Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages

Abstract

The recognition of mycobacterial cell wall components causes macrophages to secrete tumor necrosis factor alpha (TNF-alpha) and other cytokines that are essential for the development of a protective inflammatory response. We show that toll-like receptors are required for the induction of TNF-alpha in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88 (a signaling component required for toll-like receptor signaling) in a mouse macrophage cell line blocks TNF-alpha production induced by M. tuberculosis. We identify toll-like receptor-2 (TLR2) as the specific toll-like receptor required for this induction by showing that expression of an inhibitory TLR2 (TLR2-P681H) blocks TNF-alpha production induced by whole M. tuberculosis. Further, we show that TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarrabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. Thus, although many mycobacterial cell wall fractions are identified to be inflammatory, all require TLR2 for induction of TNF-alpha in macrophages. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria.

Figure 1

M. tuberculosis-induced TNF-α production depends on TLR2. RAW-TT10 cells transiently transfected with a control plasmid (Top), MyD88-dominant negative (MyD88-DN, Middle), or TLR2-P681H (Bottom) were analyzed for induction of TNF-α after stimulation with virulent M. tuberculosis (H37Rv), avirulent M. tuberculosis (H37Ra), or LPS. The thin, solid lines indicate TNF-α produced in cells not expressing the transgene, and the bold lines indicate TNF-α produced in cells expressing the indicated transgene. The dotted lines indicate background, unstimulated TNF-α production. In each case, the y axis represents relative cell number.

Figure 2

Expression of TLR2 is sufficient to mediate M. tuberculosis-induced NF-κB activation in CHO cells. The relative activity of a NF-κB-luciferase reporter was assessed in CHO cells transiently expressing the indicated constructs before stimulation (open bars) or after stimulation with heat-killed virulent (hatched bars, H37Rv) or avirulent (filled bars, H37Ra) M. tuberculosis.

Figure 3

TNF-α production induced by LAM depends on TLR2. RAW-TT10 cells transiently transfected with a control plasmid, MyD88-dominant negative (MyD88-DN), wild-type TLR2, or dominant negative TLR2 (TLR2-P681H) were analyzed for induction of TNF-α after stimulation with AraLAM. Histograms are formatted as described in Fig. 1.

Figure 4

Induction of TNF-α in response to M. tuberculosis total lipids depends on TLR2. RAW-TT10 cells transiently transfected with a control plasmid, MyD88-dominant negative (MyD88-DN), wild-type TLR2, or dominant negative TLR2 (TLR2-P681H) were analyzed for induction of TNF-α after stimulation with M. tuberculosis lipids. Histograms are formatted as described in Fig. 1.

Figure 5

TNF-α produced in response to mAGP depend on TLR2. RAW-TT10 cells transiently transfected with a control plasmid, MyD88-dominant negative (MyD88-DN), wild-type TLR2, or dominant negative TLR2 (TLR2-P681H) were analyzed for induction of TNF-α after stimulation with mAGP. Histograms are formatted as described in Fig. 1. Similar results were obtained in the presence or absence of polymyxin B.