Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors

Abstract

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.

Fig 1. Hepatic expression of inflammatory genes in WT and HO-1 KO mice after scAAV9 vectors injection.

(A) The qPCR for Mcp-1 3 days post-1^st injection of scAAV9 vectors. (B) The qPCR for Mcp-1 3 days after scAAV9 re-administration. (C) The Luminex assay for MCP-1 protein 3 days after scAAV9 re-administration. (D) The qPCR for Tnfa 3 days post-1^st injection of scAAV9 vectors. (E) The qPCR for Tnfa 3 days after scAAV9 re-administration. (F) The Luminex assay for TNFα protein 3 days after scAAV9 re-administration. Values represent means ± SEM (n = 3-4/group). p<0.05 * vs. appropriate WT, † vs. appropriate untreated, $ vs. appropriate scAAV-empty.

Fig 2. Immunofluorescent staining of macrophages in the livers of WT and HO-1 KO mice.

Representative fluorescent images of F4/80 marker (green, left panel). Representative fluorescent images of CD11b marker (green, right panel). Scale bar = 100 μm.

Fig 3. Flow cytometric analysis of two populations of hepatic macrophages in WT and HO-1 KO mice after scAAV9 vectors injection.

(A) Gating strategy and representative dot plots demonstrating F4/80^lowCD11b^high (MDMs) and F4/80^highCD11b^low (KCs). Flow cytometric analysis of KCs (B) and MDMs (C) 3 days after first and 3 days after second administration of scAAV9 vectors. Values represent means ± SEM (n = 3–4 mice/group). *p < 0.05 vs. appropriate WT; † p < 0.05 vs. appropriate untreated; # p < 0.05 vs. AAV9-GFP after the 1^st administration.

Fig 4. Plasma neutralizing activity and transgene expression in liver macrophages after scAAV9-GFP transduction.

(A) Virus neutralization test in plasma samples collected from WT and HO-1 KO mice 3 days after the 1^st administration and 3 days after the 2^nd administration of scAAV9-GFP. Flow cytometric analysis of GFP-positive (GFP⁺) KCs (B) and MDMs (C) in the livers of WT and HO-1 KO mice 3 days after the 1^st administration and 3 days after the 2^nd administration of scAAV9-GFP. (D) The qPCR assessing the number of vector genomes in the livers of WT and HO-1 KO mice 3 days after the 1^st administration and 3 days after the 2^nd administration of scAAV9-GFP. Values represent means ± SEM (n = 3-4/group). *p < 0.05 vs. appropriate WT; † p < 0.05 vs. appropriate untreated; # p < 0.05 vs. AAV9-GFP after the 1^st administration.

Fig 5. Functional assessment of mouse KCs in culture.

(A) Phase contrast and fluorescence microscopy images of KCs fluorescently labelled with DiI-AcLDL after 1 hour and 16 hours of incubation. (B) Production of TNFα in the supernatant of KCs stimulated with LPS for 1 hour and 21 hours in FBS-free conditions. (C) The qPCR for Il10 after 24 hours stimulation with CpG ODN 1585. (D) Confocal microscopy images demonstrating internalization of scAAV9 particles by KCs 2 hours after addition of 10⁴ MOI of scAAV9-empty (left panel). * p < 0.05 vs. appropriate untreated control; † p < 0.05 vs. appropriate 1 hour treatment.

Fig 6. Expression of genes associated with TLR9 signaling in primary culture of KCs exposed to scAAV9 vectors.

The qPCR for (A) Tlr9, (B) Myd88, (C) Ifna, (D) Ifnb, (E) Tnfa and (F) Il10 at 2 and 6 hours after in vitro exposure of WT and HO-1 KO KCs to 10⁴ MOI of scAAV9-empty or scAAV9-GFP. * p < 0.05 vs. appropriate WT; $ p < 0.05 vs. untreated HO-1 KO.