLacZ, adenoviral vector encoding LacZ gene. ND, not detected. Results are expressed as mean ± SD (n = 5).

FIG. 3

Human IL-1Ra gene transfer to primary rat hepatocytes. (A) The levels of human IL-1Ra produced from AdIL-1Ra transfected co-cultured rat hepatocytes. MOI was based on the hepatocyte number (*P < 0.0001 versus MOI 0, 1, 5, and 25). (B) Time course of human IL-1Ra production from co-cultured rat hepatocytes after MOI 125 transfection. (C) Normalized urea and (D) rat albumin synthesis from co-cultured rat hepatocytes without transfection (MOI 0) and with transfection (MOI 125). Results are presented as normalized to non-transfected co-cultures from the same day. IL-1Ra, interleukin-1 receptor antagonist; MOI, multiplicity of infection; AdIL-1Ra, adenoviral vector encoding human IL-1Ra gene. Results are expressed as mean ± SD (n = 3).

FIG. 4

Human IL-1Ra gene transfer to reverted TTNT cells. (A) Human IL-1Ra production from AdIL-1Ra transfected TTNT cells. *P < 0.0001 versus MOIs 0, 1, 5, and 25. (B) Time course of human IL-1Ra production after MOI 125 transfection. *P < 0.005 versus day 1. IL-1Ra, interleukin-1 receptor antagonist; MOI, multiplicity of infection; AdIL-1Ra, adenoviral vector encoding human IL-1Ra gene. Results are expressed as mean ± SD (n = 3).

FIG. 5

Human IL-1Ra levels at the inlet and outlet of bio-artificial liver device after 10 h of extracorporeal perfusion. *P < 0.05 versus the same group [no-cell rIL-1Ra(+), rat AdIL-1Ra(+) or TTNT AdIL-1Ra(+)] at the inlet, ^‡P < 0.05 versus rat AdIL-1Ra(+) and TTNT AdIL-1Ra(+) in the same group (inlet or outlet). Rat AdIL-1Ra(−), co-cultured rat hepatocytes with no transfection; no-cell rIL-1Ra(+), bioreactor without cells, but recombinant human IL-1Ra was continuously infused from a venous line; rat AdIL-1Ra(+), co-cultured rat hepatocytes transfected with adenoviral vector encoding human IL-1Ra; TTNT AdIL-1Ra(+), TTNT cells transfected with adenoviral vector encoding human IL-1Ra. IL-1Ra, interleukin-1 receptor antagonist. ND, not detected. Results are expressed as mean ± SD.

FIG. 6

Effect of bio-artificial liver treatment on hepatic enzymes in fulminant hepatic failure rats. AST (A) and ALT (B) levels 24 h after hepatitis induction. Rat AdIL-1Ra(−), co-cultured rat hepatocytes without transfection; no-cell rIL-1Ra(+), bioreactor without cells, but recombinant human IL-1Ra was continuously infused from a venous line; rat AdIL-1Ra(+), co-cultured rat hepatocytes transfected with adenoviral vector encoding human IL-1Ra; TTNT Ad(+), TTNT cells transfected with adenoviral vector encoding human IL-1Ra. IL-1Ra, interleukin-1 receptor antagonist. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

FIG. 7

Effects of bio-artificial liver treatment on the plasma level of IL-6 and the hepatic tissue level of IL-1β in fulminant hepatic failure rats. Levels of IL-6 in plasma 24 h after hepatitis induction (A) and IL-1β in hepatic tissue at the end of extracorporeal BAL perfusion (11 h after hepatitis induction) (B). *P < 0.01 versus no-cell group. **P < 0.05 versus no-cell group and rat AdIL-1Ra(+) group. IL-6, interleukin-6; IL-1, interleukin-1; Rat AdIL-1Ra(+), co-cultured rat hepatocytes transfected with adenoviral vector encoding human IL-1Ra; TTNT AdIL-1Ra(+), TTNT cells transfected with adenoviral vector encoding human IL-1Ra. IL-1Ra; interleukin-1 receptor antagonist. Results are expressed as mean ± SD.

FIG. 8

Effect of bio-artificial liver treatment on animal survival in fulminant hepatic failure rats. (A) Survival curves of no-cell, rat AdIL-Ra(−), no-cell rIL-1Ra(+), and rat AdIL-1Ra(+). (B) Survival curves of no-cell and TTNT AdIL-1Ra(+). Rat AdIL-1Ra(−), co-cultured rat hepatocytes with no transfection; no-cell rIL-1Ra(+), bioreactor without cells, but recombinant human IL-1Ra was continuously infused from a venous line; rat AdIL-1Ra(+), co-cultured rat hepatocytes transfected with adenoviral vector encoding human IL-1Ra; TTNT AdIL-1Ra(+), TTNT cells transfected with adenoviral vector encoding human IL-1Ra. IL-1Ra, interleukin-1 receptor antagonist; GalN, D-galactosamine; BAL, bio-artificial liver.