Molecular Immunology 43 (2006) 1020–1028 Induction of immunomodulator transcriptional responses by cholera toxin Atabak R. Royaee a , Rasha Hammamieh b , Chanaka Mendis b , Rina Das b , Marti Jett a,b , David C. H. Yang a,∗ a Department of Chemistry, Georgetown University, 654 Reiss Science Bldg, Washington, DC 20057, USA b Department of Molecular Pathology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA Received 30 March 2005 Available online 14 July 2005 Abstract Cholera toxin (CT) is the causative agent of cholera, binds to GM1 glycosphingolipids, induces the production of cellular cAMP and is also a very powerful mucosal adjuvant. Although the mechanism of the CT induction of cAMP production is well understood, molecular mechanisms of the adjuvanticity of cholera toxin are yet to be delineated. Here, we examined the interaction of CT with human lymphocytes and monocytes by analyzing the host transcriptional profiles using cDNA arrays. The time courses of the transcriptional activations and repressions of affected genes in lymphocytes and monocytes in response to cholera toxin were determined. CT induced the expression of IL-8 and MIP-1 early in the CT exposure. VEGF, TIMP1, HIF-1α, MMP11, hek 8, MCP1, IL-6, GCP 2, urokinase plasminogen activator, and TNF-α receptor were upregulated after 4 h CT treatment. These genes showed increased expression for 48 h. MRP-14, MRP-8A increased expression after 16h CT treatment. RT-PCR and real-time PCR using cDNA specific primers confirmed the CT induction and repression of selected genes. The results suggest that immunomodulatory genes were among the genes that were affected the most by CT, and induction of these genes may contribute to the CT adjuvanticity. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cholera toxin; Gene expression; Transcription; Adjuvant; Inflammation; cDNA arrays; cAMP; IL-8; VEGF; RT-PCR; Real time PCR 1. Introduction Cholera toxin (CT) is the major virulent factor of Vib- rio cholerae. CT belongs to AB toxins. The CT subunit A catalyzes ADP-ribosylation of the  subunit of Gs protein while the pentameric CT subunit B binds to the cell sur- face GM1 glycosphingolipid receptors. ADP-ribosylated Gs continuously activates adenylate cyclase and results the accu- mulation of cellular cAMP, consequently, the salt imbalance and drastic loss of water. CT has been used extensively as an adenylate cyclase acti- vator to demonstrate the cellular action of cAMP. Regulation of gene expression at the transcriptional level by cAMP is well known. CT has also been recognized as one of the ∗ Corresponding author. Tel.: +1 202 687 6090; fax: +1 202 687 6209. E-mail address: yangdc@georgetown.edu (D.C. H. Yang). most powerful mucosal adjuvants (Elson, 1989; Rappuoli et al., 1999; Freytag and Clements, 2005). The heat-labile enterotoxin (LT) from E. coli is structurally and function- ally very similar to CT and shares with CT its adjuvanticity (Clements et al., 1988). Administration of protein antigens together with CT results in the abrogation of oral tolerance, the induction of local and systemic humoral immunity and the induction of cytotoxic T cell responses (Snider, 1995). Part of the strong adjuvant activity is evidently due to the stimulation of the development of Th2 cells by CT through the suppressed expression of the counterregulatory cytokine IL-12 that reduces the development of Th1 cells (Braun et al., 1999). Genes that could be affected by CT through mecha- nisms other than the cAMP production have been reported such as pro-inflammatory effects by altering the expres- sion of interleukins (Braun et al., 1999) and macrophage 0161-5890/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2005.05.008