title: Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival.
abstract: We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.
authors.affiliation: Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Aprajita
authors.initials: A
authors.lastname: Sinha
authors.forename: Anjana
authors.initials: A
authors.lastname: Singh
authors.forename: Vijaya
authors.initials: V
authors.lastname: Satchidanandam
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Natarajan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000502:physiology*; D020013:Calcium Signaling / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D015536:Down-Regulation / Q000276:immunology; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000502:physiology; D007424:Intracellular Fluid / Q000201:enzymology / Q000276:immunology / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009163:Mycobacterium bovis / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000254:growth & development* / Q000276:immunology / Q000472:pathogenicity; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D013482:Superoxide Dismutase / Q000502:physiology; D000072105:Superoxide Dismutase-1; D015854:Up-Regulation / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; D017382:Reactive Oxygen Species; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D011493:Protein Kinase C