Apoptosis in Acute Shigellosis is Associated with Increased Production of Fas/Fas Ligand, Perforin, Caspase-1, and Caspase-3 but Reduced Production of Bcl-2 and Interleukin-2

Infection and Immunity. Jun, 2002  |  Pubmed ID: 12011015

Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.

Neutrophil Elastase Targets Virulence Factors of Enterobacteria

Nature. May, 2002  |  Pubmed ID: 12018205

Shigellae cause bacillary dysentery, a bloody form of diarrhoea that affects almost 200 million people and causes nearly 2 million deaths per year. Shigella invades the colonic mucosa, where it initiates an acute inflammation, rich in neutrophils, that initially contributes to tissue damage and eventually resolves the infection. Neutrophils are phagocytic cells that kill microorganisms but it is unclear how neutrophils control pathogenic bacteria expressing virulence factors that manipulate host cells. In contrast to other cells, neutrophils prevent the escape of Shigella from phagocytic vacuoles in which the bacteria are killed. Here we identify human neutrophil elastase (NE) as a key host defence protein: NE degrades Shigella virulence factors at a 1,000-fold lower concentration than that needed to degrade other bacterial proteins. In neutrophils in which NE is inactivated pharmacologically or genetically, Shigella escapes from phagosomes, increasing bacterial survival. NE also preferentially cleaves virulence factors of Salmonella and Yersinia. These findings establish NE as the first neutrophil factor that targets bacterial virulence proteins.

Caspase-1 Activation by Salmonella

Trends in Cell Biology. Apr, 2003  |  Pubmed ID: 12667758

Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system.

The ShiA Protein Encoded by the Shigella Flexneri SHI-2 Pathogenicity Island Attenuates Inflammation

Cellular Microbiology. Nov, 2003  |  Pubmed ID: 14531895

Shigella spp. are the aetiologic agents of dysentery, a severe diarrhoeal syndrome characterized by acute inflammation in the colon. The inflammatory response, which includes recruitment of polymorphonuclear leukocytes (PMN), damages the colonic mucosa and exacerbates the infection. Shigella encodes a pathogenicity island (PAI), SHI-2, which is localized in a region of the chromosome linked to the induction of inflammation. Surprisingly, SHI-2 deletion mutants induce a stronger inflammatory response than wild-type Shigella as measured by increased villus blunting, increased PMN infiltration and induction of apoptosis in a rabbit ileal loop model of shigellosis. Mutational analysis mapped the hyper-inflammatory phenotype to a single gene, shiA. Similar to SHI-2 deletion mutants, infection with a shiA mutant strain induces dramatically elevated levels of inflammation when compared to the wild-type strain. Furthermore, infection with a wild-type strain containing multiple copies of shiA results in fewer infiltrating PMN and apoptotic cells, as well as preservation of a normal villus architecture at the site of infection, thus acting in a dominant fashion over the pro-inflammatory mechanisms of Shigella. The molecular mechanism of action of ShiA is independent of any in vitro phenotype associated with Shigella virulence. Our data suggest that ShiA allows Shigella to attenuate the host inflammatory response in a novel manner.

Introduction: Forum in Immunology on Neutrophils

Microbes and Infection / Institut Pasteur. Nov, 2003  |  Pubmed ID: 14613771

Neutrophil Extracellular Traps Kill Bacteria

Science (New York, N.Y.). Mar, 2004  |  Pubmed ID: 15001782

Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.

Toll-like Receptors Are Temporally Involved in Host Defense

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2004  |  Pubmed ID: 15034062

Toll-like receptors (TLRs) are evolutionarily conserved proteins that recognize microbial molecules and initiate host defense. To investigate how TLRs work together to fight infections, we tested the role of TLRs in host defense against the Gram-negative bacterial pathogen, Salmonella. We show that TLR4 is critical for early cytokine production and killing of bacteria by murine macrophages. Interestingly, later on, TLR2, but not TLR4, is required for macrophage responses. Myeloid differentiation factor 88, an adaptor protein directly downstream of TLRs, is required for both early and late responses. TLR4, TLR2, and myeloid differentiation factor 88 are involved in murine host defense against Salmonella in vivo, which correlates with the defects in host defense observed in vitro. We propose a model where the sequential activation of TLRs tailors the immune response to different microbes.

How Do Neutrophils and Pathogens Interact?

Current Opinion in Microbiology. Feb, 2004  |  Pubmed ID: 15036142

Many pathogens can manipulate macrophages after phagocytosis yet are efficiently killed by neutrophils. This poses the question of whether neutrophils have mechanisms that enable them to specifically recognise pathogens and have pathogens evolved mechanisms to modulate neutrophil function? Here, we review recent work on neutrophils and their interaction with four different bacteria: Staphylococcus aureus, Helicobacter pylori, Anaplasma phagocytophilum and members of the Enterobacteriae family.

MyD88, but Not Toll-like Receptors 4 and 2, is Required for Efficient Clearance of Brucella Abortus

Infection and Immunity. Aug, 2005  |  Pubmed ID: 16041030

It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88(-/-) mice do not clear the bacteria as efficiently as wild-type, TLR4(-/-), TLR2(-/-), or TLR4/TLR2(-/-) mice.

Proinflammatory Effects of Bacterial Recombinant Human C-reactive Protein Are Caused by Contamination with Bacterial Products, Not by C-reactive Protein Itself

Circulation Research. Nov, 2005  |  Pubmed ID: 16254214

Intravenous administration to human volunteers of a commercial preparation of recombinant human C-reactive protein (CRP) produced in Escherichia coli was recently reported in this journal to induce an acute phase response of serum amyloid A protein (SAA) and of CRP itself, and to activate the coagulation system. The authors concluded that CRP is probably a mediator of atherothrombotic disease. Here we confirm that this recombinant CRP preparation was proinflammatory both for mouse macrophages in vitro and for mice in vivo, but show that pure natural human CRP had no such activity. Furthermore mice transgenic for human CRP, and expressing it throughout their lives, maintained normal concentrations of their most sensitive endogenous acute phase reactants, SAA and serum amyloid P component (SAP). The patterns of in vitro cytokine induction and of in vivo acute phase stimulation by the recombinant CRP preparation were consistent with contamination by bacterial products, and there was 46.6 EU of apparent endotoxin activity per mg of CRP in the bacterial product, compared with 0.9 EU per mg of our isolated natural human CRP preparation. The absence of any proinflammatory activity in natural CRP for macrophages or healthy mice strongly suggests that the in vivo effects of the recombinant preparation observed in humans were attributable to proinflammatory bacterial products and not human CRP.

Human Neutrophils Kill Bacillus Anthracis

PLoS Pathogens. Nov, 2005  |  Pubmed ID: 16292357

Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

An Endonuclease Allows Streptococcus Pneumoniae to Escape from Neutrophil Extracellular Traps

Current Biology : CB. Feb, 2006  |  Pubmed ID: 16488875

Streptococcus pneumoniae (pneumococcus) is the most common cause of community-acquired pneumonia, with high morbidity and mortality worldwide. A major feature of pneumococcal pneumonia is an abundant neutrophil infiltration . It was recently shown that activated neutrophils release neutrophil extracellular traps (NETs), which contain antimicrobial proteins bound to a DNA scaffold. NETs provide a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly. Here, we show that pneumococci are trapped but, unlike many other pathogens, not killed by NETs. NET trapping in the lungs, however, may allow the host to confine the infection, reducing the likelihood for the pathogen to spread into the bloodstream. DNases are expressed by many Gram-positive bacterial pathogens, but their role in virulence is not clear. Expression of a surface endonuclease encoded by endA is a common feature of many pneumococcal strains. We show that EndA allows pneumococci to degrade the DNA scaffold of NETs and escape. Furthermore, we demonstrate that escaping NETs promotes spreading of pneumococci from the upper airways to the lungs and from the lungs into the bloodstream during pneumonia.

Neutrophil Extracellular Traps Capture and Kill Candida Albicans Yeast and Hyphal Forms

Cellular Microbiology. Apr, 2006  |  Pubmed ID: 16548892

Neutrophils phagocytose and kill microbes upon phagolysosomal fusion. Recently we found that activated neutrophils form extracellular fibres that consist of granule proteins and chromatin. These neutrophil extracellular traps (NETs) degrade virulence factors and kill Gram positive and negative bacteria. Here we show for the first time that Candida albicans, a eukaryotic pathogen, induces NET-formation and is susceptible to NET-mediated killing. C. albicans is the predominant aetiologic agent of fungal infections in humans, particularly in immunocompromised hosts. One major virulence trait of C. albicans is its ability to reversibly switch from singular budding cells to filamentous hyphae. We demonstrate that NETs kill both yeast-form and hyphal cells, and that granule components mediate fungal killing. Taken together our data indicate that neutrophils trap and kill ascomycetous yeasts by forming NETs.

ShiA Abrogates the Innate T-cell Response to Shigella Flexneri Infection

Infection and Immunity. Apr, 2006  |  Pubmed ID: 16552062

Shigella spp. are the causative agent of bacillary dysentery. Infection results in acute colonic injury due to the host inflammatory response. The mediators of the damage, infiltrating polymorphonuclear leukocytes (PMN), also resolve the infection. Shigella flexneri's virulence effectors are encoded on its large virulence plasmid and on pathogenicity islands in the chromosome. The SHI-2 pathogenicity island encodes the virulence factor ShiA, which down-regulates Shigella-induced inflammation. In the rabbit ileal loop model, infection with a shiA null strain (DeltashiA) induces a more severe inflammation than wild-type infection. Conversely, a Shigella strain that overexpresses ShiA (ShiA+) is less inflammatory than the wild-type strain. To determine the host responses modulated by ShiA, we performed infection studies using the mouse lung model, which recapitulates the phenotypes observed in the rabbit ileal loop model. Significantly, ShiA+ strain-infected mice cleared the bacteria and survived infection, while wild-type- and DeltashiA strain-infected mice could not clear the bacteria and ultimately died. Surprisingly, microarray analysis of infected lungs revealed the regulation of genes involved in innate T-cell responses to infection. Immunohistochemistry showed that wild-type- and DeltashiA strain-infected animals have greater numbers of PMN and T cells in their lungs over the course of infection than ShiA+ strain-infected animals. These results suggest that the T-cell innate response is suppressed by ShiA in Shigella infections.

Caspase-1-mediated Activation of Interleukin-1beta (IL-1beta) and IL-18 Contributes to Innate Immune Defenses Against Salmonella Enterica Serovar Typhimurium Infection

Infection and Immunity. Aug, 2006  |  Pubmed ID: 16861683

Caspase-1 (Casp-1) mediates the processing of the proinflammatory cytokines interleukin-1beta (IL-1beta) and IL-18 to their mature forms. Casp-1-deficient mice succumb more rapidly to Salmonella challenge than do wild-type animals. Both Casp-1 substrates, IL-18 and IL-1beta, are relevant for control of Salmonella enterica serovar Typhimurium. We used IL-18-/- and IL-1beta-/- mice in addition to administration of recombinant IL-18 to Casp-1-/- mice to demonstrate that IL-18 is important for resistance to the systemic infection but not for resistance to the intestinal phase of the infection. This suggests that IL-1beta is critical for the intestinal phase of the disease. Thus, we show that Casp-1 is essential for host innate immune defense against S. enterica serovar Typhimurium and that Casp-1 substrates are required at distinct times and anatomical sites.

How Do Microbes Evade Neutrophil Killing?

Cellular Microbiology. Nov, 2006  |  Pubmed ID: 16939535

Many microbial pathogens evolved to circumvent the attack of neutrophils, which are essential effector cells of the innate immune system. Here we review six major strategies that pathogenic bacteria and fungi use to evade neutrophil defences: (i) turning on survival and stress responses, (ii) avoiding contact, (iii) preventing phagocytosis, (iv) surviving intracellularly, (v) inducing cell death and (vi) evading killing by neutrophil extracellular traps. For each category we give examples and further focus on one particular pathogenic microbe in more detail. Pathogens include Candida albicans, Cryptococcus neoformans, Yersinia ssp., Helicobacter pylori, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae.

Novel Cell Death Program Leads to Neutrophil Extracellular Traps

The Journal of Cell Biology. Jan, 2007  |  Pubmed ID: 17210947

Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We 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 process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

Capsule and D-alanylated Lipoteichoic Acids Protect Streptococcus Pneumoniae Against Neutrophil Extracellular Traps

Cellular Microbiology. May, 2007  |  Pubmed ID: 17217430

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.

What is the Role of Toll-like Receptors in Bacterial Infections?

Seminars in Immunology. Feb, 2007  |  Pubmed ID: 17280841

Innate immunity relies on signalling by Toll-like receptors (TLRs) to alert the immune system of the presence of invading bacteria. TLR activation leads to the release of cytokines that allow for effective innate and adaptive immune responses. However, the contribution of different TLRs depends on the site of the infection and the pathogen. This review will describe the involvement of TLRs in the development of three different bacterial infections as well as our current understanding of the role of TLRs during microbial pathogenesis.

Chronic Granulomatous Disease (CGD) and Complete Myeloperoxidase Deficiency Both Yield Strongly Reduced Dihydrorhodamine 123 Test Signals but Can Be Easily Discerned in Routine Testing for CGD

Clinical Chemistry. May, 2007  |  Pubmed ID: 17384005

The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD).

Netting Bacteria in Sepsis

Nature Medicine. Apr, 2007  |  Pubmed ID: 17415369

Beneficial Suicide: Why Neutrophils Die to Make NETs

Nature Reviews. Microbiology. Aug, 2007  |  Pubmed ID: 17632569

Neutrophils are one of the main types of effector cell in the innate immune system and were first shown to effectively kill microorganisms by phagocytosis more than 100 years ago. Recently, however, it has been found that stimulated neutrophils can also produce extracellular structures called neutrophil extracellular traps (NETs) that capture and kill microorganisms. This Progress article gives an overview of the structure, function and generation of NETs, and their role in infections.

Shigella Flexneri Phagosomal Escape is Independent of Invasion

Infection and Immunity. Oct, 2007  |  Pubmed ID: 17664266

Infections with Salmonella enterica serovar Typhimurium and Shigella flexneri result in mucosal inflammation in response to epithelial cell invasion and macrophage cytotoxicity. These processes are mediated by type III secretion systems encoded in homologous virulence loci in the two species, namely, Salmonella pathogenicity island 1 (SPI-1), carried in the genome, and the Shigella entry region (SER), carried in a large virulence plasmid. Here we show that SPI-1 can functionally complement a deletion of SER in S. flexneri, restoring invasion of epithelial cells, macrophage cytotoxicity, and phagosomal escape. Furthermore, S. flexneri phagosomal escape requires the SER and another gene(s) carried on the large virulence plasmid. We demonstrate that the processes of invasion and phagosomal escape can be uncoupled in S. flexneri.

Single Residue Determines the Specificity of Neutrophil Elastase for Shigella Virulence Factors

Journal of Molecular Biology. Apr, 2008  |  Pubmed ID: 18295791

Human neutrophil elastase (NE) is a key host defense protease that cleaves virulence factors of Gram-negative bacteria. NE and cathepsin G (CG) are chymotrypsin-like serine proteases with sequence and structural similarities, and both are abundant in neutrophil granules. Unlike NE, CG does not cleave virulence factors of enteric bacteria. Through structure-function analysis, we identified regions in NE that are essential for cleaving Shigella virulence proteins. NE residues at eight different positions were replaced with analogous amino acids in CG or with alanine. Functional analysis of recombinant mutant proteins showed that a single residue at position 98 and multiple amino acid stretches in the three different regions 58A-61, 163-181, and 216-224 determine NE specificity. These NE mutants cleaved the CG-specific, but not the NE-specific, synthetic peptide substrate and did not degrade Shigella virulence factors. Interestingly, exchanging the amino acid at position 98 in CG for the NE equivalent enabled this CG mutant to cleave Shigella virulence factors. Analysis of the NE proteolytic products of the Shigella virulence factor IpaB shows that NE has specific cleavage sites. These results indicate that Shigella virulence factor specificity maps to a distinct region close to NE's active site.

The Capsule Sensitizes Streptococcus Pneumoniae to Alpha-defensins Human Neutrophil Proteins 1 to 3

Infection and Immunity. Aug, 2008  |  Pubmed ID: 18474654

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified alpha-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial alpha-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 microg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to alpha-defensins. The proposed mechanisms of alpha-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by D-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, alpha-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

A Toll-like Receptor 2-integrin Beta3 Complex Senses Bacterial Lipopeptides Via Vitronectin

Nature Immunology. Jul, 2008  |  Pubmed ID: 18516040

Toll-like receptor 2 (TLR2) initiates inflammation in response to bacterial lipopeptide (BLP). However, the molecular mechanisms enabling the detection of BLP by TLR2 are unknown. Here we investigated the interaction of BLP with human serum proteins and identified vitronectin as a BLP-recognition molecule. Vitronectin and its receptor, integrin beta(3), were required for BLP-induced TLR2-mediated activation of human monocytes. Furthermore, monocytes from patients with Glanzmann thrombasthenia, which lack integrin beta(3), were completely unresponsive to BLP. In addition, integrin beta(3) formed a complex with TLR2 and this complex dissociated after BLP stimulation. Notably, vitronectin and integrin beta(3) coordinated responses to other TLR2 agonists such as lipoteichoic acid and zymosan. Our findings show that vitronectin and integrin beta(3) contribute to the initiation of TLR2 responses.

Superoxide Dismutase 1 Regulates Caspase-1 and Endotoxic Shock

Nature Immunology. Aug, 2008  |  Pubmed ID: 18604212

Caspase-1 serves an essential function in the initiation of inflammation by proteolytically maturing the cytokines interleukin 1 beta and interleukin 18. Several Nod-like receptors activate caspase-1 in response to microbial and 'danger' signals by assembling cytosolic protein complexes called 'inflammasomes'. We show here that superoxide dismutase 1 (SOD1) regulates caspase-1 activation. In SOD1-deficient macrophages, higher superoxide production decreased the cellular redox potential and specifically inhibited caspase-1 by reversible oxidation and glutathionylation of the redox-sensitive cysteine residues Cys397 and Cys362. Conversely, hypoxic conditions abrogated caspase-1 inhibition. In vivo, SOD1-deficient mice produced less caspase-1-dependent cytokines and were less susceptible to lipopolysaccharide-induced septic shock. Our findings identify a physiological post-translational mechanism in the control of caspase-1-mediated inflammatory processes.

Fungal and Bacterial Killing by Neutrophils

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19089391

Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.

IpaB-IpgC Interaction Defines Binding Motif for Type III Secretion Translocator

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2009  |  Pubmed ID: 19478065

The delivery of virulence factors into host cells through type III secretion systems is essential for enterobacterial pathogenesis. Molecular chaperones bind specifically to virulence factors in the bacterial cytosol before secretion. Invasion plasmid gene C (IpgC) is a chaperone that binds 2 essential virulence factors of Shigella: invasion plasmid antigens (Ipa) B and C. Here, we report the crystal structure of IpgC alone and in complex with the chaperone binding domain (CBD) of IpaB. The chaperone captures the CBD in an extended conformation that is stabilized by conserved residues lining the cleft. Analysis of the cocrystal structure reveals a sequence motif that is functional in the IpaB translocator class from different bacteria as determined by isothermal titration calorimetry. Our results show how translocators are chaperoned and may allow the design of inhibitors of enterobacterial diseases.

Restoration of NET Formation by Gene Therapy in CGD Controls Aspergillosis

Blood. Sep, 2009  |  Pubmed ID: 19541821

Chronic granulomatous disease (CGD) patients have impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, resulting in poor antimicrobial activity of neutrophils, including the inability to generate neutrophil extracellular traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy persons. The aim of this study was to determine whether gene therapy restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by gene therapy in a patient with CGD restores neutrophil elimination of A nidulans conidia and hyphae and is associated with rapid cure of preexisting therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.

NETs: a New Strategy for Using Old Weapons

Trends in Immunology. Nov, 2009  |  Pubmed ID: 19699684

As key players in the host innate immune response, neutrophils are recruited to sites of infection and constitute the first line of defense. They employ three strategies to eliminate invading microbes: microbial uptake, the secretion of antimicrobials, and the recently described release of Neutrophil Extracellular Traps (NETs). Composed of decondensed chromatin and antimicrobial proteins, NETs bind and kill a variety of microbes including bacteria, fungi, and parasites. In addition to using a repertoire of known antimicrobials, NETs incorporate histones into the antimicrobial arsenal. Furthermore, NETs may contribute to microbial containment by forming a physical barrier and a scaffold, to enhance antimicrobial synergy while minimizing damage to host tissues. Their role in innate immunity is only now being uncovered.

Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense Against Candida Albicans

PLoS Pathogens. Oct, 2009  |  Pubmed ID: 19876394

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

Sepsis: the Dark Side of Histones

Nature Medicine. Nov, 2009  |  Pubmed ID: 19893552

Mouse Neutrophil Extracellular Traps in Microbial Infections

Journal of Innate Immunity. 2009  |  Pubmed ID: 20375576

Neutrophil extracellular traps (NETs) play an important role in innate immunity to microbial infections. NETs have been described in several species, but the molecular details of NET formation and their role in infection has not been addressed, partly because we lack optimal experimental models. Here we describe tools to investigate NET formation in neutrophils isolated from mice. Upon in vitro stimulation of wild-type mouse neutrophils with PMA, we analyzed 3 important steps in the process of NET formation: reactive oxygen species (ROS) production, NET cell death and NET release. As expected, neutrophils from NADPH oxidase-deficient mice failed to produce ROS and did not die nor release NETs upon stimulation. We found that neutrophils from several mouse strains produced NETs with different efficiency and that NET formation correlated with the amount of ROS produced. Activation with Candida albicans also resulted in ROS production and NET cell death. The hyphal form of this fungus induced NETs more effectively than the yeast form. With this work, we provide tools to study in vitro NET assembly in the mouse system.

Impairment of Neutrophil Extracellular Trap Degradation is Associated with Lupus Nephritis

Proceedings of the National Academy of Sciences of the United States of America. May, 2010  |  Pubmed ID: 20439745

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients' sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.

Inflammasome Activation in NADPH Oxidase Defective Mononuclear Phagocytes from Patients with Chronic Granulomatous Disease

Blood. Sep, 2010  |  Pubmed ID: 20495074

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent infections and deregulated inflammatory responses. CGD is caused by mutations in subunits of the NADPH oxidase, an enzyme that generates reactive oxygen species in phagocytes. To elucidate the contribution of the proinflammatory protease caspase-1 to aberrant inflammatory reactions in CGD, we analyzed cells isolated from patients with defects in the phagocyte oxidase subunits p22phox, p47phox or gp91phox. We report that mononuclear phagocytes from CGD patients activated caspase-1 and produced biologically active interleukin-1beta (IL-1beta) in response to danger signals. Notably, caspase-1 activation and IL-1beta secretion from CGD monocytes was elevated in asymptomatic patients and strongly increased in patients with noninfectious inflammatory conditions. Treatment with IL-1 receptor antagonist reduced IL-1 production in monocytes ex vivo and during medical therapy. Our results identify phagocyte oxidase defective monocytes as a source of elevated IL-1 and provide a potential therapeutic option to ameliorate inflammatory conditions associated with CGD.

Protein Refolding is Required for Assembly of the Type Three Secretion Needle

Nature Structural & Molecular Biology. Jul, 2010  |  Pubmed ID: 20543831

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from alpha-helix into beta-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

Mutant Superoxide Dismutase 1-induced IL-1beta Accelerates ALS Pathogenesis

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2010  |  Pubmed ID: 20616033

ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS-linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1beta in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1-mediated inflammation. Notably, mutant SOD1 induced IL-1beta correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1beta or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1beta as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.

Neutrophil Elastase and Myeloperoxidase Regulate the Formation of Neutrophil Extracellular Traps

The Journal of Cell Biology. Nov, 2010  |  Pubmed ID: 20974816

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.

Neutrophil Antimicrobial Proteins Enhance Shigella Flexneri Adhesion and Invasion

Cellular Microbiology. Aug, 2010  |  Pubmed ID: 20331641

Shigella flexneri is an enteric pathogen that causes massive inflammation and destruction of the human intestinal epithelium. Neutrophils are the first cells of the innate immune system recruited to the site of infection. These cells can attack microbes by phagocytosis, Neutrophil Extracellular Trap (NET) formation and degranulation. Here, we investigated how neutrophil degranulation affects virulence and show that exposure of Shigella to granular proteins enhances infection of epithelial cells. During this process, cationic granular proteins bind to the Shigella surface causing increased adhesion which ultimately leads to hyperinvasion. This effect is mediated by changes in the surface charge, since a lipopolysaccharide (LPS) mutant with a negative surface shows enhanced hyperinvasion compared with wild-type Shigella. We propose that Shigella evolved to use host defence molecules to enhance its virulence and subvert the innate immune system.

Activation of the Raf-MEK-ERK Pathway is Required for Neutrophil Extracellular Trap Formation

Nature Chemical Biology. Feb, 2011  |  Pubmed ID: 21170021

The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

Glutamate Utilization Promotes Meningococcal Survival in Vivo Through Avoidance of the Neutrophil Oxidative Burst

Molecular Microbiology. Sep, 2011  |  Pubmed ID: 21777301

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.

Neutrophil Elastase Enhances Sputum Solubilization in Cystic Fibrosis Patients Receiving DNase Therapy

PloS One. 2011  |  Pubmed ID: 22174830

Cystic fibrosis patients suffer from chronic lung infection and inflammation due to the secretion of viscous sputum. Sputum viscosity is caused by extracellular DNA, some of which originates from the release of neutrophil extracellular traps (NETs). During NET formation neutrophil elastase (NE) partially processes histones to decondense chromatin. NE is abundant in CF sputum and is thought to contribute to tissue damage. Exogenous nucleases are a palliative treatment in CF as they promote sputum solubilization. We show that in a process reminiscent of NET formation, NE enhances sputum solubilization by cleaving histones to enhance the access of exogenous nucleases to DNA. In addition, we find that in Cf sputum NE is predominantly bound to DNA, which is known to downregulate its proteolytic activity and may restrict host tissue damage. The beneficial role of NE in CF sputum solubilization may have important implications for the development of CF therapies targeting NE.

Neutrophil Function: From Mechanisms to Disease

Annual Review of Immunology. Mar, 2011  |  Pubmed ID: 22224774

Neutrophils are the most abundant white blood cells in circulation, and patients with congenital neutrophil deficiencies suffer from severe infections that are often fatal, underscoring the importance of these cells in immune defense. In spite of neutrophils' relevance in immunity, research on these cells has been hampered by their experimentally intractable nature. Here, we present a survey of basic neutrophil biology, with an emphasis on examples that highlight the function of neutrophils not only as professional killers, but also as instructors of the immune system in the context of infection and inflammatory disease. We focus on emerging issues in the field of neutrophil biology, address questions in this area that remain unanswered, and critically examine the experimental basis for common assumptions found in neutrophil literature. Expected final online publication date for the Annual Review of Immunology Volume 30 is March 19, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Myeloperoxidase is Required for Neutrophil Extracellular Trap Formation: Implications for Innate Immunity

Blood. Jan, 2011  |  Pubmed ID: 20974672

The granule enzyme myeloperoxidase (MPO) plays an important role in neutrophil antimicrobial responses. However, the severity of immunodeficiency in patients carrying mutations in MPO is variable. Serious microbial infections, especially with Candida species, have been observed in a subset of completely MPO-deficient patients. Here we show that neutrophils from donors who are completely deficient in MPO fail to form neutrophil extracellular traps (NETs), indicating that MPO is required for NET formation. In contrast, neutrophils from partially MPO-deficient donors make NETs, and pharmacological inhibition of MPO only delays and reduces NET formation. Extracellular products of MPO do not rescue NET formation, suggesting that MPO acts cell-autonomously. Finally, NET-dependent inhibition of Candida albicans growth is compromised in MPO-deficient neutrophils. The inability to form NETs may contribute in part to the host defense defects observed in completely MPO-deficient individuals.