Regulação da expressão de genes por ExoU durante a infecção por Pseudomonas aeruginosa: papel de PAFR, NF-κB e Nrf2

• Autor(a) principal: Lima, Carolina Diettrich Mallet de
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14366
• topic: Pseudomonas aeruginosa; ExoU; Type III Secretion System; Inflammation; NF-κ; Interleukin8; PAFR; Nrf2; SOD1; Pseudomonas aeruginosa; ExoU; Sistema de secreção do tipo III; Inflamação; NF-κ; Interleucina 8; PAFR; Nrf2; SOD1; Pseudomonas aeruginosa; Células epiteliais; Células endoteliais; Infecções por pseudômonas; Sistemas de secreção bacterianos; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA

ExoU, a cytotoxin injected into host cytosol by the <i>Pseudomonas aeruginosa</i> type III secretion system, exhibits a strong proinflammatory activity characterized by an important neutrophil recruitment to the infected tissues. To evaluate the mechanisms underlying the neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We showed that ExoU activates NF-κB and induces IL-8 in epithelial and endothelial cells infected with <i>P. aeruginosa</i>. Using an animal model of acute pneumonia, we showed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage of mice treated with the NF-κB inhibitor Bay 11-7082 before infection. Subsequent studies suggested that NF-κB activation occurs not only in ExoU-injected cells but also in non-infected cells of the neighborhood, possibly indicating a wide-ranging phenomenon. We next identified the key factor of NF-κB activation by ExoU: PAF receptor, PAFR. PAFR binds to PAF molecules produced by ExoU phospholipase A2 activity and stimulates the NF-κB canonic pathway, as characterized by IKK and Akt-mediated IκBα phosphorylation and degradation and p65 nuclear translocation. Additionally, reduced ExoU-mediated p65 nuclear translocation was observed in lungs of mice PAFR-/- or treated with the PAF antagonist WEB 2086. Since NF-κB can regulate PAFR transcription, we investigated the role of ExoU, PAFR and NF-κB in PAFR expression to evaluate a potential mechanism of amplification of response to ExoU. In fact, ExoU promotes p65 occupation of the three κB sites in PAFR promoter, inducing PAFR expression. These <i>in vitro</i> results were confirmed by <i>in vivo</i> assays, since the treatment with Bay 11-7082 or WEB 2086 was able to significantly reduce PAFR expression in lungs of infected mice. NF-κB can also regulate a number of other cellular processes and, since ExoU induces oxidative stress, we evaluated the effect of ExoU in superoxide dismutase 1 (SOD1) expression. The <i>in vitro</i> e <i>in vivo</i> assays showed that ExoU PLA2 activity induces SOD1 expression, in a NF-κB-dependent mechanism. Moreover, ExoU activates the transcriptional factor Nrf2 that is also necessary to SOD1 induction. Together, our data show that ExoU activates the transcriptional factors NF-κB e Nrf2 and induces the expression of genes related to inflammatory and oxidative stress responses. Furthermore, NF-κB activation by ExoU results from the interaction between PAF produced by the infected cells and PAFR, a receptor that can be expressed by infected and non-infected cells. In these cells, NF-κB activation stimulates PAFR expression, highlighting an important mechanism of amplification of response that can affect all cells of the neighborhood.