Cicia, Donatella (2024) Dietary TRPM8 ligands in inflammation-triggered gut diseases. [Tesi di dottorato]

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Abstract

Background: The interplay between diet, host genetics, microbiota, and immune system has a key role in the development and progression of gut chronic inflammatory conditions, such as inflammatory bowel diseases (IBDs) and colitis associated colorectal cancer (CAC). Therefore, the search for dietary components that may further enhance immune function in sub-clinical situations or prevent specific immune-related chronic diseases may lead to new therapeutic strategies. The transient receptor potential (TRP) cation channel, subfamily melastatin (M), member 8 (TRPM8), is an ion-gated receptor mainly permeable to calcium. Here, we explored the possible contribution of TRPM8 to intestinal inflammation and inflammation-triggered colon tumorigenesis. Materials and Methods: A set of natural compounds was tested for TRPM8 affinity by using bioinformatic tools, and the flavonoid luteolin was identified as a novel and selective TRPM8 antagonist. In vitro, the experiments were conducted on bone marrow derived macrophages (BMDMs) obtained from wild type (WT), Trpm8-/- or IL-10fl/fl LysMCre mice femurs and tibias. TRPM8 expression was evaluated in BMDMs by immunofluorescence. Intracellular calcium measurements were conducted in cells loaded with Ca2+-sensitive fluorescent dye FLUO-4AM. Pro-inflammatory mediators [i.e., interleukin (IL) -6, IL-1b and tumor necrosis factor-a (TNFa)] were quantified in supernatant collected from BMDM cultures, using commercial ELISA kits. Seahorse extracellular flux assay, NMR and GC-MS metabolomics analysis were performed to analyze the metabolic reprogramming of macrophages. In a different set of experiments, BMDMs were pre-treated with luteolin, before assessing calcium transients, cytokine production, and metabolic profile as previously stated. Luteolin was also orally administered in the dextran sodium sulfate (DSS) model of colitis in vivo. Further, to evaluate the TRPM8 involvement in colon carcinogenesis, its expression and correlation with the survival rate was analyzed in patients with CRC patients. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted. Then, the functional role of TRPM8 in inflammation-triggered colon cancer was assessed in the AOM/DSS model of CAC in WT and Trpm8-/- mice. Results: TRPM8 mediates LPS- evoked Ca2+ influx in macrophages leading to their activation. TRPM8 is selectively blocked by the dietary flavonoid luteolin (IC50: 3.006 M), which induced a pro-tolerogenic phenotype in pro-inflammatory BMDMs. Accordingly, genetic deletion of Trpm8 in BMDMs caused a deficit in the activation of pro-inflammatory metabolic and transcriptional reprogramming, leading to reduced production of key pro-inflammatory mediators. We proved that TRPM8 anti-inflammatory effect was dependent on lactate which in turn induces downstream epigenetic modifications to regulate IL-10 gene expression. In vivo, oral administration of luteolin ameliorated intestinal inflammation through an impairment in the innate immune response. In CRC patients, TRPM8 is overexpressed in colon tumor specimens, and specifically in CD326+ tumor cell fraction. In such patients TRPM8 high expression was related to lower survival rate, Wnt–Frizzled signaling hyperactivation and APC (adenomatous polyposis coli) down‐regulation. In a colitis‐associated model of colon cancer, the genetic deletion of TRPM8 reduced tumor development. Conclusions: Overall, this PhD thesis unmasks the potential of targeting TRPM8 through specific nutrient interventions to regulate immune function in sub-clinical scenarios or to treat inflammatory diseases, primarily driven by chronic immune responses, such as IBD or CAC. Moreover, human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival.

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