Human neutrophils ≠ murine neutrophils: Does it matter?

Human and murine neutrophils differ with respect to representation in blood, receptors, nuclear morphology, signaling pathways, granule proteins, NADPH oxidase regulation, magnitude of oxidant and hypochlorous acid production, and their repertoire of secreted molecules. These differences often matter and can undermine extrapolations from murine studies to clinical care, as illustrated by several failed therapeutic interventions based on mouse models. Likewise, coevolution of host and pathogen undercuts fidelity of murine models of neutrophil-predominant human infections. However, murine systems that accurately model the human condition can yield insights into human biology difficult to obtain otherwise. The challenge for investigators who employ murine systems is to distinguish models from pretenders and to know when the mouse provides biologically accurate insights. Testing with human neutrophils observations made in murine systems would provide a safeguard but is not always possible. At a minimum, studies that use exclusively murine neutrophils should have accurate titles supported by data and restrict conclusions to murine neutrophils and not encompass all neutrophils. For now, the integration of evidence from studies of neutrophil biology performed using valid murine models coupled with testing in vitro of human neutrophils combines the best of both approaches to elucidate the mysteries of human neutrophil biology.

Neutrophils at work.

In this Review we discuss data demonstrating recently recognized aspects of neutrophil homeostasis in the steady state, granulopoiesis in 'emergency' conditions and interactions of neutrophils with the adaptive immune system. We explore in vivo observations of the recruitment of neutrophils from blood to tissues in models of blood-borne infections versus bacterial invasion through epithelial linings. We examine data on novel aspects of the activation of NADPH oxidase and the heterogeneity of phagosomes and, finally, consider the importance of two neutrophil-derived biological agents: neutrophil extracellular traps and ectosomes.

HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Neutrophil dysfunction in the pathogenesis of cystic fibrosis.

Polymorphonuclear neutrophils (PMNs) figure prominently in host defense against infection and in noninfectious inflammation. Mobilized early in an inflammatory response, PMNs mediate immediate cellular defense against microbes and orchestrate events that culminate in cessation of inflammation and restoration of homeostasis. Failure to terminate the inflammatory response and its causes can fuel exuberant inflammation characteristic of many human diseases, including cystic fibrosis (CF), an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator. CF affects multiple end organs, with persistent bacterial infection and chronic neutrophilic inflammation in airways predominating the clinical picture. To match the diverse microbial challenges that they may encounter, PMNs possess a variety of antimicrobial systems to slow or kill invading microorganisms confined in their phagosomes. Prominent among PMN defense systems is their ability to generate hypochlorous acid, a potent microbicide, by reacting oxidants generated by the NADPH oxidase with myeloperoxidase (MPO) released from azurophilic granules in the presence of chloride (Cl-). Products of the MPO-H2O2-Cl system oxidize susceptible biomolecules and support robust antimicrobial action against many, but not all, potential human pathogens. Underscoring that the MPO-H2O2-Cl system is integral to optimal host defense and proper regulation of inflammation, individuals with defects in any component of this system, as seen in chronic granulomatous disease or MPO deficiency, incur increased rates or severity of infection and signs of dysregulated inflammatory responses. We focus attention in this review on the molecular basis for and the clinical consequences of defects in the MPO-H2O2-Cl system because of the compromised Cl transport seen in CF. We will discuss first how the MPO-H2O2-Cl system in healthy PMNs participates in host defense and resolution of inflammation and then review how a defective MPO-H2O2-Cl system contributes to the increased susceptibility to infection and dysregulated inflammation associated with the clinical manifestations of CF.

Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation.

Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.

Cathepsin G Degrades Staphylococcus aureus Biofilms.

Polymorphonuclear leukocytes (PMN) phagocytose and kill individual bacteria but are far less efficient when challenged with bacterial aggregates. Consequently, growth within a biofilm affords Staphylococcus aureus some protection but PMN penetrate S. aureus biofilms and phagocytose bacteria, suggesting that enzymes released through neutrophil degranulation degrade biofilms into fragments small enough for phagocytosis. Here we show that the capacity of PMN to invade biofilms depended largely on the activity of secreted cathepsin G.

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN).

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal ß-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a ß-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal ß-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

Lysis of human neutrophils by community-associated methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes infections associated with extensive tissue damage and necrosis. In vitro, human neutrophils fed CA-MRSA lyse by an unknown mechanism that is inhibited by necrostatin-1, an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1). RIPK-1 figures prominently in necroptosis, a specific form of programmed cell death dependent on RIPK-1, RIPK-3, and the mixed-lineage kinase-like protein (MLKL). We previously reported that necrostatin-1 inhibits lysis of human neutrophils fed CA-MRSA and attributed the process to necroptosis. We now extend our studies to examine additional components in the programmed cell death pathway to test the hypothesis that neutrophils fed CA-MRSA undergo necroptosis. Lysis of neutrophils fed CA-MRSA was independent of tumor necrosis factor α, active RIPK-1, and MLKL, but dependent on active RIPK-3. Human neutrophils fed CA-MRSA lacked phosphorylated RIPK-1, as well as phosphorylated or oligomerized MLKL. Neutrophils fed CA-MRSA possessed cytoplasmic complexes that included inactive caspase 8, RIPK-1, and RIPK-3, and the composition of the complex remained stable over time. Together, these data suggest that neutrophils fed CA-MRSA underwent a novel form of lytic programmed cell death via a mechanism that required RIPK-3 activity, but not active RIPK-1 or MLKL, and therefore was distinct from necroptosis. Targeting the molecular pathways that culminate in lysis of neutrophils during CA-MRSA infection may serve as a novel therapeutic intervention to limit the associated tissue damage.

Structure of human promyeloperoxidase (proMPO) and the role of the propeptide in processing and maturation.

Myeloperoxidase (MPO) is synthesized by neutrophil and monocyte precursor cells and contributes to host defense by mediating microbial killing. Although several steps in MPO biosynthesis and processing have been elucidated, many questions remained, such as the structure-function relationship of monomeric unprocessed proMPO versus the mature dimeric MPO and the functional role of the propeptide. Here we have presented the first and high resolution (at 1.25 Å) crystal structure of proMPO and its solution structure obtained by small-angle X-ray scattering. Promyeloperoxidase hosts five occupied glycosylation sites and six intrachain cystine bridges with Cys-158 of the very flexible N-terminal propeptide being covalently linked to Cys-319 and thereby hindering homodimerization. Furthermore, the structure revealed (i) the binding site of proMPO-processing proconvertase, (ii) the structural motif for subsequent cleavage to the heavy and light chains of mature MPO protomers, and (iii) three covalent bonds between heme and the protein. Studies of the mutants C158A, C319A, and C158A/C319A demonstrated significant differences from the wild-type protein, including diminished enzymatic activity and prevention of export to the Golgi due to prolonged association with the chaperone calnexin. These structural and functional findings provide novel insights into MPO biosynthesis and processing.

Biosynthesis of human myeloperoxidase.

Members of Chordata peroxidase subfamily [1] expressed in mammals, including myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO), express conserved motifs around the heme prosthetic group essential for their activity, a calcium-binding site, and at least two covalent bonds linking the heme group to the protein backbone. Although most studies of the biosynthesis of these peroxidases have focused on MPO, many of the features described occur during biosynthesis of other members of the protein subfamily. Whereas MPO biosynthesis includes events typical for proteins generated in the secretory pathway, the importance and consequences of heme insertion are events uniquely associated with peroxidases. This Review summarizes decades of work elucidating specific steps in the biosynthetic pathway of human MPO. Discussion includes cotranslational glycosylation and subsequent modifications of the N-linked carbohydrate sidechains, contributions by molecular chaperones in the endoplasmic reticulum, cleavage of the propeptide from proMPO, and proteolytic processing of protomers and dimerization to yield mature MPO. Parallels between the biosynthesis of MPO and TPO as well as the impact of inherited mutations in the MPO gene on normal biosynthesis will be summarized. Lastly, specific gaps in our knowledge revealed by this review of our current understanding will be highlighted.

Pondering neutrophil extracellular traps with healthy skepticism.

The authors engage in a dialogue that evaluates critically the state of the study of neutrophil extracellular traps (NETs), a phenomenon currently the object of considerable interest, with the goal of identifying those aspects that merit clarification in order to assign the process its proper place in our current understanding of cell biology. Since the seminal observations in the Zychlinsky laboratory that described the extrusion of filaments of nuclear DNA associated with histones and granule proteins from neutrophils stimulated in vitro, many investigators have examined the phenomenon of NET formation in numerous and diverse settings. However, an overview of work in this rapidly growing field prompts several fundamental questions about NETs, including their precise composition, the mechanisms by which they arise, their clinical relevance, and the interrelationship of those observed in vitro and in vivo. In this discussion, the authors challenge interpretation of data from some experimental settings and provide recommendations for specific studies that would address the concerns raised, improve understanding of the biological relevance of NETs, and strengthen the field.

Methylotroph Infections and Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by a defect in production of phagocyte-derived reactive oxygen species, which leads to recurrent infections with a characteristic group of pathogens not previously known to include methylotrophs. Methylotrophs are versatile environmental bacteria that can use single-carbon organic compounds as their sole source of energy; they rarely cause disease in immunocompetent persons. We have identified 12 infections with methylotrophs (5 reported here, 7 previously reported) in patients with CGD. Methylotrophs identified were Granulibacter bethesdensis (9 cases), Acidomonas methanolica (2 cases), and Methylobacterium lusitanum (1 case). Two patients in Europe died; the other 10, from North and Central America, recovered after prolonged courses of antimicrobial drug therapy and, for some, surgery. Methylotrophs are emerging as disease-causing organisms in patients with CGD. For all patients, sequencing of the 16S rRNA gene was required for correct diagnosis. Geographic origin of the methylotroph strain may affect clinical management and prognosis.

Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus.

The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target.

Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pose a significant threat to human health. Polymorphonuclear leukocytes (PMN) are the first responders during staphylococcal infection, but 15-50% of the initial ingested inoculum survives within the PMN phagosome and likely contributes directly or indirectly to disease pathogenesis. We hypothesize that surviving intracellular CA-MRSA undermine effective phagocyte-mediated defense by causing a decrease in macrophage uptake of PMN containing viable S. aureus and by promoting PMN lysis. In support of this hypothesis, PMN harboring viable CA-MRSA strain USA300 (PMN-SA) upregulated the "don't eat me" signal CD47, remained bound to the surface, and were inefficiently ingested by macrophages. In addition, coculture with PMN-SA altered the macrophage phenotype. Compared to macrophages fed USA300 alone, macrophages challenged with PMN-SA produced more IL-8 and less IL-1 receptor antagonist, TNF-α, activated caspase-1, and IL-1ß. Although they exhibited some features of apoptosis within 3 h following ingestion of S. aureus, including phosphatidylserine exposure and mitochondrial membrane depolarization, PMN-SA had sustained levels of proliferating cell nuclear Ag expression, absence of caspase activation, and underwent lysis within 6 h following phagocytosis. PMN lysis was dependent on receptor-interacting protein 1, suggesting that PMN-SA underwent programmed necrosis or necroptosis. These data are the first demonstration, to our knowledge, that bacteria can promote sustained expression of proliferating cell nuclear Ag and that human PMN undergo necroptosis. Together, these findings demonstrate that S. aureus surviving within PMN undermine the innate immune response and may provide insight into the pathogenesis of S. aureus disease.

How methicillin-resistant Staphylococcus aureus evade neutrophil killing.

PURPOSE OF REVIEW: Methicillin-resistant strains of the important human pathogen Staphylococcus aureus pose a significant public health threat in the community, as they are easily transmitted, especially prone to cause invasive disease, and infect otherwise healthy individuals. The mechanistic basis for the ability of these organisms to evade the innate immune responses remains incompletely defined. RECENT FINDINGS: The success of pathogens such as S. aureus rests, in part, on their capacity to overcome neutrophil-mediated host defense to establish infection and cause human disease. S. aureus has the potential to thwart effective neutrophil chemotaxis, and phagocytosis, and succeeds in evading killing by neutrophils. Furthermore, S. aureus surviving within neutrophils promotes neutrophil cytolysis, with release of host-derived molecules that promote local inflammation. Here, we provide a brief overview of our understanding of the mechanisms by which S. aureus - including methicillin-resistant S. aureus - avoids neutrophil-mediated host defense and causes disease. SUMMARY: Understanding the molecular mechanisms by which S. aureus avoids neutrophil-mediated responses and initiates signaling cascades that culminate in neutrophil lysis will provide insights prerequisite to the development of novel targets for treating staphylococcal infections.

Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases.

BACKGROUND: The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for measuring oxidants released extracellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. SCOPE OF REVIEW: Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O2(-) and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. MAJOR CONCLUSIONS: Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. GENERAL SIGNIFICANCE: The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Myeloperoxidase in human neutrophil host defence.

Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by-products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism.