Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses

“Primer sequences were designed using the Operon Oligo Analysis Tool (http:// www.operon.com/technical/toolkit.aspx) and tested for sequence specificity using the Basic Local Alignment Search Tool at NCBI (Altschul et al., 1997).”

“LPS typically must bind LPS-binding protein, which is then bound by CD14 and delivered to TLR4 complexed with MD2 (Kawai and Akira, 2010).”

“It is problematic that GCs, which are universally considered anti-inflammatory and immunosuppressive (Boumpas et al., 1993; De Bosscher et al., 2003), would be elevated during a fight/flight emergency, when the chances of injury and infection increase.”

“It is problematic that GCs, which are universally considered anti-inflammatory and immunosuppressive (Boumpas et al., 1993; De Bosscher et al., 2003), would be elevated during a fight/flight emergency, when the chances of injury and infection increase.”

“Interestingly, repeated social defeat stress up-regulated protein expression of CD14 and TLR4 on microglia (Wohleb et al., 2011).”

“, 2002b) and ex vivo (Frank et al., 2007) and of the CNS structures that are sensitive to IS-induced priming effects, it yields a sufficient number of microglia to conduct ex vivo experiments.”

“The present results are consistent with a growing body of evidence demonstrating the permissive effects of stress and GCs in neuroinflammatory processes (Sorrells and Sapolsky, 2007).”

“These results are consistent with a prior study showing that adrenalectomy potentiates the central proinflammatory cytokine response observed immediately after termination of the stressor (Nguyen et al., 1998).”

“Further, peripheral injection of a b –adrenergic receptor agonist was sufficient to induce central proinflammatory cytokines (Johnson et al., 2005).”

“, 1997), however microglia do not express the progesterone receptor (Sierra et al., 2008) suggesting that the effects of RU486 observed here were mediated predominantly through the GR.”

“2007), toll-like receptor 4 (TLR4) (Wohleb et al., 2011) and F4/80 antigen (Nair and Bonneau, 2006), suggesting that stress may shift the neuroimmune microenvironment towards a pro-inflammatory immunophenotype.”

“In addition, stress-induced GCs could modify later LPS-induced signaling at the receptor level (GR; (Oakley and Cidlowski, 2011), plasma binding proteins (corticosteroid binding globulin; (Henley and Lightman, 2011), and/or metabolic enzymes (11b -hydroxysteroid dehydrogenase Type I and II; (Seckl, 1997).”

“…et al., 2011; Munhoz et al., 2006), the pro-inflammatory challenge (i.e. LPS) was administered in vivo, which necessarily convolutes the GC effects of the stressor with the GC effects of LPS. Stress-induced GCs could modulate LPS-induced GC production (HPA axis; (Beishuizen and Thijs, 2003).”

“In addition, GCs are sufficient to sensitize the neuroinflammatory as well as the microglial response to pro-inflammatory challenges (Frank et al., 2010; Munhoz et al., 2010).”

“As observed in the CNS, stressors also sensitize and potentiate the peripheral proinflammatory immune response to proinflammatory stimuli (Bailey et al., 2007, 2009; Johnson et al., 2002b, 2003; Powell et al., 2009; Stark et al., 2001).”

“The phenomenon of cross-sensitization between the neuroinflammatory sequelae of stress and pro-inflammatory immune activation has been well characterized (Anisman, 2009; Tilders and Schmidt, 1999).”

“Stress-induced microglial activation and potentiation of neuroinflammatory processes has been blocked by a glucocorticoid (GC) receptor antagonist (de Pablos et al., 2006; Espinosa-Oliva et al., 2011; Munhoz et al., 2006; Nair and Bonneau, 2006) suggesting that GCs may mediate stressor-induced sensitization of neuroin…”

“We have shown that a severe acute stressor as used here induces pro-inflammatory cytokines in the CNS (hippocampus, hypothalamus and cortex) within hours of stressor termination (O’Connor et al., 2003) and is blocked by peripheral administration of b -adrenergic receptor antagonists (Johnson et al.”

“Of note, microglia express a- and b-adrenergic receptors (Mori et al., 2002).”

“Several studies have also shown that acute and chronic stress modulates the immunophenotype of microglia as indicated by up-regulation of MHCII (de Pablos et al., 2006; Frank et al., ll rights reserved. sychology and Neuroscience, 0309-0345, USA.”

“In addition, stress-induced GCs could modify later LPS-induced signaling at the receptor level (GR; (Oakley and Cidlowski, 2011), plasma binding proteins (corticosteroid binding globulin; (Henley and Lightman, 2011), and/or metabolic enzymes (11b -hydroxysteroid dehydrogenase Type I and II; (Seckl, 1997).”

“An organism’s innate immune response to pathogens involves coordinated and bidirectional communication between central and peripheral immune response systems (Blalock, 2005).”

“A considerable literature has attempted to understand the physiological function of stress-induced GCs, in particular as they relate to host defense mechanisms (Munck and Naray-Fejes-Toth, 1994).”

“Importantly, TLR4 is expressed by microglia (Carpentier et al., 2008).”

“In addition, GCs are sufficient to sensitize the neuroinflammatory as well as the microglial response to pro-inflammatory challenges (Frank et al., 2010; Munhoz et al., 2010).”

“Hippocampal microglia were isolated using a Percoll density gradient as previously described (Frank et al., 2006).”

“Louis, Mo, USA) replacement in their drinking water since this method has been shown to mimic the normal circadian pattern of CORT secretion (Jacobson et al., 1988).”

“…consistent with several studies showing that acute and chronic stressors sensitize the neuroinflammatory response to both peripheral and central immunologic challenges (de Pablos et al., 2006; Espinosa-Oliva et al., 2011; Frank et al., 2007; Johnson et al., 2002b, 2003, 2004; Munhoz et al., 2006).”

“LPS typically must bind LPS-binding protein, which is then bound by CD14 and delivered to TLR4 complexed with MD2 (Kawai and Akira, 2010).”

“Though a highly effective GR antagonist, RU486 also antagonizes the progesterone receptor (Sarkar, 2002).”

“As observed in the CNS, stressors also sensitize and potentiate the peripheral proinflammatory immune response to proinflammatory stimuli (Bailey et al., 2007, 2009; Johnson et al., 2002b, 2003; Powell et al., 2009; Stark et al., 2001).”

“Of note, CD163 can be upregulated on activated microglia under pathological conditions (Borda et al., 2008; Holfelder et al., 2011) and thus CD163 may not be a suitable marker to differentiate microglia from other CNS macrophages given that the stressor used here activates microglia (Frank et al.,…”

“Of note, CD163 can be upregulated on activated microglia under pathological conditions (Borda et al., 2008; Holfelder et al., 2011) and thus CD163 may not be a suitable marker to differentiate microglia from other CNS macrophages given that the stressor used here activates microglia (Frank et al.,…”

“As observed in the CNS, stressors also sensitize and potentiate the peripheral proinflammatory immune response to proinflammatory stimuli (Bailey et al., 2007, 2009; Johnson et al., 2002b, 2003; Powell et al., 2009; Stark et al., 2001).”

“If a pro-inflammatory stimulus (LPS) is administered immediately before stress exposure, stress exhibits an anti-inflammatory effect, including inhibition of LPS-induced increases in brain IL-1b (Goujon et al., 1995).”

“As observed in the CNS, stressors also sensitize and potentiate the peripheral proinflammatory immune response to proinflammatory stimuli (Bailey et al., 2007, 2009; Johnson et al., 2002b, 2003; Powell et al., 2009; Stark et al., 2001).”

“Indeed, a precedent for this possibility has been established in the field of learning and memory, which has found instances where GCs can amplify the effects of catecholamines on LTP (Joels et al., 2009).”

“The phenomenon of cross-sensitization between the neuroinflammatory sequelae of stress and pro-inflammatory immune activation has been well characterized (Anisman, 2009; Tilders and Schmidt, 1999).”

“However, several studies have shown that catecholamines are sufficient to induce an anti-inflammatory immunophenotype in the CNS (Gleeson et al., 2010; McNamee et al., 2010a,b, 2009).”

“A subsequent study showed that central, but not peripheral b -adrenergic receptors mediate the effects of acute stress on central pro-inflammatory cytokines (Johnson et al., 2008).”

“Bilateral ADX was aseptically performed under halothane anesthesia (Halocarbon Laboratories, River Edge, NJ, USA) as previously described (Der-Avakian et al., 2005).”

“Of note, CD163 can be upregulated on activated microglia under pathological conditions (Borda et al., 2008; Holfelder et al., 2011) and thus CD163 may not be a suitable marker to differentiate microglia from other CNS macrophages given that the stressor used here activates microglia (Frank et al.,…”

“It is noteworthy that severe acute stress as used in the present investigation induces a rapid (within minutes of stress onset) and profound increase in serum GCs (Fleshner et al., 1993) in the absence of any pro-inflammatory challenge.”

“…stress-induced GCs could modify later LPS-induced signaling at the receptor level (GR; (Oakley and Cidlowski, 2011), plasma binding proteins (corticosteroid binding globulin; (Henley and Lightman, 2011), and/or metabolic enzymes (11b -hydroxysteroid dehydrogenase Type I and II; (Seckl, 1997).”

“Acute tail shock 2 h Rats/SD IHC of MHC-II expression mRNA expression in LPSstimulated freshly isolated microglia (ex vivo) " Number of MHC-II microglia " Iba-1, MHC-II, CD11b, IL-1b, IL-6, and NF-kBI/ mRNA $ CD163 or TNF/ mRNA HPC [46,47]”

“…psychological stress, in vivo, primes microglia for exaggerated responses to LPS ex vivo and that this is mediated by glucocorticoids, based on the ability of both adrenalectomy and inhibition of GC receptor function with the antagonist RU486 (Frank et al., 2012) to prevent this primed response.”

“Interestingly, treatment with RU486, an antagonist of glucocorticoid receptor, or ADX blocked the sensitization of the microglial pro-inflammatory response induced by stress footshock (Frank et al., 2012).”

“…priming and trafficking, subsequent alterations in microglia phenotype and, ultimately resulting in neuroinflammation (Delpech et al., 2015; Frank et al., 2007, 2012; Johnson et al., 2005; Tynan et al., 2010; Wohleb et al., 2014b), which can subsequently lead to a number of psychiatric…”

“Prolonged exposure to glucocorticoids can also reduce the sensitivity of peripheral immune cells to anti-inflammatory feedback, which leads to reduced inflammatory regulation (Frank et al., 2012; Sorrells and Sapolsky, 2007).”

“, 2012 [59] Stress exposure resulted in a potentiated pro-inflammatory cytokine response (IL-1 , IL-6, NF BI ) to LPS in isolated”

“Importantly, subsequent inflammatory or toxic stimuli result in aggravation of neuronal injury [119, 120, 163].”

“Bilateral ADX was aseptically performed under isoflurane anesthesia as previously described (Frank et al., 2012).”

“Either pharmacological antagonism of GC receptors or adrenal ectomization blocks stress-induced microglia priming and decreases the LPS-induced microglial pro-inflammatory responses (Frank et al., 2012), suggesting that GC may play a critical role in stress-triggered microglia alteration.”

“Blocking either the β-adrenergic (Wohleb et al, 2011, 2013; Hanke et al, 2012) or GC receptor (Frank et al, 2012) during stress prevents stress-induced microglial activation/priming.”

“In addition, microglial response to stress proteins is guided by their glucocorticoids receptors (Frank et al., 2012) and nor-adrenaline activated alpha/beta-adrenergic receptor signaling pathways (Blandino et al.”

“For instance, microglia acutely isolated frommice subjected to acute stress (by inescapable tail shock) showed a primed response to LPS challenge by producing higher levels of IL-1βmRNA ex vivo [100], and the specific loss of expression of GR in microglia leads to a blunted inflammatory response in vitro and to a decr…”

“Indeed, several stress-induced mediators have been implicated in neuroinflammatory priming including glucocorticoids (Frank et al., 2012), catecholamines (Wohleb et al., 2011) and pro-inflammatory cytokines (Johnson et al., 2004).”

“…studies have revealed microglia activation and neuroinflammatory signaling occurs following prolonged stress exposure (Johnson et al., 2005; Frank et al., 2007a, 2012; Tynan et al., 2010; Wohleb et al., 2011, 2012, 2013; Bian et al., 2012; Hinwood et al., 2012, 2013;…”

“Insulin resistance in this model also chronically elevates corticosterone, which, like chronic stress [93], contributes to microglial priming, increasing brain IL-1 and TNF responses [94].”

“When priming does occur in microglia or peripheral macrophages, these sensitised cells do not produce proinflammatory or antiinflammatory products, but, if further stimulated, they produce high levels of proinflammatory products [69-75].”

“…alterations in glucocorticoids have demonstrated that glucocorticoid receptors modulate the ability of LPS to direct a pro-inflammatory response in part by modulating the activity of MKP-1, IkBa, IL-1Ra and subunits of NFkB (Madrigal et al., 2001; Sorrells et al., 2009; Frank et al., 2010, 2012).”

“Hence, depressive illness alone may not lead to neuronal death but it may augment the neurodegenerative or debilitating effects of other illnesses or insults, such as cerebral stroke (Frank et al., 2012; Espinosa-Garcia et al., 2017).”

“Acute stressors induce microglial activation in many brain regions [34, 54], and the effects of chronic stress on microglial activation are heterogeneous due to multiple variables, including the stress paradigm (timing and types of stressors) [30, 55–58] and the age of animals [59].”

“Treatment in vivo with RU486 and adrenalectomized inhibited the microglial pro-inflammatory response, indicating that stress-induced GCs are able to sensitize the microglial pro-inflammatory function (185, 186).”

“Bilateral ADX was aseptically performed under halothane anesthesia (Halocarbon Laboratories, River Edge, N.J., USA) as previously described (Frank et al., 2012).”

“significantly by adrenalectomy or RU486 treatment (Frank et al., 2012).”

“It is important to note that basal levels of GCs were maintained in the ADX subjects—thus, only the stress-induced increases from baseline were prevented.”

“Moreover, we have recently shown that pharmacological (RU486) and surgical (adrenalectomy) suppression of stress-induced GCs blunts the stress-induced sensitization of the microglial pro-inflammatory response to LPS ex vivo (Frank et al., 2012).”

“These results were substantiated in studies that showed GCs released following inescapable shock primed microglial neuroinflammatory responses (Frank et al., 2012).”

“…as well as the sickness response, to a subsequent immune challenge (de Pablos et al., 2014; de Pablos et al., 2006; Espinosa-Oliva et al., 2011; Frank et al., 2007, 2012; Johnson et al., 2002, 2003, 2004; Munhoz et al., 2006; Weber et al., 2013, 2015; Wohleb et al., 2012; Wohleb et al., 2011).”

“Acute (97, 98) or chronic (99–101) exposure to exogenous or endogenous GCs increases NLRP3 mRNA expression in microglia.”

“For instance, stress-induced exposure to glucocorticoids increases the microglial inflammatory response to subsequent exposure to inflammation-inducing lipopolysaccharide (LPS) in rats (Frank et al., 2010; Frank et al., 2012).”

“Moreover, microglial priming by acute or chronic stress was shown to predispose the brain to neurodegeneration and to affect disease progression as well (Frank et al., 2012; Ramaglia et al., 2012).”

“In terms of brain cytokines, glucocorticoids contribute to the increase in levels of brain PGE2 [65] and facilitate future inflammatory responses by contributing to the priming of microglia [124, 125] and the infiltration of circulating monocytes into the brain [126].”

“24 h after termination of the stressor, hippocampal microglia were isolated and challenged with LPS. Consistent with our prior studies (Frank et al., 2007, 2012; Weber et al., 2013), pre-exposure to stress potentiated the proinflammatory response of microglia to LPS.”

“Twenty Four hours after inescapable tail-shock, LPS injection significantly increases hippocampal CORT and IL-1β compared to vehicle controls (76).”

“Interestingly, the same lab also reported that prior exposure to one tail-shock session can induce sensitization (priming) of the immune response to endotoxin that might be mediated by glucocorticoid released during tail-shocks (Frank et al., 2010, 2012; Johnson et al., 2002b, 2003).”

“This sensitizing effect was further blocked by a GR antagonist, indicating that GR signaling is essential (Frank et al., 2012).”

“One week following stress exposure, when serum CORT concentrations had returned to baseline in female rats, hippocampal microglia were isolated and challenged ex vivo with LPS.”

“Similar studies have been able to reproduce this exacerbated pro-inflammatory response by exposing animals to exogenous GC rather than chronic stress (Frank et al., 2010, 2012, 2014).”

“Pretreatment of rats with the glucocorticoid receptor antagonist RU486 or adrenalectomy before stress exposure prevented subsequent microglial proinflammatory IL-1β response to lipopolysaccharide (LPS) stimulation ex vivo (60).”

“NLRP3 could be involved in GCs pro-inflammatory actions, as GCs potentiates the LPS-induced pro-inflammatory cytokine IL-1β (45, 46).”

“Microglia that have been activated or ‘primed’ exhibit lower thresholds for cytokine release (Frank et al., 2012), and priming was evident in cells isolated from sedentary db/db mice (Erion et al., 2014).”

“, decreased synaptic density) and functional connectivity as psychosis develops (Frank et al., 2012; Frick et al., 2013; Hannestad et al., 2012; Schafer et al., 2012a; Schafer et al., 2012b; van Berckel et al., 2008; Zhan et al., 2014).”

“We have demonstrated that blocking GR activity during a stressor with RU486 prevents stress-induced sensitization to a subsequent immune challenge in vivo, and the priming of microglia observed ex vivo (Frank et al., 2012).”

“A number of paradoxical results with GCs (Busillo et al., 2011; Dhabhar and McEwen, 1999; Frank et al., 2010c; Frank et al., 2012; Hermoso et al., 2004; Lim et al., 2007; MacPherson et al., 2005) suggest that GCs do not universally exert anti-inflammatory actions (examples follow).”

“Microglia also have a circadian dependency of response to glucocorticoids in stressinduced neuroinflammation that explains their diurnal rhythm response to inflammatory stimuli [108, 110].”

“corticoids blocked the stress-induced increases of cytokine production after LPS treatment [50].”

“In the latter case, glucocorticoids modulate microglial activation induced by stressors (120, 121) and suppress hippocampal and amygdala eCB signaling (122).”

“…response to subsequent immune challenges (de Pablos et al., 2006; Espinosa-Oliva et al., 2011; Frank et al., 2007; Frank et al., 2018; Frank et al., 2012; Johnson et al., 2003; Johnson et al., 2004; Munhoz et al., 2006; Wohleb et al., 2011), suggesting that stressors sensitize or…”

“Several studies have also shown that chronic stress and GCs can modulate the immunophenotype of CNS macrophages and microglia [14, 18, 19], augment the microglial proinflammatory response to LPS [20, 21], and enhance the TNF-α-mediated increase of Toll-like receptor (TLR) [22].”

“Moreover, hippocampal microglia isolated from rats exposed to tail-shock stress produce significantly higher levels of IL-1b and the proinflammatory cytokines IL-6 and tumor necrosis factor-a (TNF-a) in response to lipopolysaccharide (LPS) (Frank et al., 2012).”

“However, this notion is being updated as prior stress sensitizes proinflammatory responses to subsequent LPS (Johnson et al., 2002; Munhoz et al., 2006; Hains et al., 2011), and the proinflammatory effects of stress have been linked to CORT signaling (Munhoz et al., 2010; Frank et al., 2012).”

“Similarly, chronic restraint stress caused a significant increase in activated microglia and number of microglia in multiple brain regions (Tynan et al., 2010; Hinwood et al., 2012), and inescapable stress potentiates the microglial response to immune stimuli (Frank et al., 2012).”

“This notion is supported both by findings of stress-mediated priming of immune cells such as microglia in animals (Frank et al., 2012), and by increased IL-6 responses to LPS in vitro after exposure to a double-blind and balanced crossover design.”

“These observations rest primarily on data from studies of psychological stress (Frank et al., 2007) and direct administration of exogenous adrenal steroids (Frank et al., 2012).”