1. Alcohol and opioids activate microglia via TLR-4, NF-κB and p-AKT/PI3K (phosphoinositide 3
kinase) pathways in rat hypothalamic microglial culture
N. Thaker; P. Shrivastava; D.K. Sarkar
The Endocrine Program, Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 8901.
Introduction
Materials and Methods
Results
Sprague-Dawley pregnant female rats were obtained from Charles Rivers laboratories (Wilmington, MA) and
were inbred at Rutgers animal facilities as per ICUAC protocol. Primary microglial cultures were grown
using hypothalamus of PND-1 rat as described by Boyadjieva et al 2010.
Purity of microglial cultures were analyzed by IBA1 (microglial marker) staining. These cells were treated
ethanol (50mM), DAMGO (μ-opioid receptor agonist, 50μM), DPDPE (δ-opioid receptor agonist, 10nM),
and combination of ethanol DAMGO+Ethanol (DA+E), DPDPE+Ethanol (DP+E) opioid receptor agonists
and opioid antagonists (Naltrexone ((NTX),10ng/ml) and Naltrindole ((NTD),10ng/ml) for MOR and DOR
respectively, for 24 hours. To assess activation of TLR4 and the signaling pathways involved, protein and
RNA was isolated from microglia for western blot and Q-RT-PCR respectively. TNF-α levels were analyzed
by multiplex ELISA in microglial supernatants.
Binge alcohol drinking is a major cause of neurotoxicity and occurs through different pathways
including neural apoptosis, excitotoxicity, and neuroinflammation, but the effects of alcohol
exposure on neuroimmune responses are poorly understood. Microglia are key immune mediator
cells in brain and are known to respond to inflammation. Activation of microglia, by various drugs
of abuse occurs via toll like receptor 4 (TLR4).
Toll-like receptors (TLRs) contribute to neuroinflammation and signaling cytokines that act on
multiple interleukins, TNF-α/TLRs. The family of IL,TNF-α /TLRs signal through kinases
(p-AKT/PI3K), that activate the pro-inflammatory transcription factor nuclear factor kappa light-
chain-enhancer of activated B cells (NF- kB) increasing expression of cytokines, oxidases, and
other genes associated with innate immune responses. Studies have shown that both ethanol and
opioids activate TLR4 which results in the release of glial neuroinflammatory cytokines and
neurotoxic substances. Recent evidence suggests that opioid receptors µ and δ act differentially
with alcohol-induced activation.
Here we investigate the differential role of MOR and DOR agonists in alcohol induced microglial
activation and how they affect TLR-4 signaling pathways. We hypothesize that alcohol and MOR
agonist may promote TLR-4 downstream pathways (p-AKT/PI3K), increase NF-κB signaling, and
increase TNF-α production, whereas DOR agonist might inhibit the TLR-4 signaling pathways in
alcohol induced microglial activation suggesting a promising therapeutic target for alcohol abuse.
p-AKT expression in microglia cellsControl
EtO
H
DAM
G
O
DPDPE
DA+E
DP+E
NTD+E
NTX+E
0.0
0.5
1.0
1.5
2.0
2.5
*
#
###
*

aa
p-AKT/betaactin
pixeldensity
Microglial marker and opioid receptor staining of primary microglial culture.
Ethanol and MOR agonist along with
ethanol activates TLR4 downstream pAkt
pathway in microglia and DOR agonist
inhibits pAkt expression.
C
ontrol
E
tO
H
D
A
M
G
O
D
P
D
P
E
D
A
+E
D
P
+E
N
TD
+E
N
TX
+E
0.0
0.5
1.0
1.5
2.0
NF-kB expression in microglia cells
**
***
***


aaa###
NF-kB/betaactinexpression
pixeldensity
β-Actin
p-Akt
Fig 1:Images representing staining of primary microglial culture 10X. (A) IBA1 (B) µ-opioid receptor
(MOR) (C) δ-opioid receptor (DOR).
Fig 3: Expression of pAKT in ethanol activated microglial
cells via TLR4. Data expressed as Mean ±SEM (n=6). * P<
0.05 as compared to control, # P<0.05, ## P<0.01 as
compared to Ethanol, aa P<0.01 as compared to DAMGO,
µ P<0.001 as compared to DA + E.
Fig 4: Expression of NF-κB in ethanol activated microglial cells
via TLR4. Data expressed as Mean ±SEM (n=6). ** P<0.01, ***
P<0.001 as compared to control, # P<0.05, ## P<0.01 as
compared to Ethanol, aaa P<0.001 as compared to DAMGO, µ
P<0.001 as compared to DA + E, δ P<0.001 as compared to DP
+ E.MOR agonist enhances ethanol induced TNF-α
mRNA expression in microglia
Fig5 TNF-α mRNA expression in microglial cells. Effects of
ethanol (50mM) with or without MOR agonist (DAMGO, 50μM)
or DOR agonist (DPDPE,10nM) treatments for 24h on TNF-α
mRNA expression in microglial cells in cultures. The mRNA
expression was measured by QRT-PCR methods. * P<0.05, **
P<0.01, vs. control.
MOR agonist increases but DOR agonist
attenuates ethanol induced NF-κB
expression in microglia.
MOR agonist increases but DOR agonist
reduces, levels of pro-inflammatory cytokine
TNF- α in ethanol induce activated microglia
supernatant.
Summary and Conclusion
NF-κB
β-Actin
A B C
MOR agonist up-regulates ethanol-induced
TLR4 expression and DOR agonist down-
regulates ethanol-induced TLR4 expression
in microglial culture.
Fig 2: TLR4 mRNA expression in primary microglial culture treated with ethanol
(50 mM) and MOR (DAMGO, 50µM) and DOR (DPDPE, 10nM) opioid agonists
and MOR antagonist (NTX, 10ng/ml), DOR antagonist (NTD, 10ng/ml) with and
without ethanol. Cells treatment time 24 hours. Cells treatment time 24 hours.
Data expressed as Mean ±SEM (n=6), * P<0.05, ** P<0.01, as compared to control,
#P<0.05, as compared to ethanol.
Fig.6. Effects of ethanol (50 mM) with or without MOR
agonist (DAMGO, 50μM) or DOR agonist
(DPDPE,10nM) and LPS (10ng/ml) on TNF-α levels in
microglial cells culture media following 24 h incubation.
The TNF-α levels were quantified by Multiplex ELISA.
Data were expressed as Mean ±SEM (n=6). ***
P<0.001as compared to control, ###P<0.001 as compared
to Ethanol.
Fig7: Schematic diagrams showing proposed
mechanisms by which ethanol and opioids (MOR,
DOR) activate microglia via TLR-4 signaling
pathways.
 We show that MOR agonist upregulated ethanol activated TLR-4 mRNA transcripts in microglial cells.
In contrast, DOR agonist downregulated ethanol activated TLR-4 mRNA expression in microglia.
 MOR agonist increased ethanol-induced TLR-4 downstream p-Akt kinase protein expression but DOR
agonist inhibited p-Akt kinase expression.
 MOR agonist increased ethanol-induced pro-inflammatory transcription factor NF-κB protein expression
but DOR significantly attenuated the expression in microglia.
 MOR agonist increases TNF-α mRNA expression in ethanol-induced microglia. MOR agonist increased
ethanol-induced pro-inflammatory cytokine, TNF-α levels in microglial supernatant whereas, DOR
agonist inhibited the release of inflammatory cytokines in microglial supernatant suggesting an anti-
inflammatory effect.
 These findings suggest that ethanol-induces neuroinflammation through TLR-4 receptors, via p-Akt and
NF-κB activation which further activates TNF-α expression and its release which is associated with
immune responses. Our results demonstrate that MOR and DOR opioid agonists play differential roles in
ethanol-induced inflammation through TLR-4 activation in primary microglial culture.