Nearly 50 years ago, the discovery of interferon prompted the notion that host cells innately respond to viral invasion. Since that time, technological advances have allowed this response to be extensively characterized and dissected in vitro. However, these advances have only recently been applied to highly complex, in vivo biological systems. To this end, we exploited high-titer adenovirus (Ad) vectors to globally investigate the innate immune response to nonenveloped viral infection in vivo. Our results indicated a potent cellular transcriptome response shortly after infection, with global assessments revealing significant dysregulation in ϳ15% of the measured transcripts derived from Ad vector-transduced tissue. Bioinformatics-based transcriptome analysis revealed a complex innate response to Ad infection, with induction of proinflammatory responses (and suppression of metabolism and mitochondrial genes) akin to those observed when mice are challenged with lipopolysaccharide. Despite this commonality, there were many unique aspects of the Ad-dependent transcriptome response, including the upregulation of several RNA regulatory mechanisms and apoptosisrelated pathways, accompanied by the suppression of lysosomal and endocytic genes. Our results also implicated the Toll-like receptors (TLRs) in these responses, prompting specific investigations into this pathway. By using MyD88KO mice, our results confirmed that Ad-induced dysregulation of five functionally related gene clusters are significantly dependent on this TLR adaptor gene. MyD88 deficiency also resulted in significantly diminished, although not abolished, adaptive and acute-phase immune responses to Ad, confirming the transcriptome data, as well as specifically identifying MyD88 as a significant Ad immunity amplifier and regulator in vivo.For the past 100 years, a significant body of research has sought to understand the mammalian innate immune system, with recent efforts focusing on understanding its molecular workings (5). The discovery of a new family of "pathogen-sensing" genes, the Toll-like receptors (TLRs) and the deciphering of their role in mammalian innate immunity has been an especially robust area of recent investigation (2, 28). However, critical issues remain unanswered, chief among which is the intracellular innate response to viral infections in vivo. While recent studies have sought to investigate intracellular viral immunity and TLR involvement, almost all have relied on tissue culture systems, with little or no attention paid to in vivo models.
Excessive complement activation can result in extreme tissue damage and systemic inflammatory responses, similar to innate immune responses rapidly elicited after systemic adenovirus (Ad) injections. To determine if Ad interactions with the complement system impact upon Ad-induced innate immune responses, we injected Ad into complement-deficient, C3-knockout mice (C3-KO) or wild-type mice (WT) and quantitatively compared multiple anti-Ad innate immune responses in both strains of mice. In Ad-treated WT mice, we noted rapid increases in plasma KC levels (1 h post injection), followed by increases in IL-6, IFN-gamma, RANTES, IL-12(p40), IL-5, G-CSF, and GM-CSF and subsequently thrombocytopenia. Conversely, in Ad-treated C3-KO mice, many of these inflammatory responses were significantly blunted, including the avoidance of Ad-induced thrombocytopenia. Global liver transcriptome responses in Ad-treated WT mice were assessed by RT-PCR-validated gene array analysis and were found to be also significantly affected by the lack of complement activity in Ad-treated C3-KO mice. Finally, our results confirmed the ability of high dose Ads to transduce hepatocytes despite a lack of complement activity. In summary, Ad interactions with the mammalian complement system are significant and likely initiate and/or exacerbate many of the inflammatory responses noted after systemic Ad injections.
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