Enhancement of fibrinogen-triggered pro-coagulant activation of monocytes in vitro by matrix metalloproteinase-9

<p>Abstract</p> <p>Background</p> <p>Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown.</p> <p>Results</p> <p>Fibrinogen and its digestion fragment D induced pro-coagulant activation of monocytes as assessed in a cellular coagulation assay by reductions in clotting times. Pro-coagulant activation was reversed by blocking antibodies against Mac-1 or LFA-1. Pre-exposure of monocytes to the p38 MAPK inhibitor SB 202190 and the MEK1.2 inhibitor U0126 led to significant increasees in coagulation times whereas blocking JNKII with its inhibitor had no such effect. Blocking NFκB with MG-132 also inhibited pro-coagulant activation of monocytes by fibrinogen. A selective inhibitor of matrix metalloproteinase-9 increased times to clot formation whereas other matrix metalloproteinase inhibitors did not significantly interfere with fibrinogen-augmented clot formation in this assay. Treatment of monocytes with fibrinogen increased concentrations of matrix metalloproteinase-9 immunoreactivity in their supernatants.</p> <p>Conclusions</p> <p>Fibrinogen induces monocyte pro-coagulant activation in an integrin-, nuclear factor κB-, p38 MAPK-, and MEK1.2-dependent manner. Activation of monocytes by fibrinogen increases metalloproteinase-9 secretion, metalloproteinase-9 itself enhances monocyte coagulation by an autocrine mechanism. Results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways.</p

Reversal of murine alcoholic steatohepatitis by pepducin-based functional blockade of interleukin-8 receptors.

OBJECTIVE: Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs. DESIGN: Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes. RESULTS: Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1β mRNA. CONCLUSIONS: We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.Wellcome Trust Career Re-entry Fellowship (103077/Z/13/Z) to NCK, Christian Doppler Research Society to HT and European Research Council (FP7/2007- 2013) to AKThis is the final version of the article. It first appeared from the BMJ Group via http://dx.doi.org/10.1136/gutjnl-2015-31034

Heparan sulfate proteoglycan-dependent neutrophil chemotaxis toward PR-39 cathelicidin

Cathelicidins are mammalian proteins containing a C-terminal cationic antimicrobial domain. Porcine PR-39 cathelicidin affects leukocyte biology. Mechanisms of action may involve alteration of heparan sulfate proteoglycan-dependent functions in inflammatory cells. It was tested whether PR-39 affects human neutrophil migration and if such effects involve heparan sulphate proteoglycans. Neutrophils were from forearm venous blood of healthy donors. Migration was tested in modified Boyden chamber assays. Involvement of heparan sulfate proteoglycans was tested by their chemical modification and by the use of specific antibodies. PR-39 induced migration in neutrophils in a concentration dependent manner. Modification of heparan sulfate proteoglycans with sodium chlorate inhibited migration whereas chemotaxis toward the chemoattractant formyl-Met-Leu-Phe was not affected. Removal of heparan sulfates or chondroitin sulfates from the surface of neutrophils by heparinase or chondroitinase inhibited migration toward PR-39. In conclusion, antimicrobial PR-39 stimulates human neutrophil chemotaxis in a heparan sulfate proteoglycan-dependent manner. Involvment of syndecans is likely as both heparinase and chondroitinase were abrogating. Data suggest active participation of heparan sulfate proteoglycans of neutrophils in cathelicidin peptide-mediated regulation of the antimicrobial host defense

A meta-analysis of controlled trials of recombinant human activated protein C therapy in patients with sepsis

BACKGROUND: Meta-analysis of two randomised controlled trials in severe sepsis performed with recombinant human activated protein C may provide further insight as to the therapeutic utility of targeting the clotting cascade in this syndrome. METHODS: In search for relevant studies published, two randomized clinical trials were found eligible. RESULTS: The studies, PROWESS and ADDRESS, enrolled a total of 4329 patients with risk ratio (RR) and 95% confidence interval (CI) data for effect on 28-day mortality relative to control treatment of 0.92 (0.83–1.02) suggesting that recombinant human activated protein C is not beneficial in severe sepsis. In PROWESS, 873 of 1690 patients presented with low risk, and 2315 of 2639 patients in ADDRESS as defined by APACHE II score < 25. In this low-risk stratum, no effect of recombinant human activated protein C administration on 28-day mortality was observed. This observation appears to be consistent and homogenous. Heterogeneity between the two studies, however, was seen in patients with APACHE II score ≥ 25 in whom recombinant activated protein C was effective in PROWESS (n = 817; RR 0.71, CI 0.59–0.85) whereas a tendency toward harm was present in ADDRESS (n = 324; RR 1.21, CI 0.85–1.74). Even though the overall treatment effect in this high-risk population was still in favour of treatment with recombinant activated protein C (n = 1141; RR 0.80, CI 0.68–0.94), the observed heterogeneity suggests that the efficacy of recombinant human activated protein C is not robust. Not unlikely, the adverse tendency observed could have become significant with higher statistical power would ADDRESS not have been terminated prematurely. CONCLUSION: This meta-analysis, therefore, raises doubts about the clinical usefulness of recombinant activated protein C in patients with severe sepsis and an APACHE II score ≥ 25 which can only be resolved by another properly designed clinical trial

GPR35 promotes glycolysis, proliferation, and oncogenic signaling by engaging with the sodium potassium pump.

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.European Research Council Consolidator Grant n° 648889 to A.K. Scientia Fellowship (FP7-PEOPLE-2013-COFUND) grant agreement n° 609020 to G.S. Addenbrooke’s Charitable Trust (ACT 25/16A) to J.E.E. UniNA and Compagnia Di San Paolo ‘STAR program for young researchers’ fellowship to E.P

Activation of the GPR35 pathway drives angiogenesis in the tumour microenvironment

Funder: NIHR Cambridge BRCObjective: Primary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers. Design: Mice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays. Results: Here, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors. Conclusions: Activation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages’ ability to create a tumour-permissive environment

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

Unresolved endoplasmic reticulum (ER) stress in the epithelium can provoke intestinal inflammation. Hypomorphic variants of ER stress response mediators, such as X-box–binding protein 1 (XBP1), confer genetic risk for inflammatory bowel disease. We report here that hypomorphic Xbp1 function instructs a multilayered regenerative response in the intestinal epithelium. This is characterized by intestinal stem cell (ISC) expansion as shown by an inositol-requiring enzyme 1α (Ire1α)–mediated increase in Lgr5+ and Olfm4+ ISCs and a Stat3-dependent increase in the proliferative output of transit-amplifying cells. These consequences of hypomorphic Xbp1 function are associated with an increased propensity to develop colitis-associated and spontaneous adenomatous polyposis coli (APC)–related tumors of the intestinal epithelium, which in the latter case is shown to be dependent on Ire1α. This study reveals an unexpected role for Xbp1 in suppressing tumor formation through restraint of a pathway that involves an Ire1α- and Stat3-mediated regenerative response of the epithelium as a consequence of ER stress. As such, Xbp1 in the intestinal epithelium not only regulates local inflammation but at the same time also determines the propensity of the epithelium to develop tumors

A purine metabolic checkpoint that prevents autoimmunity and autoinflammation.

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation