Phagosome dynamics during phagocytosis by neutrophils

The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil-pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.     

Inside the phagosome: A bacterial perspective

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.     

Phagocytosis: elegant complexity

Phagocytosis requires receptor-mediated recognition of particles, usually in the guise of infectious agents and apoptotic cells. Phagosomes fuse with lysosomes to generate phagolysosomes, which play a key role in enzymatic digestion of the internalized contents into component parts. Recent findings indicate that a simple paradigm of a single cognate receptor interaction that guides the phagosome to phagolysosome formation belies the complexity of combinatorial receptor recognition and diversity of phagosome function. In fact, phagosomes are comprised of hundreds of proteins that play a key role in deciphering the contents of the phagosome and in defining host response. In this review we discuss how the challenge of recognizing diverse molecular patterns is met by combinatorial interactions between phagocytic receptors. Furthermore, these combinations are dynamic and both sculpt the balance between a proinflammatory or anti-inflammatory response and direct phagosome diversity. We also indicate an important role for genetically tractable model organisms in defining key components of this evolutionarily conserved process.     

Salmonella sensing of anti-microbial mechanisms to promote survival within macrophages

Summary:  Salmonella enterica is a facultative intracellular pathogen that replicates within macrophages. The interaction of this pathogen with mammalian cells is a complex process involving hundreds of bacterial products that are sensed by and alter mammalian hosts. Numerous bacterial genes and their protein products have been identified that are required for Salmonella to resist killing by host innate immunity and to modify host processes. Many of these genes are regulated by a specific bacterial sensor, the PhoQ protein, which responds to the acidified phagosome environment. PhoQ is a sensor histidine kinase, which when activated in vivo within acidified macrophage phagosomes, regulates cell surface modifications that promote resistance to antimicrobial peptides and oxidative stress, alter the phagosome to promote intracellular survival, and reduce innate immune recognition. In this review, we discuss mechanisms by which Salmonella interacts with macrophages and focus in detail on recent reports describing the role of antimicrobial peptides and pH in PhoQ activation.     

The regulation of phagosome maturation in Dictyostelium

Macropinocytosis (fluid uptake) and phagocytosis (particle uptake) are processes that result in the formation of intracellular membrane enclosed vacuoles termed macropinosomes and phagosomes, respectively. Macropinosomes and phagosomes are modified by fission and fusion reactions with the endo-lysosomal pathway that eventually transform these vacuoles into a lysosomal environment. Many human bacterial pathogens, including species of Mycobacteria, Legionella, and Chlamydia, are thought to survive by disrupting the normal membrane trafficking events that usually result in the formation of phago-lysosomes and death of the microorganism. In addition, a number of important pathogens facilitate homotypic phagosome fusion in order to generate an intracellular environment conducive for survival. A greater understanding of the regulation of phagosomal maturation and fusion will be critical in designing new therapies to treat infections caused by intracellular pathogens. The genetically tractable phagocyte, D. discoideum, has proven extremely useful in dissecting the signaling pathways regulating macropinocytosis, phagocytosis, phagosomal maturation and phagosome–phagosome fusion. A body of knowledge has accumulated and demonstrates important roles for Rab GTPases, the cytoskeleton, phosphoinositide metabolism and pH regulation in regulating phagosome maturation. This review will summarize the current state of knowledge.     

Myeloperoxidase: friend and foe

Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.     

Microbes and the fate of neutrophils

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults—typically 10¹¹ per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.     

A functional NADPH oxidase prevents caspase involvement in the clearance of phagocytic neutrophils

Neutrophils play a prominent role in host defense. Phagocytosis of bacteria leads to the formation of an active NADPH oxidase complex that generates reactive oxygen species for bactericidal purposes. A critical step in the resolution of inflammation is the uptake of neutrophils by macrophages; however, there are conflicting reports on the mechanisms leading to the apoptosis of phagocytic neutrophils. The aim of this study was to clarify the role of effector caspases in these processes. Caspase activity was measured by DEVDase activity assays or immunofluorescence detection of active caspase-3. With normal human and wild-type murine neutrophils there was no caspase activation following phagocytosis of Staphylococcus aureus. However, caspase activity was observed in phagocytic neutrophils with a defective NADPH oxidase, including neutrophils isolated from X-linked gp91(phox) knockout chronic granulomatous disease mice. These results indicate that a functional NADPH oxidase and the generation of oxidants in the neutrophil phagosome prevent the activation of the cytoplasmic caspase cascade.     

The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.     

Rickettsia conorii entry into Vero cells.

The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.     

The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma

For decades, the vacuole that surrounds particles engulfed by phagocytosis was believed to originate from the plasma membrane. Conversion of the nascent phagosome into a microbicidal organelle was thought to result from the subsequent, orderly fusion of early endosomes, late endosomes, and ultimately, lysosomes with the original plasma membrane-derived vacuole. This conventional model has been challenged, if not superseded, by a revolutionary model that regards phagosome formation as resulting from the particle sliding into the endoplasmic reticulum via an opening at the base of the phagocytic cup. The merits and implications of these two hypotheses are summarized here and analyzed in light of recent results.     

Effect of plasma and matrix proteins on defensin-induced impairment of phagocytic killing by adherent neutrophils.

Infection is too often associated with prosthetic devices. Increased susceptibility to infection at these surgical sites appears to be associated with defective local phagocytic killing. The mechanisms for neutrophil down-regulation, however, continue to be obscure. We have recently demonstrated that cytotoxic substances are released from granulocytes associated with materials. One group of releasants, the cationic human neutrophil peptide(s) (also called defensins) not only impairs the antimicrobial capacity of the granulocyte that releases it but also impairs bystander phagocytes. Because plasma or matrix proteins soon become associated with implants, we investigated the interactive effect of adding these proteins, singly and in combination, on the microbicidal effect of bystander cells. Some plasma/matrix proteins (whole plasma, albumin, fibrinogen, and fibronectin) strongly interfered with the anti-microbicidal effects generated by neutrophil-polystyrene interaction. Other proteins (vitronectin and laminin) were without effect. These results suggest that protein composition at the prosthetic implant site could have a significant effect on infectivity, depending on whether neutrophils releasants were attenuated. In the absence of attenuation, the local environment would be hostile to host defenses, permitting bacterial survival and proliferation.     

Mycobacteria,metals, and the macrophage

Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here, we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies.     

Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells

Most antimicrobial peptides have been discovered based on activity-guided purification procedures, which used assays to determine their antimicrobial activity. Nevertheless, recent studies have shown that antimicrobial peptides also exert a range of other functions. Based on these observations, antimicrobial peptides are now not only implicated in host defense against infection but also in other immune reactions, inflammation, and wound-repair processes. The activities of neutrophil defensins and the cathelicidin hCAP-18/LL-37, antimicrobial peptides that are abundantly expressed in the human neutrophil, are the subject of an increasing number of studies. Exposure to neutrophil defensins and hCAP-18/LL-37 results in increases in mediator expression and release, chemotaxis, and proliferation of inflammatory and epithelial cells and fibroblasts, and the mechanisms underlying these effects have been partly elucidated. This review is focused on the effects of neutrophil defensins and hCAP-18/LL-37 on airway epithelial cells.     

Neutrophils and keratinocytes in innate immunity--cooperative actions to provide antimicrobial defense at the right time and place

The human neutrophil is a professional phagocyte of fundamental importance for defense against microorganisms, as witnessed by the life-threatening infections occurring in patients with neutropenia or with defects that result in decreased microbicidal activity of the neutrophil. Likewise, the skin and mucosal surfaces provide important barriers against infections. Traditionally, these major defense systems, the epithelial cells and the neutrophils, have been viewed as limited in their armory: The epithelial cells provide defense by constituting a physical barrier, and the neutrophils provide instant delivery of preformed antimicrobial substances or on-the-spot assembly of the multicomponent reduced nicotinamide adenine dinucleotide phosphate oxidase from stored components for the generation of reactive oxygen metabolites. Recent research has shown that epithelial cells are highly dynamic and able to generate antimicrobial peptides in response not only to microbial infection itself but more importantly, to the growth factors that are called into play when the physical barrier is broken, and the risk of microbial infection is imminent. Likewise, the neutrophil changes its profile of actively transcribed genes when it diapedeses into wounded skin. This results in generation of signaling molecules, some of which support the growth and antimicrobial potential of keratinocytes and epithelial cells. This paper will highlight some recent advances in this field.     

In vitro model of adhesion and invasion by Bacillus piliformis.

An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium. Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers. A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B. piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome. To confirm that host cell phagocytosis was involved in entry of B. piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B. piliformis, and evaluated for bacterial internalization by double-fluorescence labeling. The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function. To examine the role of B. piliformis in internalization, growth of live and Formalin-killed bacteria was compared. Dead bacteria were not internalized, suggesting that B. piliformis actively participates in internalization. B. piliformis appears to enter host cells by a bacterially directed phagocytic process. The in vitro system described should prove invaluable in further investigations of B. piliformis pathogenic mechanisms.     

Innate Immunity Against Moulds: Lessons Learned from Invertebrate Models

The emergence over the past two decades of invasive mycoses as a significant problem in immunocompromised patients underscores the importance of deciphering innate immunity against filamentous fungi. However, the complexity and cost of traditionally used mammalian model hosts presents a bottleneck that has limited the rate of advances in this field. In contrast, invertebrate model hosts have several important advantages, including simple immune systems, genetic tractability, and amenity to high-throughput experiments. The application of these models to studies of host-pathogen interactions is contingent on two tenets: (1) host innate defenses are preserved across widely disparate taxa, and (2) similar fungal virulence factors are operative in insects and in mammals. Validation of these principles paved the way for the use of invertebrates as facile models for studying invasive mould infections. These studies have helped shape our understanding of human pattern recognition receptors, phagocytic cell function and antimicrobial proteins, and their roles in host defense against filamentous fungi.     

Intracellular survival of Streptococcus pyogenes in polymorphonuclear cells results in increased bacterial virulence

It has recently been shown that survival within phagocytic cells constitutes an additional strategy used by Streptococcus pyogenes to evade the host defenses. Here we provide evidence that S. pyogenes can escape from the phagosome into the cytoplasm of phagocytic cells. Furthermore, intracellular bacteria seem to undergo phenotypic switching that results in much more virulent microorganisms.