Development of two methods for detection of IL-23 stimulation and inhibition

Abstract

The study aimed to develop and compare two different methods for the detection of interleukin 23 (IL-23) stimulation and inhibition. The presence of IL-23 signaling can be determined from various points of the signaling pathway and the presented methods have two different parameters for detecting the stimulation and inhibition. IL-23 is a proinflammatory cytokine that utilizes the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway to transduce signals. JAK family includes JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) proteins. The IL-23 signaling pathway begins with cytokine binding to its receptor on the cell surface. Intracellular TYK2 and JAK2 bind to the receptor and activate each other by phosphorylation. Activated TYK2 and JAK2 phosphorylate the tails of the receptor, forming docking sites for the STAT3 pair. STATs localize to the tail of the receptor where they are in turn phosphorylated by JAKs. Phosphorylated STAT3s dissociate from the receptor and dimerize before translocating to the nucleus to regulate the expression of cytokine-responsive genes. Flow cytometric assay takes advantage of phospho flow technology and measures pSTAT3 signal levels from whole blood samples after stimulation with IL-23. ELISA-based assay detects the IL-23 induced production of interleukin 22 (IL-22) in peripheral blood mononuclear cells by measuring IL- 22 concentration from cell culture supernatants. Inhibition of IL-23 signaling in these experiments is studied with brepocitinib. Brepocitinib is an adenosine triphosphate competitive inhibitor that shown effective inhibition against TYK2, JAK1, and JAK2. IL-23 stimulation was detectable with both proposed methods when using fold change values between cytokine-stimulated and unstimulated samples as readout. In the flow cytometric assay, the stimulation was detected by using the mean values of the phosphorylated STAT3 signal. In the ELISA-based assay, the IL-22 signal level was affected by dilution ratios and it was observed that additional stimulation with interleukin 2 improved the IL-22 signal level in the sample. Brepocitinib inhibited IL-23 signaling in both experiments. Inhibition was calculated as the amount of brepocitinib that is needed for 50 % inhibition.