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5‐Aminoethylbenzimdazole Suppresses Lipopolysaccharide (LPS)/Interferon Gamma (IFNγ)‐Induced Inflammatory Responses in Macrophages
Author(s) -
Chen Xin,
Smith Amanda,
Plummer Christopher,
Lei Wei
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04532
Subject(s) - lipopolysaccharide , tumor necrosis factor alpha , nitric oxide , macrophage , kinase , tec , nitric oxide synthase , interferon gamma , chemistry , interferon , tyrosine kinase , immunology , immune system , microbiology and biotechnology , biology , signal transduction , biochemistry , in vitro , ionosphere , physics , organic chemistry , astronomy
Tec family non‐receptor tyrosine kinases, including TEC, BTK, ITK/EMT/TSK, BMX and TXK/RLK, are expressed in a variety of immune cells. Previous studies have demonstrated that Tec kinases play pivotal roles in inflammatory disorders such as allergic asthma and atopic dermatitis. Several 5‐aminomethylbenzimdazoles (5‐AMBD) have been reported as highly potent inhibitors of ITK as well as other TEC kinases and showed strong activities on reducing IL‐2 and IL‐4 production. However, the impact of 5‐AMBD on macrophage‐mediated inflammatory responses remains unclear. The goal of this study was to determine the effect of 5‐AMBD on the inflammatory responses in macrophages stimulated with lipopolysaccharide (LPS) and interferon‐gamma (IFNγ). RAW 264.7 cells (a murine macrophage cell line) were stimulated with 10 ng/mL LPS and 1 ng/mL IFNγ for 10 min, then treated with various concentrations of 5‐AMBD. The cell viability and production of nitric oxide (NO) were measured 20 hr after treatment. The total RNA was extracted from cells treated for 4 hr, and then the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNFα), and interleukin 1 beta (IL‐1β) were evaluated using quantitative real‐time PCR. The impact of 5‐AMBD on the activation of nuclear factor kappa B (NF‐κB) was investigated using the RAW 264.7 cells stably expressing the NF‐kB luciferase reporter. 5‐AMBD dose‐dependently reduced the NO production with no significant toxicity to the macrophages. Treatment of 100 nM and 1 μM 5‐AMBD significantly inhibited the expression of iNOS by 50% and 95%, respectively. Meanwhile, 5‐AMBD (1 μM) also reduced the expression of IL‐1β and TNFα. Furthermore, we found that 5‐AMBD dose‐dependently diminished the activation of NF‐κB at 4 hr and 20 hr after treatment. These findings demonstrate that 5‐AMBD has a strong anti‐inflammatory activity in macrophages, and suggest that TEC kinases could be potential drug targets for inflammatory diseases. Support or Funding Information Work supported by Small Pharmacy Awards for Research and Collaboration from the Presbyterian College School of Pharmacy.