ABSTRACT
Following the TLR-dependent initiation phase of acute systemic proinflammatory responses such as sepsis, an adaptive phase represses or activates a specific pattern of gene expression until the inflammation resolves. Here, we used the THP-1 sepsis cell model of bacterial LPS/endotoxin tolerance to show that TLR4-induced miR-146a supports the feed-forward adaptive processes that silence transcription and disrupt translation of acute proinflammatory genes. First, we found that miR-146a regulates a pathway that promotes the binding of transcription repressor RelB to the TNF-α promoter, a step known to precede histone and DNA modifications, which generate facultative heterochromatin to silence acute proinflammatory genes. However, once RelB binding occurred, miR-146a inhibition could not reverse compacted chromatin, and endotoxin tolerance persisted. Second, we observed that miR-146a regulates a pathway that supports assembly of the translation repressor complex of TNF-α by preventing the interaction of the RNA-binding protein effector Ago2 and RBM4. We also determined that once endotoxin tolerance is established, and specific genes have been reprogrammed, transcription and translation disruption can be reversed only by simultaneously depleting RelB and inhibiting miR-146a. Thus, miR-146a induction supports the TLR4-dependent shift from initiation to gene-specific repression at two levels. Our results also imply that therapies designed to reverse endotoxin tolerance as potential therapies for sepsis should be directed at the transcription and translation pathways of reprogramming.
Subject(s)
Gene Silencing , Macrophages/metabolism , MicroRNAs/physiology , Toll-Like Receptor 4/metabolism , Transcription, Genetic , Tumor Necrosis Factor-alpha/genetics , Blotting, Western , Chromatin Immunoprecipitation , Endotoxins/pharmacology , Enzyme-Linked Immunosorbent Assay , Heterochromatin/genetics , Histones/metabolism , Humans , Immune Tolerance , Immunoprecipitation , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sepsis/genetics , Toll-Like Receptor 4/genetics , Transcription Factor RelB/antagonists & inhibitors , Transcription Factor RelB/genetics , Transcription Factor RelB/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Three-dimensional collagen gel contraction is the standard assay utilized for functionally quantifying a variety of cell types, in particular smooth muscle cells (SMCs) and myofibroblasts. Here, we have developed a method to effectively reduce the three-dimensional parameters of the standard collagen gel into a single, linear measurement. Cell/collagen suspensions that are cast into glass capillary tubes provide several advantages over the well plate format, such as eliminating the need for digital imaging equipment and software to quantify the amount of cellular contraction. In addition, capillary tube gels require significantly fewer cells and far less reagents than standard methods.
