Glucocorticoid Dose Dependency on Gene Expression in Vocal Fold Fibroblasts and Macrophages

Abstract

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

Dose‐Dependent Glucocorticoid Regulation of Transcription Factors in Vocal Fold Fibroblasts and Macrophages

“…Risks of GC injection on muscular and mucosal atrophy have also been reported, 8,9 and more recent data suggest a pro‐fibrotic response to GCs in the larynx 10 . In that regard, GCs have divergent effects on fibrosis across organs 11–14 In vitro data from our group confirmed both anti‐ and pro‐fibrotic effects of GCs in human vocal fold (VF) fibroblasts and macrophages 10,15 . Despite their primary anti‐inflammatory role, GCs increased several inflammatory genes in macrophages 15 .…”

“…Investigation of multiple mechanisms underlying the diverse functions of GCs is critical to optimize GC‐based therapies. Our prior work using HVOX and THP‐1 cells elucidated different GC functions dependent on cell type, cytokine stimuli, and GC concentrations 15 . The current study sought to provide further insight regarding molecular mechanisms related to these differential GC functions.…”

“…We recently reported differential effects of GCs on fibrotic and inflammatory gene expression in human VF fibroblasts and macrophages dependent on cell type, cytokine stimuli, and GC concentration 15 . Inflammatory genes such as TNF , IL1B , and PTGS2 , were downregulated by GCs in IFN‐γ‐ and TNF‐α‐stimulated cells.…”

“…In contrast, connective tissue growth factor (CTGF), a mediator of fibrosis, 23 was upregulated by GCs in macrophages and VF fibroblasts under TNF‐α, IFN‐γ, and IL4 stimulation. Interestingly, higher concentrations of GCs were required to upregulate CTGF, as well as a major transcription target of GR (glucocorticoid‐induced leucine zipper; GILZ) relative to concentrations required to inhibit inflammatory genes ( CXCL10 , CXCL11 , TNF , PTGS2 , IL1B ) and other fibrotic genes ( LOX , TGM2 ) 15 …”

“…In addition, we interrogated relationships between GC concentration‐dependent regulation of Ser203/Ser211 phosphorylation and transcription. GC concentrations required to increase Ser211‐phosphorylated GR (S211‐pGR) levels relative to a housekeeping gene and Ser203‐phosphorylated GR (S203‐pGR) were compared to previously reported GC concentrations required to inhibit and promote inflammatory and fibrotic gene expression 15 …”