NOD2 mediates inflammatory responses of primary murine glia to Streptococcus pneumoniae

Abstract

It is now widely accepted that resident central nervous system (CNS) cells such as microglia and astrocytes initiate and/or augment inflammation following trauma or infection. However, the mechanisms by which glial cells perceive microbial challenges are only now becoming apparent. We have recently demonstrated that microglia and astrocytes constitutively express nucleotide-binding oligomerization domain-2 (NOD2), a member of the novel nucleotide-binding domain leucine-rich repeat region-containing family of proteins (NLR) that functions as an intracellular receptor for a minimal motif present in all bacterial peptidoglycans. Furthermore, we have shown that this NLR is essential for glial responses to gram-negative pathogens and in vivo CNS inflammation elicited by these organisms. In the present study, we have established that intact Streptococcus pneumoniae, the major causative agent for gram-positive bacterial meningitis in adults, is a potent stimulus for the activation of the pivotal inflammatory transcription factor NF-kB and production of inflammatory cytokines in primary murine microglia and astrocytes. We demonstrate that NOD2 is essential for the maximal responses of these cells to intact S. pneumoniae but not cellular lysates. Finally, we have shown that this cytosolic pattern recognition receptor is required for the elevated inflammatory mediator levels, astrogliosis, and demyelination, following in vivo administration of this gram-positive CNS pathogen. As such, we suggest that NOD2 plays a critical role in the establishment of the lethal inflammation associated with streptococcal meningitis.

FIGURE 1

Inflammatory cytokine responses of glia to intact S. pneumoniae are significantly lower in the absence of NOD2 expression. Microglia (Panels A and B) or astrocytes (Panels C and D) (2 × 10⁶ cells per well) from wild type (NOD2+/+) and NOD2 knockout (NOD2-/-) animals were untreated or exposed to viable S. pneumoniae (MOI, of 25:1, 75:1, 250:1 bacteria to each glial cell). At 24 hrs following bacterial challenge culture supernatants were isolated and assayed for the presence of IL-6 (Panels A and C) or TNF-α (Panels B and D) by specific capture ELISA. Data are presented as the culture supernatant cytokine 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 separate experiments +/- SEM. Asterisks indicate statistically significant differences in cytokine production between cells derived from wild type and NOD2 deficient animals (p < 0.05).

FIGURE 2

Microglial cytokine responses to S. pneumoniae 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 wild type (NOD2+/+) (Panels A and C) or NOD2 knockout (NOD2-/-) (Panels B and C) animals were untreated or exposed to either viable S. pneumoniae (MOI, of 25:1, 75:1, 250:1 bacteria to each glial cell) or lysates derived from an equal number of bacteria. At 24 hrs following bacterial challenge culture supernatants were isolated and assayed for the presence of IL-6 and TNF-α by specific capture ELISA. Data are presented as the culture supernatant cytokine 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 +/- SEM. Asterisks indicate statistically significant differences in cytokine production between cells treated with intact bacteria or bacterial lysates (p < 0.05).

FIGURE 3

Astrocyte cytokine responses to S. pneumoniae 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 wild type (NOD2+/+) (Panels A and C) or NOD2 knockout (NOD2-/-) (Panels B and C) animals were untreated or exposed to either viable S. pneumoniae (MOI, of 25:1, 75:1, 250:1 bacteria to each glial cell) or lysates derived from an equal number of bacteria. At 24 hrs following bacterial challenge culture supernatants were isolated and assayed for the presence of IL-6 and TNF-α by specific capture ELISA. Data are presented as the culture supernatant cytokine 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 +/- SEM. Asterisks indicate statistically significant differences in cytokine production between cells treated with intact bacteria or bacterial lysates (p < 0.05).

FIGURE 4

Activation of NF-kB following exposure to intact S. pneumoniae is significantly lower in glia deficient in the expression of NOD2. Microglia (Upper immunoblot) or astrocytes (Lower immunoblot) (2 × 10⁶ cells per well) from wild type (NOD2+/+) and NOD2 knockout (NOD2-/-) animals were untreated (0) or exposed to viable S. pneumoniae (MOI, of 25:1, 75:1, 250:1 bacteria to each glial cell). At 2 hrs following bacterial challenge nuclear protein isolates were prepared and assayed for the presence of NF-kB p65 (RelA) by immunoblot analysis. Fold increases in nuclear RelA levels following bacterial challenge were determined by densitometric analysis and are indicated below the bands. The immunoblots shown are representative of three separate experiments.

FIGURE 5

In the absence of NOD2 expression, intracerebral S. pneumoniae administration fails to elicit rapid elevations in inflammatory mediator levels and is associated with higher bacterial burdens within the CNS. Vehicle (0) or S. pneumoniae (1 × 10⁶ bacteria) was administered via i.c. injection into C57BL/6 wild type (NOD2+/+) and NOD2 deficient (NOD2-/-) mice. At 1 (D1), 3 (D3), and 5 (D5), days post-infection tissue homogenates were isolated for measurement of TNF-α (Panel A) and MIP-1α (Panel B) protein expression by specific capture ELISA, and assessment of bacterial burden (Panel C). Data are presented as the means of triplicate determinations of samples from four animals in each group +/- SEM. Asterisk indicates significant difference between levels in infected NOD2+/+ and NOD2-/- mice, and pound symbol indicates significant difference from uninfected animals (p < 0.05).

FIGURE 6

S. pneumoniae-induced astrogliosis and demyelination is attenuated in the absence of NOD2 expression. Vehicle or S. pneumoniae (1 × 10⁶ bacteria) was administered via intracerebral injection into C57BL/6 wild type (NOD2+/+) and NOD2 deficient (NOD2-/-) mice. At day 1 (D1), 3 (D3), and 5 (D5), post-infection brain tissue was perfused in situ, isolated, and prepared for analysis by immunofluorescence and lightfield microscopy. Panel A shows astrocyte-associated GFAP (green) and cell nuclei-associated DAPI (blue) immunofluorescence in representative coronal cortical fields (40× objective) from one of the four animals in each group while Panel C shows representative Luxol blue staining of the corpus callosum in coronal fields (10× objective).