Distinct roles of Ca2+ mobilization and G protein usage on regulation of Toll-Like Receptor Function in Human and Murine Mast Cells

Toll-like receptors (TLRs) expressed in mast cells play important roles in orchestrating host defence against bacterial pathogens. Previous studies demonstrated that TLR2 agonist tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) stimulates both degranulation and cytokine production in human mast cells but only induces cytokine production in murine mast cells. To determine the molecular basis for this difference, we utilized a human mast cell line LAD 2, murine lung and bone marrow-derived mast cells (MLMC and BMMC). We found that Pam3Cys caused a sustained Ca2+ mobilization and degranulation in LAD 2 mast cells but not in MLMC or BMMC. Despite these differences, Pam3Cys stimulated equivalent chemokine CCL2 generation in all mast cell types tested. Cyclosporin A (CsA), an inhibitor of Ca 2+/calcineurin-mediated nuclear factor of activated T cells (NFAT) activation, blocked chemokine production in LAD 2 but not in MLMC or BMMC. In contrast, inhibitors of nuclear factor kappa B (NF-κB) completely blocked CCL2 production in MLMC and BMMC but not in LAD 2 mast cells. Pertussis toxin and U0126, which, respectively, inhibit Gαi, extracellular signal-regulated kinase (ERK) phosphorylation substantially inhibited Pam 3Cys-induced CCL2 generation in LAD 2 mast cells but had little or no effect on chemokine generation in MLMC and BMMC. These findings suggest that TLR2 activation in human LAD 2 mast cells and MLMC/BMMC promotes the release of different classes of mediators via distinct signalling pathways that depend on Ca2+ mobilization and G protein usage. © 2006 Blackwell Publishing Ltd

Platelet-Activating Factor-Induced Chemokine Gene Expression Requires NF-κB Activation and Ca2+/calcineurin Signaling Pathways: Inhibition by Receptor Phosphorylation and β-Arrestin Recruitment

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing earboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-κB activation and CCL2 production. PAF caused translocation of β-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (AST-PAFR). Interestingly, PAF induced significantly higher NF-κB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing ΔST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-κB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-κB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and β-arrestin recruitment inhibit CCL2 production by blocking both NF-κB activation and Ca 2+/calcineurin-dependent signaling pathway

A Requirement for slc15a4 in Imiquimod-Induced Systemic Inflammation and Psoriasiform Inflammation in Mice

There is competing evidence that plasmacytoid dendritic cells (pDC), the most potent source of IFN-I, may initiate psoriasis. We targeted pDC function using the slc15a4 loss-of-function mouse whose pDC are unresponsive to TLR agonists. slc15a4 treated with the topical TLR7-agonist imiquimod (IMQ) demonstrated decreased epidermal thickening 24 hours post-treatment which was more pronounced by day 5 as compared to wildtype mice. These findings were specific to the acute IMQ model and not the protracted IL23 model that drives inflammation downstream of TLR activation. Systemically, slc15a4 was required for IMQ-induced weight loss and cutaneous accumulation of CD4+ and Siglec H+, but not CD11b+ cells. Consistent with this phenotype and the function of slc15a4, induction of IFN-I was virtually absent systemically and via cutaneous gene expression. Induction of other inflammatory cytokines (cytokine storm) was modestly blunted in slc15a4 except for inflammasome-associated genes consistent with slc15a4 being required for TLR7-mediated (but not inflammasome-mediated) inflammation downstream of IMQ. Surprisingly, only IFN-I gene expression was suppressed within IMQ-treated skin. Other genes including conserved psoriasiform trademark gene expression were augmented in slc15a4 versus littermate controls. Taken together, we have identified a role for slc15a4 but not canonical psoriasiform genes in the imiquimod model of psoriasiform dermatitis

Proteome Analysis Implicates Adaptive Changes in Metabolism and Body Wall Musculature of Caenorhabditis elegans Dauer Larva

Dauer larva is an alternative developmental stage of Caenorhabditis elegans (C. elegans) that occurs when the environmental condition is unfavorable for growth. Little is known regarding how the proteome of dauer larvae respond to  poor environmental growth conditions. Such knowledge is expected to help understand the survival mechanism(s) of dauer larvae. In order to uncover the proteome differences between dauer larvae and normally developed third stage larvae (L3),  an L2 stage larvae was starved to create the dauer larvae and this proteome was compared with that of the L3 larvae. Results showed that proteins involved in muscle assembly and fatty acid oxidation are increased in dauer larvae, while proteins involved in maintaining regular organismic activity such as reproduction, translation and apoptotic processes are decreased. The protein expression profile also suggested that the glyoxylate cycle is preferentially utilized during dauer arrest over the tricarboxylic acid (TCA) cycle and significant structural rearrangement occurs on the hypodermis, body wall musculature, and pharynx.

A requirement for slc15a4 in imiquimod-induced systemic inflammation and psoriasiform inflammation in mice

Abstract There is competing evidence that plasmacytoid dendritic cells (pDC), the most potent source of IFN-I, may initiate psoriasis. We targeted pDC function using the slc15a4 feeble loss-of-function mouse whose pDC are unresponsive to TLR agonists. slc15a4 feeble treated with the topical TLR7-agonist imiquimod (IMQ) demonstrated decreased epidermal thickening 24 hours post-treatment which was more pronounced by day 5 as compared to wildtype mice. These findings were specific to the acute IMQ model and not the protracted IL23 model that drives inflammation downstream of TLR activation. Systemically, slc15a4 was required for IMQ-induced weight loss and cutaneous accumulation of CD4+ and Siglec H+, but not CD11b+ cells. Consistent with this phenotype and the function of slc15a4, induction of IFN-I was virtually absent systemically and via cutaneous gene expression. Induction of other inflammatory cytokines (cytokine storm) was modestly blunted in slc15a4 feeble except for inflammasome-associated genes consistent with slc15a4 being required for TLR7-mediated (but not inflammasome-mediated) inflammation downstream of IMQ. Surprisingly, only IFN-I gene expression was suppressed within IMQ-treated skin. Other genes including conserved psoriasiform trademark gene expression were augmented in slc15a4 feeble versus littermate controls. Taken together, we have identified a role for slc15a4 but not canonical psoriasiform genes in the imiquimod model of psoriasiform dermatitis

Distinct roles of Ca(2+) mobilization and G protein usage on regulation of Toll-like receptor function in human and murine mast cells

Toll-like receptors (TLRs) expressed in mast cells play important roles in orchestrating host defence against bacterial pathogens. Previous studies demonstrated that TLR2 agonist tripalmitoyl-S-glycero-Cys-(Lys)(4) (Pam(3)Cys) stimulates both degranulation and cytokine production in human mast cells but only induces cytokine production in murine mast cells. To determine the molecular basis for this difference, we utilized a human mast cell line LAD 2, murine lung and bone marrow-derived mast cells (MLMC and BMMC). We found that Pam(3)Cys caused a sustained Ca(2+) mobilization and degranulation in LAD 2 mast cells but not in MLMC or BMMC. Despite these differences, Pam(3)Cys stimulated equivalent chemokine CCL2 generation in all mast cell types tested. Cyclosporin A (CsA), an inhibitor of Ca(2+)/calcineurin-mediated nuclear factor of activated T cells (NFAT) activation, blocked chemokine production in LAD 2 but not in MLMC or BMMC. In contrast, inhibitors of nuclear factor kappa B (NF-κB) completely blocked CCL2 production in MLMC and BMMC but not in LAD 2 mast cells. Pertussis toxin and U0126, which, respectively, inhibit Gα(i,) extracellular signal-regulated kinase (ERK) phosphorylation substantially inhibited Pam(3)Cys-induced CCL2 generation in LAD 2 mast cells but had little or no effect on chemokine generation in MLMC and BMMC. These findings suggest that TLR2 activation in human LAD 2 mast cells and MLMC/BMMC promotes the release of different classes of mediators via distinct signalling pathways that depend on Ca(2+) mobilization and G protein usage