COX-1 mediates IL-33-induced extracellular signal-regulated kinase activation in mast cells: Implications for aspirin sensitivity.

BACKGROUND: Classical FcεRI-induced mast cell (MC) activation causes synthesis of arachidonic acid (AA)-derived eicosanoids (leukotriene [LT] C4, prostaglandin [PG] D2, and thromboxane A2), which mediate vascular leak, bronchoconstriction, and effector cell chemotaxis. Little is known about the significance and regulation of eicosanoid generation in response to nonclassical MC activation mechanisms. OBJECTIVES: We sought to determine the regulation and significance of MC-derived eicosanoids synthesized in response to IL-33, a cytokine critical to innate type 2 immunity. METHODS: We used an ex vivo model of mouse bone marrow-derived mast cells and an IL-33-dependent in vivo model of aspirin-exacerbated respiratory disease (AERD). RESULTS: IL-33 potently liberates AA and elicits LTC4, PGD2, and thromboxane A2 production by bone marrow-derived mast cells. Unexpectedly, the constitutive function of COX-1 is required for IL-33 to activate group IVa cytosolic phospholipase A2 with consequent AA release for synthesis of all eicosanoids, including CysLTs. In contrast, COX-1 was dispensable for FcεRI-driven CysLT production. Inhibition of COX-1 prevented IL-33-induced phosphorylation of extracellular signal-related kinase, an upstream effector of cytosolic phospholipase A2, which was restored by exogenous PGH2, implying that the effects of COX-1 required its catalytic function. Administration of a COX-1-selective antagonist to mice completely prevented the generation of both PGD2 and LTC4 in a model of AERD in which MC activation is IL-33 driven. CONCLUSIONS: MC-intrinsic COX-1 amplifies IL-33-induced activation in the setting of innate type 2 immunity and might help explain the phenomenon of therapeutic desensitization to aspirin by nonselective COX inhibitors in patients with AERD.

Role of lipid mediators and control of lymphocyte responses in type 2 immunopathology.

Type 2 immunopathology is a cardinal feature of allergic diseases and involves cooperation between adaptive immunity and innate effector responses. Virtually all cell types relevant to this pathology generate leukotriene and/or prostaglandin mediators that derive from arachidonic acid, express receptors for such mediators, or both. Recent studies highlight prominent functions for these mediators in communication between the innate and adaptive immune systems, as well as amplification or suppression of type 2 effector responses. This review focuses on recent advances and insights, and highlights existing and potential therapeutic applications of drugs that target these mediators or their receptors, with a special emphasis on their regulation of the innate and adaptive lymphocytes relevant to type 2 immunopathology.

Endogenous prostaglandin E2 amplifies IL-33 production by macrophages through an E prostanoid (EP)2/EP4-cAMP-EPAC-dependent pathway.

When activated through toll-like receptors (TLRs), macrophages generate IL-33, an IL-1 family member that induces innate immune responses through ST2 signaling. LPS, a TLR4 ligand, induces macrophages to generate prostaglandin E2 (PGE2) through inducible COX-2 and microsomal PGE2 synthase 1 (mPGES-1) (1). We demonstrate that IL-33 production by bone marrow-derived murine macrophages (bmMFs) requires the generation of endogenous PGE2 and the intrinsic expression of EP2 receptors to amplify NF-κB-dependent, LPS-induced IL-33 expression via exchange protein activated by cAMP (EPAC). Compared with WT cells, bmMFs lacking either mPGES-1 or EP2 receptors displayed reduced LPS-induced IL-33 levels. A selective EP2 agonist and, to a lesser extent, EP4 receptor agonist potentiated LPS-induced IL-33 generation from both mPGES-1-null and WT bmMFs, whereas EP1 and EP3 receptor agonists were inactive. The effects of PGE2 depended on cAMP, were mimicked by an EPAC-selective agonist, and were attenuated by EPAC-selective antagonism and knockdown. LPS-induced p38 MAPK and NF-κB activations were necessary for both IL-33 production and PGE2 generation, and exogenous PGE2 partly reversed the suppression of IL-33 production caused by p38 MAPK and NF-κB inhibition. Mice lacking mPGES-1 showed lower IL-33 levels and attenuated lung inflammation in response to repetitive Alternaria inhalation challenges. Cumulatively, our data demonstrate that endogenous PGE2, EP2 receptors, and EPAC are prerequisites for maximal LPS-induced IL-33 expression and that exogenous PGE2 can amplify IL-33 production via EP2 and EP4 receptors. The ubiquitous induction of mPGES-1-dependent PGE2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders.

Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation.

Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by ß7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. ß7High MCs expand and mature during lung inflammation as part of a TGF-ß-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-ß-mediated development and found that epithelial cells directly elicit TGF-ß-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.

Lipid Profile of Activated Macrophages and Contribution of Group V Phospholipase A2.

Macrophages activated by Interleukin (IL)-4 (M2) or LPS+ Interferon (IFN)γ (M1) perform specific functions respectively in type 2 inflammation and killing of pathogens. Group V phospholipase A2 (Pla2g5) is required for the development and functions of IL-4-activated macrophages and phagocytosis of pathogens. Pla2g5-generated bioactive lipids, including lysophospholipids (LysoPLs), fatty acids (FAs), and eicosanoids, have a role in many diseases. However, little is known about their production by differentially activated macrophages. We performed an unbiased mass-spectrometry analysis of phospholipids (PLs), LysoPLs, FAs, and eicosanoids produced by Wild Type (WT) and Pla2g5-null IL-4-activated bone marrow-derived macrophages (IL-4)BM-Macs (M2) and (LPS+IFNγ)BM-Macs (M1). Phosphatidylcholine (PC) was preferentially metabolized in (LPS+IFNγ)BM-Macs and Phosphatidylethanolamine (PE) in (IL-4)BM-Macs, with Pla2g5 contributing mostly to metabolization of selected PE molecules. While Pla2g5 produced palmitic acid (PA) in (LPS+IFNγ)BM-Macs, the absence of Pla2g5 increased myristic acid (MA) in (IL-4)BM-Macs. Among eicosanoids, Prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) were significantly reduced in (IL-4)BM-Macs and (LPS+IFNγ)BM-Macs lacking Pla2g5. Instead, the IL-4-induced increase in 20-carboxy arachidonic acid (20CooH AA) was dependent on Pla2g5, as was the production of 12-hydroxy-heptadecatrienoic acid (12-HHTrE) in (LPS+IFNγ)BM-Macs. Thus, Pla2g5 contributes to PE metabolization, PGE2 and PGD2 production independently of the type of activation, while in (IL-4)BM-Macs, Pla2g5 regulates selective lipid pathways and likely novel functions.

Harmful and protective roles of group V phospholipase A2: Current perspectives and future directions.

Group V Phospholipase A2 (Pla2g5) is a member of the PLA2 family of lipid-generating enzymes. It is expressed in immune and non-immune cell types and is inducible during several pathologic conditions serving context-specific functions. In this review, we recapitulate the protective and detrimental functions of Pla2g5 investigated through preclinical and translational approaches. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

Expression of the ARPC4 subunit of human Arp2/3 severely affects mycobacterium tuberculosis growth and suppresses immunogenic response in murine macrophages.

BACKGROUND: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/3) protein complex, severely affects the pathogen's growth. TEM studies display shedding of the mycobacterial outer-coat. Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization. CONCLUSIONS/SIGNIFICANCE: With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics.

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP.

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.