Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus

Abstract

Background: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.

Results: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

Conclusions: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

Figure 1

The CA-MRSA isolates LAC (USA300) and MW2 (USA400) stimulated less TNF secretion by RAW264.7 murine macrophages when exposed to daptomycin (DAP) than when exposed to vancomycin (VAN). LAC or MW2 were added to RAW264.7 cells at a final concentration of 10⁵ to 10⁷ CFU/mL (retrospective confirmation) in the presence of either vancomycin or daptomycin at 20 μg/mL. Cells were incubated for 18 hours; supernatants were collected and analyzed for TNF content by an enzyme-linked immunosorbent assay (ELISA). Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF macrophage production by macrophages not stimulated with bacteria.

Figure 2

Ketamine inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 10⁵ to 10⁷ CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, ketamine (100 μM) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF macrophage production by macrophages not stimulated with bacteria.

Figure 3

MK-801 inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 10⁵ to 10⁷ CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, MK-801 (150 μΜ) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF production by macrophages not stimulated with bacteria.

Figure 4

No additive or synergistic effects of combinations of MK-801 and ketamine on macrophage TNF secretion, in response to antibiotic-treated CA-MRSA strain MW2, were seen. Bacteria were added at a final concentration of 10⁵ to10⁷ CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, either ketamine at 100 μM, MK-801 at 150 μΜ, or both were added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Lane 1 (control) represents the mean TNF production by macrophages not stimulated with bacteria. The mean includes wells exposed to ketamine, MK-801, both ketamine and MK-801, and neither. In the absence of bacteria, TNF secretion was minimal and was not affected by ketamine and/or MK-801. Lanes 2 -5 depict mean TNF secretion by macrophages exposed to vancomycin-treated MW2 alone (lane 2), MW2 + ketamine (lane 3), MW2 + MK-801 (lane 4), or MW2 + ketamine + MK-801 (lane 5). The "*" on bars 3, 4, 5 indicates that they are statistically different (p < 0.05) from bars 1 and 2. The "**" on bar 2 indicates significantly higher TNF production (p < 0.05).

Figure 5

APV inhibited and NMDA augmented TNF secretion by RAW264.7 murine macrophages stimulated with the antibiotic-treated CA-MRSA isolate, MW2. Bacteria were added at a final concentration of 10⁵ to10⁷ CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. One hour prior to stimulation, APV ("low" concentration of 300 μM or "high" concentration of 3 mM), ketamine (100 μM), or NMDA (30 μM) were added, alone or in combination, as indicated. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. The control lane represents the mean TNF macrophage production by macrophages not stimulated with bacteria. The mean includes wells exposed to APV, ketamine, or NMDA alone or in combination. In the absence of bacteria, TNF secretion was minimal and was not affected by APV, ketamine, or NMDA. Lanes 0-9 depict mean TNF secretion by macrophages exposed to vancomycin-treated MW2 alone (lane 0) or in the presence of the indicated concentrations of APV, ketamine, and/or NMDA (lanes 1-9). TNF secretion was reduced by approximately 30-40% when macrophages were pre-incubated with APV, ketamine, or APV + ketamine (lanes 1-5). The magnitude of inhibition by ketamine and high-dose APV was similar and there were no additive or synergistic effect observed with combinations of ketamine and APV. Addition of NMDA (30 μΜ) led to a substantial increase in the amount of TNF secreted in response to the MW2 strain (lane 9), and this augmented response was blocked by both APV and ketamine. The "*" on "0.Control" and "8.NMDA+lo_APV" bars indicates significance at p < 0.05. The "**" on "0.Control_MW2" and "9.NMDA" bars indicates differences between the pretreated wells, and that TNF production after MRSA stimulation with NMDA substrate (9.NMDA) is significantly higher than that at the baseline MRSA stimulation (0.Control_MW2) at p < 0.05.

Figure 6

TNF suppression by ketamine was tested at concentrations of 10 μM, 50 μM, 100 μM and 150 μM in the presence of vancomycin at 20 μg/mL and RAW264 macrophage stimulation with either LAC (upper panel) or MW2 (lower panel) CA-MRSA strains. Bacteria were added at a final concentration of 10⁵ to10⁷ CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. The inoculation time was 18 hours. Results are depicted as percentile reduction with 95% confidence intervals shown, i.e., the percent of TNF reduction that occurs after the specific concentration of ketamine was added.

Figure 7

TNF suppression by ketamine was tested after 6, 10, 14, 18 and 24 hours exposure in the presence of vancomycin at 20 μg/mL and RAW264 macrophage stimulation with either LAC (upper panel) or MW2 (lower panel) CA-MRSA strains. Bacteria were added at a final concentration of 10⁵ to10⁷ CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. The ketamine concentration was 100 μM. Results are depicted as percentile reduction with 95% confidence intervals, i.e., the percent of TNF reduction at the specific exposure time that occurs in comparison to inoculation without ketamine at the same time. The "*" indicates statistically significant difference at p < 0.05.