Inhibition of IL-17 Release by the Novel Anti-Inflammatory Drug Vidofludimus Involves Attenuation of STAT3 and NF-kappa B Signaling Pathways in Murine Splenocytes and Hapten-Induced Colitis

Abstract

IL-17 Secretion: Murine Splenocytes Vidofludimus Inhibits the Nuclear Binding of STAT3 in a Dose Dependent Fashion: Murine Splenocytes Vidofludimus Does Not Affect the Expression of IκB-α: Murine Splenocytes Vidofludimus Attenuates Phospho-p65 Staining: Murine TNBS Colitis The STAT3 and NF-kappa B (NF-κB) pathways play a prominent role in IL-17 production and intestinal inflammation. We have shown that a novel drug named Vidofludimus (Vido, 4SC-101, SC12267) inhibited colonic IL-17 and murine colitis [Inflammatory Bowel Diseases, 2010]. Aims: The aims of our study were: 1) to determine if Vido inhibited IL-17 production by murine splenocytes, 2) to evaluate if Vido inhibited key components of the STAT3 and NF-κB signaling pathways in splenocytes and 3) to determine if Vido inhibited phosphorylated (phospho) STAT3 and NF-κB in the colons of mice with hapten (TNBS)-induced colitis. Methods: Splenocytes were collected from BALB/c mice. These cells were stimulated with IL-23 or IL-1β (10 ng/ml) alone, or in combination. Vido (25 to 100 μM), or vehicle, were added along with the cytokines. IL-17 secretion was determined by ELISA at Vidofludimus (4SC-101, SC12267) is a proprietary compound of 4SC AG Vidofludimus Inhibits Cytokine-Induced IL-17 Secretion: Murine Splenocytes Synergistic activation by IL-23 + IL-1β Vidofludimus Attenuates Phospho-AKT1 Expression: Murine Splenocytes Effects of Vidofludimus on Signaling Pathways: Murine Splenocytes 48 hours. Cytosolic extracts from splenocytes were used for western blots of phospho JAK2, STAT3 and AKT1 (STAT3 pathway proteins). Phospho I-κB and I-κB degradation were evaluated as indices of NF-κB pathway activation. Extracts were also collected from stimulated splenocytes for the evaluation of STAT3 and NF-κB nuclear binding by ELISA. Immunohistochemistry for phospho STAT3 and p65 was done on samples from BALB/c mice that underwent colitis induction with TNBS ± oral treatment for six days with Vido (50-200 mg/kg). Using coded slides, the staining for these proteins in the mucosa/submucosa was graded on a six point intensity scale. Results: IL-23 and IL-1β alone did not induce significant IL-17 production (< 30 pg/ml), but combined cytokine treatment induced a synergistic stimulation of IL-17 secretion (630 pg/ml). IL-17 production was blocked by Vido in a dose dependent fashion (IC50 = 58.1 μM). At 10 to 60 minutes after cytokine stimulation, Vido reduced the expression of phospho proteins (JAK2, STAT3, and AKT1) involved in STAT3 signaling, and also inhibited the nuclear binding of STAT3 (IC50 = 53.9 μM). Vido did not reduce the phosphorylation or degradation of I-κB, but at 100 μM inhibited the nuclear binding of p65 by 47%. Evidence of increased phospho STAT3 and p65 immunostaining was found in leukocytes within the colons of TNBS-treated mice. The staining was attenuated in mice treated with Vido. Phospho STAT3 staining scores (2-3 mice per group) were: 1.3 ± 0.8 (no TNBS), 4.3 ± 0.9 (Vehicle/TNBS), 1.1 ± 0.3 (Vido-50 mg/kg), 1.2 ± 0.7 (Vido-100 mg/kg) and 0.3 ± 0 (Vido-200 mg/kg/TNBS). Similar METHODS immuno-staining scores were found for phospho p65. Summary: Vidofludimus inhibited STAT3 and NF-κB signaling pathways in murine splenocytes. It also normalized the enhanced phospho STAT3 and p65 immunostaining associated with murine colitis. Conclusion: The inhibition of STAT3 and NF-κB pathways by Vidofludmus reduces IL-17 production and contributes to the established anti-colitis activity of this drug. Splenocyte Cultures: Spleens were rapidly harvested from BALB/c mice. Splenocytes were collected by standard techniques, and erythrocytes were removed by using RBC lysis buffer. For the IL-17 secretion studies, we plated 2 x 106 cells/ml in 4 ml of RPMI 1640 medium containing 10% FBS and 5 x 105 β-mercaptoethanol. Therefore, there were 8 x 106 cells per well in the standard six-well plates. We utilized two wells (1.6 x 107 cells) per treatment condition in these studies. Splenocytes were cultured for 48 hours at 37 degrees C. Then, the culture media was collected for the measurement of IL-17 by ELISA (R&D systems, Minneapolis, MN). In subsequent studies (western blots, transcription factor binding assays), splenocytes were plated at a density of 1 x 107 cells/ml in 4 ml of RPMI 1640 medium. Therefore,