Prophylactic and therapeutic targeting of the neurokinin-1 receptor limits neuroinflammation in a murine model of pneumococcal meningitis - PubMed (original) (raw)
Prophylactic and therapeutic targeting of the neurokinin-1 receptor limits neuroinflammation in a murine model of pneumococcal meningitis
Vinita S Chauhan et al. J Immunol. 2011.
Abstract
There is increasing evidence that the tachykinin substance P (SP) can augment inflammatory immune responses within the CNS. We have recently demonstrated that resident CNS cells express high-affinity receptors for this neuropeptide (neurokinin-1 receptors [NK-1R]), and we have shown that SP can significantly augment glial inflammatory responses to clinically relevant Gram-negative bacteria. Furthermore, we provided evidence that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation following in vivo exposure to these pathogens. In this study, we demonstrate that SP similarly enhances inflammatory glial responses to the major Gram-positive causative agent of bacterial meningitis, Streptococcus pneumoniae, and show that endogenous SP/NK-1R interactions play a critical role in the development of CNS inflammation in an in vivo model of pneumococcal meningitis. Importantly, we provide the first demonstration, to our knowledge, that pharmacological targeting of the NK-1R not only prevents the development of damaging inflammation when administered prophylactically, but can also limit or reverse neuroinflammation associated with an established streptococcal CNS infection when delivered therapeutically. We show that an NK-1R antagonist attenuates increases in CNS inflammatory cytokine levels and decreases in immunosuppressive cytokine production associated with an ongoing S. pneumoniae infection. Furthermore, we demonstrate that such a therapeutic intervention reverses infection-associated gliosis and demyelination in the absence of changes in CNS bacterial burden. Together, these results suggest that targeting SP/NK-1R interactions is a strategy worthy of further study for the treatment of microbially induced neuroinflammation.
Figures
FIGURE 1
S. pneumoniae infection-induced changes in BBB permeability are abolished in the absence of SP/NK-1R interactions. S. pneumoniae (1 × 107 bacteria; S. pneu) or vehicle was administered i.c. to C57BL/6 wild-type (WT) and NK-1 receptor deficient (NK1R−/−) mice (n = 4). In addition, a group of wild type mice received the NK-1 receptor antagonist L703,606 (5 mg/kg s.c.) at days −1, 0, and +1 days relative to bacterial challenge (Antag). At 72 hours post infection, brain tissue was isolated and BBB integrity was assessed by Evan's blue dye content (Panel A), SP levels were determined by EIA (Panel B), and bacterial burden was determined by colony counting (Panel C). Asterisk indicates a significant difference from uninfected animals of the same strain while pound symbols denote significant differences between infected WT animals and antagonist treated infected WT animals or infected NK1−/− mice (n = 4, p < 0.05).
FIGURE 2
S. pneumoniae infection-induced changes in pro- and anti-inflammatory mediator levels within the CNS are significantly reduced in the absence of SP/NK-1R interactions. S. pneumoniae (1 × 107 bacteria; S. pneu) or vehicle was administered i.c. to C57BL/6 wild-type (WT) and NK-1 receptor deficient (NK1R−/−) mice (n = 4). In addition, a group of wild type mice received L703,606 (5 mg/kg s.c.) at days −1, 0, and +1 days relative to bacterial challenge (Antag). At 72 hours post infection, brain tissue was isolated and levels of TNF-α (Panel A), IL-6 (Panel B) and IL-10 (Panel C) were assessed by specific capture ELISA. Differences in cytokine production between infected and uninfected WT mice, or between infected or uninfected NK1R−/− mice, were compared statistically and asterisks indicate a significant difference from uninfected animals of that mouse strain. In addition, differences in cytokine production between infected WT mice and antagonist treated infected WT animals or infected NK1R−/− mice were compared statistically and pound symbols denote statistically significant differences (n = 4, p < 0.05).
FIGURE 3
Prophylactic NK-1 receptor antagonist treatment abolishes the astrogliosis and behavioral changes associated with in vivo S. pneumoniae CNS infection. Panel A: S. pneumoniae (1 × 107 bacteria; S. pneu) or vehicle was administered i.c. to C57BL/6 wild-type (WT) and NK-1 receptor deficient (NK1R−/−) mice (n = 4). In addition, a group of wild type mice received L703,606 (5 mg/kg s.c.) at days −1, 0, and +1 days relative to bacterial challenge (Antag). At 72 hours post infection, brain tissue was perfused in situ, isolated, and prepared for immunofluorescent microscopy. Micrographs show representative GFAP immunofluorescence of the corpus callosum (20X objective) in coronal cortical fields from one of the three animals in each group. Panel B: S. pneumoniae (1 × 107 bacteria; S. pneu) or vehicle was administered i.c. to wild-type mice that were untreated or treated with L703,606 (5 mg/kg, s.c.) at days −1, 0, and +1 days relative to bacterial challenge (S. pneu + Antag) (n = 4). Locomotor activity was assessed at 1, 2, and 3 days post-infection using a radial arm maze and reported as the average number of maze arms visited in 8 minute recording periods. Asterisks indicate significant differences from uninfected animals and pound symbols denote significant differences between _S. pneumoniae_-challenged animals and infected animals treated with the NK-1 receptor antagonist (n = 4, p < 0.05).
FIGURE 4
SP/NK-1 receptor interactions augment microglial responses to S. pneumoniae. Panel A: S. pneumoniae (1 × 107 bacteria; S. pneu +) or vehicle (−) was administered i.c. to wild-type mice (n = 4). At 72 hours post infection, NK-1 receptor levels in protein isolates derived from whole brain (B) or equal numbers of isolated microglia (M) or astrocytes (A) were assessed by immunoblot analysis. NK-1 receptor expression in HeLa cells is shown as a positive control (C). Panel B: Isolated primary murine microglia (MG) or astrocytes (AST) were untreated (0) or exposed to S. pneumoniae (S. pneu: MOI of 1, 5 and 10 bacteria to each glial cell) in vitro and NK-1 receptor levels were assessed at 12 hours post-infection by immunoblot analysis. NK-1 receptor expression in HeLa cells is shown as a positive control (+) and expression of a loading control irrelevant protein is also shown (LC) (n = 4). Panel C: S. pneumoniae (1 × 107 bacteria; S. pneu) or vehicle was administered i.c. into C57BL/6 wild-type (WT) and NK-1 receptor deficient (NK1R−/−) mice (n = 4). In addition, a group of wild type mice received L703,606 (5 mg/kg s.c.) at days −1, 0, and +1 days relative to bacterial challenge (Antag). At 72 hours post infection, brain tissue was isolated and the relative proportion of cells expressing the microglia/macrophage markers CD11b and F4/80 was determined by flow cytometry. Asterisks indicate a significant difference from uninfected animals of the same strain while pound symbols denote significant differences between infected WT animals and antagonist treated infected WT animals or infected NK1R−/− mice. Panel D: Isolated primary murine microglia (MG) or astrocytes (AST) were untreated (0) or challenged in vitro with S. pneumoniae (S. pneu: MOI of 5 and 10 bacteria to each glial cell) in the presence or absence of SP (5 nM) and IL-6 production was assessed at 12 hours post-infection by specific capture ELISA. Data is presented as the mean of triplicate determinations of samples from three separate experiments +/− SEM. Asterisks indicate a statistically significant difference in IL-6 levels in the absence or presence of SP (p < 0.05).
FIGURE 5
Therapeutic intervention with a NK-1 receptor antagonist attenuates S. pneumoniae CNS infection-associated disease severity. S. pneumoniae (1 × 107 bacteria) was administered i.c. to wild-type mice (n = 5). Beginning at 3 days post-infection, mice were treated daily with L-703,606 (5 mg/kg s.c.; +Antag) or vehicle (−Antag) (n = 5). Animal weight (Panel B) and body temperature (Panel D) was measured at 0, 2, 4, 6, and 8 days post-infection. Asterisks indicate significant differences from uninfected animals and pound symbols denote significant differences between _S. pneumoniae_-challenged animals and infected mice treated with the NK-1 receptor antagonist. In parallel experiments, brain tissue was isolated from mice at each of these time points and BBB permeability was assessed by Evan's blue dye content (Panel C) while bacterial burden was determined by colony counting (Panel A). Asterisks indicate significant differences in BBB permeability versus that seen at day 0. No significant differences were seen in bacterial burden or BBB permeability between _S. pneumoniae_-challenged animals and infected mice treated with the NK-1 receptor antagonist (n = 5, p < 0.05).
FIGURE 6
Bacterially induced increases in CNS demyelination and astrogliosis are markedly reduced by therapeutic intervention with an NK-1R antagonist. S. pneumoniae (S. pneu: 1 × 107 bacteria) was administered i.c. to wild-type mice. Beginning at 3 days post-infection, mice were treated daily with L-703,606 (5 mg/kg s.c.; +Antag) or vehicle (Uninfected). At 8 days post-infection, brain tissue was perfused in situ, isolated, and prepared for histochemical analysis by lightfield microscopy or immunofluorescent microscopy. Left panels show representative Luxol fast blue (LFB) staining of the corpus callosum in coronal fields (10X objective) from one of the four animals in each group. Right panels show representative GFAP immunofluorescence of the corpus callosum (20X objective) in coronal cortical fields from one of the four animals in each group.
FIGURE 7
S. pneumoniae infection-induced changes in pro- and anti-inflammatory mediator levels within the CNS are attenuated by therapeutic intervention with an NK-1R antagonist. S. pneumoniae (1 × 107 bacteria) was administered i.c. to wild-type mice (n = 5). Beginning at 3 days post-infection, mice were treated daily with L-703,606 (5 mg/kg s.c.; +Antag) or vehicle (−Antag). At 0, 2, 4, 6, and 8 days post-infection, brain tissue was isolated and levels of MCP-1 (Panel A), IL-6 (Panel B), TNF-α (Panel C), and IL-10 (Panel D) were assessed by specific capture ELISA. Asterisks indicate a significant difference from uninfected WT animals while pound symbols denote significant differences between untreated infected animals and antagonist treated infected mice (n = 5, p < 0.05).
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