Chronic exposure to exogenous glucocorticoids primes microglia to pro-inflammatory stimuli and induces NLRP3 mRNA in the hippocampus

Summary

Chronic stress as well as chronic treatment with glucocorticoids (GCs) primes the neuroinflammatory response to a subsequent pro-inflammatory challenge. However, it remains unclear whether chronic GCs sensitize the response of key CNS immune substrates (i.e. microglia) to pro-inflammatory stimuli. In the present set of studies, male Sprague-Dawley rats underwent sham surgery or were adrenalectomized and then treated with varying concentrations of corticosterone (CORT; 0, 25, 50, and 75 μg/ml) administered in their drinking water. After 10 days of CORT exposure, whole hippocampus was collected and expression of glial activation markers measured or hippocampal microglia were isolated and challenged with LPS to probe for CORT-induced sensitization of pro-inflammatory responses. Chronic CORT exposure increased the gene expression of NLRP3, Iba-1, MHCII, and NF-κBIα in a concentration dependent manner. Chronic CORT (75 μg/ml) exposure potentiated the microglial proinflammatory response (TNFα, IL-1β, IL-6 and NLRP3) to LPS compared to the microglial response of sham surgery animals treated with vehicle. The present set of results demonstrate that chronic exposure to GCs primes microglia to pro-inflammatory stimuli and add to a growing body of evidence suggesting that a permissive function of GCs is that of an endogenous danger signal or alarmin.

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).