Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Abstract

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus (S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus. In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) enhances TLR2 expression in microglia, whereas interleukin-1beta has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-alpha, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-alpha-induced TLR2 expression. Similar results were observed with the IKK-2 and IkappaB-alpha inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-alpha was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-alpha knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-alpha knockout mice. Collectively, these results indicate that in response to S. aureus, TNF-alpha acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.

Fig. 1

Toll-like receptor 2 (TLR2) mRNA expression in primary microglia is augmented by tumor necrosis factor-alpha (TNF-α). Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were treated with various concentrations of interleukin-1β (IL-1β) or TNF-α (ng/mL) either alone or in combination for 6 h, whereupon total RNA was isolated and examined for TLR2 expression by qRT-PCR. Gene expression levels were calculated after normalizing TLR2 signals against the housekeeping gene GAPDH and are presented as the fold-induction in mRNA expression of cytokine-treated microglia compared with unstimulated cells. Significant differences between unstimulated and cytokine-treated microglia are denoted with asterisks (*p < 0.05). Results represent the average of three independent experiments (mean ± SEM).

Fig. 2

Tumor necrosis factor-alpha (TNF-α) enhances Toll-like receptor 2 (TLR2) protein expression in microglia. Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were treated with various concentrations of interleukin-1β (IL-1β) or TNF-α (ng/mL) either alone or in combination for 24 h, whereupon whole cell extracts (40 μg of protein per sample) were prepared and analyzed for TLR2 expression by western blotting. Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments.

Fig. 3

The broad-spectrum nuclear factor-kappa B inhibitor caffeic acid phenethyl ester (CAPE) attenuates tumor necrosis factor-alpha (TNF-α) -dependent Toll-like receptor 2 (TLR2) expression in a dose-dependent manner. Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were pre-treated with various concentrations of CAPE for 1 h prior to TNF-α stimulation (100 ng/mL) for 24 h, whereupon whole cell extracts (40 μg of protein per sample) were prepared and analyzed for TLR2 expression by western blotting. Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments.

Fig. 4

Evidence for the involvement of nuclear factor-kappa B signaling pathways in regulating the tumor necrosis factor-alpha (TNF-α) -dependent increase in Toll-like receptor 2 (TLR2) expression. Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were pre-treated for 1 h with various concentrations of the IKK-2 inhibitor SC-514 prior to TNF-α stimulation (100 ng/mL) for 24 h, whereupon whole cell extracts (40 μg of protein per sample) were prepared and analyzed for TLR2 expression by western blotting (a). Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments. In (b), microglia were pre-treated with SC-514 (50 μmol/L) for 1 h prior to TNF-α stimulation (100 ng/mL), whereupon cell viability was assessed using a standard MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay. Raw OD₅₇₀ absorbance values are reported (mean ± SD). Significant differences between unstimulated and treated microglia are denoted with asterisks (**p < 0.001).

Fig. 5

The IκB-α inhibitor BAY 11-7082 inhibits the tumor necrosis factor-alpha (TNF-α) -dependent increase in Toll-like receptor 2 (TLR2) expression without effecting cell viability. Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were pre-treated for 1 h with various concentrations of the IκB-α inhibitor BAY 11-7082 prior to TNF-α stimulation (100 ng/mL) for 24 h, whereupon whole cell extracts (40 μg of protein per sample) were prepared and analyzed for TLR2 expression by western blotting (a). Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments. In (b), microglia were pre-treated with BAY 11-7082 (10 μmol/L) for 1 h prior to TNF-α stimulation (100 ng/mL), whereupon cell viability was assessed using a standard MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay. Raw OD₅₇₀ absorbance values are reported (mean ± SD).

Fig. 6

The tumor necrosis factor-alpha (TNF-α)-dependent increase in microglial Toll-like receptor 2 (TLR2) expression is not influenced by either p38 mitogen-activated protein kinase (MAPK) or protein kinase C (PKC) pathways. Microglia were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were pre-treated for 1 h with various concentrations of the p38 MAPK or PKC inhibitors SB202190 (a) or bisindolylmaleimide (BIM; b), respectively. Subsequently, cells were stimulated with TNF-α (100 ng/mL) for 24 h, whereupon whole cell extracts (40 μg of protein per sample) were prepared and analyzed for TLR2 expression by western blotting. Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments.

Fig. 7

Tumor necrosis factor-alpha (TNF-α) is essential for the induction of microglial Toll-like receptor 2 (TLR2) expression in response to Staphylococcus aureus. Primary microglia isolated from TNF-α wild-type (WT) or knockout (KO) mice were seeded into six-well plates at 2 × 10⁶ cells per well and incubated overnight. The following day, cells were stimulated with 10⁷ colony forming units (CFU) of heat-inactivated S. aureus for 24 h, whereupon whole cell extracts were prepared (40 μg of protein per sample) and analyzed for TLR2 expression by western blotting. Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of two independent experiments.

Fig. 8

Tumor necrosis factor-alpha (TNF-α) plays a critical role in augmenting toll-like receptor 2 (TLR2) expression in vivo in a model of Staphylococcus aureus-induced brain abscess. TNF-α wild-type (WT) and knockout (KO) mice (n = 4–6 per group) received an intracerebral injection of S. aureus on day 0 and were killed at the indicated day post-infection, whereupon total RNA was isolated from brain abscesses for examination of TLR2 mRNA (a) and protein expression (b) by qRT-PCR and western blotting, respectively. For qRT-PCR, gene expression levels were calculated after normalizing TLR2 signals against the housekeeping gene GAPDH and are presented as the fold-induction in mRNA expression compared with uninfected animals. Significant differences between TNF-α WT and KO mice are denoted with asterisks (*p < 0.05, **p < 0.001). For western blots, brain abscess homogenates from four individual TNF-α WT or KO mice at day 3 post-infection were analyzed (40 μg of protein per sample). Blots were stripped and re-probed with an antibody specific for β-actin to verify uniformity in gel loading. Results are representative of two independent experiments.

Fig. 9

Augmenting Toll-like receptor 2 (TLR2) receptor levels with tumor necrosis factor-alpha (TNF-α) treatment has no effect on mi-croglial phagocytosis of Staphylococcus aureus. C57BL/6 primary microglia were seeded onto 12 mm coverslips at 2 × 10⁵ cells per coverslip and incubated overnight in 24-well plates. The following day, cells were pre-treated with 100 ng/mL of recombinant mouse TNF-α for 24 h to augment TLR2 protein expression. Subsequently, microglia were incubated with 4 × 10⁶ heat-inactivated S. aureus-green fluorescence protein (green) for 3 h and visualization of intracellular bacteria was detected using fluorescence microscopy (40×). Hoechst 33342 (blue) was used to visualize nuclei. Results are representative of three independent experiments.