RESUMO
In Mycobacterium avium infections, macrophages play a critical role in the host defense response. Apoptosis inhibitor of macrophage (AIM), also known as CD5L, may represent a novel supportive therapy against various diseases, including metabolic syndrome and infectious diseases. The mechanisms of AIM include modulating lipid metabolism in macrophages and other host cells. We investigated the role of AIM in M. avium infections in vitro and in vivo. In a mouse model of M. avium pneumonia, foamy macrophages were induced 6 wk after infection. The bacteria localized in these macrophages. Flow cytometric analysis also confirmed that the percentage of CD11chighMHCclassIIhigh interstitial and alveolar macrophages, a cell surface marker defined as foamy macrophages, increased significantly after infection. AIM in alveolar lavage fluid and serum gradually increased after infection. Administration of recombinant AIM significantly increased the number of bacteria in the lungs of mice, accompanied by the induction of inflammatory cytokine and iNOS expression. In mouse bone marrow-derived macrophages, the mRNA expression of AIM after M. avium infection and the amount of AIM in the supernatant increased prior to the increase in intracellular bacteria. Infected cells treated with anti-AIM Abs had fewer bacteria and a higher percentage of apoptosis-positive cells than infected cells treated with isotype control Abs. Finally, AIM in the sera of patients with M. avium-pulmonary disease was measured and was significantly higher than in healthy volunteers. This suggests that AIM production is enhanced in M. avium-infected macrophages, increasing macrophage resistance to apoptosis and providing a possible site for bacterial growth.
Assuntos
Infecção por Mycobacterium avium-intracellulare , Mycobacterium avium , Camundongos , Animais , Macrófagos/fisiologia , Infecção por Mycobacterium avium-intracellulare/complicações , Infecção por Mycobacterium avium-intracellulare/microbiologia , Macrófagos Alveolares/microbiologia , ApoptoseRESUMO
BACKGROUND: Mycobacterium avium is associated with pulmonary disease in otherwise healthy adults. Several clarithromycin-refractory cases have been reported, including some cases caused by clarithromycin-susceptible strains. OBJECTIVES: To characterize the reason for the discrepancy between clinical response and antibiotic susceptibility results. METHODS: We conducted population analysis of clarithromycin-tolerant and heteroresistant subpopulations of M. avium cultured in vitro and in homogenates of infected lungs of mice. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for 28 M. avium and two M. kansasii strains. Mice were intranasally infected with M. avium and treated with or without clarithromycin (100 mg/kg) thrice weekly. They were sacrificed on day 35 and the bacteria in lung homogenates were tested for clarithromycin resistance. Population analysis assays were performed based on colony growth on plates containing two-fold dilutions of clarithromycin. RESULTS: The MBC/MIC ratios were ≥8 in all 28 strains of M. avium tested. In the population analysis assay, several colonies were observed on the plates containing clarithromycin concentrations above the MIC (2-64 mg/L). No growth of M. kansasii colonies was observed on the plates containing clarithromycin concentrations ≥2 mg/L. M. avium in the homogenates of infected lungs showed clearer clarithromycin-resistant subpopulations than in vitro, regardless of clarithromycin exposure. CONCLUSION: M. avium shows intrinsic heterogeneous resistance (heteroresistance) to clarithromycin. This may explain the observed discrepancies between clarithromycin susceptibility testing results and clinical response to clarithromycin treatment. Further studies are needed to confirm a link between heteroresistance and clinical outcomes.
Assuntos
Claritromicina , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Mycobacterium avium , Claritromicina/farmacologia , Claritromicina/uso terapêutico , Animais , Camundongos , Mycobacterium avium/efeitos dos fármacos , Pulmão/microbiologia , Feminino , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , HumanosRESUMO
Telomere length is thought to be a biomarker of biological aging. This study examined whether telomere length was associated with urinary concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative stress, and antioxidative trace elements in 73 female Japanese university students (age: 19.2 ± 0.7 years). We quantified 8-OHdG and selenium in urine by liquid chromatography-mass spectrometry and inductively coupled plasma-mass spectrometry, respectively. Telomere length and urinary concentrations of other essential trace elements (molybdenum, cobalt, and chromium) that were previously measured in the same study participants, were used in this study. We used multiple linear regression analysis to examine the associations of telomere length with urinary 8-OHdG and essential trace element concentrations (covariates: urinary cotinine concentration, age, BMI, and drinking status). The geometric means (geometric standard deviation) of 8-OHdG and selenium were 3.4 (1.5) and 31 (1.3) µg/g creatinine, respectively. Telomere length was not associated with urinary 8-OHdG concentration, but was negatively associated with urinary selenium concentration. In conclusion, telomere length was not associated with urinary 8-OHdG concentration in the young women in this study. Longitudinal studies should be conducted to clarify the association between telomere shortening rate and oxidative stress level.
Assuntos
Oligoelementos , 8-Hidroxi-2'-Desoxiguanosina , Adolescente , Adulto , Biomarcadores , Desoxiguanosina , Feminino , Humanos , Japão , Estresse Oxidativo , Estudantes , Telômero , Universidades , Adulto JovemRESUMO
In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived macrophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macrophages, which may contribute to improved survival in L. pneumophila pneumonia.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Legionella pneumophila/efeitos dos fármacos , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Metformina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Legionella pneumophila/imunologia , Doença dos Legionários/genética , Doença dos Legionários/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , FosforilaçãoRESUMO
In light of the increasing number of clinical cases resistant to traditional monotherapies and the lack of novel antimicrobial agents, combination therapy is an appealing solution for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the efficacy of anti-MRSA agents, such as vancomycin (VAN), daptomycin (DAP), and linezolid (LZD), in conjunction with 13 beta-lactams and non-beta-lactams. We assessed the in vitro activities of the various combinations against 40 MRSA strains based on the maximum synergistic effect (MSE), an index calculated from the MIC change with a combination agent. Nearly all the anti-MRSA agents, which were combined with beta-lactams as well as VAN and DAP, showed a synergistic effect with arbekacin. VAN also exhibited varying degrees of synergy depending on the type of beta-lactam, whereas DAP and LZD showed similar synergy with different beta-lactams. These effects were confirmed by antibiotic kill curves, except for the apparent interaction between LZD and beta-lactams. The MSE results were analyzed according to strain characteristics including susceptibility to combination agents, staphylococcal cassette chromosome mec type, and presence of the blaZ gene; however, no obvious correlations were observed. In a fluorescence binding assay, the fluorescence intensity of boron-dipyrromethene (BODIPY)-VAN decreased, whereas that of BODIPY-DAP increased in combination with a beta-lactam agent. These findings suggest that beta-lactam combinations are promising treatment options for MRSA infections and that the type of beta-lactam combined with VAN is important for the synergistic effect.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamas/farmacologia , Antibacterianos/uso terapêutico , Daptomicina/farmacologia , Daptomicina/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Humanos , Linezolida/farmacologia , Linezolida/uso terapêutico , Resistência a Meticilina/efeitos dos fármacos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Vancomicina/farmacologia , Vancomicina/uso terapêutico , beta-Lactamas/uso terapêuticoRESUMO
Community-acquired pneumonia is a common disease with considerable morbidity and mortality, for which Streptococcus pneumoniae is accepted as a leading cause. Although ß-lactam-plus-macrolide combination therapy for this disease is recommended in several guidelines, the clinical efficacy of this strategy against pneumococcal pneumonia remains controversial. In this study, we examined the effects of ß-lactam-plus-macrolide combination therapy on lethal mouse pneumococcal pneumonia and explored the mechanisms of action in vitro and in vivo We investigated survival, lung bacterial burden, and cellular host responses in bronchoalveolar lavage fluids obtained from mice infected with pneumonia and treated with ceftriaxone, azithromycin, or both in combination. Although in vitro synergy was not observed, significant survival benefits were demonstrated with combination treatment. Lung neutrophil influx was significantly lower in the ceftriaxone-plus-azithromycin-treated group than in the ceftriaxone-treated group, whereas no differences in the lung bacterial burden were observed on day 3 between the ceftriaxone-plus-azithromycin-treated group and the ceftriaxone-treated group. Notably, the analysis of cell surface markers in the ceftriaxone-plus-azithromycin combination group exhibited upregulation of presumed immune checkpoint ligand CD86 and major histocompatibility complex class II in neutrophils and CD11b-positive CD11c-positive (CD11b(+) CD11c(+)) macrophages and dendritic cells, as well as downregulation of immune checkpoint receptors cytotoxic-T lymphocyte-associated antigen 4 and programmed death 1 in T helper and T regulatory cells. Our data demonstrate that the survival benefits of ceftriaxone-plus-azithromycin therapy occur through modulation of immune checkpoints in mouse pneumococcal pneumonia. In addition, immune checkpoint molecules may be a novel target class for future macrolide research.
Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Pneumonia Pneumocócica/tratamento farmacológico , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Carga Bacteriana/efeitos dos fármacos , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Infecções Comunitárias Adquiridas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos CBA , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/mortalidade , Pneumonia Pneumocócica/patologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/patogenicidade , Análise de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α(+) DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.
Assuntos
Antígenos/metabolismo , Citosol/metabolismo , Células Dendríticas/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Animais , Transporte Biológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Heat shock protein 90 (HSP90) is a molecular chaperone required for efficient antigen presentation and cross-presentation. In addition, HSP90 was recently reported to interact with and stabilize the activation-induced cytidine deaminase (AID) and plays a critical role in immunoglobulin gene hypermutation and class switch recombination. In mice and humans, there are two HSP90 isoforms, HSP90α and HSP90ß, but the in vivo role of each isoform remains largely unknown. Here we have analyzed humoral immune responses in HSP90α-deficient mice. We found that HSP90α deficiency did not affect AID protein expression. B cell development and maturation, as well as immunoglobulin gene hypermuation and class switch, occurred normally in HSP90α-deficient mice. However, antibody production to a T-dependent antigen was elevated in the mutant mice and this was associated with enhanced MHC class II antigen presentation to T helper cells by dendritic cells. Our results reveal a previously unidentified inhibitory role for HSP90α isoform in MHC class II antigen presentation and the humoral immune response. Along with our recent finding that HSP90α is required for antigen cross-presentation, these results suggest that HSP90α controls the balance of humoral and cellular immunity by dictating the fate of presentation of exogenous antigen.
Assuntos
Linfócitos B/imunologia , Proteínas de Choque Térmico HSP90/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apresentação de Antígeno/genética , Antígenos/imunologia , Antígenos/metabolismo , Citidina Desaminase/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Hibridomas , Imunidade Humoral/genética , Switching de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
Priming of CD8(+) T cells requires two signals, one produced by T-cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non-covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen-presenting cells (APC) to be presented by MHC I molecules to CD8(+) T cells, thus, signal 1 has been well characterized in this pathway of cross-presentation. Signal 2 is not fully understood, although there are reports that Toll-like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross-priming by a mycobacterial HSP70-antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP-mediated cross-priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross-priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4-dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden.
Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , Neoplasias/imunologia , Ovalbumina/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/imunologia , Receptores Toll-Like/imunologiaRESUMO
Extracellular HSP90 associated with Ag peptides have been demonstrated to efficiently cross-prime T cells, following internalization by dendritic cells (DCs). In addition, the nature of cell-associated Ags required for cross-priming is implicated as peptides and proteins chaperoned by heat shock protein (HSP). However, the role of endogenous HSP in DCs during cross-presentation remains elusive. In this paper, we show that endogenous HSP90 is essential for cross-presentation of both soluble and cell-associated Ags in DCs. Cross-presentation of soluble OVA and OVA-loaded transporter associated with Ag processing-1-deficient cells by bone marrow-derived DCs and DC-like cell line DC2.4 was profoundly blocked by HSP90 inhibitors, whereas presentation of endogenously expressed OVA was only partially suppressed. Assays using small interfering RNA and heat shock factor-1-deficient DCs (with defective expression of HSP90alpha) revealed the pivotal role of HSP90alpha in cross-presentation. The results suggest that in addition to HSP90 in Ag donor cells, endogenous HSP90 in DCs plays an essential role during Ag cross-presentation and, moreover, points to a link between heat shock factor-1-dependent induction of HSP90alpha within DC and cytotoxic T cell immunity.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Animais , Apresentação de Antígeno/genética , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Apresentação Cruzada/genética , Proteínas de Choque Térmico HSP90/deficiência , Proteínas de Choque Térmico HSP90/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais/imunologia , SolubilidadeRESUMO
Introduction. Clostridioides difficile infection (CDI) causes toxin-mediated enteropathy, such as antibiotic-associated diarrhoea and pseudomembranous colitis. Rho-glucosylating toxin A (TcdA) and toxin B (TcdB) have been clearly implicated in pathogenesis, whereas the virulence of binary toxin (CDT) is still debated.Hypothesis statement. We hypothesized that CDT is involved in the host immune response and plays a pivotal role in establishing virulence by modulating pro-inflammatory cytokine production; this is achieved through the integral Toll-like receptor (TLR) signalling pathways.Aim. The aim of the present study was to determine whether and how CDT impacts macrophages compared to TcdA or TcdB by examining the induction of CXC chemokine ligand 2 (CXCL2) and tumour necrosis factor-α (TNF-α), both of which are crucial in mediating local and systematic inflammatory responses.Methodology. RAW264.7 cells or transfected human embryonic kidney (HEK) 293 T cells were incubated with TcdA, TcdB, or CDT. In some experiments, a neutralizing antibody against TLR2 or TLR4, or myeloid differentiation 88 inhibitory peptide were added. The amount of CXCL2 and TNF-α secreted was then measured.Results. In RAW264.7 macrophages, CXCL2 and TNF-α were produced via the Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) pathway in a TcdA, TcdB, or CDT dose-dependent manner. Interleukin-8 secretion was induced in TLR4/MD2/CD14-transfected, but not in TLR2-transfected, HEK 293 T cells following TcdB or CDT exposure.Conclusion. Our results showed that C. difficile toxins, including CDT, enhanced macrophage-mediated CXCL2 and TNF-α production via TLR2 and TLR4, indicating that CDT affects host immune responses.
Assuntos
Toxinas Bacterianas/farmacologia , Quimiocina CXCL2/metabolismo , Clostridioides difficile/patogenicidade , Macrófagos/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , VirulênciaRESUMO
In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.
Assuntos
Bacteriemia , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos , Biofilmes , Cromossomos , Humanos , Incidência , Japão/epidemiologia , Staphylococcus aureus Resistente à Meticilina/genética , Plasma , Infecções Estafilocócicas/epidemiologiaRESUMO
Mycobacterium avium complex is a causative organism for refractory diseases. In this study, we examined the effects of N-acetyl-cysteine on M. avium infection in vitro and in vivo. N-acetyl-cysteine treatment suppressed the growth of M. avium in A549 cells in a concentration-dependent manner. This effect was related to the induction of the antibacterial peptide human ß-defensin-2. In a mouse model, N-acetyl-cysteine treatment significantly reduced the number of bacteria in the lungs and induced murine ß-defensin-3. In interleukin-17-deficient mice, the effects of N-acetyl-cysteine disappeared, indicating that these mechanisms may be mediated by interleukin-17. Moreover, an additional reduction in bacterial load was observed in mice administered N-acetyl-cysteine in combination with clarithromycin. Our findings demonstrate the potent antimycobacterial effects of N-acetyl-cysteine against M. avium by inducing antimicrobial peptide, suggesting that N-acetyl-cysteine may have applications as an alternative to classical treatment regimens.
Assuntos
Acetilcisteína/farmacologia , Antituberculosos/farmacologia , Infecção por Mycobacterium avium-intracellulare/tratamento farmacológico , Mycobacterium avium/efeitos dos fármacos , beta-Defensinas/metabolismo , Células A549 , Acetilcisteína/uso terapêutico , Animais , Antituberculosos/uso terapêutico , Carga Bacteriana/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Interleucina-17/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Camundongos , Mycobacterium avium/crescimento & desenvolvimento , Infecção por Mycobacterium avium-intracellulare/metabolismo , Infecção por Mycobacterium avium-intracellulare/microbiologia , Células RAW 264.7 , Transdução de SinaisRESUMO
Streptococcus pneumoniae may colonize the nasopharynx, and as pneumococcal colonization causes invasive diseases and the subsequent transmission, reducing bacterial burden in the nasal cavity is critical. Hochu-ekki-to (TJ-41) is a traditional Japanese herbal medicine that exerts immunomodulatory effects in host cells. In this study, we investigated the potency of TJ-41 in modulating pneumococcal colonization clearance by activating host immunity. Mice, intranasally inoculated with pneumococci, were treated orally with TJ-41. During colonization, TJ-41 treatment significantly reduced pneumococcal burden and increased macrophage population in the nasopharynx. Furthermore, interleukin 17A production was significantly enhanced after TJ-41 treatment. In vitro experiment using nasal-derived cells revealed that pneumococcal antigen exposure upregulated the transcription of interleukin 17A in the TJ-41-treated group compared with that in the control group. Macrophages activated by killed bacteria were significantly increased in the presence of TJ-41 in an interleukin 17A-dependent manner. Moreover, TJ-41 enhanced phagocytosis, inhibited bacterial growth, and improved the antigen-presenting capacity of macrophages. Our results demonstrate that TJ-41 accelerates the clearance of pneumococcal nasopharyngeal colonization via macrophage activation. Subsequent production of interleukin 17A provides an additional benefit to effector cells.
Assuntos
Medicamentos de Ervas Chinesas , Infecções Pneumocócicas , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicina Herbária , Interleucina-17 , Japão , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/tratamento farmacológico , Streptococcus pneumoniaeRESUMO
IL-17 plays a critical role in the immunological control of various infectious diseases; its function has been investigated in the removal of both extracellular and intracellular bacteria. Our group previously revealed the importance of IL-17 in neutrophil migration following Legionella infection by using IL-17AF knockout mice; however, aside from neutrophil infiltration, alternative causes for the reduced survival of these mice have not been characterized. In this study, we found that γδ T cells in IL-17AF knockout mice were markedly increased and produced the cytotoxic substances granzyme B and perforin. Moreover, the elimination of γδ T cells from these mice, via an anti-TCRδ Ab, caused a substantial reduction in the level of lactate dehydrogenase in bronchoalveolar lavage fluid, indicating that γδ T cells contribute to lung tissue damage. Moreover, although cells lysed by cytotoxic substances are typically eliminated by phagocytic cells, in IL-17AF knockout mice, lung homeostasis was not maintained because of a decrease in phagocytic cells that impaired the clearance of dead cells. Our results indicate that increased γδ T cells in IL-17AF knockout mice help eliminate Legionella by releasing cytotoxic substances and lysing infected cells; however, this results in tissue damage due to insufficient removal of dead cells by phagocytic cells. This study enhances our understanding of the protective response against Legionella and provides insights into γδ T cell-mediated protective immunity against various infectious diseases.
Assuntos
Interleucina-17/metabolismo , Legionella/imunologia , Fagocitose/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Feminino , Imunidade Celular , Interleucina-17/genética , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Linfócitos T/metabolismoRESUMO
Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host's innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases.
Assuntos
Coinfecção/imunologia , Imunidade Inata , Macrófagos/imunologia , Infecções por Orthomyxoviridae/imunologia , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígeno B7-2/metabolismo , Carga Bacteriana , Coinfecção/microbiologia , Coinfecção/mortalidade , Coinfecção/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Vírus da Influenza A , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/virologia , Macrófagos/microbiologia , Camundongos , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/microbiologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Fagocitose , Vacinas Pneumocócicas/administração & dosagem , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/mortalidade , Pneumonia Pneumocócica/virologia , Streptococcus pneumoniae , Taxa de Sobrevida , Vacinação , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.
Assuntos
Interleucina-17/deficiência , Interleucina-17/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Pneumonia Estafilocócica/imunologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Interleucina-17/genética , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/imunologia , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/mortalidade , Taxa de SobrevidaRESUMO
Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Epitopos de Linfócito T/uso terapêutico , Proteínas de Choque Térmico HSC70/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Antígenos de Neoplasias/imunologia , Antígenos CD4 , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/imunologia , Células Tumorais CultivadasRESUMO
In this study, we analyzed interferon (IFN)-γ-producing cells and M1/M2 macrophage polarization in Legionella pneumophila pneumonia following anti-Gr-1 antibody treatment. Anti-Gr-1 treatment induced an M1-to-M2 shift of macrophage subtypes in the lungs and weakly in the peripheral blood, which was associated with increased mortality in legionella-infected mice. CD8+ T lymphocytes and natural killer cells were the dominant sources of IFN-γ in the acute phase, and anti-Gr-1 treatment reduced the number of IFN-γ-producing CD8+ T lymphocytes. In the CD3-gated population, most Gr-1-positive cells were CD8+ T lymphocytes in the lungs and lymph nodes (LNs) of infected mice. Additionally, the number of IFN-γ-producing Gr-1+ CD8+ T lymphocytes in the lungs and LNs increased 2 and 4 days after L. pneumophila infection, with anti-Gr-1 treatment attenuating these populations. Antibody staining revealed that Gr-1+ CD8+ T lymphocytes were Ly6C-positive cells rather than Ly6G, a phenotype regarded as memory type cells. Furthermore, the adoptive transfer of Gr-1+ CD8+ T lymphocytes induced increases in IFN-γ, M1 shifting and reduced bacterial number in the Legionella pneumonia model. These data identified Ly6C+ CD8+ T lymphocytes as a source of IFN-γ in innate immunity and partially associated with reduced IFN-γ production, M2 polarization, and high mortality in anti-Gr-1 antibody-treated mice with L. pneumophila pneumonia.