RESUMO
AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL at 4.5 Å resolution reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to-head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.
Assuntos
Proteínas de Bactérias/química , Chaperonas Moleculares/química , Multimerização Proteica , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Domínios Proteicos , Streptococcus pneumoniae/metabolismoRESUMO
Asthma is an allergic airway disease (AAD) characterized by eosinophilic inflammation, mucus hypersecretion, and airway hyper responsiveness, and it is caused by dysregulated immune responses. Conversely, regulatory T cells (Tregs) control aberrant immune responses and maintain homeostasis. Recent evidence suggests that Streptococcus pneumoniae, including its components as well as a live attenuated mutant, and pneumococcal infection induce Tregs and can thus potentially be harnessed therapeutically for asthma treatment. Previously, a pep27 deletion mutant (Δpep27) demonstrated a significantly attenuated virulence in a sepsis model, and Δpep27 immunization induced serotype-nonspecific protection against S. pneumoniae infection, as well as influenza virus, possibly via an immune tolerance mechanism. Here, the potential of Δpep27 immunization for asthma protection was studied. Mice were immunized intranasally with Δpep27 before or after ovalbumin sensitization and subsequent challenge. Δpep27 immunization suppressed hallmark features of AAD, including antigen-specific type 2 helper T cell cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, and goblet cell hyperplasia. Thus, a Δpep27 vaccine may be highly feasible as a preventive or therapeutic agent for asthma.
Assuntos
Asma/tratamento farmacológico , Vacinas Pneumocócicas/farmacologia , Streptococcus pneumoniae/genética , Administração Intranasal , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/prevenção & controle , Proteínas de Bactérias/genética , Líquido da Lavagem Broncoalveolar , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Mutação , Ovalbumina/toxicidade , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologiaRESUMO
During influenza pandemics, secondary pneumococcal infections cause excessive mortality. However, the current 13-valent pneumococcal conjugate vaccine, PCV13, provides only limited protection against secondary infection. Therefore, a more effective pneumococcal vaccine is required to protect against secondary pneumococcal infections. Here, intranasal immunization with an attenuated pneumococcal pep27 mutant provides protection from influenza virus infection, and also from secondary pneumococcal challenge. These results indicate that mucosal immunity might be an effective way to reduce the morbidity and mortality due to secondary pneumococcal infections during influenza pandemics.
Assuntos
Infecções por Orthomyxoviridae/prevenção & controle , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/imunologia , Administração Intranasal , Animais , Proteínas de Bactérias/genética , Peso Corporal , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos BALB C , Mutação , Infecções por Orthomyxoviridae/complicações , Vacinas Pneumocócicas/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Fatores de Virulência/genéticaRESUMO
Background: Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Methods: Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. Results: In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Conclusions: Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways.
Assuntos
Interleucina-17/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Via de Sinalização Wnt/imunologia , Administração Intranasal/métodos , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Imunização/métodos , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Streptococcus pneumoniae/imunologia , Células Th17/imunologia , Vacinação/métodosRESUMO
Pep27 from Streptococcus pneumoniae is reported to initiate pneumococcal autolysis, thereby constituting a major virulence factor. Although a few antisera recognizing Pep27 have been reported, no monoclonal, well-characterized antibody for Pep27 has been developed. Here we screened two single-chain antibody variable fragments (scFv) using a phage display from a large human synthetic scFv library to select clones E2 and F9. Dissociation constants (Kd) of E2 and F9 were 1.1⯵M and 0.50⯵M, respectively. E2 and F9 did not cross-react with other pneumococcal and unrelated proteins. The epitopes of Pep27 were localized to residues 24, 26 and 27 by alanine scanning. Molecular docking analysis supported the experimentally investigated epitope. The E2 and F9 clones specifically detected Pep27 in an environment mimicking in vivo conditions, demonstrated in human serum. The scFv clones characterized here represent molecular tools for the detection of pneumococcal diseases with potential for further improvement in affinity.
Assuntos
Anticorpos Monoclonais/imunologia , Peptídeos/imunologia , Infecções Pneumocócicas/imunologia , Anticorpos de Cadeia Única/imunologia , Streptococcus pneumoniae/imunologia , Fatores de Virulência/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Peptídeos/química , Anticorpos de Cadeia Única/química , Streptococcus pneumoniae/química , Fatores de Virulência/químicaRESUMO
BACKGROUND INFORMATION: Adipose tissue regulates energy metabolism by means of adipocyte hypertrophy and/or the differentiation of pre-existing adipocytes. Excessive production of some cytokines in adipose tissue is known to be a negative regulator of adipocyte differentiation, and the resulting impaired adipogenesis contributes to disorders like insulin resistance. IFN-α is a key immunoregulatory cytokine in the development of type 1 diabetes, lipid disorders and insulin resistance; however, its effect on adipogenesis remains unknown. METHOD: We examined the effect of IFN-α on adipocyte differentiation and its mechanisms. The effect of IFN-α on adipogenesis was evaluated by Western blotting, qRT-PCR, flow cytometric analysis and Oil Red O staining. We also investigated the role of STAT1 in adipogenesis using gene silencing analysis. RESULTS: IFN-α inhibited the accumulation of lipid droplets and the expression of adipogenesis related genes. The inhibition of adipocyte differentiation by IFN-α occurred in the early stages of differentiation. IFN-α arrested the cell cycle at the G0/G1 phase and regulated the expression of CDK2 and p21. These results were confirmed in MEF cells. Treatment with IFN-α increased STAT1 phosphorylation, and STAT1 siRNA or inhibitor prevented IFN-α from inhibiting the expression of PPARγ and C/EBPα as well as cell cycle progression in 3T3-L1 cells. CONCLUSION: We suggest that IFN-α inhibits adipocyte differentiation during the early stage of adipogenesis by regulating the expression of PPARγ and C/EBPα as well as the cell cycle through JAK/STAT1 signaling pathways. GENERAL SIGNIFICANCE: Our study provides new insights into possible mechanisms of the anti-adipogenetic effects of IFN-α.
Assuntos
Adipogenia , Interferon-alfa/farmacologia , Janus Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Células 3T3 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ciclo Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismoRESUMO
Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Streptococcus pneumoniae/imunologia , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Técnicas Biossensoriais , Imunoensaio , Simulação de Acoplamento Molecular , Ligação Proteica , Anticorpos de Cadeia Única/isolamento & purificação , Streptococcus pneumoniae/químicaRESUMO
BACKGROUND INFORMATION: The 53kDa protein pneumolysin (PLY) is the main virulence factor of Streptococcus pneumoniae, a leading cause of invasive pneumococcal diseases. PLY forms pores in cholesterol-containing membranes, thereby interfering with the function of cells. Bone destruction is a serious matter in chronic inflammatory diseases such as septic arthritis and osteomyelitis. S. pneumoniae is increasingly being recognized as a common cause of septic arthritis, but its pathogenesis is poorly defined. METHOD: We examined the effect of PLY on osteoblast differentiation and its mechanisms of action. The effect of PLY on osteoblast differentiation was evaluated by qRT-PCR, ALP activity assay, flow cytometric analysis, and Western blotting. We also examined the role of PLY-induced autophagy in osteoblast differentiation using RNA interference analysis. RESULTS: PLY inhibited osteoblast differentiation by decreasing the expression of osteoblast marker genes such as Runx2 and OCN, along with ALP activity. ROS production was increased by PLY during osteoblast differentiation. PLY induced autophagy through ROS-mediated regulation of AMPK and mTOR, which downregulated the expression of Sp1 and subsequent inhibition of differentiation. Treatment with autophagy inhibitors or Atg5 siRNA alleviated the PLY-induced inhibition of differentiation. CONCLUSION: The results suggest that PLY inhibits osteoblast differentiation by downregulation of Sp1 accompanied by induction of autophagy through ROS-mediated regulation of the AMPK/mTOR pathway. GENERAL SIGNIFICANCE: This study proposes a molecular mechanism for inhibition of osteoblast differentiation in response to PLY.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteossarcoma/genética , Fator de Transcrição Sp1/genética , Estreptolisinas/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Streptococcus pneumoniae/química , Streptococcus pneumoniae/patogenicidade , Estreptolisinas/administração & dosagem , Serina-Treonina Quinases TOR/metabolismoRESUMO
Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Regulação Enzimológica da Expressão Gênica , Streptococcus pneumoniae/enzimologia , Estreptolisinas/toxicidade , Fatores de Virulência/metabolismo , Álcool Desidrogenase/genética , Animais , Proteínas de Bactérias/toxicidade , Sobrevivência Celular , Tolerância a Medicamentos , Deleção de Genes , Macrófagos/microbiologia , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos ICR , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidade , Virulência , Fatores de Virulência/genéticaRESUMO
Senescence, which is irreversible cell cycle arrest, is induced by various types of DNA damage, including genotoxic stress. Senescent cells show dysregulation of tumor suppressor genes and other regulators of cellular proliferation. Activating transcription factor 3 (ATF3) plays a pleiotropic role in biological processes through genotoxic stress. In this study, we examined the effects of acrylamide (ACR), a genotoxic carcinogen, on cellular senescence and the molecular mechanisms of ATF3 function in macrophages. Treatment of macrophages with ACR at low concentrations (<1.0 mM) resulted in senescence-like morphology and an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity. Exposure of macrophages to ACR led to stress-induced, telomerase-independent senescence. In addition, ACR treatment for 1, 3, or 5 days showed a concentration-dependent increase in ATF3 expression and G0/G1 phase arrest. To better understand the role of ATF3 in controlling the senescence response to ACR, SA-ß-gal activity was examined using ATF3 knockdown and overexpression. ACR-mediated senescence was significantly decreased by knockdown of ATF3, whereas it was increased with ATF3 overexpression. We found that ATF3 regulated p53 and p21 levels. ATF3 also played an important role in regulating intracellular reactive oxygen species (ROS) production in response to ACR treatment. Moreover, phosphorylation of p38 and JNK kinases, which were activated during ATF3-mediated senescence, was observed in ACR-treated macrophages. Taken together, these results suggest that ATF3 contributes to ACR-induced senescence by enhancing ROS production, activating p38 and JNK kinases, and promoting the ATF3-dependent expression of p53, resulting in regulation of cellular senescence in macrophages.
Assuntos
Acrilamida/toxicidade , Carcinógenos/toxicidade , Senescência Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/fisiologia , Fibroblastos , MAP Quinase Quinase 4/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Telomerase , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Activating transcription factor-3 (ATF3) is known as a suppressor of cytokine production after exposure to lipopolysaccharide or during gram-negative bacterial infection. However, the mechanism by which ATF3 regulates innate immunity against gram-positive bacterial infection, particularly Streptococcus pneumoniae, remains unknown. METHODS: The wild-type and ATF3 knock-out (KO) mice were infected intranasally (i.n) or intraperitoneally with S. pneumoniae, and bacterial colonization or survival rate was determined. Pneumococcal pneumonia was induced by i.n infection, and ATF3 level was determined by Western blot. ATF3 KO cells or ATF3 siRNA transfection were used to determine expression of ATF3 downstream genes. Enzyme-linked immunosorbent assay was used to examine cytokines levels. RESULTS: ATF3 was highly expressed in various cell lines in vitro and in many organs in vivo. Pneumolysin was a novel inducer of ATF3. Pneumococcal infection induced ATF3, which subsequently stimulated production of cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and interferon [IFN]-γ). ATF3-mediated cytokine induction protected the host from pneumococcal infection. In the pneumonia infection model, the bacterial clearance of wild-type mice was more efficient than those of ATF3 KO mice. CONCLUSIONS: Taken together, we can conclude that ATF3 regulates innate immunity positively upon pneumococcus infection by enhancing TNF-α, IL-1ß, and IFN-γ expression and modulating bacterial clearance.
Assuntos
Fator 3 Ativador da Transcrição/genética , Citocinas/biossíntese , Resistência à Doença/genética , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/metabolismo , Streptococcus pneumoniae , Fator 3 Ativador da Transcrição/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Citocinas/genética , Modelos Animais de Doenças , Resistência à Doença/imunologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/mortalidade , Regiões Promotoras Genéticas , Ligação Proteica , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Neoplasias Pulmonares/metabolismo , Infecções Pneumocócicas/metabolismo , Serina Endopeptidases/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Endopeptidase Clp , Humanos , Neoplasias Pulmonares/genética , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/microbiologia , Serina Endopeptidases/genética , Complexo Shelterina , Streptococcus pneumoniae/genética , Proteínas de Ligação a Telômeros/genética , Virulência/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspA's C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA(+) and PspA(-) strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA(-) mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA(-) strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA(-) mutant. Also, there was much greater C3 deposition onto the PspA(-) pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína C-Reativa/metabolismo , Complemento C3/metabolismo , Fosforilcolina/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Ligação Competitiva , Proteína C-Reativa/antagonistas & inibidores , Proteína C-Reativa/imunologia , Complemento C3/antagonistas & inibidores , Complemento C3/imunologia , Meios de Cultura , Humanos , Camundongos , Fosforilcolina/química , Fosforilcolina/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/imunologiaRESUMO
Conventional immunization methods such as intramuscular injections lack effective mucosal protection against pathogens that enter through the mucosal surfaces. Moreover, conventional therapy often leads to adverse events and compromised immunity, followed by complicated outcomes, leading to the need to switch to other options. Thus, a need to develop safe and effective treatment with long-term beneficial outcomes to reduce the risk of relapse is mandatory. Mucosal vaccines administered across mucosal surfaces, such as the respiratory or intestinal mucosa, to prompt robust localized and systemic immune responses to prevent the public from acquiring pathogenic diseases. Mucosal immunity contains a unique immune cell milieu that selectively identify pathogens and limits the transmission and progression of mucosal diseases, such as allergic dermatitis and inflammatory bowel disease (IBD). It also offers protection from localized infection at the site of entry, enables the clearance of pathogens on mucosal surfaces, and leads to the induction of long-term immunity with the ability to shape regulatory responses. Regulatory T (Treg) cells have been a promising strategy to suppress mucosal diseases. To find advances in mucosal treatment, we investigated the therapeutic effects of intranasal pep27 mutant immunization. Nasal immunization protects mucosal surfaces, but nasal antigen presentation appears to entail the need for an adjuvant to stimulate immunogenicity. Here, a novel method is developed to induce Tregs via intranasal immunization without an adjuvant to potentially overcome allergic diseases and gut and lung inflammation using lung-gut axis communication in animal models. The implementation of the pep27 mutant for these therapies should be preceded by studies on Treg resilience through clinical translational studies on dietary changes.
RESUMO
Ethanol exposure has deleterious effects on the central nervous system. Although several mechanisms for ethanol-induced damage have been suggested, the precise mechanism underlying ethanol-induced neuronal cell death remains unclear. Recent studies indicate that the p75 neurotrophin receptor (p75NTR) has a critical role in the regulation of neuronal survival. This study was designed to examine the role of p75NTR in ethanol-induced apoptotic signaling in neuroblastoma cells. Ethanol caused highly increased level of p75NTR expression. The use of small interfering RNA to inhibit p75NTR expression markedly attenuated ethanol-induced cell cycle arrest and apoptosis. DNA binding activity of Sp1 was increased by ethanol, whereas inhibition of Sp1 activity by mithramycin, a Sp1 inhibitor, or short hairpin RNA suppressed ethanol-induced p75NTR expression. In addition, inhibitors of casein kinase 2 (CK2) and extracellular signal-regulated kinase (ERK) augmented ethanol-induced p75NTR expression. Our results also demonstrate that inhibition of ERK and CK2 caused a further increase in the activation of the p75NTR proximal promoter induced by ethanol. This increased activation was partially suppressed by the deletion of the Sp1 binding sites. These results suggest that Sp1-mediated p75NTR expression is regulated at least in part by ERK and CK2 pathways. The present study also showed that treatment with ethanol resulted in significant increases in the expression of p21, but not the levels of p53 and p53 target genes such as Bax, Puma, and Bcl-2. Furthermore, the inhibition of p75NTR expression or Sp1 activity suppressed ethanol-induced p21 expression, cell cycle arrest, and apoptosis. These data suggest that ethanol increases p75NTR expression, and CK2 and ERK signaling inversely regulate Sp1-mediated p75NTR expression in ethanol-treated neuroblastoma cells. Thus, our study provides more insight into the mechanisms underlying ethanol actions.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Proteínas do Tecido Nervoso/genética , Receptores de Fator de Crescimento Neural/genética , Fator de Transcrição Sp1/fisiologia , Alcoolismo/patologia , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Vaccines help protect people from infections. However, Coronavirus 2019 (COVID-19) vaccinees often still become infected with COVID-19 variants (breakthrough infections) and may go on to suffer from long COVID symptoms due to short-lasting immunity and less-effective protection provided by available vaccines. Moreover, the current COVID-19 vaccines do not prevent viral transmission and ward off only about 15% of breakthrough infections. To prepare more effective vaccines, it is essential to predict the viral strains that will be circulating based on available epidemiological data. The World Health Organization recommends in advance which influenza strains are expected to be prevalent during influenza season to guide the production of influenza vaccines by pharmaceutical companies. However, future emerging COVID-19 strain(s) have not been possible to predict since no sound epidemiological information has been established. Thus, for more effective protection, immune stimulators alone or in combination with vaccines would be preferable to protect people from COVID-19 infection. One of those remedies would be ginseng, which has been used for potentiating immunity in the past.
RESUMO
Atopic dermatitis (AD) is an inflammatory disease driven in part by type 2 helper T (Th2) cytokines and skin barrier disruption alleviating the entry of allergens. Thymic stromal lymphopoietin (TSLP), an epithelial cellâderived cytokine, is known to aggravate AD symptoms by activating Th2. In addition, regulatory T cells (Tregs) inhibit inflammatory cells such as Th2. However, the relationship between TSLP and Tregs in AD is unclear. A murine dermatitis model was induced by applying oxazolone to the ear skin of mice. Prophylactic and therapeutic responses were analyzed by immunizing mice intranasally with a pneumococcal pep27 mutant (Δpep27 mutant), attenuated strain by reducing the virulence of a pathogen. Intranasal immunization with a pneumococcal pep27 mutant could elicit anti-inflammatory Treg-relevant factors and epithelial barrier genes (loricrin, involucrin, filaggrin, and small proline-rich repeat proteins). Thus, pneumococcal pep27-mutant immunization suppressed epidermal collapse, IgE, TSLP, and upregulation of Th2 expression by upregulating Treg activity. In contrast, Treg inhibition aggravated AD symptoms through the upregulation of TSLP and Th2 and the repression of epithelial barrier function compared with that of the noninhibited pneumococcal Δpep27-mutant group. Taken together, immunization with pneumococcal Δpep27 mutant upregulated Treg and epithelial barrier function and inhibited TSLP and Th2 to relieve AD symptoms.
Assuntos
Dermatite Atópica , Camundongos , Animais , Linfócitos T Reguladores , Linfopoietina do Estroma do Timo , Regulação para Cima , Citocinas/metabolismo , ImunizaçãoRESUMO
Allergic rhinitis (AR), one of the most common inflammatory diseases, is caused by immunoglobulin E (IgE)-mediated reactions against inhaled allergens. AR involves mucosal inflammation driven by type 2 helper T (Th2) cells. Previously, it was shown that the Streptococcus pneumoniae pep27 mutant (Δpep27) could prevent and treat allergic asthma by reducing Th2 responses. However, the underlying mechanism of Δpep27 immunization in AR remains undetermined. Here, we investigated the role of Δpep27 immunization in the development and progression of AR and elucidated potential mechanisms. In an ovalbumin (OVA)-induced AR mice model, Δpep27 alleviated allergic symptoms (frequency of sneezing and rubbing) and reduced TLR2 and TLR4 expression, Th2 cytokines, and eosinophil infiltration in the nasal mucosa. Mechanistically, Δpep27 reduced the activation of the NLRP3 inflammasome in the nasal mucosa by down-regulating the Toll-like receptor signaling pathway. In conclusion, Δpep27 seems to alleviate TLR signaling and NLRP3 inflammasome activation to subsequently prevent AR.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Rinite Alérgica , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Imunização , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Ovalbumina , Rinite Alérgica/induzido quimicamente , Rinite Alérgica/metabolismo , Rinite Alérgica/prevenção & controle , Células Th2RESUMO
Inflammatory bowel disease (IBD) is a highly prevalent gut inflammatory disorder. Complicated clinical outcomes prolong the use of conventional therapy and often lead to compromised immunity followed by adverse events and high relapse rates. Thus, a profound medical intervention is required. Previously, intranasal immunization of pneumococcal pep27 mutant (Δpep27) exhibited long-lasting protection against immune-related disorders. System biology analysis has predicted an inverse correlation between Δpep27 immunization and gastroenteritis. Recently, we established that Δpep27-elicited Tregs repressed Wnt5a expression and enhanced barrier integrity, suggesting the restoration of immunological tolerance. Therefore, we evaluated whether Δpep27 can alleviate IBD. Δpep27 dose-dependent response was analyzed in dextran sulfate sodium-induced mice using transcriptome analysis. Pro- and anti-inflammatory signatures were cross-correlated by quantitative PCR and western blot analyses. To address the hierarchy regulating the activity of caspase-14, an undefined marker in IBD, and regulatory T cells (Tregs), antibody-based neutralization studies were conducted. Fecal microbiome profiles were analyzed by 16S rRNA pyrosequencing. Δpep27 significantly attenuated dextran sulfate sodium-induced oxidative stress parameters, proinflammatory cytokines, caspase-14 expression level, and upregulated tight junction, anti-inflammatory genes IL-10 and TGF-ß1 via upregulation of Tregs to restore healthy gut microbiota. Neutralization studies unveiled that ∆pep27 had a remedial effect via Treg upregulation. Caspase-14, being an important mediator in the pathogenesis of IBD, can be an alternate therapeutic target in IBD. ∆pep27-increased Tregs repressed caspase-14 expression and reversed gut microbial dysbiosis, aiding to re-establish immunological tolerance.