Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus
Introduction
Chronic stress primes the neuroinflammatory response to both peripheral and central pro-inflammatory challenges (Audet et al., 2011, de Pablos et al., 2006, Espinosa-Oliva et al., 2011, Munhoz et al., 2006, Wohleb et al., 2012) and primes the pro-inflammatory response of microglia to LPS ex vivo (Wohleb et al., 2011). Consistent with these stress-induced priming effects, chronic stress modulates the immunophenotype of microglia as evidenced by the up-regulation of MHCII (de Pablos et al., 2006, Espinosa-Oliva et al., 2011), TLR4 (Wohleb et al., 2011), F4/80 antigen (Nair and Bonneau, 2006) and Iba-1 expression (Hinwood et al., 2012, Tynan et al., 2010). Notably, stress-induced glucocorticoids (GCs) appear to play a pivotal role in chronic stress-induced neuroinflammatory priming (de Pablos et al., 2006, Espinosa-Oliva et al., 2011, Munhoz et al., 2006) as well as the stress-induced modulation of microglia immunophenotype (de Pablos et al., 2006, Espinosa-Oliva et al., 2011, Nair and Bonneau, 2006). Consistent with these stress studies, chronic administration of GCs is sufficient to prime neuroinflammatory responses to a subsequent pro-inflammatory challenge (Kelly et al., 2012, Munhoz et al., 2010). However, it is unknown whether chronic GCs sensitize the response of key CNS innate immune substrates such as microglia to pro-inflammatory stimuli.
An emerging literature suggests that GCs modulate key pro-inflammatory pathways, which may serve as the basis for how stress and GCs prime pro-inflammatory immune responses (Frank et al., 2013). Of particular relevance here, GCs induce the expression of the NLRP (nucleotide-binding domain, leucine-rich repeat, pyrin domain containing protein) 3 inflammasome, which is the only known inflammasome requiring a priming stimulus that is modulated by GCs (Busillo et al., 2011). NLRP3 inflammasome assembly and activation requires a priming stimulus, which induces NLRP3 transcription, and a secondary stimulus, which induces the formation of the NLRP3 molecular scaffold. The formation and activation of the NLRP3 inflammasome in turn leads to the formation and release of active, mature IL-1β (Hornung and Latz, 2010). Busillo et al. (2011) found that GCs induce NLRP3 at both the mRNA and protein level in THP-1 cells, bone marrow-derived macrophages, and primary human monocytes in vitro, thereby priming NLRP3 inflammasome formation to a subsequent stimulus such as ATP, and potentiating the pro-inflammatory cytokine response. IL-1β is critical to the inflammatory response (Basu et al., 2004) and the production and release of mature IL-1β requires inflammasome formation and activation (Lamkanfi and Kanneganti, 2010). Therefore, GC-induction of NLRP3 could serve as a mechanism of stress- and GC-induced priming of neuroinflammatory processes. However, neither the effects of GCs on NLRP3 in vivo, in brain, or in microglia have been examined.
In the present study, we explored whether (1) microglia serve as a neuroimmune substrate of chronic GC-induced priming and (2) chronic GC exposure modulates the NLRP3 inflammasome. Prior studies have shown that stress primes neuroinflammatory processes in several brain regions including the frontal cortex, hypothalamus, and hippocampus (Johnson et al., 2002). In the present study, the hippocampus was chosen for study because of the deleterious effects of neuroinflammatory processes on hippocampus dependent cognitive function (Barrientos et al., 2012).
Section snippets
Animals
Male Sprague-Dawley rats (60–90 days old; Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA) were pair-housed with food and water available ad libitum. The colony was maintained at 25 °C on a 12-h light/dark cycle (lights on at 0700 h). All experimental procedures were conducted in accord with the University of Colorado Institutional Animal Care and Use Committee.
Adrenalectomy (ADX)
Bilateral ADX was aseptically performed under halothane anesthesia (Halocarbon Laboratories, River Edge, NJ, USA) as previously
Serum and hippocampal CORT levels
Chronic CORT treatment induced a significant change in both serum (F3, 18 = 27.77, p < 0.0001) and hippocampal (F3, 18 = 32.03, p < 0.0001) CORT (Fig. 1). In serum (Fig. 1A), CORT treatment for 10 days resulted in a concentration dependent increase in CORT levels. CORT levels in vehicle treated intact animals did not significantly differ from levels observed in ADX animals treated with 25 μg/ml CORT, indicating that a concentration of 25 μg/ml CORT restores CORT levels to basal levels. In hippocampus (
Discussion
An emerging literature has demonstrated that chronic stress and GCs modulate the immunophenotype of CNS macrophages and microglia (de Pablos et al., 2006, Espinosa-Oliva et al., 2011, Hinwood et al., 2012, Munhoz et al., 2010, Nair and Bonneau, 2006, Wohleb et al., 2011). Likewise, the present results show that chronic exposure to exogenous GCs up-regulates the expression of the macrophage/microglia activation antigens MHCII and Iba-1, replicating the results of prior studies (de Pablos et al.,
Role of the funding source
The funding source had no role in study design, data collection, analysis or interpretation of the data. The manuscript was prepared independently from the funding source and the funding source did not influence the decision to submit the paper for publication.
Conflict of interest
None declared.
Acknowledgement
The present work was supported by grant R21MH096224 from the National Institute of Mental Health.
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