Activation of the neutrophil respiratory burst by plasma from periodontitis patients is mediated by pro-inflammatory cytokines

Aim: To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. Materials and Methods: Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-a to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. Results: Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p<0.05). This difference was maintained after depletion of albumin and Ig. Plasma from patients contained higher mean levels of IL-8, GM-CSF and IFN-a. Individual neutralizing antibodies against IL-8, GM-CSF or IFN-a inhibited the direct stimulatory effect of patients' plasma, whereas the ability to prime for fMLP-stimulated superoxide production was only inhibited by neutralization of IFN-a. The stimulating and priming effects of control plasma were unaffected by antibody neutralization. Conclusions: This study demonstrates that plasma cytokines may have a role in inducing the hyperactive (IL-8, GM-CSF, IFN-a) and hyper-reactive (IFN-a) neutrophil phenotype seen in periodontitis patients.

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

Dehdyroepiandrosterone sulfate directly activates protein kinase C-β to increase human neutrophil superoxide generation

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-ß (PKC-ß) in a cell-free assay. Enhanced PKC-ß activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-ß acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.

Infection with Anaplasma phagocytophilum activates the phosphatidylinositol 3-Kinase/Akt and NF-κB survival pathways in neutrophil granulocytes

Anaplasma phagocytophilum, a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-?B signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum-induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-?B in A. phagocytophilum-infected neutrophils. Activation of NF-?B leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum-infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum-infected neutrophils.

The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bis-hydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E-2)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.

IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC

Characterization of neutrophil function in Papillon-Lefèvre syndrome

Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.

Técnica para segmentação automática de imagens microscópicas de componentes sanguíneos e classificação diferencial de leucócitos baseada em lógica fuzzy

Automatic detection of blood components is an important topic in the field of hematology. The segmentation is an important stage because it allows components to be grouped into common areas and processed separately and leukocyte differential classification enables them to be analyzed separately. With the auto-segmentation and differential classification, this work is contributing to the analysis process of blood components by providing tools that reduce the manual labor and increasing its accuracy and efficiency. Using techniques of digital image processing associated with a generic and automatic fuzzy approach, this work proposes two Fuzzy Inference Systems, defined as I and II, for autosegmentation of blood components and leukocyte differential classification, respectively, in microscopic images smears. Using the Fuzzy Inference System I, the proposed technique performs the segmentation of the image in four regions: the leukocyte’s nucleus and cytoplasm, erythrocyte and plasma area and using the Fuzzy Inference System II and the segmented leukocyte (nucleus and cytoplasm) classify them differentially in five types: basophils, eosinophils, lymphocytes, monocytes and neutrophils. Were used for testing 530 images containing microscopic samples of blood smears with different methods. The images were processed and its accuracy indices and Gold Standards were calculated and compared with the manual results and other results found at literature for the same problems. Regarding segmentation, a technique developed showed percentages of accuracy of 97.31% for leukocytes, 95.39% to erythrocytes and 95.06% for blood plasma. As for the differential classification, the percentage varied between 92.98% and 98.39% for the different leukocyte types. In addition to promoting auto-segmentation and differential classification, the proposed technique also contributes to the definition of new descriptors and the construction of an image database using various processes hematological staining

Estudo da resposta Th17 no transplante renal alogênico: contribuição do eixo quimiotático CCR6/CCL20 e dos polimorfismos gênicos em IL17A e IL17RA

Kidney transplantation is the best treatment for patients who have lost kidney function. Renal transplant patients require accurate immunosuppressive drugs to prevent rejection. In this process T helper cells of the immune system perform key role in the immune response to the graft, and recently the Th17 cells has been investigated by production of IL-17 potent proinflammatory cytokine whose role in the rejection has also been described. Increased of Th17 cell expression has an important association with the development of rejection in renal microenvironment, however the likely mechanism is not well understood. This study aimed to evaluate the Th17 response from the influence of the chemotactic axis CCR6/CCL20 and genetic variants in IL-17 and IL-17RA. We conducted a case-control study involving 148 patients transplanted at the University Hospital Onofre Lopes/UFRN in which assessed by immunohistochemistry protein expression of IL-17 and chemokines CCR6/CCL20 and by PCR-RFLP genetic variants in IL17A and IL17RA. Our results showed no influence of genetic polymorphisms on the outcome of the graft or the protein expression of IL-17. In renal graft microenvironment found several sources producing IL-17: tubular epithelial cells, glomerular cells, neutrophils and cell interstitial infiltration, in turn the expression of chemotactic axis CCR6/CCL20 was restricted to the tubular epithelium cells. There was a slight positive linear correlation between the presence of IL-17 and expression of chemotactic axis CCR6/CCL20 in the microenvironment of renal graft. Therefore, we believe that, combined with our results, further studies with increased "n" sample and greater control over the variables involved in obtaining the renal specimen, can determine more clearly the influence of chemotactic axis CCR6 / CCL20 and polymorphisms in cytokines related to Th17 profile on the control of this cell subtype response in rejection processes to renal allograft.

Estudo das alterações hematológicas e urinárias em cães em diferentes estágios de disfunção renal e avaliação do biomarcador cistatina C

Chapter 2 - Cystatin C is a cationic protein is not glycosylated, produced a steady state for all nucleated and present in biological fluids cells being freely filtered by the glomeruli and almost completely catabolized in the proximal tubule, it is a promising early renal dysfunction marker. This study aimed to determine and compare the serum concentration of cystatin C biomarker in 86 dogs. The animals were divided into four groups according to serum creatinine levels: G1 - up. 1.4 mg / dL (23 animals), G2 - 1.5-2.0 mg / dL (16 animals), G3 - 2.1 to 5.0 mg / dL (24 animals) and G4 - above 5.1 mg / dL (23 animals). There was the measurement of the parameters used in the clinical routine of small animals such as urea, urinary gamma glutamyl transferase, proteinuria, alkaline phosphatase, sodium, potassium, chloride, calcium, phosphorus, calcium/phosphorus ratio and cystatin C. There was no statistical difference for urea, proteinuria, phosphorus, calcium/phosphorus, potassium and cystatin C, however, the other showed no statistical difference. Based on the results we can infer that cystatin C was not a good early indicator of kidney disease in dogs. Chapter 3 - This study aimed to determine the hematological and urinalysis elements such as density, proteinuria, cylinders and pH in 86 dogs The animals were divided into four stages according to serum creatinine levels: I - up to 1.4 mg/dL (23 animals), II - 1.5-2.0 mg/dL (16 animals), III from 2.1 to 5.0 mg/dL (24 animals) and IV - above 5.1 mg/dL (23 animals). In stage III, IV there was anemia normocytic normochromic type. Stage II had a leukocytosis frame by neutrophilia with a regenerative left shift and stage III and IV detour degenerative left. The density remained within the reference values all stages. Proteinuria showed statistical significance for the classification 2+ (1.0 g/L), between stage I and II, II and IV. Only the cylinder granular statistical difference in the classification 2+ between stage II and III, and 3+ between stage I and III. The prevailing pH was acid. The haematological values compared to serum creatinine stages showed the changes in hemoglobin and packed cell volume erythrocytes become more pronounced as serum creatinine values rise , this is also the behavior of neutrophils rods and proteinuria.

Improving the use of G-CSF during chemotherapy using physiological mathematical modelling : a quantitative systems pharmacology approach

La diminution des doses administrées ou même la cessation complète d'un traitement chimiothérapeutique est souvent la conséquence de la réduction du nombre de neutrophiles, qui sont les globules blancs les plus fréquents dans le sang. Cette réduction dans le nombre absolu des neutrophiles, aussi connue sous le nom de myélosuppression, est précipitée par les effets létaux non spécifiques des médicaments anti-cancéreux, qui, parallèlement à leur effet thérapeutique, produisent aussi des effets toxiques sur les cellules saines. Dans le but d'atténuer cet impact myélosuppresseur, on administre aux patients un facteur de stimulation des colonies de granulocytes recombinant humain (rhG-CSF), une forme exogène du G-CSF, l'hormone responsable de la stimulation de la production des neutrophiles et de leurs libération dans la circulation sanguine. Bien que les bienfaits d'un traitement prophylactique avec le G-CSF pendant la chimiothérapie soient bien établis, les protocoles d'administration demeurent mal définis et sont fréquemment déterminés ad libitum par les cliniciens. Avec l'optique d'améliorer le dosage thérapeutique et rationaliser l'utilisation du rhG-CSF pendant le traitement chimiothérapeutique, nous avons développé un modèle physiologique du processus de granulopoïèse, qui incorpore les connaissances actuelles de pointe relatives à la production des neutrophiles des cellules souches hématopoïétiques dans la moelle osseuse. À ce modèle physiologique, nous avons intégré des modèles pharmacocinétiques/pharmacodynamiques (PK/PD) de deux médicaments: le PM00104 (Zalypsis®), un médicament anti-cancéreux, et le rhG-CSF (filgrastim). En se servant des principes fondamentaux sous-jacents à la physiologie, nous avons estimé les paramètres de manière exhaustive sans devoir recourir à l'ajustement des données, ce qui nous a permis de prédire des données cliniques provenant de 172 patients soumis au protocol CHOP14 (6 cycles de chimiothérapie avec une période de 14 jours où l'administration du rhG-CSF se fait du jour 4 au jour 13 post-chimiothérapie). En utilisant ce modèle physio-PK/PD, nous avons démontré que le nombre d'administrations du rhG-CSF pourrait être réduit de dix (pratique actuelle) à quatre ou même trois administrations, à condition de retarder le début du traitement prophylactique par le rhG-CSF. Dans un souci d'applicabilité clinique de notre approche de modélisation, nous avons investigué l'impact de la variabilité PK présente dans une population de patients, sur les prédictions du modèle, en intégrant des modèles PK de population (Pop-PK) des deux médicaments. En considérant des cohortes de 500 patients in silico pour chacun des cinq scénarios de variabilité plausibles et en utilisant trois marqueurs cliniques, soient le temps au nadir des neutrophiles, la valeur du nadir, ainsi que l'aire sous la courbe concentration-effet, nous avons établi qu'il n'y avait aucune différence significative dans les prédictions du modèle entre le patient-type et la population. Ceci démontre la robustesse de l'approche que nous avons développée et qui s'apparente à une approche de pharmacologie quantitative des systèmes (QSP). Motivés par l'utilisation du rhG-CSF dans le traitement d'autres maladies, comme des pathologies périodiques telles que la neutropénie cyclique, nous avons ensuite soumis l'étude du modèle au contexte des maladies dynamiques. En mettant en évidence la non validité du paradigme de la rétroaction des cytokines pour l'administration exogène des mimétiques du G-CSF, nous avons développé un modèle physiologique PK/PD novateur comprenant les concentrations libres et liées du G-CSF. Ce nouveau modèle PK a aussi nécessité des changements dans le modèle PD puisqu’il nous a permis de retracer les concentrations du G-CSF lié aux neutrophiles. Nous avons démontré que l'hypothèse sous-jacente de l'équilibre entre la concentration libre et liée, selon la loi d'action de masse, n'est plus valide pour le G-CSF aux concentrations endogènes et mènerait en fait à la surestimation de la clairance rénale du médicament. En procédant ainsi, nous avons réussi à reproduire des données cliniques obtenues dans diverses conditions (l'administration exogène du G-CSF, l'administration du PM00104, CHOP14). Nous avons aussi fourni une explication logique des mécanismes responsables de la réponse physiologique aux deux médicaments. Finalement, afin de mettre en exergue l’approche intégrative en pharmacologie adoptée dans cette thèse, nous avons démontré sa valeur inestimable pour la mise en lumière et la reconstruction des systèmes vivants complexes, en faisant le parallèle avec d’autres disciplines scientifiques telles que la paléontologie et la forensique, où une approche semblable a largement fait ses preuves. Nous avons aussi discuté du potentiel de la pharmacologie quantitative des systèmes appliquées au développement du médicament et à la médecine translationnelle, en se servant du modèle physio-PK/PD que nous avons mis au point.

Mechanism of Superoxide Mediated Regulation of Particle Uptake and Exocytosis by a GPI-anchored Superoxide Dismutase C in Dictyostelium

Dictyostelium discoideum is a simple model organism that can be used to study endocytic pathways such as phagocytosis and macropinocytosis because of its homology to cells of the mammalian innate immune system, namely macrophages and neutrophils. Consequently, Dictyostelium can also be used to study the process of exocytosis. In our laboratory, we generated Dictyostelium cells lacking superoxide dismutase SodC. Our data suggest that cells that lack SodC are defective in macropinocytosis and exocytosis when compared to wild type cells. In this study I describe a regulatory mechanism of macropinocytosis by SodC via regulation of RasG, which in turn controls PI3K activation and thus macropinocytosis. Our results show that proper metabolism of superoxide is critical for efficient particle uptake, for the proper trafficking of internalized particles, and a timely exocytosis of fluid uptake in Dictyostelium cells.

Characterising granuloma regression and liver recovery in a murine model of schistosomiasis japonica

For hepatic schistosomiasis the egg-induced granulomatous response and the development of extensive fibrosis are the main pathologies. We used a Schistosoma japonicum-infected mouse model to characterise the multi-cellular pathways associated with the recovery from hepatic fibrosis following clearance of the infection with the anti-schistosomal drug, praziquantel. In the recovering liver splenomegaly, granuloma density and liver fibrosis were all reduced. Inflammatory cell infiltration into the liver was evident, and the numbers of neutrophils, eosinophils and macrophages were significantly decreased. Transcriptomic analysis revealed the up-regulation of fatty acid metabolism genes and the identification of Peroxisome proliferator activated receptor alpha as the upstream regulator of liver recovery. The aryl hydrocarbon receptor signalling pathway which regulates xenobiotic metabolism was also differentially up-regulated. These findings provide a better understanding of the mechanisms associated with the regression of hepatic schistosomiasis.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β2-agonists, corticosteroids, and critical illness

Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. 

Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. 

Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β₂-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. 

Results β₂-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β₂-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β₂-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. 

Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.