Evaluation of an anti-tumor necrosis factor therapeutic in a mouse model of Niemann-Pick C liver disease

Abstract

Background: Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease.

Methodology/principal findings: Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown.

Conclusions/significance: Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.

Figure 1. Effect of NPC1 knockdown and anti-TNF treatment on NPC1 protein levels, whole body weight, liver weight, and hepatic cholesterol content.

A: Expression of NPC1 in livers of mice injected for 9 weeks with mismatched (MM) and NPC1-specific ASOs and subjected to treatment (TX) with saline or anti-TNF. β-Actin was used as a loading control. Whole body weight (B), relative liver weight (C) and hepatic unesterified cholesterol content (D) in mice injected with mismatched (MM) and NPC1-specific ASOs and subjected to treatment (TX) with saline or anti-TNF. Each bar represents the mean ± SD of 5 animals in each treatment group. The lettering (a, b) shows statistically dissimilar groups. Samples with two letters represent values that are intermediate to the statistical groups and are thus not considered significantly different from either group.

Figure 2. Hepatic formation and clustering of foamy macrophages in NPC1 knockdown mice.

Masson's trichrome stained liver sections from mice injected for 9 weeks with mismatched (MM) and NPC1-specific ASOs and subjected to treatment with saline or anti-TNF. Images were photographed at 200X magnification.

Figure 3. Effect of NPC1 knockdown and anti-TNF treatment on the number of foamy macrophages and hepatocytes.

Quantification of foamy macrophages (A) and hepatocytes (B) in H&E stained liver sections from mice injected for 9 weeks with mismatched (MM) and NPC1-specific ASOs and subjected to treatment (TX) with saline or anti-TNF. Each bar represents the mean ± SD number of cells per field from 3 animals in each treatment group. The lettering (a, b) shows statistically dissimilar groups.

Figure 4. Effect of NPC1 knockdown and anti-TNF treatment on hepatic apoptosis.

Liver sections from mice injected for 9 weeks with mismatched (MM) and NPC1-specific ASOs and subjected to treatment (TX) with saline or anti-TNF were subjected to fluorometric TUNEL staining. Apoptotic nuclei were quantified. Each bar represents the mean ± SD number of cells per field from 5 animals in each treatment group. The lettering (a, b) shows statistically dissimilar groups. Samples with two letters represent values that are intermediate to the statistical groups and are thus not considered significantly different from either group.

Figure 5. Stellate cell activation after NPC1 knockdown and anti-TNF treatment.

Liver sections from mice injected for 9 weeks with mismatched (MM) and NPC1-specific ASOs and subjected to treatment with saline or anti-TNF were subjected to α-smooth muscle actin immunohistochemistry (A). Immunohistochemical reaction product was quantified (B). Each bar represents the mean± SD relative area of brown reaction product per field from 5 animals in each treatment group. The lettering (a, b) shows statistically dissimilar groups. Samples with two letters represent values that are intermediate to the statistical groups and are thus not considered significantly different from either group.