Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia.
BACKGROUND
Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb).


METHODS
Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.


RESULTS
We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.


CONCLUSIONS
These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.
Yang
Chul-Su
CS
Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, S. Korea. ironwater514@gmail.com
Lee
Hye-Mi
HM
Lee
Ji-Yeon
JY
Kim
Jeong-Ah
JA
Lee
Sung Joong
SJ
Shin
Dong-Min
DM
Lee
Young-Ho
YH
Lee
Dong-Seok
DS
El-Benna
Jamel
J
Jo
Eun-Kyeong
EK
D000818:Animals; D002460:Cell Line; D002493:Central Nervous System Diseases / Q000378:metabolism* / Q000382:microbiology*; D053762:Interleukin-12 Subunit p40 / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism; D037181:Lectins, C-Type; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000472:pathogenicity*; D019255:NADPH Oxidases / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099181:Dectin-1
D053762:Interleukin-12 Subunit p40; D053583:Interleukin-1beta; D015850:Interleukin-6; D037181:Lectins, C-Type; D008565:Membrane Proteins; D048052:Mitogen-Activated Protein Kinase 3; D019255:NADPH Oxidases; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D000099181:Dectin-1; C493486:Tlr2 protein, mouse; C060505:neutrophil cytosolic factor 1