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1.
Microorganisms ; 11(1)2023 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-36677464

RESUMEN

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1ß but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1ß upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

2.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34507997

RESUMEN

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.


Asunto(s)
Lesión Renal Aguda/prevención & control , Anemia Hemolítica/prevención & control , Bacillus anthracis/química , Pared Celular/química , Complemento C5/antagonistas & inhibidores , Peptidoglicano/toxicidad , Sepsis/complicaciones , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Anemia Hemolítica/etiología , Anemia Hemolítica/patología , Animales , Carbunco/microbiología , Carbunco/patología , Femenino , Hemólisis , Masculino , Papio , Sepsis/inducido químicamente
3.
Microorganisms ; 8(7)2020 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-32668703

RESUMEN

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis, the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown. In this study, we investigated the role of complement C3 deposition on anthrax particles for neutrophil recognition of bacterium and/or its cell wall peptidoglycan, an abundant pathogen-associated molecular pattern that supports anthrax sepsis. C3 opsonization and recognition by complement receptors accounted for 70-80% of the affinity interactions between neutrophils and anthrax particles at subphysiologic temperatures. In contrast, C3 supported up to 50% of the anthrax particle ingestion under thermophysiologic conditions. Opsonin-dependent low affinity interactions and, to a lower extent, opsonin-independent mechanisms, provide alternative entry routes. Similarly, C3 supported 58% of peptidoglycan-induced degranulation and, to a lower extent, 23% of bacterium-induced degranulation. Interestingly, an opsonin independent mechanism mediated by complement C5, likely through C5a anaphylatoxin, primes azurophilic granules in response to anthrax particles. Overall, we show that C3 deposition supports anthrax recognition by neutrophils but is dispensable for pathogen ingestion and neutrophil degranulation, highlighting immune recognition redundancies that minimize the risk of pathogen evasion.

4.
Blood Adv ; 3(16): 2436-2447, 2019 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-31416821

RESUMEN

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis. We hypothesized that immune recognition of PGN by monocytes is critical for procoagulant responses to PGN and investigated whether and how PGN induces TF expression in primary human monocytes. We found that PGN induced monocyte TF expression in a large cohort of healthy volunteers similar to lipopolysaccharide stimulation. Both immune and procoagulant responses to PGN involve intracellular recognition after PGN internalization, as well as surface signaling through immune Fcγ receptors (FcγRs). In line with our hypothesis, blocking immune receptor function, both signaling and FcγR-mediated phagocytosis, significantly reduced but did not abolish PGN-induced monocyte TF expression, indicating that FcγR-independent internalization contributes to intracellular recognition of PGN. Conversely, when intracellular PGN recognition is abolished, TF expression was sensitive to inhibitors of FcγR signaling, indicating that surface engagement of monocyte immune receptors can promote TF expression. The primary procoagulant responses to PGN were further amplified by proinflammatory cytokines through paracrine and autocrine signaling. Despite intersubject variability in the study cohort, dual neutralization of tumor necrosis factor-α and interleukin-1ß provided the most robust inhibition of the procoagulant amplification loop and may prove useful for reducing coagulopathies in gram-positive sepsis.


Asunto(s)
Carbunco/inmunología , Coagulación Sanguínea/inmunología , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Peptidoglicano/inmunología , Transducción de Señal , Biomarcadores , Coagulación Sanguínea/efectos de los fármacos , Brefeldino A/farmacología , Citometría de Flujo , Interacciones Huésped-Patógeno/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/inmunología , Monocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tromboplastina/metabolismo
5.
Blood ; 132(8): 849-860, 2018 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-29921614

RESUMEN

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.


Asunto(s)
Carbunco/metabolismo , Bacillus anthracis , Coagulación Sanguínea/efectos de los fármacos , Coagulación Intravascular Diseminada/sangre , Monocitos/metabolismo , Animales , Carbunco/patología , Coagulación Intravascular Diseminada/inducido químicamente , Coagulación Intravascular Diseminada/patología , Factor XIIa/metabolismo , Femenino , Masculino , Monocitos/patología , Papio , Papio anubis , Precalicreína/metabolismo
6.
Microb Pathog ; 121: 9-21, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29704667

RESUMEN

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1ß (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.


Asunto(s)
Células Epiteliales Alveolares/microbiología , Bacillus anthracis/patogenicidad , Quimiocinas/metabolismo , Perfilación de la Expresión Génica , Esporas Bacterianas/patogenicidad , Carbunco/genética , Carbunco/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Quimiocinas/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Monocitos/metabolismo , Monocitos/microbiología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Factor Plaquetario 4/genética , Factor Plaquetario 4/metabolismo , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/metabolismo , Regulación hacia Arriba
7.
Infect Immun ; 86(5)2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29531132

RESUMEN

We showed that human IgG supported the response by human innate immune cells to peptidoglycan (PGN) from Bacillus anthracis and PGN-induced complement activation. However, other serum constituents have been shown to interact with peptidoglycan, including the IgG-like soluble pattern recognition receptor serum amyloid P (SAP). Here, we compared the abilities of SAP and of IgG to support monocyte and complement responses to PGN. Utilizing in vitro methods, we demonstrate that SAP is superior to IgG in supporting monocyte production of cytokines in response to PGN. Like IgG, the response supported by SAP was enhanced by phagocytosis and signaling kinases, such as Syk, Src, and phosphatidylinositol 3-kinase, that are involved in various cellular processes, including Fc receptor signaling. Unlike IgG, SAP had no effect on the activation of complement in response to PGN. These data demonstrate an opsonophagocytic role for SAP in response to PGN that propagates a cellular response without propagating the formation of the terminal complement complex.


Asunto(s)
Bacillus anthracis/inmunología , Inmunidad Innata/inmunología , Inmunoglobulina G/inmunología , Peptidoglicano/inmunología , Componente Amiloide P Sérico/inmunología , Humanos
8.
PLoS One ; 13(2): e0193207, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29474374

RESUMEN

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2.


Asunto(s)
Bacillus anthracis/química , Inmunidad Innata/efectos de los fármacos , Peptidoglicano/farmacología , Staphylococcus aureus/química , Receptor Toll-Like 2/inmunología , Animales , Bacillus anthracis/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Peptidoglicano/química , Peptidoglicano/inmunología , Staphylococcus aureus/inmunología , Receptor Toll-Like 2/genética
9.
Proc Natl Acad Sci U S A ; 114(31): E6390-E6399, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28720697

RESUMEN

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced "oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.

10.
Cell ; 166(3): 624-636, 2016 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-27374331

RESUMEN

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.


Asunto(s)
Hexoquinasa/metabolismo , Inflamasomas/metabolismo , Peptidoglicano/metabolismo , Receptores Inmunológicos/metabolismo , Acetilación , Acetilglucosamina/metabolismo , Animales , Bacillus anthracis/metabolismo , Pared Celular/metabolismo , Células Dendríticas/metabolismo , Glucólisis , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Biológicos , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Potasio/metabolismo
11.
Microbes Infect ; 18(10): 615-626, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27320392

RESUMEN

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores escape from the alveolus to regional lymph nodes, germinate and enter the circulatory system to cause disease. The roles of carrier cells and the effects of B. anthracis toxins in this process are unclear. We used a human lung organ culture model to measure spore uptake by antigen presenting cells (APC) and alveolar epithelial cells (AEC), spore partitioning between these cells, and the effects of B. anthracis lethal toxin and protective antigen. We repeated the study in a human A549 alveolar epithelial cell model. Most spores remained unassociated with cells, but the majority of cell-associated spores were in AEC, not in APC. Spore movement was not dependent on internalization, although the location of internalized spores changed in both cell types. Spores also internalized in a non-uniform pattern. Toxins affected neither transit of the spores nor the partitioning of spores into AEC and APC. Our results support a model of spore escape from the alveolus that involves spore clustering with transient passage through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.


Asunto(s)
Carbunco/patología , Antígenos Bacterianos/metabolismo , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/metabolismo , Pulmón/microbiología , Ganglios Linfáticos/microbiología , Movimiento , Esporas Bacterianas/patogenicidad , Células Presentadoras de Antígenos/microbiología , Sangre/microbiología , Línea Celular , Células Epiteliales/microbiología , Humanos , Modelos Teóricos , Técnicas de Cultivo de Órganos
12.
Arthritis Rheumatol ; 66(9): 2521-31, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24891301

RESUMEN

OBJECTIVE: To assess disease features in Sle1.Yaa mice with genetic interleukin-6 (IL-6) deficiency. METHODS: Sera and tissues were collected from C57BL/6 (B6), Sle1.Yaa, and Sle1.Yaa.IL-6(-/-) mice and analyzed for various features of disease. Using serum samples, autoantibody specificities were determined by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence, cytokine production was analyzed by Luminex and ELISA, and levels of blood urea nitrogen were determined by ELISA. Renal, lung, and salivary gland tissue sections were evaluated for pathologic changes. Lymphocyte phenotypes, including CD4+ T cell cytokine production, and those of follicular and extrafollicular T helper subsets, germinal center B cells, and plasma cells, were determined using flow cytometry. RESULTS: IL-6 deficiency not only ameliorated autoantibody production and renal disease in this model, but also effectively reduced inflammation of lungs and salivary glands. Furthermore, IL-6 deficiency abrogated differentiation of Th1 and extrafollicular T helper cells, germinal center B cells, and plasma cells in the spleen and eliminated renal T cells with IL-17, interferon-γ, and IL-21 production potential. CONCLUSION: Our findings highlight IL-6-mediated T cell aberrations in Yaa-driven autoimmunity and support the concept of therapeutic IL-6/IL-6 receptor blockade in systemic lupus erythematosus and Sjögren's syndrome by impairing the production of autoantibodies and lymphocytic infiltration of the kidneys, lungs, and salivary glands.


Asunto(s)
Interleucina-6/deficiencia , Lupus Eritematoso Sistémico/inmunología , Linfocitos/inmunología , Nefritis/inmunología , Síndrome de Sjögren/inmunología , Animales , Citometría de Flujo , Interleucina-6/sangre , Pulmón/inmunología , Pulmón/patología , Lupus Eritematoso Sistémico/patología , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis/patología , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Glándulas Salivales/inmunología , Glándulas Salivales/patología , Síndrome de Sjögren/patología , Células TH1/inmunología , Células TH1/patología
13.
Thromb Res ; 133 Suppl 1: S28-31, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24759136

RESUMEN

Sepsis is a potent activator of the hemostatic and complement systems. While local activation of these proteolytic cascades contributes to the host defense, their uncontrolled systemic activation has major tissue damaging effects that lead to multiple organ failure and death. We have extensively studied the activation of complement and coagulation cascades in experimental sepsis using baboons challenged with live bacteria, such as Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus and Bacillus anthracis, or with the bacterial product peptidoglycan. We observed that these challenges rapidly induce disseminated intravascular coagulation and robust complement activation. We applied a potent C3 convertase inhibitor, compstatin, which prevented sepsis-induced complement activation, reduced thrombocytopenia, decreased the coagulopathic responses, and preserving the endothelial anticoagulant properties. Overall, our work demonstrates that live bacteria and bacterial products activate the complement and coagulation cascades, and that blocking formation of complement activation products, especially during the organ failure stage of severe sepsis could be a potentially important therapeutic strategy.


Asunto(s)
Coagulación Sanguínea , Proteínas del Sistema Complemento/inmunología , Sepsis/sangre , Sepsis/inmunología , Animales , Activación de Complemento , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/sangre , Coagulación Intravascular Diseminada/etiología , Coagulación Intravascular Diseminada/inmunología , Humanos , Inmunidad Innata , Papio , Sepsis/complicaciones
14.
J Immunol ; 191(6): 3319-27, 2013 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-23966633

RESUMEN

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer--a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an FcγR-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain-like receptors.


Asunto(s)
Proteínas Portadoras/inmunología , Fagocitosis/inmunología , Componente Amiloide P Sérico/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Western Blotting , Citometría de Flujo , Humanos
15.
Infect Immun ; 81(10): 3693-702, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23876807

RESUMEN

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.


Asunto(s)
Bacillus anthracis/metabolismo , Toxinas Bacterianas/farmacología , Citocinas/metabolismo , Leucocitos/efectos de los fármacos , Peptidoglicano/farmacología , Adulto , Bacillus anthracis/química , Células Cultivadas , Citocinas/genética , Humanos , Leucocitos/metabolismo , Persona de Mediana Edad , Peptidoglicano/genética , Peptidoglicano/metabolismo , Adulto Joven
16.
Blood ; 122(4): 571-9, 2013 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-23733338

RESUMEN

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbß3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.


Asunto(s)
Bacillus anthracis/inmunología , Proteínas del Sistema Complemento/fisiología , Peptidoglicano/inmunología , Activación Plaquetaria , Receptores de IgG/fisiología , Bacillus anthracis/química , Plaquetas/inmunología , Proteínas del Sistema Complemento/metabolismo , Humanos , Inmunoglobulina G/fisiología , Peptidoglicano/metabolismo , Peptidoglicano/farmacología , Fosfatidilserinas/metabolismo , Plasma/metabolismo , Plasma/fisiología , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Receptores de IgG/metabolismo
17.
J Immunol ; 189(5): 2423-31, 2012 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-22815288

RESUMEN

Gram-positive bacteria are an important public health problem, but it is unclear how they cause systemic inflammation in sepsis. Our previous work showed that peptidoglycan (PGN) induced proinflammatory cytokines in human cells by binding to an unknown extracellular receptor, followed by phagocytosis leading to the generation of NOD ligands. In this study, we used flow cytometry to identify host factors that supported PGN binding to immune cells. PGN binding required plasma, and plasma from all tested healthy donors contained IgG recognizing PGN. Plasma depleted of IgG or of anti-PGN Abs did not support PGN binding or PGN-triggered cytokine production. Adding back intact but not F(ab')2 IgG restored binding and cytokine production. Transfection of HEK293 cells with FcγRIIA enabled PGN binding and phagocytosis. These data establish a key role for anti-PGN IgG and FcγRs in supporting inflammation to a major structural element of Gram-positive bacteria and suggest that anti-PGN IgG contributes to human pathology in Gram-positive sepsis.


Asunto(s)
Anticuerpos Antibacterianos/fisiología , Mediadores de Inflamación/fisiología , Peptidoglicano/inmunología , Receptores de IgG/fisiología , Sepsis/inmunología , Sepsis/microbiología , Bacillus anthracis/inmunología , Sitios de Unión/inmunología , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Monocitos/inmunología , Monocitos/microbiología , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/microbiología , Neutrófilos/patología , Peptidoglicano/metabolismo , Sepsis/patología , Staphylococcus aureus/inmunología
18.
J Immunol ; 186(7): 3841-5, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21357534

RESUMEN

The cell wall of bacteria induces proinflammatory cytokines in monocytes and neutrophils in human blood. The nature of the stimulating component of bacterial cell walls is not well understood. We have previously shown polymeric peptidoglycan (PGN) has this activity, and the cytokine response requires PGN internalization and trafficking to lysosomes. In this study, we demonstrate that peptidoglycan monomers such as muramyl dipeptide and soluble peptidoglycan fail to induce robust cytokine production in immune cells, although they activate the nucleotide-binding oligomerization domain proteins in transfected cell models. We further show that lysosomal extracts from immune cells degrade intact peptidoglycan into simpler products and that the lysosomal digestion products activate the nucleotide-binding oligomerization domain proteins. We conclude that naive innate immune cells recognize PGN in its polymeric form rather than monomers such as muramyl dipeptide and require PGN lysosomal hydrolysis to respond. These findings offer new opportunities in the treatment of sepsis, especially sepsis arising from Gram-positive organisms.


Asunto(s)
Inmunidad Innata , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peptidoglicano/química , Peptidoglicano/inmunología , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Bacillus anthracis/inmunología , Células HEK293 , Humanos , Hidrólisis , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiología , Lisosomas/inmunología , Lisosomas/metabolismo , Lisosomas/microbiología , Monocitos/microbiología , Neutrófilos/microbiología , Proteína Adaptadora de Señalización NOD1/biosíntesis , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/biosíntesis , Proteína Adaptadora de Señalización NOD2/metabolismo , Peptidoglicano/metabolismo , Polímeros/química , Polímeros/metabolismo , Transporte de Proteínas/inmunología
19.
Sci Signal ; 3(122): ra38, 2010 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-20484116

RESUMEN

The activation and fusion of macrophages and of osteoclasts require the adaptor molecule DNAX-activating protein of 12 kD (DAP12), which contains immunoreceptor tyrosine-based activation motifs (ITAMs). TREM2 (triggering receptor expressed on myeloid cells-2) is the main DAP12-associated receptor in osteoclasts and, similar to DAP12 deficiency, loss of TREM2 in humans leads to Nasu-Hakola disease, which is characterized by bone cysts and dementia. Furthermore, in vitro experiments have shown that deficiency in DAP12 or TREM2 leads to impaired osteoclast development and the formation of mononuclear osteoclasts. Here, we demonstrate that the ligation of TREM2 activated phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the guanine nucleotide exchange factor Vav3; induced the mobilization of intracellular calcium (Ca(2+)) and the reorganization of actin; and prevented apoptosis. The signaling adaptor molecule DAP10 played a key role in the TREM2- and DAP12-dependent recruitment of PI3K to the signaling complex. Src homology 2 (SH2) domain-containing inositol phosphatase-1 (SHIP1) inhibited TREM2- and DAP12-induced signaling by binding to DAP12 in an SH2 domain-dependent manner and preventing the recruitment of PI3K to DAP12. These results demonstrate a previously uncharacterized interaction of SHIP1 with DAP12 that functionally limits TREM2- and DAP12-dependent signaling and identify a mechanism through which SHIP1 regulates key ITAM-containing receptors by directly blocking the binding and activation of PI3K.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Glicoproteínas de Membrana/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Receptores Inmunológicos/fisiología , Animales , División Celular/fisiología , Línea Celular , Activación Enzimática , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Endogámicos C57BL , Osteoclastos/citología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Transducción de Señal , Dominios Homologos src
20.
Blood ; 115(23): 4699-706, 2010 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-20351305

RESUMEN

Src homology 2 domain-containing inositol 5-phosphatase (SHIP(-/-)) animals display an age-related increase in interleukin-6 (IL-6), a decrease in B lymphopoiesis, and an elevation in myelopoiesis. We investigated the origin of the IL-6 production and show that it is largely produced by peritoneal and splenic macrophages. IL-6 production by these macrophages is not a direct result of the loss of SHIP: IL-6 production is not spontaneous, is absent from bone marrow-derived macrophages, declines with prolonged culture of macrophages, and requires a stimulus present in vivo. The IL-6-rich peritoneal cavity of SHIP(-/-) mice shows more than 700-fold more immunoglobulin G (IgG) than wild-type, approximately 20% of which is aggregated or in an immune complex and contains B220(+) cells that secrete IgG. The SHIP-deficient peritoneal macrophages show evidence of IgG receptor stimulation. Animals lacking both the signal-transducing gamma-chain of IgG receptors and SHIP or Ig and SHIP produce less IL-6. The data indicate a feed-forward process in which peripheral macrophages, responding through IgG receptors to secreted IgG, produce IL-6, to support further B-cell production of IgG. Because of the proinflammatory phenotype of SHIP(-/-) animals, these findings emphasize the importance of IL-6-neutralizing strategies in autoimmune and proinflammatory diseases.


Asunto(s)
Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Interleucina-6/inmunología , Mielopoyesis/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Envejecimiento/genética , Envejecimiento/inmunología , Animales , Formación de Anticuerpos/genética , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos B/metabolismo , Células Cultivadas , Inmunoglobulina G/biosíntesis , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inositol Polifosfato 5-Fosfatasas , Interleucina-6/genética , Interleucina-6/metabolismo , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Mielopoyesis/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores de IgG/genética , Receptores de IgG/inmunología , Receptores de IgG/metabolismo
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