Effects of Poly‐Substituted Pyrroles on Pro‐Inflammatory Signaling in RAW264.7 Macrophages

Abstract

Inflammation is the body’s natural means of protection against infection and the restoration to homeostasis after cellular and tissue damage from injury or metabolic stress. A multitude of complex signaling pathways are necessary for the activation of pro‐inflammatory signaling in macrophages, and the dysregulation of these pathways can contribute to disease states linked to chronic inflammation making them attractive targets for therapeutics. Our laboratory explores the potential of a class of poly‐substituted pyrrole compounds that are known microtubule depolymerizers to regulate the inflammatory response in macrophages. Previous work from our laboratory demonstrated that JG‐03‐14 (3,5‐dibromo‐4‐(3,4‐dimethoxyphenyl)‐1H‐pyrrole‐2‐carboxylic_acid ethyl ester) and its refined analog, NT‐07‐16, which possesses an additional methoxy group at the 2‐position of the phenyl ring decrease the production of pro‐inflammatory cytokines in lipopolysaccharide (LPS)‐activated RAW264.7 murine macrophages. Our results demonstrated that the reduction in pro‐inflammatory mediators is due, in part, to a decrease in the nuclear translocation of NFκB, a critical transcription factor for pro‐inflammatory signaling. These results are supported since microtubule dynamics is thought to be involved in NFκB nuclear translocation. Here we expand the scope of our previous work by exploring the effects of NT‐07‐16, and a potentially more potent stereoisomer mix of the NT analog, NT‐07‐45 on other pro‐inflammatory signaling pathways in the RAW264.7 macrophages. Recent studies have shown that exposure to NT‐07‐45 decreases the production of the antiviral and pro‐inflammatory cytokine interferon‐beta (IFN‐β) in macrophages. In addition, the NT analog alters the phosphorylation of the interferon regulatory factor (IRF3) which decreases its activation and could affect its ability to enter the nucleus and induce IFN‐β production. Our recent work has also found that exposure of RAW264.7 macrophages to the NT analogs alters the phosphorylation, and possibly the activation, of c‐Jun N‐terminal kinase (JNK), an important MAP‐Kinase (MAPK) involved in macrophage inflammatory signaling. These findings indicate that the decrease in the production of pro‐inflammatory mediators observed in RAW264.7 macrophages exposed to polysubstituted pyrroles, may be due to the inability of several transcription factors involved in the inflammatory response to efficiently translocate into the nucleus. While much is still to be learned about the anti‐inflammatory mechanisms of these pyrrole compounds it appears that the effects may go beyond their microtubule‐depolymerizing activity.