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

Whole-genome expression Profiling in skin reveals sYK as a Key regulator of inflammation in experimental epidermolysis Bullosa acquisita

Because of the morbidity and limited therapeutic options of autoimmune diseases, there is a high, and thus far, unmet medical need for development of novel treatments. Pemphigoid diseases, such as epidermolysis bullosa acquisita (EBA), are prototypical autoimmune diseases that are caused by autoantibodies targeting structural proteins of the skin, leading to inflammation, mediated by myeloid cells. To identify novel treatment targets, we performed cutaneous genome-wide mRNA expression profiling in 190 out-bred mice after EBA induction. Comparison of genome-wide mRNA expression profiles in diseased and healthy mice, and construction of a co-expression network identified Sykb (spleen tyrosine kinase, SYK) as a major hub gene. Aligned, pharmacological SYK inhibition protected mice from experimental EBA. Using lineage-specific SYK-deficient mice, we identified SYK expression on myeloid cells to be required to induce EBA. Within the predicted co-expression network, interactions of Sykb with several partners (e.g., Tlr13, Jdp2, and Nfkbid) were validated by curated databases. Additionally, novel gene interaction partners of SYK were experimentally validated. Collectively, our results identify SYK expression in myeloid cells as a requirement to promote inflammation in autoantibody-driven pathologies. This should encourage exploitation of SYK and SYK-regulated genes as potential therapeutic targets for EBA and potentially other autoantibody-mediated diseases

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity

Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS

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

Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors

Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches

Neutrophils in animal models of autoimmune disease

Neutrophils have traditionally been thought to play only a peripheral role in the genesis of many autoimmune and inflammatory diseases. However, recent studies in a variety of animal models suggest that these cells are central to the initiation and propagation of autoimmunity. The use of mouse models, which allow either deletion of neutrophils or the targeting of specific neutrophil functions, has revealed the many complex ways these cells contribute to autoimmune/inflammatory processes. This includes generation of self antigens through the process of NETosis, regulation of T-cell and dendritic cell activation, production of cytokines such as BAFF that stimulate self-reactive B-cells, as well as indirect effects on epithelial cell stability. In comparing the many different autoimmune models in which neutrophils have been examined, a number of common underlying themes emerge - such as a role for neutrophils in stimulating vascular permeability in arthritis, encephalitis and colitis. The use of animal models has also stimulated the development of new therapeutics that target neutrophil functions, such as NETosis, that may prove beneficial in human disease. This review will summarize neutrophil contributions in a number of murine autoimmune/inflammatory disease models. © 2016 Elsevier Ltd

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