RESUMO
Legionella species are Gram-negative intracellular bacteria that evolved in soil and freshwater environments, where they infect and replicate within various unicellular protozoa. The primary virulence factor of Legionella is the expression of a type IV secretion system (T4SS), which contributes to the translocation of effector proteins that subvert biological processes of the host cells. Because of its evolution in unicellular organisms, T4SS effector proteins are not adapted to subvert specific mammalian signaling pathways and immunity. Consequently, Legionella pneumophila has emerged as an interesting infection model for investigating immune responses against pathogenic bacteria in multicellular organisms. This review highlights recent advances in our understanding of mammalian innate immunity derived from studies involving L. pneumophila. This includes recent insights into inflammasome-mediated mechanisms restricting bacterial replication in macrophages, mechanisms inducing cell death in response to infection, induction of effector-triggered immunity, activation of specific pulmonary cell types in mammalian lungs, and the protective role of recruiting monocyte-derived cells to infected lungs.
Assuntos
Imunidade Inata , Legionella pneumophila , Doença dos Legionários , Legionella pneumophila/imunologia , Legionella pneumophila/patogenicidade , Humanos , Animais , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Fagócitos/imunologia , Fagócitos/microbiologia , Sistemas de Secreção Tipo IV/imunologia , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Monócitos/imunologia , Monócitos/microbiologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Interações Hospedeiro-Patógeno/imunologiaRESUMO
BACKGROUND: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo. METHODS: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR. RESULTS: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05). CONCLUSION: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling.
Assuntos
Glucose , Legionella pneumophila , Macrófagos , Proteína Adaptadora de Sinalização NOD1 , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Animais , Humanos , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Legionella pneumophila/imunologia , Glucose/metabolismo , Cobaias , Masculino , Interleucina-6/metabolismo , Doença dos Legionários/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células U937 , Fator de Necrose Tumoral alfa/metabolismo , CamundongosRESUMO
Legionella pneumophila is an intracellular pathogen that can cause severe pneumonia after the inhalation of contaminated aerosols and replication in alveolar macrophages. Several pattern recognition receptors (PRRs) have been identified that contribute to the recognition of L. pneumophila by the innate immune system. However, the function of the C-type lectin receptors (CLRs), which are mainly expressed by macrophages and other myeloid cells, remains largely unexplored. Here, we used a library of CLR-Fc fusion proteins to search for CLRs that can bind the bacterium and identified the specific binding of CLEC12A to L. pneumophila. Subsequent infection experiments in human and murine macrophages, however, did not provide evidence for a substantial role of CLEC12A in controlling innate immune responses to the bacterium. Consistently, antibacterial and inflammatory responses to Legionella lung infection were not significantly influenced by CLEC12A deficiency. Collectively, CLEC12A is able to bind to L. pneumophila-derived ligands but does not appear to play a major role in the innate defense against L. pneumophila.
Assuntos
Interações Hospedeiro-Patógeno , Imunidade Inata , Lectinas Tipo C , Legionella pneumophila , Doença dos Legionários , Receptores Mitogênicos , Animais , Humanos , Camundongos , Lectinas Tipo C/metabolismo , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Receptores Mitogênicos/imunologiaRESUMO
Flagellin-induced NAIP/NLRC4 inflammasome activation and pyroptosis are critical events restricting Legionella pneumophila infection. However, the cellular and molecular dynamics of the in vivo responses against this bacterium are still unclear. We have found temporal coordination of two independent innate immunity pathways in controlling Legionella infection, the inflammasome activation and the CCR2-mediated Mo-DC recruitment. Inflammasome activation was an important player at the early stage of infection by lowering the numbers of bacteria for an efficient bacterial clearance conferred by the Mo-DC at the late stage of the infection. Mo-DC emergence highly depended on CCR2-signaling and dispensed inflammasome activation and pyroptosis. Also, Mo-DC compartment did not rely on the inflammasome machinery to deliver proper immune responses and was the most abundant cytokine-producing among the monocyte-derived cells in the infected lung. Importantly, when the CCR2- and NLRC4-dependent axes of response were simultaneously ablated, we observed an aggravated bacterial burden in the lung of infected mice. Taken together, we showed that inflammasome activation and CCR2-mediated immune response interplay in distinct pathways to restrict pulmonary bacterial infection. These findings extend our understanding of the in vivo integration and cooperation of different innate immunity arms in controlling infectious agents.
Assuntos
Células Dendríticas , Inflamassomos , Legionella pneumophila , Doença dos Legionários , Monócitos , Animais , Camundongos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Legionella pneumophila/imunologia , Doença dos Legionários/genética , Doença dos Legionários/imunologia , Macrófagos , Camundongos Knockout , Monócitos/metabolismo , Receptores CCR2/metabolismoRESUMO
Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-ß. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts.
Assuntos
Eucariotos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Legionella pneumophila/metabolismo , Doença dos Legionários/imunologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , Eucariotos/genética , Vesículas Extracelulares , Humanos , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1 , Doença dos Legionários/microbiologia , Receptores Imunológicos , Transdução de SinaisRESUMO
BACKGROUND: Myeloid-derived suppressor cells (MDSCs) and cyclooxy-genase-2 (COX-2)/Prostaglandin E2 (PGE2) axis are important contributors to sepsis-induced immune-suppression. The purpose of present study is to explore whether COX-2 inhibitor can improve immunological disorder after sepsis via regulating MDSCs. METHODS: A ''two-hit'' model reflecting clinical sepsis development was performed. Cecal ligation and puncture (CLP) and Legionella pneumophila infection were used as the first and the second hit, respectively. NS398, a selective COX-2 inhibitor, was utilized to treat septic mice. The motality, bacterial counts in the lung, systematic inflammatory reaction and CD4 + T cells response after sepsis were assessed, so as the frequency and function of MDSCs. In some experiments, the number of MDSCs was manipulated by adoptive transfer or neutralizing antibody before induction of secondary infection. RESULTS: Mice surviving CLP showed a marked expansion and activation of MDSCs in spleen, accompanied by suppressed proliferating capability, impaired secreting functionand increased apoptosis of CD4 + T cells. Majority of CLP survivors became succumbed to L. pneumophila invasion, associated with defective bacteria elimination ability. NS398 treatment was found to ameliorate these adverse outcomes significantly. CONCLUSION: MDSCs contribute greatly to the sepsis-induced immune dysfunction. Inhibiting COX-2 may become a promising therapy that targets MDSCs-induced immunosuppression.
Assuntos
Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Doença dos Legionários/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Nitrobenzenos/uso terapêutico , Sepse/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/imunologia , Ceco/cirurgia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Citocinas/sangue , Modelos Animais de Doenças , Hipersensibilidade Tardia , Tolerância Imunológica/efeitos dos fármacos , Legionella pneumophila , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/imunologia , Nitrobenzenos/farmacologia , Sepse/imunologia , Sepse/microbiologia , Baço/citologia , Baço/imunologia , Sulfonamidas/farmacologiaRESUMO
Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.
Assuntos
Proteínas do Sistema Complemento/imunologia , Legionella pneumophila/genética , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Acetiltransferases/genética , Acetiltransferases/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Feminino , Genoma Bacteriano , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/imunologia , Legionella pneumophila/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , FilogeniaRESUMO
Legionella pneumophila is an intracellular pathogen that can cause Legionnaire's disease by invading alveolar epithelial cells and macrophages. The major outer membrane protein (MOMP) plays an important role in the interaction between bacteria and host cells. However, the role of MOMP in the process of L. pneumophila invasion of macrophages and its working mechanism remain unknown. We aimed to explore the effects of MOMP on phagocytosis and chemotaxis of RAW 264.7 macrophages. The chemotactic activity, toxicity, and phagocytosis of RAW 264.7 cocultured with different concentrations of MOMP were determined by Transwell, CCK-8, and neutral red uptake assays, respectively. Target genes were detected by double-luciferase and pull down assays. qRT-PCR and Western blot were performed to analyze the expression of several important proteins involved in the immune response pathway, including coronin-1, interleukins (IL-10), forkhead transcription factor 1 (FOXO1), nucleotide-binding oligomerization domain protein (NOD) 1, NOD2, and receptor-interacting protein (RIP) 2. After coculturing with MOMP, cytological observation indicated a decrease of phagocytosis and a marked increase of chemotaxis in RAW 264.7 macrophages. The phagocytosis degree of RAW 264.7 macrophage varied with the concentration gradient of MOMP in a time-dependent manner. MOMP could increase the expression levels of MCP-1, IL-10, NOD2, and RIP2 and decrease the expression levels of FOXO1 and coronin-1 in cell culture supernatants. In addition, we found that FOXO1 could promote its transcription by binding to the promoter of coronin-1. The results of the present study suggested that MOMP could inhibit phagocytosis and facilitate chemotaxis of RAW 264.7 macrophage, which might be associated with the FOXO1/coronin-1 axis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína Forkhead Box O1/metabolismo , Legionella pneumophila/fisiologia , Doença dos Legionários/imunologia , Macrófagos/imunologia , Proteínas dos Microfilamentos/metabolismo , Porinas/metabolismo , Animais , Proteínas de Bactérias/imunologia , Quimiocina CCL2/metabolismo , Quimiotaxia , Regulação da Expressão Gênica , Interleucina-10/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Fagocitose , Porinas/imunologia , Células RAW 264.7 , Transdução de SinaisRESUMO
Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.
Assuntos
Doença dos Legionários/imunologia , Macrófagos/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Humanos , Legionella pneumophila/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Moléculas com Motivos Associados a Patógenos/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Patients are susceptible to immunosuppression in late-stage of sepsis, in which myeloid-derived suppressor cells (MDSCs) is an important contributor. This study aims to investigate whether all-trans-retinoic acid (ATRA), which has been proved to inhibit MDSCs generation in cancer, will ameliorate sepsis-induced immuno-suppression through modulating MDSCs. METHODS: A clinically relevant "two-hit'' model of sepsis, the cecal ligation and puncture (CLP) model and secondary pneumonia model, were established in mice. The effects of ATRA on the mortality, the bacterial burden, the expansion and activity of CLP-induced MDSCs, as well as the function of CD4+ T cells were evaluated. RESULTS: In CLP model, ATRA was found to reduce frequency of MDSCs in spleen of mice and inhibit activity of MDSCs by regulating the generation and activity of arginase-1 and iNOS, and the secretion of immune-supressive cytokines. ATRA administration eventually reduced mortality of secondary infection by Legionella pneumophila in CLP-surviving mice, which might be associated with the restoration of CD4+ T cells proliferating and secreting activity. CONCLUSION: ATRA can restore CD4+ T cells dysfunction in sepsis by modulating the expansion and function of MDSCs and therefore provides a potential therapy that targets the immunosuppressive state of sepsis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Legionella pneumophila/imunologia , Células Supressoras Mieloides/imunologia , Sepse/imunologia , Tretinoína/farmacologia , Animais , Arginase/metabolismo , Citocinas/metabolismo , Doença dos Legionários/imunologia , Doença dos Legionários/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Sepse/microbiologia , Sepse/patologiaRESUMO
TOLLIP (Toll-interacting protein) is an intracellular adaptor protein with diverse actions throughout the body. In a context- and cell type-specific manner, TOLLIP can function as an inhibitor of inflammation and endoplasmic-reticulum stress, an activator of autophagy, or a critical regulator of intracellular vacuole trafficking. The distinct functions of this protein have been linked to innate immune responses and lung epithelial-cell apoptosis. TOLLIP genetic variants have been associated with a variety of chronic lung diseases, including idiopathic pulmonary fibrosis, asthma, and primary graft dysfunction after lung transplantation, and with infections, such as tuberculosis, Legionella pneumonia, and respiratory viruses. TOLLIP exists in a delicate homeostatic balance, with both positive and negative effects on the trajectory of pulmonary diseases. This translational review summarizes the genetic and molecular associations that link TOLLIP to the development and progression of noninfectious and infectious pulmonary diseases. We highlight current limitations of in vitro and in vivo models in assessing the role of TOLLIP in these conditions, and we describe future approaches that will enable a more nuanced exploration of the role of TOLLIP in pulmonary conditions. There has been a surge in recent research evaluating the role of this protein in human diseases, but critical mechanistic pathways require further exploration. By understanding its biologic functions in disease-specific contexts, we will be able to determine whether TOLLIP can be therapeutically modulated to treat pulmonary diseases.
Assuntos
Asma/genética , Rejeição de Enxerto/genética , Fibrose Pulmonar Idiopática/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Asma/imunologia , Asma/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Fibrose Pulmonar Idiopática/imunologia , Fibrose Pulmonar Idiopática/patologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Doença dos Legionários/genética , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Doença dos Legionários/patologia , Transplante de Pulmão , Camundongos , MicroRNAs/genética , MicroRNAs/imunologia , Infecções por Respirovirus/genética , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/patologia , Infecções por Respirovirus/virologia , Transdução de Sinais , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Legionella pneumophila, an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8+ T cells are directly associated with protection against L. pneumophila, with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila, they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8+ CTLs. Of the 13 PAL peptides tested, PAL92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL92-100-specific CD8+ T cells play a central role in the host defense response against L. pneumophila.
Assuntos
Proteínas da Membrana Bacteriana Externa/administração & dosagem , Vacinas Bacterianas/administração & dosagem , Epitopos , Legionella pneumophila/imunologia , Doença dos Legionários/prevenção & controle , Pulmão/imunologia , Fragmentos de Peptídeos/administração & dosagem , Proteoglicanas/administração & dosagem , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Imunização , Doença dos Legionários/imunologia , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Proteoglicanas/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/microbiologiaRESUMO
BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacterium and the cause of an atypical pneumonia in humans - legionnaire's disease. Immunological assessment of bacterial antigens clarifies the way that host may develop protection against the pathogen. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS in combination with immunogenic proteins and looked into the result of bacterial challenge. METHODS: LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) and either of recombinant FlaA or PAL separately. Afterwards, specific serum IgG was assessed by ELISA. Mice were challenged intravenously with sublethal dose of L. pneumpphila then splenocytes were cultured. Cytokine responses of splenocytes were analyzed by ELISA. RESULTS: Polysaccharide antigen did not elicit significant serum IgG. Combination of the dLPS with recombinant FlaA and PAL led to risen IgG and its subclasses (IgG1, IgG2a and IgG2b) against polysaccharide. Mice immunized with combination of the dLPS and recombinant proteins showed significant elevation of cytokine responses in splenocyte culture after being challenged with L. pneumophila. CONCLUSIONS: Our results suggest that combination of polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with Th-1 stimulating protein antigens. Although not covalently bond, Legionella detoxified LPS combination with recombinant FlaA and PAL effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.
Assuntos
Vacinas Bacterianas/imunologia , Legionella pneumophila , Doença dos Legionários/prevenção & controle , Linfócitos T/imunologia , Animais , Flagelina/genética , Imunidade Celular , Imunização , Doença dos Legionários/imunologia , Lipopolissacarídeos , Lipoproteínas , Camundongos , Camundongos Endogâmicos BALB C , Peptidoglicano , VacinaçãoRESUMO
Legionella pneumophila is a flagellated pathogenic bacterium that causes atypical pneumonia called Legionnaires' disease. The flagellum plays a key role in the pathogenesis of L. pneumophila in the host. The protein FlgL forms a junction between the flagellar hook and filament and has been reported to elicit the host humoral immune response. To provide structural insights into FlgL-mediated junction assembly and FlgL-based vaccine design, we performed structural and serological studies on L. pneumophila FlgL (lpFlgL). The crystal structure of a truncated lpFlgL protein that consists of the D1 and D2 domains was determined at 3.06 Å resolution. The D1 domain of lpFlgL adopts a primarily helical, rod-shaped structure, and the D2 domain folds into a ß-sandwich structure that is affixed to the upper region of the D1 domain. The D1 domain of lpFlgL exhibits structural similarity to the flagellar filament protein flagellin, allowing us to propose a structural model of the lpFlgL junction based on the polymeric structure of flagellin. Furthermore, the D1 domain of lpFlgL exhibited substantially higher protein stability than the D2 domain and was responsible for most of the antigenicity of lpFlgL, suggesting that the D1 domain of lpFlgL would be a suitable target for the development of an anti-L. pneumophila vaccine.
Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/química , Proteínas de Bactérias/imunologia , Cristalografia por Raios X , Humanos , Imunidade Humoral , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Legionella pneumophila is a Gram-negative intracellular bacterium and causes legionnaire's disease an -atypical pneumonia in humans. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS and O-antigen polysaccharide in combination with bovine serum albumin (BSA) and explored the immunological responses of mice to the bacterial infection. LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. O-polysaccharide antigen (OPS) obtained by acetic acid treatment of LPS. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) or OPS with BSA separately. Pure polysaccharide antigens did not elicit significant serum IgG against LPS. Combination of the dLPS and OPS with BSA resulted in risen IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide. Mice were challenged intravenously with sublethal dose of L. pneumpphila. Then, splenocytes were cultured and cytokine responses of splenocytes to pathogenic Legionella was studied by ELISA. Mice immunized with combination of the dLPS or OPS and BSA showed significant elevation of cytokine responses to pathogenic L. pneumophila. Our results suggest that combination of the polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with a protein antigen which is capable of eliciting cell-mediated responses. Although not covalently bond, Legionella polysaccharides combined with BSA effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.
Assuntos
Doença dos Legionários , Lipopolissacarídeos , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias , Doença dos Legionários/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Legionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays, Legionella species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of Legionella infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking.
Assuntos
Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Amoeba/genética , Amoeba/imunologia , Amoeba/patogenicidade , Células Eucarióticas/imunologia , Células Eucarióticas/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Legionella/classificação , Legionella/genética , Legionella/imunologia , Legionella pneumophila/genética , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Doença dos Legionários/patologiaRESUMO
Mucosal-associated invariant T (MAIT) cells are activated in a TCR-dependent manner by antigens derived from the riboflavin synthesis pathway, including 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), bound to MHC-related protein-1 (MR1). However, MAIT cell activation in vivo has not been studied in detail. Here, we have found and characterized additional molecular signals required for optimal activation and expansion of MAIT cells after pulmonary Legionella or Salmonella infection in mice. We show that either bone marrow-derived APCs or non-bone marrow-derived cells can activate MAIT cells in vivo, depending on the pathogen. Optimal MAIT cell activation in vivo requires signaling through the inducible T cell costimulator (ICOS), which is highly expressed on MAIT cells. Subsequent expansion and maintenance of MAIT-17/1-type responses are dependent on IL-23. Vaccination with IL-23 plus 5-OP-RU augments MAIT cell-mediated control of pulmonary Legionella infection. These findings reveal cellular and molecular targets for manipulating MAIT cell function under physiological conditions.
Assuntos
Antígenos de Bactérias/imunologia , Interleucina-23/imunologia , Legionella/imunologia , Doença dos Legionários/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , VacinaçãoRESUMO
Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires' disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip-/- mice demonstrated significantly fewer bacterial colony-forming unit and increased cytokine responses from BAL fluid. Tollip-/- macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16-0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Pulmão/metabolismo , Adulto , Idoso , Animais , Carga Bacteriana , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Legionella pneumophila/crescimento & desenvolvimento , Legionella pneumophila/imunologia , Doença dos Legionários/genética , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Transdução de SinaisRESUMO
Introducción: Legionella pneumophila se sitúa entre los principales agentes causales de neumonía adquirida en la comunidad y de origen nosocomial. La inhalación de aerosoles potencialmente contaminados con la bacteria, producto de la colonización de redes y otros sistemas que utilizan agua, representa un peligro para la salud de los individuos expuestos. Objetivo: evaluar la viabilidad de L. pneumophila en muestras de agua almacenadas en diferentes intervalos de tiempo para el diagnóstico por cultivo microbiológico de Legionella spp. Métodos: Se contaminaron artificialmente muestras de agua con dos cepas de L. pneumophila de serogrupos diferentes y la conformación de una mezcla de ellas, para un total de 15 muestras. Los frascos contaminados fueron procesados a las 24 h, 72 h, 7 días, 14 días y 21 días. Se realizó cultivo microbiológico según ISO 11731: 2004 y PNO 03-013: 2015. Resultados: Se demostró viabilidad de la bacteria en muestras almacenadas hasta 21 días. El método de concentración por filtración resultó tener los mayores recobrados del microorganismo. Conclusiones: El tiempo de almacenamiento de las muestras afecta la viabilidad de L. pneumophila. Sienta las bases para estudios posteriores de robustez del diagnóstico de L. pneumophila como parte del servicio que presta el Centro de Investigaciones Científicas de la Defensa Civil en los programas de prevención y control Legionella spp. en instalaciones de interés turístico e industrial(AU)
Introduction: Legionella pneumophila is one of the main causative agents of community- and hospital-acquired pneumonia. Inhalation of sprays potentially contaminated with the bacterium, due to the colonization of networks and other systems using water, is a hazard to the health of exposed individuals. Objective: Evaluate the viability of L. pneumophila in samples of water stored at various time intervals for the microbiological culture diagnosis of Legionella spp. Methods: Water samples were artificially contaminated with two strains of L. pneumophila from different serogroups and a mixture of them, for a total of 15 samples. The contaminated vessels were processed at 24 h, 72 h, 7 d, 14 d and 21 d. Microbiological culture was performed in compliance with ISO 11731: 2004 and PNO 03-013: 2015. Results: The bacterium was found to be viable in samples stored up to 21 days. The filtration concentration method obtained the greatest amount of the microorganism. Conclusions: Storage time of the samples affects the viability of L. pneumophila. The study lays the foundations for further research about the validity of L. pneumophila diagnosis as part of the service offered by the Civil Defense Scientific Research Center in Legionella spp. prevention and control programs for tourist and industrial facilities(AU)
Assuntos
Humanos , Doença dos Legionários/imunologia , Amostras de Água , Viabilidade Microbiana/imunologia , Pneumonia/microbiologia , ComunicaçãoRESUMO
BACKGROUND: Legionella can cause Legionnaires' disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100. METHODS: We evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs. RESULTS: We found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death. CONCLUSIONS: These observations suggest a mechanism of Legionnaires' disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.