RESUMEN
Short-chain fatty acids (SCFAs) are products of microbial fermentation that are important for intestinal epithelial health. Here, we describe that SCFAs have rapid and reversible effects on toll-like receptor (TLR) responses in epithelial cells. Incubation of HEK293 or HeLa epithelial cells with the SCFAs butyrate or propionate at physiological concentrations enhanced NF-κB activation induced by TLR5, TLR2/1, TLR4, and TLR9 agonists. NF-κB activation in response to tumor necrosis factor α (TNFα) was also increased by SCFAs. Comparative transcript analysis of HT-29 colon epithelial cells revealed that SCFAs enhanced TLR5-induced transcription of TNFα but dampened or even abolished the TLR5-mediated induction of IL-8 and monocyte chemotactic protein 1. SCFAs are known inhibitors of histone deacetylases (HDACs). Butyrate or propionate caused a rapid increase in histone acetylation in epithelial cells, similar to the small molecule HDAC inhibitor trichostatin A (TSA). TSA also mimicked the effects of SCFAs on TLR-NF-κB responses. This study shows that bacterial SCFAs rapidly alter the epigenetic state of host cells resulting in redirection of the innate immune response and selective reprograming of cytokine/chemokine expression.
RESUMEN
The Mycobacterium bovis BCG vaccine is the only tuberculosis (TB) vaccine available, yet it provides limited protection against pulmonary TB in adults and fails to protect against TB reactivation. We hypothesized that immunity against Mycobacterium tuberculosis "resuscitation-promoting factors" (Rpfs), which are small bacterial proteins that promote proliferation of dormant mycobacteria, may be relevant in the human immune response to M. tuberculosis. In previous unpublished work, we found that Rpfs Rv0867c and Rv2389c induced gamma interferon (IFN-γ) production in the blood of TB patients' healthy household contacts in several different African populations. Here we examine these two dominant Rpf antigens in more detail and define the nature of the responding T-cell subsets. Multiparameter cytokine profiling showed that Rv2389c and, to a lesser extent, Rv0867c were recognized by mycobacterium-responsive healthy Dutch individuals; peptide-scanning revealed several epitopes, including a single immunodominant epitope in Rv2389c. Rv0867c and, to a lesser extent, Rv2389c Rpf-specific T-cell responses were maintained for decades in long-term M. tuberculosis nonprogressors. Prominent Rv0867c-specific double- and single-cytokine-producing CD8(+) T-cell subset responses were found, including a large population of CD8(+) effector memory and effector T-cell subsets. We conclude that M. tuberculosis Rpf antigens are important targets in the human immune response to M. tuberculosis and represent interesting TB vaccine candidate antigens.
Asunto(s)
Proteínas Bacterianas/inmunología , Citocinas/inmunología , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Anciano , Citocinas/metabolismo , Mapeo Epitopo , Epítopos de Linfocito T/inmunología , Humanos , Países BajosRESUMEN
Mycobacterium tuberculosis DosR regulon-encoded antigens are highly immunogenic in M. tuberculosis-infected humans and are associated with latent tuberculosis infection. We have investigated the hypothesis that infection with or exposure to nontuberculous mycobacteria (NTM) can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens since responsiveness has been observed in non-M. tuberculosis-exposed but purified protein derivative-responsive individuals. M. tuberculosis DosR regulon-encoded antigen-specific T-cell responses were studied in peripheral blood mononuclear cells (PBMCs) of NTM-infected/exposed individuals. BLASTP was used to determine the presence of M. tuberculosis DosR regulon-encoded protein orthologs among environmental mycobacteria and nonmycobacteria. Significant gamma interferon production was observed in PBMCs from NTM-infected/exposed individuals in response to M. tuberculosis DosR regulon-encoded antigens. DosR regulon-encoded protein orthologs were prominently present in tuberculous and environmental mycobacteria and surprisingly also in nonmycobacteria. The ubiquitous presence of the highly conserved DosR master regulator protein Rv3133c suggests that this is a general adaptive bacterial response regulator. We report a first series of M. tuberculosis antigens to which cross-reactive immunity is induced by NTM infection/exposure. The high conservation of M. tuberculosis DosR regulon-encoded antigens most likely enables them to induce cross-reactive T-cell responses.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones por Mycobacterium/inmunología , Mycobacterium/inmunología , Proteínas Quinasas/inmunología , Adulto , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Reacciones Cruzadas , Proteínas de Unión al ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Mycobacterium/genética , Infecciones por Mycobacterium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Proteínas Quinasas/genética , Regulón/genética , Regulón/inmunología , Prueba de TuberculinaRESUMEN
During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen.
Asunto(s)
Antígenos Bacterianos/genética , Mycobacterium tuberculosis/genética , Linfocitos T/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Administración por Inhalación , Animales , Antígenos Bacterianos/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Femenino , Humanos , Inmunización Secundaria , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Ácido Láctico/administración & dosificación , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Nanopartículas/administración & dosificación , Polietileneimina/administración & dosificación , Polietileneimina/farmacología , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Vacunas contra la Tuberculosis/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas de ADN/genética , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
Mycobacterium tuberculosis is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. As most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including post-exposure/therapeutic vaccines. The current bacille Calmette-Guérin (BCG) vaccine and all new generation TB vaccines being developed and tested are essentially designed as prophylactic vaccines. Unfortunately, these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here, we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during latent stage infection may lead to the identification of new antigenic targets for anti-TB strategies. We will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on novel groups of late-stage specific genes, encoded amongst others by the M. tuberculosis dormancy (dosR) regulon, and summarise recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for vaccine development against TB.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/uso terapéutico , Tuberculosis/microbiología , Tuberculosis/prevención & control , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Genoma , Humanos , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Regulón/genética , Regulón/inmunología , Tuberculosis/inmunologíaRESUMEN
Mycobacterium tuberculosis (M. tuberculosis) is one of the worlds' most successful and sophisticated pathogens. It is estimated that over 2 billion people today harbour latent M. tuberculosis infection without any clinical symptoms. Since most new cases of active tuberculosis (TB) arise from this (growing) number of latently infected individuals, urgent measures to control TB reactivation are required, including more effective drugs and new TB vaccines. The currently widely used BCG vaccines, as well as most new generation TB-vaccines that are being developed are designed as prophylactic or as BCG-booster vaccines. Unfortunately, many of these vaccines are unlikely to be effective in individuals already latently infected with M. tuberculosis. Here we argue that detailed analysis of M. tuberculosis genes that are switched on predominantly during the latent stage of infection may lead to the identification of new M. tuberculosis targets for drug and vaccine development. First, we will describe essential host-pathogen interactions in TB with particular emphasis on TB latency and persistent infection. Subsequently, we will focus on a novel group of late-stage specific genes, encoded by the M. tuberculosis dormancy (dosR) regulon, and summarize recent studies describing human T-cell recognition of these dormancy antigens in relation to (latent) M. tuberculosis infection. We will discuss the possible relevance of these new classes of antigens for new TB intervention strategies.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Antígenos Bacterianos/inmunología , Antituberculosos/uso terapéutico , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Tuberculosis/inmunología , Tuberculosis/metabolismo , Tuberculosis/microbiología , Vacunas contra la Tuberculosis/uso terapéuticoRESUMEN
Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB), despite its variable protective efficacy. Relatively little is known about the immune response profiles following BCG vaccination in relation to protection against TB. Here we tested whether BCG vaccination results in immune responses to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens are targeted by the immune system during persistent Mycobacterium tuberculosis infection and have been associated with immunity against latent M. tuberculosis infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis showed at least 97% amino acid sequence homology, with 41 out of 48 genes being identical. Transcriptional profiling of 14 different BCG strains, under hypoxia and nitric oxide exposure in vitro, revealed a functional DosR regulon similar to that observed in M. tuberculosis. Next, we assessed human immune responses to a series of immunodominant TB latency antigens and found that BCG vaccination fails to induce significant responses to latency antigens. Similar results were obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency antigens were observed in individuals with suspected exposure to TB (as indicated by positive gamma interferon responses to TB-specific antigens ESAT-6 and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency antigens. Since immune responses to TB latency antigens have been associated with control of latent M. tuberculosis infection, our findings support the development of vaccination strategies incorporating DosR regulon antigens to complement and improve the current BCG vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacuna BCG/administración & dosificación , Proteínas Bacterianas/inmunología , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/fisiología , Regulón/inmunología , Tuberculosis/prevención & control , Adolescente , Adulto , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Vacuna BCG/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bovinos , Femenino , Humanos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Regulón/genética , Tuberculosis/inmunología , Tuberculosis/microbiología , VacunaciónRESUMEN
During stationary growth or in vitro conditions mimicking relevant aspects of latency, the HspX protein (Rv2031c) is specifically upregulated by Mycobacterium tuberculosis. In this study we compared T-cell responses against HspX and the secreted M. tuberculosis protein Ag85B (Rv1886c) in tuberculosis (TB) patients, tuberculin skin test-positive individuals, M. bovis BCG-vaccinated individuals, and healthy negative controls. Gamma interferon responses to HspX were significantly higher in M. tuberculosis-exposed individuals than in M. tuberculosis-unexposed BCG vaccinees. In contrast, no such differences were found with respect to T-cell responses against Ag85B. Therefore, BCG-based vaccines containing relevant fragments of HspX may induce improved responses against this TB latency antigen. To identify relevant major histocompatibility complex class I- and class II-restricted HspX-specific T-cell epitopes, we immunized HLA-A2/K(b) and HLA-DR3.Ab(0) transgenic (tg) mice with HspX. Two new T-cell epitopes were identified, p91-105 and p31-50, restricted via HLA-A*0201 and HLA-DRB1*0301, respectively. These epitopes were recognized by human T cells as well, underlining the relevance of HspX T-cell recognition both in vivo and in vitro. In line with the data in humans, BCG immunization of both tg strains did not lead to T-cell responses against HspX-derived epitopes, whereas nonlatency antigens were efficiently recognized. These data support the notion that BCG vaccination per se does not induce T-cell responses against the latency antigen, HspX. Thus, we suggest that subunit vaccines incorporating HspX and/or other latency antigens, as well as recombinant BCG strains expressing latency antigens need to be considered as new vaccines against TB.
Asunto(s)
Antígenos Bacterianos/metabolismo , Vacuna BCG/inmunología , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/inmunología , Linfocitos T/metabolismo , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Humanos , Mycobacterium bovis/inmunología , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/genética , VacunaciónRESUMEN
Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one K(d)-restricted T-cell epitope could be identified. BALB/c and (B6D2)F(1) mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.
Asunto(s)
Antígenos Bacterianos/inmunología , Mycobacterium tuberculosis/inmunología , Regulón/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Mapeo Epitopo , Epítopos de Linfocito B , Epítopos de Linfocito T , Citometría de Flujo , Vectores Genéticos , Epítopos Inmunodominantes , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Subgrupos Linfocitarios/inmunología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Plásmidos/genética , Vacunas contra la Tuberculosis/genética , Vacunas de ADN/genéticaRESUMEN
The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular , Proliferación Celular , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/fisiología , Regulón , Estadística como Asunto , Prueba de TuberculinaRESUMEN
We have investigated the role of p75NTR in inflammation in experimental allergic encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS). Induction of EAE in C57/BL6 wild-type mice resulted in expression of p75NTR in endothelial cells in the CNS. In contrast to the clinical manifestation of EAE observed in wild-type C57/BL6 mice, mice deficient for p75NTR (p75NTR knockout mice) developed severe or lethal disease and concomitant increased levels of inflammation in the CNS. Our findings suggest a physiological significant role for p75NTR in CNS endothelial cells during inflammation and involvement in preservation of blood-brain barrier integrity during a severe infiltrative attack.