Impact of combination antiretroviral therapy in the NOD.c3c4 mouse model of autoimmune biliary disease

Abstract

Background & aims: The NOD.c3c4 mouse model develops autoimmune biliary disease characterized by spontaneous granulomatous cholangitis, antimitochondrial antibodies and liver failure. This model for primary biliary cirrhosis (PBC) has evidence of biliary infection with mouse mammary tumour virus (MMTV), suggesting that the virus may have a role in cholangitis development and progression of liver disease in this mouse model. We tested the hypothesis that MMTV infection is associated with cholangitis in the NOD.c3c4 mouse model by investigating whether antiretroviral therapy impacts on viral levels and liver disease.

Methods: NOD.c3c4 mice were treated with combination antiretroviral therapy. Response to treatment was studied by measuring MMTV RNA in the liver, liver enzyme levels in serum and liver histology using a modified Ishak score.

Results: Combination therapy with the reverse transcriptase inhibitors, tenofovir and emtricitabine, resulted in a significant reduction in serum liver enzyme levels, attenuation of cholangitis and decreased MMTV levels in the livers of NOD.c3c4 mice. Furthermore, treatment with the retroviral protease inhibitors, lopinavir and ritonavir, in addition to the reverse transcriptase inhibitors, resulted in further decrease in MMTV levels and attenuation of liver disease in this model.

Conclusions: The attenuation of cholangitis with regimens containing the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the protease inhibitors, lopinavir and ritonavir, suggests that retroviral infection may play a role in the development of cholangitis in this model.

Keywords: NOD.c3c4; autoimmune biliary disease; combination antiretroviral therapy; mouse mammary tumour virus; primary biliary cirrhosis.

Figure 1

Serum alkaline phosphatase levels in NOD.c3c4 receiving no antiretroviral therapy. (A) Serial measurement of serum alkaline phosphatase in the NOD.c3c4 mice showing variance in individual levels as well as a reduction mean levels as mice aged, mainly from 8 to 12 weeks (P < 0.0001, 1 way anova). (B) Reduction in serum alkaline phosphatase levels from baseline levels between 8 and 12 weeks, showing diminished reduction in NOD.c3c4 vs. NOD.GFP (Data shown as means ± SEM, ***P < 0.001, t-test).

Figure 2

Histological features in NOD.c3c4 mice receiving no antiretroviral therapy. (A–C) Representative histological of images demonstrating total Ishak scores of (A) 0, (B) 3, and (C) 5 (Haematoxylin and Eosin, upper panel -100× magnification, lower panel - 300× magnification). (D) NOD.c3c4 mice have a variable penetrance of cholangitis and necroinflammation. As the mice age, a trend was observed for an increased variability in all histological scores as well as a gradual increase of necroinflammatory and total Ishak score in the older mice (Data shown as means ± SEM).

Figure 3

Histological improvement in the liver following 12 weeks of antiretroviral therapy. NOD.c3c4 mice treated with antiretroviral therapy showed significant reduction in necroinflammation (P < 0.001), cholangitis (P = 0.0001) and total Ishak Score (P < 0.0005, one way anova). Combinations that included Truvada with or without Kaletra had no evidence of cholangitis and combination regimens incorporating Kaletra demonstrated improved necroinflammation, cholangitis and total Ishak score [Combivir (zidovudine and lamivudine), Truvada (tenofovir and emtricitabine), Kaletra (lopinavir and ritonavir); data shown as means ± SEM, *P < 0.05, **P < 0.01 and ***P < 0.001 vs. placebo using Dunn's multiple comparison test].

Figure 4

Antiretroviral regimens associated with reduction in serum liver enzyme levels from baseline. (A) After 4 weeks of treatment, only mice receiving Truvada and Kaletra experienced a significant mean reduction in alkaline phosphatase levels as compared to placebo, whereas by the end of 12 weeks therapy mice treated with regimens containing Truvada with or without Kaletra experienced significant reduction. (B) A significant reduction in serum alanine transaminase levels was observed in mice receiving Truvada and Kaletra but not Combivir and Kaletra [Combivir (zidovudine and lamivudine), Truvada (tenofovir and emtricitabine), Kaletra (lopinavir and ritonavir); data shown as means ± SEM; *P < 0.01 vs. placebo by Dunn's multiple comparison test].

Figure 5

Antiretroviral therapy associated with reduced hepatic MMTV RNA and biliary epithelial cell-associated MMTV Gag expression. (A) Antiretroviral therapy modulated MMTV RNA levels in the NOD.c3c4 liver after 12 weeks therapy (P < 0.0005, 1 way anova). A significant reduction in mean hepatic MMTV RNA levels was seen with Truvada alone, whereas a trend for decreased viral load was observed with regimens containing Kaletra [Combivir (zidovudine and lamivudine), Truvada (tenofovir and emtricitabine), Kaletra (lopinavir and ritonavir); data shown as means ± SEM, *P < 0.01 vs. placebo by Dunn's multiple comparison test]. (B) Mice receiving placebo showed MMTV p27 Gag protein reactivity in biliary epithelial cells. This reactivity was attenuated in mice treated with Combivir and Kaletra. [Haematoxylin, 200× magnification with 400× in insets showing staining in biliary epithelial cells].

Figure 6

Variations in MMTV pol gene were observed after 12 weeks Combivir (zidovudine and lamivudine) therapy. Alignment of amino acid sequence 136–198 of MMTV Pol P03365.2 using ClustalW alignment (MacVector 11.1 software) showing the amino acid variations W150R, R176G, Y183H, M188V and L192P in five clones derived from two mice treated with Combivir (zidovudine and lamivudine) that were not observed in control mice on placebo. Variants G160S and D181N were observed in mice receiving placebo and antiretroviral therapy.