Long-Term Liver Expression of an Apolipoprotein A-I Mimetic Peptide Attenuates Interferon-Alpha-Induced Inflammation and Promotes Antiviral Activity

Abstract

Apolipoprotein A-I mimetic peptides are amphipathic alpha-helix peptides that display similar functions to apolipoprotein A-I. Preclinical and clinical studies have demonstrated the safety and efficacy of apolipoprotein A-I mimetic peptides in multiple indications associated with inflammatory processes. In this study, we evaluated the effect of the long-term expression of L37pA in the liver by an adeno-associated virus (AAV-L37pA) on the expression of an adeno-associated virus encoding interferon-alpha (AAV-IFNα). Long-term IFNα expression in the liver leads to lethal hematological toxicity one month after AAV administration. Concomitant administration of AAV-L37pA prevented the lethal toxicity since the IFNα expression was reduced one month after AAV administration. To identify the mechanism of action of L37pA, a genomic and proteomic analysis was performed 15 days after AAV administration when a similar level of IFNα and interferon-stimulated genes were observed in mice treated with AAV-IFNα alone and in mice treated with AAV-IFNα and AAV-L37pA. The coexpression of the apolipoprotein A-I mimetic peptide L37pA with IFNα modulated the gene expression program of IFNα, inducing a significant reduction in inflammatory pathways affecting pathogen-associated molecular patterns receptor, dendritic cells, NK cells and Th1 immune response. The proteomic analysis confirmed the impact of the L37pA activity on several inflammatory pathways and indicated an activation of LXR/RXR and PPPARα/γ nuclear receptors. Thus, long-term expression of L37pA induces an anti-inflammatory effect in the liver that allows silencing of IFNα expression mediated by an adeno-associated virus.

Keywords: adeno-associated virus; antiviral; apolipoprotein A-I mimetic peptide; liver; type I Interferon.

Figure 1

Coadministration of AAV-L37 avoids the lethal toxicity induced by AAV-IFNα. C57BL/6 mice (N = 6) were left untreated (Mock) or received 1.2x10¹¹ vg/mouse of AAV-IFNα alone (AAV-IFNα) or combined with 9.5x10¹¹ vg/mouse AAV-L37pA and the body weight (A) and survival (B) was followed up for 120 days. At day 30, 60, and 120, serum levels of IFNα were determined by ELISA (C) and complete blood counts were analyzed with a Hemavet Hematology Analyzer. (D) WBC, white blood cells; RBC, red blood cells; PLT, platelets; HCT, hematocrit. Mean ± SEM is represented. **p < 0.01 ***p < 0.001 ****p < 0.0001. Body weight was analyzed by Extra Sum-of-Square F test after non-linear regression with a third order polynomial least square fit. Survival was analyzed by Log-Rank test. IFNα concentrations were analyzed by nested one-way ANOVA followed by Dunnett’s multiple comparisons test. **** indicate p < 0.0001 for AAV-IFNα compared to the other two experimental groups. Red blood counts were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 2

Comparable AAV infection and expression IFN related genes in the liver two weeks after AAV administration. C57BL/6 mice (N = 3) were left untreated (Mock) or received 1.2x10¹¹ vg/mouse of AAV-IFNα alone (AAV-IFNα) or combined with 9.5x10¹¹ vg/mouse AAV-L37pA. Two weeks later, mice were sacrificed and DNA, RNA and proteins from livers were purified. (A) Schematic representation of the experimental setting. (B) AAV viral genome was quantified by quantitative PCR of total DNA. (C) The expression of the AAV transgene mIFNα and the interferon-stimulated genes, interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and 2’-5’-oligoadenylate synthetase (2,5-OAS) were determined by RT-qPCR. Mean ± SEM is represented. n.s, non-significant; ***p < 0.001 One-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 3

Coadministration of AAV-IFNα and AAV-L37pA modify the gene expression program modulated by AAV-IFNα in the liver. mRNA was isolated from the liver as described in Figure 2A and differentially expressed genes were analyzed by microarray analysis. (A) Volcano plot of differentially expressed genes in the liver of mice untreated (Mock), treated with AAV-IFNα or AAV-IFNα and AAV-L37pA. (B) Top significantly affected pathways based on Ingenuity pathway analysis (IPA). The horizontal bars denote the different pathways based on the z-score. Orange color indicates upregulation, while blue color indicates downregulation. (C) Heat map representing the relative expression of most differentially expressed genes. (D) Heat map representing the relative expression of genes contributing to the pathways with a negative Z-score. (E) Heat map representing the relative expression of genes contributing to the pathways with a positive Z-score. (F) Validation of relevant differentially expressed genes by RT-qPCR. Mean ± SEM is represented. *p < 0.05, **p < 0.001, ***p < 0.001 One-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 4

Long-term expression of L37pA modifies the AAV-IFNα effect in the liver at the protein level. Total proteins were isolated from the liver as described in Figure 2A and proteomic analysis was performed. (A) Volcano plot of proteins different levels in the liver of mice untreated (Mock), treated with AAV-IFNα or AAV-IFNα and AAV-L37pA. (B) Top significantly affected pathways based on Ingenuity pathway analysis (IPA). The horizontal bars denote the different pathways based on the z-score. Orange color indicates upregulation, while blue color indicates downregulation. (C) Heat map representing the relative protein levels contributing to the top significantly affected pathways. (D) Top significantly affected upstream regulators based on Ingenuity pathway analysis (IPA). The horizontal bars denote the different pathways based on the Z-score. Orange color indicates upregulation, while blue color indicates downregulation. (E) Heat map representing the relative protein levels contributing to the top significantly affected upstream regulators.

Figure 5

Long-term expression of AAV-IFNα and AAV-L37pA does not induce alterations in the liver. C57BL/6 mice (N = 6) were left untreated (mock) or received 1.2x10¹¹ vg/mouse of AAV-IFNα combined with 9.5x10¹¹ vg/mouse AAV-L37pA. Mice were followed for 120 days and then sacrificed. (A) The histology of the liver was analyzed by hematoxylin and Eosin staining. (B) Aspartate transaminase (AST) serum levels. (C) Representative image of intrahepatic T lymphocytes identified by immunohistochemistry against CD3 and quantification of T lymphocytes in the liver. (D) Representative image of intrahepatic macrophages identified by immunohistochemistry against anti-F4/80 and quantification of macrophages in the liver. Mean ± SEM is represented. n.s, non-significant. Student t-test.

Figure 6

Long-term effect on lipid accumulation in the liver and on plasma lipoprotein profile. C57BL/6 mice (N = 6) were left untreated (mock) or received 1.2x10¹¹ vg/mouse of AAV-IFNα combined with 9.5x10¹¹ vg/mouse AAV-L37pA. Mice were followed for 120 days and then sacrificed. (A) Lipid accumulation in the liver was analyzed by Fat Red staining. Representative images. (B) Lipoprotein profile of mice after fast protein liquid chromatography followed by cholesterol determination in the different fractions.