Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococci can counteract the action of neutrophils with an antiphagocytic capsule and through electrochemical repulsion of antimicrobial peptides via addition of positive charge to the surface. Pneumococci are captured, but not killed in neutrophil extracellular traps (NETs). Here, we study the role of the polysaccharide capsule and lipoteichoic acid (LTA) modification on pneumococcal interaction with NETs. Expression of capsule (serotypes 1, 2, 4 and 9V) significantly reduced trapping by NETs, but was not required for resistance to NET-mediated killing. Pneumococci contain a dlt operon that mediates the incorporation of d-alanine residues into LTAs, thereby introducing positive charge. Genetic inactivation of dltA in non-encapsulated pneumococci rendered the organism sensitive to killing by antimicrobial components present in NETs. However, the encapsulated dltA mutant remained resistant to NET-mediated killing in vitro. Nevertheless, in a murine model of pneumococcal pneumonia, the encapsulated dltA-mutant strain was outcompeted by the wild-type upon invasion into the lungs and bloodstream. This suggests a non-redundant role for LTA alanylation in pneumococcal virulence at the early stage of invasive disease when capsule expression has been shown to be low.     

Neutrophil extracellular traps: casting the NET over pathogenesis

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identified. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.     

Streptococcus pneumoniae serotypes in children with acute pneumonia and pleurisy

The study of pneumococci of different serotypes, isolated from patients with acute pneumonia and pleuritis and from healthy children was carried out. Among the pneumococcal serotypes causing pneumonia and pleuritis in children serotypes 1, 6, 19, 14 and 3 were most widely spread and constituted 62.3% of all isolated pneumococci. In young children cases of acute pneumonia and pleuritis were more often induced by serotypes 6 and 14 and in older children, by serotypes 1 and 3. In patients with uncomplicated pneumonia and pleuritis differences in the detected serotypes of pneumococci were observed, and the disease course differed in severity. Serotypes 14, 3 and 3 induced destructive processes in the lungs more often than other serotypes. Monitoring of the sensitivity of pneumococci to antibiotics showed that most of the strains retained high sensitivity to penicillin and ampicillin. In most cases the detected resistant pneumococcal strains belonged to serogroup 19.     

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.     

T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.     

Innate immune collectin surfactant protein D simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA-protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D-NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA-protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D-DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.     

中性粒细胞胞外诱捕网在炎症相关疾病中的研究进展

中性粒细胞是抵御病原体入侵机体的第一道防线,通过趋化和吞噬作用使病原体失活,从而进行免疫防御,杀灭病原体。研究证实,中性粒细胞通过吞噬病原体、分泌抗微生物蛋白颗粒来杀灭病原微生物。2004年Brinkmann发现了一种中性粒细胞新型抗感染机制,即中性粒细胞经病原体活化刺激后释放中性粒细胞胞外诱捕网(neutrophil extracellular trap,NET)至细胞外。NET是由双链DNA染色质和镶嵌在染色质上的抗菌蛋白构成的纤维网格状结构,通过网罗、捕获而杀灭病原体。诸多研究表明,NET在炎症相关疾病中起重要作用,其生成和降解会影响急慢性炎性疾病的病理过程。本文主要从NET的特征、产生机制、抗菌作用及其在炎性相关疾病中的作用等方面着手,概述其最新研究进展,为炎性疾病的治疗及其药物开发提供新的思路和方向。     

NETosis occurs independently of neutrophil serine proteases

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.     

IL-18 improves the early antimicrobial host response to pneumococcal pneumonia.

To determine the role of endogenous IL-18 during pneumonia, IL-18 gene-deficient (IL-18(-/-)) mice and wild-type (WT) mice were intranasally inoculated with Streptococcus pneumoniae, the most common causative agent of community-acquired pneumonia. Infection with S. pneumoniae increased the expression of IL-18 mRNA and was associated with elevated concentrations of both precursor and mature IL-18 protein within the lungs. IL-18(-/-) mice had significantly more bacteria in their lungs and were more susceptible for progressing to systemic infection at 24 and 48 h postinoculation. Similarly, treatment of WT mice with anti-IL-18 was associated with enhanced outgrowth of pneumococci. In contrast, the clearance of pneumococci from lungs of IL-12(-/-) mice was unaltered when compared with WT mice. Furthermore, anti-IL-12 did not influence bacterial clearance in either IL-18(-/-) or WT mice. These data suggest that endogenous IL-18, but not IL-12, plays an important role in the early antibacterial host response during pneumococcal pneumonia.     

Pneumococcal immune evasion: ZmpC inhibits neutrophil influx

Neutrophil recruitment is essential in clearing pneumococcal infections. The first step in neutrophil extravasation involves the interaction between P‐selectin on activated endothelium and P‐Selectin Glycoprotein 1 (PSGL‐1) on neutrophils. Here, we identify pneumococcal Zinc metalloproteinase C as a potent inhibitor of PSGL‐1. ZmpC degrades the N‐terminal domain of PSGL‐1, thereby disrupting the initial rolling of neutrophils on activated human umbilical vein endothelial cells. Furthermore, mice infected with wild‐type strain in the model of pneumococcal pneumonia showed lower lungs neutrophil infiltration compare to animals infected with ZmpC mutant. In addition, we confirmed the association of zmpC with serotype 8 and 11A and found it to be associated with serotype 33F as well. In conclusion, wereport PSGL‐1 as a novel target for ZmpC and show that ZmpC inhibits neutrophil extravasation during pneumococcal pneumonia.     

Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection.     

Novel vaccine strategies with protein antigens of Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.     

Urokinase receptor is necessary for adequate host defense against pneumococcal pneumonia

Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the beta(2) integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for beta(2) integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a beta(2) integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR(-/-)), uPA-deficient (uPA(-/-)), and wild-type (Wt) mice were intranasally inoculated with 10(5) CFU S. pneumoniae. uPAR(-/-) mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA(-/-) mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a beta(2) integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.     

Streptococcus pneumoniae induced p38 MAPK- and NF-kappaB-dependent COX-2 expression in human lung epithelium

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.     

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high¹. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.     

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.     

Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL

During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr(-/-) mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDL-specific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.     

Neutrophil intrinsic and extrinsic regulation of NETosis in health and disease

Neutrophil extracellular traps (NETs) evolved to protect the host against microbial infections and are formed by a web-like structure of DNA that is decorated with antimicrobial effectors. Due to their potent inflammatory functions, NETs also cause tissue damage and can favor and/or aggravate inflammatory diseases. This multipronged activity of NETs requires that the induction, release, and degradation of NETs are tightly regulated. Here we describe the key pathways that are intrinsic to neutrophils and regulate NETosis, and we review the most recent findings on how neutrophil extrinsic factors participate in the formation of NETs. In particular, we emphasize how bystander cells contribute to modifying the capacity of neutrophils to undergo NETosis. Finally, we discuss how these neutrophil extrinsic processes can be harnessed to protect the host against the excessive inflammation elicited by uncontrolled NET release.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.