RESUMEN
Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.
Asunto(s)
Tolerancia Inmunológica , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Sepsis/inmunología , Transcripción Genética , beta-Glucanos/inmunología , Diferenciación Celular , Metilación de ADN , Epigenómica , Redes Reguladoras de Genes , Código de Histonas , Humanos , Inmunidad Innata , Memoria Inmunológica , Macrófagos/citología , Monocitos/citología , Sepsis/genéticaRESUMEN
Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes ß-glucan in the fungal cell wall. C. albicans ß-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how ß-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates ß-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated ß-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated ß-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.
Asunto(s)
Candida albicans/patogenicidad , Candidiasis/inmunología , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Virulencia/fisiología , beta-Glucanos/inmunología , Animales , Candida albicans/inmunología , Candida albicans/metabolismo , Candidiasis/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , beta-Glucanos/metabolismoRESUMEN
Acinetobacter baumannii has emerged as an important nosocomial pathogen, particularly for patients in intensive care units and with invasive indwelling devices. The most recent clinical isolates are resistant to several classes of clinically important antibiotics, greatly restricting the ability to effectively treat critically ill patients. The bacterial envelope is an important driver of A. baumannii disease, both at the level of battling against antibiotic therapy and at the level of protecting from host innate immune function. This review provides a comprehensive overview of key features of the envelope that interface with both the host and antimicrobial therapies. Carbohydrate structures that contribute to protecting from the host are detailed, and mutations that alter these structures, resulting in increased antimicrobial resistance, are explored. In addition, protein complexes involved in both intermicrobial and host-microbe interactions are described. Finally we discuss regulatory mechanisms that control the nature of the cell envelope and its impact on host innate immune function.
Asunto(s)
Acinetobacter baumannii , Pared Celular/inmunología , Farmacorresistencia Bacteriana Múltiple/genética , Glucolípidos , Virulencia/genética , Acinetobacter baumannii/citología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/inmunología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas , Pared Celular/microbiología , Infección Hospitalaria , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Genes Bacterianos , Glucolípidos/inmunología , Glucolípidos/metabolismo , Interacciones Microbiota-Huesped , Humanos , Inmunidad Innata , Canales Iónicos/genética , Canales Iónicos/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Interacciones Microbianas , Polisacáridos Bacterianos , Porinas/genética , Porinas/metabolismo , Sistemas de Secreción Tipo II/genética , Sistemas de Secreción Tipo II/metabolismo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , beta-Glucanos/inmunología , beta-Glucanos/metabolismoRESUMEN
Masking the immunogenic cell wall epitope ß(1,3)-glucan under an outer layer of mannosylated glycoproteins is an important virulence factor deployed by Candida albicans during infection. Consequently, increased ß(1,3)-glucan exposure (unmasking) reveals C. albicans to the host's immune system and attenuates its virulence. We have previously shown that activation of the Cek1 MAPK pathway via expression of a hyperactive allele of an upstream kinase (STE11ΔN467) induced unmasking. It also increased survival of mice in a murine disseminated candidiasis model and attenuated kidney fungal burden by ≥33 fold. In this communication, we utilized cyclophosphamide-induced immunosuppression to test if the clearance of the unmasked STE11ΔN467 mutant was dependent on the host immune system. Suppression of the immune response by cyclophosphamide reduced the attenuation in fungal burden caused by the STE11ΔN467 allele. Moreover, specific depletion of neutrophils via 1A8 antibody treatment also reduced STE11ΔN467-dependent fungal burden attenuation, but to a lesser extent than cyclophosphamide, demonstrating an important role for neutrophils in mediating fungal clearance of unmasked STE11ΔN467 cells. In an effort to understand the mechanism by which Ste11ΔN467 causes unmasking, transcriptomics were used to reveal that several components in the Cek1 MAPK pathway were upregulated, including the transcription factor CPH1 and the cell wall sensor DFI1. In this report we show that a cph1ΔΔ mutation restored ß(1,3)-glucan exposure to wild-type levels in the STE11ΔN467 strain, confirming that Cph1 is the transcription factor mediating Ste11ΔN467-induced unmasking. Furthermore, Cph1 is shown to induce a positive feedback loop that increases Cek1 activation. In addition, full unmasking by STE11ΔN467 is dependent on the upstream cell wall sensor DFI1. However, while deletion of DFI1 significantly reduced Ste11ΔN467-induced unmasking, it did not impact activation of the downstream kinase Cek1. Thus, it appears that once stimulated by Ste11ΔN467, Dfi1 activates a parallel signaling pathway that is involved in Ste11ΔN467-induced unmasking.
Asunto(s)
Candida albicans/inmunología , Candidiasis/prevención & control , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Neutrófilos/inmunología , Factores de Transcripción/metabolismo , Virulencia , beta-Glucanos/inmunología , Animales , Candidiasis/inmunología , Candidiasis/microbiología , Pared Celular , Proteínas Fúngicas/genética , Ratones , Ratones Endogámicos ICR , Neutrófilos/microbiología , Factores de Transcripción/genéticaRESUMEN
Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by genetic defects in leukocyte NADPH oxidase, which has both microbicidal and immunomodulatory roles. Hence, CGD is characterized by recurrent bacterial and fungal infections as well as aberrant inflammation. Fungal cell walls induce neutrophilic inflammation in CGD; yet, underlying mechanisms are incompletely understood. This study investigated the receptors and signaling pathways driving aberrant proinflammatory cytokine production in CGD neutrophils activated by fungal cell walls. Although cytokine responses to ß-glucan particles were similar in NADPH oxidase-competent and NADPH oxidase-deficient mouse and human neutrophils, stimulation with zymosan, a more complex fungal particle, induced elevated cytokine production in NADPH oxidase-deficient neutrophils. The dectin-1 C-type lectin receptor, which recognizes ß-glucans (1-3), and TLRs mediated cytokine responses by wild-type murine neutrophils. In the absence of NADPH oxidase, fungal pathogen-associated molecular patterns engaged additional collaborative signaling with Mac-1 and TLRs to markedly increase cytokine production. Mechanistically, this cytokine overproduction is mediated by enhanced proximal activation of tyrosine phosphatase SHP2-Syk and downstream Card9-dependent NF-κB and Card9-independent JNK-c-Jun. This activation and amplified cytokine production were significantly decreased by exogenous H2O2 treatment, enzymatic generation of exogenous H2O2, or Mac-1 blockade. Similar to zymosan, Aspergillus fumigatus conidia induced increased signaling in CGD mouse neutrophils for activation of proinflammatory cytokine production, which also used Mac-1 and was Card9 dependent. This study, to our knowledge, provides new insights into how NADPH oxidase deficiency deregulates neutrophil cytokine production in response to fungal cell walls.
Asunto(s)
Aspergillus fumigatus/fisiología , Enfermedad Granulomatosa Crónica/inmunología , Lectinas Tipo C/metabolismo , Antígeno de Macrófago-1/metabolismo , NADPH Oxidasa 2/metabolismo , Neutrófilos/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Animales , Antígenos Fúngicos/inmunología , Células Cultivadas , Citocinas/metabolismo , Enfermedad Granulomatosa Crónica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2/genética , FN-kappa B/metabolismo , Activación Neutrófila , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Receptor Cross-Talk , Transducción de Señal , beta-Glucanos/inmunologíaRESUMEN
Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.
Asunto(s)
Candida albicans/inmunología , Pared Celular/inmunología , Células Epiteliales/inmunología , Mucosa Intestinal/inmunología , beta-Glucanos/inmunología , Células CACO-2 , Candida albicans/metabolismo , Candida albicans/fisiología , Línea Celular Tumoral , Pared Celular/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HT29 , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Intestinal/microbiología , Fosforilación/inmunología , Quinasa Syk/inmunología , Quinasa Syk/metabolismo , Zimosan/inmunología , Zimosan/metabolismo , beta-Glucanos/metabolismoRESUMEN
Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.
Asunto(s)
Saccharomyces cerevisiae/inmunología , Infecciones Estafilocócicas/inmunología , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Coagulasa/administración & dosificación , Coagulasa/genética , Coagulasa/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Saccharomyces cerevisiae/química , Infecciones Estafilocócicas/microbiología , Vacunas Estafilocócicas/administración & dosificación , Vacunas Estafilocócicas/genética , Staphylococcus aureus/genética , Células TH1/inmunología , Células Th17/inmunología , Vacunación , beta-Glucanos/administración & dosificación , beta-Glucanos/inmunologíaRESUMEN
The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.
Asunto(s)
Aspergilosis/inmunología , Interacciones Huésped-Patógeno , Macrófagos/fisiología , Neutrófilos/fisiología , beta-Glucanos/inmunología , Animales , Aspergillus fumigatus , Ratones , Fagocitosis , Fagosomas , Esporas Fúngicas , Talaromyces , Pez CebraRESUMEN
BACKGROUND: An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal ß-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo. METHODS: Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array. RESULTS: Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1ß, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia. CONCLUSIONS: Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Microglía/efectos de los fármacos , Microglía/inmunología , beta-Glucanos/inmunología , Animales , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , beta-Glucanos/farmacologíaRESUMEN
Sirtuin 1 (SIRT1) has been described to modify immune responses by modulation of gene transcription. As transcriptional reprogramming is the molecular substrate of trained immunity, a de facto innate immune memory, we investigated the role of SIRT1 in the induction of trained immunity. We identified various SIRT1 genetic single nucleotide polymorphisms affecting innate and adaptive cytokine production of human peripheral blood mononuclear cells (PBMCs) in response to various stimuli on the one hand, and in vitro induction of trained immunity on the other hand. Furthermore, inhibition of SIRT1 upregulated pro-inflammatory innate cytokine production upon stimulation of PBMCs. However, inhibition of SIRT1 in vitro had no effect on cytokine responses upon induction of trained immunity, while activation of SIRT1 mildly modified trained immunity responses. In conclusion, SIRT1 modifies innate cytokine production by PBMCs in response to various microbes, but has only a secondary role for BCG and ß-glucan-induced trained immunity responses.
Asunto(s)
Genotipo , Inflamación/inmunología , Leucocitos Mononucleares/inmunología , Mycobacterium bovis/inmunología , Sirtuina 1/metabolismo , Inmunidad Adaptativa , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunidad Innata , Inmunización , Memoria Inmunológica , Mediadores de Inflamación/metabolismo , Polimorfismo de Nucleótido Simple , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , beta-Glucanos/inmunologíaRESUMEN
Trained immunity is a form of innate immune memory best described in mice and humans. Clear evidence of the evolutionary conservation of trained immunity in teleost fish is lacking. Given the evolutionary position of teleosts as early vertebrates with a fully developed immune system, we hypothesize that teleost myeloid cells show features of trained immunity common to those observed in mammalian macrophages. These would at least include the ability of fish macrophages to mount heightened responses to a secondary stimulus in a nonspecific manner. We established an in vitro model to study trained immunity in fish by adapting a well-described culture system of head kidney-derived macrophages of common carp. A soluble NOD-specific ligand and a soluble ß-glucan were used to train carp macrophages, after which cells were rested for 6 d prior to exposure to a secondary stimulus. Unstimulated trained macrophages displayed evidence of metabolic reprogramming as well as heightened phagocytosis and increased expression of the inflammatory cytokines il6 and tnf-α. Stimulated trained macrophages showed heightened production of reactive oxygen and nitrogen species as compared with the corresponding stimulated but untrained cells. We discuss the value of our findings for future studies on trained immunity in teleost fish.
Asunto(s)
Carpas/inmunología , Riñón Cefálico/inmunología , Macrófagos/inmunología , Animales , Evolución Biológica , Células Cultivadas , Reprogramación Celular , Proteínas de Peces/metabolismo , Inmunidad , Inmunización , Interleucina-6/metabolismo , Mamíferos , Oxigenasas/inmunología , Fagocitosis , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , beta-Glucanos/inmunologíaRESUMEN
Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.
Asunto(s)
Hongos/inmunología , Micosis/inmunología , beta-Glucanos/inmunología , Inmunidad Adaptativa , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Inmunidad , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Noqueados , Micosis/genética , Micosis/microbiología , Receptor EphA2/genética , Receptor EphA2/inmunologíaRESUMEN
BACKGROUND: Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) ß-glucans. METHODS: Major surface glycoprotein was shown to greatly reduce ß-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc ß-glucan. RESULTS: The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis ß-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. CONCLUSIONS: Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory ß -glucan components of the organisms.
Asunto(s)
Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Glicoproteínas de Membrana/metabolismo , Pneumocystis carinii/inmunología , Neumonía por Pneumocystis/inmunología , beta-Glucanos/metabolismo , Animales , Proteínas Fúngicas/metabolismo , Lectinas Tipo C/genética , Macrófagos/microbiología , Ratones , Pneumocystis/inmunología , Células RAW 264.7 , ARN Mensajero/genética , Saccharomyces cerevisiae/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , beta-Glucanos/inmunologíaRESUMEN
Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.
Asunto(s)
Aspergillus fumigatus/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Complemento C3/inmunología , Polisacáridos Fúngicos/farmacología , Macrófagos/efectos de los fármacos , Suero/inmunología , Esporas Fúngicas/inmunología , Aspergilosis/genética , Aspergilosis/inmunología , Aspergilosis/microbiología , Aspergillus fumigatus/química , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/microbiología , Pared Celular/química , Pared Celular/inmunología , Activación de Complemento/efectos de los fármacos , Complemento C3/genética , Citocinas/biosíntesis , Citocinas/inmunología , Polisacáridos Fúngicos/inmunología , Polisacáridos Fúngicos/aislamiento & purificación , Galactosa/análogos & derivados , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Celular , Inmunidad Humoral , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/inmunología , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Mananos/inmunología , Mananos/aislamiento & purificación , Mananos/farmacología , Proteínas Opsoninas/farmacología , Fagocitosis/efectos de los fármacos , Cultivo Primario de Células , Unión Proteica , Especies Reactivas de Oxígeno , Suero/química , Suero/microbiología , Esporas Fúngicas/química , beta-Glucanos/inmunología , beta-Glucanos/aislamiento & purificación , beta-Glucanos/farmacologíaRESUMEN
The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the ß-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.
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Pared Celular/enzimología , Criptococosis/inmunología , Cryptococcus neoformans/enzimología , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Evasión Inmune/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Animales , Pared Celular/inmunología , Células Cultivadas , Criptococosis/microbiología , Criptococosis/patología , Cryptococcus neoformans/patogenicidad , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Femenino , Proteínas Fúngicas/genética , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Transporte de Proteínas , beta-Glucanos/inmunologíaRESUMEN
Mast cells (MCs) are engaged in host defense against various pathogens as they are equipped with pattern recognition receptors (PRRs). Among PRRs expressed on MCs, there are also molecules recognizing components of the fungal cell wall, which are able to induce cellular activation and response. However, little information is available concerning the MC activation by various fungal-derived components. The aim of the study was to determine whether curdlan, a model fungal particle of ß-(1,3)-glucan, can directly stimulate tissue MCs. We demonstrated that curdlan triggers MCs to initiate pro-inflammatory response as it activates these cells to synthesize essential pro-inflammatory and/or immunoregulatory factors. We also showed that curdlan serves as a potent chemoattractant for MCs and stimulates those cells to generate reactive oxygen species (ROS). Finally, we documented that curdlan induces MC response via Dectin-1. Our observations support the idea that MCs serve as important sentinels modulating immune response during fungal infection.
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Degranulación de la Célula/inmunología , Quimiotaxis de Leucocito/inmunología , Lectinas Tipo C/inmunología , Mastocitos/inmunología , beta-Glucanos/inmunología , Animales , Femenino , Ratas , Ratas Wistar , Especies Reactivas de OxígenoRESUMEN
Vaccination constitutes one of the major breakthroughs in human medicine. At the same time, development of more immunogenic vaccine alternatives to using aluminium-based adjuvants is one of the most important phases of vaccination development. Among different sources of carbohydrate polymers, including plants, microbes and synthetic sources tested, glucans were found to be the most promising vaccine adjuvant, as they alone stimulate various immune reactions including antibody production without any negative side effects. The use of glucan particles as a delivery system is a viable option based on the documented efficient antigen loading and receptor-targeted uptake in antigen-presenting cells. In addition to particles, soluble glucans can be used as novel hydrogels or as direct immunocyte-targeting delivery systems employing novel complexes with oligodeoxynucleotides. This review focuses on recent advances in glucan-based vaccine development from glucan-based conjugates to a glucan-based delivery and adjuvant platform.
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Adyuvantes Inmunológicos , Sistemas de Liberación de Medicamentos/métodos , Hidrogeles/uso terapéutico , Vacunas/inmunología , beta-Glucanos/inmunología , Animales , Presentación de Antígeno , Humanos , Oligodesoxirribonucleótidos , Vacunación , beta-Glucanos/uso terapéuticoRESUMEN
Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.
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Agaricales/química , Criptococosis/prevención & control , Polisacáridos Fúngicos/inmunología , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , beta-Glucanos/inmunología , Animales , Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Factores Inmunológicos/farmacología , Lectinas Tipo C/inmunología , Enfermedades Pulmonares Fúngicas , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Fagocitos/microbiología , Transducción de Señal , beta-Glucanos/farmacologíaRESUMEN
As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.
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Enfermedades de los Peces/prevención & control , Carpa Dorada , Infecciones por Herpesviridae/veterinaria , Herpesviridae/inmunología , Inmunización/veterinaria , Alcaloides Solanáceos/inmunología , Vacunas Virales/inmunología , beta-Glucanos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Acuicultura , Enfermedades de los Peces/virología , Carpa Dorada/inmunología , Carpa Dorada/virología , Herpesviridae/fisiología , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/virología , Inmunidad Innata , Inmunidad Mucosa , Inmunización/métodos , Propiolactona , Escopolamina/administración & dosificación , Escopolamina/inmunología , Alcaloides Solanáceos/administración & dosificación , Tasa de Supervivencia , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/administración & dosificación , Replicación Viral , beta-Glucanos/administración & dosificaciónRESUMEN
Arthritis in a genetically susceptible SKG strain of mice models a theoretical paradigm wherein autoimmune arthritis arises because of interplay between preexisting autoreactive T cells and environmental stimuli. SKG mice have a point mutation in ZAP-70 that results in attenuated TCR signaling, altered thymic selection, and spontaneous production of autoreactive T cells that cause arthritis following exposure to microbial ß-glucans. In this study, we identify Nod2, an innate immune receptor, as a critical suppressor of arthritis in SKG mice. SKG mice deficient in Nod2 (Nod2-/-SKG) developed a dramatically exacerbated form of arthritis, which was independent of sex and microbiota, but required the skg mutation in T cells. Worsened arthritis in Nod2-/-SKG mice was accompanied by expansion of Th17 cells, which to some measure coproduced TNF, GM-CSF, and IL-22, along with elevated IL-17A levels within joint synovial fluid. Importantly, neutralization of IL-17A mitigated arthritis in Nod2-/-SKG mice, indicating that Nod2-mediated protection occurs through suppression of the Th17 response. Nod2 deficiency did not alter regulatory T cell development or function. Instead, Nod2 deficiency resulted in an enhanced fundamental ability of SKG CD4+ T cells (from naive mice) to produce increased levels of IL-17 and to passively transfer arthritis to lymphopenic recipients on a single-cell level. These data reveal a previously unconsidered role for T cell-intrinsic Nod2 as an endogenous negative regulator of Th17 responses and arthritogenic T cells. Based on our findings, future studies aimed at understanding a negative regulatory function of Nod2 within autoreactive T cells could provide novel therapeutic strategies for treatment of patients with arthritis.