Transcriptional profiling of murine CD4+ T cells following treatment with the supercooling compound icilin

The synthetic supercooling drug, icilin, and its primary receptor target, the cation channel transient receptor potential (TRP) melastatin-8 (TRPM8), have been described as potent negative regulators of inflammation in the colon. The aim of this study was to determine whether the anti-inflammatory action of icilin could potentially be used to treat autoimmune neuroinflammatory disorders, such as multiple sclerosis (MS). During experimental autoimmune encephalomyelitis (EAE)—a CD4+ T cell–driven murine model of MS—we found that both wild-type (WT) and TRPM8-deficient EAE mice were protected from disease progression during icilin treatment, as evidenced by delays in clinical onset and reductions in neuroinflammation. In vitro, icilin potently inhibited the proliferation of murine and human CD4+ T cells, with the peripheral expansion of autoantigen-restricted T cells similarly diminished by the administration of icilin in mice. Attenuation of both TRPM8-/- and TRP ankyrin-1-/- T cell proliferation by icilin was consistent with the WT phenotype, which suggests a mechanism that is independent of these channels. In addition, icilin treatment altered the expressional profile of activated CD4+ T cells to one that was indicative of restricted effector function and limited neuroinflammatory potential. These findings identify a potent anti-inflammatory role for icilin in lymphocyte-mediated neuroinflammation and highlight clear pleiotropic effects of the compound beyond classic TRP channel activation. Purified murine WT CD4+ T cells were treated with 100 µM icilin (n=3 from n=3 mice) or the vehicle DMSO (n=3 from the same n=3 mice) for 24 h. T cells were stimulated with 10 µg/mL anti-mouse CD3 and 10 µg/mL anti-mouse CD28 monoclonal antibodies for 24 h before addition of icilin or vehicle. Total RNA was extracted by using an RNeasy Mini Kit, and RNA quality and RNA integrity number was measured with Agilent RNA Nano Chips on a 2100 Bioanalyzer. A total of 0.25 µg of RNA with an RNA integrity number of ≥8 was used for each sample. RNA was labeled by using the GeneChip Whole Transcript Express Labeling Assay and hybridized to GeneChip Mouse Gene 2.0 ST Arrays (Affymetrix) at 45°C for 16 h at the SACRI Microarray and Genomics Facility (University of Calgary) (n=6 total array samples). Arrays were stained and washed by using GeneChip Fluidics 450 per manufacturer protocols and scanned with a GeneChip Scanner 3000 7G System (Affymetrix). Probe cell intensity data files were generated and summarized with GeneChip Command Console Software, and Robust Multi-Array Average was used to log 2 transform and normalize data. Altered gene expression was analyzed by Significance Analysis of Microarrays software, with genes that were differentially expressed ≥1.5-fold with a false discovery rate of <0.1 deemed significant.