Maren von Köckritz-Blickwede

Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation.

These days it has been increasingly recognized that mast cells (MCs) are critical components of host defense against pathogens. In this study, we have provided the first evidence that MCs can kill bacteria by entrapping them in extracellular structures similar to the extracellular traps described for neutrophils (NETs). We took advantage of the ability of MCs to kill the human pathogen Streptococcus pyogenes by a phagocytosis-independent mechanism in order to characterize the extracellular antimicrobial activity of MCs. Close contact of bacteria and MCs was required for full antimicrobial activity. Immunofluorescence and electron microscopy revealed that S pyogenes was entrapped by extracellular structures produced by MCs (MCETs), which are composed of DNA, histones, tryptase, and the antimicrobial peptide LL-37. Disruption of MCETs significantly reduced the antimicrobial effect of MCs, suggesting that intact extracellular webs are critical for effective inhibition of bacterial growth. Similar to NETs, production of MCETs was mediated by a reactive oxygen species (ROS)-dependent cell death mechanism accompanied by disruption of the nuclear envelope, which can be induced after stimulation of MCs with phorbol-12-myristate-13-acetate (PMA), H(2)O(2), or bacterial pathogens. Our study provides the first experimental evidence of antimicrobial extracellular traps formation by an immune cell population other than neutrophils.

Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

Statins Enhance Formation of Phagocyte Extracellular Traps

Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.

Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps

The formation of extracellular traps (ETs) by phagocytic cells has been recognized as a novel and important mechanism of the host innate immune response against infections. ETs are formed by different host immune cells such as neutrophils, mast cells, and eosinophils after stimulation with mitogens, cytokines, or pathogens themselves, in a process dependent upon induction of a reactive-oxygen-species-mediated signaling cascade. ETs consist of nuclear or mitochondrial DNA as a backbone with embedded antimicrobial peptides, histones, and cell-specific proteases and thereby provide a matrix to entrap and kill microbes and to induce the contact system. This review summarizes the latest research on ETs and their role in innate immunity and host innate defense. Attention is also given to mechanisms by which certain leading bacterial pathogens have evolved to avoid entrapment and killing in these specialized structures.

M1 Protein Allows Group A Streptococcal Survival in Phagocyte Extracellular Traps through Cathelicidin Inhibition

M1 protein contributes to Group A Streptococcus (GAS) systemic virulence by interfering with phagocytosis and through proinflammatory activities when released from the cell surface. Here we identify a novel role of M1 protein in the stimulation of neutrophil and mast cell extracellular trap formation, yet also subsequent survival of the pathogen within these DNA-based innate defense structures. Targeted mutagenesis and heterologous expression studies demonstrate M1 protein promotes resistance to the human cathelicidin antimicrobial peptide LL-37, an important effector of bacterial killing within such phagocyte extracellular traps. Studies with purified recombinant protein fragments mapped the inhibition of cathelicidin killing to the M1 hypervariable N-terminal domain. A survey of GAS clinical isolates found that strains from patients with necrotizing fasciitis or toxic shock syndrome were significantly more likely to be resistant to cathelicidin than GAS M types not associated with invasive disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections.

Immunological Mechanisms Underlying the Genetic Predisposition to Severe Staphylococcus aureus Infection in the Mouse Model

Host genetic variations play a significant role in conferring predisposition to infection. In this study, we examined the immune mechanisms underlying the host genetic predisposition to severe Staphylococcus aureus infection in different mouse strains. Whereas C57BL/6 mice were the most resistant in terms of control of bacterial growth and survival, A/J, DBA/2, and BALB/c mice were highly susceptible and succumbed to infection shortly after bacterial inoculation. Other strains (C3H/HeN, CBA, and C57BL/10) exhibited intermediate susceptibility levels. Susceptibility of mice to S. aureus was associated with an inability to limit bacterial growth in the kidneys and development of pathology. Resistance to S. aureus in C57BL/6 mice was dependent on innate immune mechanisms because Rag2-IL2Rgamma(-/-) C57BL/6 mice, which are deficient in B, T, and NK cells, were also resistant to infection. Indeed, neutrophil depletion or inhibition of neutrophil recruitment rendered C57BL/6 mice completely susceptible to S. aureus, indicating that neutrophils are essential for the observed resistance. Although neutrophil function is not inhibited in A/J mice, expression of neutrophil chemoattractants KC and MIP-2 peaked earlier in the kidneys of C57BL/6 mice than in A/J mice, indicating that a delay in neutrophil recruitment to the site of infection may underlie the increased susceptibility of A/J mice to S. aureus.

M Protein and Hyaluronic Acid Capsule Are Essential for In Vivo Selection of covRS Mutations Characteristic of Invasive Serotype M1T1 Group A Streptococcus

ABSTRACT The initiation of hyperinvasive disease in group A Streptococcus (GAS) serotype M1T1 occurs by mutation within the covRS two-component regulon (named covRS for control of virulence regulatory sensor kinase), which promotes resistance to neutrophil-mediated killing through the upregulation of bacteriophage-encoded Sda1 DNase. To determine whether other virulence factors contribute to this phase-switching phenomenon, we studied a panel of 10 isogenic GAS serotype M1T1 virulence gene knockout mutants. While loss of several individual virulence factors did not prevent GAS covRS switching in vivo, we found that M1 protein and hyaluronic acid capsule are indispensable for the switching phenotype, a phenomenon previously attributed uniquely to the Sda1 DNase. We demonstrate that like M1 protein and Sda1, capsule expression enhances survival of GAS serotype M1T1 within neutrophil extracellular traps. Furthermore, capsule shares with M1 protein a role in GAS resistance to human cathelicidin antimicrobial peptide LL-37. We conclude that a quorum of GAS serotype M1T1 virulence genes with cooperative roles in resistance to neutrophil extracellular killing is essential for the switch to a hyperinvasive phenotype in vivo. IMPORTANCE The pathogen group A Streptococcus (GAS) causes a wide range of human infections ranging from the superficial “strep throat” to potentially life-threatening conditions, such as necrotizing fasciitis, also known as “flesh-eating disease.” A marked increase in the number of cases of severe invasive GAS infection during the last 30 years has been traced to the emergence and spread of a single clone of the M1T1 serotype. Recent studies have shown that GAS serotype M1T1 bacteria undergo a genetic “switch” in vivo to a hypervirulent state that allows dissemination into the bloodstream. The present study was undertaken to identify specific GAS serotype M1T1 virulence factors required for this switch to hypervirulence. The surface-anchored GAS M1 protein and hyaluronic acid capsule are found to be essential for the switching phenotype, and a novel role for capsule in GAS resistance to host defense peptides and neutrophil extracellular killing is revealed. The pathogen group A Streptococcus (GAS) causes a wide range of human infections ranging from the superficial “strep throat” to potentially life-threatening conditions, such as necrotizing fasciitis, also known as “flesh-eating disease.” A marked increase in the number of cases of severe invasive GAS infection during the last 30 years has been traced to the emergence and spread of a single clone of the M1T1 serotype. Recent studies have shown that GAS serotype M1T1 bacteria undergo a genetic “switch” in vivo to a hypervirulent state that allows dissemination into the bloodstream. The present study was undertaken to identify specific GAS serotype M1T1 virulence factors required for this switch to hypervirulence. The surface-anchored GAS M1 protein and hyaluronic acid capsule are found to be essential for the switching phenotype, and a novel role for capsule in GAS resistance to host defense peptides and neutrophil extracellular killing is revealed.

A new pharmacological agent (AKB-4924) stabilizes hypoxia inducible factor-1 (HIF-1) and increases skin innate defenses against bacterial infection

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 increases HIF-1 levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinetobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.

Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program

ABSTRACT The complex response of murine macrophages to infection with Streptococcus pyogenes was investigated at the level of gene expression with a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encode molecules involved in the immune response and in inflammation, transcription, signaling, apoptosis, the cell cycle, electron transport, and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype), such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, and as well as the up-regulation of anti-inflammatory mediators, such as IL-1 decoy receptor and IL-10, associated with alternative macrophage activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation, was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine involved in the alternative activation pathway, was up-regulated in S. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated that S. pyogenes induces an atypical activation program in macrophages, with some but not all features of the classical or alternative activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxidase, as p47phox was up-regulated in infected cells. Indeed, the in vivo and in vitro killing of S. pyogenes was markedly diminished in the absence of functional phagocyte (p47phox−/−) but not in the absence of iNOS (iNOS−/−). An understanding of how macrophages respond to S. pyogenes at the molecular level may facilitate the development of new therapeutic paradigms.

Influences of Chloride and Hypochlorite on Neutrophil Extracellular Trap Formation

Background The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl−) and hydrogen peroxide (H2O2), and its product hypochlorite (HOCl) in NETosis. Findings In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl−, a substrate for MPO, also reduced NETosis. While exogenous addition of H2O2 and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils. Conclusions Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl− homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.