Rapamycin Augments the NMDA-Mediated TNF Suppression of MRSA-Stimulated RAW264.7 Murine Macrophages

Thomas Spentzas,Elizabeth Meals,B Keith English,Fabio Savorgnan,Rebekah K H Shappley

doi:10.1155/2012/542727

Thomas Spentzas, Elizabeth Meals + Show 3 more

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https://doi.org/10.1155/2012/542727

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Abstract

Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves N-methyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical community-acquired- (CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF induction-suppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10–15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition.

Highlights

Sepsis is a major cause of morbidity and mortality, with an estimated incidence in the pediatric population of 0.56 cases per 1000 annually
Nmethyl-D-aspartic acid (NMDA) pathway inhibition or induction modulates the tumor necrosis factor (TNF) response of RAW264.7 murine macrophages stimulated by CA-Methicillin-resistant Staphylococcus aureus (MRSA) wild-type strains MW2 and LAC
Stimulated macrophage TNF secretion without exposure to ketamine, AP5 (APV), LY-294002, or NMDA substrate served as the control

Summary

Introduction

Sepsis is a major cause of morbidity and mortality, with an estimated incidence in the pediatric population of 0.56 cases per 1000 annually. Macrophage cells are the main source of proinflammatory cytokines, including interleukins such as IL-1β, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF) secreted in response to bacterial stimulation [4, 5]. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves Nmethyl-D-aspartic acid (NMDA) receptor antagonism. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. Rapamycin exhibited a concentration-dependent TNF inductionsuppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Ketamine-induced suppression of TNF secretion was intensified 10–15% when rapamycin was added, but not when LY294002 was added. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition

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