MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms

Targeting vascular endothelial growth factor receptor 2 and protein kinase d1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-alpha -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways

Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9(-/-) neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release

Investigating Strategies for Sustainable Vegetable Food Crop System in Three Agro Ecological Zones of the Humid Tropics Area of Cameroon

Vegetable cultivation remains an essential component of local people’s livelihoods. However, marked trend shifts in the varieties of vegetables due to large-scale commercial vegetable farming of exotic varieties in the broader market economy have resulted in the gradual disappearance of biodiversity involving vital species. The present study examined the situation of vegetable crop farming in three agro-ecological zones of Cameroon. Data were collected from a random sample of 235 respondents (177 farming households and 58 farm input wholesalers) by means of structured questionnaires. Vegetables were observed in both single and mixed cropping systems in all agro-ecological zones. Traditional vegetables such as African nightshade, waterleaf and Fluted pumpkin (okomobong) dominated in the Buea and Ebolowa areas. Over 51% of the vegetable farmers were women, although there was a rising population of men farming particularly in the Bafoussam area. Farmers remarked that pests and diseases followed by lack of good seeds were the main obstacles to vegetable farming. The study therefore points to the need for modification of the microenvironment and changing farming practices. Hence, strategies to fight poverty and malnutrition in Cameroon should include the promotion of traditional leafy and fruit vegetables by providing good quality seeds and variety screening trials

Screening for cytokine production induced by rMASP-1, and the dose dependence of IL-6, IL-8, and MCP-1.

<p>The cells were treated with 2 µM rMASP-1 for 6 hours (mRNA level) or 24 hours (protein level). Seven cytokines were assessed by qPCR and xMAP technology. (0: no effect, +: significant induction, (+): tendency for induction, not confirmed by ELISA.) (<b>A</b>) The cells were treated with 0, 0.25, 0.5, 1, 2 µM rMASP-1 for 24 hours, and then, the supernatants were collected. IL-6 (<b>B</b>), IL-8 (<b>C</b>), and MCP-1 (<b>D</b>) production were determined by sandwich ELISA kits according to the manufacturers' protocol. The values were calculated as the mean (+/−SEM) of three independent experiments.</p

The signaling pathways of IL-6 and IL-8 production induced by rMASP-1.

<p>The cells were pre-incubated with signaling pathway inhibitors for 30 minutes and then treated with 2 µM rMASP-1 for 3 (<b>A, B</b>) or 24 (<b>C, D</b>) hours. Supernatants were collected and analyzed with commercial IL-6 (<b>A, C</b>) and IL-8 (<b>B, D</b>) ELISA kits. C1-Inhibitor (C1Inh) was applied differently; it was premixed with rMASP-1 for 30 minutes and then, the mixture was added to the cells. Each panel was calculated as the mean (+/−SEM) of 3 independent experiments using HUVECs from different donors. The significance of the differences among rMASP-1 and other treatments is shown. *: p<0.05, **: p<0.01.</p

The kinetics of IL-6 and IL-8 production induced by rMASP-1.

<p>HUVECs were treated with 2 µM rMASP-1, or with 10 ng/mL TNFα (only at protein level) for different periods. The samples were analyzed for IL-6 (<b>A, B, C</b>) and IL-8 (<b>D, E, F</b>) by qPCR (<b>A, D</b>) and ELISA (<b>B, C, E, F</b>). qPCR results were plotted as percentage of non-treated control. The results of ELISA were calculated from the standard curve, and plotted as concentration values (<b>B, E</b>). To compare the effects of rMASP-1 and of TNFalpha, we calculated the magnitude of the increase of the values (treated/non-treated) at each time-point (<b>C, F</b>). All data are presented as the mean (+/−SEM) of three independent experiments.</p

The migration of PMNs activated by the rMASP-1-treated HUVEC supernatant.

<p>HUVEC cells were treated with 2 µM rMASP-1, or left untreated for 30 minutes. Then, the medium was changed to rMASP-1 free HBSS for 2.5 hours and then MASP-SN and UNT-SN were collected. Residual rMASP-1 concentration of MASP-SN was checked by LPAPR-AMC fluorescent substrate assay, where the Control column shows the rMASP-1 concentration of the rMASP-1 treated HUVEC supernatant before changed to HBSS (<b>A</b>). PMNs were isolated from venous blood collected from 2 healthy volunteers. 10?5 cells were seeded in 3-µm pore size transwell inserts and placed for an hour into the wells containing the MASP-SN, UNT-SN or HBSS with/without 2 ng/ml IL-8. The percentage of transmigrated/total cells was calculated as the mean (+/−SEM) of 3 different chemotaxis assays (<b>B</b>). The significance of the differences among rMASP-1 and other treatments is shown. *: p<0.05, ns: non-significant.</p

rMASP-1 treatment of HUVEC activates CREB and JNK signaling pathways.

<p>The cells were treated or not treated with 2 µM rMASP-1 for 25 minutes and then, fixed with ice cold methanol-acetone (1∶1), labeled overnight at 4°C with 1∶200 diluted rabbit anti-human Phosopho-CREB antibody, and stained with goat anti-rabbit (1∶500) Alexa 568 (left column) and Hoechst nuclear staining (right column). The figure depicts one out of 5 similar, independent experiments. The bars represent 50 µm (<b>A</b>). The images were obtained by Olympus IX-81 inverted fluorescence microscope equipped with 40× UPLF objective (NA = 0,75), and an Olympus DP70 digital camera. The mean intensity of red fluorescence in the nuclear and perinuclear region was evaluated using the AnalySIS software. The mean (+/−SEM) differences between the indicated regions of 5 experiments are shown (<b>B</b>). Cells were seeded on 6-well plates and treated for 25 minutes. Cells were then lysed and Western-blots were performed. The membranes were probed for CREB and phospho-CREB (<b>C</b>) or JNK and phospho-JNK (<b>D</b>). Two representative phospho-CREB and phospho-JNK images are shown (linear intensity adjusted). The graphs were calculated from unadjusted values of phospho- and total protein ratios from 3 independent experiments. The significance of the differences among rMASP-1 and the other treatments is shown. *: p<0.05, **: p<0.01, ***: p<0.001, ns: non-significant.</p