Objective Glucocorticoids (GCs) modulate multiple cellular activities including inflammatory and fibrotic responses. Outcomes of GC treatment for laryngeal disease vary, affording opportunity to optimize treatment. In the current study, three clinically employed GCs were evaluated to identify optimal in vitro concentrations at which GCs mediate favorable anti‐inflammatory and fibrotic effects in multiple cell types. We hypothesize a therapeutic window will emerge as a foundation for optimized therapeutic strategies for patients with laryngeal disease. Study Design In vitro. Methods Human vocal fold fibroblasts and human macrophages derived from THP‐1 monocytes were treated with 0.03–1000 nM dexamethasone, 0.3–10,000 nM methylprednisolone, and 0.3–10,000 nM triamcinolone in combination with interferon‐γ, tumor necrosis factor‐α, or interleukin‐4. Real‐time polymerase chain reaction was performed to analyze inflammatory (CXCL10, CXCl11, PTGS2, TNF, IL1B) and fibrotic (CCN2, LOX, TGM2) genes, and TSC22D3, a target gene of GC signaling. EC50 and IC50 to alter inflammatory and fibrotic gene expression was calculated. Results Interferon‐γ and tumor necrosis factor‐α increased inflammatory gene expression in both cell types; this response was reduced by GCs. Interleukin‐4 increased LOX and TGM2 expression in macrophages; this response was also reduced by GCs. GCs induced TSC22D3 and CCN2 expression independent of cytokine treatment. EC50 for each GC to upregulate CCN2 was higher than the IC50 to downregulate other genes. Conclusion Lower concentrations of GCs repressed inflammatory gene expression and only moderately induced genes involved in fibrosis. These data warrant consideration as a foundation for optimized clinical care paradigms to reduce inflammation and mitigate fibrosis. Level of Evidence NA Laryngoscope, 133:1169–1175, 2023
ObjectiveVariable outcomes of glucocorticoid (GC) therapy for laryngeal disease are putatively due to diverse interactions of the GC receptor (GR) with cell signaling pathways, limited consideration regarding concentration‐dependent effects, and inconsistent selection of GCs. In the current study, we evaluated the concentration‐dependent effects of three frequently administered GCs on transcription factors with an emphasis on the phosphorylation of GR at Ser203 and Ser211 regulating the nuclear translocation of GR. This study provides foundational data regarding the diverse functions of GCs to optimize therapeutic approaches.Study designIn vitro.MethodsHuman vocal fold fibroblasts and THP1‐derived macrophages were treated with different concentrations of dexamethasone, methylprednisolone, and triamcinolone in combination with IFN‐γ, TNF‐α, or IL4. Phosphorylated STAT1, NF‐κB family molecules, and phosphorylated STAT6 were analyzed by Western blotting. Ser211‐phosphorylated GR (S211‐pGR) levels relative to GAPDH and Ser203‐phosphorylated GR (S203‐pGR) were also analyzed.ResultsGCs differentially altered phosphorylated STAT1 and NF‐κB family molecules in different cell types under IFN‐γ and TNF‐α stimuli. GCs did not alter phosphorylated STAT6 in IL4‐treated macrophages. The three GCs were nearly equivalent. A lower concentration of dexamethasone increased S211‐pGR/GAPDH ratios relative to increased S211‐pGR/S203‐pGR ratios regardless of cell type and treatment.ConclusionThe three GCs employed in two cell lines had nearly equivalent effects on transcription factor regulation. Relatively high levels of Ser203‐phosphorylation at low GC concentrations may be related to concentration‐dependent differential effects of GCs in the two cell lines.Level of EvidenceNA Laryngoscope, 133:2704–2711, 2023
Objectives/HypothesisGlucocorticoids (GC)s are commonly employed to treat vocal fold (VF) pathologies. However, VF atrophy has been associated with intracordal GC injections. Dexamethasone‐induced skeletal muscle atrophy is well‐documented in other tissues and believed to be mediated by increased muscle proteolysis via upregulation of Muscle Ring Finger (MuRF)‐1 and Atrogin‐1. Mechanisms of dexamethasone‐mediated VF atrophy have not been described. This pilot study employed in vitro and in vivo models to investigate the effects of dexamethasone on VF epithelium, thyroarytenoid (TA) muscle, and TA‐derived myoblasts. We hypothesized that dexamethasone will increase atrophy‐associated gene expression in TA muscle and myoblasts and decrease TA muscle fiber size and epithelial thickness.Study DesignIn vitro, pre‐clinical.MethodsTA myoblasts were isolated from a female Sprague–Dawley rat and treated with 1 μM dexamethasone for 24‐h. In vivo, 15 New Zealand white rabbits were randomly assigned to three treatment groups: (1) bilateral intracordal injection of 40 μL dexamethasone (10 mg/ml; n = 5), (2) volume‐matched saline (n = 5), and (3) untreated controls (n = 5). Larynges were harvested 7‐days post‐injection. Across in vivo and in vitro experimentation, MuRF‐1 and Atrogin‐1 mRNA expression were measured via RT‐qPCR. TA muscle fiber cross‐sectional area (CSA) and epithelial thickness were also quantified in vivo.ResultsDexamethasone increased MuRF‐1 gene expression in TA myoblasts. Dexamethasone injection, however, did not alter atrophy‐associated gene expression, TA CSA, or epithelial thickness in vivo.ConclusionDexamethasone increased atrogene expression in TA myoblasts, providing foundational insight into GC induced atrophic gene transcription. Repeated dexamethasone injections may be required to elicit atrophy in vivo.Level of EvidenceNA Laryngoscope, 133:2264–2270, 2023
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