1C). Pharmacokinetic analysis after the administration of rIA confirmed these data, as recombinant IFNα (rIFNα) presented a sharp decay in mouse plasma levels while the concentration of rIA decreased slowly (Supporting Information Fig. 1D). Interestingly, we found that after hydrodynamic
administration of pIA, all circulating IA produced by the liver was incorporated into HDL particles (Supporting Information Fig. 3A,B) and that, as a consequence, the HDL fraction of plasma displayed antiviral activity. In contrast, in mice treated with pIFN, antiviral activity was only found in lipoprotein-depleted serum (Supporting Information Fig. 3C). After intravenous injection of rIA, only a minor fraction (10%) of this protein Abiraterone purchase was detected in isolated HDLs (Supporting Information Fig. 3D,E) Native ApoA-I has strong liver tropism.16 Thus, we reasoned that linkage of IFNα to ApoA-I
might result STI571 purchase in targeting IFNα to the liver. To test this hypothesis, we analyzed the distribution of IFNα by ELISA in different organs (liver, brain, lung, heart, kidney, and spleen) at 5 and 150 minutes following intravenous (IV) administration of 1.6 μg of rIA or rIFNα. At 5 minutes, IFNα immunoreactivity was mostly detected in kidney and spleen, whereas IA was predominantly accumulated in the liver at 150 minutes postinjection. In the case of rIFNα, the cytokine was barely detectable at this timepoint in all organs NADPH-cytochrome-c2 reductase examined (Fig. 1A,B). We then quantitated hepatic interferon-stimulated genes (ISGs) messenger RNA (mRNA) levels 24 hours after IV injection of 10,000 U of rIFNα or the same antiviral units
of purified HDLs containing IA (HDL-IA) or 24 hours after administration of 70,000 U of rIFN or rIA. ISGs activation was significantly greater when using HDL-IA (Fig. 1C) or rIA (Fig. 1D), suggesting preferential signaling to the liver when IFNα was linked to ApoA-I. Confirming these data, hepatic expression of ISGs at day 3 following injection of pIA or pIFN was higher with the former treatment (Fig. 1E). We also found that at day 3 after hydrodynamic injection of pIA or pALF, the expression of ISGs in the liver tended to be higher following pIA administration (for ubiquitin-specific peptidase 18 [USP18] differences reached statistical significance) (Fig. 1F) despite the fact that the serum concentration of IA was half that of ALF at this timepoint (Supporting Information Fig. 1C). Studies using L929 mouse fibroblasts incubated with rIFNα or the same antiviral units of HDL-IA or of rIA showed that the phosphorylation of STAT-1 and -2 was similar in both cases (Fig. 2A). However, the administration of 70,000 IU of rIA was able to protect 50% of the mice against a lethal challenge with EMCV, whereas 100% of mice treated with the same antiviral units of rIFNα succumbed (Fig. 2B).