Quantitative proteomics and phosphoproteomics reveal glucocorticoid stimulation of TLR and Rho GTPase signaling in neutrophil-like cells

Glucocorticoids are corticosteroid hormones that are commonly used for treating systemic inflammatory diseases and acute infections. Immunosuppressive effects of glucocorticoids have been studied in many cell types, particularly macrophages and T cells. Despite the importance and abundance of neutrophils in the human immune system, glucocorticoid responses remain understudied in neutrophils.

Here, we perform quantitative mass spectrometry-based proteomics of primary neutrophils and neutrophil-like cells differentiated from human HL-60 promyelocyte cells. Primary neutrophils exhibited CK2 kinase activation and increase phosphorylation of HSP90 following 2-h incubation, highlighting potential effects of short-term ex vivo handling. Proteome and flow cytometry analysis show that neutrophil-like cells share features of neutrophils. Quantitative proteomics and phosphoproteomics of neutrophil-like cells treated with two synthetic glucocorticoid compounds, the clinical drugs dexamethasone and prednisolone, identify higher numbers of significantly regulated proteins and phosphosites compared to parental HL-60 cells. Glucocorticoid treatments modulated toll-like receptor signaling and CXCR4 serine phosphorylation. In addition, we identify RIPOR2 as a glucocorticoid-regulated protein associated with Rho GTPase signaling networks and actin cytoskeletal remodeling in neutrophils and neutrophil-like cells, though its exact functional role requires further investigation.

Our results not only reveal unconventional regulatory mechanisms of glucocorticoids in the human immune system but also provide valuable resources for discovering novel glucocorticoid-responsive protein targets in neutrophils.