RESUMO
Staphylococcus aureus is a human-adapted pathogen that replicates by asymptomatically colonizing its host. S. aureus is also the causative agent of purulent skin and soft tissue infections as well as bloodstream infections that result in the metastatic seeding of abscess lesions in all organ tissues. Prolonged colonization, infection, disease relapse, and recurrence point to the versatile capacity of S. aureus to bypass innate and adaptive immune defenses as well as the notion that some hosts fail to generate protective immune responses. Here, we find a genetic trait that provides protection against this pathogen. Mice lacking functional H2-O, the equivalent of human HLA-DO, inoculated with a mouse-adapted strain of S. aureus, efficiently decolonize the pathogen. Further, these decolonized animals resist subsequent bloodstream challenge with methicillin-resistant S. aureus. A genetic approach demonstrates that T-cell dependent B cell responses are required to control S. aureus colonization and infection in H2-O-deficient mice. Reduced bacterial burdens in these animals correlate with increased titers and enhanced phagocytic activity of S. aureus-specific antibodies. H2-O negatively regulates the loading of high affinity peptides on major histocompatibility class II (MHC-II) molecules. Thus, we hypothesize that immune responses against S. aureus are derepressed in mice lacking H2-O because more high affinity peptides are presented by MHC-II. We speculate that loss-of-function HLA-DO alleles may similarly control S. aureus replication in humans.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Camundongos , Staphylococcus aureus/imunologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Classe II/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , HumanosRESUMO
Poly-ß-(1-6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qß as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Camundongos , Staphylococcus aureus/imunologia , Coelhos , Vacinas Antiestafilocócicas/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Feminino , Staphylococcus aureus Resistente à Meticilina/imunologia , Acetilglucosamina/imunologia , Humanos , Epitopos/imunologia , Camundongos Endogâmicos BALB CRESUMO
To increase the effectiveness of methicillin-resistant Staphylococcus aureus vaccines (MRSA), a new generation of immune system stimulating adjuvants is necessary, along with other adjuvants. In some vaccines, monophosphoryl lipid A (MPLA) as a toll-like receptor 4 agonist is currently used as an adjuvant or co-adjuvant. MPLA could increase the immune response and vaccine immunogenicity. The current investigation assessed the immunogenicity and anti-MRSA efficacy of recombinant autolysin formulated in MPLA and Alum as co-adjuvant/adjuvant. r-Autolysin was expressed and purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE. Then, the vaccine candidate formulation in MPLAs and Alum was prepared. To investigate the immunogenic responses, total IgG, isotype (IgG1 and IgG2a) levels, and cytokines (IL-4, IL-12, TNF-α, and IFN-γ) profiles were evaluated by ELISA. Also, the bacterial burden in internal organs, opsonophagocytosis, survival rate, and pathobiology changes was compared among the groups. Results demonstrated that mice immunized with the r-Autolysin + Alum + MPLA Synthetic and r-Autolysin + Alum + MPLA Biologic led to increased levels of opsonic antibodies, IgG1, IgG2a isotype as well as increased levels of cytokines profiles, as compared with other experimental groups. More importantly, mice immunized with MPLA and r-Autolysin exhibited a decrease in mortality and bacterial burden, as compared with the control group. The highest level of survival was seen in the r-Autolysin + Alum + MPLA Synthetic group. We concluded that both MPLA forms, synthetic and biological, are reliable candidates for immune response improvement against MRSA infection.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos , Modelos Animais de Doenças , Imunoglobulina G , Lipídeo A , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Vacinas Antiestafilocócicas , Animais , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeo A/administração & dosagem , Lipídeo A/farmacologia , Camundongos , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Adjuvantes Imunológicos/administração & dosagem , Feminino , Citocinas/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/imunologia , Desenvolvimento de Vacinas , Compostos de Alúmen/administração & dosagem , Camundongos Endogâmicos BALB C , Adjuvantes de Vacinas , HumanosRESUMO
IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte de Cátions , Citocinas , Staphylococcus aureus Resistente à Meticilina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Infecções Estafilocócicas , Receptor 4 Toll-Like , Humanos , Proteínas de Bactérias/imunologia , Caspase 1/metabolismo , Proteínas de Transporte de Cátions/imunologia , Citocinas/metabolismo , Inflamassomos/metabolismo , Ferro/metabolismo , Staphylococcus aureus Resistente à Meticilina/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções Estafilocócicas/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
In this study, we examined the effect of Il9 deletion on macrophages in methicillin-resistant Staphylococcus aureus (MRSA) infection. MRSA-infected mice were employed for the in vivo experiments, and RAW264.7 cells were stimulated with MRSA for the in vitro experiments. Macrophage polarization was determined by flow cytometry and quantitative real-time PCR; macrophage phagocytosis was assessed by flow cytometry and laser scanning confocal microscopy; cell apoptosis was assessed by flow cytometry and western blotting. Il9 deletion markedly elevated macrophage phagocytosis and M2 macrophages in MRSA infection, which was accompanied by elevated expression of Il10 and Arg1 and reduced expression of Inos, tumor necrosis factor-α (Tnfα), and Il6. Il9 deletion also inhibited macrophage apoptosis in MRSA infection, which was manifested by elevated B-cell lymphoma 2 (BCL-2) protein level and reduced protein levels of cleaved cysteine protease 3 (CASPASE-3) and BCL2-Associated X (BAX). Both the in vivo and in vitro experiments further showed the activation of phosphoinositide 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB) signaling pathway in MRSA infection and that the regulation of Il9 expression may be dependent on Toll-like receptor (TLR) 2/PI3K pathway. The above results showed that Il9 deletion exhibited a protective role against MRSA infection by promoting M2 polarization and phagocytosis of macrophages and the regulation of Il9 partly owing to the activation of TLR2/PI3K pathway, proposing a novel therapeutic strategy for MRSA-infected pneumonia.
Assuntos
Interleucina-9 , Staphylococcus aureus Resistente à Meticilina , Fagocitose , Pneumonia Estafilocócica , Animais , Camundongos , Interleucina-9/genética , Interleucina-9/metabolismo , Macrófagos/metabolismo , Staphylococcus aureus Resistente à Meticilina/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Pneumonia Estafilocócica/tratamento farmacológico , Pneumonia Estafilocócica/imunologiaRESUMO
Intracellular Methicillin-Resistant Staphylococcus aureus (MRSA) remains a major factor of refractory and recurrent infections, which cannot be well addressed by antibiotic therapy. Here, we design a cellular infectious microenvironment-activatable polymeric nano-system to mediate targeted intracellular drug delivery for macrophage reprogramming and intracellular MRSA eradication. The polymeric nano-system is composed of a ferrocene-decorated polymeric nanovesicle formulated from poly(ferrocenemethyl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) (PFMMA-b-PMPC) copolymer with co-encapsulation of clofazimine (CFZ) and interferon-γ (IFN-γ). The cellular-targeting PMPC motifs render specific internalization by macrophages and allow efficient intracellular accumulation. Following the internalization, the ferrocene-derived polymer backbone sequentially undergoes hydrophobic-to-hydrophilic transition, charge reversal and Fe release in response to intracellular hydrogen peroxide over-produced upon infection, eventually triggering endosomal escape and on-site cytosolic drug delivery. The released IFN-γ reverses the immunosuppressive status of infected macrophages by reprogramming anti-inflammatory M2 to pro-inflammatory M1 phenotype. Meanwhile, intracellular Fe2+-mediated Fenton reaction together with antibiotic CFZ contributes to increased intracellular hydroxyl radical (â¢OH) generation. Ultimately, the nano-system achieves robust potency in ablating intracellular MRSA and antibiotic-tolerant persisters by synchronous immune modulation and efficient â¢OH killing, providing an innovative train of thought for intracellular MRSA control.
Assuntos
Antibacterianos , Macrófagos , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Interferon gama , Macrófagos/imunologia , Metalocenos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/imunologia , Polímeros/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/imunologia , Nanoestruturas/uso terapêuticoRESUMO
Extracellular vesicles (EVs) released from bacteria are enclosed particles carrying biological active molecules. They have been shown to play a role in bacterial communications and delivery of virulence factors to the host cells. Staphylococcus aureus is an opportunistic pathogen causing a variety of infections ranging from impetigo to septicaemia. The EVs released from S. aureus have a high potential to be used for vaccine development against S. aureus infections. However, it is important to clearly understand the impact of SaEVs on the host's immune response. Our study demonstrated that purified EVs from a clinical isolated methicillin-resistant S. aureus (SaEVs) significantly stimulated proinflammatory cytokine production in mouse immune cells and induced host cell death. An impairment of cytokine production in the Toll-like receptor (TLR)-silenced macrophages suggested that SaEVs stimulate proinflammatory response via TLRs 2, 4 and 9. In mouse infection model, the results demonstrated that SaEV immunization did not provide protective effect. In contrast, all SaEV-immunized mice died within Day 1 after methicillin-resistant S. aureus (MRSA) infection. After MRSA infection for 3â h, the production of IL-6, TNF-α and IL-17 in the spleen of SaEV-immunized mice was significantly higher than that of control mice. On Day 5 after the second immunization, total IgE in the serum was significantly enhanced, and a high titre of Th2-related cytokines was remarkably induced after ex vivo stimulation of the spleen cells with SaEVs. These results suggested that MRSA-derived EVs act as an immunostimulant that induces inflammatory response and IgE-mediated hypersensitivity after MRSA infection.
Assuntos
Citocinas/imunologia , Vesículas Extracelulares/imunologia , Hipersensibilidade Imediata/etiologia , Hipersensibilidade Imediata/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Estafilocócicas/complicações , Animais , Citocinas/genética , Vesículas Extracelulares/genética , Feminino , Humanos , Hipersensibilidade Imediata/genética , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/imunologia , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Staphylococcus aureus is an important human opportunistic pathogen that can have a major influence on public health. Here, we aimed to evaluate different aspects of the immune response to a novel multi-epitope fusion protein (HMS) based on HlaH35L, MntC, and SACOL0723 proteins in comparison to the individual antigens. For this purpose, specific total IgG, IgG1, and IgG2a isotypes and the cytokines related to Th1, Th2, and Th17 were assessed. The Bio-efficiency of the fusion protein was evaluated by opsonic killing activity. The HMS fusion protein elicited a high specific IgG level and also induced a higher level of Th1, Th2, and Th17-related cytokines which were more polarized towards the Th1 and Th17 compared to individual antigens. The HMS-specific antisera also significantly promoted phagocytosis of S. aureus COL strain by mouse macrophages. In conclusion, the fusion protein might be an effective vaccine for potential protective immunity against a lethal infection of S. aureus in mice.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Citocinas/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Linfócitos T/imunologiaRESUMO
Th9 is a subset of CD4+ T cells that mainly secrete IL-9. Th9/IL-9 participates in immune response during Staphylococcus aureus and methicillin-resistant Staphylococcus aureus pneumonia (MRSA) infection. Here, we collected bronchoalveolar lavage fluid (BALF) from 30 children with MRSA pneumonia (MRSA group) and 10 children with bronchial foreign bodies (Control group). RT-PCR, ELISA and flow cytometry were used to detect the expression of miR-155 and IL-9 in BALF and the number of Th9 cells. CD4+ T cells isolated from BALF of MRSA and Control group were transfected with miR-155 mimic or inhibitor, and then induced Th9 cell differentiation. The results showed that the expression of miR-155 and IL-9 were significantly increased in BALF and Th9 cell of MRSA group, as well as the number of Th9 cells. miR-155 mimic upregulated IL-9 mRNA expression, IL-9 secretion and increased number of Th9 cells. On the contrary, miR-155 inhibitor inhibited IL-9 mRNA expression, IL-9 secretion and decreased number of Th9 cells. The dual luciferase assays demonstrated miR-155 can target binding to SIRT1 3'UTR. Moreover, overexpression of SIRT1 could reverse the effect of miR-155 mimic on IL-9 expression level, Th9 cell number and transcription factors PU.1 and IRF4 expression. In conclusion, miR-155 regulates Th9 differentiation in children with MRSA by targeting SIRT1.
Assuntos
Diferenciação Celular/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , MicroRNAs/genética , Sirtuína 1/genética , Infecções Estafilocócicas/etiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Regiões 3' não Traduzidas , Biomarcadores , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Suscetibilidade a Doenças/imunologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunofenotipagem , Masculino , Interferência de RNA , Infecções Estafilocócicas/metabolismoRESUMO
Staphylococcus aureus is an opportunistic pathogen causing high morbidity and mortality. Since multi-drug resistant S. aureus lineages are nowadays omnipresent, alternative tools for preventive or therapeutic interventions, like immunotherapy, are urgently needed. However, there are currently no vaccines against S. aureus. Surface-exposed and secreted proteins are regarded as potential targets for immunization against S. aureus infections. Yet, many potential staphylococcal antigens of this category do not elicit protective immune responses. To obtain a better understanding of this problem, we compared the binding of serum IgGs from healthy human volunteers, highly S. aureus-colonized patients with the genetic blistering disease epidermolysis bullosa (EB), or immunized mice to the purified S. aureus peptidoglycan hydrolases Sle1, Aly and LytM and their different domains. The results show that the most abundant serum IgGs from humans and immunized mice target the cell wall-binding domain of Sle1, and the catalytic domains of Aly and LytM. Interestingly, in a murine infection model, these particular IgGs were not protective against S. aureus bacteremia. In contrast, relatively less abundant IgGs against the catalytic domain of Sle1 and the N-terminal domains of Aly and LytM were almost exclusively detected in sera from EB patients and healthy volunteers. These latter IgGs may contribute to the protection against staphylococcal infections, as previous studies suggest that serum IgGs protect EB patients against severe S. aureus infection. Together, these observations focus attention on the use of particular protein domains for vaccination to direct potentially protective immune responses towards the most promising epitopes within staphylococcal antigens.
Assuntos
Imunoglobulina G/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , N-Acetil-Muramil-L-Alanina Amidase/imunologia , Infecções Estafilocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Domínio Catalítico/genética , Domínio Catalítico/imunologia , Parede Celular/genética , Parede Celular/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoglobulina G/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/química , Peptidoglicano/genética , Peptidoglicano/imunologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/prevenção & controleRESUMO
Antibiotic-resistant Staphylococcus aureus strains constitute a major public health concern worldwide and are responsible for both health care- and community-associated infections. Here, we establish a robust and easy-to-implement model of oral S. aureus infection using Drosophila melanogaster larvae that allowed us to follow the fate of S. aureus at the whole-organism level as well as the host immune responses. Our study demonstrates that S. aureus infection triggers H2O2 production by the host via the Duox enzyme, thereby promoting antimicrobial peptide production through activation of the Toll pathway. Staphylococcal catalase mediates H2O2 neutralization, which not only promotes S. aureus survival but also minimizes the host antimicrobial response, hence reducing bacterial clearance in vivo. We show that while catalase expression is regulated in vitro by the accessory gene regulatory system (Agr) and the general stress response regulator sigma B (SigB), it no longer depends on these two master regulators in vivo. Finally, we confirm the versatility of this model by demonstrating the colonization and host stimulation capabilities of S. aureus strains belonging to different sequence types (CC8 and CC5) as well as of two other bacterial pathogens, Salmonella enterica serovar Typhimurium and Shigella flexneri. Thus, the Drosophila larva can be a general model to follow in vivo the innate host immune responses triggered during infection by human pathogens. IMPORTANCE The pathogenicity of methicillin-resistant S. aureus (MRSA) strains relies on their ability to produce a wide variety of tightly regulated virulence factors. Current in vivo models to analyze host-pathogen interactions are limited and difficult to manipulate. Here, we have established a robust and reliable model of oral S. aureus infection using Drosophila melanogaster larvae. We show that S. aureus stimulates host immunity through the production of reactive oxygen species (ROS) and antimicrobial peptide (AMP) and that ROS potentialize AMP gene expression. S. aureus catalase plays a key role in this complex environment and acts in vivo independently from SigB and Agr control. We propose that fly larvae can provide a general model for studying the colonization capabilities of human pathogens.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Staphylococcus aureus Resistente à Meticilina/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Espécies Reativas de Oxigênio/imunologia , Animais , Modelos Animais de Doenças , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Regulação Bacteriana da Expressão Gênica , Larva/imunologia , Larva/microbiologia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , VirulênciaRESUMO
Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell-independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice were acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 was expressed in neutrophils and activated by IL-12 via a JAK2-dependent pathway. We demonstrate that STAT4 was required for multiple neutrophil functions, including IL-12-induced ROS production, chemotaxis, and production of the neutrophil extracellular traps. Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 resulted in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.
Assuntos
Staphylococcus aureus Resistente à Meticilina/imunologia , Neutrófilos/imunologia , Fator de Transcrição STAT4/metabolismo , Infecções Estafilocócicas/imunologia , Animais , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interleucina-12/metabolismo , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Fator de Transcrição STAT4/genética , Infecções Estafilocócicas/microbiologiaRESUMO
Neddylation, a posttranslational modification in which NEDD8 is covalently attached to target proteins, has emerged as an endogenous regulator of innate immunity. However, the role of neddylation in methicillin-resistant Staphylococcus aureus (MRSA) infection remains unknown. In this study, we found that neddylation was activated after MRSA infection in vivo and in vitro. Inhibition of neddylation with MLN4924 promoted injury of liver and kidneys in C57BL/6 mice with MRSA bloodstream infection and increased mortality. Blockade of neddylation, either pharmacologically (MLN4924, DI591) or through the use of Uba3 small interfering RNA, inhibited Cullin3 neddylation and promoted Nrf2 accumulation, thus reducing reactive oxygen species (ROS) induction and bacterial killing ability in mouse peritoneal macrophages. In summary, our findings suggest that activation of neddylation in macrophages plays a critical protective role against MRSA infection by increasing ROS production, partially by signaling through the NEDD8-Cullin3-Nrf2-ROS axis. Furthermore, our results may provide a new non-antibiotic treatment strategy for MRSA infection through targeting of neddylation.
Assuntos
Macrófagos/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Espécies Reativas de Oxigênio/imunologia , Infecções Estafilocócicas/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3RESUMO
Activation of the endoplasmic reticulum stress sensor, IRE1α, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases in which neutrophils are a key immune component. However, the specific role of IRE1α in regulating neutrophil effector function has not been studied. In this study, we show that infection-induced IRE1α activation licenses neutrophil antimicrobial capacity, including IL-1ß production, formation of neutrophil extracellular traps (NETs), and methicillin-resistant Staphylococcus aureus (MRSA) killing. Inhibition of IRE1α diminished production of mitochondrial reactive oxygen species and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in CASPASE-2 or neutrophil IRE1α were highly susceptible to MRSA infection and failed to effectively form NETs in the s.c. abscess. IRE1α activation enhanced calcium influx and citrullination of histone H3 independently of mitochondrial reactive oxygen species production, suggesting that IRE1α coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1α-CASPASE-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1α in neutrophil antibacterial function.
Assuntos
Endorribonucleases/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Neutrófilos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Voluntários Saudáveis , Humanos , Interleucina-1beta/biossíntese , Camundongos , Transdução de Sinais/imunologiaRESUMO
Worldwide the increase in multi-resistant bacteria due to misuse of traditional antibiotics is a growing threat for our health. Finding alternatives to traditional antibiotics is thus timely. Probiotic bacteria have numerous beneficial effects and could offer safer alternatives to traditional antibiotics. Here, we use the nematode Caenorhabditis elegans (C. elegans) to screen a library of different lactobacilli to identify potential probiotic bacteria and characterize their mechanisms of action. We show that pretreatment with the Lactobacillus spp. Lb21 increases lifespan of C. elegans and results in resistance towards pathogenic methicillin-resistant Staphylococcus aureus (MRSA). Using genetic analysis, we find that Lb21-mediated MRSA resistance is dependent on the DBL-1 ligand of the TGF-ß signaling pathway in C. elegans. This response is evolutionarily conserved as we find that Lb21 also induces the TGF-ß pathway in porcine epithelial cells. We further characterize the host responses in an unbiased proteome analysis and identify 474 proteins regulated in worms fed Lb21 compared to control food. These include fatty acid CoA synthetase ACS-22, aspartic protease ASP-6 and vitellogenin VIT-2 which are important for Lb21-mediated MRSA resistance. Thus, Lb21 exerts its probiotic effect on C. elegans in a multifactorial manner. In summary, our study establishes a mechanistic basis for the antimicrobial potential of lactobacilli.
Assuntos
Doenças dos Animais/metabolismo , Doenças dos Animais/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Resistência à Doença , Staphylococcus aureus Resistente à Meticilina , Neuropeptídeos/metabolismo , Probióticos , Infecções Estafilocócicas/veterinária , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Resistência à Doença/imunologia , Interações entre Hospedeiro e Microrganismos , Interações Hospedeiro-Patógeno , Ligantes , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Probióticos/administração & dosagem , Transdução de SinaisRESUMO
Staphylococcus aureus (SA), especially the methicillin-resistant variant (MRSA), is becoming a serious threat to human health in hospitals and communities, making the development of an effective vaccine urgent. Alpha-hemolysin (Hla) is a key virulence factor and also a good target for the development of SA vaccines. However, the epitopes in Hla recognized by human immunity are not characterized in detail, which hinders the design of epitope-based human vaccines against SA. In this study, we collected sera from volunteers in a phase 1b clinical trial of a novel recombinant five-antigen SA vaccine (NCT03966040). Using a Luminex-based assay, we characterized the human serologic response against Hla, and identified Hla121-138 as a neutralizing epitope. In addition, we successfully produced ferritin nanoparticles carrying the neutralizing Hla121-138 epitope (EpNP) in E. coli. EpNP presented as homogenous nanoparticles in aqueous solution. Immunization with EpNP elicited potent hemolysis-neutralizing antibodies and conferred significant protection in a mouse model of SA skin infection. Our data suggest that EpNP, carrying the neutralizing epitope Hla121-138, is a good candidate for a vaccine against SA.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Estafilocócicas/prevenção & controle , Animais , Toxinas Bacterianas/antagonistas & inibidores , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Feminino , Proteínas Hemolisinas/antagonistas & inibidores , Humanos , Nanopartículas Metálicas/química , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/microbiologia , Vacinação , Fatores de VirulênciaRESUMO
The skin innate immune response to methicillin-resistant Staphylococcus aureus (MRSA) culminates in the formation of an abscess to prevent bacterial spread and tissue damage. Pathogen recognition receptors (PRRs) dictate the balance between microbial control and injury. Therefore, intracellular brakes are of fundamental importance to tune the appropriate host defense while inducing resolution. The intracellular inhibitor suppressor of cytokine signaling 1 (SOCS-1), a known JAK/STAT inhibitor, prevents the expression and actions of PRR adaptors and downstream effectors. Whether SOCS-1 is a molecular component of skin host defense remains to be determined. We hypothesized that SOCS-1 decreases type I interferon production and IFNAR-mediated antimicrobial effector functions, limiting the inflammatory response during skin infection. Our data show that MRSA skin infection enhances SOCS-1 expression, and both SOCS-1 inhibitor peptide-treated and myeloid-specific SOCS-1 deficient mice display decreased lesion size, bacterial loads, and increased abscess thickness when compared to wild-type mice treated with the scrambled peptide control. SOCS-1 deletion/inhibition increases phagocytosis and bacterial killing, dependent on nitric oxide release. SOCS-1 inhibition also increases the levels of type I and type II interferon levels in vivo. IFNAR deletion and antibody blockage abolished the beneficial effects of SOCS-1 inhibition in vivo. Notably, we unveiled that hyperglycemia triggers aberrant SOCS-1 expression that correlates with decreased overall IFN signatures in the infected skin. SOCS-1 inhibition restores skin host defense in the highly susceptible hyperglycemic mice. Overall, these data demonstrate a role for SOCS-1-mediated type I interferon actions in host defense and inflammation during MRSA skin infection.
Assuntos
Interferon Tipo I/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Proteína 1 Supressora da Sinalização de Citocina/imunologia , Animais , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pele/imunologia , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologia , Proteína 1 Supressora da Sinalização de Citocina/metabolismoRESUMO
Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of ß-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that ß-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.
Assuntos
Antibacterianos/farmacologia , Cefoxitina/farmacologia , Células Dendríticas/imunologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Infecções Estafilocócicas/metabolismo , Animais , Células da Medula Óssea , Interleucina-12/biossíntese , Interleucina-23/biossíntese , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Transdução de Sinais/fisiologia , Infecções Estafilocócicas/imunologiaRESUMO
Methicillin-resistant Staphylococcus aureus (SA) bacteremia is responsible for over 10,000 deaths in the hospital setting each year. Both conventional CD4+ T cells and γδ T cells play protective roles in SA infection through secretion of IFN-γ and IL-17. However, the role of other unconventional T cells in SA infection is largely unknown. Natural killer T (NKT) cells, a subset of innate-like T cells, are activated rapidly in response to a wide range of self and microbial lipid antigens presented by MHC I-like molecule CD1d. NKT cells are divided into two groups, invariant NKT (iNKT) and type II NKT cells, based on TCR usage. Using mice lacking either iNKT cells or both types of NKT cells, we show that both NKT cell subsets are activated after systemic SA infection and produce IFN-γ in response to SA antigen, however type II NKT cells are sufficient to control bacterial burden and inflammatory infiltrate in infected organs. This protective capacity was specific for NKT cells, as mice lacking mucosal associated invariant T (MAIT) cells, another innate-like T cell subset, had no increased susceptibility to SA systemic infection. We identify polar lipid species from SA that induce IFN-γ production from type II NKT cells, which requires both CD1d-TCR engagement and IL-12 production by antigen presenting cells. We also demonstrate that a population of T cells enriched for type II NKT cells are increased in PBMC of SA bacteremic patients compared to healthy controls. Therefore, type II NKT cells perform effector functions that enhance control of SA infection prior to conventional T cell activation and recognize SA-derived lipid antigens. As CD1d is highly conserved in humans, these CD1d-restricted SA lipid antigens could be used in the design of next generation SA vaccines targeting cell-mediated immunity.
Assuntos
Imunidade Celular , Staphylococcus aureus Resistente à Meticilina/imunologia , Células T Matadoras Naturais/imunologia , Infecções Estafilocócicas/imunologia , Transferência Adotiva , Adulto , Idoso , Animais , Antígenos CD1d/metabolismo , Carga Bacteriana , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/microbiologia , Células T Matadoras Naturais/transplante , Fenótipo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controleRESUMO
The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague-Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.