Marcel Schouten

Inflammation, endothelium, and coagulation in sepsis

Sepsis is a systemic response to infection, and symptoms are produced by host defense systems rather than by the invading pathogens. Amongst the most prominent features of sepsis, contributing significantly to its outcome, is activation of coagulation with concurrent down‐regulation of anticoagulant systems and fibrinolysis. Inflammation‐induced coagulation on its turn contributes to inflammation. Another important feature of sepsis, associated with key symptoms such as hypovolemia and hypotension, is endothelial dysfunction. Under normal conditions, the endothelium provides for an anticoagulant surface, a property that is lost in sepsis. In this review, data about the interplay between inflammation and coagulation in sepsis are summarized with a special focus on the influence of the endothelium on inflammation‐induced coagulation and vice versa. Possible procoagulant properties of the endothelium are described, such as expression of tissue factor (TF) and von Willebrand factor and interaction with platelets. Possible procoagulant roles of microparticles, circulating endothelial cells and endothelial apoptosis, are also discussed. Moreover, the important roles of the endothelium in down‐regulating the anticoagulants TF pathway inhibitor, antithrombin, and the protein C (PC) system and inhibition of fibrinolysis are discussed. The influence of coagulation on its turn on inflammation and the endothelium is described with a special focus on protease‐activated receptors (PARs). We conclude that the relationship between endothelium and coagulation in sepsis is tight and that further research is needed, for example, to better understand the role of activated PC signaling via PAR‐1, the role of the endothelial PC receptor herein, and the role of the glycocalyx.

The Receptor for Advanced Glycation End Products Impairs Host Defense in Pneumococcal Pneumonia

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that is expressed ubiquitously in the lungs. Engagement of RAGE leads to activation of multiple intracellular signaling pathways, including NF-κB and subsequent transcription of several proinflammatory mediators. To determine the role of RAGE in the innate immune response to S. pneumoniae pneumonia, RAGE-deficient (RAGE−/−) and wild-type mice were intranasally inoculated with S. pneumoniae. S. pneumoniae pneumonia resulted in an up-regulation of constitutively present RAGE expression in lung tissue, especially in the interalveolar septae. RAGE−/− mice showed an improved survival, which was accompanied by a lower bacterial load in the lungs at 16 h and a decreased dissemination of the bacteria to blood and spleen at 16 and 48 h after inoculation. RAGE−/− macrophages showed an improved killing capacity of S. pneumoniae in vitro. Lung inflammation was attenuated in RAGE−/− mice at 48 h after inoculation, as indicated by histopathology and cytokine/chemokine levels. Neutrophil migration to the lungs was mitigated in the RAGE−/− mice. In addition, in RAGE−/− mice, activation of coagulation was diminished. Additional studies examining the effect of RAGE deficiency on the early (6-h) inflammatory response to S. pneumoniae did not reveal an early accelerated or enhanced immune response. These data suggest that RAGE plays a detrimental role in the host response to S. pneumoniae pneumonia by facilitating the bacterial growth and dissemination and concurrently enhancing the pulmonary inflammatory and procoagulant response.

Role Played by Toll-Like Receptors 2 and 4 in Lipoteichoic Acid—Induced Lung Inflammation and Coagulation

BACKGROUND The cell wall of Streptococcus pneumoniae consists of lipoteichoic acid (LTA), which is released when pneumococci are killed by either the host immune system or antibiotic treatment. Release of excessive amounts of LTA has been implicated in the toxic sequelae of severe gram-positive infection by virtue of its proinflammatory properties. Several in vitro studies have shown that LTA is recognized by Toll-like receptor (TLR) 2 and CD14. Our objective here was to investigate the inflammatory properties of S. pneumoniae LTA in vivo and the role played by TLR2, TLR4, and CD14 therein. METHODS Wild-type (WT), TLR2 knockout (KO), TLR4 KO, TLR2x4 double-KO, and CD14 KO mice were intranasally inoculated with highly purified pneumococcal LTA. RESULTS LTA induced a dose-dependent inflammatory response and activation of the coagulation and fibrinolytic pathways in a TLR2-dependent fashion. Surprisingly, TLR4 KO mice also displayed a somewhat diminished pulmonary inflammatory and coagulant response compared with WT mice, possibly as a result of absent TLR4 signaling through LTA-induced release of endogenous mediators. CONCLUSION Pneumococcal LTA induces a profound inflammatory response and activation of the coagulation pathway in the lungs in vivo through a TLR2-dependent route, which likely is amplified by endogenous TLR4 ligands.

Lipopolysaccharide Inhibits Th2 Lung Inflammation Induced by House Dust Mite Allergens in Mice

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 μg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.

Sepsis, Coagulation, and Antithrombin: Old Lessons and New Insights

Current insights in the pathogenesis of multiple organ dysfunction in patients with sepsis point to a pivotal role of inflammation and coagulation. One of the most important mechanisms contributing to the activation of coagulation in sepsis is the downregulation of physiologic anticoagulant systems, such as the antithrombin pathway. More than 20 years ago, Eberhard Mammen already hypothesized that coagulation activation and antithrombin were important factors in patients with sepsis. Abundant experimental and clinical studies have supported that notion in recent years. The better understanding of the pathogenesis of coagulation activation and the role of natural anticoagulants in sepsis has led to the development of anticoagulant factor concentrates, such as antithrombin concentrate. Clinical studies indicate that these interventions may have a role in the (supportive) treatment of patients with sepsis, mostly based on surrogate outcomes, but ongoing studies will have to confirm a beneficial effect in reducing mortality.

Natural anticoagulants limit lipopolysaccharide-induced pulmonary coagulation but not inflammation

Pulmonary coagulopathy and hyperinflammation may contribute to an adverse outcome in sepsis. The present study determines the effects of natural inhibitors of coagulation on bronchoalveolar haemostasis and inflammation in a rat model of endotoxaemia. Male Sprague-Dawley rats were randomised to treatment with normal saline, recombinant human activated protein C (APC), plasma-derived antithrombin (AT), recombinant human tissue factor pathway inhibitor (TFPI), heparin or recombinant tissue plasminogen activator (tPA). Rats were intravenously injected with lipopolysaccharide (LPS), which induced a systemic inflammatory response and pulmonary inflammation. Blood and bronchoalveolar lavage were obtained at 4 and 16 h after LPS injection, and markers of coagulation and inflammation were measured. LPS injection caused an increase in the levels of thrombin–AT complexes, whereas plasminogen activator activity was attenuated, both systemically and within the bronchoalveolar compartment. Administration of APC, AT and TFPI significantly limited LPS-induced generation of thrombin–AT complexes in the lungs, and tPA stimulated pulmonary fibrinolytic activity. However, none of the agents had significant effects on the production of pulmonary cytokines, chemokines, neutrophil influx and myeloperoxidase activity. Natural inhibitors of coagulation prevent bronchoalveolar activation of coagulation, but do not induce major alterations of the pulmonary inflammatory response in rat endotoxaemia.

Early activation of coagulation after allergen challenge in patients with allergic asthma

Asthma is characterized by allergic airway inflammation which is associated with bronchial hyperreponsiveness and airway obstruction [1]. Recent evidence indicates that activation of coagulation within the airways in asthma may aggravete inflammation [2]. Asthma patients were found to have elevated concentrations of thrombin, thrombin-antithrombin complexes (TATc) and soluble tissue factor and reduced activated protein C (APC)/thrombin ratios in induced sputum [3,4]. However, knowledge on coagulation activation in the lower airways in asthma in humans is limited, especially with regard to the acute impact of an allergen challenge. We therefore determined activation of coagulation in the bronchoalveolar space and the acute effect of a segmental allergen challenge hereon in asthma patients as compared to healthy controls. Our study population has been described previously [5]. In short, thirteen allergic asthmatic subjects and nine healthy volunteers were included. Patients had a positive skin prick test for house dust mite allergens, grass pollen or both. Patients had not experienced an exacerbation of asthma during at least 2 months and had not used bronchodilators for at least 8 h before the investigations. None of the subjects had experienced recent airway infection or used anti-inflammatory or anticoagulant drugs. The study was approved by the Internal Review Board of the Academic Medical Center Amsterdam and written informed consent was obtained from all participants. Intracutaneous dose-response series with house dust mite or grass pollen (ALK Abello, Nieuwegein, The Netherlands) were performed to determine the concentration that produced a 10 mm wheal response 15 min after injection. Asthmatic subjects underwent an intrabronchial challenge with 1 mL of this allergen concentration – brought to a final volume of 5 mL with saline – whereas controls were challenged with the highest concentration applied in the patient group. Levels of lipopolysaccharide in the allergen solution were < 1.3 pg mL )1 in all

CD44 is protective during hyperoxia-induced lung injury.

Patients with acute lung injury or respiratory distress syndrome often require supplemental oxygen to maintain tissue oxygenation; however, this treatment can cause or worsen lung inflammation. CD44 is a transmembrane adhesion molecule that is present on a wide variety of cell types, including leukocytes and parenchymal cells, and is an important player in leukocyte trafficking. The aim of this study was to determine the role of CD44 during hyperoxia-induced (> 95% oxygen) acute lung injury. Whereas all wild-type mice survived the 72-hour observation period, 37.5% of CD44 knockout (KO) mice died. CD44 deficiency was associated with a profound influx of neutrophils into the bronchoalveolar space, in the presence of similar or even lower neutrophil numbers in lung parenchyma, suggesting that CD44 is important for containing neutrophils in the pulmonary interstitium during hyperoxia. In addition, CD44 deficiency resulted in increased IL-6 and keratinocyte-derived chemokine release into bronchoalveolar lavage fluid (BALF). CD44 KO mice further displayed evidence for increased vascular leak and injury of type II respiratory epithelial cells. CD44 protected against bronchial epithelial cell death, as shown by increased epithelial cell necrosis and a trend toward increased BALF nucleosome levels in CD44 KO mice. CD44 can bind and internalize hyaluronic acid (HA), which acts proinflammatory. Concentrations of HA increased in BALF from CD44 KO but not wild-type mice during hyperoxia. These data suggest that CD44 protects against hyperoxia-induced lung injury and mortality by a mechanism that at least in part relies on its ability to clear HA from the bronchoalveolar space.

CCAAT/enhancer-binding protein δ facilitates bacterial dissemination during pneumococcal pneumonia in a platelet-activating factor receptor-dependent manner

CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ−/− mice are relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection. Moreover, expression of platelet-activating factor receptor (PAFR), which is known to potentiate bacterial translocation of Gram-positive bacteria, was significantly reduced during infection in C/EBPδ−/− mice compared with WT controls. Importantly, cell stimulation experiments revealed that C/EBPδ potentiates PAFR expression induced by lipoteichoic acid and pneumococci. Thus, C/EBPδ exaggerates bacterial dissemination during Streptococcus pneumoniae-induced pulmonary infection, suggesting an important role for PAFR-dependent bacterial translocation.

Osteopontin Impairs Host Defense During Pneumococcal Pneumonia

BACKGROUND Streptococcus pneumoniae is the most frequently isolated pathogen responsible for community-acquired pneumonia. Osteopontin is involved in inflammation during both innate and adaptive immunity. METHODS To determine the role of osteopontin in the host response during pneumococcal pneumonia, osteopontin knockout (KO) and normal wild-type (WT) mice were intranasally infected with viable S. pneumoniae. RESULTS Pneumonia was associated with a rapid increase in pulmonary osteopontin concentrations in WT mice from 6 h onward. Osteopontin KO mice showed a prolonged survival relative to WT mice, which was accompanied by diminished pulmonary bacterial growth and reduced dissemination to distant body sites. In addition, at 48 h after infection pulmonary inflammation was decreased in osteopontin KO mice as reflected by lower inflammation scores and reduced chemokine concentrations. In contrast to pneumococcal pneumonia, osteopontin deficiency did not influence bacterial growth in primary pneumococcal sepsis induced by direct intravenous infection, suggesting that the detrimental effect of osteopontin on antibacterial defense during pneumonia primarily is exerted in the pulmonary compartment. Moreover, recombinant osteopontin stabilized S. pneumoniae viability in vitro. CONCLUSIONS These results suggest that the pneumococcus misuses osteopontin in the airways for optimal growth and to cause invasive disease after entering the lower airways.