Prdx6 (peroxiredoxin 6), a bifunctional protein with both GSH peroxidase and PLA2 (phospholipase A2) [aiPLA2 (acidic calcium-independent PLA2)] activities, is responsible for the metabolism of lung surfactant phospholipids. We propose that the aiPLA2 activity of the enzyme is regulated through phosphorylation. Incubation of isolated rat alveolar type II cells (AECII) with PMA, a PKC (protein kinase C) agonist, had no effect on Prdx6 expression but led to ~75 % increase in aiPLA2 activity that was abolished by pretreatment of cells with the MAPK (mitogen-activated protein kinase) inhibitors, SB202190 or PD98059. Prdx6 phosphorylation after incubation of AECII with PMA was demonstrated by autoradiography after immunoprecipitation with either anti-phosphothreonine or -phosphoserine antibodies. In vitro, several active isoforms of ERK (extracellular-signal-regulated kinase) and p38 phosphorylated Prdx6, resulting in an 11-fold increase in aiPLA2 activity. The increased activity was calcium-independent and was abolished by the aiPLA2 inhibitors, surfactant protein A and hexadecyl-3-trifluorethylglycero-sn-2-phospho-methanol (MJ33). The peroxidase activity of Prdx6 was unaffected by phosphorylation. Mass spectroscopic analysis of in vitro phosphorylated Prdx6 showed a unique phosphorylation site at Thr-177 and mutation of this residue abolished protein phosphorylation and the increase in MAPK-mediated activity. These results show that the MAPKs can mediate phosphorylation of Prdx6 at Thr-177 with a consequent marked increase in its aiPLA2 activity.

Previous studies have established that phagocytes are key cells of the pulmonary innate immune defense against A. fumigatus, an opportunistic fungus responsible of invasive pulmonary aspergillosis. Macrophages detect A. fumigatus via Toll-like receptors 2 and 4 (TLR2 and -4) and respond by the MyD88-NF-B-dependent synthesis of inflammatory mediators. In the present study, we demonstrate that respiratory epithelial cells also sense A. fumigatus and participate in the host defense. Thus, the interaction of respiratory epithelial cells with germinating but not resting conidia of A. fumigatus results in interleukin (IL)-8 synthesis that is controlled by phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2. Using MyD88-dominant negative transfected cells, we also show that IL-8 production is not dependent on the TLR-MyD88 pathway, although the MyD88 pathway is activated by A. fumigatus and leads to NF-B activation. Thus, our results provide evidence for the existence of two independent signaling pathways activated in respiratory epithelial cells by A. fumigatus, one that is MyD88-dependent and another that is My88-independent and involved in IL-8 synthesis.Aspergillus fumigatus is an opportunistic fungus responsible for invasive pulmonary aspergillosis (IPA), 2 a lifethreatening disease that usually only occurs in immunocompromised patients as inhalation of spores by those hosts results in fungal growth inside the lung and hematogenous dissemination (1, 2).The mechanisms of host resistance to IPA are not completely understood. It is generally accepted that macrophages and neutrophils represent the first two lines of innate host defense against A. fumigatus (3, 4). Specifically, alveolar macrophages phagocyte and kill conidia, whereas neutrophils lyse hyphae of germinated spores (5). However, it is now recognized that epithelial cells also play an important role in the innate defense (6 -8). Rather than just representing a physical barrier to prevent pathogen gaining unwanted access to essential organs, the epithelial layer provides a surface where the host can interact with pathogens. The airway epithelium senses micro-organisms and responds accordingly by increasing its defense. This response consists of the synthesis and release of antimicrobial peptides, cytokines, and chemokines. As do macrophages and neutrophils, epithelial cells can also sense microbial products through pattern recognition receptors that bind conserved molecular patterns expressed by micro-organisms.Toll-like receptors (TLRs) have been identified as a major class of pattern recognition receptors (9 -11). Recognition of pathogen-associated molecular patterns by TLRs triggers a cascade of cellular signals that culminates in the activation of NF-B, which leads to inflammatory gene expression and ultimately to the clearance of the infectious agents (11-14). There is accumulating evidence, based on in vitro and in vivo studies, that supports a role for TLRs in A. fumigatus sensing. Findings from various studies demonstrated that A. fumigatus recognit...

Young cystic fibrosis (CF) patients' airways are mainly colonized by Staphylococcus aureus, while Pseudomonas aeruginosa predominates in adults. However, the mechanisms behind this infection switch are unclear. Here, we show that levels of type-IIA-secreted phospholipase A2 (sPLA2-IIA, a host enzyme with bactericidal activity) increase in expectorations of CF patients in an age-dependent manner. These levels are sufficient to kill S. aureus, with marginal effects on P. aeruginosa strains. P. aeruginosa laboratory strains and isolates from CF patients induce sPLA2-IIA expression in bronchial epithelial cells from CF patients (these cells are a major source of the enzyme). In an animal model of lung infection, P. aeruginosa induces sPLA2-IIA production that favours S. aureus killing. We suggest that sPLA2-IIA induction by P. aeruginosa contributes to S. aureus eradication in CF airways. Our results indicate that a bacterium can eradicate another bacterium by manipulating the host immunity.

Maximal resection of tumor while preserving the adjacent healthy tissue is particularly important for larynx surgery, hence precise and rapid intraoperative histology of laryngeal tissue is crucial for providing optimal surgical outcomes. We hypothesized that deep-learning based stimulated Raman scattering (SRS) microscopy could provide automated and accurate diagnosis of laryngeal squamous cell carcinoma on fresh, unprocessed surgical specimens without fixation, sectioning or staining. Methods : We first compared 80 pairs of adjacent frozen sections imaged with SRS and standard hematoxylin and eosin histology to evaluate their concordance. We then applied SRS imaging on fresh surgical tissues from 45 patients to reveal key diagnostic features, based on which we have constructed a deep learning based model to generate automated histologic results. 18,750 SRS fields of views were used to train and cross-validate our 34-layered residual convolutional neural network, which was used to classify 33 untrained fresh larynx surgical samples into normal and neoplasia. Furthermore, we simulated intraoperative evaluation of resection margins on totally removed larynxes. Results : We demonstrated near-perfect diagnostic concordance (Cohen's kappa, κ > 0.90) between SRS and standard histology as evaluated by three pathologists. And deep-learning based SRS correctly classified 33 independent surgical specimens with 100% accuracy. We also demonstrated that our method could identify tissue neoplasia at the simulated resection margins that appear grossly normal with naked eyes. Conclusion : Our results indicated that SRS histology integrated with deep learning algorithm provides potential for delivering rapid intraoperative diagnosis that could aid the surgical management of laryngeal cancer.

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A 2 (PLA 2 ) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA 2 activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA 2 activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA 2 activity) showed K m 0.35 mM and V max 138 nmol/min/mg of protein. SP-A inhibited PLA 2 activity non-competitively with K i 10 g/ml and was Ca 2؉ -independent. Activity at pH 7.4 was ϳ50% less, and inhibition by SP-A was partially dependent on Ca 2؉ . Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni 2؉ -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca 2؉ dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA 2 activity of Prdx6 by SP-A.Pulmonary surfactant, a lipoprotein complex lining the lung surface, consists of phospholipids, specific proteins, and other lipid components. It is synthesized by alveolar type II epithelial cells, assembled in lamellar bodies, the intracellular surfactant storage organelle, and secreted into the alveolar space and terminal airways where it functions to reduce surface tension and stabilize alveoli (1). Dipalmitoylphosphatidylcholine (DPPC), 2 the major phospholipid of surfactant, is the critical component for the surface-tension-lowering function (2). DPPC is cleared from the alveolar space predominantly through endocytosis by type II cells with a minor contribution from alveolar macrophages. Under normal physiological conditions, clearance and secretion of DPPC appear to be coordinately regulated (3).Peroxiredoxins (Prdxs) are a recently described superfamily of Se-independent peroxidases that are distributed in all phyla (4). They are classified according to the number (1 or 2) of conserved cysteine (Cys) residues directly involved in peroxidase catalysis. Of the six mammalian peroxiredoxins, Prdx6 has a single conserved Cys, whereas Prdx1-5 are 2-Cys enzymes. Prdx6 is expressed in various tissues, but is especially enriched in lung and brain (5, 6). By immunocytochemistry, Prdx6 in lung is present in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium (6), and subcellular fractionation of lungs has demonstrated the presence of this protein in lamellar body, lysosomal, and cytosolic...

BackgroundType 2 diabetes is characterized by dyslipidemia and the accumulation of lipids in non-adipose tissue, including skeletal muscle. Puerarin, which is a natural isoflavonoid isolated from the root of the plant Pueraria lobata, has been shown to have antidiabetic activity. However, the lipid-reducing effect of puerarin, in particular in skeletal muscle, has not yet been addressed.MethodsWe examined the effect of puerarin on mitochondrial function and the oxidation of fatty acids in the skeletal muscle of high-fat diet/streptozotocin-induced diabetic rats.ResultsPuerarin effectively alleviated dyslipidemia and decreased the accumulation of intramyocellular lipids by upregulating the expression of a range of genes involved in mitochondrial biogenesis, oxidative phosphorylation, the detoxification of reactive oxygen species, and the oxidation of fatty acids in the muscle of diabetic rats. Also, the effect of puerarin on mitochondrial biogenesis might partially involve the function of the μ-opioid receptor. In addition, puerarin decreased the trafficking of fatty acid translocase/CD36 to the plasma membrane to reduce the uptake of fatty acids by myocytes. In vitro studies confirmed that puerarin acted directly on muscle cells to promote the oxidation of fatty acids in insulin-resistant myotubes treated with palmitate.ConclusionsPuerarin improved the performance of mitochondria in muscle and promoted the oxidation of fatty acids, which thus prevented the accumulation of intramyocellular lipids in diabetic rats. Our findings will be beneficial both for elucidating the mechanism of the antidiabetic activity of puerarin and for promoting the therapeutic potential of puerarin in the treatment of diabetes.

There is a considerable body of evidence supporting the role of secretory type II-A phospholipase A2 (sPLA2-IIA) as an effector of the innate immune response. This enzyme also exhibits bactericidal activity especially toward Gram-positive bacteria. In this study we examined the ability of sPLA2-IIA to kill Bacillus anthracis, the etiological agent of anthrax. Our results show that both germinated B. anthracis spores and encapsulated bacilli were sensitive to the bactericidal activity of recombinant sPLA2-IIA in vitro. In contrast, nongerminated spores were resistant. This bactericidal effect was correlated to the ability of sPLA2-IIA to hydrolyze bacterial membrane phospholipids. Guinea pig alveolar macrophages, the major source of sPLA2-IIA in an experimental model of acute lung injury, released enough sPLA2-IIA to kill extracellular B. anthracis. The production of sPLA2-IIA was significantly inhibited by B. anthracis lethal toxin. Human bronchoalveolar lavage fluids from acute respiratory distress syndrome patients are known to contain sPLA2-IIA; bactericidal activity against B. anthracis was detected in a high percentage of these samples. This anthracidal activity was correlated to the levels of sPLA2-IIA and was abolished by an sPLA2-IIA inhibitor. These results suggest that sPLA2-IIA may play a role in innate host defense against B. anthracis infection and that lethal toxin may help the bacteria to escape from the bactericidal action of sPLA2-IIA by inhibiting the production of this enzyme.

Background: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density Lipoproteins (HDL) has potent antiinflammatory effects in various cell types, its effect on inflammatory responses in macrophages remain ill defined. Methods & Results: Pre-incubation of human and murine macrophages in vitro with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-B1 or CD36. These pro-inflammatory effects are confirmed in vivo in peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. In line, innate responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Native and reconstituted HDL enhances Toll Like Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using microarray analysis and macrophages from NF-kB luciferase mice, we observed that HDL induces NF-kB activation. Western blot and ChIP-PCR analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice for the upstream activation pathways, we show that the observed HDL effects are independent of the upstream kinases IKK, NIK and CKII. Furthermore, using STAT1 knock-out mice we observed that also STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12 secretion. On the other hand, using pharmacological inhibitors, we show that HDL enhances ADAM protease activity, thereby mediating TNF release. Conclusion and Clinical Relevance: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC, NF-kB and STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials.