RESUMEN
Serum amyloid A (SAA) is an acute phase inflammatory protein that we previously described as a robust biomarker of colorectal inflammation in patients with ulcerative colitis (UC) in clinical remission. However, what induces SAA expression in UC remains unclear. This study demonstrates that SAA is significantly expressed in the intestinal tract of UC mouse models when compared with C-reactive protein, another inflammatory biomarker. Moreover, interleukin-6 and tumor necrosis factor-α were found to promote SAA1 expression, as were Toll-like receptor ligands flagellin and lipopolysaccharide. Furthermore, results suggested that the nuclear factor-kappa B (NF-κB) pathway may be involved in the promotion of SAA1 expression by flagellin, which was inhibited by treatment with 5-aminosalicylic acid (5-ASA). Therefore, the flagellin/NF-κB/SAA1 axis may represent one of the mechanisms by which 5-ASA suppresses intestinal inflammation.
Asunto(s)
Colitis Ulcerosa , Proteína Amiloide A Sérica , Animales , Colitis Ulcerosa/tratamiento farmacológico , Células Epiteliales/metabolismo , Flagelina/uso terapéutico , Humanos , Inflamación/patología , Mesalamina/uso terapéutico , Ratones , FN-kappa B/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. The major bacterial cause of COPD exacerbations is non-typeable Haemophilus influenzae (NTHi). 25 to over 80% of cases are associated with NTHi. This susceptibility to infection involves a defective production of interleukin (IL)-22 which plays an important role in mucosal defense. Prophylactic administration of flagellin, a Toll-like receptor 5 (TLR5) agonist, protects healthy mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbations. Mice chronically exposed to cigarette smoke (CS), which presented COPD symptoms, were infected with NTHi and intraperitoneally treated with recombinant flagellin following a prophylactic or therapeutic protocol. Compared with control, cigarette smoke-exposed mice treated with flagellin showed a lower bacterial load in the airways, the lungs and the blood. This protection was associated with an early neutrophilia, a lower production of pro-inflammatory cytokines and an increased IL-22 production. Flagellin treatment decreased the recruitment of inflammatory cells and the lung damages related to exacerbation. Morover, the protective effect of flagellin against NTHi was altered by treatment with anti-IL-22 blocking antibodies in cigarette smoke-exposed mice and in Il22-/- mice. The effect of flagellin treatment did not implicated the anti-bacterial peptides calgranulins and defensin-ß2. This study shows that stimulation of innate immunity by a TLR5 ligand is a potent antibacterial treatment in CS-exposed mice, suggesting innovative therapeutic strategies against acute exacerbation in COPD.
Asunto(s)
Flagelina/uso terapéutico , Infecciones por Haemophilus/prevención & control , Humo/efectos adversos , Receptor Toll-Like 5/agonistas , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Citocinas/análisis , Flagelina/genética , Flagelina/metabolismo , Flagelina/farmacología , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/patología , Haemophilus influenzae/aislamiento & purificación , Interleucinas/deficiencia , Interleucinas/genética , Interleucinas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/etiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Nicotiana , Receptor Toll-Like 5/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Interleucina-22RESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Flagelina/uso terapéutico , Inmunoterapia , Nanopartículas/uso terapéutico , Neoplasias/terapia , Terapia Fototérmica , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Línea Celular Tumoral , Femenino , Flagelina/administración & dosificación , Flagelina/inmunología , Células HEK293 , Humanos , Inmunosenescencia/efectos de los fármacos , Inmunosenescencia/efectos de la radiación , Inmunoterapia/métodos , Rayos Infrarrojos/uso terapéutico , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Neoplasias/inmunología , Neoplasias/patología , Terapia Fototérmica/métodos , Receptor Toll-Like 5/antagonistas & inhibidores , Receptor Toll-Like 5/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de la radiación , Vibrio vulnificus/inmunologíaAsunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/terapia , Flagelina/inmunología , Flagelina/uso terapéutico , Inmunidad Innata/inmunología , Neutrófilos/inmunología , Neumonía Viral/inmunología , Neumonía Viral/terapia , Receptor Toll-Like 5/inmunología , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , Betacoronavirus/inmunología , COVID-19 , Proteínas de Unión al Calcio/metabolismo , Quimioterapia Adyuvante , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/prevención & control , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasa I/farmacología , Desoxirribonucleasa I/uso terapéutico , Flagelina/administración & dosificación , Humanos , Inmunidad Innata/efectos de los fármacos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/patología , Virus de la Influenza A/inmunología , Interferón beta/biosíntesis , Interferón beta/inmunología , Interleucina-18/inmunología , Ratones , Modelos Inmunológicos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Pandemias/prevención & control , Péptidos/uso terapéutico , Neumonía Viral/complicaciones , Neumonía Viral/prevención & control , Pseudomonas aeruginosa/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2 , Receptor Toll-Like 5/agonistasRESUMEN
BACKGROUND: Sepsis is the leading cause of death among critically ill patients, and no specific therapeutic agent is currently approved for the treatment of sepsis. METHODS: We assessed the effects of flagellin administration on survival, bacterial burden, and tissue injury after sepsis. In addition, we examined the effects on phagocytosis and bacterial killing in monocytes/macrophages. RESULTS: Therapeutic administration of flagellin increased bacterial clearance, decreased organ inflammation and injury, and reduced immune cell apoptosis after experimental sepsis, in a Toll-like receptor 5 (TLR5)-dependent manner. Macrophages, but not neutrophils, mediated the beneficial effects of flagellin on experimental sepsis, and flagellin induced macrophage polarization into M1 in septic mice. Flagellin treatment could directly enhance phagocytosis and bacterial killing of macrophages, but not neutrophils. Subsequent studies demonstrated that flagellin could promote phagosome formation and increase reactive oxygen species (ROS) levels in macrophages. Finally, we found that the expression of TLR5 was significantly elevated on the surface of circulating monocytes, but not neutrophils, from patients with sepsis. Higher expression levels of TLR5 on monocytes were associated with increased mortality, documented bacteremia, and higher Sequential Organ Failure Assessment scores of the septic patients. Moreover, flagellin treatment rescued the impaired phagocytosis and bacterial killing ability of monocytes/macrophages from patients who died of sepsis. CONCLUSIONS: These novel findings not only established the potential value of application of flagellin as an immunoadjuvant in treating sepsis, but also provided new insights into targeted therapeutic strategy on the basis of monocyte TLR5 expression in septic patients.
Asunto(s)
Flagelina/farmacología , Sepsis/tratamiento farmacológico , Receptor Toll-Like 5/análisis , Animales , Carga Bacteriana/estadística & datos numéricos , Modelos Animales de Enfermedad , Flagelina/uso terapéutico , Inflamación/sangre , Inflamación/metabolismo , Inflamación/fisiopatología , Ratones , Factores Protectores , Sepsis/fisiopatología , Análisis de Supervivencia , Receptor Toll-Like 5/sangreRESUMEN
BACKGROUND: Flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system. METHODS: The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR. RESULTS: The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine. CONCLUSIONS: The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos Bacterianos/inmunología , Escherichia coli/genética , Flagelina/genética , Flagelina/inmunología , Salmonella typhimurium/genética , Adyuvantes Inmunológicos/uso terapéutico , Adyuvantes Farmacéuticos , Antígenos Bacterianos/genética , Citocinas/metabolismo , ADN Bacteriano/genética , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/uso terapéutico , Flagelina/uso terapéutico , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Vectores Genéticos , Leucocitos Mononucleares/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéutico , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Análisis de Secuencia , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas de ADN/genética , Vacunas de ADN/uso terapéuticoRESUMEN
As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin (tFL) with deletion of the hypervariable regions was adopted as a carrier-adjuvant by chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a heterobifunctional polyethylene glycol (PEG) linker. After booster immunization in mice without any extra adjuvants, the M312-PEG-tFL conjugates elicited M312-specific antibody titers 100-1000 times higher than M312 and 10-100 times higher than the physical mixture of M312 and tFL. The elicited specific antibodies could recognize the native parasites, and the immunofluorescence assay (IFA) titer was 2100 for M312-P5k-tFL, which was about 7 times higher than M312. Furthermore, the IFA titers of the conjugates were comparable to the positive control of complete Freund's adjuvant (CFA). Compared to M312, the M312-PEG-tFL conjugates enhanced the proliferation index, lymphocyte activation, and memory T-cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could slightly trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more-efficient innate immune response was provoked by the M312-PEG-tFL conjugates, as determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen to tFL could induce strong humoral and moderate cellular immune responses. Thus, conjugation of antigen to tFL as a potent carrier-adjuvant is an effective strategy for developing a promising protein-based vaccine.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Flagelina/uso terapéutico , Inmunidad Humoral , Vacunas contra la Malaria/uso terapéutico , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Vacunas Conjugadas/uso terapéutico , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Formación de Anticuerpos , Flagelina/química , Flagelina/farmacología , Inmunidad Celular , Vacunas contra la Malaria/química , Vacunas contra la Malaria/farmacología , Malaria Falciparum/inmunología , Ratones Endogámicos BALB C , Vacunas Conjugadas/química , Vacunas Conjugadas/farmacologíaRESUMEN
Salmonella Typhimurium, engineered to express flagellin B, recently demonstrated unprecedented tumor control through a TLR-dependent mechanism. Here, we review new observations that support the potential of utilizing modified bacteria to enhance antitumor immunity. We also discuss the implications of these findings for clinical applications, including immune checkpoint blockade therapies.
Asunto(s)
Flagelina/inmunología , Flagelina/uso terapéutico , Inmunoterapia/métodos , Neoplasias/terapia , Salmonella typhimurium/inmunología , Animales , Flagelina/genética , Humanos , Neoplasias/inmunología , Salmonella typhimurium/genética , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa.
Asunto(s)
Infecciones por Salmonella/prevención & control , Vacunas contra la Salmonella/uso terapéutico , Salmonella typhimurium , África del Sur del Sahara , Animales , Anticuerpos Antibacterianos/sangre , Modelos Animales de Enfermedad , Femenino , Flagelina/inmunología , Flagelina/uso terapéutico , Glicoconjugados/inmunología , Glicoconjugados/uso terapéutico , Humanos , Inmunoglobulina G/sangre , Ratones , Antígenos O/inmunología , Antígenos O/uso terapéutico , Análisis de Regresión , Infecciones por Salmonella/inmunología , Vacunas contra la Salmonella/inmunología , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/uso terapéuticoRESUMEN
Prophylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment of Streptococcus pneumoniae respiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treat S. pneumoniae-infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection against S. pneumoniae dissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.
Asunto(s)
Antibacterianos/uso terapéutico , Flagelina/uso terapéutico , Infecciones Neumocócicas/tratamiento farmacológico , Receptor Toll-Like 5/agonistas , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Amoxicilina/uso terapéutico , Animales , Femenino , Inmunidad Innata/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/patogenicidadRESUMEN
BACKGROUND: The present study was designed to observe the effects of the bacterial component flagellin on anti-sepsis protection through TLR-5, VCAN and IL-1RN. METHODS: A clinically relevant model of sepsis was induced by cecal ligation and puncture (CLP). An in vitro culture of endothelial cells was analyzed. RESULTS: Flagellin induced anti-sepsis protection through inhibition of inflammation and induction of endothelial proliferation by down-regulating the expression of TLR 3, TLR 4, and IL-1RN and promoting the expression of VCAN in mice 24 h post-CLP. In vitro, flagellin promoted the proliferation of endothelial cells. These effects could be inhibited by transfection of endothelial cells with VCAN siRNA or IL-1RN over-expression constructs. VCAN expression decreased after transfection of the cells with an IL-1RN over-expression construct and increased after transfection of the cells with an IL-1RN siRNA construct. IL-1RN expression remained unchanged after transfection of the cells with VCAN over-expression or siRNA constructs. CONCLUSIONS: These data suggest that flagellin pretreatment promoted anti-sepsis protection through the TLR-5, IL-1RN and VCAN pathway. This pathway is necessary to mediate endothelial repair and thereby promote survival following sepsis challenge.
Asunto(s)
Antiinflamatorios/uso terapéutico , Flagelina/uso terapéutico , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Sepsis/tratamiento farmacológico , Receptor Toll-Like 5/inmunología , Versicanos/inmunología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/análisis , Proteína Antagonista del Receptor de Interleucina 1/genética , Pulmón/efectos de los fármacos , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Sepsis/genética , Sepsis/inmunología , Sepsis/microbiología , Receptor Toll-Like 5/análisis , Receptor Toll-Like 5/genética , Versicanos/análisis , Versicanos/genéticaRESUMEN
Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory ("N2"; IL-10(+) IL-12(-)) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil ("N1"; IL-10(-)IL-12(+)) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.
Asunto(s)
Flagelina/farmacología , Factores Inmunológicos/farmacología , Interleucina-10/fisiología , Interleucina-12/fisiología , Neutrófilos/inmunología , Infecciones por Pseudomonas/prevención & control , Sepsis/prevención & control , Animales , Quemaduras/complicaciones , Quemaduras/tratamiento farmacológico , Quemaduras/inmunología , Polaridad Celular , Femenino , Flagelina/uso terapéutico , Inmunidad Innata , Factores Inmunológicos/uso terapéutico , Interleucina-10/antagonistas & inhibidores , Interleucina-12/antagonistas & inhibidores , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Infecciones por Pseudomonas/etiología , Sepsis/etiología , Receptores Toll-Like/metabolismoRESUMEN
Radiation-induced acute intestinal toxicity remains a major limitation to the delivery of tumoricidal doses of colorectal irradiation. Recent reports indicate that Toll-like receptor (TLR) agonists TLR4 and TLR5 protect against toxicity due to intestinal irradiation. The phenotype (M1 or M2) of macrophages expressing TLRs may play a role in tissue repair. The aim was to investigate whether administration of TLR4 agonist lipopolysaccharide (LPS) or TLR5 agonist flagellin after irradiation modified the recruitment and phenotype of colonic macrophages and improved tissue damage. Rats were exposed to single 20- or 27-Gy doses of colorectal irradiation. TLR4 agonist LPS or TLR5 agonist flagellin (at 50 or 200 µg/rat) was administered i.p. 3 days after irradiation. Flow cytometric analysis, immunostaining, and real-time polymerase chain reaction analysis were used to assess the M1/M2 phenotype and crypt cell proliferation 7 days after irradiation. Irradiation (20 and 27 Gy) increased TLR4⺠and TLR5⺠macrophage frequency in the mucosa. LPS or flagellin administration maintained this elevated frequency after the 27-Gy irradiation. LPS and flagellin drove macrophages toward the anti-inflammatory M2 phenotype by increasing Arg1 and CD163 expression and microenvironmental effector molecules (C-C motif chemokine 22, transforming growth factor-ß1, and interleukin-10). Proliferating cell nuclear antigen immunostaining, Ki67 expression, and antimicrobial factor Reg3γ showed that the M2 shift correlated with epithelial regeneration. In conclusion, administration of either LPS or flagellin after colorectal irradiation may provide effective protection against epithelial remodeling. This tissue repair was associated with an M2 macrophage shift. Using TLR agonists to moderately activate innate immunity should be considered as a strategy for protecting healthy tissue from irradiation.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Colon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Macrófagos/efectos de los fármacos , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 5/agonistas , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Colon/inmunología , Colon/fisiología , Colon/efectos de la radiación , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/radioterapia , Relación Dosis-Respuesta en la Radiación , Flagelina/uso terapéutico , Mucosa Intestinal/inmunología , Mucosa Intestinal/fisiología , Mucosa Intestinal/efectos de la radiación , Ligandos , Lipopolisacáridos/uso terapéutico , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/efectos de la radiación , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Masculino , Terapia Molecular Dirigida , Traumatismos Experimentales por Radiación/inmunología , Traumatismos Experimentales por Radiación/metabolismo , Ratas , Ratas Sprague-Dawley , Recto/efectos de los fármacos , Recto/inmunología , Recto/fisiología , Recto/efectos de la radiación , Regeneración/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 5/metabolismoRESUMEN
Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar. Seu processo fisiopatológico é desencadeado pela expressão de fatores de virulência, que proporcionam sua invasão e sobrevivência nas células do hospedeiro, ativando o sistema imune inato e adaptativo da mucosa intestinal. Trabalhos recentes têm salientado a importância do sistema de secreção e da flagelina bacteriana como agonista de receptores da imuninade inata dos macrófagos, em especial alguns dos receptores do tipo NLR. Uma vez que esta espécie de E. coli também é capaz de expressar flagelina e fazer a montagem completa do flagelo e do sistema de secreção do tipo III, a nossa proposta foi avaliar o papel da flagelina e do sistema de secreção de EIEC na resposta imune dos macrófagos murinos. Para isso, utilizamos três cepas de EIEC: a cepa selvagem; a cepa mutante no gene responsável pela síntese da flagelina; e a cepa sem o plasmídio de virulência plnv, deficiente no sistema de secreção, para a infecção de macrófagos peritoniais de camundongos C57BI/6, caspase-1-/-, IPAF-/- e ASC-/-. Neste estudo foi possível observar que o escape bacteriano e a morte dos macrófagos infectados por EIEC, assim como a ativação da caspase-1 e posterior secreção de IL-1ß é independente da flagelina bacteriana, mas dependente do sistema de secreção, além disso, a ativação da caspase-1 de macrófagos infectados por EIEC é dependente do receptor IPAF e parcialmente da proteína adaptadora ASC. Assim, no nosso modelo, a ativação da caspase-1 dos macrófagos infectados por EIEC parece estar envolvida com o processamento e secreção de IL-1ß e, possivelmente na secreção de IL-18, mas não na morte celular. No modelo de infecção in vivo, o sistema de secreção bacteriano foi importante para a sobrevivência bacteriana no hospedeiro, assim como para a indução de uma resposta inflamatória no local da infecção. Ainda, a caspase-1 parece ter um papel importante para o controle da infecção in vivo por EIEC, podendo assim contribuir para uma resposta imune protetora do hospedeiro
Enteroinvasive Escherichia coli (EIEC) is one of the etiologic agents responsible for bacillary dysentery. The pathophysiological process induced by this bacteria is triggered by the expression of virulence factors that provide the invasion and survival in host cells, resulting in activation of innate and adaptive immune system present on intestinal mucosa. Recent studies have emphasized the importance of the secretion system and bacterial flagellin as agonist of innate immune receptors present in macrophage, especially NLR (Nod like receptors). Then, our proposal was evaluate the role of flagellin (f1iC) and secretion system of EIEC in the induction of immune response of murine macrophages using the EIEC strains wild type (WT), mutant flagellin gene (f1iC), and a strain deficient in secretion system (DSS) for infection of peritoneal macrophages of C57Bl/6, caspase-1-/-, IPAF-/- and ASC-/-- mice. In this study we observed that the bacterial escape and death of infected macrophages with EIEC, the caspase-1 activation and subsequent IL-1ß secretion is independent of bacterial flagellin, but dependent of secretion system, moreover, the caspase-1 activation in infected macrophages is IPAF-dependent and partially dependent of the adapter protein ASC. Thus, in our model, the caspase-1 activation in EIEC infected macrophages seems to be involved with the processing and secretion of IL-1ß and possibly with the secretion of IL-18, but not involved with cell death. In the infection model in vivo, bacterial secretion system was important for bacterial survival in the host, as well as for the inflammatory response induction at the infection site. In addition, caspase-1 seems to have an important role to the control of in vivo infection by EIEC and can contribute to a protective immune response of the host
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Proteínas Inflamatorias de Macrófagos/agonistas , Escherichia coli/patogenicidad , Flagelina , Flagelina/uso terapéutico , Activación de Macrófagos/efectos de los fármacos , Diarrea , Piroptosis , Inflamación , Macrófagos/patologíaRESUMEN
This study was aimed to investigate the prophylactic effect of Toll like receptor (TLR)5 agonist flagellin on acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its possible mechanism. The animal model with allo-HSCT aGVHD was established by using purebred mice (male mouse C57BL/6 as donor, female mouse BALB/c as recipient) with complete-unidentical major histocompatibility antigen. The recipient mice were randomly divided into 3 groups: group 1 in which mice were injected with high purity (95%) flagellin before and after allo-HSCT respectively, group 2 in which mice received allo-HSCT without injection of flagellin, group 3 in which mice were radiated alone. The aGVHD features of mice in group 1 and 2 were observed and compared. The results showed that the typical symptoms of aGVHD appeared in transplanted mice. The death peak of mice in group 2 appeared at day 4-5 after transplantation. The aGVHD symptoms were obviously alleviated and the mean survival time was prolonged significantly in mice group 1 as compared with mice in group 2 (P < 0.05). The comparison of WBC count in peripheral blood of mice in 3 groups before transplantation showed no significant difference (P > 0.05), while WBC count of mice in group 1 and 2 showed the significant difference at days 14 and 21 after transplantation (P < 0.05). The pathological appearances of aGVHD in mice of group 1 were obviously reduced as compared with mice in group 2. The flow cytometric detection of Treg cell/CD4(+) T cell levels at different time before and after transplantation demonstrated that the Treg cell level in mice of group 1 at weeks 2-4 after transplantation significantly increased as compared with mice in group 2 (P < 0.05). It is concluded that flagellin can effectively prevent the aGVHD occurrence after allo-HSCT, reduce the symptoms and pathological changes of aGVHD, obviously prolong mean survival time of mice in group 1. The mechanism of flagellin effect may be associated to increase of Treg cell level in mice after allo-HSCT.
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Flagelina/uso terapéutico , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/métodos , Animales , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores , Receptor Toll-Like 6/agonistas , Trasplante HomólogoRESUMEN
BACKGROUND: The ectodomain of matrix protein 2 (M2e) is a promising candidate for a broadly protective influenza A vaccine because it is highly conserved and antibodies to M2e are protective in animal models. STF2.4xM2e (VAX102) is a recombinant fusion protein that links four tandem copies of the M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand used as an adjuvant. The objectives of this first-in-human study were to assess the safety and immunogenicity of VAX102 given as a prime-boost regimen to healthy adults. METHODS: Sixty subjects 18-49 years old were enrolled in a multicenter, double-blind, randomized, placebo-controlled trial (Study 1). Based on pre-clincial data, initial design included doses starting at 10 µg, with an escalation plan. After reactogenicity was noted at the 10 µg dose, the trial was redesigned to evaluate 0.3, 1.0, and 3 µg doses. Following this study, 16 subjects were enrolled in Study 2, an open label, low dose study, to evaluate doses of 0.03 and 0.1 µg. In both trials, vaccine or placebo was given intramuscularly (i.m.) at 0 and 28 days. Clinical and laboratory safety assessments took place 1 and 7 days after immunization. Immune responses to M2e and flagellin were assessed by ELISA at 7, 14 and 28 days after each dose. Seroconversion was defined as a serum IgG anti-M2e antibody value ≥0.174 µg/ml and a fourfold rise in concentration. RESULTS: Doses of 0.03-1 µg were safe and well tolerated in all subjects. Doses of 0.03 and 0.1 µg produced limited immunogenicity (38% and 75% respectively), after the second dose of vaccine. Doses of 0.3 and 1.0 µg were immunogenic in 18 (75%) of 24 vaccinees after the first dose and 23 (96%) after the second dose. In the 1.0 µg group, the geometric mean M2e antibody concentration was 0.4 µg/ml after the first dose and 1.7 µg/ml after the booster dose. M2e antibody concentrations and seroconversion rates were not significantly different at higher doses (p>0.05). Immune response to flagellin was robust but did not appear to interfere with M2e antibody responses after the booster dose. Following the first injection of VAX102 at higher doses (3 and 10 µg), self-limited but severe symptoms were noted in some subjects and were associated with elevated levels of C-reactive protein. Although not directly measured, this reaction was believed to be mediated by cytokine release. CONCLUSIONS: VAX102 was safe and induced high antibody levels to M2e at 0.3 and 1.0 µg doses. The TLR5 ligand, S. typhimurium flagellin, is a novel approach to adjuvant-like activity through activation of innate immunity, and when fused to multiple copies of the M2e protein, the vaccine was able to induce a fourfold rise in antibody in humans, to a previously non-immunogenic, highly-conserved portion of the influenza virus. Clinical correlates of protection that may be afforded by M2e antibody in humans are a future focus of investigation.
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Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Adolescente , Adulto , Anticuerpos/sangre , Proteína C-Reactiva/análisis , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Flagelina/inmunología , Flagelina/uso terapéutico , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Masculino , Persona de Mediana Edad , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND AND AIMS: Radiotherapy for neoplastic disease is associated with significant adverse enteric effects associated with excessive cell death. Ionising radiation induces cell death by a mechanism that is dependent on JNK (c-jun N-terminal kinase) pathway signalling. Additionally, it is known that cells exposed to extracellular bacterial products such as flagellin, pleiotropically activate a number of innate immune pathways, including that of JNK. The JNK pathway controls its own activity by inducing the transcription of mitogen-activated protein kinase phosphatase-7 (MKP-7) which directly targets phosphorylated JNK, thus functioning as a negative feedback loop. Previously, it has been shown that flagellin limits ionising radiation-induced mortality in mice, but the cellular mechanism of protection remained unknown. METHODS: Wild-type C57BL/6 or tlr5(-/-) C57BL/6 were injected with flagellin 2 h before exposure to irradiation, and their intestines were examined for apoptosis. Candidate proteins mediating cytoprotection from irradiation were identified by expression profiling. One of these candidates, MKP-7, was cloned and packaged into adenovirus particles, used to infect cultured cells, and examined for the extent to which its activity reduced cellular apoptosis by flow cytometry or immunoblot analysis. RESULTS: Flagellin pretreatment protected mice from radiation-induced intestinal mucosal injury and apoptosis via a Toll-like receptor 5 (TLR5)-dependent mechanism. Expression profiling of flagellin-treated mice showed upregulation of MKP-7, an inducible repressor of the JNK pathway. MKP-7 expression reached a maximum at 2 h after flagellin treatment, coinciding with suppression of phosphorylated JNK and JNK pathway inhibition. Furthermore, constitutive MKP-7 expression protected cultured cells from radiation-induced apoptosis. CONCLUSIONS: Flagellin is a promising adjuvant for suppressing ionising radiation-induced injury. MKP-7 activity exhibits cytoprotective effects, and is thus a candidate cellular molecule for limiting the damaging effect of radiotherapy on the gastreointestinal system.
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Flagelina/uso terapéutico , Mucosa Intestinal/efectos de la radiación , Proteína Quinasa 7 Activada por Mitógenos/fisiología , Traumatismos Experimentales por Radiación/prevención & control , Protectores contra Radiación/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Factor Inductor de la Apoptosis/antagonistas & inhibidores , Células Cultivadas , Citoprotección/genética , Evaluación Preclínica de Medicamentos/métodos , Flagelina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Fosforilación/efectos de la radiación , ARN Mensajero/genética , Traumatismos Experimentales por Radiación/enzimología , Traumatismos Experimentales por Radiación/patología , Protectores contra Radiación/farmacología , Receptor Toll-Like 5/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The bacterial protein flagellin can trigger immune responses to infections by interacting with TLR5 on APCs, and Ag-flagellin fusion proteins can act as effective vaccines. We report that flagellin-related peptides containing a His-tag and sequence related to conserved N-motif (aa 85-111) of FliC flagellin, purportedly involved in the interaction of flagellin with TLR5, can be used to target delivery of liposomal Ag to APCs in vitro and in vivo. When engrafted onto liposomes, two flagellin-related peptides, denoted as 9Flg and 42Flg, promoted strong liposome binding to murine bone marrow-derived dendritic cells and CD11c(+) splenocytes, and cell binding correlated with expression of TLR5. Liposomes engrafted with 9Flg or 42Flg induced functional MyD88-dependent maturation of dendritic cells in vivo. The vaccination of mice with 9Flg liposomes containing OVA induced OVA-specific T cell priming, increased the number of Ag-responsive IFN-gamma-producing CD8(+) T cells, and increased Ag-specific IgG(1) and IgG(2b) in serum. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA-derived plasma membrane vesicles, engrafted with 9Flg or 42Flg, potently inhibited tumor growth/metastasis and induced complete tumor regression in the majority of mice challenged with the syngeneic B16-OVA melanoma, in the lung and s.c. tumor models. Strong antitumor responses were also seen in studies using the s.c. P815 tumor model. Therefore, vaccination with Ag-containing liposomes engrafted with 9Flg or 42Flg is a powerful strategy to exploit the innate and adaptive immune systems for the development of potent vaccines and cancer immunotherapies.
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Antígenos de Neoplasias/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Flagelina/inmunología , Flagelina/uso terapéutico , Melanoma Experimental/inmunología , Melanoma Experimental/prevención & control , Péptidos/inmunología , Péptidos/uso terapéutico , Aminas/inmunología , Aminas/uso terapéutico , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/inmunología , Células CHO , Vacunas contra el Cáncer/inmunología , Línea Celular , Línea Celular Tumoral , Secuencia Conservada/inmunología , Cricetinae , Cricetulus , Sistemas de Liberación de Medicamentos/métodos , Células Hep G2 , Humanos , Liposomas , Mastocitoma/inmunología , Mastocitoma/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/inmunología , Ácido Nitrilotriacético/uso terapéutico , Péptidos/síntesis químicaRESUMEN
Antibiotics are routinely used to control bacterial infection, but the acquisition of acquired immunity following successful treatment has rarely been examined. We developed a model that allows visualization of acquired immunity during and following antibiotic treatment of typhoid. Pathogen-specific humoral and cellular immune responses were activated rapidly in antibiotic-treated mice, but were not sustained after successful antibiotic treatment and did not confer protection to secondary infection. In marked contrast, pathogen-specific Th1 and Ab responses matured over several weeks following immunization with a live vaccine strain. The deficiency in protective immunity following antibiotic treatment could be overcome by administering flagellin during antibiotic therapy. Thus, development of protective immunity is hindered by rapid therapeutic elimination of bacteria, but can be overcome by providing additional inflammatory and/or antigenic stimuli.