Investigating the effect of Glucocorticoids on Macrophage Plasticity: Optimizing Identification of THP-1 macrophage differentiation into M1/M2 phenotypes by flow cytometry

Abstract:

Synthetic glucocorticoids (GCs) have been utilized within the medical field for over 70 years for their broad versatility to treat inflammatory conditions. However, the underlying mechanism involving synthetic GCs still remains elusive within the inflammatory response. Macrophages, an essential first responder within the innate immune system, are vital to maintaining resident tissue homeostasis; external cues like cytokines and damaged/pathogenic associated molecular patterns greatly influence their phenotypic behavior. Macrophages display a wide spectrum of polarized phenotypes, from classically activated (M1) pro-inflammatory/cytotoxic macrophage phenotypes to alternatively activated (M2) anti-inflammatory/wound repair macrophage phenotypes. Recent studies report increased M2 macrophage populations in diseases like systemic sclerosis-associated interstitial lung disease (SSc-ILD), idiopathic Pulmonary Fibrosis, and post-COVID lung tissue samples. Due to nearly40% of patients with COVID-19 developing a form of acute respiratory distress syndrome (ARDS), GCs have become a common treatment for respiratory inflammation, though little is known about how GCs affect macrophage plasticity and whether such treatment is efficacious for the patient. To better elucidate how synthetic GCs affect THP-1 macrophage plasticity, several techniques will be used to identify THP-1 macrophage differentiation and M1/M2 phenotypes including: 1) immunofluorescent staining of THP-1 monocyte surface marker (CD14) and macrophage marker (CD68) 2) Flow cytometry analysis of macrophage surface receptors (M1: MCH class II and CD80) (M2:CD163, and CD206); and 3) Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) to measure the expression of key genes related to THP-1 macrophage phenotypes (M1: Nos2/iNOS, Ccl5/Rantes, and Tnf )(M2: Arg1, Retnla, and Chil3). The goal of this study is to gain a better molecular insight into how GC affects immune cells with the hope of shedding light on the efficacy of such therapeutic treatments when it comes to lung inflammation.