Expression of cPLA2γ mRNA and protein differs the response of PBMC from severe and non-severe asthmatics to bacterial lipopolysaccharide and house dust mite allergen.

Chronic inflammation in asthmatics is initiated/exacerbated by many environmental factors, such as bacterial lipopolysaccharide and allergens. Phospholipase A2 and histone acetyltransferase/deacetylases are enzymes involved in inflammatory process, particularly in lipid inflammatory mediators production and control of transcription of many inflammatory genes, respectively. The aim of the study was to identify differences in the inflammatory process in patients with severe and non-severe asthma, taking as a criterion expression of two groups of enzymes: phospholipases A2 and histone acetyltransferases/deacetylases. Thirty-two patients with severe, non-severe atopic to house dust mite asthmatics and 14 healthy volunteers were recruited. Peripheral blood mononuclear cells were stimulated with Dermatophagoides pteronyssinus allergen (nDer p1) and bacterial lipopolysaccharide (LPS). The expression of phospholipases A2 and histone acetyltransferases and deacetylases were assessed using TaqMan Low Density Array Cards. The protein expression was analyzed with immunoblot. Increased expression of phospholipase A2 Group IVC (PLA2G4C) and cytosolic phospholipase A2 gamma (cPLA2γ) protein was observed in peripheral blood mononuclear cells (PBMC) from severe asthmatics in response to LPS and nDer p1, compared to non-severe asthmatics. nDer p1-stimulated PBMC from severe asthmatics exhibit induced expression of HDAC1 and similar trend was observed in protein concentration. Decreased expression of EP300 occurred in PBMC of severe asthmatics. PBMC from non-severe asthmatics showed decreased expression of HDAC2 and PLA2G15 after LPS treatment. In conclusion, in response to LPS and dust mite allergen, PBMC from severe and non-severe asthmatics modulate expression of selected phospholipase A2, histone acetyltransferases and deacetylases, while increased expression of cPLA2γ characterizes PBMC response from severe asthmatics.

Current knowledge on autoantigens and autoantibodies in psoriasis.

In the past decades, clinical and experimental evidence has demonstrated that psoriasis is an immune-mediated inflammatory disease of the skin that occurs in genetically susceptible individuals. Psoriasis also shows clear autoimmune pathomechanisms, but specific cellular targets for the onset and maintenance of psoriatic lesions were not established until 2014. Since then, four psoriasis autoantigens were discovered, namely cathelicidin LL-37, melanocytic ADAMTSL5, lipid antigen PLA2G4D and keratin 17. Autoreactive T cells against these autoantigens were found in a number of patients with moderate-to-severe plaque psoriasis. Moreover, the discovery of autoantibodies against LL-37 and ADAMTSL5 and their strong association with psoriatic arthritis (PsA) suggest a potential role of these autoantibodies in the pathogenesis of PsA. This review discusses the current studies on psoriatic autoantigens and the associated circulating autoantibodies and their mechanisms involved in the development and maintenance of psoriatic plaques. Recent autoimmune evidence fuelled the discussion on psoriasis as an autoimmune skin disorder and has the potential to develop new treatment strategies with protective and therapeutic antigen-targeted methods.

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a.

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

The step further to understand the role of cytosolic phospholipase A2 alpha and group X secretory phospholipase A2 in allergic inflammation: pilot study.

Allergens, viral, and bacterial infections are responsible for asthma exacerbations that occur with progression of airway inflammation. cPLA2 α and sPLA2X are responsible for delivery of arachidonic acid for production of eicosanoids-one of the key mediators of airway inflammation. However, cPLA2 α and sPLA2X role in allergic inflammation has not been fully elucidated. The aim of this study was to analyze the influence of rDer p1 and rFel d1 and lipopolysaccharide (LPS) on cPLA2 α expression and sPLA2X secretion in PBMC of asthmatics and in A549 cell line. PBMC isolated from 14 subjects, as well as A549 cells, were stimulated with rDer p1, rFel d1, and LPS. Immunoblotting technique was used to study the changes in cPLA2 α protein expression and ELISA was used to analyze the release of sPLA2X. PBMC of asthmatics released more sPLA2X than those from healthy controls in the steady state. rDer p1 induced more sPLA2X secretion than cPLA2 α protein expression. rFel d1 caused decrease in cPLA2 α relative expression in PBMC of asthmatics and in A549 cells. Summarizing, Der p1 and Fel d1 involve phospholipase A2 enzymes in their action. sPLA2X seems to be one of important PLA2 isoform in allergic inflammation, especially caused by house dust mite allergens.

Phospholipase A2 regulates eicosanoid class switching during inflammasome activation.

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

Citrus allergy from pollen to clinical symptoms.

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and ß) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.

Cytosolic phospholipase A2α blockade abrogates disease during the tissue-damage effector phase of experimental autoimmune encephalomyelitis by its action on APCs.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

Cytosolic phospholipase A2α gene silencing in the myeloid lineage alters development of Th1 responses and reduces disease severity in collagen-induced arthritis.

OBJECTIVE: Several lines of evidence implicate cytosolic phospholipase A(2)α (cPLA(2)α) as a critical enzyme in inflammatory disorders, including rheumatoid arthritis. Since cells from the myeloid compartment regulate local and systemic disease pathogenesis, the present study was undertaken to examine the effect of cPLA(2)α inhibition in experimental arthritis, using a delivery system tailored to target monocyte functions by RNA interference (RNAi). METHODS: Mice with collagen-induced arthritis (CIA) were injected intravenously with an anti-cPLA(2)α small interfering RNA (siRNA) sequence (siPLA2) formulated as lipoplexes with the RPR209120/DOPE cationic liposome and a carrier DNA. The clinical course of joint inflammation was assessed, and the immunologic balance was analyzed by measuring T helper cell frequencies and cytokine expression. Biodistribution studies of siRNA were also performed. RESULTS: Weekly systemic injection of siPLA2 lipoplexes significantly reduced the incidence and severity of CIA, in both preventive and curative settings, as compared with findings in control animals. Histologic scores for inflammation and cartilage damage were reduced. The clinical effect was associated with local inhibition of tumor necrosis factor α secretion and lower cPLA(2)α expression and activity. The siPLA2 lipoplexes enabled triggering of in vivo RNAi-mediated gene silencing of cPLA(2)α in CD11b+ cells recovered from the spleen. While the treatment had no effect on anti-type II collagen (anti-CII) antibodies, CII-specific T helper cells producing interferon-γ, but not interleukin-17, in draining lymph node cells were decreased. CONCLUSION: Our findings indicate that systemic RNAi-mediated cPLA(2)α gene silencing in CD11b+ cells is effective in the treatment of CIA, and Th1 suppression is one of the potential underlying mechanisms, whereas Th17 suppression is not.

Pulmonary surfactant phosphatidylglycerol inhibits Mycoplasma pneumoniae-stimulated eicosanoid production from human and mouse macrophages.

Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38(MAPK) and p44/42(ERK1/2) MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD(2) and PGE(2) from RAW264.7 cells and thromboxane B(2) (TXB(2)) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42(ERK1/2) or inactivation of cytosolic phospholipase A(2)α (cPLA(2)α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.

A ceramide analog inhibits cPLA(2) activity and consequent PGE(2) formation in LPS-stimulated macrophages.

Prostaglandin E(2) (PGE(2)) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE(2) production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE(2) production in macrophages. Inhibition of PGE(2) production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA(2)α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA(2)α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE(2) production in LPS-stimulated macrophages by direct interaction with cPLA(2), and suggest that ceramide may similarly counteract C1P effect on cPLA(2) activity in cells. The suppression of PGE(2) production is suggested to contribute to the anti-inflammatory action of PCERA-1.

Cytosolic phospholipase a2 activation by Candida albicans in alveolar macrophages: role of dectin-1.

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

Calcium-independent phospholipase A2beta-Akt signaling is involved in lipopolysaccharide-induced NADPH oxidase 1 expression and foam cell formation.

Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.

Calcium-independent phospholipase A2 beta is dispensable in inflammasome activation and its inhibition by bromoenol lactone.

Calcium-independent phospholipase A2 (iPLA2) has been suggested to play an important role in the activation of caspase-1 induced by lipopolysaccharides (LPS). Here, we used pharmacological and genetic approaches to study the role of iPLA 2 in the activation of caspase-1. Bromoenol lactone (BEL), an inhibitor that was originally used to support a role for iPLA2 in the secretion of IL-1 beta, prevented caspase-1 activation induced by LPS and ATP as described, and also activation triggered by Salmonella infection and cytosolic flagellin, which rely on the Nlrc4 inflammasome. Analysis of BEL enantiomers showed that the S-BEL form was more effective than R-BEL in inhibiting the inflammasome, suggesting a role for iPLA2 . However, caspase-1 activation and IL-1 beta secretion and their inhibition by BEL were unimpaired in macrophages deficient in iPLA2 beta. BEL was originally identified as an inhibitor of serine proteases. Consistent with the latter, the serine proteases inhibitors TPCK, TLCK and AAF-cmk prevented the activation of the Nlrc4 and Nlrp3 inflammasomes while pan-cathepsin inhibitors were ineffective. These results indicate that iPLA2 beta is not critical for caspase-1 activation as currently proposed. Instead, the results suggest that serine protease(s) targeted by BEL may play a critical role in the activation of the inflammasome triggered by microbial stimuli.

In vitro allergen challenge of peripheral blood induces differential gene expression in mononuclear cells of asthmatic patients: inhibition of cytosolic phospholipase A2alpha overcomes the asthma-associated response.

BACKGROUND: Existing treatments for asthma are not effective in all patients and disease exacerbations are common, highlighting the need for increased understanding of disease mechanisms and novel treatment strategies. The leukotriene pathway including the enzyme responsible for arachidonic acid release from cellular phospholipids, cPLA(2)alpha, is a major contributor to asthmatic responses and an attractive target in asthma therapies. OBJECTIVE: The study reported here investigates (a) the differential effects of in vitro exposure of peripheral blood mononuclear cells (PBMCs) to allergen between asthma and healthy subjects, and (b) the contribution of cPLA(2)alpha to these differences in gene expression. METHODS: In vitro responses of asthma (N=26) and healthy (N=11) subject PBMC samples to allergen stimulation in the presence and absence of cPLA(2)alpha inhibition or 5-lipoxygenase inhibition were compared at the gene expression level using oligonucleotide arrays and at the protein level using ELISA. RESULTS: Subject samples within both asthma and healthy groups showed allergen-dependent cytokine production and allergen-dependent gene expression changes, although transcriptional profiling identified 153 genes that were modulated significantly differently by allergen between asthma and healthy subjects. Among these were genes previously associated with asthma, but the majority (about 80%) have not previously been associated with asthma. CONCLUSIONS: Transcriptional profiling elucidated novel gene expression differences between the asthmatic and healthy subject samples. Although 5-lipoxygenase inhibition did not significantly affect allergen-modulated gene expression, the inhibition of cPLA(2)alpha activity affected many of the allergen-dependent, asthma-associated gene expression changes.

iPLA2beta: front and center in human monocyte chemotaxis to MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca(2+)-independent phospholipase (iPLA(2)beta) and cytosolic phospholipase (cPLA(2)alpha), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA(2)beta is recruited to the membrane-enriched pseudopod. In contrast, cPLA(2)alpha is recruited to the endoplasmic reticulum. Although iPLA(2)beta or cPLA(2)alpha antisense oligodeoxyribonucleotide (ODN)-treated monocytes display reduced speed, iPLA(2)beta also regulates directionality and actin polymerization. iPLA(2)beta or cPLA(2)alpha antisense ODN-treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA(2)beta and cPLA(2)alpha regulate monocyte migration from different intracellular locations, with iPLA(2)beta acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

Activation of cytosolic phospholipase A2alpha in resident peritoneal macrophages by Listeria monocytogenes involves listeriolysin O and TLR2.

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.