Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses.
We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.
Means
T K
TK
Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA.
Jones
B W
BW
Schromm
A B
AB
Shurtleff
B A
BA
Smith
J A
JA
Keane
J
J
Golenbock
D T
DT
Vogel
S N
SN
Fenton
M J
MJ
D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis / Q000502:physiology; D000995:Antitubercular Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D029721:Drosophila Proteins; D005260:Female; D005786:Gene Expression Regulation; D008050:Lipid A / Q000031:analogs & derivatives / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051218:Lymphocyte Antigen 96; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D016676:Macrophages, Alveolar / Q000166:cytology / Q000187:drug effects / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D008647:Mesocricetus; D051379:Mice; D008809:Mice, Inbred C3H; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000276:immunology; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014376:Tuberculosis / Q000401:mortality / Q000517:prevention & control; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics
D000954:Antigens, Surface; D000995:Antitubercular Agents; D029721:Drosophila Proteins; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C092845:E 5531; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse