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INVESTIGADORES

ERRASTI Andrea Emilse

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Título:

NMDA antagonist ketamine induces a regulatory macrophage program on human monocytes.

Autor/es:

NOWAK W; ESTECHO IG; DARAY FM; CARRERA SILVA EA; ERRASTI AE

Lugar:

Cancún

Reunión:

Congreso; XII Congress of the Latin American Association of Immunology (ALAI) and XXIII Congress of the Mexican Society of Immunology (SMI).; 2018

Institución organizadora:

ALAI-SMI

Resumen:

Introduction: The NMDA antagonist, ketamine, discoveredmore than 50 years ago, is still used as an anesthetic, but recently a newindication emerged for this old drug as a fast and potent antidepressant(Potter and Choudhury, 2014). The biology of this effect is poorly understoodbut new research implicates that one of the main targetsof its action is theimmune response (Laudanski et al., 2015; Kiraly et al., 2017). Strong evidence demonstratesthat inflammation plays an important role in the pathophysiology of depression (Romanet al., 2013; Haapakoski et al., 2016; Mechawar and Savitz, 2016). In the samesense, macrophages are key innate players cells promoting inflammation orregulatory signals. This balance depends on the environmental stimuli as wellas the cytokine milieu. Pro-inflammatory (M1) program is induced byinflammatory stimuli such as INFγ, while an anti-inflammatory (M2) profile couldbe induced by type-2 cytokines such as IL-4 and IL-10 (Mantovaniand Locati, 2009; Gordon S., 2003). The aim of the present study was to elucidate the effect ofketamine on human macrophage differentiation and polarization, the type of receptorinvolved, andthe gene program elicited by this drug.Methods: 10-15 ml of blood was drawn fromhealthy volunteers and mononuclear cellular fraction was obtained by Ficollgradient centrifugation. CD14+ monocytes were isolated using a positive selectionkit (Stemcell) and 1.5 x 105 cells were seeded in 48-well plateswith 10% FBS supplemented RPMI medium. After 7 days of culture, cells wereharvested for phenotype characterization and gene expression analysis. Ketamine (0.1, 1 and 10 µM) was added after 6 days of macrophagedifferentiation and stimulated with LPS (1 ng/ml) to analyze activation markersand cytokine production by flow cytometry and ELISA, respectively. On the other hand, ketamine (0.1, 1 and 10µM) was added from day 0 of CD14+ cell culture and differentiation andpolarization markers were analyzed. Also, in independent experiments, MK-801(10 µM, an NMDA receptor selective antagonist) or NBQX (10 µM, an AMPA receptorselective antagonist) were added from day 0.Gene expression analysis to discriminate pro-inflammatory and regulatorymacrophages was performed by qPCR.LPS (1 ng/ml) + INFγ (50 ng/ml), IL-4 (40 ng/ml) or dexamethasone(0.1µM) were used as positive controls for macrophage polarization.Results: Ketamine treatment, 30 minutes before the additionof LPS on day 6, dampens macrophage activation determined by lower expressionof CD80 and HLADR. Moreover, when ketamine was added to monocyte cultures fromday 0 and then stimulated overnight with LPS at day 6, expression of CD80 andHLADR as well as TNF-α production was also significantly reduced.Ketamine treatmentof CD14+ monocytes from day 0 of culture strongly induced the expression ofCD163 and MERTK with intermediate levels of CD64, compatible with a regulatoryM2c-like phenotype.The NMDA receptor antagonist, MK-801, induced asimilar macrophage phenotype as ketamine in contrast to the AMPAreceptor antagonist, NBQX.Gene expression analysisdemonstrate that ketamine up-regulates mRNA levels of TGM2 and CCL22, genes relatedto an M2 profile. Quite the opposite, ketamine had no effect on mRNA levels ofCXCL10, a gene related to an M1 profile. Interestingly, ketamine promoted theup-regulation of mRNA levels of mTOR pathway related genes, SGK1 and eIF4B.Conclusions: The present study demonstrates thatketamine dampens the acute inflammatory response elicited by LPS on differentiatedmacrophages as well as induces a regulatory program on human monocytes,promoting an M2c-like phenotype in these cells. Moreover, this regulatoryprogram appears to be mediated by the NMDA receptor. Furthermore, ketamineinduces a particular gene expression profile involving components of the mTORpathway.

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