Differential effects of a toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli c ontain distinct ligands that activate cells via the mammalian Toll-like rec eptor (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-kappaB activation, Coexpression of both dominant ne gative proteins blocked virtually all Mtb-induced NF-kappaB activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induce d macrophage activation was not augmented by overexpression of ectopic MD-2 . Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded norm ally to Mtb, We also observed that the lipid A-like antagonist E5531 specif ically 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 inhibite d Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence o f the inhibition of TNF-alpha production by E5531, In contrast, E5531 did n ot inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-sti mulated peritoneal macrophages from TLR2- and TLR4-deficient animals produc ed 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-induc ed activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages, Together, these data suggest that Mtb-indu ced TNF-alpha production is largely dependent on TLR signaling. In contrast , Mtb-induced NO production may be either TLR independent or mediated by TL R proteins in a MyD88-independent manner.