Publications by authors named "Ilse Jongerius"

37 Publications

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Am J Hum Genet 2021 08 13;108(8):1367-1384. Epub 2021 Jul 13.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, 6525EX, the Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525GA, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10), FHR-2 (p = 1.47 × 10), FHR-3 (p = 1.05 × 10) and FHR-4A (p = 1.22 × 10) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10 and p = 2.81 × 10, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387287PMC
August 2021

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Thromb Haemost 2021 Jun 25. Epub 2021 Jun 25.

Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.
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http://dx.doi.org/10.1055/s-0041-1731051DOI Listing
June 2021

Future Biomarkers for Infection and Inflammation in Febrile Children.

Front Immunol 2021 17;12:631308. Epub 2021 May 17.

Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Emma Children's Hospital, Amsterdam UMC, Amsterdam, Netherlands.

Febrile patients, suffering from an infection, inflammatory disease or autoimmunity may present with similar or overlapping clinical symptoms, which makes early diagnosis difficult. Therefore, biomarkers are needed to help physicians form a correct diagnosis and initiate the right treatment to improve patient outcomes following first presentation or admittance to hospital. Here, we review the landscape of novel biomarkers and approaches of biomarker discovery. We first discuss the use of current plasma parameters and whole blood biomarkers, including results obtained by RNA profiling and mass spectrometry, to discriminate between bacterial and viral infections. Next we expand upon the use of biomarkers to distinguish between infectious and non-infectious disease. Finally, we discuss the strengths as well as the potential pitfalls of current developments. We conclude that the use of combination tests, using either protein markers or transcriptomic analysis, have advanced considerably and should be further explored to improve current diagnostics regarding febrile infections and inflammation. If proven effective when combined, these biomarker signatures will greatly accelerate early and tailored treatment decisions.
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http://dx.doi.org/10.3389/fimmu.2021.631308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165271PMC
September 2021

Pulmonary Procoagulant and Innate Immune Responses in Critically Ill COVID-19 Patients.

Front Immunol 2021 14;12:664209. Epub 2021 May 14.

Center for Experimental and Molecular Medicine, Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.

Rationale: Systemic activation of procoagulant and inflammatory mechanisms has been implicated in the pathogenesis of COVID-19. Knowledge of activation of these host response pathways in the lung compartment of COVID-19 patients is limited.

Objectives: To evaluate local and systemic activation of coagulation and interconnected inflammatory responses in critically ill COVID-19 patients with persistent acute respiratory distress syndrome.

Methods: Paired bronchoalveolar lavage fluid and plasma samples were obtained from 17 patients with COVID-19 related persistent acute respiratory distress syndrome (mechanical ventilation > 7 days) 1 and 2 weeks after start mechanical ventilation and compared with 8 healthy controls. Thirty-four host response biomarkers stratified into five functional domains (coagulation, complement system, cytokines, chemokines and growth factors) were measured.

Measurements And Main Results: In all patients, all functional domains were activated, especially in the bronchoalveolar compartment, with significantly increased levels of D-dimers, thrombin-antithrombin complexes, soluble tissue factor, C1-inhibitor antigen and activity levels, tissue type plasminogen activator, plasminogen activator inhibitor type I, soluble CD40 ligand and soluble P-selectin (coagulation), next to activation of C3bc and C4bc (complement) and multiple interrelated cytokines, chemokines and growth factors. In 10 patients in whom follow-up samples were obtained between 3 and 4 weeks after start mechanical ventilation many bronchoalveolar and plasma host response biomarkers had declined.

Conclusions: Critically ill, ventilated patients with COVID-19 show strong responses relating to coagulation, the complement system, cytokines, chemokines and growth factors in the bronchoalveolar compartment. These results suggest a local pulmonary rather than a systemic procoagulant and inflammatory "storm" in severe COVID-19.
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http://dx.doi.org/10.3389/fimmu.2021.664209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160522PMC
June 2021

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

Cancer Immunol Res 2021 Jul 14;9(7):790-810. Epub 2021 May 14.

Department of Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, New York.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8287091PMC
July 2021

It takes two to thrombosis: Hemolysis and complement.

Blood Rev 2021 Apr 30:100834. Epub 2021 Apr 30.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Switzerland; Department for BioMedical Research, University of Bern, Switzerland. Electronic address:

Thromboembolic events represent the most common complication of hemolytic anemias characterized by complement-mediated hemolysis such as paroxysmal nocturnal hemoglobinuria and autoimmune hemolytic anemia. Similarly, atypical hemolytic uremic syndrome is characterized by hemolysis and thrombotic abnormalities. The main player in the development of thrombosis in hemolytic diseases is suggested to be the complement system. However, the release of extracellular hemoglobin and heme by hemolysis itself can also drive procoagulant responses. Both, complement activation and hemolysis promote the activation of neutrophils resulting in the formation of neutrophil extracellular traps and induce inflammation and vascular damage which all together might (synergistically) lead to hypercoagulability. In this review we aim to summarize the current knowledge on the role of complement activation and hemolysis in the onset of thrombosis in hemolytic diseases. This review will discuss the interplay between different biological systems and neutrophil activation contributing to the pathogenesis of thrombosis. Finally, we will combine this fundamental knowledge and address the pathophysiology of hemolysis in prototypical complement-driven diseases.
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http://dx.doi.org/10.1016/j.blre.2021.100834DOI Listing
April 2021

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Thromb Haemost 2021 May 3. Epub 2021 May 3.

Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.
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http://dx.doi.org/10.1055/a-1497-9573DOI Listing
May 2021

A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

Front Immunol 2021 30;12:660194. Epub 2021 Mar 30.

Department of Ophthalmology, University Hospital Regensburg, Regensburg, Germany.

Inflammation is a common denominator of diseases. The complement system, an intrinsic part of the innate immune system, is a key driver of inflammation in numerous disorders. Recently, a family of proteins has been suggested to be of vital importance in conditions characterized by complement dysregulation: the human Factor H (FH) family. This group of proteins consists of FH, Factor H-like protein 1 and five Factor H-related proteins. The FH family has been linked to infectious, vascular, eye, kidney and autoimmune diseases. In contrast to FH, the functions of the other highly homologous proteins are largely unknown and, hence, their role in the different disease-specific pathogenic mechanisms remains elusive. In this perspective review, we address the major challenges ahead in this emerging area, including 1) the controversies about the functional roles of the FH protein family, 2) the discrepancies in quantification of the FH protein family, 3) the unmet needs for validated tools and 4) limitations of animal models. Next, we also discuss the opportunities that exist for the immunology community. A strong multidisciplinary approach is required to solve these obstacles and is only possible through interdisciplinary collaboration between biologists, chemists, geneticists and physicians. We position this review in light of our own perspective, as principal investigators of the SciFiMed Consortium, a consortium aiming to create a comprehensive analytical system for the quantitative and functional assessment of the entire FH protein family.
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http://dx.doi.org/10.3389/fimmu.2021.660194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044877PMC
October 2021

Biomarkers to predict infection and infection-related complications during chemotherapy-induced neutropenia in acute myeloid leukaemia: a pilot study.

Br J Haematol 2021 06 8;193(5):1008-1012. Epub 2021 Apr 8.

Department of Blood Cell Research, Division Research and Landsteiner Laboratory of Amsterdam UMC, Sanquin Blood Supply, Amsterdam, the Netherlands.

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http://dx.doi.org/10.1111/bjh.17422DOI Listing
June 2021

Molecular Basis for Bordetella pertussis Interference with Complement, Coagulation, Fibrinolytic, and Contact Activation Systems: the Cryo-EM Structure of the Vag8-C1 Inhibitor Complex.

mBio 2021 03 23;12(2). Epub 2021 Mar 23.

Sir William Dunn School of Pathology, Oxford, United Kingdom

Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, a C1 inhibitor (C1-INH), is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, virulence-associated gene 8 (Vag8), produced by the whooping cough pathogen, , was shown to bind to C1-INH and interfere with its function. Here, we present the structure of the Vag8-C1-INH complex determined using cryo-electron microscopy at a 3.6-Å resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens, where Vag8 sequesters the reactive center loop of C1-INH, preventing its interaction with the target proteases. The structure of a 10-kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The structure reveals that C1-INH is sequestered in an inactivated state by burial of the reactive center loop in Vag8. By so doing, the bacterium is able to simultaneously perturb the many pathways regulated by C1-INH. Virulence mechanisms such as the one described here assume more importance given the emerging evidence about dysregulation of contact activation, coagulation, and fibrinolysis leading to COVID-19 pneumonia.
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http://dx.doi.org/10.1128/mBio.02823-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092270PMC
March 2021

Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use.

Pharmaceuticals (Basel) 2021 Jan 11;14(1). Epub 2021 Jan 11.

Department of Immunopathology, Sanquin Research, Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.
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http://dx.doi.org/10.3390/ph14010054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826800PMC
January 2021

Therapeutic Lessons to be Learned From the Role of Complement Regulators as Double-Edged Sword in Health and Disease.

Front Immunol 2020 10;11:578069. Epub 2020 Dec 10.

Sanquin Research, Department of Immunopathology, and Landsteiner Laboratory, Amsterdam University Medical Centre, Amsterdam Infection and Immunity Institute, Amsterdam, Netherlands.

The complement system is an important part of the innate immune system, providing a strong defense against pathogens and removing apoptotic cells and immune complexes. Due to its strength, it is important that healthy human cells are protected against damage induced by the complement system. To be protected from complement, each cell type relies on a specific combination of both soluble and membrane-bound regulators. Their importance is indicated by the amount of pathologies associated with abnormalities in these complement regulators. Here, we will discuss the current knowledge on complement regulatory protein polymorphisms and expression levels together with their link to disease. These diseases often result in red blood cell destruction or occur in the eye, kidney or brain, which are tissues known for aberrant complement activity or regulation. In addition, complement regulators have also been associated with different types of cancer, although their mechanisms here have not been elucidated yet. In most of these pathologies, treatments are limited and do not prevent the complement system from attacking host cells, but rather fight the consequences of the complement-mediated damage, using for example blood transfusions in anemic patients. Currently only few drugs targeting the complement system are used in the clinic. With further demand for therapeutics rising linked to the wide range of complement-mediated disease we should broaden our horizon towards treatments that can actually protect the host cells against complement. Here, we will discuss the latest insights on how complement regulators can benefit therapeutics. Such therapeutics are currently being developed extensively, and can be categorized into full-length complement regulators, engineered complement system regulators and antibodies targeting complement regulators. In conclusion, this review provides an overview of the complement regulatory proteins and their links to disease, together with their potential in the development of novel therapeutics.
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http://dx.doi.org/10.3389/fimmu.2020.578069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758290PMC
June 2021

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants.

J Immunol 2020 10 26;205(7):1778-1786. Epub 2020 Aug 26.

Department of Immunopathology, Sanquin Research, Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, the Netherlands;

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.
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http://dx.doi.org/10.4049/jimmunol.2000368DOI Listing
October 2020

Pre-existing antibody-mediated adverse effects prevent the clinical development of a bacterial anti-inflammatory protein.

Dis Model Mech 2020 09 28;13(9). Epub 2020 Sep 28.

Department of Medical Microbiology, University Medical Center Utrecht, 3584CX Utrecht, The Netherlands

Bacterial pathogens have evolved to secrete strong anti-inflammatory proteins that target the immune system. It was long speculated whether these virulence factors could serve as therapeutics in diseases in which abnormal immune activation plays a role. We adopted the secreted chemotaxis inhibitory protein of (CHIPS) as a model virulence factor-based therapeutic agent for diseases in which C5AR1 stimulation plays an important role. We show that the administration of CHIPS in human C5AR1 knock-in mice successfully dampens C5a-mediated neutrophil migration during immune complex-initiated inflammation. Subsequent CHIPS toxicology studies in animal models were promising. However, during a small phase I trial, healthy human volunteers showed adverse effects directly after CHIPS administration. Subjects showed clinical signs of anaphylaxis with mild leukocytopenia and increased C-reactive protein concentrations, which are possibly related to the presence of relatively high circulating anti-CHIPS antibodies and suggest an inflammatory response. Even though our data in mice show CHIPS as a potential anti-inflammatory agent, safety issues in human subjects temper the use of CHIPS in its current form as a therapeutic candidate. The use of staphylococcal proteins, or other bacterial proteins, as therapeutics or immune-modulators in humans is severely hampered by pre-existing circulating antibodies.
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http://dx.doi.org/10.1242/dmm.045534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541340PMC
September 2020

Gain-of-function mutation in complement C2 protein identified in a patient with aHUS.

J Allergy Clin Immunol 2020 10 28;146(4):916-919.e11. Epub 2020 Feb 28.

Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2020.02.014DOI Listing
October 2020

The Role of Complement in Transfusion-Related Acute Lung Injury.

Transfus Med Rev 2019 10 18;33(4):236-242. Epub 2019 Oct 18.

Sanquin Research, Department of Experimental Immunohematology, Amsterdam and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of acute respiratory distress occurring within 6 hours of blood transfusion. TRALI is one of the leading causes of transfusion-related fatalities and specific therapies are unavailable. Neutrophils are recognized as the major pathogenic cells, whereas T regulatory cells and dendritic cells appear to be important for protection against TRALI. The pathogenesis, however, is complex and incompletely understood. It is frequently postulated that the complement system plays an important role in the TRALI pathogenesis. In this article, we assess the evidence regarding the involvement of complement in TRALI from both human and animal studies. We hypothesize about the potential connection between the complement system and neutrophils in TRALI. Additionally, we draw parallels between TRALI and other acute pulmonary disorders of acute lung injury and acute respiratory distress syndrome regarding the involvement of complement. We conclude that, even though a role for complement in the TRALI pathogenesis seems plausible, studies investigating the role of complement in TRALI are remarkably limited in number and also present conflicting findings. Different types of TRALI animal models, diverse experimental conditions, and the composition of the gastrointestinal microbiota may perhaps all be factors which contribute to these discrepancies. More systematic studies are warranted to shed light on the contribution of the complement cascade in TRALI. The underlying clinical condition of the patient, which influences the susceptibility to TRALI, as well as the transfusion factor (antibody-mediated vs non-antibody-mediated), will be important to take into consideration when researching the contribution of complement. This should significantly increase our understanding of the role of complement in TRALI and may potentially result in promising new treatment strategies.
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http://dx.doi.org/10.1016/j.tmrv.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127679PMC
October 2019

Complement C3 inhibition by compstatin Cp40 prevents intra- and extravascular hemolysis of red blood cells.

Haematologica 2020 31;105(2):e57-e60. Epub 2020 Jan 31.

Sanquin Research, Department of Immunopathology, Amsterdam and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

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http://dx.doi.org/10.3324/haematol.2019.216028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012486PMC
April 2021

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

Haematologica 2019 02 27;104(2):403-416. Epub 2018 Sep 27.

Department of Immunohaematology and Blood Transfusion, Leiden University Medical Center

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.
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http://dx.doi.org/10.3324/haematol.2018.201665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355480PMC
February 2019

Reference Intervals of Factor H and Factor H-Related Proteins in Healthy Children.

Front Immunol 2018 2;9:1727. Epub 2018 Aug 2.

Sanquin Diagnostic Services, Amsterdam, Netherlands.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.
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http://dx.doi.org/10.3389/fimmu.2018.01727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082937PMC
October 2019

Detection of opsonizing antibodies directed against a recently circulating Bordetella pertussis strain in paired plasma samples from symptomatic and recovered pertussis patients.

Sci Rep 2018 08 13;8(1):12039. Epub 2018 Aug 13.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Correlates of protection (CoPs) against the highly contagious respiratory disease whooping cough, caused by Bordetella pertussis, remain elusive. Characterizing the antibody response to this pathogen is essential towards identifying potential CoPs. Here, we evaluate levels, avidity and functionality of B. pertussis-specific-antibodies from paired plasma samples derived from symptomatic and recovered pertussis patients, as well as controls. Natural infection is expected to induce protective immunity. IgG levels and avidity to nine B. pertussis antigens were determined using a novel multiplex panel. Furthermore, opsonophagocytosis of a B. pertussis clinical isolate by neutrophils was measured. Findings indicate that following infection, B. pertussis-specific antibody levels of (ex-) pertussis patients waned, while the avidity of antibodies directed against the majority of studied antigens increased. Opsonophagocytosis indices decreased upon recovery, but remained higher than controls. Random forest analysis of all the data revealed that 28% of the opsonophagocytosis index variances could be explained by filamentous hemagglutinin- followed by pertussis toxin-specific antibodies. We propose to further explore which other B. pertussis-specific antibodies can better predict opsonophagocytosis. Moreover, other B. pertussis-specific antibody functions as well as the possible integration of these functions in combination with other immune cell properties should be evaluated towards the identification of CoPs against pertussis.
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http://dx.doi.org/10.1038/s41598-018-30558-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089961PMC
August 2018

Virulence Associated Gene 8 of Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor.

Front Immunol 2018 4;9:1172. Epub 2018 Jun 4.

Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands.

is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen . Activation of the contact system by may contribute to its pathogenicity and virulence.
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http://dx.doi.org/10.3389/fimmu.2018.01172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994690PMC
August 2019

Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor.

Emerg Microbes Infect 2018 Mar 21;7(1):39. Epub 2018 Mar 21.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), 3721 MA, Bilthoven, The Netherlands.

Whooping cough, caused by Bordetella pertussis, has resurged and presents a global health burden worldwide. B. pertussis strains unable to produce the acellular pertussis vaccine component pertactin (Prn), have been emerging and in some countries represent up to 95% of recent clinical isolates. Knowledge on the effect that Prn deficiency has on infection and immunity to B. pertussis is crucial for the development of new strategies to control this disease. Here, we characterized the effect of Prn production by B. pertussis on human and murine dendritic cell (DC) maturation as well as in a murine model for pertussis infection. We incubated human monocyte-derived DCs (moDCs) with multiple isogenic Prn knockout (Prn-KO) and corresponding parental B. pertussis strains constructed either in laboratory reference strains with a Tohama I background or in a recently circulating clinical isolate. Results indicate that, compared to the parental strains, Prn-KO strains induced an increased production of pro-inflammatory cytokines by moDCs. This pro-inflammatory phenotype was also observed upon stimulation of murine bone marrow-derived DCs. Moreover, RNA sequencing analysis of lungs from mice infected with B. pertussis Prn-KO revealed increased expression of genes involved in cell death. These in vitro and in vivo findings indicate that B. pertussis strains which do not produce Prn induce a stronger pro-inflammatory response and increased cell death upon infection, suggesting immunomodulatory properties for Prn.
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http://dx.doi.org/10.1038/s41426-018-0039-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861065PMC
March 2018

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

PLoS Pathog 2017 Jul 24;13(7):e1006531. Epub 2017 Jul 24.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.
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http://dx.doi.org/10.1371/journal.ppat.1006531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542704PMC
July 2017

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

Front Microbiol 2016 20;7:2004. Epub 2016 Dec 20.

Department of Medical Microbiology, University Medical Center UtrechtUtrecht, Netherlands; Centre for Infectious Disease Control, National Institute for Public Health and the EnvironmentBilthoven, Netherlands.

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.
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http://dx.doi.org/10.3389/fmicb.2016.02004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167704PMC
December 2016

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

J Mol Med (Berl) 2015 Apr 18;93(4):395-402. Epub 2015 Feb 18.

Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands.

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.
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http://dx.doi.org/10.1007/s00109-015-1259-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366546PMC
April 2015

Nonfunctional variant 3 factor H binding proteins as meningococcal vaccine candidates.

Infect Immun 2014 Mar 30;82(3):1157-63. Epub 2013 Dec 30.

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Neisseria meningitidis is a human-specific pathogen and leading cause of meningitis and septicemia. Factor H binding protein (fHbp), a virulence factor which protects N. meningitidis from innate immunity by binding the human complement regulator factor H (fH) with high affinity, is also a key antigen in vaccines being developed to prevent meningococcal disease. fHbp can be divided into three variant groups (V1, V2, and V3) that elicit limited immunological cross-reactivity. The interaction of fH with fHbp could impair the immunogenicity of this antigen by hindering access to the antigenic epitopes in fHbp, providing the rationale for the development of nonfunctional fHbps as vaccine candidates. Here, we characterized the two nonfunctional V3 fHbps, fHbp(T286A) and fHbp(E313A), which each contains a single amino acid substitution that leads to a marked reduction in affinity for fH without affecting the folding of the proteins. The immunogenicity of the nonfunctional fHbps was assessed in transgenic mice expressing a single chimeric fH containing domains of human fH involved in binding to fHbp. No differences in anti-V3 fHbp antibody titers were elicited by the wild-type V3 fHbp, V3 fHbp(T286A), and V3 fHbp(E313A), demonstrating that the nonfunctional fHbps retain their immunogenicity. Furthermore, the nonfunctional V3 fHbps elicit serum bactericidal activity that is equivalent to or higher than that observed with the wild-type protein. Our findings provide the basis for the rational design of next-generation vaccines containing nonfunctional V3 fHbps.
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http://dx.doi.org/10.1128/IAI.01183-13DOI Listing
March 2014

Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface.

PLoS Pathog 2013 12;9(12):e1003816. Epub 2013 Dec 12.

Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.
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http://dx.doi.org/10.1371/journal.ppat.1003816DOI Listing
September 2014

Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

PLoS Pathog 2013 1;9(8):e1003528. Epub 2013 Aug 1.

Sir William Dunn School of Pathology, University of Oxford, United Kingdom.

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.
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http://dx.doi.org/10.1371/journal.ppat.1003528DOI Listing
March 2014

Staphylococcal Ecb protein and host complement regulator factor H enhance functions of each other in bacterial immune evasion.

J Immunol 2013 Aug 17;191(4):1775-84. Epub 2013 Jul 17.

Department of Bacteriology and Immunology, Haartman Institute, Research Programs Unit, Immunobiology, University of Helsinki, FIN-00014 Helsinki, Finland.

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19-20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19-20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.
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http://dx.doi.org/10.4049/jimmunol.1300638DOI Listing
August 2013

Staphylococcus aureus virulence is enhanced by secreted factors that block innate immune defenses.

J Innate Immun 2012 7;4(3):301-11. Epub 2012 Feb 7.

Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Staphylococcus aureus is a leading human pathogen that causes a large variety of diseases. In vitro studies have shown that S. aureus secretes several small proteins that block specific elements of the host innate immune system, but their role in bacterial pathogenicity is unknown. For instance, the extracellular complement-binding protein (Ecb) impairs complement activation by binding to the C3d domain of C3. Its homolog, the extracellular fibrinogen-binding protein (Efb), is known to block both complement activation and neutrophil adhesion to fibrinogen. Here, we show that targeted inactivation of the genes encoding Ecb and Efb strongly attenuates S. aureus virulence in a murine infection model: mice experienced significantly higher mortality rates upon intravenous infection with wild-type bacteria (79%) than with an isogenic ΔEcbΔEfb mutant (21%). In addition, Ecb and Efb are both required for staphylococcal persistence in host tissues and abscess formation in the kidneys (27% for wild-type vs. 7% for the ΔEcbΔEfb mutant). During staphylococcal pneumonia, Ecb and Efb together promote bacterial survival in the lungs (p = 0.03) and block neutrophil influx into the lungs. Thus, Ecb and Efb are essential to S. aureus virulence in vivo and could be attractive targets in future vaccine development efforts.
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http://dx.doi.org/10.1159/000334604DOI Listing
August 2012
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