The role of LPCAT in regulating cellular inflammatory responses to toll-like receptor ligands

Abstract

A novel lipid modifying enzyme, lysophosphatidylcholine acyltransferase (LPCAT), has been shown to regulate pro-inflammatory cytokine responses to lipopolysaccharide (LPS), a key molecule in initiating sepsis. Current research has shown that phospholipid metabolism may influence cell signalling, since that is dependent on the clustering of the receptor molecules into membrane microdomains (lipid rafts). LPCAT therefore, may be a target for novel anti-sepsis therapies. This study aimed to determine if LPCAT is essential in the regulation of pro-inflammatory cytokine responses to Gram-positive cell bacterial components, such as peptidoglycan (PG), lipoteichoic acid (LTA) and synthetic tripalmitoylated lipopeptide Pam3CSK4 in monocytes and lung epithelial cells. Inhibition of LPCAT activity consistently reduced TNF-α, IL-6 and IL-8 protein and mRNA levels in monocytes stimulated with all microbial ligands tested. Furthermore, cytokine production observed in monocytes primed with IFN-γ prior to ligand stimulation was twice that seen in unprimed cells, yet still inhibition of LPCAT significantly decreased the amplified inflammatory response. Lung epithelial cells, BEAS-2B, were more immunologically responsive to Pam3CSK4 where it consistently induced a high secretion of IL-8 and IL-6. However, whilst inhibition of LPCAT demonstrated a reduction in cytokine secretion, it was not a predominant as observed in monocytes indicating that LPCAT may have a lesser role in these cells.The secondary aim was to characterise the expression levels of 5 LPCAT enzymes in diverse cell types to ascertain if there is differential expression of the LPCATs, which may help to explain the altered effects that LPCAT inhibition produces on inflammatory cytokine production between cell types. Data in this report did not demonstrate one particular LPCAT iso-form to be highly expressed by monocytes, however literature has suggested that LPCAT2 might be the enzyme to modulate the inducible phospholipid remodeling pathways in innate immune cells. Further studies on inducible LPCAT expression in human cell lines is required to support these ideas.Previous studies have observed LPCAT to regulate translocation of TLR4 into membrane lipid raft domains and subsequent down-stream inflammatory responses. Although the translocation of TLR2 into membrane lipid rafts has been researched, its regulation by LPCAT has not, thus it was investigated in this study. Complications with experiments, however, did not allow this to be adequately assessed but, literature suggests this is the likely mechanism that LPCAT elicits its immuno-regulatory effect in response to microbial stimuli.The present study provided evidence that LPCAT influences the complex network of cell signalling involved in microbial responses, underlying it’s importance in inflammatory responses and potentially offering a target for novel anti-sepsis therapies