Lysophosphatidylcholine acyltransferase 2 (LPCAT2) co-localises with TLR4 and regulates macrophage inflammatory gene expression in response to LPS.

Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system.

Heat-killed probiotic bacteria differentially regulate colonic epithelial cell production of human ß-defensin-2: dependence on inflammatory cytokines.

The inducible antimicrobial peptide human ß-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1ß and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1ß-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1ß-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1ß-induced IL-10. LcS upregulated IL-1ß-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1ß-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1ß-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1ß-stimulation, whereas LF further suppressed IL-1ß-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.