Chromium allergy is a common occupational skin disease mediated by chromium (VI)-specific T cells that induce delayed-type hypersensitivity in sensitized individuals. Additionally, chromium (VI) can act as an irritant. Both responses critically require innate immune activation, but if and how chromium (VI) elicits this signal is currently unclear. Using human monocytes, primary human keratinocytes, and murine dendritic cells we show that chromium (VI) compounds fail to trigger direct proinflammatory activation but potently induce processing and secretion of IL-1β. IL-1β release required priming by phorbol-ester or toll-like receptor stimulation and was prevented by inhibition of K efflux, NLRP3 depletion or caspase-1 inhibition, identifying chromium (VI) as a hapten activator of the NLRP3 inflammasome. Inflammasome activation was initiated by mitochondrial reactive oxygen species production triggered by chromium (VI), as indicated by sensitivity to treatment with the ROS scavenger N-acetyl cysteine and a coinciding failure of K efflux, caspase-1, or NLRP3 inhibition to prevent mitochondrial reactive oxygen species accumulation. IL-1β release further correlated with cytotoxicity that was secondary to reactive oxygen species, K efflux, and NLRP3 activation. Trivalent chromium was unable to induce mitochondrial reactive oxygen species production, inflammasome activation, and cytotoxicity, suggesting that oxidation state-specific differences in mitochondrial reactivity may determine inflammasome activation and allergic/irritant capacity of different chromium compounds.
Background: Eosinophils appear to contribute to the efficacy of immunotherapy and their frequency was suggested as a predictive biomarker. Whether this observation could be transferred to patients treated with targeted therapy remains unknown. Methods: Blood and serum samples of healthy controls and 216 patients with advanced melanoma were prospectively and retrospectively collected. Freshly isolated eosinophils were phenotypically characterized by flow cytometry and co-cultured in vitro with melanoma cells to assess cytotoxicity. Soluble serum markers and peripheral blood counts were used for correlative studies. Results: Eosinophil-mediated cytotoxicity towards melanoma cells, as well as phenotypic characteristics, were similar when comparing healthy donors and patients. However, high relative pre-treatment eosinophil counts were significantly associated with response to MAPKi (p = 0.013). Eosinophil-mediated cytotoxicity towards melanoma cells is dose-dependent and requires proximity of eosinophils and their target in vitro. Treatment with targeted therapy in the presence of eosinophils results in an additive tumoricidal effect. Additionally, melanoma cells affected eosinophil phenotype upon co-culture. Conclusion: High pre-treatment eosinophil counts in advanced melanoma patients were associated with a significantly improved response to MAPKi. Functionally, eosinophils show potent cytotoxicity towards melanoma cells, which can be reinforced by MAPKi. Further studies are needed to unravel the molecular mechanisms of our observations.
Background The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. Methods RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. Results In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. Conclusions Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.
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