Toll-like receptor 2 modulates the proinflammatory milieu in Staphylococcus aureus-induced brain abscess

Abstract

Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that plays an important role in innate immune recognition of conserved structural motifs on a wide array of pathogens, including Staphylococcus aureus. To ascertain the functional significance of TLR2 in the context of central nervous system (CNS) parenchymal infection, we evaluated the pathogenesis of S. aureus-induced experimental brain abscess in TLR2 knockout (KO) and wild-type (WT) mice. The expression of several proinflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor alpha, and macrophage inflammatory protein-2, was significantly attenuated in brain abscesses of TLR2 KO mice compared to WT mice during the acute phase of infection. Conversely, interleukin-17 (IL-17), a cytokine produced by activated and memory T cells, was significantly elevated in lesions of TLR2 KO mice, suggesting an association between innate and adaptive immunity in brain abscess. Despite these differences, brain abscess severity in TLR2 KO and WT animals was similar, with comparable mortality rates, bacterial titers, and blood-brain barrier permeability, implying a role for alternative PRRs. Expression of the phagocytic PRRs macrophage scavenger receptor type AI/AII and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was increased in brain abscesses of both TLR2 KO and WT mice compared to uninfected animals. However, LOX-1 induction in brain abscesses of TLR2 KO mice was significantly attenuated compared to WT animals, revealing that the TLR2-dependent signal(s) influence LOX-1 expression. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition in the CNS which occurs, in part, through engagement of TLR2 and highlight the importance of receptor redundancy for S. aureus detection in the CNS.

FIG. 1.

TLR2 is important for regulating TNF-α production during the acute phase of experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for TNF-α mRNA (A) and protein (B) expression by qRT-PCR and ELISA, respectively. For qRT-PCR, the level of TNF-α expression was calculated after normalizing signals against the “housekeeping” gene GAPDH and is presented in relative mRNA expression units (mean ± standard deviation [SD] [error bar] of four independent experiments). ELISA results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of TNF-α (in picograms) per milligram of protein (mean ± SD; representative of four independent experiments). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05; **, P < 0.001).

FIG. 2.

MIP-2 expression is attenuated during the acute stage of brain abscess development in TLR2 KO mice compared to WT mice. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for MIP-2 protein expression by ELISA. Results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of MIP-2 (in picograms) per milligram of protein (± standard deviations [error bars]). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of four independent experiments.

FIG. 3.

TLR2 regulates iNOS induction in experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU), whereupon brain abscess protein extracts (40 μg per sample) were prepared at the indicated day postinfection (day 1 [D1] or day 3 [D3] postinfection) and analyzed for iNOS expression by Western blotting as described in Materials and Methods. Blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading. Results are presented from two or three individual animals per group and are representative of three independent experiments.

FIG. 4.

The absence of TLR2 signaling leads to augmented IL-17 expression throughout brain abscess development. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for IL-17 protein expression by ELISA. Results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of IL-17 (in picograms) per milligram of protein (± standard deviations [SD] [error bars]). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of two independent experiments.

FIG. 5.

The expression of the pattern recognition receptor CD14 is attenuated in TLR2 KO mice. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods, whereupon brain abscess homogenates were prepared (n = 4 to 6 animals per group for each time point) at the indicated intervals and analyzed for CD14 mRNA and protein expression by qRT-PCR (A) and Western blotting (B), respectively. (B) Western blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading with results from two or three individual mice per group presented at either day 1 (D1) or 3 (D3) postinfection as well as uninfected animals. Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of three independent experiments.

FIG. 6.

TLR2 is not pivotal for bacterial containment in experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods. Animals (n = 4 to 6 per group) were euthanized at the indicated time points, and the number of viable organisms associated with brain abscesses was determined by quantitative culture. Titers are expressed as the mean log₁₀ CFU per milliliter of brain abscess homogenate (± standard deviation [SD] [error bar]). Results are representative of eight independent experiments.

FIG. 7.

Expression of the phagocytic PRR LOX-1 is influenced by TLR2-dependent signal(s) in experimental brain abscesses. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (10⁴ CFU) as described in Materials and Methods, whereupon total RNA was extracted from brain abscess tissues (n = 4 animals per group for each time point) at the indicated intervals and analyzed for macrophage scavenger receptor 1 (MSR) (A) and LOX-1 (B) expression by qRT-PCR. Gene expression levels were calculated after normalizing target signals (MSR or LOX-1) against the “housekeeping” gene GAPDH and are presented as the change in mRNA induction compared to uninfected animals. Significant differences between uninfected versus S. aureus-infected TLR2 WT and KO mice (*, P < 0.05; **, P < 0.001) and significant differences between brain abscesses of TLR2 WT and KO animals (#, P < 0.05) are also indicated. Results are representative of three independent experiments.