Elsevier

Brain, Behavior, and Immunity

Volume 41, October 2014, Pages 90-100
Brain, Behavior, and Immunity

Microglial NLRP3 inflammasome activation mediates IL-1β-related inflammation in prefrontal cortex of depressive rats

https://doi.org/10.1016/j.bbi.2014.04.007Get rights and content

Abstract

Depression is an inflammatory disorder. Pro-inflammatory cytokine interleukin-1 beta (IL-1β) may play a pivotal role in the central nervous system (CNS) inflammation of depression. Here, we investigated IL-1β alteration in serum, cerebrospinal fluid (CSF) and prefrontal cortex (PFC) of chronic unpredictable mild stress (CUMS)-exposed rats, a well-documented model of depression, and further explored the molecular mechanism by which CUMS procedure induced IL-1β-related CNS inflammation. We showed that 12-week CUMS procedure remarkably increased PFC IL-1β mRNA and protein levels in depressive-like behavior of rats, without significant alteration of serum and CSF IL-1β levels. We found that CUMS procedure significantly caused PFC nuclear factor kappa B (NF-κB) inflammatory pathway activation in rats. The intriguing finding in this study was the induced activation of nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome with the increased IL-1β maturation in PFC of CUMS rats, suggesting a new grade of regulatory mechanism for IL-1β-related CNS inflammation. Moreover, microglial activation and astrocytic function impairment were observed in PFC of CUMS rats. The increased co-location of NLRP3 and ionized calcium binding adaptor molecule 1 (Iba1) protein expression supported that microglia in glial cells was the primary contributor for CUMS-induced PFC NLRP3 inflammasome activation in rats. These alterations in CUMS rats were restored by chronic treatment of the antidepressant fluoxetine, indicating that fluoxetine-mediated rat PFC IL-1β reduction involves both transcriptional and post-transcriptional regulatory mechanisms. These findings provide in vivo evidence that microglial NLRP3 inflammasome activation is a mediator of IL-1β-related CNS inflammation during chronic stress, and suggest a new therapeutic target for the prevention and treatment of depression.

Introduction

Major depressive disorder (MDD) is a major public health concerns in modern society (Kupfer et al., 2012). More studies have shown that depression is an inflammatory disorder (Maes, 2011), but, litter is known about the molecular mechanisms involved in the central nervous system (CNS) inflammation. Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is demonstrated to participate in inflammatory responses in the brain, thereby leading to cellular damage in stress-related neuropsychiatric diseases including MDD (Gadek-Michalska et al., 2013). Clinical studies show that IL-1β levels in plasma or CSF are increased in depressed patients (Levine et al., 1999, Owen et al., 2001). However, there is some evidence that IL-1β levels in periphery or CSF are unchanged in patients with MDD (Brambilla and Maggioni, 1998, Dowlati et al., 2010, Kagaya et al., 2001, Martinez et al., 2012). Consistently, 9-week procedure of unpredictable chronic mild stress can not alter peripheral IL-1β levels in mice (Farooq et al., 2012). In contrast, peripheral IL-1β expression is increased in mice exposed to 3-week procedure of chronic mild stress (Mormede et al., 2002). It seems that the results of periphery or CSF IL-1β levels under the depressed conditions are controversial.

Prefrontal cortex (PFC) is critical for translation of emotional information into stressful action (de Kloet et al., 2005, McKlveen et al., 2013), and participates in neural mechanisms underlying stress adaptation and pathology (McKlveen et al., 2013). PFC IL-1β mRNA and protein levels are significantly increased in patients with MDD and suicidal behavior (Pandey et al., 2012). Increased IL-1β mRNA expression is also detected in cortex of rats exposed to chronic mild stress (You et al., 2011). These observations indicate that there may be a positive relationship between PFC IL-1β and MDD. However, the mechanism by which psychological stress induces PFC IL-1β alteration associated depression remains elusive. The glial cells, especially microglia and astrocyte, are the major source of CNS innate immunity and CNS-derived IL-1β (Ransohoff and Brown, 2012). Patients with MDD show the elevated microglial density in PFC (Steiner et al., 2008). Reduction of reactive astroglia is observed in PFC of young patients with MDD (Miguel-Hidalgo et al., 2000). Similarly, chronic unpredictable stress reduces glial metabolism and astrocyte marker glial fibrillary acidic protein (GFAP) mRNA expression in PFC of rats (Banasr et al., 2010). Moreover, chronic stress increases the number of microglia marker ionized calcium binding adaptor molecule 1 (Iba1) positive cells in PFC of mice susceptible to anhedonia (Couch et al., 2013). These observations may suggest a susceptible role of PFC glial cells in IL-1β-related CNS inflammation of chronic stress and depression.

The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is found to be a pivotal mediator of IL-1β function (Haneklaus et al., 2013). This inflammasome, composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, is a multiprotein complex that mediates the activation of caspase-1, which in turn cleaves pro-IL-1β to form the mature IL-1β (Haneklaus et al., 2013). The NLRP3 inflammasome couples with the nuclear factor kappa B (NF-κB) inflammatory pathway to mediate IL-1β transcription and function (Bauernfeind et al., 2009), inducing CNS innate immunity and inflammation (Jha et al., 2010, Liu et al., 2013). The activation of the NLRP3 inflammasome is detected in rat cerebral cortex of traumatic brain injury (Liu et al., 2013) and in glial cells of CNS inflammatory disease (Ransohoff and Brown, 2012). Caspase-1 dominant-negative inhibitor is over-expressed in PFC of MDD patients with inflammation (Shelton et al., 2011). Recently, the NLRP3 inflammasome is demonstrated to link cytokine, psychological stress and depression (Alcocer-Gomez et al., 2014, Iwata et al., 2013, Maslanik et al., 2013), indicating that this inflammasome may have potential to induce IL-1β-related CNS inflammation in depression. Therefore, it is intriguing to investigate the role of PFC NLRP3 inflammasome in CNS inflammation of depression.

Chronic unpredictable mild stress (CUMS) in rats as a well-documented model of depression (Willner, 1997), is a potentially reliable model to study depressive stress-induced neuroinflammation (Farooq et al., 2012). In this study, we detected IL-1β levels in serum, CSF and PFC to clarify pathological alteration of IL-1β in CUMS rats. Furthermore, we focused on investigating whether CUMS procedure activates PFC NLRP3 inflammasome to increase PFC IL-1β expression in rats, and explored the changes of PFC microglia and astrocyte to find out which of them should be the contributor for PFC NLRP3 inflammasome activation and IL-1β-related CNS inflammation in CUMS rats. Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7), Toll-like receptor 2 (TLR2) and TLR4 are the important mediators of psychosocial stress-related CNS inflammation (Barden, 2006, Weber et al., 2013) and inducers of the NLRP3 inflammasome activation (Babelova et al., 2009), therefore we explored their possible alterations in PFC of CUMS rats.

On the other hand, antidepressants are found to block the effects of inflammatory cytokines including IL-1β on the brain of patients with MDD (Hannestad et al., 2011). Fluoxetine as an effective clinical antidepressant (a selective serotonin reuptake inhibitor) effectively improves depressive behavior and inflammation in CUMS rats in our and other previous studies (Grippo et al., 2006, Pan et al., 2007, Pan et al., 2010, Pan et al., 2013). It reduces inflammation induced by serotonin (Bianchi et al., 1994) and inhibits NF-κB signaling in intestinal epithelial cells exposed to dextran sulfate sodium (Koh et al., 2011). Furthermore, fluoxetine decreases microglial release of glutamate and D-serine to promote cortical neuronal viability following ischemic insult (Dhami et al., 2013), prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of dopaminergic neurons by inhibiting microglial activation (Chung et al., 2011), and reduces inflammatory response in lipopolysaccharides-stimulated microglial cells (Liu et al., 2011), indicating that fluoxetine may have the anti-inflammatory action in glial cells. We therefore investigated the effects of fluoxetine on CUMS-induced these inflammatory alterations in rats. This study may further support the hypothesis that microglial NLRP3 inflammasome activation may be a mediator of IL-1β-related CNS inflammation in depression.

Section snippets

Animals

Male Wistar rats, weighting 180–220 g were purchased from the Suzhou Industrial Park AyeMatt Technology Co., Ltd. (Suzhou, China, Certificate No. SCXK(Su)2009-0001) and housed in plastic cages with a 12:12-h light–dark cycle under constant temperature of 22–24 °C and relative humidity (50–60%). They were fed standard chow ad libitum and allowed 4 weeks of acclimatization to the laboratory environment. The mean body weight was about 300 g before experiments. All the procedures were in strict

CUMS-induced depressive-like behavior in rats is reversed by chronic fluoxetine treatment

Before antidepressant fluoxetine treatment, we established rat animal model of anhedonia induced by CUMS procedure for the evaluation of the effectiveness of depression. As reported previously by us and others (Pan et al., 2010, Pan et al., 2013, Willner, 1997, Willner et al., 1987), 6-week CUMS procedure caused anhedonia behavior (measured as a reduction of sucrose solution intake, −27.4%, p < 0.001) with significant decrease of body weight gain (p < 0.01) compared with Non-CUMS rats (Fig. 1).

Discussion

IL-1β as a pivotal mediator is involved in stress-induced neuronal inflammatory response (Koo and Duman, 2008, Norman et al., 2010). In this study, 12-week CUMS procedure was found to up-regulate PFC IL-1β expression in depressive-like behavior of rats, without significant alteration of serum and CSF IL-1β levels. We also found that CUMS procedure caused activation of the NF-κB pathway and NLRP3 inflammasome with over-expression of P2RX7 and TLR2 in PFC of rats. Moreover, microglial NLRP3

Competing Interests

The authors have declared that no competing interests exist.

Acknowledgment

This study is supported by grants 81025025, 81001671 and 81373788 from the National Natural Science Foundation of China.

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    These authors contributed equally to this work.

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