RESEARCH ARTICLE

Astrocytes Inhibit Nitric Oxide-DependentCa21 Dynamics in Activated Microglia:

Involvement of ATP Released ViaPannexin 1 Channels

Juan A. Orellana, Trinidad D. Montero, and Rommy von Bernhardi

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca21 and produce high amounts of nitricoxide (NO). However, whether NO, Ca21 dynamics, and gliotransmitter release are reciprocally modulated is not fully under-stood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aimwas to address if astrocytes could regulate NO-dependent Ca21 dynamics and ATP release in LPS-stimulated microglia. Gri-ess assays and Fura-2AM time-lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca21]ithat depended on the sequential activation of iNOS, COXs, and EP1 receptor. TGFb1 released by astrocytes inhibited theabovementioned responses and also abolished LPS-induced ATP release by microglia. Luciferin/luciferase assays and dyeuptake experiments showed that release of ATP from LPS-stimulated microglia occurred via pannexin 1 (Panx1) channels, butnot connexin 43 hemichannels. Moreover, in LPS-stimulated microglia, exogenous ATP triggered activation of purinergic P2Y1

receptors resulting in Ca21 release from intracellular stores. Interestingly, TGFb1 released by astrocytes inhibited ATP-induced Ca21 response in LPS-stimulated microglia to that observed in control microglia. Finally, COX/EP1 receptor signalingand activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS-stimulated microglia. Thus, Ca21 dynamics depended on the inflammatory profile of microglia and could be modulated byastrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the developmentof new treatments to reduce inflammatory cytotoxicity in several brain pathologies.

GLIA 2013;61:2023–2037Key words: neuroinflammation, calcium, glia

Introduction

An increased body of evidence shows that the innate

immune response exerts a dichotomous role in the cen-

tral nervous system (CNS). Under physiological conditions,

microglia exhibit a resting phenotype associated with produc-

tion of anti-inflammatory and neurotrophic factors, whereas

in response to a wide variety of insults they shift to an acti-

vated phenotype (Block et al., 2007). In this state, rather

than serving protective functions, microglia activation possibly

becomes a detrimental process leading to further recruitment

of other cells involved in the innate immune response (von

Bernhardi, 2007) that promote disease progression, character-

ized by synaptic dysfunction and even cell death as occur in

chronic neurodegenerative disorders (Block et al., 2007; Ram-

irez et al., 2008). Under these conditions, microglia exhibit

an elevated intracellular Ca21 concentration ([Ca21]i) and

increased production of nitric oxide (NO) and prostaglandins

(Farber and Kettenmann, 2006; Saha and Pahan, 2006),

processes associated with neuronal damage in neuroinflamma-

tory conditions (Anrather et al., 2011; Ha et al., 2008). On

the contrary, most of evidence indicate that astrocytes, the

largest glial cell population of the brain, could support

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22573

Published online October 7, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Oct 18, 2012, Accepted for publication Aug 19, 2013.

Address correspondence to Juan A. Orellana, Departamento de Neurolog�ıa, Escuela de Medicina, Pontificia Universidad Cat�olica de Chile, Marcoleta 391,

Santiago, Chile. E-mail: jaorella@uc.cl or Rommy von Bernhardi, Departamento de Neurolog�ıa, Escuela de Medicina, Pontificia Universidad Cat�olica de Chile,

Marcoleta 391, Santiago, Chile. E-mail: rvonb@med.puc.cl

From the Departamento de Neurolog�ıa; Escuela de Medicina, Pontificia Universidad Cat�olica de Chile, Santiago, Chile.

Additional Supporting Information may be found in the online version of this article.

VC 2013 Wiley Periodicals, Inc. 2023

neuronal survival during brain inflammation (Escartin and

Bonvento, 2008). Indeed, astrocytes regulate the extracellular

concentration of glutamate, K1 and H1 express a large reper-

toire of neurotransmitter receptors and release several neurotro-

phic factors, functions that make them essential protagonists in

brain processing and memory acquisition (Haydon and

Carmignoto, 2006).

Microglia and astrocytes respond to any injury with a

spatial and temporal distinctive pattern of activation in the

inflamed CNS, their crosstalk appearing to be critical for

neuronal fate (Liu et al., 2011). On the other hand, several

studies show that intracellular Ca21 dynamics plays a critical

role on intercellular communication among glial cells, and

serves as a sensitive system to mediate release of gliotransmit-

ters, cytokines and growth factors, representing the primary

pathway by which glial cells respond to neural activity (Farber

and Kettenmann, 2006; Koizumi, 2010). Although microglia

and astrocytes appear to activate each other, whether the

crosstalk among microglia and astrocytes could modulate

Ca21 dynamics and gliotransmitter release during neuroin-

flammation is not fully understood. Several studies have

shown signaling interactions among astrocytes and microglia,

including the fact that astrocytes inhibit several features of

activated microglia such as release of NO and TNFa (Smits

et al., 2001; Tichauer et al., 2007; von Bernhardi and Euge-

nin 2004). TGFb is recognized as a putative factor released

by astrocytes associated with inhibition of microglial cell

inflammatory activation (Herrera-Molina and von Bernhardi

2005). Supporting this idea, TGFb reduces the expression of

several proteins involved in antigen presentation and produc-

tion of inflammatory cytokines, NO and oxygen free radicals

(Herrera-Molina and von Bernhardi 2005). Nevertheless,

whether NO could modulate Ca21 dynamics and gliotrans-

mitter release in activated microglia is not fully understood.

More importantly, if astrocytes could potentiate or suppress

the abovementioned signaling is unknown. Therefore, our

aim was to address if astrocytes could regulate the

NO-dependent Ca21 dynamics and gliotransmitter release in

activated microglia. Here, we show that lipopolysaccharide

(LPS)-induced [Ca21]i increase and ATP release depends on

NO and the activation of COXs and EP1 receptor in micro-

glia. Moreover, TGFb released by astrocytes inhibited the

responses and abolished the LPS-induced changes in ATP-

dependent intracellular Ca21 dynamics in microglia.

Materials and Methods

Reagents and AntibodiesHorseradish peroxidase (HRP)-conjugated anti-rabbit IgG was pur-

chased from Pierce (Rockford, IL). Gap26 and 10panx1 mimetic

peptides were obtained from NeoMPS, SA. (Strasbourg, France).

HEPES, DMEM, DNAse I, poly-L-lysine, A740003, MRS2179,

BAPTA, polyclonal anti-Cx43 antibody, Brilliant blue G (BBG),

oATP, xestospongin B, ethidium (Etd) bromide, and probenecid

(Prob) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal

bovine serum (FBS) was obtained from Hyclone (Logan, UT). Peni-

cillin, streptomycin, polyclonal anti-Panx1 antibody (PI488000),

goat anti-mouse Alexa Fluor 488 and goat anti-mouse Alexa Fluor

555 were obtained from Invitrogen (Carlsbad, CA). Normal goat

serum (NGS) was purchased from Zymed (San Francisco, CA).

Cx43E2, a Cx43 hemichannel antibody (Orellana et al., 2011) was

generously made available by Dr. Jean X. Jiang (University of Texas

Health Science Center).

Cell Cultures

MICROGLIAL CELL CULTURES. Microglia cultures were pre-

pared from cortex of P2 Sprague-Dowley rats as previously described

(Orellana et al., 2010). Briefly, dissected meninges were carefully

peeled off and cortices were mechanically dissociated. Cells were

seeded into 100-mm-diameter culture dishes at a density of 5x106

cells/dish in DMEM, containing 10% FBS. The medium was

changed at 1 and 3 days in vitro (DIV) and microglia were collected

at 10 DIV by orbital shaking. Microglia were seeded onto 35-mm

dishes (2x106 cells/dish) or onto glass coverslips placed in 24-well

plastic plates (5x105 cells/well) in DMEM containing 10% FBS.

ASTROCYTE CULTURES. These cultures were prepared with the

protocol for microglia mentioned before. Cells were seeded onto 35-

mm plastic dishes (Nunclon) or onto glass coverslips (Gassalem,

Limeil-Brevannes, France) placed in 16-mm 24-well plastic plates

(Nunclon) at the density of 5x105 cells/dish or 1x105 cells/well, respec-

tively, in DMEM, supplemented with penicillin (5 U/mL), streptomy-

cin (5 lg/mL), and 10% FBS. After 8–10 DIV, 1 lM cytosine-

arabinoside was added during 3 days to eliminate proliferating micro-

glia. Medium was changed twice a week and cultures were used after 3

weeks. Cultures contained >95% GFAP (1) and S100b (1) cells.

Cell Treatments and Conditioned MediaCultures of microglia, astrocytes or mixed glial cells (1:4 micro-

glia:astrocytes) were stimulated with 1 lg/mL LPS (E. coli.,

O111:B4, Sigma) for 24–96 h. In addition to LPS stimulation, in

different experiments microglia were co-treated with 1 lM L-N6

(iNOS selective inhibitor), 15 lM indometacine (nonselective COXs

inhibitor), 15 lM sc-560 (inhibitor of COX1), 5 lM ns-398

(COX2 inhibitor), 20 lM sc-19220 (EP1 prostanoid receptor antag-

onist) or 0.1-1 ng/mL TGFb1. To obtain conditioned media (CM)

from astrocytes, cells were seeded (2x106 cells in 35 mm dishes) in

DMEM containing 10% FCS under control condition (non stimu-

lated CM) or stimulated with 1 lg/mL LPS for 96 h. Astrocyte

CMs were collected after 96 h of stimulation, filtered (0.22 lm

pore), and stored at 220�C until use. CM were heated at 80�C for

10 min to activate TGFb1 (Munger et al., 1997; Vincent et al.,

1997). When needed, to neutralize TGFb1, CM were incubated

with a neutralizing antibody specific for TGFb1 (3 lg/mL, R&D)

at room temperature for 4 h, before being added to cell cultures

(Vincent et al., 1997). Accordingly, in some experiments cultured

microglia were stimulated with 1 lg/mL LPS and incubated with

2024 Volume 61, No. 12

CM alone or with 10 lM SIS3 (a specific Smad3 inhibitor). A sum-

mary of all pharmacological agents used in this study and their

actions are included in Table 1.

siRNA TransfectionThree unique 27mer siRNA duplexes against rat Panx1 or Cx43

were predesigned and obtained from Origene (Rockville, MD).

siRNA (10 nM) was transfected with Oligofectamine (Invitrogen)

according to the Origene application guide for Trilencer-27 siRNA.

Negligible cell death was detected after transfection (data not

shown). Sequences for siRNAs against rat Panx1 were: siRNA-

Panx1-A: rArGrArCrUrArArArUrArUrGrGrArArArCrGrArGrUrArA

GT; siRNA-Panx1-B: rGrGrArArArCrCrUrGrArGrUrUrUrArGrAr

UrCrArCrUrGAG; siRN-Panx1-C: rGrGrArCrUrUrCrArArArGrAr

UrUrUrGrGrArCrCrUrGrAGC. Sequences for siRNAs against rat

Cx43 were: siRNA-Cx43-B: rGrGrArArGrArGrArArGrCrUrArArAr

CrArArGrArArArGAA, siRNA-Cx43-C: rArGrArCrUrCrArCrArAr

ArUrArCrArGrArUrUrUrGrArATC.

Determination of NitritesNitrites, a stable downstream product of NO released by cells, was

determined in culture media by the Griess assay. Cultures with

3x104 cells were maintained at control condition, or stimulated with

1 lg/mL LPS. For determination of NO2-, 50 lL of medium was

mixed with 10 ll EDTA:H2O 1:1 (0.5 M, pH 8.0) and 60 lL of

freshly prepared Griess reagent (20 mg N-[1-naphtyl]-ethylenedia-

mine and 0.2 g sulphanilamide dissolved in 20 mL of 5% phos-

phoric acid, w/v). Calibration curves were established with 1–80 lM

NaNO2. Absorbency was measured at 570 nm in a microplate auto

reader (ANTHOS 2010, Anthos Labtec Instrument).

Immunofluorescence and Confocal MicroscopyCells grown on coverslips were fixed with 2% paraformaldehyde at

room temperature for 30 min and then washed three times with PBS.

They were incubated in 0.1 M PBS-glycine, three times for 5 min each

and then in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min.

Fc receptors were masked by incubation in Fc-Block (1:100) solution at

room temperature for 45 min. Microglia and astrocytes were identified

with Iba1 and GFAP, respectively. Cells were incubated at room tem-

perature for 2 h with anti-GFAP monoclonal antibody (IgG1, 1:500)

or anti-Iba1 polyclonal antibody (1:400) diluted in 0.1% PBS-Triton

X-100 with 2% NGS. After three rinses with 0.1% PBS-Triton X-100,

cells were incubated at room temperature for 50 min with goat anti-

mouse IgG Alexa Fluor 355 (1:1,500) and goat anti-rabbit IgG Alexa

Fluor 488 (1:1,500). After several washes, coverslips were mounted in

Fluoromount and examined with a confocal laser-scanning microscope

(Olympus, Fluoview FV1000, Tokyo, Japan).

Western Blot AnalysisCultures were rinsed twice with PBS (pH 7.4) and harvested by

scraping with a rubber policeman in ice-cold PBS containing 5 mM

EDTA, Halt (78440) and M-PER protein extraction cocktail

(78501) according to manufacturer instructions (Pierce, Rockford,

IL). The cellular suspension was sonicated on ice. Proteins were

measured using the Bio-Rad protein assay. Aliquots of cell lysates

(100 lg of protein) were resuspended in Laemli’s sample buffer,

TABLE 1: Pharmacological Agents Used in This Study

Agent Action

SIS3 Specific Smad3 inhibitor

aTGFb1 Neutralizing antibody specific for TGFb1

L-N6 Selective inhibitor of iNOS

Indometacin Nonselective inhibitor of COX 1 and 2

NS-398 Selective COX 2 inhibitor

SC-560 Selective COX 1 inhibitor

SC 19220 EP1 Prostanoid receptor antagonist

BAPTA-AM Intracellular calcium chelator

probenecid Panx1 hemichannel blocker

Gap26 Mimetic peptide against Cx43 hemichannels

Cx43E2 Antibody against Cx43 hemichannels

xestospongin B IP3 receptor inhibitor

MRS2179 Selective P2Y1 inhibitor

A740003 Selective P2X7 inhibitor

BBG Nonselective P2X7 inhibitor

oATP Selective P2X7 inhibitor

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2025

separated in an 8% sodium dodecyl sulfate polyacrylamide gel elec-

trophoresis and electro-transferred to nitrocellulose sheets. Nonspe-

cific protein binding was blocked by incubation of nitrocellulose

sheets in PBS-BLOTTO (5% nonfat milk in PBS) for 30 min. Blots

were then incubated with primary antibody at room temperature for

1 h or at 4�C overnight, followed by four 15 min washes with PBS.

Blots were incubated with goat anti-rabbit antibody conjugated to

HRP. Immunoreactivity was detected by enhanced chemilumines-

cence (ECL) detection using the SuperSignal kit (Pierce, Rockford,

IL) according to the manufacturer�s instructions.

Intracellular Ca21 ImagingCells plated on glass coverslips were loaded with 5 lM Fura-2-AM

in DMEM without serum at 37�C for 45 min and then washed

three times in Locke’s solution (154 mM NaCl, 5.4 mM KCl,

2.3 mM CaCl2, and 5 mM HEPES, pH 7.4) followed by de-

esterification at 37�C for 15 min. The experimental protocol for

[Ca21]i imaging involved data acquisition every 5 s (emission at 510

nm) at 340- and 380-nm excitation wavelengths using an Olympus

BX 51W1I upright microscope with a 40x water immersion objec-

tive. Changes were monitored using an imaging system equipped

with a Retiga 1300I fast-cooled monochromatic digital camera (12-

bit) (Qimaging, Burnaby, BC, Canada), monochromator for fluoro-

phore excitation, and METAFLUOR software (Universal Imaging,

Downingtown, PA) for image acquisition and analysis. Analysis

involved determination of pixels assigned to each cell. The average

pixel value allocated to each cell was obtained with excitation at each

wavelength and corrected for background. Due to the low excitation

intensity, no bleaching was observed even when cells were illumi-

nated for a few minutes. The ratio was obtained after dividing the

340-nm by the 380-nm fluorescence image on a pixel-by-pixel base

(R5F340 nm/F380 nm). Fura-2AM in situ calibration to obtain the

intracellular Ca21 concentration ([Ca21]i) of a single cell was per-

formed according to the equation of Grynkiewicz and colleagues

(Grynkiewicz et al., 1985): [Ca21]i 5 Kd b(R2Rmin)/(Rmax2R),

where Kd 5 224 nM is the dissociation constant of Fura-2AM and

R is the measured F340/F380 ratio. The Rmin was determined by

using 10 mM EGTA 1 10 lM A23187 in Ca21-free Locke’s solu-

tion and Rmax was obtained by using 5 lM ionomycin and 10 mM

CaCl2. b was calculated as the ratio Fmin/Fmax at 380 nm.

Measurement of Extracellular ATP ConcentrationCells were grown in 6-well plates for 15 days, after which they were

incubated in Locke’s solution at 37�C for 30 min. Cells were then

treated with 10 mM glucose for 10 min, and ATP concentration in

the extracellular solution was measured using a luciferin/luciferase

bioluminescence assay kit (Sigma-Aldrich). Baseline measurements

were performed on separate cultures using standard Locke’s solution.

The amount of ATP in each sample was calculated from standard

curves and normalized for the protein concentration as determined

by the BCA assay (Pierce).

Dye Uptake and Time-Lapse Fluorescence ImagingFor “snapshot” experiments, control and treated cells were exposed

to 5 lM Etd bromide at 37�C for 10 min. Then, cells were washed

with Hank�s balanced salt solution (137 mM NaCl; 5.4 mM KCl;

0.34 mM Na2HPO4; 0.44 mM KH2PO4, 1.2 mM CaCl2, pH 7.4),

fixed at room temperature with 2% paraformaldehyde for 30 min

and washed three times with PBS. Afterwards, they were incubated

in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min and

incubated with isolectin GS-IB4-488 (1:100) for 3 h, followed by

four PBS washes. Coverslips were mounted in Fluoromount and

examined with an upright microscope equipped with epifluorescence.

Images of Etd uptake were analyzed with the image J program (NIH

software). For time-lapse fluorescence imaging, cells plated on glass

coverslips were washed twice in Hank�s balanced salt solution. Then,

cells were exposed to Locke’s solution (154 mM NaCl, 5.4 mM

KCl, 2.3 mM CaCl2, 5 mM HEPES, pH 7.4) with 5 lM Etd. Flu-

orescence intensity was recorded in selected cells (ROIs, regions of

interest). Images were captured every 15 s with a QImaging Retiga

13001 fast-cooled monochromatic digital camera (12-bit) (Qimag-

ing, Burnaby, BC, Canada). Metafluor software (version 6.2R5, Uni-

versal Imaging Co., Downingtown, PA, USA) was used for off-line

image analysis and fluorescence quantification. To test for changes in

slope, regression lines were fitted to points before and after various

treatments using Excel program, and mean values of slopes were

compared using GraphPad Prism software and expressed as AU/min.

Cells were pre-incubated with channel blockers, synthetic mimetic

peptides, Gap26 (200 lM; VCYDKSFPISHVR, first extracellular

loop domain of Cx43) and 10panx1 (200 lM, WRQAAFVDSY,

extracellular loop domain of Panx1), for 15 min before experiments

were performed.

Data Analysis and StatisticsFor each data group, results were expressed as mean 6 standard

error (SEM); n refers to the number of independent experiments.

For statistical analysis, each treatment was compared with its respec-

tive control, and significance was determined using a one-way

ANOVA followed, in case of significance, by a Tukey post-hoc test.

Results

TGFb1 Released by Astrocytes Inhibits LPS-InducedNO Production by MicrogliaUnder inflammatory conditions, microglial cell activation

results among other changes, on increased expression of

inducible nitric oxide synthase (iNOS) and NO release (Fie-

bich et al., 1998; Zielasek et al., 1992). Furthermore, micro-

glia exhibit a long-lasting increase in basal [Ca21]i after

stimulation with LPS (Hoffmann et al., 2003), a well-

characterized and widely accepted agent from Gram negative

bacteria known to induce microglial activation (Kloss et al.,

2001). In order to evaluate whether astrocytes could modulate

the release of NO and [Ca21]i by microglia subjected to

inflammatory conditions; microglia, astrocytes or mixed glial

cell cultures were stimulated with 1 lg/mL LPS (Fig. 1A–F).

After stimulation with LPS, microglia in pure cultures became

round shaped and showed shorter and thicker processes with

an increased cell body size in mixed glial cell cultures and

astrocytes became more fibrillary in appearance (Fig. 1A–F).

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To assess the release of NO, nitrites, a stable derivative of

NO, were measured as previously reported (Tichauer et al.,

2007). Under control conditions, microglia did not exhibit a

significant variation in nitrite levels (Fig. 1G), whereas after

LPS stimulation for 24 h, they showed a �2-fold increase in

nitrites that increased over time up to ~11-fold after 168 h of

stimulation (Fig. 1G). Interestingly, cultures of mixed glial

cells or astrocytes exhibited a �4-fold increase in nitrites

reaching a plateau at 48 h of LPS stimulation that persisted

for at least 120 h (Fig. 1H,I). These results suggest that astro-

cytes appear to inhibit the increased NO production by

microglia after long periods of LPS stimulation (Fig. 1J).

Supporting this view, the proportion of astrocytes on the

mixed glial cell culture was inversely correlated with the

observed NO production by the culture after LPS stimulation

(Fig. 1K).

To determine whether the inhibitory effect of astrocytes

on LPS-induced NO production occurs directly or by soluble

factors released by them, microglia were incubated with con-

ditioned medium (CM) from LPS-stimulated or nonstimu-

lated astrocytes. Interestingly, CM collected from astrocytes

after 96 h of LPS stimulation, reduced LPS-induced nitrite

production by microglia to levels similar to those observed

under control conditions (Figs. 1G and 2A). These data sug-

gest that in LPS-stimulated mix glial cell cultures, the major

source of NO production were astrocytes, because they appear

to almost fully inhibit NO production by microglia (Fig.

1H). Moreover, CM from nonstimulated astrocytes partially

inhibited LPS-induced NO production by microglia, suggest-

ing that both LPS-stimulated and nonstimulated astrocytes

were able to inhibit NO production by microglia (Fig. 2A).

Astrocyte CM collected at different times of LPS stimulation

(24-168 h) exhibited a similar inhibitory effect (not shown).

It has been previously shown that TGFb1 reduces LPS-

induced NO release by microglia (Vincent et al., 1997).

Accordingly, we investigated whether TGFb1 was the soluble

FIGURE 1: Astrocytes inhibit LPS-induced NO production by microglia. A–F: Representative confocal images depicting Iba1 (green) andGFAP (red) immunolabeling in microglia cultures (A–B), microglia-astrocyte co-cultures (mixed glial cultures; C–D) or astroglial cultures(E–F) subjected to control conditions or stimulated with LPS for 96 h. Calibration bar 5 50 lm. G–I: Nitrite levels in microglia cultures(MG, G) under control conditions (white circles) or after LPS treatment (black circles), mixed glial (MG:AS, 1:4, H) or astrocyte cultures(AS, I). J: Nitrite production under control conditions (white bars) or after 96 h of LPS treatment (black bars) in mixed glial cultures(MG:AS, 1:4), astrocyte (AS) or microglia cultures (MG). K: Nitrite production after LPS treatment of mixed cultures with the followingratios of microglia:astrocytes: 1:4 (white circles), 2:3 (light gray circles), 4:1 (white squares) and 5:0 (black squares). *P < 0.05, LPS com-pared with control; #P < 0.05, MG compared to AS after LPS treatment. Averages were obtained from at least three independentexperiments in triplicate.

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2027

factor released from astrocytes that inhibited LPS-induced

nitrite production by microglia. At 96 h of LPS stimulation,

microglia exhibited a �9-fold increase in nitrites over control

conditions that was conspicuously reduced to �2-fold

increase by CM from LPS-stimulated astrocytes and partially

reduced to �5-fold increase by CM from nonstimulated

astrocytes (Fig. 2B). The CM-induced inhibition on NO pro-

duction by LPS-stimulated microglia did not occur when the

CM was immunoneutralized with an antibody specific for

active TGFb1 (Fig. 2B). Similar results in nitrite levels were

obtained when microglia were co-treated with LPS plus SIS3,

a specific inhibitor of Smad3 (Fig. 2B), a key protein

involved in the intracellular signaling triggered by the activa-

tion of the TGFb receptor. Because these results strongly sug-

gested that TGFb1 was the soluble factor released from

astrocytes in this system, we evaluated if addition of recombi-

nant TGFb1 could mimic the CM-induced inhibition on

NO production. Thus, when microglia were co-treated with

LPS plus 0.1 or 1 ng/ml TGFb1, they exhibited nitrite levels

similar to control microglia (Fig. 2B). These data strongly

indicate that TGFb1 released from astrocytes inhibits NO

release by microglia stimulated with LPS. Accordingly, we

observed that after 96 h of stimulation, LPS-treated astrocytes

exhibited an increased production of TGFb1 compared with

nonstimulated astrocytes (not shown). This result explains

why CM from LPS-treated astrocytes was more effective than

CM from non stimulated astrocytes for reducing LPS-

induced NO production by microglia (Fig. 2B).

Because 1 lM LN-6, a specific iNOS blocker, greatly

reduced the �8-fold increase of nitrites in LPS-stimulated

microglia to control levels (Fig. 2B), we performed a western

blot analysis to determine whether CM-induced inhibition on

nitrite production depended on changes in iNOS protein lev-

els. CM from LPS-stimulated astrocytes completely inhibited

the �3.5-fold increase of iNOS protein level induced by LPS

in microglia, whereas CM from nonstimulated astrocytes did

FIGURE 2: TGFb1 released by astrocytes inhibits LPS-induced nitrite production by microglia. A: NO production by microglial cell cul-tures after increasing periods of LPS treatment alone (96 h, black circles) or in combination with conditioned media (CM) from astrocytesstimulated with LPS (96 h, white diamonds) or CM from nonstimulated astrocytes (gray diamonds). B: NO production by microglia undercontrol conditions or stimulated with LPS (96 h) alone or in combination with CM from nonstimulated or LPS stimulated (96 h) astro-cytes. In addition, NO production by microglia stimulated with LPS (96 h) plus TGFb1 (0.1 and 1 ng/ml) or L-N6 (1 lM) was analyzed.Immunoneutralization of TGFb1 (aTGFb1) or SIS3 treatment abolished the reduction on NO production induced by the CM from LPS-stimulated astrocytes. *P < 0.05, **P < 0.005, for treatments compared with control; ##P < 0.005; compared with LPS stimulation. Aver-ages were obtained from at least four independent experiments in triplicate. C: Protein level of iNOS in microglia cultures under controlcondition (Lane 1), stimulated with LPS for 96 h (Lane 2) alone, or in combination with CM from nonstimulated (Lane 3) or 96 h LPS-stimulated astrocytes (Lane 4). SIS3 treatment (Lane 5) or immunoneutralization of TGFb1 with aTGFb1 (Lane 6) abolished the reductionon iNOS levels induced by CM from LPS-stimulated astrocytes. In addition, protein levels of iNOS in microglia stimulated with LPS plusTGFb1 (0.1 ng/mL, Lane 7) are shown. Protein expression was normalized by the corresponding level of a-tubulin. D: Quantification ofprotein levels of iNOS from three independent experiments. *P < 0.05 for treatments compared with control; #P < 0.05; for treatmentscompared with LPS stimulation.

2028 Volume 61, No. 12

not produce a similar inhibition (Fig. 2C,D). Supporting the

regulatory role of astroglial TGFb1 on iNOS expression,

CM-induced inhibition of iNOS did not occur when LPS-

stimulated microglia were exposed to CM previously immu-

noneutralized for TGFb1, or in microglia co-treated with

LPS plus SIS3 (Fig. 2C,D). These data, along with the fact

that TGFb1 reduced iNOS levels to control values in LPS-

stimulated microglia (Fig. 2C,D), suggest that CM-induced

inhibition of NO release occurs by a TGFb1-dependent inhi-

bition of iNOS expression.

To elucidate whether astroglial modulation of LPS-

induced NO release was correlated with changes in microglial

activation, we used confocal microscopy to evaluate the expres-

sion of CD68 (marker for macrophage function) and major

histocompatibility complex II (MHC II) (marker for antigen

presentation activity). Under control conditions, microglia

exhibited low levels of CD68 or MHC II (Fig. 3A,M),

whereas they shown a robust increase in the expression of

both markers after LPS stimulation for 96 h (Fig. 3E,Q;

Supp. Info. Fig. 1). Importantly, CM from LPS-stimulated

astrocytes changed the LPS-induced increase in CD68 and

MHC II expression to levels similar to those observed under

control conditions (Fig. 3I,U; Supp. Info. Fig. 1). These data

indicate that astrocytes inhibited microglial cell activation

induced by LPS.

Increased Basal Intracellular Ca21 in LPS-StimulatedMicroglia Depends on iNOS/PGE2 Pathway and IsInhibited by TGFb1 Released by AstrocytesAs mentioned, microglia exhibit a long-lasting increase in

basal [Ca21]i after LPS stimulation (Hoffmann et al., 2003).

However, whether astrocytes could modulate this process

remains to be elucidated. We examined the effect of CM

from LPS-stimulated or nonstimulated astrocytes in [Ca21]i

of LPS-stimulated microglia. Measurement of Fura-2AM ratio

(340/380) showed that control microglia exhibited a low

[Ca21]i (Fig. 4A,C). At 96 h of LPS stimulation, microglia

exhibited a �5-fold increase in basal [Ca21]i compared with

the control condition; increase that was abolished by CM

from LPS-stimulated astrocytes and was partially reduced to a

�3-fold increase by CM from nonstimulated astrocytes (Fig.

4B,C). Importantly, the CM-induced inhibition on basal

Ca21 signal did not occur in LPS-stimulated microglia

exposed to CM that was previously immunoneutralized for

FIGURE 3: LPS-induced increase in CD68 and MHCII expression is inhibited by astrocytes. A–L: Representative confocal images depictingIba1 (green) and CD-68 (red) immunolabeling of microglia under control condition (A–D), stimulated with LPS for 96 h alone (E–H) or incombination with CM from LPS stimulated astrocytes (I–L). Insets of representative microglia (*) labeled for Iba1, CD68, or merged arealso shown for Panels A, E, and I. M–X: Representative confocal images depicting Iba1 (green) and MHCII (red) immunolabeling of micro-glia under control condition (M–P), stimulated with LPS for 96 h alone (Q–Y) or in combination with CM from LPS stimulated astrocytes(U-X). The respective insets of representative microglia (*) with staining for Iba1, MHCII or merge are also shown for panels M, Q and U.Scale bar 5 8 lm.

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2029

TGFb1, or in microglia co-stimulated with LPS and SIS3

(Fig. 4C). As expected, recombinant TGFb1 reduced the

�5-fold increase in basal [Ca21]i in LPS-stimulated microglia

to control values (Fig. 4C), indicating that CM-induced inhi-

bition in basal [Ca21]i depended on the TGFb1 secreted by

astrocytes.

Because both the increase in NO production and the

basal [Ca21]i were inhibited by TGFb1, we examined

whether both responses were linked. It has been previously

shown that NO increases COX2 activity and prostaglandin E2

(PEG2) production by macrophages (Salvemini et al., 1993),

whereas the latter could further increase [Ca21]i via its action

on the G protein-coupled PEG2 receptor 1 (EP1 receptor)

(Woodward et al., 2011). iNOS and COXs inhibition by

L-N6 and indometacin, respectively, reduced the �5-fold

increase on basal [Ca21]i observed in LPS-stimulated micro-

glia to control values (Fig. 4C). To determine which COX

was involved in the response, we employed sc-560 and

ns-398, specific inhibitors for COX1 and COX2, respectively.

Thus, sc-560 reduced to �3-fold the increase of basal [Ca21]i

observed in LPS-stimulated microglia, whereas ns-398 com-

pletely abolished it (Fig. 4C). Importantly, inhibition of EP1

receptor with sc-19220 reduced [Ca21]i to control values in

LPS-stimulated microglia (Fig. 4C). All this evidence indicates

that the increased [Ca21]i found in LPS-stimulated microglia

depended on the activation of the iNOS/COXs pathway and

activation of the PGE2 receptor EP1.

TGFb1 Released by Astrocytes Inhibits LPS-InducedIncrease in ATP Release Via Pannexin1 ChannelsMicroglia exposed to inflammatory conditions, including

exposure to LPS, show an increased release of the gliotransmit-

ter ATP (Ferrari et al., 1997; Fujita et al., 2008; Kim et al.,

2007). We examined whether the increase in [Ca21]i observed

in LPS-stimulated microglia could be associated with increased

release of ATP. As shown by ATP measurement with the

FIGURE 4: TGFb1 released by astrocytes inhibits increased basal intracellular Ca21 and ATP release in LPS-stimulated microglia. A, B:Representative fluorescence micrographs of basal Fura-2AM ratio (pseudo-colored scale) in microglia under control condition (A) orstimulated with LPS by 96 h (B). C, D: Averaged data of [Ca21]i (C) and ATP release (D) by microglia under control conditions or stimu-lated with LPS (96 h) alone or in combination with the following blockers: 1 lM L-N6; 15 lM indometacin (indomet); 1 lM sc-560; 5 lMns-398; 20 lM sc-19220. In addition, basal Fura-2AM ratio and ATP release were analyzed in microglia stimulated with LPS (96 h) in com-bination with CM from astrocytes nonstimulated or stimulated with LPS (96 h). Immunoneutralization of TGFb1 (aTGFb1) or SIS3 treat-ment abolished the reduction on basal [Ca21]i and ATP release induced by CM from LPS-stimulated astrocytes. In addition, basal [Ca21]iand ATP release by microglia stimulated with LPS (96 h) in combination with TGFb1 (0.1 ng/mL) were analyzed. **P < 0.005, treatmentscompared with control; ##P < 0.005, #P < 0.05; treatments compared with LPS stimulation. Averages were obtained from at least fourindependent experiments in triplicate. Scale bar 5 25 lm.

2030 Volume 61, No. 12

luciferin/luciferase bioluminescence assay, LPS-stimulated

microglia exhibited a �5-fold increase on ATP release com-

pared with control conditions (Fig. 4D). The increase was

abolished by CM from LPS-stimulated astrocytes and partially

reduced to a �2-fold increase by CM from nonstimulated

astrocytes (Fig. 4D). In addition, the CM-induced inhibition

on ATP release did not occur in LPS-stimulated microglia

exposed to CM immunoneutralized for active TGFb1 or in

microglia co-stimulated with LPS plus SIS3 (Fig. 4D). Nota-

bly, as occurred with changes in basal [Ca21]i, TGFb1 and

inhibition of iNOS, COX1, COX2 or the PGE2 receptor EP1

abolished the �5-fold increase on ATP release in LPS-

stimulated microglia (Fig. 4D).

Recently, it has been demonstrated that two of the path-

ways for ATP release by microglia are connexin hemichannels

and pannexin channels (Higashi et al., 2011; Orellana et al.,

2011). They are plasma membrane channels that enable dif-

fusional exchange between the intra- and extracellular com-

partments, allowing cellular release of relevant quantities of

autocrine/paracrine signaling molecules (MacVicar and

Thompson, 2010; Stout et al., 2004). Given that these chan-

nels are known to be opened by the rise of [Ca21]i, (Locovei

et al., 2006; Wang et al., 2012), and because LPS-induced

ATP release in microglia was prevented by BAPTA (Fig. 4D),

we examined if connexin hemichannels or pannexin channels

participate in the ATP release induced by LPS. First, we

examined the functional activity of these channels in LPS-

stimulated microglia by measuring the rate of ethidium (Etd)

uptake. Etd only crosses the plasma membrane in healthy

cells by passing through specific large channels, including

connexin hemichannels and pannexin channels. Upon bind-

ing to intracellular nucleic acids, Etd becomes fluorescent,

indicating channel opening when appropriate blockers are

employed (Schalper et al., 2008). LPS-stimulated microglia

exhibited a �3.5-fold increase in Etd uptake compared with

control conditions (Fig. 5A–C). Uptake was strongly inhib-

ited by CM from LPS-stimulated astrocytes, but not by CM

immunoneutralized for active TGFb1, or in microglia co-

stimulated with LPS and SIS3 (Fig. 5C). Microglia express

connexin 43 (Cx43) hemichannels and pannexin 1 (Panx1)

channels. The possible role of Panx1 channels in LPS-induced

Etd uptake and ATP release was studied using Probenecid

and the mimetic peptide 10panx1 with an amino acid

sequence homologous to the second loop of Panx1 (Pelegrin

and Surprenant 2006; Silverman et al., 2008). Probenecid

(500 lM) and 10panx1 (200 lM) nearly abolished the

increase in LPS-induced Etd uptake and ATP release by

microglia (Fig. 5C,D).

To confirm the involvement of Panx1 channels in the

above phenomenon, we downregulated Panx1 protein expres-

sion using siRNA. Three siRNAs (siRNA A, B, and C) for

Panx1 were tested by transfection of microglia cultures. Each

siRNA reduced Panx1 protein expression, as determined by

Western blot (Supp. Info. Fig. 2). siRNA C for Panx1 was

the most effective in reducing Panx1 protein expression in

microglia cultures (Supp. Info. Fig. 2A). Thus, siRNA C to

Panx1 was used in the subsequent experiments. As expected,

downregulation of Panx1 with siRNA strongly reduced the

increase in LPS-induced Etd uptake and ATP release by

microglia (Fig. 5C,D). In contrast, Cx43E2, an antibody that

blocks Cx43 hemichannels (Orellana et al., 2011) and the

mimetic peptide Gap26 homologous to the second loop of

Cx43; (Evans and Leybaert, 2007) did not reduce LPS-

induced Etd uptake and ATP release (Fig. 5C,D). Further-

more, to rule out the involvement of Cx43 hemichannels in

LPS-induced Etd uptake and ATP release, we downregulated

Cx43 by using two siRNAs (siRNA B and C). siRNA C to

Cx43 was the most effective in reducing Cx43 protein expres-

sion (Supp. Info. Fig. 2B) and therefore it was used in the

subsequent experiments. In agreement with the abovemen-

tioned results, downregulation of Cx43 with siRNA did not

abolish the increase in LPS-induced Etd uptake and ATP

release in microglia (Fig. 5C,D), indicating that Cx43 hemi-

channels were not involved in this phenomenon.

Importantly, in microglia preloaded with the intracellular

Ca21 chelator BAPTA-AM (5 lM), LPS-induced Etd uptake

was almost totally abrogated (Fig. 5C). Altogether, these

results indicate that astrocytes inhibited Panx1-dependent ATP

release induced by LPS, possibly by reducing [Ca21]i in

microglia.

LPS-Induced Changes in ATP-Dependent Ca21

Dynamics Are Inhibited by TGFb1 Released byAstrocytesTo study the impact of astrocytes on receptor mediated Ca21

signaling on activated microglia, we elicited transient increases

in [Ca21]i with ATP, known to activate both ionic and

metabotropic purinergic receptors, P2X and P2Y, respectively.

In control microglia, exposure to 500 lM ATP induced a

small transient peak in [Ca21]i followed by a second gradual

increment (Fig. 6A). However, upon application of 500 lM

ATP, LPS-stimulated microglia showed a single prominent

peak of [Ca21]i without the second gradual increment (Fig.

6B). In both control and LPS-stimulated microglia, transient

peaks of [Ca21]i secondary to ATP stimulation, were strongly

inhibited by 5 lM BAPTA or after blockade of P2Y1 or IP3

receptors with 10 lM MRS2179 or 5 lM xestospongin B,

respectively (Fig. 6C,E). These results suggest that upon ATP

application, the early peaks in [Ca21]i in microglia came

from Ca21 released from intracellular stores via activation of

P2Y1 and IP3 receptors. The involvement of P2X receptors

on the above response was ruled out by the lack of inhibition

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2031

after exposure to 200 lM oATP, a general P2X receptor

blocker, or 10 lM A740003 and 10 lM brilliant blue G

(BBG), both P2X7 receptor blockers (Fig. 6E). In contrast,

the second increase in [Ca21]i observed upon ATP stimula-

tion in control microglia was abolished by 10 lM A740003,

suggesting the involvement of P2X7 receptors on this response

(Fig. 6D).

Interestingly, the ATP-induced peak of [Ca21]i was

almost entirely inhibited in LPS-stimulated microglia when

they were treated with CM from LPS-stimulated astrocytes

(Fig. 7A,B). In those conditions, LPS-stimulated microglia

shown [Ca21]i responses that were similar to those observed

in control microglia. Moreover, CM-induced inhibition on

ATP-induced [Ca21]i did not occur in LPS-stimulated micro-

glia exposed to CM immunoneutralized for active TGFb1, or

in microglia co-stimulated with LPS plus SIS3 (Fig. 7B).

Furthermore, as observed for the changes in basal [Ca21]i and

ATP release, TGFb1 and the inhibition of iNOS, COX1,

COX2 or the PGE2 receptor EP1 turned the ATP-induced

[Ca21]i to control values in LPS-stimulated microglia (Fig. 7B).

LPS-Induced Nitrite Production Depends onActivation of COXs, EP1 Receptors, Panx1Channels, and P2 ReceptorsFinally, we examined whether LPS-induced NO production

by microglia could have a self-perpetuating mechanism,

involving activation of COXs, PGE2 receptor EP1 and further

activation of P2 receptors via ATP released through Panx1

channels. Sc-560 and ns-398, inhibitors of COX1 and COX2,

respectively, as well as inhibition of EP1 receptor with

FIGURE 5: Increased ATP release observed in LPS-stimulated microglia occurs via Panx1 channels. A, B: Representative immunofluores-cence images depicting IB4 (white) labeling and Etd (red) nuclei-staining from dye uptake experiments (10 min exposure to Etd) in micro-glia cultures under control conditions (A) or treated with LPS for 96 h (B). High magnification inserts of microglia stained for IB4 and Etdare also shown. C, D: Averages of Etd uptake rate (C) and ATP release (D) by microglia under control condition or stimulated with LPS(96 h) alone or in combination with the following treatments: 0.1 ng/ml TGFb1; 5 lM BAPTA; 500 lM Probenecid (Prob); 200 lM10panx1; siRNAPanx1; 200 lM Gap26; 1:500 Cx43E2 and siRNACx43. TGFb1 immunoneutralization (aTGFb1) or SIS3 treatment preventedthe reduction on Etd uptake induced by CM from LPS-stimulated astrocytes. *P < 0.05, treatments compared with control; #P < 0.05;treatments compared with LPS stimulation. Averages were obtained from at least four independent experiments in triplicate. Scale bar5 20 lm.

2032 Volume 61, No. 12

sc-19220 and 5 lM BAPTA, partially reduced LPS-induced

NO production by microglia (Fig. 8). These results indicate

that increased levels of [Ca21]i likely associated with activa-

tion of COXs and PGE2 receptor EP1 were necessary to sus-

tain LPS-induced nitrite production by microglia. Moreover,

NO production in LPS-stimulated microglia was strongly

reduced by Probenecid (500 lM), 10panx1 (200 lM) or

siRNA against Panx1 (Fig. 8). In contrast, Gap26, Cx43E2

and siRNA against Cx43 did not reduce NO production

(Fig. 8). In addition, inhibition of P2Y1 and P2X7 receptors

by MRS2179 and A740003, respectively, partially reduced

LPS-induced NO production by microglia. Therefore, this

evidence support the idea that activation of P2 receptors via

ATP released through Panx1 channels appears to be crucial

for preserving high levels of NO production by LPS-

stimulated microglia.

Discussion

Our work demonstrates that astrocytes inhibit the inflamma-

tory profile triggered by LPS in microglia. TGFb1 released by

astrocytes reduced LPS-induced increase of [Ca21]i and ATP

release in microglia. Moreover, LPS-induced increase in [Ca21]i

required the involvement of iNOS, COX1, COX2, and the

PGE2 receptor EP1. The increase in [Ca21]i was related to

ATP release through the opening of Panx1 channels. In addi-

tion, TGFb1 released by astrocytes also abolished LPS-induced

changes in ATP-dependent intracellular Ca21 dynamics in

microglia. Interestingly, intracellular Ca21 linked to COX/EP1

receptor signaling and activation of P2 receptors through ATP

released through Panx1 channels were crucial to preserve the

NO production observed in LPS-stimulated microglia.

Previous studies have demonstrated that LPS increases

expression of iNOS and NO production via NF-kB pathway

FIGURE 6: ATP-dependent Ca21 dynamics in microglia depends on their inflammatory profile. A–D: Plots of relative changes in [Ca21]iover time induced by 500 lM ATP (gray vertical line) on microglia under control condition (A) or stimulated with LPS by 96 h (B). Theeffect of 10 lM MRS2179 and 10 lM A740003 on ATP-triggered Ca21 signal of LPS-stimulated microglia (C) or control microglia (D),respectively. E: Average data of maximal [Ca21]i intensity during the transient peak in control or LPS-stimulated microglia exposed toATP alone or in combination with the following blockers: 5 lM BAPTA; 5 lM xestospongin (XeB); 10 lM MRS2179; 10 lM A740003; 10lM Brilliant blue G (BBG), and 200 lM oxidized ATP (oATP). #P < 0.05, blockers compared with control; *P < 0.05, LPS treatment com-pared with control; &P < 0.05, blockers compared with LPS treatment. Averages were obtained from at least four independent experi-ments in triplicate.

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2033

in microglia both in vitro and in vivo (Han et al., 2001; Pos-

sel et al., 2000). More importantly, astrocytes are able to

inhibit this LPS-induced response by the release of TGFb(Vincent et al., 1997; Vincent et al., 1996). In agreement

with these studies, we found that as the proportion of astro-

cytes increased on a mixed glial cell culture, NO production

induced by LPS stimulation was progressively reduced. As

indicated also by previous studies (Vincent et al., 1997;

Vincent et al., 1996), by using SIS3 and TGFb1 immuno-

neutralization, we determined that the activation of the

TGFb1 pathway was necessary for the inhibitory effect of

astrocytes on NO production and iNOS expression. However,

our data indicated that this phenomenon was extended to

other properties of activated microglia, including Ca21

dynamics and ATP release, and was closely associated with

them by specific activation pathways. In fact, by using selec-

tive inhibitors of iNOS, COX1, COX2 and PGE2 receptor

EP1, we shown that LPS-induced increase in basal [Ca21]i on

microglia involved the participation of several elements start-

ing with NO production and ending with further activation

of PGE2 receptor EP1 (Fig. 9). Supporting this idea, it is

known that NO activates COXs enzymes (Salvemini et al.,

1993), which further produce prostaglandins and the activation

of prostanoid receptors (Woodward et al., 2011). Relevant to

this point, we found that LPS-induced increase in basal [Ca21]i

on microglia depended on PGE2 receptor EP1, which has been

detected in microglia (Li et al., 2011) and trigger release of

Ca21 from intracellular stores (Woodward et al., 2011).

Currently, ATP is considered to be an essential transmit-

ter in the intercellular communication among glial cells and

neurons, and can be released through membrane channels and

vesicles (Fields and Burnstock 2006). Moreover, ATP has been

implicated in the activation and chemotaxis-related features of

microglia upon inflammatory stimulation (Davalos et al.,

2005; Farber and Kettenmann 2006). Here, we demonstrated

that LPS-induced ATP release by microglia occurred via Panx1

channels and depended on intracellular Ca21 levels, EP1

receptor, COXs and iNOS. Accordingly, the LPS-induced

FIGURE 8: Increased NO production by LPS-stimulated microgliadepends on COXs and Panx1 channel activation. Nitrite levels inmicroglia cultures under control conditions or stimulated withLPS (96 h) alone or in combination with the following blockers: 1lM sc-560; 5 lM ns-398, 20 lM sc-19220, 5 lM BAPTA; 500 lMProbenecid (Prob); 200 lM 10panx1; siRNAPanx1; 200 lM Gap26;1:500 Cx43E2 and siRNACx43. *P < 0.05, **P < 0.005 treatmentscompared with LPS stimulation. Averages were obtained from atleast four independent experiments in triplicate.

FIGURE 7: LPS-induced changes in ATP-dependent Ca21 dynamics are inhibited by astroglial TGFb1. A: Relative changes in ATP-triggered Ca21 signal ([Ca21]i) over time in microglia stimulated with LPS (96 h) in combination with CM collected from astrocytes stimu-lated with LPS (96 h). Photomicrographs of time-lapse images showing changes in Fura-2AM ratio (pseudo-colored scale) are also shown.B: Averaged data of maximal [Ca21]i intensity during the ATP-induced transient peak by microglia under control conditions or stimulatedwith LPS (96 h) alone or in combination with the following blockers: 1 lM L-N6; 15 lM indometacin; 1 lM sc-560; 5 lM ns-398, 20 lMsc-19220. In addition, maximal [Ca21]i intensity during the ATP-induced peak were analyzed in microglia stimulated with LPS (96 h) incombination with CM from non stimulated astrocytes or astrocytes stimulated with LPS (96 h). TGFb1 immunoneutralization (aTGFb1) orSIS3 treatment fully prevented the reduction in maximal [Ca21]i induced by CM from LPS-stimulated astrocytes. The maximal [Ca21]i dur-ing the ATP-induced peak by microglia stimulated with LPS (96 h) in combination with TGFb1 (0.1 ng/mL) is also shown. **P < 0.005,treatments compared with control; #P < 0.05, ##P < 0.005; treatments compared with LPS stimulation. Averages were obtained from atleast four independent experiments in triplicate.

2034 Volume 61, No. 12

increase in ATP release was not detected after Panx1 channel

blockade with probenecid and 10panx1, or in microglia treated

with siRNA for Panx1. In agreement with this interpretation,

an increase in Etd uptake was observed in LPS-stimulated

microglia that was totally inhibited by Panx1 channel blockers

(e.g., probenecid and 10panx1) and downregulation of Panx1.

In contrast, Cx43 hemichannel blockers (e.g., Gap26 and

Cx43E2) or downregulation of Cx43 had no effect on Etd

uptake, indicating that channels composed by Panx1 were the

major contributors to this response. These results are consist-

ent with previous patch clamp and dye uptake experiments

showing that microglia exposed to inflammatory conditions

express functional single plasma membrane channels formed

by Panx1 (Orellana et al., 2011; Takeuchi et al., 2006).

Because LPS-induced Panx1 channel activity was inhibited by

blockers of iNOS, COXs, EP1 receptor and intracellular Ca21

levels, activation of Panx1 channels likely occurred down-

stream on the signaling pathway triggered by LPS (Fig. 9).

Our experiments with BAPTA indicated that intracellular

Ca21 levels were critical for ATP release and channel activity,

which is coherent with previous studies showing that ATP

released via Panx1 channels is mediated by rising [Ca21]i

(Locovei et al., 2006). Remarkably, as already observed for

other LPS-induced responses, astrocytes were able to reduce

the LPS-induced increase of Panx1 channel activity and ATP

release through the inhibitory effect of TGFb1.

As already mentioned, intracellular Ca21 dynamics serve

as a tight sensitive system to mediate intercellular communi-

cation among glial cells through the release of gliotransmit-

ters, cytokines and growth factors (Farber and Kettenmann,

2006; Koizumi, 2010). Because LPS stimulation of microglia

resulted in further release of ATP, we evaluated whether this

transmitter could affect Ca21 dynamics. Microglia express

two families of ATP receptors, G-protein coupled-type P2

receptors (P2Y receptors) and ionotropic P2 receptors (P2X

receptors). Previous studies with microglia revealed that ATP

(500 lM) produce a biphasic [Ca21]i response: the release of

stored Ca21 (first spike) and Ca21 influx from the extracellu-

lar medium (second shoulder) (Ferrari et al., 1996; Moller

et al., 2000; Verderio and Matteoli, 2001). The first spike in

the ATP-induced [Ca21]i response depend on P2Y receptors,

whereas the second shoulder response occurs via P2X7 recep-

tor activation. Accordingly, upon acute ATP stimulation con-

trol microglia exhibited an intracellular Ca21 increase

associated with P2Y1 and P2X7 receptors, whereas LPS-

stimulated microglia exhibited an intracellular Ca21 profile

characterized by activation of only P2Y1 receptors. The latter

acute ATP-induced Ca21 dynamics were inhibited by

FIGURE 9: Astroglial modulation of ATP-induced Ca21 dynamics in LPS-stimulated microglia. A: Under control conditions, extracellularATP and its derivates activate both P2X7 (1) and P2Y1 (2) receptors in resting microglia. Activation of P2Y7 receptors lead to a directincrease of [Ca21]i, whereas activation of P2Y1 receptors trigger the induction of IP3 receptors (3) and further release Ca21 stored in theendoplasmic reticulum (4). B: Upon LPS stimulation, microglia respond with intracellular signal transduction leading to iNOS activation,NO production, COXs activation and further production of PGE2 by an unknown mechanism (1). PGE2 released by microglia binds itsEP1 metabotropic receptors (2) produces the release of Ca21 from intracellular stores (3). The later increases [Ca21]i, a known conditionthat induces opening of Panx1 channels and release of ATP through them (4). ATP released via Panx1 channels and its degradation toADP activate P2Y1 (5) receptors, which induces activation of IP3 receptors (6) and further release of Ca21 stored in the endoplasmicreticulum (7). The later induces an unknown self-perpetuating mechanism (see discussion), in which high levels of [Ca21]i could reactivateiNOS, COXs, EP1 metabotropic receptors, and Panx1 channels (8). Astrocytes stimulated with LPS release TGFb1 (9), which inhibits LPS-induced intracellular signal transduction causing iNOS activation (10). An alternative negative feedback loop is the inhibitory effect thatATP could have on Panx1 channels (11). Finally, paracrine release of ATP from microglia could act on neighboring or distant microglia,resulting in an additional feed-forward mechanism (not depicted).

Orellana et al: Astrocytes Inhibit Inflammatory Activation of Microglia

December 2013 2035

BAPTA, MRS2179, and xestospongin B, but not by P2X

receptor blockers, revealing the involvement of the metabo-

tropic P2Y1 receptors, IP3 receptors and intracellular Ca21

stores. Given that ADP is the major ligand for P2Y1 recep-

tors, and because they participate in microglia Ca21 dynamics

(De Simone et al., 2010), in our system, ADP derived from

ATP degradation likely triggered the P2Y1-dependent changes

in [Ca21]i evoked by acute ATP administration.

Interestingly, the above described Ca21 response associ-

ated to P2Y1 receptors were completely inhibited when LPS-

stimulated microglia were treated with blockers of iNOS,

COXs or EP1 receptors, showing a similar Ca21 dynamic

profile to control microglia. These data indicate that Ca21

dynamics triggered by ATP depended on the inflammatory

profile of microglia. When components of LPS signaling were

inhibited, microglia behavior changed to a control profile. In

agreement with this idea, TGFb1 released by astrocytes

changed the Ca21 dynamic profile of LPS-stimulated micro-

glia to that observed in control microglia. TGFb1 has been

involved as a protective agent in several brain disorders by

reducing microglial activation (Flores and von Bernhardi

2012; Herrera-Molina et al., 2012; Uribe-San Martin et al.,

2009), in agreement with our results. Despite that in our sys-

tem, besides TGFb1 released from astrocytes, participation of

other factors released by astrocytes could not be ruled out.

Little is known about how the crosstalk among astrocytes

and microglia could modulate neuronal fate. Nevertheless,

gliotransmitter release depending on Ca21 dynamics stands up

as a possible mechanism for long-distance communication and

stimulation. Here, we showed that astrocytes could modulate

Ca21 dynamics and ATP release in microglia through the

release of factors that alter intracellular signaling (Fig. 9).

Moreover, our data showed that intracellular Ca21 linked to

COX/EP1 receptor signaling and P2 receptor activation, likely

via ATP released through Panx1 channels, were crucial to pre-

serve the NO production observed in LPS-stimulated micro-

glia. The latter could induce a self-perpetuating mechanism,

in which high levels of [Ca21]i could reactivate COXs, EP1

metabotropic receptors and Panx1 channels (Fig. 9). There-

fore, ATP released from microglia could stimulate distant

microglia in a paracrine manner, resulting in microglia Ca21

responses that could depend on the microglia inflammatory

profile (Fig. 9). If so, the activation of purinergic P2Y recep-

tors could be turned off in part by diffusion of ATP to distal

regions as well as by desensitization of P2Y1 receptors and

degradation of extracellular ATP by exonucleases. In addition,

astrocytes could modulate and suppress this self-perpetuating

mechanism triggered by inflammatory conditions in microglia.

In parallel, an alternative negative feedback loop is the inhibi-

tory effect that could be exerted by ATP on Panx1 channels as

previously described (Qiu and Dahl, 2009).

Future studies will be required to determine whether

astrocytes could inhibit the inflammatory profile of microglia

in vivo, leading to effective neuroprotection in diverse brain

pathologies. Understanding the mechanisms underlying glial

interaction can contribute to the knowledge on neuronal fate

in neurodegenerative conditions and open novel pharmaco-

logical strategies for therapeutic treatment of neurodegenera-

tive diseases.

Acknowledgment

Grant sponsor: CONICYT; Grant number: 79090028.

Grant sponsor: FONDECYT; Grant number: 11121133,

1090353, 1131025.

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