RESUMO
Cystic fibrosis is a life-threatening genetic disorder caused by mutations in the CFTR chloride channel. Clinically, over 90% of patients with cystic fibrosis succumb to pulmonary complications precipitated by chronic bacterial infections, predominantly by Pseudomonas aeruginosa and Staphylococcus aureus. Despite the well-characterized gene defect and clearly defined clinical sequelae of cystic fibrosis, the critical link between the chloride channel defect and the host defense failure against these specific pathogens has not been established. Previous research from us and others has uncovered that neutrophils from patients with cystic fibrosis are defective in phagosomal production of hypochlorous acid, a potent microbicidal oxidant. Here we report our studies to investigate if this defect in hypochlorous acid production provides P. aeruginosa and S. aureus with a selective advantage in cystic fibrosis lungs. A polymicrobial mixture of cystic fibrosis pathogens (P. aeruginosa and S. aureus) and non-cystic fibrosis pathogens (Streptococcus pneumoniae, Klebsiella pneumoniae, and Escherichia coli) was exposed to varied concentrations of hypochlorous acid. The cystic fibrosis pathogens withstood higher concentrations of hypochlorous acid than did the non-cystic fibrosis pathogens. Neutrophils derived from F508del-CFTR HL-60 cells killed P. aeruginosa less efficiently than did the wild-type counterparts in the polymicrobial setting. After intratracheal challenge in wild-type and cystic fibrosis mice, the cystic fibrosis pathogens outcompeted the non-cystic fibrosis pathogens and exhibited greater survival in the cystic fibrosis lungs. Taken together, these data indicate that reduced hypochlorous acid production due to the absence of CFTR function creates an environment in cystic fibrosis neutrophils that provides a survival advantage to specific microbes-namely, S. aureus and P. aeruginosa-in the cystic fibrosis lungs.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Animais , Camundongos , Neutrófilos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ácido Hipocloroso/metabolismo , Staphylococcus aureus/metabolismo , Fibrose Cística/patologia , Pulmão/patologia , Fibrose , Pseudomonas aeruginosa , Infecções por Pseudomonas/microbiologiaRESUMO
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.
Assuntos
Anti-Infecciosos , Triagem , Espécies Reativas de Oxigênio/metabolismo , NADP/metabolismo , Macrófagos/metabolismo , Anti-Infecciosos/metabolismo , Via de Pentose Fosfato/fisiologiaRESUMO
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.
Assuntos
NADPH Oxidases , Neutrófilos , Humanos , Camundongos , Animais , NADPH Oxidases/metabolismo , Transdução de SinaisRESUMO
The text extracted from the initial paragraph of a paper coauthored by Zanvil Cohn, one of the pioneers in the study of leukocyte biology, highlights two phenomena that stimulated investigations of innate immunity in the middle of the last century, namely phagocytosis and intracellular antimicrobial activity. Although many features of phagocytosis have been characterized since that time, fundamental aspects of the antimicrobial action of neutrophils remain unknown. The report by Ashby et al. provides a refined and nuanced look at the interface between an ingested microbe, Staphylococcus aureus, and HOCl generated by the myeloperoxidase (MPO)-H2 O2 -chloride system in neutrophil phagosomes and represents a holistic approach to the analysis of bactericidal mechanisms that recognizes contributions from both phagocyte and its ingested prey.
Assuntos
Neutrófilos , Peroxidase , Antibacterianos/farmacologia , Cloretos/farmacologia , Ácido Hipocloroso , Fagocitose , FagossomosRESUMO
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.
Assuntos
Fibrose Cística , Transtornos Leucocíticos , Cloretos , Humanos , Peróxido de Hidrogênio , Ácido Hipocloroso , Inflamação , Neutrófilos/microbiologia , PeroxidaseRESUMO
Neutrophils (PMN) regulate inflammation in many ways, including communication with other immune cells via extracellular vesicles (EVs). EVs released by human neutrophils activated with N-formylmethionyl-leucyl-phenylalanine (fMLF) (PMN-fMLF EVs) had an outside-out orientation and contained functionally important neutrophil plasma membrane proteins, including flavocytochrome b558, and enzymatically active granule proteins, elastase, and myeloperoxidase. Treatment of naïve PMN with PMN-fMLF EVs primed fMLF-stimulated NADPH oxidase activity, increased surface expression of the complement receptors CD11b/CD18 and CD35, the specific granule membrane protein CD66, and flavocytochrome b558 , and promoted phagocytosis of serum-opsonized Staphylococcus aureus. The primed oxidase activity reflected increased surface expression of flavocytochrome b558 and phosphorylation of SER345 in p47phox , two recognized mechanisms for oxidase priming. Taken together, these data demonstrate that stimulated PMN released EVs that altered the phenotype of naïve phagocytes by priming of the NADPH oxidase activity and augmenting phagocytosis, two responses that are integral to optimal PMN host defense.
Assuntos
Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Humanos , NADPH Oxidases/metabolismo , FenótipoRESUMO
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.
Assuntos
Catepsina G , Neutrófilos , Fagocitose , Infecções Estafilocócicas , Staphylococcus aureus , Biofilmes , Humanos , Neutrófilos/imunologiaRESUMO
Optimal innate immune response to infection includes eradication of potential pathogens, resolution of associated inflammation, and restitution of homeostasis. Phagocytosing human polymorphonuclear leukocytes (hPMN) undergo accelerated apoptosis, a process referred to as phagocytosis-induced cell death (PICD) and an early step in their clearance from inflammatory sites. Among human pathogens that modulate hPMN apoptosis, Neisseria gonorrhoeae delays PICD, which may contribute to the exuberant neutrophilic inflammation that characterizes gonorrhea. To elucidate the mechanisms underlying delayed PICD, we compared features of hPMN cell death that followed phagocytosis of N. gonorrhoeae FA1090 wild-type (GC) or serum-opsonized zymosan (OPZ), a prototypical stimulus of PICD. Phosphatidylserine externalization required NADPH oxidase activity after ingestion of GC or OPZ, and annexin V staining and DNA fragmentation were less after phagocytosis of GC compared to OPZ. Caspase 3/7 and caspase 9 activities after phagocytosis of GC were less than that seen after ingestion of OPZ, but caspase 8 activity was the same after ingestion of GC or OPZ. When hPMN sequentially ingested GC followed by OPZ, both caspase 3/7 and 9 activities were less than that seen after OPZ alone, and the inhibition was dose dependent for GC, suggesting that ingestion of GC actively inhibited PICD. Sequential phagocytosis did not block caspase 8 activity, mitochondrial depolarization, or annexin V/propidium iodide staining compared to responses of hPMN fed OPZ alone, despite inhibition of caspases 3/7 and 9. Taken together, these data suggest that active inhibition of the intrinsic pathway of apoptosis contributes to the delay in PICD after hPMN ingestion of N. gonorrhoeae.
Assuntos
Apoptose/imunologia , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Neutrófilos/imunologia , Fagocitose , Caspases/imunologia , Fragmentação do DNA , Gonorreia/patologia , Humanos , Neutrófilos/patologiaRESUMO
Persistent neutrophilic inflammation is a hallmark of cystic fibrosis (CF). However, the mechanisms underlying this outstanding pathology remain incompletely understood. Here, we report that CFTR in myeloid immune cells plays a pivotal role in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr-/-) mice and congenic wild-type (WT) mice were challenged peritoneally with zymosan particles at different doses, creating aseptic peritonitis with varied severity. A high-dose challenge resulted in significantly higher mortality in Mye-Cftr-/- mice, indicating an intrinsic defect in host control of inflammation in mice whose myeloid cells lack CF. The low-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr-/- mice, reflected by a significant overproduction of proinflammatory cytokines, including neutrophil chemokines MIP-2 and KC, and sustained accumulation of neutrophils. Tracing neutrophil mobilization in vivo demonstrated that myeloid CF mice recruited significantly more neutrophils than did WT mice. Pulmonary challenge with zymosan elicited exuberant inflammation in the lung and recapitulated the findings from peritoneal challenge. To determine the major type of cell that was primarily responsible for the over-recruitment of neutrophils, we purified and cultured ex vivo zymosan-elicited peritoneal neutrophils and macrophages. The CF neutrophils produced significantly more MIP-2 than did the WT counterparts, and peripheral blood neutrophils isolated from myeloid CF mice also produced significantly more MIP-2 after zymosan stimulation in vitro. These data altogether suggest that CFTR dysfunction in myeloid immune cells, especially neutrophils, leads to hyperinflammation and excessive neutrophil mobilization in the absence of infection. Thus, dysregulated inflammation secondary to abnormal or absent CFTR in myeloid cells may underlie the clinically observed neutrophilic inflammation in CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística/imunologia , Macrófagos Peritoneais/imunologia , Neutrófilos/imunologia , Animais , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Mutação com Perda de Função , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Mutantes , Neutrófilos/patologia , Zimosan/toxicidadeRESUMO
Neisseria gonorrhoeae is a Gram-negative diplococcus that is responsible for the sexually transmitted infection gonorrhea, a high-morbidity disease in the United States and worldwide. Over the past several years, N. gonorrhoeae strains resistant to antibiotics used to treat this infection have begun to emerge across the globe. Thus, new treatment strategies are needed to combat this organism. Here, we utilized N. gonorrhoeae transcriptomic data sets, including those obtained from natural infection of the human genital tract, to infer the first global gene coexpression network of this pathogen. Interrogation of this network revealed genes central to the network that are likely critical for gonococcal growth, metabolism, and virulence, including genes encoding hypothetical proteins expressed during mucosal infection. In addition, network analysis revealed overlap in the response of N. gonorrhoeae to incubation with neutrophils and exposure to hydrogen peroxide stress in vitro Network analysis also identified new targets of the gonococcal global regulatory protein Fur, while examination of the network neighborhood of genes allowed us to assign additional putative categories to several proteins. Collectively, the characterization of the first gene coexpression network for N. gonorrhoeae described here has revealed new regulatory pathways and new categories for proteins and has shown how processes important to gonococcal infection in both men and women are linked. This information fills a critical gap in our understanding of virulence strategies of this obligate human pathogen and will aid in the development of new treatment strategies for gonorrhea.IMPORTANCE Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, a disease with high morbidity worldwide with an estimated 87 million cases annually. Current therapeutic and pharmacologic approaches to treat gonorrhea have been compromised by increased antibiotic resistance worldwide, including to the most recent FDA-approved antibiotic. New treatment strategies are urgently needed to combat this organism. In this study, we used network analysis to interrogate and define the coordination of pathways and processes in N. gonorrhoeae An analysis of the gonococcal network was also used to assign categories to genes and to expand our understanding of regulatory strategies. Network analysis provides important insights into pathogenic mechanisms of this organism that will guide the design of new strategies for disease treatment.
RESUMO
Venous blood provides a ready source of large numbers of unstimulated granulocytes and mononuclear cells. Exploiting the differences in the relative densities of the leukocytes circulating in venous blood, one can separate leukocytes from erythrocytes as well as isolate the individual leukocyte populations in high purity for use in ex vivo studies. For selected functional studies, such as transcriptional analysis or cytokine quantitation, addition of an immunomagnetic negative selection step to the standard isolation protocol can yield highly purified human neutrophils.
Assuntos
Separação Celular , Neutrófilos/citologia , Neutrófilos/metabolismo , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Dextranos , Ficoll , Humanos , Contagem de LeucócitosRESUMO
BACKGROUND Pseudopropionibacterium propionicum was called Propionibacterium propionicum until a recent taxonomy change in 2016. Diseases caused by P. propionicum resemble actinomycosis and thus differ dramatically from the infectious syndromes caused by common cutaneous Propionibacterium spp. However, if treating physicians are not familiar with P. propionicum and its clinical presentations, it is possible for them to regard it as a skin contaminant such as Cutibacterium acnes (formerly Propionibacterium acnes). CASE REPORT A 71-year-old man with past surgical history of right pneumonectomy was admitted with right chest wall abscess and right empyema. The chest wall abscess was drained surgically, and the empyema was drained via a chest tube. The abscess culture took 5 days to grow beaded branching Gram-positive rods, and 15 days to identify them as P. propionicum. The patient received 17 days of ceftriaxone and 4 weeks of doxycycline. However, he experienced a relapse of the chest wall abscess and right empyema 4 months after discontinuation of doxycycline. Cultures from the chest wall abscess and empyema grew P. propionicum again. We treated him with ceftriaxone for 6 months followed by minocycline for 7 months along with adequate drainage. CONCLUSIONS It is important to recognize that P. propionicum can cause thoracic actinomycosis and will likely require the prolonged treatment course typical for actinomycotic disease, which is 2 to 8 weeks of intravenous antibiotic therapy followed by 6 to 12 months of oral antibiotic therapy.
Assuntos
Abscesso/microbiologia , Actinomicose/microbiologia , Propionibacteriaceae/isolamento & purificação , Doenças Torácicas/microbiologia , Parede Torácica/microbiologia , Actinomicose/diagnóstico , Idoso , Humanos , MasculinoRESUMO
The phagocyte NADPH oxidase possesses a transmembrane electron transferase comprised of gp91phox (aka NOX2) and p22phox and two multicomponent cytosolic complexes, which in stimulated phagocytes translocate to assemble a functional enzyme complex at plasma or phagosomal membranes. The NOX2-centered NADPH oxidase shuttles electrons from cytoplasmic NADPH to molecular oxygen in phagosomes or the extracellular space to produce oxidants that support optimal antimicrobial activity by phagocytes. Additionally, NOX2-generated oxidants have been implicated in both autocrine and paracrine signaling in a variety of biological contexts. However, when interpreting experimental results, investigators must recognize the complexity inherent in the biochemistry of oxidant-mediated attack of microbial targets and the technical limitations of the probes currently used to detect intracellular oxidants.
Assuntos
NADPH Oxidase 2/metabolismo , Oxirredução , Fagócitos/imunologia , Fagócitos/metabolismo , Fagossomos/metabolismo , Transdução de Sinais , Animais , Doença Granulomatosa Crônica/etiologia , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Humanos , Peróxido de Hidrogênio/metabolismo , Neutrófilos/metabolismo , Oxidantes/metabolismo , FagocitoseRESUMO
Neutrophils serve as the circulating cells that respond early and figure prominently in human host defense to infection and in inflammation in other settings. Optimal oxidant-dependent antimicrobial activity by neutrophils relies on the ability of stimulated phagocytes to utilize a multicomponent NADPH oxidase to generate oxidants. The frequent, severe, and often fatal infections experienced by individuals with chronic granulomatous disease (CGD), an inherited disorder in which one of the NADPH oxidase components is absent or dysfunctional, underscore the link between a functional phagocyte NADPH oxidase and robust host protection against microbial infection.The history of the discovery and characterization of the normal neutrophil NADPH oxidase and the saga of recognizing CGD and its underlying causes together illustrate how the observations of astute clinicians and imaginative basic scientists synergize to forge new understanding of both basic cell biology and pathogenesis of human disease.In this chapter, we review the events in the stepwise evolution of our understanding of the phagocyte NADPH oxidase, both in the context of normal human neutrophil function and in the setting of CGD. The phagocyte oxidase complex employs a heterodimeric transmembrane protein composed of gp91phox and p22phox to relay electrons from NADPH to molecular oxygen, while other cofactors contribute to localization and regulation of the activity of the assembled oxidase. The b-type cytochrome gp91phox, also known as NOX2, serves as the catalytic component of this multicomponent enzyme complex. Although many of the features of the composition and regulation of the phagocyte oxidase may apply as well to NOX2 expressed in non-phagocytes and to other members of the NOX protein family, exceptions exist and pose special challenges to investigators exploring the biology of NADPH oxidases.
Assuntos
Doença Granulomatosa Crônica/etiologia , Doença Granulomatosa Crônica/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Animais , Biomarcadores , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Gerenciamento Clínico , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/terapia , Humanos , NADPH Oxidases/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Oxirredução , Fagócitos/patologia , Papel do Médico , Explosão Respiratória , Transdução de SinaisRESUMO
All members of the NOX protein family contain a unique b-type cytochrome that mediates the electron transport that characterizes the activity of the multicomponent oxidase complexes. Referred to as cytochrome b558, because of its signature spectral absorbance at 558 nm in reduced-minus-oxidized difference spectroscopy, or cytochrome b(-245), because of its very low midpoint potential of -245 mV at pH 7.0, the protein possesses two stacked inequivalent hemes ligated by pairs of histidine residues in membrane helices h3 and h5. In a flavin-dependent fashion, cytochrome b558 shuttles electrons from cytoplasmic NADPH across membranes to molecular oxygen and thereby generates superoxide anion. By performing reduced-minus-oxidized difference spectroscopy and using the millimolar extinction coefficient, E 559-540 nm = 21.6 cm-1 mM-1, one can calculate the amount of cytochrome b558 in intact cells or partially purified membrane preparations. Measurements in samples where cytochrome b558 is relatively high and the presence of unrelated heme-containing proteins low, as in neutrophils, are straightforward. However, low levels of cytochrome b558 expression combined with an abundance of mitochondria and other sources of heme proteins make spectral detection of cytochrome b558 in non-phagocytic cells extremely challenging.
Assuntos
NADPH Oxidases/química , Análise Espectral , Linhagem Celular , Grupo dos Citocromos b/química , Humanos , Isoenzimas , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Análise Espectral/métodosRESUMO
Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1ß, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1ß has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.
Assuntos
Coinfecção/imunologia , Interleucina-17/imunologia , Leishmania major/fisiologia , Leishmaniose Cutânea/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Animais , Coinfecção/microbiologia , Coinfecção/parasitologia , Coinfecção/patologia , Feminino , Humanos , Interleucina-17/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leishmania major/genética , Leishmania major/isolamento & purificação , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Células Th17/imunologiaRESUMO
Microbial infection elicits robust immune responses that initially depend on polymorphonuclear neutrophils (PMN), which ingest and kill invading bacteria. However, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) remain viable within PMN and prompt their lysis with concomitant release of damage-associated molecular patterns and proinflammatory cytokines that promote additional inflammation. Here, we show that ultrapure human PMN (>99.8% pure) that have ingested CA-MRSA released interleukin (IL)-1ß but not IL-18. The ingested CA-MRSA needed to be viable, and phagocytosis alone was insufficient to stimulate IL-1ß secretion from PMN fed CA-MRSA. In contrast to PMN response to the canonical NLRP3 inflammasome agonist nigericin, IL-1ß secretion by PMN fed CA-MRSA occurred independently of NLRP3 inflammasome or caspase-1 activation and required instead active receptor-interacting protein kinase 3 (RIPK3) but not RIPK1. Furthermore, inhibition of neutrophil serine proteases blocked pro-IL-1ß cleavage in PMN fed CA-MRSA. Taken together, our data suggest that with respect to secretion of IL-1ß and IL-18, PMN differ from human macrophages and exhibit agonist-specific responses. After phagocytosis of CA-MRSA, human PMN secreted IL-1ß through a previously unrecognized mechanism dependent on RIPK3 and serine proteases but independent of canonical NLRP3 inflammasome and caspase-1 activation.
Assuntos
Interleucina-1beta/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina Proteases/metabolismo , Staphylococcus aureus/metabolismo , Caspase 1/metabolismo , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
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.