Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Differential Macrophage Subsets in Muscle Damage Induced by a K49-PLA2 from Bothrops jararacussu Venom Modulate the Time Course of the Regeneration Process.

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.

ATP-Gated P2X7 Receptors Require Chloride Channels To Promote Inflammation in Human Macrophages.

Immune cells of myeloid origin show robust expression of ATP-gated P2X7 receptors, two-transmembrane ion channels permeable to Na+, K+, and Ca2+ Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1ß and IL-18. In this study, we show that ATP generates facilitating cationic currents in monocyte-derived human macrophages and permeabilizes the plasma membrane to polyatomic cationic dyes. We find that antagonists of PLA2 and Cl- channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1ß release. Our data demonstrate significant differences in the actions of ATP in murine and human macrophages and suggest that PLA2 and Cl- channels mediate innate immunity downstream of P2X7 receptors in human macrophages.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Dectin-1/2-induced autocrine PGE2 signaling licenses dendritic cells to prime Th2 responses.

The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from Schistosoma mansoni, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE2), which subsequently-in an autocrine manner-induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE2 synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1-independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2-/-, and to a lesser extent Dectin-1-/- mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following S. mansoni infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE2-OX40L through which Th2 immune responses are induced.

Secreted PLA2 group X orchestrates innate and adaptive immune responses to inhaled allergen.

Phospholipase A2 (PLA2) enzymes regulate the formation of eicosanoids and lysophospholipids that contribute to allergic airway inflammation. Secreted PLA2 group X (sPLA2-X) was recently found to be increased in the airways of asthmatics and is highly expressed in airway epithelial cells and macrophages. In the current study, we show that allergen exposure increases sPLA2-X in humans and in mice, and that global deletion of Pla2g10 results in a marked reduction in airway hyperresponsiveness (AHR), eosinophil and T cell trafficking to the airways, airway occlusion, generation of type-2 cytokines by antigen-stimulated leukocytes, and antigen-specific immunoglobulins. Further, we found that Pla2g10-/- mice had reduced IL-33 levels in BALF, fewer type-2 innate lymphoid cells (ILC2s) in the lung, less IL-33-induced IL-13 expression in mast cells, and a marked reduction in both the number of newly recruited macrophages and the M2 polarization of these macrophages in the lung. These results indicate that sPLA2-X serves as a central regulator of both innate and adaptive immune response to proteolytic allergen.

Safety of essential bee venom pharmacopuncture as assessed in a randomized controlled double-blind trial.

ETHNOPHARMACOLOGICAL RELEVANCE: While bee venom (BV) pharmacopuncture use is common in Asia, frequent occurrence of allergic reactions during the treatment process is burdensome for both practitioner and patient. AIM OF THE STUDY: This study compared efficacy and safety in isolated and purified essential BV (eBV) pharmacopuncture filtered for phospholipase A2 (PLA2) and histamine sections, and original BV to the aim of promoting safe BV pharmacopuncture use. MATERIALS AND METHODS: In in vitro, we examined the effect of BV and eBV on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages, and clinically, 20 healthy adults aged 20-40 years were randomly allocated and administered eBV 0.2mL and BV pharmacopuncture 0.2mL on left and right forearm, respectively, and physician, participant, and outcome assessor were blinded to treatment allocation. Local pain, swelling, itching, redness, wheals, and adverse reactions were recorded by timepoint. RESULTS: eBV and BV exhibited similar inhibitory effects on NO production. Also, in comparison between eBV and BV pharmacopuncture administration areas on each forearm, eBV displayed significantly lower local pain at 24h post-administration (P=0.0062), and less swelling at 30min (P=0.0198), 2 (P=0.0028), 24 (P=0.0068), and 48h post-administration (P=0.0253). eBV also showed significantly less itching at 24 (P=0.0119), 48 (P=0.0082), and 96h (P=0.0141), while redness was significantly less at 30min (P=0.0090), 6 (P=0.0005), and 24h (P<0.0001). Time-by-treatment interactions were statistically significant for itching and redness (P<0.001, and P<0.001, respectively), and all original BV pharmacopuncture administered regions showed a tendency toward more severe itching and redness in later measurements. CONCLUSIONS: eBV and BV displayed comparable anti-inflammatory effects, and eBV pharmacopuncture presented less local allergic reactions.

Cytosolic phospholipase A2 contributes to innate immune defense against Candida albicans lung infection.

BACKGROUND: The lung is exposed to airborne fungal spores, and fungi that colonize the oral cavity such as Candida albicans, but does not develop disease to opportunistic fungal pathogens unless the immune system is compromised. The Group IVA cytosolic phospholipase A2 (cPLA2α) is activated in response to Candida albicans infection resulting in the release of arachidonic acid for eicosanoid production. Although eicosanoids such as prostaglandins and leukotrienes modulate inflammation and immune responses, the role of cPLA2α and eicosanoids in regulating C. albicans lung infection is not understood. METHODS: The responses of cPLA2α(+/+) and cPLA2α(-/-) Balb/c mice to intratracheal instillation of C. albicans were compared. After challenge, we evaluated weight loss, organ fungal burden, and the recruitment of cells and the levels of cytokines and eicosanoids in bronchoalveolar lavage fluid. The ability of macrophages and neutrophils from cPLA2α(+/+) and cPLA2α(-/-) mice to recognize and kill C. albicans was also compared. RESULTS: After C. albicans instillation, cPLA2α(+/+) mice recovered a modest weight loss by 48 h and completely cleared fungi from the lung by 12 h with no dissemination to the kidneys. In cPLA2α(-/-) mice, weight loss continued for 72 h, C. albicans was not completely cleared from the lung and disseminated to the kidneys. cPLA2α(-/-) mice exhibited greater signs of inflammation including higher neutrophil influx, and elevated levels of albumin and pro-inflammatory cytokines/chemokines (IL1α, IL1ß, TNFα, IL6, CSF2, CXCL1, CCL20) in bronchoalveolar lavage fluid. The amounts of cysteinyl leukotrienes, thromboxane B2 and prostaglandin E2 were significantly lower in bronchoalveolar lavage fluid from C. albicans-infected cPLA2α(-/-) mice compared to cPLA2α(+/+) mice. Alveolar macrophages and neutrophils from uninfected cPLA2α(-/-) mice exhibited less killing of C. albicans in vitro than cells from cPLA2α(+/+) mice. In addition alveolar macrophages from cPLA2α(-/-) mice isolated 6 h after instillation of GFP-C. albicans contained fewer internalized fungi than cPLA2α(+/+) macrophages. CONCLUSIONS: The results demonstrate that cPLA2α contributes to immune surveillance and host defense in the lung to prevent infection by the commensal fungus C. albicans and to dampen inflammation.

Lysophosphatidylcholine plays critical role in allergic airway disease manifestation.

Phospholipase A2 (sPLA2), pivotal for allergic and inflammatory response, hydrolyses phosphatidylcholine (PC) to lysophosphatidylcholine (LPC). In present study, the role of LPC in allergic airway disease manifestation was studied using mouse model. Balb/c mice were immunized using cockroach extract (CE) and LPC release was blocked by sPLA2 inhibitor. Airway hyperresponse (AHR), lung-histology, total and differential leukocyte count (TLC&DLC), Th2 type cytokines, sPLA2 activity and LPC levels in bronchoalveolar lavage fluid (BALF) were measured. Exogenous LPC was given to the mice with or without CE sensitization, to demonstrate its role in allergic airway disease manifestation. Anti-CD1d antibody was given to study the involvement of natural killer T (NKT) cells in LPC induced response. AHR, lung-inflammation, TLC, DLC, Th2 type cytokines, sPLA2 activity and LPC levels were increased on CE challenge. sPLA2 activity and LPC release was blocked by sPLA2-inhibitor, which decreased AHR, and inflammatory parameters. Exogenous LPC with or without CE sensitization increased above parameters. CE challenge or LPC exposure increased LY49C(+)TCRß(+) NKT cells in BALF and spleen, which was reduced by anti-CD1d antibody, accompanied with reduction in AHR and allergic airway inflammation parameters. Conclusively, LPC induces allergic airway disease manifestation and it does so probably via CD1d-restricted LY49C(+)TCRß(+) NKT cells.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

BACKGROUND: The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. METHODS: Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2ß, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. RESULTS: The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2ß or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis. CONCLUSION: iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

Consequences of periodic α-to-ß(3) residue replacement for immunological recognition of peptide epitopes.

Oligomers that contain both α- and ß-amino acid residues, or "α/ß-peptides", have emerged as promising mimics of signal-bearing polypeptides that can inhibit or augment natural protein-protein interactions. α/ß-Peptides that contain a sufficient proportion of ß residues evenly distributed along the sequence can be highly resistant to enzymatic degradation, which is favorable with regard to in vivo applications. Little is known, however, about recognition of α/ß-peptides by the immune system. Prior studies have focused almost entirely on examples that contain a single ß residue; such α/ß-peptides frequently retain the immunological profile of the analogous α-peptide. We have conducted α-peptide vs α/ß-peptide comparisons involving higher ß residue content, focusing on molecules with αααß and ααßαααß backbone repeat patterns. Among analogues of an 18-mer derived from the Bim BH3 domain and an 8-mer derived from secreted phospholipase-2 (sPLA2), we find that recognition by antibodies raised against the prototype α-peptide is suppressed by periodic α → ß replacements. Complementary studies reveal that antibodies raised against Bim BH3- or sPLA2-derived α/ß-peptides fail to recognize prototype α-peptides displaying identical side chain repertoires. Because polypeptides containing d-α-amino acid residues are of growing interest for biomedical applications, we included the enantiomer of the sPLA2-derived α-peptide in these studies; this d-peptide is fully competent as a hapten, but the resulting antibodies do not cross react with the enantiomeric peptide. Among analogues of the 9-mer CD8(+) T-cell viral epitope GP33, we observe that periodic α → ß replacements suppress participation in the MHC I + peptide + T-cell receptor ternary complexes that activate cytotoxic T-lymphocytes, due in part to disruption of MHC binding.

Structure-based grafting, mutation, and optimization of peptide inhibitors to fit in the active pocket of human secreted phospholipase A2: find new use of old Peptide agents with anti-inflammatory activity.

Phospholipase A2 (PLA2) is a key enzyme in the production of diverse mediators of inflammatory conditions, which possesses an open active pocket that is physicochemically compatible with a variety of small-molecule substrates and peptide inhibitors. Although various peptides and peptide analogues have been identified to have inhibitory activity against PLA2 originated from animals and plants, only very few were designed for human secreted PLA2 (hsPLA2), an attractive target of inflammatory arthritis. Considering that the catalytic domains of PLA2 family members across different species are highly conserved in primary sequence, advanced structure, and biological function, in this study, we proposed a synthetic pipeline to implement structure-based grafting, mutation, and optimization of peptide ligands from the snake PLA2-peptide complex crystal structures into the active pocket of apo hsPLA2 structure to computationally generate a large number of potential peptide inhibitors for hsPLA2, and the hsPLA2 inhibitory potency of few highly promising candidates arising from the theoretical analysis was determined. As might be expected, three peptides FLSFK, FLVYK, and FISYR showed relatively high inhibitory capability against hsPLA2, and other three ALSYK, LVFYA, and KGAILGFM were also modestly potent as they can suppress the enzymatic activity with observable doses. Further, the designed peptide FLVYK with highest potency was carried out with structure-guided modification based on its atomic interactions with hsPLA2 using the computationally modeled structure data, consequently resulting in a dual-point mutant ELIYK with significantly increased activity.

[Emerging roles of phospholipase A2s in mast cell biology].

  Tissue-resident mast cells are derived from circulating committed progenitors, which are originated from pluripotent hematopoietic stem cells in bone marrow. These progenitors migrate into extravascular tissues, where they undergo differentiation and maturation into tissue-specific mast cell phenotypes. When activated by antigen or microenvironmental factors, mast cells release various biologically active products, including pre-formed mediators stored in secretory granules, de novo synthesized lipid mediators, and newly transcribed cytokines and chemokines, thereby promoting anaphylactic inflammation as well as other acute and chronic inflammatory diseases. Here, I will highlight the newest understanding of the phospholipase A2 (PLA2)-driven lipid networks in the maturation and effector functions of mast cells and attendant allergic responses. Group III secreted PLA2, the sole mammalian homolog of the potent extrinsic anaphylaxis inducer bee venom PLA2, regulates mast cell maturation through the paracrine prostaglandin D2 (PGD2) circuit. While cytosolic PLA2α is essential for the generation of PGD2 and leukotriene C4 by mast cells, it is also functionally coupled, through the arachidonic acid transfer mechanism, with PGE2 synthase in stromal fibroblasts to provide anti-anaphylactic PGE2. In addition, the roles of two particular mast cell maturation-responsible genes, NDRG1 and NLRP3, in mast cells will be discussed.

Lysosomal phospholipase A2: a novel player in host immunity to Mycobacterium tuberculosis.

Phospholipases catalyze the cleavage of membrane phospholipids into smaller bioactive molecules. The lysosomal phospholipase A2 (LPLA2 ) is specifically expressed in macrophages. LPLA2 gene deletion in mice causes lysosomal phospholipid accumulation in tissue macrophages leading to phospholipidosis. This phenotype becomes most prominent in alveolar macrophages where LPLA2 contributes to surfactant phospholipid degradation. High expression of LPLA2 in alveolar macrophages prompted us to investigate its role in host immunity against the respiratory pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we report that adaptive immune responses to M. tuberculosis were impaired in LPLA2 deficient mice. Upon aerosol infection with M. tuberculosis, LPLA2 deficient mice showed enhanced mycobacterial counts but less lung immunopathology and pulmonary inflammatory responses. Compromised T-cell priming in the lymph nodes was associated with impaired pulmonary T-cell recruitment and activation. Together with reduced Th1 type cytokine production, these results indicate that LPLA2 is indispensable for the induction of adaptive T-cell immunity to M. tuberculosis. Taken together, we identified an unexpected and novel function of a lysosomal phospholipid-degrading enzyme.

Lipid biology of the podocyte--new perspectives offer new opportunities.

In the past 15 years, major advances have been made in understanding the role of lipids in podocyte biology. First, susceptibility to focal segmental glomerulosclerosis (FSGS) and glomerular disease is associated with an APOL1 sequence variant, is expressed in podocytes and encodes apolipoprotein L1, an important component of HDL. Second, acid sphingomyelinase-like phosphodiesterase 3b encoded by SMPDL3b has a role in the conversion of sphingomyelin to ceramide and its levels are reduced in renal biopsy samples from patients with recurrent FSGS. Furthermore, decreased SMPDL3b expression is associated with increased susceptibility of podocytes to injury after exposure to sera from these patients. Third, in many individuals with membranous nephropathy, autoantibodies against the phospholipase A2 (PLA2) receptor, which is expressed in podocytes, have been identified. Whether these autoantibodies affect the activity of PLA2, which liberates arachidonic acid from glycerophospholipids and modulates podocyte function, is unknown. Fourth, clinical and experimental evidence support a role for ATP-binding cassette sub-family A member 1-dependent cholesterol efflux, free fatty acids and glycerophospolipids in the pathogenesis of diabetic kidney disease. An improved understanding of lipid biology in podocytes might provide insights to develop therapeutic targets for primary and secondary glomerulopathies.

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

Macrophage VLDL receptor promotes PAFAH secretion in mother's milk and suppresses systemic inflammation in nursing neonates.

Mother's milk is widely accepted as nutritious and protective to the newborn mammals by providing not only macronutrients but also immune-defensive factors. However, the mechanisms accounting for these benefits are not fully understood. Here we show that maternal very-low-density-lipoprotein (VLDL) receptor deletion in mice causes the production of defective milk containing diminished levels of platelet-activating factor acetylhydrolase (PAFAH). As a consequence, the nursing neonates suffer from alopecia, anaemia and growth retardation owing to elevated levels of pro-inflammatory platelet-activating factors. VLDL receptor deletion significantly impairs the expression of phospholipase A2 group 7 (Pla2g7) in macrophages, which decreases PAFAH secretion. Exogenous oral supplementation of neonates with PAFAH effectively rescues the toxicity. These findings not only reveal a novel role of VLDL receptor in suppressing inflammation by maintaining macrophage PAFAH secretion, but also identify the maternal VLDL receptor as a key genetic program that ensures milk quality and protects the newborns.

Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68.

Seasonal influenza outbreaks and recurrent influenza pandemics present major challenges to public health. By studying immunological responses to influenza in different host species, it may be possible to discover common mechanisms of susceptibility in response to various influenza strains. This could lead to novel therapeutic targets with wide clinical application. Using a mouse-adapted strain of influenza (A/HK/1/68-MA20 [H3N2]), we produced a mouse model of severe influenza that reproduces the hallmark high viral load and overexpression of cytokines associated with susceptibility to severe influenza in humans. We mapped genetic determinants of the host response using a panel of 29 closely related mouse strains (AcB/BcA panel of recombinant congenic strains) created from influenza-susceptible A/J and influenza-resistant C57BL/6J (B6) mice. Combined clinical quantitative trait loci (QTL) and lung expression QTL mapping identified candidate genes for two sex-specific QTL on chromosomes 2 and 17. The former includes the previously described Hc gene, a deficit of which is associated with the susceptibility phenotype in females. The latter includes the phospholipase gene Pla2g7 and Tnfrsf21, a member of the TNFR superfamily. Confirmation of the gene underlying the chromosome 17 QTL may reveal new strategies for influenza treatment.

New insights into pathogenesis of exercise-induced bronchoconstriction.

PURPOSE OF REVIEW: Exercise-induced bronchoconstriction (EIB) refers to acute airflow obstruction that is triggered by a period of physical exertion. Here we review recent findings about the epidemiology of EIB, immunopathology leading to EIB, and the latest understanding of the pathogenesis of EIB. RECENT FINDINGS: Longitudinal studies demonstrated that airway hyper-responsiveness to exercise or cold air at an early age are among the strongest predictors of persistent asthma. Patients that are susceptible to EIB have epithelial disruption and increased levels of inflammatory eicosanoids such as cysteinyl leukotrienes (CysLT)s. The leukocytes implicated in production of eicosanoids in the airways include both a unique mast cell population as well as eosinophils. A secreted phospholipase A(2) (sPLA(2)) enzyme that serves as a regulator of CysLT formation is present in increased quantities in asthma. Transglutaminase 2 (TGM2) is expressed at increased levels in asthma and serves as a regulator of secreted phospholipase A(2) group X (sPLA(2)-X). Further, sPLA(2)-X acts on target cells such as eosinophils to initiate cellular eicosanoid synthesis. SUMMARY: Recent studies have advanced our understanding of EIB as a syndrome that is caused by the increased production of inflammatory eicosanoids. The airway epithelium may be an important regulator of the production of inflammatory eicosanoids by leukocytes.

The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.