title: Differential signaling of inducible nitric oxide synthase induction in Mycobacterium tuberculosis infected alveolar epithelial cell line A549 in response to cytokines IFN-γ, TNF-α and IL-1β.
abstract: BACKGROUND
  In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-γ, TNF-α and IL-1β. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-κB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production.
  
  
  METHODS
  Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells.
  
  
  RESULTS
  Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-γ or a combination of three cytokines (IFN-γ, TNF-α and IL-1β) formed DNA protein complexes with probes from both -5.2kb region (specific for binding of STAT-1 protein) and -5.8kb region (specific for binding of both STAT-1 and NF-κB) of the iNOS promoter. However, TNF-α or IL-1β stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from -5.2kb region.
  
  
  CONCLUSIONS
  This differential response indicated that TNF-α/IL-1β does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-γ. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.
authors.affiliation: Dept. of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: sugataroy@gmail.com.
authors.forename: Sugata
authors.initials: S
authors.lastname: Roy
authors.affiliation: Dept. of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: sadhnas@rediffmail.com.
authors.forename: Sadhna
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Dept. of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: Monikasharma.mh@gmail.com.
authors.forename: Monika
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Dept. of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110007, India. Electronic address: mridulabose@hotmail.com.
authors.forename: Mridula
authors.initials: M
authors.lastname: Bose
keywords: Alveolar epithelial cell line A549; NF-κB; STAT-1; iNOS gene