Abstract

RationaleReplication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis.MethodsWe instilled Ad viruses ranging from 107 to 1.625×109 ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining.ResultsInstillation of high dose Ad vectors (1.625×109 ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites.ConclusionsOur results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner.

Highlights

  • Adenoviruses (Ad) are simple double stranded DNA viruses that are nonenveloped [1]

  • These results suggest that intratracheal instillation of high dose of Ad vectors induces a modest but sustained lung injury

  • It is known that Ad vectors activate innate and adaptive immunity and inflammation contributing to the pathogenesis of fibrosis, it is unclear whether intratracheal administration of Ad vectors is sufficient to induce lung injury and lung fibrosis

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Summary

Introduction

Among approximately 50 distinct serotypes, the group C (serotype 2 and 5) has been most extensively studied and has been used as vaccine vectors and gene therapy vectors [2]. Replication deficient Ad vectors can carry large DNA, are easy to produce in large quantities, and have broad cell tropism. These features make them ideal research tools to manipulate expression of candidate genes in cell culture and/or in small animals. [3,4,5], where inflammation contributes to the diseases. It is difficult to distinct the effect of the target gene from the effect of Ad vectormediated inflammation

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