Influence of Itraconazole on T‐cell Immune Deviation

Objective: The objective of this study was to determine if the presence of itraconazole can alter the activation and differentiation pattern of T‐cells. Proliferation assays utilizing immunofluorescence and flow cytometry were used to determine the presence, number, and type of T‐cell subpopulations within each experimental group. Also, we sough to verify that the presence of itraconazole can alter the production of Th2 mediated cytokines. RT‐PCR was employed to measure relative numbers of gene transcripts for the various characteristic cytokines associated with T‐cell subpopulations. Design. Prospective, controlled experiment. Subjects. Samples of whole blood were be taken from healthy volunteers. Intervention(s): Following isolation of naive T cells, cells were divided into 4 groups and immune deviated in the following fashion. Controls cell lines were activated with anti‐CD3 and CD 28 and additional cytokines and antibodies were added to deviate towards Th1 and Th2 conditions. Experimental groups consisted of similar conditions with the addition of itraconazole. Results. Successful immune deviation of controls was performed. With the addition of itraconazole to Th2 deviating conditions, IL‐5, IL‐13, and IFN gamma were all noted to decrease (both by PCR and supernatant assay). Conclusions. Itraconazole inhibits the deviation of naive T cells towards the Th2 mediated pathway. METHODS and MATERIALS Samples of whole blood were taken from healthy volunteers (n = 10). Whole blood was washed and then filtered through bead column with anti‐CD45RA beads to isolate naive T cells. Naive T cells were then divided into 6 groups specified as follows: Cells were incubated in respective solutions for 96 hours. Following incubation, T cells were then harvested into 2 groups. Group 1 cells were stained and fixed, in preparation for flow cytometry to determine the number of each T‐cell population (Th1 vs Th2). Real time PCR was then performed on the group 2 cells, to determine the relative number of mRNA transcripts for IFN‐g, IL‐4, IL‐5, and IL‐13 as compared to a reference protein. Assays were then performed on incubated supernatant to evaluate for cytokine production in each of the respective 6 groups, assaying for IL‐2, IL‐4, IL‐5, IL‐9, IL‐13, INF‐g and reference proteins. INTRODUCTION Chronic rhinosinusitis (CRS) is a chronic inflammatory condition affecting the paranasal sinuses and nasal cavities which has a significant adverse impact on the quality of life and daily functioning of its sufferers.1‐3 No one specific etiology has been defined and it is more likely that this term represents either multiple different diseases with or without various stages of severity along their spectra. Certain subsets of CRS have been strongly associated with a Th2 mediated inflammatory process and, in turn, it has been theorized that at least a portion of these subsets are associated with an abnormal reaction to the presence of fungus in the nasal cavity and paranasal sinuses.4‐9 Previous work has demonstrated the ability of itraconazole to mitigate the production of Th2 associated cytokines when non‐specific T‐cell cultures are grown in its presence. Additionally, there has been some data regarding the clinical efficacy of treating CRS patients with itraconazole.10 However, no work has been done evaluating the potential role that it may have in affecting the initial differentiation process of the Th0 cell upon activation. Several studies have related anti‐mycotics to the suppression of T cell (IL‐2) and Th2‐mediated cytokines (IL‐4 and IL‐5). Bruserad demonstrated that itraconazole suppressed the cytokine‐driven proliferation of human AML cell lines.11 Kanda et al showed results that anti‐mycotics reduced the anti‐ CD3/CD28‐induced mRNA expression and promoter activities for interleukin‐4 and interleukin‐5 in atopic and normal volunteer human T‐cells.12