RESUMEN
A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1ß, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Inflamación/inmunología , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Adulto , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/inmunologíaRESUMEN
Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⺠T-cell responses to viral and tumour antigens.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Vacuna contra Viruela/inmunología , Virus Vaccinia/inmunología , Vaccinia/prevención & control , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/virología , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Línea Celular , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Vacuna contra Viruela/administración & dosificación , Vacuna contra Viruela/genética , Vacuna contra Viruela/metabolismo , Factores de Tiempo , Transfección , Vacunación , Vacunas Sintéticas/inmunología , Vaccinia/inmunología , Vaccinia/metabolismo , Vaccinia/virología , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas Virales/genéticaRESUMEN
CD8 T cells can acquire cytokine-secreting phenotypes paralleling cytokine production from Th cells. IL-17-secreting CD8 T cells, termed Tc17 cells, were shown to promote inflammation and mediate immunity to influenza. However, most reports observed a lack of cytotoxic activity by Tc17 cells. In this study, we explored the anti-viral activity of Tc17 cells using a vaccinia virus (VV) infection model. Tc17 cells expanded during VV infection, and TCR transgenic Tc17 cells were capable of clearing recombinant VV infection. In vivo, adoptively transferred Tc17 cells lost the IL-17-secreting phenotype, even in the absence of stimulation, but they did not acquire IFN-gamma-secreting potential unless stimulated with a virus-encoded Ag. However, examination of cells following infection demonstrated that these cells acquired cytotoxic potential in vivo, even in the absence of IFN-gamma. Cytotoxic potential correlated with Fasl expression, and the cytotoxic activity of postinfection Tc17 cells was partially blocked by the addition of anti-FasL. Thus, Tc17 cells mediate VV clearance through expression of specific molecules associated with cytotoxicity but independent of an acquired Tc1 phenotype.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad/inmunología , Interleucina-17/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/inmunología , Factor de Transcripción STAT4/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Proteínas de Dominio T Box/metabolismo , Vaccinia/virologíaRESUMEN
Rapid evaluation of therapies designed to preserve ß cells in persons with type 1 diabetes (T1D) is hampered by limited availability of sensitive ß-cell health biomarkers. In particular, biomarkers elucidating the presence and degree of ß-cell stress are needed. We characterized ß-cell secretory activity and stress in 29 new-onset T1D subjects (10.6 ± 3.0 years, 55% male) at diagnosis and then 8.2 ± 1.2 weeks later at first clinic follow-up. We did comparisons with 16 matched healthy controls. We evaluated hemoglobin A1c (HbA1c), ß-cell function (random C-peptide [C] and proinsulin [PI]), ß-cell stress (PI:C ratio), and the ß-cell stress marker heat shock protein (HSP)90 and examined these parameters' relationships with clinical and laboratory characteristics at diagnosis. Mean diagnosis HbA1c was 11.3% (100 mmol/mol) and 7.6% (60 mmol/mol) at follow-up. C-peptide was low at diagnosis (P < 0.001 vs controls) and increased at follow-up (P < 0.001) to comparable with controls. PI did not differ from controls at diagnosis but increased at follow-up (P = 0.003) signifying increased release of PI alongside improved insulin secretion. PI:C ratios and HSP90 concentrations were elevated at both time points. Younger subjects had lower C-peptide and greater PI, PI:C, and HSP90. We also examined islets isolated from prediabetic nonobese diabetic mice and found that HSP90 levels were increased â¼4-fold compared with those in islets isolated from matched CD1 controls, further substantiating HSP90 as a marker of ß-cell stress in T1D. Our data indicate that ß-cell stress can be assessed using PI:C and HSP90. This stress persists after T1D diagnosis. Therapeutic approaches to reduce ß-cell stress in new-onset T1D should be considered.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Estrés Fisiológico/fisiología , Adolescente , Envejecimiento , Animales , Biomarcadores , Glucemia , Péptido C/sangre , Péptido C/metabolismo , Estudios de Casos y Controles , Niño , Diabetes Mellitus Tipo 1/genética , Femenino , Proteínas HSP90 de Choque Térmico/genética , Humanos , Masculino , Ratones , Proinsulina/genéticaRESUMEN
Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8(+) T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8(+) T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4(+) T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.