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Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease.

Inadequate supplies of donor corneas have evoked an escalating interest in corneal xenotransplantation. However, innate immune responses contribute significantly to the mechanism of xenograft rejection. We hypothesized that complement component C5 and TLR co-receptor CD14 inhibition would inhibit porcine cornea induced innate immune responses. Therefore, we measured cytokine release in human blood, induced by three forms of corneal xenografts with or without inhibitors. Native porcine cornea (NPC) induced interleukins (IL-1β, IL-2, IL-6, IL-8, IL-1ra), chemokines (MCP-1, MIP-1α, MIP-1β) and other cytokines (TNF, G-CSF, INF-γ, FGF-basic). Decellularized (DPC) and gamma-irradiated cornea (g-DPC) elevated the release of those cytokines. C5-blockade by eculizumab inhibited all the cytokines except G-CSF when induced by NPC. However, C5-blockade failed to reduce DPC and g-DPC induced cytokines. Blockade of CD14 inhibited DPC-induced cytokines except for IL-8, MCP-1, MIP-1α, and G-CSF, while it inhibited all of them when induced by g-DPC. Combined blockade of C5 and CD14 inhibited the maximum number of cytokines regardless of the xenograft type. Finally, by using the TLR4 specific inhibitor Eritoran, we showed that TLR4 activation was the basis for the CD14 effect. Thus, blockade of C5, when combined with TLR4 inhibition, may have therapeutic potential in pig-to-human corneal xenotransplantation. Statement of significance Bio-engineered corneal xenografts are on the verge of becoming a viable alternative to allogenic human-donor-cornea, but the host's innate immune response is still a critical barrier for graft acceptance. By overruling this barrier, limited graft availability would no longer be an issue for treating corneal diseases. We showed that the xenograft induced inflammation is initiated by the complement system and toll-like receptor activation. Intriguingly, the inflammatory response was efficiently blocked by simultaneously targeting bottleneck molecules in the complement system (C5) and the TLR co-receptor CD14 with pharmaceutical inhibitors. We postulate that a combination of C5 and CD14 inhibition could have a great therapeutic potential to overcome the immunologic barrier in pig-to-human corneal xenotransplantation. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. To address the shortcomings associated with corneal transplants, substantial efforts have been focused on developing new modalities such as xenotransplantion. Xenogeneic corneas are anatomically and biomechanically similar to the human cornea, yet their applications require prior decellularization to remove the antigenic components to avoid rejection. In the context of bringing decellularized corneas into clinical use, sterilization is a crucial step that determines the success of the transplantation. Well-standardized sterilization methods, such as gamma irradiation (GI), have been applied to decellularized porcine corneas (DPC) to avoid graft-associated infections in human recipients. However, little is known about the effect of GI on decellularized corneal xenografts. Here, we evaluated the radiation effect on the ultrastructure, optical, mechanical and biological properties of DPC. Transmission electron microscopy revealed that gamma irradiated decellularized porcine cornea (G-DPC) preserved its structural integrity. Moreover, the radiation did not reduce the optical properties of the tissue. Neither DPC nor G-DPC led to further activation of complement system compared to native porcine cornea when exposed to plasma. Although, DPC were mechanically comparable to the native tissue, GI increased the mechanical strength, tissue hydrophobicity and resistance to enzymatic degradation. Despite these changes, human corneal epithelial, stromal, endothelial and hybrid neuroblastoma cells grew and differentiated on DPC and G-DPC. Thus, GI may achieve effective tissue sterilization without affecting critical properties that are essential for corneal transplant survival.

Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.

eLife 10, e63586 (1-27) (2021). doi:10.7554/eLife.63586, Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3���6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 �� from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential., Published by eLife Sciences Publications, Cambridge

AIMS Heart failure (HF) is an impending complication to myocardial infarction. We hypothesized that the degree of complement activation reflects severity of HF following acute myocardial infarction. METHODS AND RESULTS The LEAF trial (LEvosimendan in Acute heart Failure following myocardial infarction) evaluating 61 patients developing HF within 48 h after percutaneous coronary intervention-treated ST-elevation myocardial infarction herein underwent a post hoc analysis. Blood samples were drawn from inclusion to Day 5 and at 42 day follow-up, and biomarkers were measured with enzyme immunoassays. Regional myocardial contractility was measured by echocardiography as wall motion score index (WMSI). The cardiogenic shock group (n = 9) was compared with the non-shock group (n = 52). Controls (n = 44) were age-matched and sex-matched healthy individuals. C4bc, C3bc, C3bBbP, and sC5b-9 were elevated in patients at inclusion compared with controls (P

The liver is both an immunologically complex and a privileged organ. The innate immune system is a central player, in which the complement system emerges as a pivotal part of liver homeostasis, immune responses, and crosstalk with other effector systems in both innate and adaptive immunity. The liver produces the majority of the complement proteins and is the home of important immune cells such as Kupffer cells. Liver immune responses are delicately tuned between tolerance to many antigens flowing in from the alimentary tract, a tolerance that likely makes the liver less prone to rejection than other solid organ transplants, and reaction to local injury, systemic inflammation, and regeneration. Notably, complement is a double-edged sword as activation is detrimental by inducing inflammatory tissue damage in, for example, ischemia-reperfusion injury and transplant rejection yet is beneficial for liver tissue regeneration. Therapeutic complement inhibition is rapidly developing for routine clinical treatment of several diseases. In the liver, targeted inhibition of damaged tissue may be a rational and promising approach to avoid further tissue destruction and simultaneously preserve beneficial effects of complement in areas of proliferation. Here, we argue that complement is a key system to manipulate in the liver in several clinical settings, including liver injury and regeneration after major surgery and preservation of the organ during transplantation.

ObjectiveThe aetiology of thoracic aortic aneurysm (TAA) is largely unknown, but inflammation is likely to play a central role in the pathogenesis. In this present study, we aim to investigate the complement receptors in TAA.MethodsAortic tissue and blood from 31 patients with non-syndromic TAA undergoing thoracic aortic repair surgery were collected. Aortic tissue and blood from 36 patients with atherosclerosis undergoing coronary artery bypass surgery or aortic valve replacement were collected and served as control material. The expression of the complement anaphylatoxin receptors C3aR1, C5aR1 and C5aR2 in aortic tissue were examined by quantitative RT-PCR and C5aR2 protein by immunohistochemistry. Colocalisation of C5aR2 to different cell types was analysed by immunofluorescence. Complement activation products C3bc and sC5b-9 were measured in plasma.ResultsCompared with controls, TAA patients had substantial (73%) downregulated gene expression of C5aR2 as seen both at the mRNA (p=0.005) level and protein (p=0.03) level. In contrast, there were no differences in the expression of C3aR1 and C5aR1 between the two groups. Immunofluorescence examination showed that C5aR2 was colocalised to macrophages and T cells in the aortic media. There were no differences in the degree of systemic complement activation between the two groups.ConclusionOur findings suggest downregulation of the C5aR2, regarded to act mainly anti-inflammatory, in electively operated TAA as compared with non-aneurysmatic aortas of patients with aortic stenosis and/or coronary artery disease. This may tip the balance towards a relative increase in the inflammatory responses induced by C5aR1 and thus enhance the inflammatory processes in TAA.

Background: Elevated interleukin-6 (IL-6) and complement activation are associated with detrimental effects of inflammation in coronary artery disease (CAD). The complement anaphylatoxins C5a and C3a interact with their receptors; the highly inflammatory C5aR1, and the C5aR2 and C3aR. We evaluated the effect of the IL-6 receptor (IL-6R)-antagonist tocilizumab on the expression of the anaphylatoxin receptors in whole blood from non-ST-elevation myocardial infarction (NSTEMI) patients. Separately, anaphylatoxin receptor expression in peripheral blood mononuclear cells (PBMC) from patients with different entities of CAD was investigated. Materials and Methods: NSTEMI patients were randomized to one dose of tocilizumab (n = 28) or placebo (n = 32) and observed for 6 months. Whole blood samples drawn at inclusion, at day 2, 3 and after 6 months were used for mRNA isolation. Plasma was prepared for analysis of complement activation measured as sC5b-9 by ELISA. Furthermore, patients with different CAD entities comprising stable angina pectoris (SAP, n = 22), non-ST-elevation acute coronary syndrome (NSTE-ACS, n = 21) and ST-elevation myocardial infarction (STEMI, n = 20) were included. PBMC was isolated from blood samples obtained at admission to hospital and mRNA isolated. Anaphylatoxin-receptor-expression was analyzed with qPCR using mRNA from whole blood and PBMC, respectively. Results: Our main findings were (i) Tocilizumab decreased C5aR1 and C5aR2 mRNA expression significantly (p < 0.001) and substantially (>50%) at day 2 and 3, whereas C3aR expression was unaffected. (ii) Tocilizumab did not affect complement activation. (iii) In analyzes of different CAD entities, C5aR1 expression was significantly increased in all CAD subgroups compared to controls with the highest level in the STEMI patients (p < 0.001). For C5aR2 and C3aR the expression compared to controls were more moderate with increased expression of C5aR2 in the STEMI group (p < 0.05) and C3aR in the NSTE-ACS group (p < 0.05). Conclusion: Expression of C5aR1 and C5aR2 in whole blood was significantly attenuated by IL-6R-inhibition in NSTEMI patients. These receptors were significantly upregulated in PBMC CAD patients with particularly high levels of C5aR1 in STEMI patients. Copyright © 2018 Orrem, Nilsson, Pischke, Kleveland, Yndestad, Ekholt, Damås, Espevik, Bendz, Halvorsen, Gregersen, Wiseth, Andersen, Ueland, Gullestad, Aukrust, Barratt-Due and Mollnes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Background: Meconium aspiration syndrome (MAS) is a severe lung condition affecting newborns and it can lead to a systemic inflammatory response. We previously documented complement activation and cytokine release in a piglet MAS model. Additionally, we showed ex vivo that meconium-induced inflammation was dependent on complement and Toll-like receptors. Objectives: To assess the efficacy of the combined inhibition of complement (C5) and CD14 on systemic inflammation induced in a forceful piglet MAS model. Methods: Thirty piglets were randomly allocated to a treatment group receiving the C5-inhibitor SOBI002 and anti-CD14 (n = 15) and a nontreated control group (n = 15). MAS was induced by intratracheal meconium instillation, and the piglets were observed for 5 h. Complement, cytokines, and myeloperoxidase (MPO) were measured by ELISA. Results: SOBI002 ablated C5 activity and the formation of the terminal complement complex in vivo. The combined inhibition attenuated the inflammasome cytokines IL-1β and IL-6 by 60 (p = 0.029) and 44% (p = 0.01), respectively, and also MPO activity in the bronchoalveolar fluid by 42% (p = 0.017). Ex vivo experiments in human blood revealed that the combined regimen attenuated meconium-induced MPO release by 64% (p = 0.008), but there was only a negligible effect with single inhibition, indicating a synergic cross-talk between the key molecules C5 and CD14. Conclusion: Combined inhibition of C5 and CD14 attenuates meconium-induced inflammation in vivo and this could become a future therapeutic regimen for MAS. © 2018 The Author(s) Published by S. Karger AG, Basel. This article is licensed under the Creative Commons AttributionNonCommercial-NoDerivatives 4.0 International License (CC BYNC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission.

Accepted manuscript version. Published version available at https://doi.org/10.1016/j.ijcard.2018.05.032. Licensed CC BY-NC-ND 4.0. Background: The inflammatory response following myocardial infarction (MI) is prerequisite for proper healing of infarcted tissue, but can also have detrimental effects on cardiac function. Interleukin (IL)-1α and IL-1β are potent inflammatory mediators and their bioactivity is tightly regulated by IL-1 receptor antagonist (IL-1ra) and soluble (s) IL-1 receptors (R). We aimed to examine whether levels of soluble regulators of IL-1 signalling are changed during ST-elevation MI (STEMI) and their associations with parameters of cardiac injury and ventricular remodelling. Methods: Plasma levels of IL-1Ra, sIL-1R1, sIL-1R2 and sIL-1R accessory protein (sIL-1RAcP) were measured by immunoassays in repeated samples from patients with STEMI (n = 255) and compared to healthy controls (n = 65). Results: IL-1Ra, sIL-1R1 and sIL-1R2 levels were all significantly elevated after STEMI, while levels of sIL-1RAcP were lower compared to controls. sIL-1R2 levels (at different time points) correlated positively with C-reactive protein, myocardial infarct size and change in indexed left ventricular end-diastolic and end-systolic volume (LVEDVi and LVESVi) measured by cardiac MR acutely and after 4 months, and negatively with LV ejection fraction. Patients with >median levels of sIL-1R2 in the acute phase were more likely to have increased change in LVEDVi and LVESVi. Importantly, sIL-1R2 remained significantly associated with change in LVEDVi and LVESVi also after adjustment for clinical covariates. Conclusion: Levels of sIL-1R2 are independently associated with parameters of LV adverse remodelling following STEMI.

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard.

The complement system, an important arm of the innate immune system, is activated in heart failure (HF). We hypothesized that HF patients are characterized by an imbalance of alternative amplification loop components; including properdin and complement factor D and the alternative pathway inhibitor factor H. These components and the activation product, terminal complement complex (TCC), were measured in plasma from 188 HF patients and 67 age- and sex- matched healthy controls by enzyme immunoassay. Our main findings were: (i) Compared to controls, patients with HF had significantly increased levels of factor D and TCC, and decreased levels of properdin, particularly patients with advanced clinical disorder (i.e., NYHA functional class IV), (ii) Levels of factor D and properdin in HF patients were correlated with measures of systemic inflammation (i.e., C-reactive protein), neurohormonal deterioration (i.e., Nt-proBNP), cardiac function, and deteriorated diastolic function, (iii) Low levels of factor H and properdin were associated with adverse outcome in univariate analysis and for factor H, this was also seen in an adjusted model. Our results indicate that dysregulation of circulating components of the alternative pathway explain the increased degree of complement activation and is related to disease severity in HF patients. © 2017 The Authors. Published by Nature Publishing Group. This is an open access article licensed under a Creative Commons Attribution 4.0 International License

Source at https://doi.org/10.1073/pnas.1612385114. Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab. © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation. K.N.E., J.D.L., B.N. and Y.T. equally contributed to this paper as a supervisor.

International audience; Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p < 0.01 for all), and closely correlated (p < 0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p < 0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood. K. Otterdal, (C) 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

International audience; Background: The pathophysiological hallmark of Rickettsia conorii (R. ă conorii) infection comprises infection of endothelial cells with ă perivascular infiltration of T-cells and macrophages. Although ă interferon (IFN)-gamma-induced protein 10 (IP-10)/CXCL10 is induced ă during vascular inflammation, data on CXCL10 in R. conorii infection is ă scarce. ă Methods: Serum CXCL10 was analyzed in two cohorts of southern European ă patients with R. conorii infection using multiplex cytokine assays. The ă mechanism of R. conorii-induced CXCL10 release was examined ex vivo ă using human whole blood interacting with endothelial cells. ă Results: (i) At admission, R. conorii infected patients had excessively ă increased CXCL10 levels, similar in the Italian (n = 32, similar to ă 56-fold increase vs controls) and the Spanish cohort (n = 38, 68-fold ă increase vs controls), followed by a marked decrease after recovery. The ă massive CXCL10 increase was selective since it was not accompanied with ă similar changes in other cytokines. (ii) Heat-inactivated R. conorii ă induced a marked CXCL10 increase when whole blood and endothelial cells ă were co-cultured. Even plasma obtained from R. conorii-exposed whole ă blood induced a marked CXCL10 release from endothelial cells, comparable ă to the levels found in serum of R. conorii-infected patients. Bacteria ă alone did not induce CXCL10 production in endothelial cells, macrophages ă or smooth muscle cells. ă Conclusions: We show a massive and selective serum CXCL10 response in R. ă conorii-infected patients, likely reflecting release from infected ă endothelial cells characterized by infiltrating T cells and monocytes. ă The CXCL10 response could contribute to T-cell infiltration within the ă infected organ, but the pathologic consequences of CXCL10 in clinical R. ă conorii infection remain to be defined. (C) 2016 Elsevier Ltd. All ă rights reserved.

Endothelial cells (EC) play a central role in inflammation. E-selectin and ICAM-1 expression are essential for leukocyte recruitmentand are good markers of EC activation. Most studies of EC activation are done in vitro using isolated mediators. The aim of thepresent study was to examine the relative importance of pattern recognition systems and downstream mediators in bacteria-inducedEC activation in a physiological relevant human model, using EC incubated with whole blood. HUVEC were incubated with humanwhole blood.Escherichia coli– andStaphylococcus aureus–induced EC activation was measured by E-selectin and ICAM-1expression using flow cytometry. The mAb 18D11 was used to neutralize CD14, and the lipid A analog eritoran was used toblock TLR4/MD2. C5 cleavage was inhibited using eculizumab, and C5aR1 was blocked by an antagonist. Infliximab andcanakinumab were used to neutralize TNF and IL-1b. The EC were minimally activated when bacteria were incubated in serum,whereas a substantial EC activation was seen when the bacteria were incubated in whole blood.E. coli–induced activation waslargely CD14-dependent, whereasS. aureusmainly caused a C5aR1-mediated response. Combined CD14 and C5 inhibitionreduced E-selectin and ICAM-1 expression by 96 and 98% forE. coliand by 70 and 75% forS. aureus.Finally, the EC activationby both bacteria was completely abolished by combined inhibition of TNF and IL-1b.E. coliandS. aureusactivated EC in aCD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1bmediating the effect.The Journal ofImmunology, 2016, 196: 2293–2299. Copyright © 2016 by The American Association of Immunologists, Inc. This article is distributed under The American Association of Immunologists, Inc.,Reuse Terms and Conditions for Author Choice articles.

Review of how targeting key upstream molecules at the recognition phase of innate immunity exert anti-inflammatory effects; a potential therapeutic regimen for inflammatory diseases., The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross-talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL-1β, and IL-6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by “dual blockade” of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad-acting therapeutic regimen against a number of diseases where innate immunity is improperly activated.

Combined inhibition of C5 and CD14 attenuates systemic inflammation in a newborn pig-model of meconium aspiration syndrome

Characterization of a novel whole blood model for the study of thrombin in complement activation and inflammation

When an artificial biomaterial (e.g., a stent or implantable pump) is exposed to blood, plasma proteins immediately adhere to the surface, creating a new interface between the biomaterial and the blood. The recognition proteins within the complement and contact activation/coagulation cascade systems of the blood will be bound to, or inserted into, this protein film and generate different mediators that will activate polymorphonuclear leukocytes and monocytes, as well as platelets. Under clinical conditions, the ultimate outcome of these processes may be thrombotic and inflammatory reactions, and consequently the composition and conformation of the proteins in the initial layer formed on the surface will to a large extent determine the outcome of a treatment involving the biomaterial, affecting both the functionality of the material and the patient's life quality. This review presents models of biomaterial-induced activation processes and describes various strategies to attenuate potential adverse reactions by conjugating bioactive molecules to surfaces or by introducing nanostructures.

The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent.

The IL-6 receptor inhibitor tocilizumab attenuated expression of C5a receptor 1 and 2 in patients with myocardial infarction

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)–induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54–95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24–48 h). Combined treatment increased median survival to 96 h (range 24–240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24–48 h] and 48 h [24–96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

Platelet activation during thrombotic events is closely associated with complement and contact system activation, which in turn leads to inflammation . Here we review the interactions between activated platelets and the complement and contact activation systems in clotting blood. Chondroitin sulfate A (CS-A), released from alpha granules during platelet activation, is a potent mediator of crosstalk between platelets and the complement system. CS-A activates complement in the fluid phase, generating anaphylatoxins that mediate leukocyte activation. No complement activation seems to occur on the activated platelet surface, but C3 in the form of C3(H2O) is bound to the surfaces of activated platelets . This finding is consistent with the strong expression of membrane-bound complement regulators present at the platelet surface. CS-A exposed on the activated platelets is to a certain amount responsible for recruiting soluble regulators to the surface. Platelet-bound C3(H2O) acts as a ligand for leukocyte CR1 (CD35), potentially enabling platelet–leukocyte interactions. In addition, platelet activation leads to the activation of contact system enzymes, which are specifically inhibited by antithrombin, rather than by C1INH, as is the case when contact activation is induced by material surfaces. Thus, in addition to their traditional role as initiators of secondary hemostasis, platelets also act as mediators and regulators of inflammation in thrombotic events.

Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets.Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase.Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte-platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist.We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.

Autoregulation of thromboinflammation on biomaterials and cells by a novel therapeutic coating technique

The ratio between C4 and C4BP adsorbed from plasma predicts cytokine generation induced by artificial polymers in contact with whole blood