RESUMEN
The benefits of mucosal vaccines over injected vaccines are difficult to ascertain, since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia virus Ankara expressing human immunodeficiency virus type 1 (HIV-1) gp120 (MVAgp120) prime and a HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (i.m.) administered MVAgp120 prime/gp120 boost to allow comparison of an i.m. immunization regimen to a mucosal vaccination regimen for the ability to protect against a low-dose rectal simian-human immunodeficiency virus (SHIV) challenge. A 3-fold higher antigen dose was required for intranasal (i.n.) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by i.m. immunization. gp120 fused to the adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced i.n. immunogenicity compared to gp120. MVAgp120 was more immunogenic after i.n. delivery than after gastric or rectal delivery. Using these optimized vaccines, an i.n. MVAgp120 prime/combined i.m. (gp120) and i.n. (gp120-Ad2F) boost regimen (i.n./i.m.-plus-i.n.) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (i.m./i.m.) in rabbits and nonhuman primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the i.m./i.m. nor the i.n./i.m.-plus-i.n. regimen protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the i.n. route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization.IMPORTANCE Mucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an i.n. prime followed by boosters administered by both i.n. and i.m. routes to induce serum antibody responses similar to those induced by i.m. prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally transmitted pathogens.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Inmunización Secundaria , Vacunación , Virus Vaccinia/inmunología , Vacunas contra el SIDA/genética , Administración Intranasal , Animales , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Inmunidad Mucosa , Ratones , Virus Vaccinia/genéticaRESUMEN
OBJECTIVE: IL-35 (interleukin-35) is an anti-inflammatory cytokine, which inhibits immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. It remains unknown whether atherogenic stimuli induce IL-35 and whether IL-35 inhibits atherogenic lipid-induced endothelial cell (EC) activation and atherosclerosis. EC activation induced by hyperlipidemia stimuli, including lysophosphatidylcholine is considered as an initiation step for monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the roles and mechanisms of IL-35 in suppressing lysophosphatidylcholine-induced EC activation. APPROACH AND RESULTS: Using microarray and ELISA, we found that IL-35 and its receptor are significantly induced during early atherosclerosis in the aortas and plasma of ApoE (apolipoprotein E) knockout mice-an atherosclerotic mouse model-and in the plasma of hypercholesterolemic patients. In addition, we found that IL-35 suppresses lysophosphatidylcholine-induced monocyte adhesion to human aortic ECs. Furthermore, our RNA-sequencing analysis shows that IL-35 selectively inhibits lysophosphatidylcholine-induced EC activation-related genes, such as ICAM-1 (intercellular adhesion molecule-1). Mechanistically, using flow cytometry, mass spectrometry, electron spin resonance analyses, and chromatin immunoprecipitation-sequencing analyses, we found that IL-35 blocks lysophosphatidylcholine-induced mitochondrial reactive oxygen species, which are required for the induction of site-specific H3K14 (histone 3 lysine 14) acetylation, increased binding of proinflammatory transcription factor AP-1 in the promoter of ICAM-1, and induction of ICAM-1 transcription in human aortic EC. Finally, IL-35 cytokine therapy suppresses atherosclerotic lesion development in ApoE knockout mice. CONCLUSIONS: IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Histonas/metabolismo , Interleucinas/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilación , Animales , Aorta/efectos de los fármacos , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucinas/farmacología , Lisina , Lisofosfatidilcolinas/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Procesamiento Proteico-Postraduccional , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE's pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10-producing B220(+)CD5(+) B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220(+)CD5(-) B cells from MOG-pσ1-treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1-induced B220(+)CD5(+) Bregs greatly ameliorated EAE. MOG-pσ1-, but not OVA-pσ1-induced IL-10-producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5(-) B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA(-/-) mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1-induced BTLA(+) Bregs, BTLA(-/-) mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.
Asunto(s)
Linfocitos B Reguladores/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Linfocitos B Reguladores/efectos de los fármacos , Linfocitos B Reguladores/fisiología , Antígenos CD5/genética , Antígenos CD5/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Linfocitos T Reguladores/patología , Linfocitos T Reguladores/fisiologíaRESUMEN
Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rß2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.
Asunto(s)
Células Endoteliales/inmunología , Interleucinas/fisiología , Adulto , Anciano , Animales , Aterosclerosis/sangre , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Estudios de Casos y Controles , Adhesión Celular , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/patología , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/inmunología , Femenino , Humanos , Leucocitos/inmunología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Sepsis/sangre , Sepsis/inmunología , Factor de Transcripción AP-1/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adulto JovenRESUMEN
A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-ß-producing Foxp3(-)CD39(+)CD4(+) T cells and IL-10-producing Foxp3(+)CD39(+)CD4(+) T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3(+)CD39(+)CD4(+) T cells were the source of secreted IL-35, whereas IL-27 production by CD11c(+) cells was inhibited. Inquiring into their relevance, CFA/I fimbriae-treated IL-27R-deficient (WSX-1(-/-)) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae-mediated protection was abated in EBI3(-/-) mice, accompanied by the loss of TGF-ß- and IL-10-producing Tregs. Adoptive transfer of C57BL/6 CD39(+)CD4(+) T cells to EBI3(-/-) mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II-specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3(-/-) CD39(+)CD4(+) T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-ß and IL-10.
Asunto(s)
Artritis Experimental/inmunología , Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Fimbrias Bacterianas/inmunología , Interleucina-27/inmunología , Interleucinas/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos CD/inmunología , Artritis Experimental/inducido químicamente , Factores de Transcripción Forkhead/inmunología , Interleucina-10/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Receptores de Citocinas/inmunología , Células Th17/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the ß-trefoil domain of C-terminus heavy chain (Hcßtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcßtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcßtre-Ad2F alone also induced elevated antibody production in contrast to Hcßtre alone. Plasma from Hcßtre + CT- and Hcßtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcßtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcßtre + CT-immunized mice, which only showed â¼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.
Asunto(s)
Anticuerpos Neutralizantes , Toxinas Botulínicas Tipo A/inmunología , Botulismo/inmunología , Proteínas de la Cápside/inmunología , Clostridium botulinum/inmunología , Administración Sublingual , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Neutralizantes/biosíntesis , Formación de Anticuerpos , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/toxicidad , Botulismo/complicaciones , Botulismo/genética , Botulismo/terapia , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Clostridium botulinum/patogenicidad , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/prevención & control , VacunaciónRESUMEN
Sjögren's Syndrome (SjS) results in loss of salivary and lacrimal gland excretion due to an autoimmune attack on these secretory glands. Conventional SjS treatments address the symptoms, but not the cause of disease. Recognizing this deficit of treatments to reverse SjS disease, studies were pursued using the fimbriae from enterotoxigenic E. coli, colonization factor antigen I (CFA/I), which has anti-inflammatory properties. To determine if CFA/I fimbriae could attenuate SjS-like disease in C57BL/6.NOD-Aec1Aec2 (SjS) females, the Lactococcus lactis (LL) 301 strain was developed to chromosomally express the cfaI operon. Western blot analysis confirmed CFA/I protein expression, and this was tested in SjS females at different stages of disease. Repeated dosing with LL 301 proved effective in mitigating salivary flow loss and in reducing anti-nuclear antibodies (ANA) and inflammation in the submandibular glands (SMGs) in SjS females and in restoring salivary flow in diseased mice. LL 301 treatment reduced proinflammatory cytokine production with concomitant increases in TGF-ß+ CD25+ CD4+ T cells. Moreover, LL 301 treatment reduced draining lymph and SMG follicular T helper (Tfh) cell levels and proinflammatory cytokines, IFN-γ, IL-6, IL-17, and IL-21. Such evidence points to the therapeutic capacity of CFA/I protein to suppress SjS disease and to have restorative properties in combating autoimmune disease.
Asunto(s)
Lactococcus lactis , Síndrome de Sjögren , Femenino , Animales , Ratones , Síndrome de Sjögren/genética , Síndrome de Sjögren/terapia , Escherichia coli , Lactococcus lactis/genética , Ratones Endogámicos NOD , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Stimulation of Ag-specific inducible Treg can enhance resolution of autoimmune disease. Conventional methods to induce Treg often require induction of autoimmune disease or subjection to infection. Reovirus adhesin, protein σ1 (pσ1), can successfully facilitate tolerance when fused to a tolerogen. We tested whether myelin oligodendrocyte glycoprotein (MOG) fused to pσ1 (MOG-pσ1) can stimulate Ag-specific Treg. We show that C57BL/6 mice treated nasally with MOG-pσ1 fail to induce MOG-specific Abs and delayed-type hypersensitivity (DTH) responses and resist EAE. Such resistance was attributed to stimulation of Foxp3(+) Treg, as well as Th2 cells. MOG-pσ1's protective capacity was abrogated in IL-10(-/-) mice, but restored when adoptively transferred with MOG-pσ1-induced Treg. As a therapeutic, MOG-pσ1 diminished EAE within 24 h of nasal application, unlike recombinant MOG (rMOG), pσ1, or pσ1+rMOG, implicating the importance of Ag specificity by pσ1-based therapeutics. MOG-pσ1-treated mice showed elevated IL-4, IL-10, and IL-28 production by CD4(+) T cells, unlike rMOG treated or control mice that produced elevated IFN-γ or IL-17, respectively. These data show the feasibility of using pσ1 as a tolerogen platform for Ag-specific tolerance induction and highlight its potential use as an immunotherapeutic for autoimmunity.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Interleucina-10/inmunología , Linfocitos T Reguladores/inmunología , Vacunación/métodos , Traslado Adoptivo , Animales , Proteínas de la Cápside/administración & dosificación , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Recuento de Células , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/diagnóstico , Femenino , Glicoproteínas/inmunología , Hipersensibilidad Tardía/inmunología , Tolerancia Inmunológica/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/administración & dosificación , Glicoproteína Asociada a Mielina/genética , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Médula Espinal/citología , Médula Espinal/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplante , Células Th2/inmunología , Células Th2/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4(+) T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT(+) NK and T(eff) cells and BTLA(+) B cells. Further characterization revealed these activated IKDCs being MHC class II(high), and upon their adoptive transfer (CD11c(+)NK1.1(+)MHC class II(high)), IKDCs, but not CD11c(+)NK1.1(+)MHC class II(intermediate/low) (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM(+) IKDCs to resolve autoimmune disease.
Asunto(s)
Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Proteínas de la Mielina/metabolismo , Proteínas Virales/metabolismo , Traslado Adoptivo , Animales , Antígenos Ly/metabolismo , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/patología , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Tolerancia Inmunológica , Interferones/metabolismo , Activación de Linfocitos , Ratones , Proteínas de la Mielina/genética , Proteínas de la Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Recurrencia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-ß, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-ß in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.
Asunto(s)
Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Lactococcus lactis/inmunología , Probióticos/administración & dosificación , Administración Oral , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Glucemia/análisis , Linfocitos T CD4-Positivos , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Proteínas Fimbrias/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ganglios Linfáticos/citología , Ratones , Ratones Transgénicos , Cultivo Primario de Células , Bazo/citología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Sjögren's syndrome (SjS), one of the most common autoimmune diseases, impacts millions of people annually. SjS results from autoimmune attack on exocrine (salivary and lacrimal) glands, and women are nine times more likely to be affected than men. To date, no vaccine or therapeutic exists to treat SjS, and patients must rely on lifelong therapies to alleviate symptoms. METHODS: Oral treatment with the adhesin from enterotoxigenic Escherichia coli colonization factor antigen I (CFA/I) fimbriae protects against several autoimmune diseases in an antigen (Ag)-independent manner. Lactococcus lactis, which was recently adapted to express CFA/I fimbriae (LL-CFA/I), effectively suppresses inflammation by the induction of infectious tolerance via Ag-specific regulatory T cells (Tregs), that produce IL-10 and TGF-ß. To test the hypothesis that CFA/I fimbriae can offset the development of inflammatory T cells via Treg induction, oral treatments with LL-CFA/I were performed on the spontaneous, genetically defined model for SjS, C57BL/6.NOD-Aec1Aec2 mice to maintain salivary flow. RESULTS: Six-week (wk)-old C57BL/6.NOD-Aec1Aec2 mice were orally dosed with LL-CFA/I and treated every 3 wks; control groups were given L. lactis vector or PBS. LL-CFA/I-treated mice retained salivary flow up to 28 wks of age and showed significantly reduced incidence of inflammatory infiltration into the submandibular and lacrimal glands relative to PBS-treated mice. A significant increase in Foxp3+ and IL-10- and TGF-ß-producing Tregs was observed. Moreover, LL-CFA/I significantly reduced the expression of proinflammatory cytokines, IL-6, IL-17, GM-CSF, and IFN-γ. Adoptive transfer of CD4+ T cells from LL-CFA/I-treated, not LL vector-treated mice, restored salivary flow in diseased SjS mice. CONCLUSION: These data demonstrate that oral LL-CFA/I reduce or halts SjS progression, and these studies will provide the basis for future testing in SjS patients.
Asunto(s)
Escherichia coli Enterotoxigénica , Lactococcus lactis , Síndrome de Sjögren , Animales , Modelos Animales de Enfermedad , Proteínas Fimbrias , Humanos , Lactococcus lactis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Modelos Genéticos , Síndrome de Sjögren/genética , Linfocitos T ReguladoresRESUMEN
BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.
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Adenoviridae/metabolismo , Toxinas Botulínicas/inmunología , Vacunación/métodos , Proteínas Virales/metabolismo , Administración Intranasal , Animales , Linfocitos B/inmunología , Toxinas Botulínicas/química , Toxinas Botulínicas Tipo A , Botulismo/inmunología , Botulismo/prevención & control , Toxina del Cólera/inmunología , Citocinas/inmunología , Inmunoensayo , Inyecciones Intramusculares , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Análisis de Supervivencia , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic TNF-α+CD8+ T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ+CD4+ T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet+CD4+ T cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.
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Diabetes Mellitus Tipo 1/prevención & control , Proteínas Fimbrias/uso terapéutico , Administración Oral , Animales , Modelos Animales de Enfermedad , Femenino , Proteínas Fimbrias/administración & dosificación , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Linfocitos T Reguladores/fisiologíaRESUMEN
BACKGROUND & AIMS: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. METHODS: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-psigma1), psigma1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined. RESULTS: A low dose of OVA-psigma1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-psigma1 than those fed psigma1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) production compared with nontolerized mice. CONCLUSIONS: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.
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Linfocitos T CD4-Positivos/inmunología , Proteínas de la Cápside/inmunología , Tolerancia Inmunológica , Mucosa Bucal/inmunología , Ovalbúmina/inmunología , Administración Oral , Animales , Formación de Anticuerpos , Epítopos , Inmunidad Mucosa , Inmunización , Interferón gamma/análisis , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB C , Mucosa Bucal/citología , Ovalbúmina/administración & dosificación , Factor de Crecimiento Transformador beta1/análisisRESUMEN
Previous studies have shown that mucosal application of interleukin-12 (IL-12) can stimulate elevated secretory immunoglobulin A (IgA) responses. Since possible exposure to plague is via Yersinia pestis-laden aerosols that results in pneumonic plague, arming both the mucosal and systemic immune systems may offer an added benefit for protective immunity. Two bicistronic plasmids were constructed that encoded the protective plague epitopes, capsular antigen (F1-Ag) and virulence antigen (V-Ag) as a F1-V fusion protein but differed in the amounts of IL-12 produced. When applied nasally, serum IgG and mucosal IgA anti-F1-Ag and anti-V-Ag titers were detectable beginning at week 6 after three weekly doses, and recombinant F1-Ag boosts were required to elevate the F1-Ag-specific antibody (Ab) titers. Following pneumonic challenge, the best efficacy was obtained in mice primed with IL-12(Low)/F1-V vaccine with 80% survival compared to mice immunized with IL-12(Low)/F1, IL-12(Low)/V, or IL-12(Low) vector DNA vaccines. Improved expression of IL-12 resulted in lost efficacy when using the IL-12(High)/F1-V DNA vaccine. Despite differences in the amount of IL-12 produced by the two F1-V DNA vaccines, Ab responses and Th cell responses to F1- and V-Ags were similar. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague, but in a dose-dependent fashion.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Interleucina-12/inmunología , Vacuna contra la Peste/inmunología , Peste/prevención & control , Proteínas Citotóxicas Formadoras de Poros/inmunología , Vacunas de ADN/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Citocinas/metabolismo , Femenino , Inmunidad Mucosa , Inmunización Secundaria , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Interleucina-12/genética , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Vacuna contra la Peste/administración & dosificación , Vacuna contra la Peste/genética , Plásmidos , Proteínas Citotóxicas Formadoras de Poros/genética , Bazo/inmunología , Análisis de Supervivencia , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3+ and Foxp3- CD39+ CD4+ T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6+ and CCR6- Foxp3+or- CD39+ CD4+ T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases.
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Artritis Experimental/terapia , Linfocitos B Reguladores/inmunología , Interleucinas/inmunología , Lactococcus lactis/inmunología , Linfocitos T Reguladores/inmunología , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Linfocitos B Reguladores/patología , Interleucinas/genética , Lactococcus lactis/genética , Masculino , Ratones , Linfocitos T Reguladores/patologíaRESUMEN
Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-ß and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.
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Antígenos CD/metabolismo , Apirasa/metabolismo , Artritis Experimental/tratamiento farmacológico , Suplementos Dietéticos , Interleucina-10/metabolismo , Leche/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Efecto Espectador/inmunología , Tolerancia Inmunológica/inmunología , Ratones , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
PURPOSE: In an effort to develop a non-lethal model of ricin toxicosis, we studied the biochemical effects of the administration of ricin in mice. METHODS: Mice received an intraperitoneal injection of ricin toxin, after which a panel of 21 biochemical parameters was determined. Effect of ricin administration on blood glucose concentration was studied in greater detail. To determine whether results of biochemical analyses correlated with therapeutic manipulations known to protect against ricin toxicosis, the effect of immunization on blood glucose values after ricin administration was studied. RESULTS: Of the biochemical parameters studied, only blood glucose and amylase values were affected by ricin administration. Because blood glucose concentration can be easily and instantaneously measured on 25 to 50 microl of blood, using handheld instruments, this parameter was further evaluated. A dose- and time-related decrease in blood glucose concentration was documented. Mice immunized with ricin had smaller changes in blood glucose concentration than did non-immunized animals after ricin administration. CONCLUSION: Blood glucose concentration may be used as a surrogate for lethal challenge as a measure of ricin toxicosis after its systemic administration.
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Sustancias para la Guerra Química/toxicidad , Hipoglucemia/inducido químicamente , Ricina/toxicidad , Animales , Animales no Consanguíneos , Biomarcadores , Glucemia , Pruebas de Química Clínica , Relación Dosis-Respuesta a Droga , Femenino , Hipoglucemia/sangre , Hipoglucemia/prevención & control , Inyecciones Intraperitoneales , Masculino , Ratones , Ricina/administración & dosificación , Ricina/inmunología , VacunaciónRESUMEN
To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-ß and interleukin-10.
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Antígenos Bacterianos/inmunología , Autoantígenos/inmunología , Proteínas Fimbrias/inmunología , Salmonella/inmunología , Linfocitos T Reguladores/inmunología , Vacunación , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/prevención & control , Humanos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/prevención & controlRESUMEN
Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+) and CD8(+) T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses.