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1.
ACS Appl Mater Interfaces ; 13(4): 4844-4852, 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33486952

RESUMO

The combination of photothermal therapy (PTT) and toll-like receptor (TLR)-mediated immunotherapy can elicit antitumor immunity and modulate the immunosuppressive tumor microenvironment (TME). Unlike other TLRs, TLR-5 is a promising target for immune activation, as its expression is well-maintained even during immunosenescence. Here, we developed a unique tumor microenvironment-regulating immunosenescence-independent nanostimulant consisting of TLR-5 adjuvant Vibrio vulnificus flagellin B (FlaB) conjugated onto the surface to an IR 780-loaded hyaluronic acid-stearylamine (HIF) micelles. These HIF micelles induced immune-mediated cell death via PTT when irradiated with a near-infrared laser. In comparison with PTT alone, the combination of in situ-generated tumor-associated antigens produced during PTT and the immune adjuvant FlaB demonstrated enhanced vaccine-like properties and modulated the TME by suppressing immune-suppressive regulatory cells (Tregs) and increasing the fraction of CD103+ migratory dendritic cells, which are responsible for trafficking tumor antigens to draining lymph nodes (DLNs). This combinatorial strategy (i.e., applying a TLR-5 adjuvant targeted to immunosenescence-independent TLR-5 and the in situ photothermal generation of tumor-associated antigens) is a robust system for next-generation immunotherapy and could even be applied in elderly patients, thus broadening the clinical scope of immunotherapy strategies.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Flagelina/uso terapêutico , Imunoterapia , Nanopartículas/uso terapêutico , Neoplasias/terapia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Flagelina/administração & dosagem , Flagelina/imunologia , Células HEK293 , Humanos , Imunossenescência/efeitos dos fármacos , Imunossenescência/efeitos da radiação , Imunoterapia/métodos , Raios Infravermelhos/uso terapêutico , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Neoplasias/imunologia , Neoplasias/patologia , Receptor 5 Toll-Like/antagonistas & inibidores , Receptor 5 Toll-Like/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Vibrio vulnificus/imunologia
3.
Nat Commun ; 11(1): 3763, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724132

RESUMO

In both animals and plants, the perception of bacterial flagella by immune receptors elicits the activation of defence responses. Most plants are able to perceive the highly conserved epitope flg22 from flagellin, the main flagellar protein, from most bacterial species. However, flagellin from Ralstonia solanacearum, the causal agent of the bacterial wilt disease, presents a polymorphic flg22 sequence (flg22Rso) that avoids perception by all plants studied to date. In this work, we show that soybean has developed polymorphic versions of the flg22 receptors that are able to perceive flg22Rso. Furthermore, we identify key residues responsible for both the evasion of perception by flg22Rso in Arabidopsis and the gain of perception by the soybean receptors. Heterologous expression of the soybean flg22 receptors in susceptible plant species, such as tomato, enhances resistance to bacterial wilt disease, demonstrating the potential of these receptors to enhance disease resistance in crop plants.


Assuntos
Flagelina/imunologia , Imunidade Vegetal , Proteínas de Plantas/imunologia , Receptores Imunológicos/imunologia , Soja/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/metabolismo , Resistência à Doença/genética , Resistência à Doença/imunologia , Epitopos/imunologia , Flagelina/genética , Flagelina/metabolismo , Evasão da Resposta Imune/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polimorfismo Genético/imunologia , Ralstonia solanacearum/imunologia , Ralstonia solanacearum/patogenicidade , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Soja/genética , Soja/metabolismo , Soja/microbiologia
4.
Proc Natl Acad Sci U S A ; 117(29): 16985-16991, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641510

RESUMO

Vertebrates, from zebra fish to humans, have an innate immune recognition of many bacterial flagellins. This involves a conserved eight-amino acid epitope in flagellin recognized by the Toll-like receptor 5 (TLR5). Several important human pathogens, such as Helicobacter pylori and Campylobacter jejuni, have escaped TLR5 activation by mutations in this epitope. When such mutations were introduced into Salmonella flagellin, motility was abolished. It was previously argued, using very low-resolution cryoelectron microscopy (cryo-EM), that C. jejuni accommodated these mutations by forming filaments with 7 protofilaments, rather than the 11 found in other bacteria. We have now determined the atomic structure of the C. jejuni G508A flagellar filament from a 3.5-Å-resolution cryo-EM reconstruction, and show that it has 11 protofilaments. The residues in the C. jejuni TLR5 epitope have reduced contacts with the adjacent subunit compared to other bacterial flagellar filament structures. The weakening of the subunit-subunit interface introduced by the mutations in the TLR5 epitope is compensated for by extensive interactions between the outer domains of the flagellin subunits. In other bacteria, these outer domains can be nearly absent or removed without affecting motility. Furthermore, we provide evidence for the stabilization of these outer domain interactions through glycosylation of key residues. These results explain the essential role of glycosylation in C. jejuni motility, and show how the outer domains have evolved to play a role not previously found in other bacteria.


Assuntos
Campylobacter jejuni/ultraestrutura , Flagelos/ultraestrutura , Flagelina/imunologia , Receptor 5 Toll-Like/imunologia , Campylobacter jejuni/imunologia , Epitopos/química , Epitopos/imunologia , Flagelos/química , Flagelos/imunologia , Flagelina/química , Humanos , Imunidade Inata
5.
Arch Virol ; 165(7): 1585-1597, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32399789

RESUMO

Groundnut bud necrosis virus (GBNV), a member of the genus Tospovirus, has an extensive host range and is associated with necrosis disease of chilli (Capsicum annuum L.), which is a major threat to commercial production. Plant growth promoting rhizobacteria (PGPR) have been investigated for their antiviral activity in several crops and for their potential use in viral disease management. However, the microbial mechanisms associated with PGPR in triggered immunity against plant viruses have rarely been studied. To understand the innate immune responses activated by Bacillus spp. against GBNV, we studied microbe-associated molecular pattern (MAMP) triggered immunity (MTI) in chilli using transient expression of the flagellin gene of Bacillus amyloliquefaciens CRN9 from Agrobacterium clones, which also induced the expression of EAS1 gene transcripts coding for epi-aristolochene synthase, which is responsible for the accumulation of capsidiol phytoalexin. In addition, the transcript levels of WRKY33 transcription factor and salicylic acid (SA)-responsive defense genes such as NPR1, PAL, PO and SAR8.2 were increased. Jasmonate (JA)-responsive genes, viz., PDF, and LOX genes, were also upregulated in chilli plants challenged with GBNV. Further analysis revealed significant induction of these genes in chilli plants treated with B. amyloliquefaciens CRN9 and benzothiadiazole (BTH). The transcript levels of defense response genes and pathogenesis-related proteins were significantly higher in plants treated with Bacillus and BTH and remained significantly higher at 72 h post-inoculation and compared to the inoculated control. The plants treated with flagellin using the agrodrench method and exogenous treatment with B. amyloliquefaciens and BTH showed resistance to GBNV upon mechanical inoculation and a reduced virus titre which was confirmed by qPCR assays. Thus, transient expression of flagellin, a MAMP molecule from B. amyloliquefaciens CRN9, is able to trigger innate immunity and restrain virus growth in chilli via induced systemic resistance (ISR) activated by both the SA and JA/ET signalling pathways.


Assuntos
Bacillus amyloliquefaciens/imunologia , Capsicum/imunologia , Flagelina/imunologia , Doenças das Plantas/virologia , Tospovirus/fisiologia , Bacillus amyloliquefaciens/fisiologia , Capsicum/genética , Capsicum/microbiologia , Capsicum/virologia , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia
6.
PLoS One ; 15(4): e0231998, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330169

RESUMO

Two experiments were conducted to evaluate the immune response of broilers vaccinated with Salmonella chitosan-nanoparticle (CNP) vaccine and challenged with Salmonella. The Salmonella CNP vaccine was synthesized with Salmonella enterica outer membrane proteins (OMPs) and flagellin proteins. In Experiment I, birds were orally gavaged with PBS or 500, 1000, or 2000µg of CNP vaccine 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live S. Enteritidis (SE). Macrophage-nitrite production 11d-post-challenge was higher (P<0.05) in the 500µg group when compared to the control. At d14 (8h-post-challenge), broilers vaccinated with 1000µg CNP had higher (P<0.05) serum anti-OMPs IgG and IgA and cloacal anti-OMP IgA amounts. At 11d-post-challenge, birds vaccinated with 1000µg CNP vaccine had greater (P<0.05) bile anti-OMP and anti-flagellin IgA amounts. At 11d-post-challenge, birds administered 1000µg CNP vaccine has increased (P<0.05) IL-1ß and IL-10 mRNA in cecal tonsils. In Experiment II, birds were orally gavaged with PBS or 1000µg CNP or a live commercial vaccine at 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live SE or S. Heidelberg (SH). Birds vaccinated with CNP showed higher (P<0.05) serum anti-OMPs IgG amounts at 8h-post-challenge. At 4d-post-SH challenge, birds vaccinated with CNP had higher (P<0.05) bile anti-flagellin IgA amounts. CNP decreased (P<0.05) anti-OMPs IgG levels in serum at 2d-post-SE challenge and 4d-post-SH or SE challenge. Salmonella Enteritidis loads in cecal content at 2d-post-challenge was decreased (P<0.05) by 65.9% in birds vaccinated with CNP, when compared to the control. Chitosan-nanovaccine had no adverse effects on bird's production performance. In conclusion, 1000µg CNP vaccine can induce a specific immune response against Salmonella and has the potential to mitigate SE cecal colonization in broiler birds.


Assuntos
Galinhas/imunologia , Quitosana/farmacologia , Vacinas contra Salmonella/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Ceco/metabolismo , Galinhas/microbiologia , Quitosana/imunologia , Flagelina/imunologia , Antígenos de Histocompatibilidade Classe II , Nanopartículas/uso terapêutico , Doenças das Aves Domésticas/imunologia , Salmonella/imunologia , Salmonella/patogenicidade , Salmonelose Animal/imunologia , Salmonella enterica/metabolismo , Salmonella enteritidis/imunologia , Vacinas/imunologia , Vacinas Atenuadas/imunologia
7.
Physiol Genomics ; 52(5): 217-221, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275178
8.
Vet Microbiol ; 243: 108633, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32273012

RESUMO

Salmonella Enteritidis (SE) is one of the most common culprits of foodborne disease in humans due to its horizontal transmission from infected animals to humans. The development of a safe vaccine against Salmonella would be important for both farm animals and humans concerning disease containment. The SE ghosts carrying FliC were genetically constructed using a special ghost plasmid pJHL184 that co-expressed FliC and the phage lysis gene E. These SE ghosts were characterized by ghost generation efficacy by increasing the culture temperature to "42 °C" in the absence of L-arabinose. This temperature change led to an ghost generation with almost complete lysis of the SE host strain in 48 hs. The expression of FliC was confirmed by Western blot analysis. Also, indirect ELISA was used to prove FliC specific antibody generation in immunized mice. The parenteral adjuvant effect of the FliC antigen was demonstrated by immunizing mice with pJHL184::flC, pJHL184 alone, or PBS alone. The mice were intramuscularly immunized at six weeks of age (n = 8) and boosted after three weeks of primary inoculation. A total of 32 mice were equally divided into four groups. Each group was treated with pJHL-ghosts alone, ghost surface displaying FliC adjuvant, and compared to the PBS and naïve control groups. The immunized mice demonstrated greater IgG and IgA antibody responses than did the PBS control group. Furthermore, the addition of the ghosts to the FliC led to a significant increase in both the humoral and cell-mediated immune responses compared to those in the ghost alone group. Besides, the in vitro antigen uptake and presentation studies revealed efficient antigen presentation on the mouse macrophage cell surfaces. This finding further corroborated the potential efficacy of immune stimulation by SE ghosts. After the virulent challenge, we observed a significant reduction in the bacterial load in the spleen and liver tissues in SE ghosts surface, displaying FliC adjuvant. Our results demonstrate a safe and effective strategy to prevent salmonellosis. They also suggest that the surface expression of flagellin (FliC) significantly enhances antigen-specific humoral and cell-mediated immune responses. This FliC expression can also enhance the protective efficacy of the bacterial ghosts-based vaccine against virulent challenge.


Assuntos
Anticorpos Antibacterianos/sangue , Flagelina/imunologia , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/prevenção & controle , Salmonella enteritidis/imunologia , Adjuvantes Imunológicos , Animais , Feminino , Flagelina/genética , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Plasmídeos/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/imunologia , Vacinas contra Salmonella/administração & dosagem , Vacinas contra Salmonella/imunologia , Salmonella enteritidis/genética
9.
Arch Virol ; 165(6): 1299-1309, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32253616

RESUMO

Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.


Assuntos
Infecções por Coronavirus/veterinária , Flagelina/administração & dosagem , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular , Colostro/química , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Feminino , Flagelina/imunologia , Imunização , Gravidez , Suínos , Doenças dos Suínos/patologia , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem
10.
Mol Cells ; 43(3): 251-263, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32131150

RESUMO

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasomedependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in antiflagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-ß (IFN-ß) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo . Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-ß, but not for other proinflammatory cytokines. In addition, we found that antiflagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-ß produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.


Assuntos
Flagelina/farmacologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Interferon beta/biossíntese , Animais , Modelos Animais de Doenças , Flagelina/antagonistas & inibidores , Flagelina/imunologia , Flagelina/isolamento & purificação , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Receptor 5 Toll-Like/imunologia , Receptor 5 Toll-Like/metabolismo
11.
J Vet Med Sci ; 82(3): 325-332, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-31996495

RESUMO

Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed a serology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of Burkholderia pseudomallei as antigens. DNA corresponding to each antigen was cloned into a pET32a vector and expressed in Escherichia coli. Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95% homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated B. pseudomallei (1:1,000) on a Western blot. Subsequently, we demonstrated that both recombinants could capture the antibodies present in goat with naturally occurring melioidosis (optimized titer 1:40) while not cross-reacting with the serum samples of goats naturally infected by Corynebacterium pseudotuberculosis or Staphylococcus aureus. Finally, an enzyme-linked immunosorbent assay (ELISA) using 20 goat serum samples without melioidosis and 10 goat serum samples with melioidosis demonstrated that the infected group has significantly higher antibody titer levels than the normal group (P<0.001) when using either OmpA or FliC as an antigen. However, the sensitivity (100%) of the assay using OmpA was superior to that (90%) from using FliC. Serological tests that are commonly used often rely on antigens from crude cell extracts, which pose risks for laboratory-acquired infections and inconsistency in their preparation; however, use of recombinant OmpA is safe; it can potentially be used as a reagent in testing for goat melioidosis.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Burkholderia pseudomallei/imunologia , Flagelina/imunologia , Doenças das Cabras/diagnóstico , Melioidose/veterinária , Animais , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/sangue , Cabras , Imunoensaio , Melioidose/diagnóstico , Melioidose/imunologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/veterinária
12.
Curr Pharm Biotechnol ; 21(4): 316-324, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31729940

RESUMO

BACKGROUND: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. METHODS: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. RESULTS: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund's adjuvant group. CONCLUSION: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.


Assuntos
Adjuvantes Imunológicos , Antígenos Virais/imunologia , Biotecnologia/métodos , Vacinas contra Papillomavirus/imunologia , Tecnologia Farmacêutica/métodos , Proteínas Estruturais Virais/imunologia , Adjuvantes Imunológicos/química , Animais , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Simulação por Computador , Citocinas/sangue , Escherichia coli/genética , Feminino , Flagelina/imunologia , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus/administração & dosagem , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Vacinação , Proteínas Estruturais Virais/genética
13.
Mol Plant Microbe Interact ; 33(2): 247-255, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31644369

RESUMO

The first layer of plant immunity is deployed by recognition of pathogen-associated molecule patterns (PAMPs) and induction of early stress responses. Flagellin is the major protein component of the flagellum. Flagellin-derived peptide fragments such as Flg22, a short active peptide derived from the highly conserved part of the N-terminal region, are recognized as PAMPs by a specific perception system present in most higher plants. Some bacteria evade the plant recognition system by altering the Flg22 region in the flagellin. Instead, a small subset of plants (i.e., solanaceous plants) can sense these bacteria by recognizing a second region, termed FlgII-28. The function of FlgII-28 has been well-documented in tomato but not in potato plants. Here, we investigated the effect of FlgII-28 on several defense responses in potato. Cytosolic calcium (Ca2+) elevation is an early defense response upon pathogenic infection. We generated transgenic potato plants expressing aequorin, a nontoxic Ca2+-activated photoprotein. The results showed that FlgII-28 induced strong cytosolic Ca2+ elevation in a dose-dependent manner, whereas the response was attenuated when a Ca2+ channel blocker was added. In addition, the FlgII-28-triggered cytosolic Ca2+ elevation was shown to subsequently promote extracellular alkalinization, reactive oxygen species production, mitogen-activated protein kinase phosphorylation, and transcriptional reprogramming of defense-related genes in potato. Interestingly, all tested defense responses caused by FlgII-28 were significantly stronger than those caused by Flg22, suggesting that FlgII-28 acts as a primary flagellar PAMP to elicit multiple defense responses in potato.


Assuntos
Flagelina , Imunidade Vegetal , Solanum tuberosum , Cálcio/metabolismo , Citosol/química , Citosol/imunologia , Flagelina/genética , Flagelina/imunologia , Regulação da Expressão Gênica de Plantas , Imunidade Vegetal/genética , Solanum tuberosum/genética , Solanum tuberosum/imunologia
14.
J Infect Dis ; 221(6): 1000-1005, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31678998

RESUMO

Human bronchial epithelial cells play a key role in airway immune homeostasis. We hypothesized that these sentinel cells can remember a previous contact with pathogen compounds and respond nonspecifically to reinfection, a phenomenon called innate immune memory. We demonstrated that their preexposure to Pseudomonas aeruginosa flagellin modify their inflammatory response to a second, nonrelated stimulus, including live pathogens or lipopolysaccharide. Using histone acetyltransferase and methyltransferase inhibitors, we showed that this phenomenon relied on epigenetic regulation. This report is a major breakthrough in the field of multimicrobial respiratory tract infections, wherein control of inflammatory exacerbations is a major therapeutic issue.


Assuntos
Memória Imunológica , Mucosa Respiratória/citologia , Epigênese Genética , Células Epiteliais/imunologia , Flagelina/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação , Lipopolissacarídeos , Estudo de Prova de Conceito , Pseudomonas aeruginosa/imunologia , RNA Mensageiro , Mucosa Respiratória/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
15.
Microb Pathog ; 138: 103697, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31465785

RESUMO

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly achieves antibiotic resistance, and thus, developing an effective vaccine is critically important for combating P. aeruginosa infection. Flagella and pili play important roles in colonization of P. aeruginosa at the burn wound site and its subsequent dissemination to deeper tissue and organs. In the present study, we evaluated protective efficacy of a trivalent vaccine containing flagellins A and B (FlaA + FlaB) + pilin (PilA) in a murine burn model of infection. "FlaA + FlaB + PilA" induced greater protection in P. aeruginosa murine burn model than the single components alone, and it showed broad immune protection against P. aeruginosa strains. Immunization with "FlaA + FlaB + PilA" induced strong opsonophagocytic antibodies and resulted in reduced bacterial loads, systemic IL-12/IL-10 cytokine expression, and increased survival after challenge with three times lethal dose fifty (LD50) of P. eruginosa strains. Moreover, the protective efficacy of "FlaA + FlaB + PilA" vaccination was largely attributed to specific antibodies. Taken together, these data further confirm that the protective effects of "FlaA + FlaB + PilA" vaccine significantly enhance efficacy compared with antibodies against either mono or divalent antigen, and that the former broadens the coverage against P. eruginosa strains that express two of the three antigens.


Assuntos
Queimaduras/microbiologia , Vacinas contra Pseudomonas , Pseudomonas aeruginosa/imunologia , Infecção dos Ferimentos/microbiologia , Animais , Modelos Animais de Doenças , Proteínas de Fímbrias/imunologia , Flagelina/imunologia , Camundongos , Infecções por Pseudomonas/prevenção & controle , Vacinação
16.
Front Immunol ; 10: 2750, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824516

RESUMO

T-helper-17 (Th17) cells are a subset of CD4+ T cells that can produce the cytokine interleukin (IL)-17 and play vital roles in protecting the host from bacterial and fungal infections, especially at the mucosal surface. These are abundant in the small intestinal lamina propria (SILP) and their differentiation are associated with the colonization of the intestinal flora. Segmented filamentous bacteria (SFB) drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the SI LP of mice. Recent work has highlighted that SFB used microbial adhesion-triggered endocytosis (MATE) to transfer SFB antigenic proteins into small intestinal epithelial cells (SI ECs) and modulate host immune homeostasis. However, which components of SFB are involved in this immune response process remains unclear. Here, we examined the roles of SFB flagellins in Th17 cells induction using various techniques, including ELISA, ELISPOT, and RNA-seq in vitro and in vivo. The results show that the immune function of SFB flagellins is similar to SFB, i.e., induces the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the SI LP. Furthermore, treatment of mice with SFB flagellins lead to a significant increase in the expression of genes associated with the IL-17 signaling pathway, such as IL-6, IL-1ß, TNF-α, IL-17A, IL-17F, and IL-22. In addition, SFB flagellins have an intimate relationship with intestinal epithelial cells, influencing the expression of epithelial cell-specific genes such as Nos2, Duox2, Duoxa2, SAA3, Tat, and Lcn2. Thus, we propose that SFB flagellins play a significant role in the involvement of SFB in the induction of intestinal Th17 cells.


Assuntos
Bactérias/imunologia , Flagelina/imunologia , Mucosa Intestinal , Intestino Delgado , Células Th17/imunologia , Animais , Aderência Bacteriana/imunologia , Citocinas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestino Delgado/citologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Masculino , Camundongos , Células Th17/citologia
17.
Nat Commun ; 10(1): 5650, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827095

RESUMO

Alterations in gut microbiota composition are associated with metabolic syndrome and chronic inflammatory diseases such as inflammatory bowel disease. One feature of inflammation-associated gut microbiotas is enrichment of motile bacteria, which can facilitate microbiota encroachment into the mucosa and activate pro-inflammatory gene expression. Here, we set out to investigate whether elicitation of mucosal anti-flagellin antibodies by direct administration of purified flagellin might serve as a general vaccine against subsequent development of chronic gut inflammation. We show, in mice, that repeated injection of flagellin elicits increases in fecal anti-flagellin IgA and alterations in microbiota composition, reduces fecal flagellin concentration, prevents microbiota encroachment, protects against IL-10 deficiency-induced colitis, and ameliorates diet-induced obesity. Flagellin's impact on the microbiota is B-lymphocyte dependent and, in humans, obese subjects exhibit increased levels of fecal flagellin and reduced levels of fecal flagellin-specific IgA, relative to normal weight subjects. Thus, administration of flagellin, and perhaps other pathobiont antigens, may confer some protection against chronic inflammatory diseases.


Assuntos
Imunidade Adaptativa , Vacinas Bacterianas/imunologia , Colite/prevenção & controle , Flagelina/imunologia , Microbioma Gastrointestinal , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Colite/genética , Colite/imunologia , Colite/microbiologia , Fezes/microbiologia , Flagelina/administração & dosagem , Flagelina/genética , Humanos , Imunoglobulina A/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/imunologia , Obesidade/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia
18.
Int J Mol Sci ; 21(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861650

RESUMO

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1ß, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.


Assuntos
Aeromonas veronii/imunologia , Vacinas Bacterianas/administração & dosagem , Doenças dos Peixes/prevenção & controle , Flagelina/metabolismo , Lactobacillus casei/fisiologia , Administração Oral , Aeromonas veronii/patogenicidade , Animais , Vacinas Bacterianas/imunologia , Carpas/imunologia , Doenças dos Peixes/imunologia , Flagelina/genética , Flagelina/imunologia , Regulação da Expressão Gênica , Imunoglobulina M/sangue , Interferon gama/genética , Interleucinas/genética , Fator de Necrose Tumoral alfa/genética
19.
J Vet Sci ; 20(6): e70, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31775197

RESUMO

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.


Assuntos
Antígenos Virais/imunologia , Flagelina/imunologia , Imunidade Humoral/fisiologia , Vírus da Diarreia Epidêmica Suína/imunologia , Vibrio vulnificus/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C
20.
mSphere ; 4(6)2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776239

RESUMO

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI.IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.


Assuntos
Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Flagelina/imunologia , Interleucina-10/metabolismo , Receptor 5 Toll-Like/metabolismo , Bexiga Urinária/imunologia , Escherichia coli Uropatogênica/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Camundongos Endogâmicos C57BL , Modelos Teóricos , Fatores de Tempo , Bexiga Urinária/microbiologia
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