220. AAV-Mediated miR122 Overexpression Rescues Hepatic Inflammatory Response Induced by Acute Iron-Overload

Abstract

Frequent blood transfusions among patients with thalassaemia and sickle cell disease can lead to acute iron overload (IO), also known as transfusional haemosiderosis. Excessive iron is initially distributed to the liver, the primary organ for iron recycling and storage, and then gradually leads to damage of other major organs such as the heart and endocrine organs. IO frequently increases hepatic inflammatory infiltration and expression of pro-inflammatory cytokines. Recently, hepcidin, a well-known iron-regulatory hormone, and iron-catalyzed reactive oxygen species were proven to be independent of the IO-mediated inflammation. These findings triggered us to further pursue the potential underlying molecular mechanisms.In our preliminary studies, we observed that IO significantly reduced the expression of liver-specific microRNA, miR122, through the inhibition of a nuclear receptor, hepatocyte nuclear factor 4 alpha (HNF4α), but not other regulators of miR122. Furthermore, we demonstrated that the chemokine (C-C motif) ligand 2 (CCL2), which recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation, is negatively regulated by miR122. Thus, we hypothesized that overexpression of hepatic miR-122 may relieve the inflammatory responses to the acute IO. To this end, a recombinant adeno-associated virus serotype 8 (rAAV8) vector was generated, in which a miR-122 expression cassette was embedded in the intron region between a Guassia Luciferase (GLuc) transgene and a chicken β-actin promoter. Another rAAV8 vector, which has the exact GLuc transgene cassette but without miR-122, was used as a negative control. C57BL/6 mice were intra-peritoneal challenged with iron-dextran at 200mg/kg mouse weight or PBS as a control, at Day 0 and Day 8. The IO group was sub-divided into three sub-groups, in which PBS, rAAV8-GLuc, or rAAV8-miR122-GLuc vectors were tail-vein injected at 10^11 viral genome/mouse, respectively, at Day 6. All mice were sacrificed at Week 4. It was evident that following systemic administration, rAAV8-miR122-GLuc vectors led to a 12-fold increase in the expression of miR122. On the other hand, only a modest increase (<2-fold) in the HNF4α expression was observed. Interestingly, although in vivo overexpression of miR-122 had no effect on serum iron indices or hepatic expression of iron-related molecules, it significantly reduced the expression of several inflammatory genes, such as IL-6 and IL-1β, in the IO mice. Western blot analysis also revealed that overexpression of miR-122 resulted in a significant reduction of CCL2 and NF-kB proteins in the IO mice. Consequently, both the hepatic inflammation scores and serum activities of AST and ALT in the IO mice were significantly reduced upon miR-122 overexpression.In sum, our model (Figure 1Figure 1) and studies should be important and informative for elucidating the mechanism of IO-induced hepatic inflammation and for developing clinical strategies to prevent them in the patients with major haemoglobinopathy.View Large Image | Download PowerPoint Slide