Abstract
BackgroundDespite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS.Methodology/Principal FindingsAfter a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings.Conclusions/SignificanceUsing a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and sepsis-induced ALI/ARDS.
Highlights
Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), is a relatively common syndrome in critically ill patients associated with high morbidity and mortality [1]
LPS-induced airway epithelial cell injury models We chose A549 cells [human pulmonary alveolar epithelial carcinoma cells (ATCC, Manassas, VA, USA)] and BEAS-2B cells [human bronchial epithelial cells (ATCC, Manassas, VA, USA)], as representative airway epithelial cell lines [14] to study the effects of the synthetic derivatives on the ability to inhibit LPS-induced effects in the airway epithelium These cells have been extensively used to study LPS-induced activation of pro-inflammtory cytokines, as an in vitro model based on the first steps in the development of sepsis-induced acute lung injury (ALI)/ARDS [14,15,16,17,18,19,20,23]
Appropriate models have been developed [26], and this strategy has been applied for the past couple of decades by the National Cancer Institute (NCI) within the Development Therapeutics Program [11]
Summary
Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), is a relatively common syndrome in critically ill patients associated with high morbidity and mortality [1]. Data from the ALIVE Study [2] showed that about 7% of all intensive care unit patients develop ALI/ARDS, and that sepsis is the most common predisposing factor. Sepsis is characterized by a systemic inflammatory state in response to circulating microbes or microbial toxins such as lipopolysaccharide (LPS) or bacterial DNA [3]. Toll-like receptors (TLRs), which function as sensors of microbial infection, recognize LPS and are critical for the initiation of inflammatory and immune defense responses [6]. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS
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