NOD2 contributes to the inflammatory responses of primary murine microglia and astrocytes to Staphylococcus aureus

Abstract

We have recently demonstrated that microglia and astrocytes express nucleotide-binding oligomerization domain-2 (NOD2), a novel cytosolic pattern recognition receptor for bacterial motifs, and we have shown that this intracellular receptor is essential for glial responses to Gram-negative pathogens. Here, we demonstrate that intact Staphylococcus aureus, a major Gram-positive causative agent of brain abscesses, activates the transcription factor NF-kappaB and is a potent stimulus for inflammatory cytokine production in primary murine microglia and astrocytes. Interestingly, we demonstrate that NOD2 is essential for maximal glial responses to intact S. aureus, but not cellular lysates. As such, this data indicates that NOD2 plays an important role in initiating inflammatory mediator production by resident brain cells following S. aureus infection and we suggest that this cytosolic receptor acts in conjunction with cell surface pattern recognition receptors to elicit maximal glial responses.

Figure 1

Activation of NF-kB following exposure to S. aureus is reduced in NOD2 deficient glia. Microglia (Panel A) or astrocytes (Panel B) (2 × 10⁶ cells per well) from NOD2+/+ and NOD2-/- animals were untreated (0) or exposed to viable S. aureus (MOI, of 25:1, 75:1, 250:1). At 2 hrs following challenge, nuclear protein isolates were assayed for the presence of NF-kB p65 (RelA) by immunoblot analysis. Nuclear RelA levels were quantified by densitometric analyses and normalized to protein loading. Average values +/- SEM are shown for each treatment group composed of three separate in vitro cell populations. Asterisks indicate statistically significant differences from corresponding uninfected cells (p < 0.05).

Figure 2

Inflammatory cytokine responses of glia to intact S. aureus are significantly lower in the absence of NOD2 expression. Microglia (Panels A and B) and astrocytes (Panels C and D) (2 × 10⁶ cells per well) from NOD2+/+ and NOD2-/- animals were untreated or exposed to viable S. aureus (MOI, of 25:1, 75:1, 250:1). At 24 hrs following challenge culture supernatants were assayed for IL-6 (Panel A) or TNF-α (Panel B) content. Data are presented as the supernatant concentrations (Left panels) and as fold increases over levels in unstimulated cells (Right panels) and are the means of triplicate determinations of samples from three experiments +/- SD. Asterisks indicate significant differences between cells derived from NOD2+/+ and NOD2-/- animals (p < 0.05).

Figure 3

Microglial cytokine responses to S. aureus lysates are significantly smaller than those elicited by intact bacteria and are not dependent on the expression of NOD2. Microglia (2 × 10⁶ cells per well) from NOD2+/+ (Panels A and C) or NOD2-/- (Panels B and C) animals were untreated or exposed to either viable S. aureus (MOI, of 25:1, 75:1, 250:1) or lysates derived from an equal number of bacteria. At 24 hrs following challenge culture supernatants were assayed for the presence of IL-6 and TNF-α. Data are presented as the supernatant concentrations (Panels A and B) and as fold increases over levels in unstimulated cells (Panel C) and are the means of triplicate determinations of samples from three separate experiments +/- SD. Asterisks indicate statistically significant differences in cytokine production between cells treated with intact bacteria or bacterial lysates (p < 0.05).

Figure 4

Astrocyte cytokine responses to S. aureus lysates are significantly smaller than those elicited by intact bacteria and are not dependent on the expression of NOD2. Astrocytes (2 × 10⁶ cells per well) from NOD2+/+ (Panels A and C) or NOD2-/- (Panels B and C) animals were untreated or exposed to either viable S. aureus (MOI, of 25:1, 75:1, 250:1) or lysates derived from an equal number of bacteria. At 24 hrs following challenge culture supernatants were assayed for the presence of IL-6 and TNF-α. Data are presented as the supernatant concentrations (Panels A and B) and as fold increases over levels in unstimulated cells (Panel C) and are the means of triplicate determinations of samples from three separate experiments +/- SD. Asterisks indicate statistically significant differences in cytokine production between cells treated with intact bacteria or bacterial lysates (p < 0.05).