Background

The immune system has evolved to recognize not only pathogenic stimuli such as bacterial components and viral nucleic acids, but also endogenous danger signals including proteins secreted from necrotic cells or expressed upon tissue damage. Both types of stimuli are sensed by pattern recognition receptors, initiating signalling cascades that trigger inflammatory responses. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor essential for the activation of the immune response to infection and tissue damage. NF-κB has an important role in the expression of a wide range of inflammatory mediators following pattern recognition receptor activation, including cytokines (such as tumour necrosis factor α [TNFα], interleukin-6 and interleukin-1), chemokines (e.g., interleukin-8 or CXCL1), proteases, growth factors and MHC-related molecules, among others. To assess the activation of this pathway downstream of TLR4, we used the commercially available cell line THP1-Blue^TM NF-κB (Invivogen). These cells are stably transfected with a construct containing a secreted embryonic alkaline phosphatase (SEAP) gene induced by the NF-κB transcription factor. The construct contains an interferon-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site, which drives the expression of the reporter gene. After cell stimulation with pathogenic or endogenous stimuli, NF-κB activation leads to the secretion of SEAP, which is then quantified using the colorimetric reagent QUANTI-Blue^TM. This is a quick and reliable method to assess activation of NF-κB downstream of toll-like receptors, as the amount of SEAP in the media correlates with the triggering of this signalling pathway. However, this is an engineered cell line that does not express a full complement of inflammatory effector molecules (see Note 5), and so whilst useful in screening for NF-κB activation, data should always be confirmed in additional experimental systems, for example in primary macrophages, or in vivo.