Investigating MAPK signaling and TLR2 expression in monocyte cells obtained from the blood of cystic fibrosis patients

Abstract

Cystic fibrosis (CF) is characterized by chronic inflammation and persistent infections, leading to significant morbidity and mortality. Understanding the molecular mechanisms underlying immune responses in CF is crucial for developing targeted therapies. This study investigates the expression and signaling pathways of Mitogen-Activated Protein Kinase (MAPK) and Toll-like Receptor 2 (TLR2) in monocyte cells derived from CF patients. Methods and Materials: In this observational study, blood samples were collected from 20 cystic fibrosis patients referred to Imam Reza Hospital clinic. Monocyte cells were isolated using the PBMC technique through MACS with Kit No. 137-117-337 from Miltenyi Biotec Company, and CD14 antibody was used for purity assessment via flow cytometry. The cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1% Penestrep, and stimulated with TLR2 from Invivogen at 0, 12, 24, and 48 hours. Total RNA was extracted using Trizol solution and a Qiagen DNA extraction kit. cDNA was synthesized from mRNA, and TLR2 gene expression was measured by Real-Time PCR. MAPK expression was assessed using Western blotting. Results: Western blot analysis revealed a significant increase in MAPK protein expression in stimulated monocyte cells compared to non-stimulated cells, with relative protein levels markedly higher in the stimulated condition. Conversely, Real-Time PCR analysis showed no significant difference in TLR2 mRNA expression between monocytes from CF patients and controls, with both groups exhibiting a mRNA fold change value of 1.0.