Adenovirus RIDalphabeta inhibits signaling from three proinflammatory ligands

Abstract

Adenoviruses (Ad) have a variety of immunoregulatory genes, many of which are clustered in a 3.5kB segment of DNA known as early region 3 (E3). Earlier observations showed that transgenic expression of Ad E3 in pancreatic islets prevented mononuclear cell infiltration in murine models of allogeneic rejection and autoimmune diabetes. Since chemokines direct immune cells to the site of insult, we were interested in determining whether chemokine production or activity might be altered by Ad E3. We studied this function mainly in a human astrocytoma cell line, U373. When U373 cells are activated by the pro-inflammatory molecule TNF-alpha, the increase in MCP-1, IL-8 and IP-10 transcripts is blocked by a recombinant Ad expressing the E3 genes under CMV promoter control. Subsequently, by using Ad vectors expressing individual E3 proteins separately, we were able to map this function to RID (for Receptor Internalization and Degradation). We showed that RID expression correlates with down-regulation of the cell surface expression of the TNF receptor 1 in several human cells including U373 cells. This observation provided the first mechanistic explanation for the inhibition of TNFalpha-induced chemokines by RID. We also analyzed the immunoregulatory activities of RID on lipopolysaccharide (LPS) and IL-1beta-mediated responses. Although both signaling pathways are strongly inhibited by RID, the chemokines upregulated by IL-1beta stimulation are only marginally inhibited. In addition, RID inhibits signaling induced by LPS without affecting the expression of the LPS receptor Toll-Like Receptor (TLR4), demonstrating that RID need not target degradation of the receptor to alter signal transduction. Taken together, our data demonstrate the inhibitory effect of RID on two additional cell surface receptor-mediated signaling pathways involved in inflammatory processes. The data suggest that RID has intracellular targets that impair signal transduction and chemokine expression without evidence of receptor down-regulation. Our results suggest that construction of Ad-vectors to express RID will improve the efficacy and reduce immunogenicity of such viral-based gene therapy protocols. These results also suggest that RID have potential therapeutic uses in inflammatory disease.