RESUMEN
Smokers are more susceptible to respiratory infections, including influenza. To explore the effect of smoking on influenza-induced responses within the nasal mucosa, we have developed a protocol using inoculation with live attenuated influenza virus (LAIV) vaccine followed by sampling of the nasal mucosa. Mucosal cell populations were harvested through superficial biopsy of the nasal inferior turbinate pre and post LAIV inoculation and analyzed using flow cytometry. The majority of nasal biopsy CD45+ immune cells at baseline were CD3+ T lymphocytes. Following LAIV, these lymphocytes increased in nonsmokers but not in smokers. A subset of individuals was negative for helper T cell marker CD4 and cytotoxic T cell marker CD8 but positive for the γδ T cell receptor (TCR). Increases in γδ TCR+ cells were greater in nonsmokers, than in smokers. Thus, LAIV-induced changes in CD3 T as well as γδ T lymphocyte percentages are suppressed in smokers compared to nonsmokers.
Asunto(s)
Vacunas contra la Influenza/inmunología , Fumar/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Biopsia , Supervivencia Celular , Femenino , Humanos , Inmunidad Mucosa , Masculino , Estudios Prospectivos , Linfocitos T/citología , Vacunas Atenuadas/inmunología , Adulto JovenRESUMEN
Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza-induced immune response, we established a model using differentiated nasal epithelial cells (NECs) from nonsmokers and smokers, co-cultured with peripheral blood monocyte-derived dendritic cells (mono-DCs) from nonsmokers. NEC/mono-DC co-cultures were infected with influenza A virus and analyzed for influenza-induced immune responses 24 hours after infection. We observed that NECs from smokers, as well as mono-DCs co-cultured with NECs from smokers, exhibited suppressed influenza-induced, interferon-related proteins interferon regulatory factor-7, Toll-like receptor-3, and retinoic acid inducible gene-1, likely because of the suppressed production of IFNα from the NECs of smokers. Furthermore, NEC/mono-DC co-cultures using NECs from smokers exhibited suppressed concentrations of T-cell/natural killer cell chemokine interferon gamma-induced protein 10 (IP-10) after infection with influenza, indicating that NECs from smokers may skew early influenza-induced Th1 responses. In contrast, NEC/mono-DC co-cultures using NEC from smokers contained increased influenza-induced concentrations of the Th2 chemokine thymic stromal lymphopoeitin (TSLP). In addition, NECs from smokers cultured alone had increased influenza-induced concentrations of the Th2 chemokine thymus and activation-regulated chemokine (TARC). Using this model, we demonstrated that in the context of infection with influenza, NECs obtained from smokers create an overall cytokine microenvironment that suppresses the interferon-mediated Th1 response and enhances the TSLP-TARC-mediated Th2 response, with the potential to modify the responses of DCs. Smoking-induced alterations in the Th1/Th2 balance may play a role in developing underlying susceptibilities to respiratory viral infections, and may also promote the likelihood of acquiring Th2 proallergic diseases.
Asunto(s)
Células Dendríticas/inmunología , Células Epiteliales/inmunología , Gripe Humana/inmunología , Mucosa Nasal/inmunología , Fumar , Células TH1/inmunología , Células Th2/inmunología , Western Blotting , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Gripe Humana/virología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/metabolismo , Células TH1/virología , Células Th2/metabolismo , Células Th2/virologíaRESUMEN
BACKGROUND: Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers. METHODS: In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses. RESULTS: CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group. CONCLUSIONS: These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.
Asunto(s)
Vacunas contra la Influenza/administración & dosificación , Células Asesinas Naturales/inmunología , Orthomyxoviridae/inmunología , Fumar/inmunología , Administración Intranasal , Adulto , Análisis de Varianza , Antígeno CD56/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Citotoxicidad Inmunológica , Femenino , Citometría de Flujo , Proteínas Ligadas a GPI/metabolismo , Granzimas/metabolismo , Humanos , Inmunidad Innata , Inmunohistoquímica , Inmunofenotipificación/métodos , Células Asesinas Naturales/virología , Antígenos Comunes de Leucocito/metabolismo , Estudios Longitudinales , Masculino , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Líquido del Lavado Nasal/inmunología , Líquido del Lavado Nasal/virología , North Carolina , Estudios Prospectivos , Receptores CXCR3/metabolismo , Receptores de IgG/metabolismo , Factores de Tiempo , Vacunas Atenuadas/administración & dosificación , Adulto JovenRESUMEN
Smokers are more susceptible to respiratory viral infections, including influenza virus, but the mechanisms mediating this effect are unknown. To determine how epithelial cells contribute to the enhanced susceptibility seen in smokers, we established an in vitro model of differentiated nasal epithelial cells (NECs) from smokers, which showed enhanced mucin expression. The NECs from smokers responded to influenza infection with greater cytotoxicity, release of interleukin-6, and viral shedding than NECs from nonsmokers. Focusing on type I interferon (IFN) expression, we observed that influenza-infected NECs from smokers produced significantly less IFN-alpha than NECs from nonsmokers. Similarly, the expression of IRF7, a key transcription factor controlling the expression of IFN-alpha, was significantly decreased in influenza-infected and IFN-beta-stimulated NECs from smokers. Furthermore, our data indicate that the DNA methylation of the IRF7 gene and expression of the DNA (cytosine-5-)-methyltransferase 1 was enhanced in NECs from smokers. To confirm these findings in vivo, we initiated a study in which smoking and nonsmoking healthy volunteers were inoculated nasally with the live-attenuated influenza virus (LAIV) vaccine, and nasal biopsies were obtained before and after the administration of LAIV. The LAIV-induced expression of IRF7 was lower in the nasal epithelium from smokers, supporting our in vitro observations. These data demonstrate that infection with influenza results in the reduced expression of transcription factor IRF7 in NECs from smokers, and that these effects may be mediated by an epigenetic modification of the IRF7 gene, thus providing a potential mechanism rendering smokers more susceptible to respiratory virus infections.