Recombinant adeno-associated virus-mediated inhibition of microRNA-21 protects mice against the lethal schistosome infection by repressing both IL-13 and transforming growth factor beta 1 pathways

Abstract

Schistosomiasis is a serious parasitic disease in humans, which can lead to liver fibrosis and death. Accumulating evidence indicated that targeting the deregulated microRNAs (miRNAs) could mitigate disease outcomes. Here, we showed that progressive hepatic schistosomiasis caused elevation of miR-21 and efficient and sustained inhibition of miR-21 by using highly hepatic tropic adeno-associated virus serotype 8 (rAAV8), which protected mice against lethal schistosome infection through attenuation of hepatic fibrosis (HF). We demonstrated an additive role of interleukin (IL)-13 and transforming growth factor beta 1 (TGF-β1) in up-regulating miR-21 expression in hepatic stellate cells (HSCs) by activation of mothers against decapentaplegic (SMAD) proteins. Furthermore, down-regulation of miR-21 in HSCs reversed HF by enhancing SMAD7 expression, thus repressing TGF-β1/Smad and IL-13/Smad pathways.

Conclusion: This study suggests the mechanism of IL-13-mediated schistosomiasis HF by up-regulation of miR-21 and highlights the potential of rAAV8-mediated miR-21 inhibition as a therapeutic intervention for hepatic fibrotic diseases, such as schistosomiasis.

Fig. 1

Down-regulation of miR-21 protected mice from lethal schistosome infection. (A) Time schedule for parasite infection and intravenous injections of various virus constructs or PBS. Mice were infected with 30 S. japonicum cercariae at day 0 and treated with various vectors at a dose of 1×10¹² virus genomes, or PBS at day 10 post-infection. The animals were subjected to an 80-day survival study. (B) Kaplan Meier Survival curves were plotted for all groups as indicated.

Fig. 2

Down-regulation of miR-21 attenuated schistosomiasis-induced hepatic fibrosis in mice. (A) Time schedule for parasite infection and intravenous injections of virus constructs or PBS and sample withdrawal. Mice were infected with 16 S. japonicum cercariae at day 0 or remained uninfected. Infected mice received various vectors at a dose of 6×10¹¹ virus genomes or PBS at day 10 post-infection. Liver samples were collected at the indicated time points. (B) RT-PCR analysis of expression levels of miR-21 in the liver samples. (C) Northern blots analysis of miR-21, miR-122, and U6 in total RNA from liver of mice after 56-day infection. Three or four biological replicates are shown. (D) Collagen content of the livers determined as hydroxyproline content. (E) Masson's trichrome staining of collagen in the liver sections. Scale bar, 200 μm. (F) Fibrosis scores measured from Masson's trichrome staining liver sections.

Fig. 3

Down-regulation of miR-21 partially reversed schistosomiasis-mediated hepatic fibrosis. (A) Time schedule for parasite infection and intravenous injections of virus constructs or PBS and sample withdrawal. Mice were infected with 25 S. japonicum cercariae or remained uninfected. The infected mice were treated with praziquantel to kill the parasites, and then received either the different vectors at a dose of 1×10¹² virus genomes or PBS at day 42 post-infection. Liver and serum samples of the mice were collected at day 70 post-infection. (B) RT-PCR analysis of expression levels of miR-21 in the liver samples. (C, D) Collagen content of the livers was measured as (C) hydroxyproline content and (D) fibrosis score determined from Masson's trichrome staining liver sections (Supporting Fig. 6A). (E) Mean area of granuloma measured from Mayer's H&E staining liver sections (Supporting Fig. 6A) using a calibrated measuring eyepiece. (F) Levels of serum ALT.

Fig. 4

MiR-21 was primarily located in the activated HSCs. (A) In situ hybridization of miR-21 in liver sections from infected mice after 56-day infection. Scale bar, 200 μm. (B) RT-PCR analysis of miR-21 expression levels in HSCs, hepatocytes, and Kupffer cells isolated from livers of three to five uninfected or infected mice after 56-day infection. (C) Hepatocytes, Kupffer cells (KC), HSCs were isolated from livers of uninfected or infected mice (56 days post-infection), and relative miR-21 expression in comparison with hepatocytes of uninfected mice was determined by qPCR.

Fig. 5

The additive role of the TGF-β1 and IL-13 in the schistosomiasis hepatic fibrosis through up-regulation of miR-21expression. (A, B) RT-PCR analysis of expression levels of TGF-β1 (A) and IL-13 (B) in the infected livers from the 4 groups described in Fig. 2A. (C, D) MiR-21 or Col1α1 expression levels in HSCs after TGF-β1 or IL-13 stimulation. Primary mouse HSCs were treated at day 3 after seeding with 0, 5, 25, 50, 100 ng/mL TGF-β1 or IL-13 for 24 hours. Total RNA was extracted for RT-PCR analysis. (E) Primary mouse HSCs were treated at day 3 after seeding with 25 ng/mL TGF-β1, 50 ng/mL IL-13 or the mixture of 25 ng/mL TGF-β1 and 50 ng/mL IL-13 for 24 hours, respectively. Total RNA was extracted for RT-PCR analysis of miR-21 expression levels.

Fig. 6

The TGF-β1- and IL-13-mediated up-regulation of miR-21 involves the SMAD pathway. (A) Phosphorylation-mediated activations of SMAD proteins were determined by western blot for HSCs isolated from the livers of three to five uninfected or infected mice at various time points. (B) Primary mouse HSCs were treated at day 3 after seeding with 25 ng/mL TGF-β1 or 50 ng/mL IL-13 for 24 hours. Total protein was extracted and analyzed for phospho-SMAD proteins by western blot. (C, D) HSCs were isolated from the livers of three to five mice from the four groups aforementioned after 56-day infection, respectively. Total RNA was extracted for RT-PCR analysis of (C) miR-21 and (D) SMAD7 expression levels. (E, F) Primary mouse HSCs were transfected with either EGFP or SMAD7 plasmids at day 2 after seeding and then treated with 25 ng/mL TGF-β1 or 50 ng/mL IL-13 at day 3 for 24 hours. Total RNA was extracted for RT-PCR analysis of (E) miR-21 and (F) Col1α1 expression levels.