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Role of purinergic P2X7 receptors in inflammatory responses in the brain and liver: a study using a mouse model of chronic ethanol and high-fat diet exposure
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Role of purinergic P2X7 receptors in inflammatory responses in the brain and liver: a study using a mouse model of chronic ethanol and high-fat diet exposure
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Content
Role of purinergic P2X7 receptors in inflammatory responses in the brain and
liver: A study using a mouse model of chronic ethanol and high-fat diet exposure
by
Daniel Freire
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTERS OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2016
ACKNOWLEDGEMENTS
I would like to thank my thesis advisor and mentor throughout my time at USC, Dr. Liana
Asatryan. She has been a constant support in both my research and the writing of this thesis. I
have learned a great deal from her these past two years, and what she has taught me will
continue to help me in my career well past this degree.
I would like to thank Dr. Daryl Davies and Dr. Curtis Okamoto for serving on my committee and
providing me with helpful advice throughout these past years.
I am thankful to all my lab members who have provided new insights into science and been a
great support throughout my time in the lab.
Finally, I am very grateful for my family for supporting me even while being far away, so that I
may achieve my degree.
ii
TABLE OF CONTENTS
Acknowledgements ii
List of Tables v
List of Figures vi
Abbreviations vii
Abstract viii
Chapter 1: Introduction 1
1.1 Alcohol abuse as a major health problem 1
1.2 Ethanol metabolism and effects in liver 2
1.3 Ethanol effects on the brain 4
1.4 Ethanol and neuroinflammation 5
1.5 ATP, purinergic receptors and ethanol action 8
Chapter 2: Aims 12
2.1 Aim 1: Investigate the role of P2X7R in inflammatory 12
response in the brain and liver in the early phases of ethanol
exposure and withdrawal
2.2 Aim 2: Investigate the effects of P2X7R antagonist on 13
the expression of pro-inflammatory markers and mediators
linked to P2X7R-signaling in the brain and liver
Chapter 3: Methods 14
3.1 Hybrid ethanol exposure 2/4 week treatment 14
3.2 Hybrid ethanol exposure plus antagonist treatment 15
3.3 Harvesting brains and liver tissues 16
3.4 Western Blotting 16
3.5 Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) 17
3.6 Custom Gene Expression RT-qPCR 17
3.7 Immunohistochemistry 18
3.8 Data Analysis 19
Chapter 4: Aim 1: Investigate the role of P2X7R in inflammatory 20
response in the brain and liver in the early phases of ethanol
exposure and withdrawal
4.1 Rationale 20
4.2 Results 21
4.2.1 Hybrid exposure alters GFAP expression 21
in hippocampus
4.2.2 Changes in inflammatory markers following 22
2 and 4 weeks Hybrid exposure
4.2.3 Changes in P2X7R expression in hippocampus 26
and amygdala after 2 and 4 weeks of Hybrid exposure
4.2.4 Hybrid model results in steatosis 27
4.2.5 Hybrid exposure for 2 and 4 weeks resulted in 29
changes in inflammatory markers in the liver
iii
4.2.6 Liver P2X7R expression decreased following 31
Hybrid exposure
4.3 Discussion 31
Chapter 5: Aim 2: Investigate the effects of a P2X7R antagonist 35
on the expression of pro-inflammatory markers and mediators
linked to P2X7R-signaling in the brain and liver
5.1 Rationale 35
5.2 Results 36
5.2.1 P2X7R antagonist inhibited changes in GFAP 36
expression in hippocampus
5.2.2 Changes in signaling and inflammatory markers 37
following Hybrid and P2X7R antagonist treatment
5.2.3 Antagonist treatment model reduced liver 39
steatosis in the Hybrid model
5.2.4 Inflammatory changes in the liver are attenuated 40
by P2X7R antagonist
5.3 Discussion 41
Chapter 6: Conclusion 45
References 47
iv
LIST OF TABLES
Table 1 List of primers for Taqman Custom Gene Expression RT-qPCR 18
v
LIST OF FIGURES
Figure 1 The role of P2X7Rs in inflammasome formation leading to 9
caspase 1 activation, cleavage of pro- IL-1β and mature
IL-1β release from cells
Figure 2 Body weights of mice post-mortem 15
Figure 3 GFAP staining of hippocampus tissue after 2 and 4 weeks 22
of ethanol exposure and withdrawal
Figure 4 RT-qPCR for inflammatory markers in hippocampus and 24
amygdala brain regions after 2 weeks of EtOH treatment
and EtOH+WD
Figure 5 RT-qPCR for inflammatory markers in hippocampus and 25
amygdala brain regions after 4 weeks of EtOH treatment
and EtOH+WD
Figure 6 mRNA expression of P2X7R for brain regions 26
Figure 7 P2X7R staining of hippocampus at 4 weeks 27
Figure 8 Western Blot P2X7R expression in brain 27
Figure 9 Liver to body weight ratio 28
Figure 10 Fat accumulation in liver following 2 and 4 weeks of ethanol 29
exposure and withdrawal
Figure 11 mRNA levels of inflammatory markers in liver following 2 30
weeks of treatment and 4 weeks of treatment
Figure 12 mRNA expression of P2X7R in liver 31
Figure 13 GFAP staining of hippocampus tissue 37
Figure 14 RT-qPCR of Neuroimmune response genes in 38
hippocampus and amygdala
Figure 15 Mice body weight throughout study 39
Figure 16 Fat accumulation in liver with antagonist treatment 40
Figure 17 mRNA expression of inflammatory markers in liver 41
vi
ABBREVIATIONS
ATP Adenosine triphosphate
ADH Alcohol dehydrogenase
AUD Alcohol use disorder
ALD Alcoholic liver disease
AD Alzheimer’s disease
BDNF Brain derived neurotrophic factor
CNS Central nervous system
A804598 N-Cyano-N"-[(1S)-1-phenylethyl]-N'-5-quinolinyl-guanidine
cAMP Cyclic adenosine monophosphate
GABA
A
R γ-aminobutyric acid receptors
GFAP Glial fibrillary acidic protein
H&E Hematoxylin and eosin
IHC Immunohistochemistry
IL Interleukin
iG Intragastric
iNOS Inducible nitric oxide synthase
JNK1 c-Jun N-terminal kinase 1
LPS Lipopolysaccharides
mRNA Messenger ribonucleic acid
MCP-1 Monocyte chemotactic protein 1
NAD Nicotinamide adenine dinucleotide
NLR NOD-like-receptor
NF-κB Nuclear factor kappa binding
NOD Nucleotide-binding oligomerization domain
NMDAR N-methyl-D-aspartate receptors
P2X7R P2X receptors
RT-qPCR Quantitative real time polymerase chain reaction
ROS Reactive oxygen species
SREBP-1 Sterol regulatory element-binding protein-1
TLR Toll-like receptors
TGF-β Transforming growth factor beta
TNF-α Tumor necrosis factor alpha
WB Western blotting
vii
ABSTRACT
Alcohol use disorder (AUD) is a problem which afflicts millions of people throughout the
world. Chronic consumption of alcohol leads to a myriad of health issues affecting several
organs systems in the body. Two major organs where alcohol has a dramatic damaging impact
are the brain and liver. Presently, there is a lack of pharmacological treatment options for
prevention or reduction of AUD. By understanding the pathway(s) for ethanol action and its
targets, especially in the liver and brain, we may hope to develop new approaches for treatment
of AUD. Recent growing evidence has shown that inflammatory responses to alcohol exposure
may be a mechanism for chronic alcohol-induced organ damage. Building evidence suggests
that ATP-activated purinergic P2X7 receptors (P2X7Rs), primarily residing on immune cells
throughout the body, play an important role in regulating inflammation and cell death. For
example, P2X7Rs have been linked to neurological diseases including neurodegenerative
pathologies and inflammatory conditions. Our laboratory is currently testing the hypothesis that
P2X7Rs play a role in alcohol-induced neuroinflammation. Initial results from this work, using a
model of intragastric (iG) ethanol exposure combined with high-fat diet (Hybrid) in C57BL/6J
mice (over 8 weeks), identified upregulation of P2X7Rs in ethanol-sensitive brain regions.
Importantly, these changes paralleled neuroinflammation and neurotoxicity in the same brain
regions. These findings served the basis for my thesis that tested the hypothesis that there is a
functional link between P2X7R-activated signaling events and ethanol-induced
inflammatory processes. Two interrelated Aims were designed to test the hypothesis. Aim 1
tested the role of P2X7R in inflammatory response in the brain and liver in the early phases of
ethanol exposure and withdrawal. Aim 2 explored the effects of a P2X7R antagonist on the
expression of pro-inflammatory markers and mediators linked to P2X7R-signaling in the brain
and liver. For Aim 1 studies, I used the Hybrid model at shorter time periods of exposure, i.e. 2
and 4 weeks. The effects of Hybrid exposure were investigated immediately after treatments as
well as 24 hours after ethanol withdrawal. In Aim 2 studies, I used the 4 week Hybrid exposure
viii
to test the effects of a P2X7R antagonist. I evaluated the inflammatory response in different
brain regions such as hippocampus and amygdala and in the liver using various techniques
including immunofluorescence, Western blotting, and RT-qPCR. The results of these studies
demonstrated that Hybrid treatment caused an inflammatory response after 2 and 4 weeks of
exposure as evidenced by the activation of astrocytes, release of cytokines, and increase in
P2X7R expression in the hippocampus and amygdala. In the liver, chronic alcohol consumption
caused steatosis and an increase in cytokines, but unexpectedly, levels of P2X7R decreased.
Furthermore, incorporation of a P2X7R antagonist into the study resulted in the attenuation of
the changes caused by the Hybrid treatment. Collectively, results from my investigation
supported the hypothesis that there is a functional link between P2X7Rs and the inflammatory
responses that arise as a result of chronic ethanol exposure.
ix
CHAPTER 1
INTRODUCTION
1.1 Alcohol abuse as a major health problem
Alcohol abuse continues to be a major worldwide concern as the most popular
consumed mind altering substance. Each year, approximately 3 million people die due to
alcohol abuse either through motor vehicle related accidents or direct overdosing. Apart from
the short term effects of alcohol intoxication, the long term effects from alcohol use disorder
(AUD) cost the United States alone almost $235 billion per year (2014). The negative health
effects from AUD include chronic pancreatitis (Yadav and Whitcomb, 2010), alcoholic liver
disease (ALD) (Rubin and Lieber, 1968), hepatitis (Szabo et al., 2010), cardiovascular disease
(Costanzo et al., 2010) and damage to the central and peripheral nervous systems (Muller et al.,
1985). Specifically in the central nervous system (CNS), alcohol has a wide range of negative
effects on the brain including: development of Wernicke encephalopathy–Korsakoff syndrome,
loss of cognitive function, behavioral changes, and reduction of brain volume (Vetreno and
Crews, 2014).
Presently, the pharmaco-social treatment options for AUD are limited due in part to
multiple targets of alcohol in the body. The most commonly used treatments include disulfiram,
naltrexone, and acamprosate (Lee and Leggio, 2014; Muller et al., 2014). Naltrexone and
acamprosate reduce the rewarding effects of alcohol and the desire for alcohol consumption
through different pathways. Naltrexone is an opioid antagonist whereas acamprosate is believed
to modulate glutamate and N-methyl-D-aspartate receptors (Muller et al., 2014). Disulfiram
functions by creating an aversion to alcohol, so if alcohol is consumed while on the medication
the patient becomes sick and experiences discomfort (Petrakis et al., 2005). Though there have
been some positive results, effectiveness of these drugs continues to be lacking and greatly
depends on patient compliance to maintain their drug therapy and social counseling, which is an
1
issue with AUD patients. As such, the hunt to develop new therapies for AUD continues to be a
primary goal of the substance abuse research community.
1.2 Ethanol metabolism and effects in liver
As with most substances, ethanol is first metabolized in the liver and is converted to
acetaldehyde through three different enzymes, i.e. alcohol dehydrogenase (ADH), cytochrome
P450 systems, and catalase. ADH is the major pathway that uses nicotinamide adenine
dinucleotide (NAD)
+
as an electron acceptor, converting it to NADH. This shift in the
NAD
+
/NADH ratio can cause an increase in the rate of fatty acid synthesis and a decrease in
mitochondrial beta-oxidation of free fatty acids. One of the results of this process is
development of steatosis, the first stage of ALD (Beier and McClain, 2010).
The cytochrome P450 systems primarily uses CYP2E1 driven by the reduction of O
2
to
H
2
O. This pathway dominates following high ethanol consumption (e.g., ethanol binge drinking),
as ethanol promotes CYP2E1 synthesis and decreases its degradation in hepatocytes (Cohen
et al., 2011). The change in pathway is believed to protect organisms as a whole, but as a
result, causes damage to the liver. A study by Abdelmegeed and colleagues reported that when
fed ethanol, mice lacking CYP2E1 had increased ratio of liver to body weights, serum
endotoxin, and hepatic levels of endobacteria, as compared to wildtype mice (Abdelmegeed et
al., 2013). CYP2E1 has also been shown to generate reactive oxygen species (ROS) in
hepatocytes through Kupffer cells (Han et al., 2016). ROS are involved in liver injury through
induction of tumor necrosis factor alpha (TNF-α) and activation of c-Jun N-terminal kinase 1
(JNK1). These two signaling pathways are involved in hepatocellular apoptosis (Xu et al., 2011).
Ethanol itself can also add to the production of ROS and oxidative stress, since metabolism of
ethanol requires a great deal of oxygen, creating hypoxia in the liver (Cohen et al., 2011). One
additional pathway of ethanol metabolism is due to catalase which oxidizes ethanol via
2
hydrogen peroxide reduction. Collectively, all three of these enzymes work rapidly to metabolize
ethanol to acetaldehyde.
Unlike the previous enzymatic reactions, the conversion of acetaldehyde to acetate (via
aldehyde dehydrogenase) is much slower, which allows for the accumulation of acetaldehyde.
Accumulation of acetaldehyde along with ethanol disrupts the intestinal barrier to allow bacterial
components into the systemic circulation (Lipnik-Strangelj, 2012). Acetaldehyde and ethanol
cause a decrease in expression of tight junction proteins between gut epithelial cells, which
increases gut permeability, allowing cytokines to spread through the liver. Lipopolysaccharides
(LPS) also leak through the gut and spread throughout the system, which eventually lead to
further cytokine production and activation of the immune system (Crews and Vetreno, 2014).
It has also been observed that ethanol increases levels of alanine aminotransferase and
aspartate aminotransferase which are both enzymes that are used as biomarkers of liver
function (Han et al., 2016). Another important indicator of liver damage is adiponectin, which
has impaired function in alcoholics. This protein works through its receptors AdipoR1/2 to
activate adenosine monophosphate (AMP) activated protein kinase which in turn inhibits
lipogenesis through sterol regulatory element-binding protein-1 (SREBP-1) and acetyl-CoA
carboxylase. However, in alcoholics, SREBP-1 can be induced by TNF-α and increases
endoplasmic reticulum stress, leading to enhanced hepatic lipogenesis (Xu et al., 2011). Using
an intragastric model of ethanol infusion in mice, Xu and colleagues reported observed an
increase in plasma alanine aminotransferase, adiponectin, and messenger ribonucleic acid
(mRNA) expression of AdipoR1, apoptotic genes, and pro-inflammatory genes (Xu et al., 2011).
Taken together, ethanol affects a variety of pathways through its metabolism and
signaling in the liver. Combination of changes in metabolic/signaling pathways and inflammatory
response may lead to substantial liver steatosis that can get aggravated and lead to liver fibrosis
and alcoholic liver damage upon chronic ethanol abuse.
3
1.3 Ethanol effects on the brain
Chronic ethanol use results in brain damage which is evident from addictive behaviors
and cognitive decrease in heavy drinkers (Sullivan and Pfefferbaum, 2005). Ethanol affects
several brain regions with the prefrontal cortex and the limbic system being the most sensitive
structures. Frontal lobes are important for regulating complex cognitive skills such as working
memory, temporal ordering, discrimination, and reversal learning that underlie judgement,
attention, risk taking, motivation, mood, and wanting. As a depressant, ethanol consumption
inhibits these cognitive skills, and these same symptoms are seen in chronic alcoholics in the
absence of ethanol in their systems. This has been attributed to a decrease in frontal cortical
thickness, of which the volume of this decrease is related to the duration and extent of ethanol
consumption (Crews and Vetreno, 2014). Ethanol causes a decrease in maturation and survival
of neurons, and inhibits proliferation of neurons, either through slowing of the cell cycle or
simply decreasing the number of cells that are proliferating (Crews and Vetreno, 2014; Nixon
and Crews, 2002). There is also a similar indirect effect from thiamine (vitamin B
1
) deficiency,
which is caused by chronic ethanol consumption. Thiamine deficiency will lead to neurotoxicity
and metabolic insults in the cerebral cortex, hippocampus and white matter (de la Monte and
Kril, 2014). Unfortunately, as lesions to the frontal areas of the brain result in poor decision
making and impulsivity, this can lead to further ethanol abuse and greater damage (Bowden et
al., 2001).
Due to its physical-chemical nature, ethanol acts non-specifically as an allosteric
modulator with multiple sites of action in the brain, affecting the activities of ionotropic,
metabotropic, and voltage gated ion channels. Depending on the particular protein target,
ethanol effects can either increase inhibition or decrease excitation. Behavioral impact of
ethanol in large arises from its action on ionotropic receptors. The most important ionotropic
receptors involving the effects of ethanol are N-methyl-D-aspartate receptors (NMDAR) and γ-
aminobutyric acid receptors (GABA
A
R). These two receptors have contrasting roles, NMDARs
4
are excitatory and GABA
A
Rs are inhibitory. As such, ethanol affects these receptors in opposing
manners, inhibiting the excitatory action of NMDARs and positively modulating GABA
A
Rs
(Chastain, 2006). Metabotropic receptors have less direct effects as they are coupled with
protein-signaling cascades. Ethanol affects several metabotropic receptors including mGlu2/3,
mGlu5, GABA
B
R, and opioid receptors, among others (Blednov and Harris, 2008; Tanchuck et
al., 2011). All these receptors are thought to contribute to the cognitive symptoms seen in
chronic alcoholics.
1.4 Ethanol and neuroinflammation
Ethanol’s chronic health effects on the brain have recently been attributed to the process
of neuroinflammation. Microglia, a type of glial macrophages, are a key component of the
inflammatory response in CNS. Microglia assist in scavenging the brain for potentially
dangerous substances, in addition to maintaining homeostasis in the brain by producing
neurotrophic factors and sequestering neurotransmitters. In response to an insult, microglia
undergo a morphological change, which is associated with their activation (Graeber, 2010).
Raivich has described five different levels of activation: resting, alert, homing, phagocytic, and
bystander activation (Marshall et al., 2013). The level of activation depends upon the type of
injury, the extent of damage, and the time to recover from injury. The differing levels of
activation can be identified by upregulation of certain cytokines or pro-inflammatory factors
(Marshall et al., 2013). As the activation progresses, the microglia become enlarged and
increase the expression of cell matrix and cell adhesion proteins, then they begin to undergo
proliferation. Highly activated microglia are characterized by a phagocytic, rounded
macrophage-like morphology (Monif et al., 2010).
There is some controversy regarding the morphological change in microglia after ethanol
exposure. Marshall et al. observed in a four day binge mouse model that microglia were only
partially activated, as evidenced by autoradiograph binding and increases in transforming
5
growth factor beta (TGF-β) and interleukin 10 (IL-10) alone (Marshall et al., 2013). Meanwhile,
the Kane lab, using five day ethanol exposure in neonatal mice, reported full activation of
microglia with concomitant increases in cytokines TNF-α, IL-1β, and monocyte chemotactic
protein 1 (MCP-1) (Drew et al., 2015). There have also been reports that microglia activate in
response to minor pathological changes from remote, sterile stimuli (Graeber, 2010).
Microglia respond to insults through changes in cell marker protein levels and the
release of pro- and anti-inflammatory cytokines. It has been shown that ionized calcium-binding
adapter molecule 1, a protein which serves as a marker for expression of microglia, is increased
in the brains of alcoholics (Crews et al., 2015). Activation of microglia through consumption of
ethanol results in an increase in nuclear factor kappa binding (NF-κB), a protein essential for
transcription of cytokines. As a result, there is an increased production of inducible nitric oxide
synthase (iNOS) and inflammatory cytokines such as IL-1β, IL-21, and TNF-α. Concomitantly,
ethanol also activates toll-like receptors (TLRs) to cause a similar signaling cascade
(Fernandez-Lizarbe et al., 2009). Under acute insult, these cytokines play a role in defending
against pathogen infection and clearing damaged cells and tissue, but prolonged release can
result in damage to the electron transport chain and release of ROS which in turn causes
neuronal apoptosis (Vetreno and Crews, 2014). On the other hand, following ethanol exposure,
brain derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) are
decreased by microglia. These compounds serve a protective role in reducing levels of
intracellular free radicals, ROS, nitrite, glutathione, and catalase. Thus, by reducing the levels of
BDNF and cAMP, it is possible to favor apoptotic conditions. Therefore, partial activation of
microglia may be beneficial to the system as a whole, but upon prolonged full activation,
microglia can cause apoptosis through immune responses.
Astrocytes, a type of glial cells in the CNS, also play a role in regulating inflammatory
responses. They are involved in providing metabolic and trophic support to neurons, modulating
synaptic activities and have a strong capacity to scavenge oxidants and suppress cellular
6
apoptosis (Ridet et al., 1997). Astrocytes are heterogeneous cells and respond to changes in
the environment. As with microglia, chronic activation results in a change in morphology of
astrocytes, i.e. reactive astrocytes. Reactive astrocytes are characterized by hypertrophy,
proliferation, and an increase in the number and size of cells expressing glial fibrillary acidic
protein (GFAP) and vimentin. GFAP and vimentin function as intermediate filaments and help
constitute the cytoskeleton (Li et al., 2008). Both these products have been shown to be
necessary for scar formation following an insult (Sofroniew and Vinters, 2010). This scar inhibits
axonal regeneration, but also serves for wound closure and to protect other regions of the brain
from the injured tissue. Mice lacking these intermediate filaments exhibited increased infarct
volume following a stroke (Robel et al., 2011). Activated astrocytes also regulate the immune
system by expressing major histocompatibility complex antigens. Antigens are then presented
to major histocompatibility complexes via astrocytes and produce cytokines (Eddleston and
Mucke, 1993).
Activation of astrocytes via ethanol has been evidenced by increases in GFAP staining
and increases in the size of the astrocyte cell soma and the width and length of the processes.
Following activation of astrocytes there is increased production of cytokines and chemokines
such as MCP-1 and IL-6 (Kane et al., 2014). This then leads to oxidative stress and lipid
peroxidation through the release of iNOS, cytochrome c oxidase subunit 2, and other ROS. Due
to the high oxygen demand of the brain, it is highly sensitive to oxidative stress. ROS then
increases intracellular Ca
2+
, inhibits responses of astrocytes to physiological agonists, and
stimulates excess glutamate secretion. An increase in calcium concentration can also produce
more ROS, causing a feed-forward loop (Gonzalez and Salido, 2009). Finally, these alterations
have been attributed to motor deficits and lack of coordination and stability (Teixeira et al.,
2014).
There is a duality to the activation of astrocytes as they can also be neuroprotective. By
adding astrocytes to a primary cortical neuron culture, Narasimhan and colleagues were able to
7
induce release of the antioxidant glutathione and attenuate ethanol induced neurodegeneration
(Narasimhan et al., 2012). Release of glutathione also assists in protecting the astrocytes
themselves from the toxic effects of ethanol (Watts et al., 2005).
As discussed above, microglia and astrocytes respond to injuries and cause
inflammatory responses, though the exact mechanism controlling these glial cells is still
unknown. TLRs and IL-1β have been suggested to play a role (Alfonso-Loeches et al., 2012;
John et al., 1999), however there remains a paucity of information on the effects of ethanol on
other downstream receptors involved in ethanol’s damaging actions.
1.5 ATP, purinergic receptors and ethanol action
Adenosine triphosphate (ATP) is universally recognized as a primary form of energy.
However, ATP can serve as a neurotransmitter in the brain, activating several ligand gated ion
channels including adenosine receptors and purinergic receptors. Three different classes make
up the purinergic receptors, P1, P2Y, and P2X receptors (P2XRs). P2XRs consist of 7 family
members, P2X1-P2X7, which have 30-50 % sequence homology (Singh et al., 2009).
Structurally, P2XRs consist of 2 transmembrane domains, N- and C-termini and a large
extracellular domain (ectodomain) (Idzko et al., 2014). All P2X receptors are permeable to small
monovalent cations. P2XRs are found on a wide variety of tissues and cell types including brain
neurons, retina, spinal cord neurons, epithelia and endothelia, skeletomuscular tissues, and
hemopoietic tissue (North, 2002). Accordingly, P2XRs serve several different functions which
include synaptic transmission, pain perception, cardiovascular modulation, immunomodulation,
and tumorigenesis (Wang and Yu, 2016).
Among the seven family members, P2X7Rs are unique in that they are activated by high
milimolar concentrations of ATP resulting in the formation of a homotrimer receptor with a large
pore allowing transmembrane fluxes of small hydrophobic molecules up to 900 Da, including
ATP (Kawate et al., 2009). P2X7Rs interact with many intracellular adaptor and signaling
8
proteins due to their unusual long C-terminus. P2X7Rs have been identified most frequently on
immune cells such as mast cells, macrophages, microglia, and dendrite cells (Monif et al.,
2010). P2X7Rs are also reported to be upregulated or involved in several inflammatory
diseases including Alzheimer’s Disease (McLarnon et al., 2006), amyotrophic lateral sclerosis
(Volonte et al., 2011), Huntington’s Disease (Diaz-Hernandez et al., 2009), behavioral disorders,
and other inflammatory diseases (Skaper et al., 2010).
Fig. 1. The role of P2X7Rs in inflammasome formation leading to caspase 1 activation,
cleavage of pro- IL-1β and mature IL-1β release from cells.
Collectively, the evidence indicates a regulatory role for P2X7R in inflammation, as the
level of ATP required to activate P2X7R is typically found near injured cells. Most evidence for
the involvement of P2X7R in inflammation comes from the relationship between P2X7R and
release of IL-1β, an inflammatory cytokine that plays a key role in response to ischemia and
CNS injury. Though the compound alone is not neurotoxic in the brain, its signaling pathway
becomes harmful in the impaired CNS where several neurotransmission systems are severely
dysregulated (Takenouchi et al., 2009). IL-1β itself also has a strong indirect effect on
inflammation as high concentrations prime the cell to susceptible neurotoxic effects. It was also
shown that the presence of IL-1β is necessary for α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid neurotoxicity (Bernardino et al., 2008b). Several studies have shown
strong evidence for the proposed relationship between P2X7R and IL-1β. Using a P2X7R
9
knock-out model, Solle and colleagues reported release of IL-1β by macrophages after
treatment with ATP was significantly less as compared to wild type control mice (Solle et al.,
2001). It is unclear exactly how P2X7R and IL-1β are related, but one proposed mechanism is
P2X7Rs activate nucleotide-binding oligomerization domain (NOD)-like-receptor (NLR)-
mediated inflammasome assembly with pro-caspase 1 proteolytic activation followed by
subsequent pro-IL-1 and pro-IL-18 cleavage and release of their active forms (Idzko et al.,
2014).
Other studies have also shown P2X7R involved with several other inflammatory
cytokines. Suzuki et al. used microglial cell cultures to show P2X7R was involved in the release
of TNF-α (Suzuki et al., 2004). Inhibitors of mitogen-activated protein kinase kinase, JNK, and
p38 were added to the cells which suppressed the production of TNF-α. Similar effects were
seen with the use of tyrosine kinase inhibitors. Treatment of microglia with a P2X7R agonist led
to reduced levels of glutamate-induced neuronal cell death. Reducing the levels of TNF-α
helped in the protection of cell death.
P2X7R is typically increased following injury to the CNS or peripheral tissues, though
few studies have shown that ethanol specifically can cause such a response. Recently, Jiang
and colleagues have shown that following chronic constriction injury to the spinal cord dorsal
horn, P2X7R expression increased along with microglial activation (Jiang et al., 2016). One
proposed mechanism for this activation is that stimulation of P2X7R, via injury to the CNS, in
turn causes the activation of microglia through changes in basal cytosolic calcium levels. This
mechanism has been supported by studies showing the addition of LPS in combination with
specific P2X7R agonists to neuronal cell cultures results in changes in the morphology of
microglia, indicating activation (Bernardino et al., 2008a). Similar effects are seen following the
addition of ionomycin, which increases cytoplasmic Ca
2+
levels. Furthermore, inhibition or
downregulation of P2X7R inhibited LPS induced microglial activation (Bianco et al., 2006).
10
Despite this large body of evidence linking P2X7Rs to neuroinflammation, the role of
P2X7R in ethanol-induced inflammatory responses in brain and liver is only beginning to be
investigated (work from Dr. Liana Asatryan) and has not been tested. As such, my thesis project
will focus on this interesting question. In support of this project, Dr. Asatryan has recently
started investigations to reveal the potential involvement of P2X7R in ethanol/mild obesity-
induced inflammatory responses using a mouse model of alcoholic liver disease developed at
the Southern California Research Center for Alcoholic Liver Disease, Pancreatitis and Cirrhosis
(Director, Tsukamoto). The findings of this work found neuroinflammatory responses and
parallel increases in P2X7R expression in this model of ethanol and high fat diet exposure
(Hybrid) (Asatryan et al., 2015; Xu et al., 2011).
11
Chapter 2
AIMS
The central goal of this thesis is to elucidate the potential role of P2X7R in regulating
ethanol-induced inflammatory responses in the brain and liver. This will be accomplished by
testing the hypothesis that Hybrid exposure induces early inflammatory responses in the brain
and liver which may be attenuated through addition of a P2X7R antagonist. In support of this
hypothesis, our laboratory has shown that 8 weeks of intragastric (iG) ethanol exposure
combined with high-fat diet in C57BL/6J mice causes an increase in pro-inflammatory markers
in the brain along with activation of microglia and astrocytes, two important glial cells in
regulating immune responses (Asatryan et al., 2015). Concomitantly, P2X7R expression in
similar ethanol-sensitive brain regions increased (Asatryan et al., 2015). Others have also
demonstrated an increased expression of P2X7R in other inflammatory diseases such as
multiple sclerosis and amyotrophic lateral sclerosis (Yiangou et al., 2006), Huntington’s Disease
(Diaz-Hernandez et al., 2009), and Alzheimer’s Disease (Parvathenani et al., 2003). Though the
exact mechanism is still unknown, evidence suggests that P2X7R regulates the post-
translational processing of pro-IL-1β into mature IL-1β (Solle et al., 2001). Mature IL-1β can
initiate upregulation of several other pro-inflammatory cytokines and ROS, thus leading to
neurodegeneration. Collectively, these findings served the basis for the hypothesis that
there is a functional link between P2X7R-activated signaling events and ethanol-induced
inflammatory processes.
Two Aims are set forth to test the hypothesis and to help understand the relationship between
P2X7Rs and ethanol induced inflammation.
2.1 Aim 1: Investigate the role of P2X7R in inflammatory response in the brain and liver in the
early phases of ethanol exposure and withdrawal.
The previous work has used 8 week exposure model where C57BL/6J mice were
exposed to chronic iG ethanol combined with high-fat diet. The current work extended these
12
studies to test the onset of inflammatory responses, i.e. effects of 2 and 4 weeks of ethanol
exposure as well as ethanol withdrawal. Others in the Asatryan laboratory have tested the
effects of P2X7Rs in both brain and liver.
The Aim 1 experiment compared differing periods of ethanol exposure and withdrawal. The
purpose of this design was to investigate any differences in expression of P2X7R or pro-
inflammatory makers in the brain and liver at different time periods. Results of Aim 1 also
informed the experimental design for Aim 2.
2.2 Aim 2: Investigate the effects of a P2X7R antagonist on the expression of pro-inflammatory
markers and mediators linked to P2X7R-signaling in the brain and liver.
Following the results from Aim 1, a 4 week exposure model was chosen to test the
effects of P2X7R specific antagonist A804598. The antagonist was administered via the iG
catheter, 3-times a week at 5 mg/kg dose. The purpose of this experimental design was to
investigate the consequence of inhibiting the P2X7R, in regards to the expression of pro-
inflammatory markers and signaling cascades in the brain and liver.
Collectively, the current studies are the first to test the effects of a P2X7R antagonist for
its effects on ethanol-induced neuroinflammation and liver damage. Overall, the results begin to
identify critical mediators linked to P2X7R-function and downstream signaling in ethanol effects
as well as identify molecular interactions of P2X7R-signaling with other neurochemical
pathways important for ethanol action. In addition, these studies set the stage for understanding
the therapeutic potential of P2X7Rs in conferring ethanol-induced brain and liver damage.
13
Chapter 3
METHODS
3.1 Hybrid ethanol exposure 2/4 week treatment
The Hybrid model of alcoholic liver disease developed at the Southern California Center
for Alcoholic Liver and Pancreatic Disease and Cirrhosis (Director Dr. Tsukamoto) combines
Western high fat diet with an established iG ethanol infusion (Ueno et al., 2012; Xu et al., 2011).
For this experiment, 8-10 weeks old C57BL/6J male mice were assigned to one of six of the
following groups (n=4): 2 or 4 week exposure of dextrose and high-fat liquid diet (“Dex”), ethanol
and high-fat liquid diet (“EtOH”), or ethanol and high-fat liquid diet followed by ethanol
withdrawal (“EtOH+WD”). First, all mice underwent surgery to place a catheter in their stomach.
After recovery from the surgery, mice were fed ad libitum solid Western diet high in cholesterol
and saturated fat (HCFD - 1% w/w cholesterol, 20%Cal lard, 17% corn oil:HCFD, Dyets Inc
#180724) for 2 weeks. After this, mice were split into either the 2 or 4 week groups with liquid
high fat diet (HFD - 35%Cal corn oil) plus either ethanol (~27 g/kg/day) at 60% of total required
calories for EtOH and EtOH+WD or isocaloric dextrose for Dex. All mice continued to consume
HCFD for the remaining 40% calories. In addition, once per week EtOH and EtOH+WD mice
were given a binge bolus dose (3.5~5g/kg) during the dark cycle after the infusion was
withdrawn for 5-6 hr. Finally, EtOH+WD were slowly weaned off of ethanol over the course of 2
days and then given no ethanol for 24 hours. BECs achieved were ~200-400 mg% or 50-100
mM. No significant changes in body weights were seen post-mortem as a result of Hybrid
treatment (Fig. 2).
14
Fig. 2. Body weights of mice post-mortem. Mean ± SEM. (n=4)
3.2 Hybrid ethanol exposure plus antagonist treatment
The Hybrid model was used following the same procedure as described above, though
all mice were treated for 4 weeks as we determined this time period showed the most significant
results. After 3 days of ethanol treatment, and three times a week following, mice were first
weighed and then injected iG through their catheters at a dose of 5 mg/kg with either the P2X7R
antagonist, N-Cyano-N"-[(1S)-1-phenylethyl]-N'-5-quinolinyl-guanidine (A804598) (Tocris
Bioscience, Bristol, UK) for “EtOH+Ant” group (n=10), or vehicle, consisting of 2% dimethyl
sulfoxide, 30% polyethylene glycol 300, and 68% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-
Aldrich, St. Louis, MO) for both “Dex+Veh” (n=10) and “EtOH+Veh” (n=10) groups. As with the
2/4 weeks study, Dex+Veh received dextrose whereas EtOH+Ant and EtOH+Veh received
ethanol. All mice were harvested 2 days after last injection of either vehicle or antagonist, while
ethanol treatment continued until sacrifice, with no withdrawal period.
15
3.3 Harvesting brains and liver tissues
Mice were sacrificed by CO
2
followed by cervical dislocation, weighed, then blood was
collected from the inferior vena cava and transcardial perfusion was performed using saline
solution through the left ventricle. Then mice were decapitated and the brain was removed and
dissected coronally into either three general sections (front, mid, rear) for immunohistochemistry
(IHC) or by specific region (hippocampus, amygdala, hypothalamus, prefrontal cortex, olfactory
bulb, and striatum) for western blotting (WB) and quantitative real-time polymerase chain
reaction (RT-qPCR). Whole livers were weighed at time of collection, then small 1 cm pieces
were cut from the median lobes for later histological evaluations, Western blot, and RT-qPCR.
Blood was centrifuged for 10 min at 10,000 rpm and serum was collected. Serum and tissues
collected for WB and RT-qPCR were snap frozen and kept at -80 °C, whereas tissues for IHC
were kept in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for two days, then transferred
to 70% ethanol.
3.4 Western Blotting
Brain sections were thawed in RIPA buffer (10 mM Tris, 140 mM NaCl, 1 mM EDTA,
1% Triton X-100, 0.1% sodium deoxycholate, pH 8.0) and homogenized by sonication. Protein
concentration was determined by bicinchoninic acid protein assay (Thermo Scientific, Houston,
TX). Once the concentration was determined, samples were appropriately aliquoted and
denatured using Laemmli buffer (Bio-Rad, Irvine, CA). Proteins were separated by 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene
fluoride membranes. Membranes were subsequently blocked with 5% blocking solution and
then incubated with primary antibodies, P2X7R (1:500, Alamone Labs, Israel) and β-Actin
(1:20,000, Sigma-Aldrich, St. Louis, MO). β-Actin was used as a normalization control.
Membranes were then imaged using ChemiDoc Touch Gel Imaging System (Bio-Rad, Irvine,
CA).
16
3.5 Quantitative Real-time Polymerase Chain Reaction (RT-qPCR)
Brain sections were thawed in Trizol (Invitrogen, Carlsbad, CA) and homogenized using
400 µm silica beads (OPS Diagnostics LLC, Lebanon, NJ) in Bullet Blender (Next Advance Inc,
Averill Park, NY). Following homogenization, RNA was isolated using RNeasy Mini Kit (Qiagen,
Valencia, CA) and then quantified using NanoDrop ND-1000 (Thermo Scientific, Houston, TX).
Once the concentration was known, 1 μg of RNA was aliquoted to convert to cDNA using High-
Capacity RNA-to-cDNA Kit (Life Technologies, Grand Island, NY). cDNA was again quantified
and purity was verified before mixing with primers and conducting RT-qPCR using ABI 7900 fast
real-time system (Applied Biosystems, Houston, TX). GAPDH was used as normalization
control. The following sets of primers were used: IL-1β forward — 5′-
TCGCTCAGGGTCACAAGAAA-3′, IL-1β reverse — 5′-CATCAGAGGCAAGGAGGAAAAC-3′;
IL-6 forward — 5′-TCGGAGGCTTAATTACACATGTTC-
3′, IL-6 reverse — 5′-CAAGTGCATCATCGTTGTTCATAC-3′; TNFα forward — 5′-
CATCTTCTCAAAATTCGAGTGACAA-3′, TNFα reverse —5′-
TGGGAGTAGACAAGGTACAACCC-3′; MCP-1 forward — 5′-
CCACTCACCTGCTGCTACTCAT-3′, MCP-1 reverse — 5′-TGGTGATCCTCTTGTAGCTCTCC-
3′; iNOS forward — 5′-CCTGGTACGGGCATTGCT-3′, iNOS reverse 5′-
GCTCATGCGGCCTCCTT-3′; P2RX7 forward — 5′-GACAAACAAAGTCACCCGGAT-3′, P2RX7
reverse 5′-CGCTCACCAAAGCAAAGCTAAT-3′; IL-22 forward — 5′-
TTTCCTGACCAAACTCAGCA-3′, IL-22 reverse 5′-TCTGGATGTTCTGGTCGTCA -3′.
3.6 Custom Gene Expression RT-qPCR
Samples were prepared in the same manner as in RT-qPCR. Primers were already
loaded onto the custom plates, so samples were mixed with master mix and water and then
loaded directly onto plates and analyzed using ABI Quant Studio OpenArray Real-Time PCR
System (Applied Biosystems, Houston, TX). See Table 1 for full list of primers.
17
Table 1. List of primers for Taqman Custom Gene Expression RT-qPCR
3.7 Immunohistochemistry
Brains and tissues were embedded in paraffin and 5 µm sections were prepared using a
rotary Microtome at the Histology Lab at the School of Pharmacy of USC. Hematoxylin and
eosin (H&E) staining on liver was performed at the Histology Lab. These slides were imaged
using a Nikon Eclipse 80i microscope and Optronics MicroFire True Color 4MP digital CCD
camera (Liver Core, USC School of Medicine). Images were saved using the PictureFrame
software. Brain sections were taken for deparaffinization, followed by fixation in acetone.
Antigen retrieval was then done using Tris-EDTA solution (10mM Tris Base, 1mM EDTA, 0.05%
Tween 20, pH 9.0) and heating using a common pressure cooker. This was followed by
permeabilization using 0.25% Triton X-100 and then blocking in 1% BSA and 10% goat serum.
After blocking, the sections were incubated with the following primary antibodies: P2X7R (1:100,
48 hrs, Alamone, Israel) and GFAP (1:2000, 2 hrs, Millipore, Temecula, CA). Slides were
mounted with Vectashield Hard Set Mounting Medium with DAPI (Vector Laboratories Inc,
Burlingame, CA). The slides were then visualized using the Nikon Diaphot 300 Inverted
Microscope and X-Cite 120LED for fluorescent illumination. Images were taken using the Nikon
18
DS-Qi1 monochrome digital camera, Nikon Digital Sight DS-U3 controller, and NIS-Elements
AR.4.40.00 software.
3.8 Data Analysis
Densitometry data for IHC was analyzed using Image J software (NIH). Fat
accumulation in H&E liver staining was analyzed using threshold adjustments in Image J to
automatically count fat deposits in liver tissues. All data presented as mean ± SEM using
GraphPAD Prism (San Diego, CA). Cell counts for positively stained cells were hand counted
using comparative brain region 10x images. Western blots were analyzed using Image Lab (Bio-
Rad, Irvine, CA). 2(-Delta Ct) method was used for the analyses of the RT-qPCR data
(Schmittgen and Livak, 2008). Significant differences between mouse groups were determined
using Student's t-test and significance set at P < 0.05. For custom gene expression RT-qPCR,
differences between mouse groups were determined using one-way ANOVA and significance
set at P ≤ 0.05.
19
Chapter 4
AIM 1: INVESTIGATE THE ROLE OF P2X7R IN INFLAMMATORY RESPONSE IN THE
BRAIN AND LIVER IN THE EARLY PHASES OF ETHANOL EXPOSURE AND
WITHDRAWAL
4.1 Rationale
As outlined above, ethanol has an adverse impact on several organ systems in the body
and the extent of the damage depends greatly on the degree and duration of the exposure.
Presently, there is no universal model of alcoholism and as this is a new field, there is presently
a large degree of variability in the literature from one study to the next, in part due to the
differing models employed from study to study. For example, Nixon and Crews showed in a 4
day binge in vivo model that ethanol inhibited neurogenesis in the hippocampus, though no cell
death was seen (Nixon and Crews, 2002). Whereas Alfonso-Loeches and colleagues found
ethanol induced apoptosis in mice drinking 10% ethanol ad libitum for 5 months (Alfonso-
Loeches et al., 2014). Despite these differing results, there is an emerging consensus that the
dosage and the duration of dosing are important factors in ethanol-induced brain damage. In
addition, repeating episodes of high doses of ethanol exposure are a contributing factor to the
adverse effects of ethanol exposure. This is in part due to sudden removal of ethanol, so called
ethanol withdrawal which is characterized by a hyperexcitability state demonstrating symptoms
such as tremors, diaphoresis, nausea/vomiting, hypertension, tachycardia, hyperthermia, and
tachypnea (Mirijello et al., 2015). These effects are a result of complex compensatory changes
in different neurotransmitter system functioning (decrease in inhibitory channels and activation
of excitatory receptors).
The previous work performed in our lab found neuroinflammatory response in the
standard protocol of 8 week exposure to Hybrid where C57BL/6J mice were receiving iG
ethanol combined with high-fat diet (Asatryan et al., 2015). Furthermore, significantly higher
extent of changes were found in the model with additional weekly binge exposures (Asatryan et
al., 2015). To understand whether changes are occurring earlier than the 8 week of Hybrid
20
treatment, in Aim 1 studies I sought to test the onset of the neuroinflammatory changes using
similar experimental models with shorter exposure time (2 and 4 weeks). I also tested the
effects of ethanol withdrawal. Overall, I predicted that the findings of my Aim 1 studies would
help me to optimize the experimental design for my Aim 2 studies where I proposed to use a
P2X7R pharmacological antagonist. In addition, as Hybrid models are generated for the studies
of alcoholic liver disease, I also harvested and tested liver tissues for changes in histological
features.
4.2 Results
4.2.1 Hybrid exposure alters GFAP expression in hippocampus
Staining of hippocampal slices with GFAP antibody showed an apparent increase in
proliferation in astrocytes in Hybrid ethanol binge groups (EtOH), compared to dextrose controls
(Dex) as early as 2 weeks of exposure. These changes are observed on the IHC images as
illustrated in Fig. 3 A. The analysis with Image J software did not show increases when
calculated as GFAP fluorescence intensity (Fig. 3 B) or cells area (Fig. 3 C). However, the
number of GFAP-positive cells significantly increased in both 2 week and 4 week EtOH groups
(Fig. 3 D). GFAP-positive cell number was also increased compared to the controls after 2 days
of ethanol withdrawal (Fig. 3 D, EtOH+WD). This increase was less compared to the EtOH
group and did not reach significance (P = 0.3).
21
Fig. 3. GFAP staining of hippocampus tissue after 2 and 4 weeks of ethanol exposure and
withdrawal. A. Immunohistochemistry images. B. Fluorescence intensity of staining
analyzed from cell bodies. C. Area of cell bodies. D. Number of GFAP-positive cells per
image field. All values reported as fold change compared to control, mean ± SEM. * P < 0.05,
** P < 0.005 compared to control (n=2)
4.2.2 Changes in inflammatory markers following 2 and 4 weeks Hybrid exposure
After homogenizing the hippocampus and amygdala, RNA was isolated and converted to
cDNA for RT-qPCR analysis. Most of the markers I analyzed in the hippocampus showed an
increase following 2 weeks of Hybrid treatment, with the exception of IL-6 (Fig. 4). IL-1β, TNF-α,
and iNOS increased in both EtOH and EtOH+WD groups, though significance was only seen in
22
EtOH+WD. MCP-1 had a significant increase in both EtOH and EtOH+WD and the increase in
the EtOH+WD group was the greatest of all the markers analyzed. IL-22 saw no change in
EtOH group and despite a large increase in the EtOH+WD group, this change was not
significant. Overall in the amygdala the results were not as profound as those seen in the
hippocampus. EtOH exposure caused a significant increase in IL-22 which was substantially
more during the EtOH+WD (Fig. 4). There was also a significant increase in IL-6 in the
EtOH+WD group.
23
Fig. 4. RT-qPCR for inflammatory markers in hippocampus and amygdala brain regions
after 2 weeks of EtOH treatment and EtOH+WD. All values reported as fold change compared
to control, mean ± SEM. * P < 0.05, ** P < 0.005, # P < 0.001 compared to control (n=4)
As presented in Fig. 5, 4 weeks of EtOH exposure resulted in similar changes in the
hippocampus as to what was seen at 2 weeks treatment. Most of the markers increased with
the exception of IL-6 and IL-22. IL-1β, TNF-α, and iNOS increased following EtOH treatment,
with significance for IL-1β and TNF- α reaching P < 0.05. Ethanol withdrawal caused greater
increase in TNF-α and MCP-1. In contrast to the changes in hippocampus, in amygdala there
24
was a significant decrease in the mRA levels for IL-1β, TNF-α, MCP-1, iNOS, and IL-22 (Fig. 5).
Lastly, no significant change was seen in EtOH+WD group compared to the controls for any of
the markers.
Fig. 5. RT-qPCR for inflammatory markers in hippocampus and amygdala brain regions
after 4 weeks of EtOH treatment and EtOH+WD. All values reported as fold change compared
to control, mean ± SEM. * P < 0.05, ♦ P < 0.01, ** P < 0.005, # P < 0.001; compared to control
(n=4)
25
4.2.3 Changes in P2X7R expression in hippocampus and amygdala after 2 and 4 weeks of
Hybrid exposure
No significant changes were found in P2X7R mRNA levels in the hippocampus region
following 2 weeks of Hybrid exposure (Fig. 6). P2X7R mRNA expression was increased
following Hybrid exposure in hippocampus and amygdala at both time points, 2 and 4 weeks, as
evidenced by RT-qPCR experiments (Fig. 6). However, only at 2 weeks is there a significant
change in the hippocampus of the EtOH+WD group and the amygdala of the EtOH.
IHC staining of the hippocampus also showed an increase in the number of P2X7R
positive cells in ethanol treated mice compared to controls (Fig. 7). As with the GFAP staining,
there seems to be recovery during withdrawal, though the change is more significant after 4
weeks of treatment (Fig. 7 B).
Results from the Western blotting identified brain region specific changes (Fig. 8).
Hippocampal P2X7R protein levels showed an increasing trend in the EtOH and EtOH+WD
groups at both time periods (2 week, EtOH P = 0.10, EtOH+WD P = 0.17; 4 week, EtOH P =
0.13, EtOH+WD P = 0.07). In the amygdala, results show a decreasing trend towards
significance of P2X7R protein expression, again in both groups at both time periods (2 week,
EtOH P = 0.07, EtOH+WD P = 0.15; 4 week, EtOH P = 0.22, EtOH+WD P = 0.07).
Fig. 6. mRNA expression of P2X7R for brain regions. All values reported as fold change
compared to control, mean ± SEM. * P < 0.05 compared to control (n=4)
26
Fig. 7. P2X7R staining of hippocampus at 4 weeks. A. Immunohistochemistry images B.
Number of P2X7R-positive cells per image field. All values reported as fold change
compared to control, mean ± SEM. * P < 0.05, compared to control, except where noted (n=1)
Fig. 8. Western Blot P2X7R expression in brain. A. Expression levels in brain regions at 2
weeks. B. Expression levels in brain regions at 4 weeks. C. Blot images of P2X7R at 2
weeks D. Blot images of P2X7R at 4 weeks. All values reported as fold change compared to
control, mean ± SEM. (n=3)
4.2.4 Hybrid model results in steatosis
Body and liver weights of mice were collected post-mortem. The Hybrid model caused
no significant changes in total body weight (Fig. 2). However, the liver weights increased (2
week treatment: Dex average liver weight = 1.48 g, EtOH = 2.12 g; 4 week treatment: Dex
27
average liver weight = 1.76 g, EtOH = 2.94 g), resulting in a time-dependent increase in liver to
body weight ratio (Fig. 9). The EtOH+WD groups also exhibited a slight recovery, though no
significant difference from their respective EtOH groups.
H&E staining of liver showed steatosis and an accumulation of Kupffer cells as well as
infiltrating leukocytes following EtOH exposure, which was more prominent at 4 weeks
compared to 2 weeks (Fig. 10). There was dramatically more steatosis after 4 weeks of
exposure in the EtOH+WD group compared to EtOH (Fig. 10 B). Abstinence following 2 weeks
of Hybrid exposure did not produce similar results.
Fig. 9. Liver to body weight ratio. Mean ± SEM. * P < 0.05, ** P < 0.005, # P < 0.001
comparing to respective time point controls, except where noted (n=4)
28
Fig. 10. Fat accumulation in liver following 2 and 4 weeks of ethanol exposure and
withdrawal. A. H&E staining of liver tissues. Accumulation of fat and infiltration of
inflammatory cells after 2 and 4 weeks of the Hybrid exposure. Arrows point to the inflammatory
foci (Kupffer cells and infiltrating lymphocytes). B. Analysis of steatosis in H&E slides. All
values reported as fold change compared to control, mean ± SEM. ** P < 0.005, # P < 0.001;
comparing to respective time point controls, except where noted (n=4)
4.2.5 Hybrid exposure for 2 and 4 weeks resulted in changes in inflammatory markers in the
liver
RT-qPCR was conducted using various primers for proteins involved with inflammation
in the liver (Fig. 11). Overall, results are specific for markers and times of exposure. At 2 weeks
IL-1β and IL-6 markers showed an increase following Hybrid treatment and subsequent
decrease during abstinence (Fig. 11 A). 2 weeks of treatment also resulted in a decrease in
MCP-1 in both groups compared to control. There was a decrease in IL-22 mRNA level
following 2 weeks of Hybrid treatment, though was only significant in the EtOH+WD group. 4
weeks of treatment resulted in a much greater increase in IL-1β mRNA in the liver, with
significance in the EtOH+WD group (Fig. 11 B). MCP-1 also increased in both EtOH and
EtOH+WD, though neither change was statistically significant. The other markers analyzed, IL-6
and IL-22, decreased following Hybrid exposure, though the changes were only statistically
significant for IL-6 in the EtOH and EtOH+WD groups.
29
Fig. 11. mRNA levels of inflammatory markers in liver following A. 2 weeks of treatment
and B. 4 weeks of treatment. All values reported as fold change compared to control, mean ±
SEM. * P < 0.05, # P < 0.001 compared to control (n=4)
30
4.2.6 Liver P2X7R expression decreased following Hybrid exposure
EtOH exposure resulted in a 0.25 and 0.5 extent of decrease in the P2X7R mRNA levels
at 2 and 4 weeks respectively, with significance reaching at 4 weeks of treatment (Fig. 12).
P2X7R mRNA levels were not significantly different from those of Dex in both 2 and 4 weeks of
EtOH+WD.
Fig. 12. mRNA expression of P2X7R in liver. All values reported as fold change compared to
control, mean ± SEM. * P < 0.05 compared to controls (n=4)
4.3 Discussion
In Aim 1 I set out to determine the early effects of Hybrid exposure, as well as
withdrawal from it. As glial cells are key regulators of inflammatory responses in the system, I
first focused on finding evidence of morphological changes to astrocytes. Following injury to the
CNS, astrocytes undergo physiological changes and increase in number in the immediate
affected area (Aschner et al., 2002). These changes are typically followed by alterations in
neuronal signaling and neurodegeneration. My results show that 2 and 4 weeks of Hybrid
exposure caused activation of astrocytes, as evidenced by an increase in the number of GFAP-
positive cells found in the hippocampal region. Importantly, these changes were observed as
early as at 2 weeks of Hybrid exposure. GFAP is widely accepted as a marker for astrocytes,
and my results coincide with others that have shown similar results following ethanol treatment
31
(Gonca et al., 2005; Gonzalez et al., 2007). Moreover, withdrawal from the Hybrid treatment
showed only a slight recovery as the number of GFAP-positive cells were not significantly
different compared to Hybrid treatment alone. These results suggest a progression of astrocytic
activation throughout chronic ethanol exposure.
There are few studies which specifically look at changes in astrocytes following differing
periods of ethanol exposure. The effects of abstinence from ethanol on neurogenesis and
neuronal proliferation are debated. Some studies show that cell proliferation further increases
during withdrawal. Glial cell proliferation in mice has been observed 2 days following abstinence
from a 4 day ethanol binge (Nixon et al., 2008). Furthermore, these new glial cells survived for
up to 2 months. However, abstinence from chronic ethanol is related to depression, which has
been attributed to reduced hippocampal neurogenesis (Stevenson et al., 2009). This has also
been confirmed through in vivo testing in mice, as treatment with an antidepressant prevented
reduction of hippocampal neurogenesis and altered depressive like behaviors. Most likely these
conflicting results suggest a widespread effect of differing exposure levels of ethanol in various
brain regions. Just as ethanol can have differing effects depending on the dosage and length of
exposure, similar variations arise during withdrawal from various ethanol exposures.
As discussed above, glial cells regulate the release and production of various
inflammatory markers. Therefore, changes in glial cells typically accompany changes in
cytokines and inflammatory proteins. My results from RT-qPCR experiments support this. In the
hippocampus, I observed an increase in IL-1β, TNF-α, MCP-1, iNOS, and IL-22. These changes
were more pronounced at the 2 week time point as compared to 4 week. This may indicate
increasing tolerance in the mice after prolonged chronic exposure to ethanol as well as
increasing binge episodes. Tolerance results as a desire for homeostasis within the system
when an exogenous effect creates an imbalance. Resulting changes are typically first seen in
the primary metabolizers of ethanol, ADH and aldehyde dehydrogenase. Acute exposure to
ethanol can result in increased ADH activity and subsequent increased locomotor activity.
32
Whereas these changes are diminished during chronic exposure, as a result of tolerance to the
effects of ethanol (Tran et al., 2015). Therefore, one might expect similar changes in pathways
downstream of alcohol metabolism, such as cytokine release. The changes I observed were not
only time dependent, but also region dependent. The hippocampus showed a general increase
in pro-inflammatory markers as a result of Hybrid, but the amygdala had a decrease. The
decrease was most prominent at 4 weeks with mRNA changes in IL-1β, TNF-α, MCP-1, and IL-
22.
Finally, alongside these changes I observed an increase in P2X7R expression in various
brain regions as evidenced by RT-qPCR, IHC, and WB, though with varying degrees of
significance. Again, the greatest increase was observed following 2 weeks of exposure, further
suggesting development of tolerance to the effects of ethanol. Furthermore, there was no
change in the other purinergic receptor analyzed, P2X4R (data not shown), indicating that the
observed effects of Hybrid on astrocytes and inflammatory markers are more specific to
P2X7Rs.
The effects of ethanol on the liver were apparent immediately upon sacrificing the mice.
Despite consistent body weights for all groups, those treated with Hybrid had liver weights
approximately 1 g greater than controls, and upon analysis their liver to body weight ratio was
also significantly greater. This is attributed to fat accumulation in the liver as a result of chronic
ethanol consumption, and this was later supported by the H&E staining showing progression of
ALD. These changes in the liver were time dependent, and were observed as early as 2 weeks
of Hybrid treatment and dramatically increased at 4 weeks of exposure. Interestingly, 2 day
withdrawal from Hybrid caused more accumulation of fat in the liver which may be related to the
increased levels of acetaldehyde, a direct and toxic metabolite of ethanol (Ceni et al., 2014).
In studying the inflammatory markers in the liver, most decreased in mRNA expression
as a result of Hybrid treatment, with the exception of IL-1β. IL-1β increased in a time dependent
manner, and while there was some recovery during abstinence after 2 weeks of treatment,
33
mRNA levels of IL-1β further increased during withdrawal of 4 weeks of treatment. This may
suggest that the progression of ALD as observed through H&E staining is mostly attributed to
increases in IL-1β. Decreases of IL-22 and MCP-1 mRNA levels at 2 weeks were reversed
following 4 weeks of treatment with a much greater increase. However, while at 2 weeks there
was change in IL-6, there was a decrease after 4 weeks of treatment. Apart from the changes in
IL-1β, the data differs from that seen in the hippocampus and amygdala. This may be explained
by additional metabolic changes that occur in the liver following chronic ethanol exposure as a
result of the progression of ALD.
I also found differences in P2X7R mRNA levels in liver and brain. Whereas in the brain
there was a general trend towards increased P2X7R, the liver P2X7R levels decreased in
Hybrid treated mice. This may further support the role of P2X7R specifically in
neuroinflammation. In addition, parallel decreases in the levels of P2X7R and several pro-
inflammatory markers, suggest a role of P2X7Rs in ethanol action in the liver. The nature of this
relationship needs more detailed investigation.
34
Chapter 5
AIM 2: INVESTIGATE THE EFFECTS OF A P2X7R ANTAGONIST ON THE EXPRESSION
OF PRO-INFLAMMATORY MARKERS AND MEDIATORS LINKED TO P2X7R-SIGNALING
IN THE BRAIN AND LIVER
5.1 Rationale
As presented in Chapter 1, the current pharmaco-social treatment options for AUD are
limited and mostly ineffective. Only recently have investigators started to focus on the
inflammatory processes induced by ethanol as a potential mechanism for organ/tissue damage.
However, the inflammatory response to ethanol is a complex process that involves many
different pathways and a myriad of proteins (Alfonso-Loeches et al., 2010; Idzko et al., 2014;
Vetreno and Crews, 2014). P2X7Rs have been shown to play an important role in the
generation of inflammatory responses during number of pathologies (Diaz-Hernandez et al.,
2009; McLarnon et al., 2006; Skaper et al., 2010; Volonte et al., 2011). In this regards,
pharmacological blockade of the P2X7R pathway ameliorated neuropathology in animal models
of neurodegenerative diseases [reviewed in (Takenouchi et al., 2010)]. For example, in a model
of Alzheimer’s Disease, P2X7R antagonist brilliant blue G reduced the levels of P2X7R
expression, attenuated gliosis, diminished the leakiness of blood-brain barrier and was
neuroprotective (Ryu and McLarnon, 2008). In vivo administration of brilliant blue G prevented
neuronal apoptosis in the mouse model of Huntington disease (Diaz-Hernandez et al., 2009).
Similarly, P2X7R inhibition has been shown to exhibit beneficial effects on other animal models
of CNS diseases including spinal cord injury and multiple sclerosis (Matute et al., 2007; Peng et
al., 2009). Furthermore, P2X7R antagonist A438709 was able to inhibit activation of Kupffer
cells leading to steatohepatitis in obese mice (Chatterjee et al., 2012).
Presently, P2X7R inhibition has not been investigated for its potential to reduce or block
ethanol and/or obesity-induced inflammatory responses. To this end, my studies began to
address this issue by testing the effect of a P2X7R antagonist on the inflammatory responses in
brain and liver caused by Hybrid exposure. There are a number of commercially available
35
P2X7R antagonists. For these studies, I used the potent P2X7R antagonist A804598 because it
is highly selective to P2X7Rs, it is equally potent on human and rodent P2X7Rs (Donnelly-
Roberts et al., 2009), readily crosses the blood-brain barrier (Able et al., 2011) and is an
effective blocker of agonist stimulated release of IL-1β (Donnelly-Roberts et al., 2009). It also
demonstrated specific and high affinity P2X7R binding throughout the brain (with Kd estimated
at ~7 nM) (Able et al., 2011). In addition, I chose the 4 week Hybrid exposure paradigm to allow
sufficient time for any meaningful effects of the antagonist which was administered only 3-times
a week. By administering the P2X7R antagonist, A804598, I hoped to attenuate the
inflammatory responses caused by P2X7R and further elucidate the role of P2X7R in ethanol-
induced inflammation.
5.2 Results
5.2.1 P2X7R antagonist inhibited changes in GFAP expression in hippocampus
IHC staining using GFAP antibody to visualize astrocytes once again showed that the
Hybrid treatment induces astrocyte proliferation (Fig. 13 A,B). Administration of A804598 at 5
mg/kg dose 3-times a week through iG catheters inhibited astrocyte proliferation as seen by
GFAP immunostaining (Fig. 13 A, D).
RT-qPCR results also showed an increase of mRNA expression of GFAP in the
amygdala following Hybrid exposure, and recovery through antagonist treatment (Fig. 14).
However, in the hippocampus I observed no significant changes in GFAP mRNA expression.
36
Fig. 13. GFAP staining of hippocampus tissue. A. Immunohistochemistry images. B.
Fluorescence intensity of cell staining. C. Cell area of cell bodies. D. Number of cells per
image field. All values reported as fold change compared to control, mean ± SEM. ** P < 0.005,
# P < 0.001 compared to control, except where noted (n=2)
5.2.2 Changes in signaling and inflammatory markers following Hybrid and P2X7R antagonist
treatment
Taqman custom array plates were used to run RT-qPCRs for a wide variety of genes
encoding inflammatory and oxidative stress markers, signaling molecules, critical components
of neurochemical cascades (total of 46 genes) in the brain. Of these my thesis is focusing on
the inflammatory panel and relevant signaling molecules. Among the cytokines, IL-1β showed
the greatest significant increase in the hippocampus and amygdala in the EtOH+Veh group and
this was attenuated significantly by the antagonist treatment (Fig. 14). MCP-1 was also
increased in both brain regions following Hybrid treatment, however significant changes were
observed in amygdala. The antagonist treatment attenuated these increases. Similar effects
were found for iNOS in the hippocampus but not amygdala. TNF-α had opposite effects in the
amygdala and hippocampus compared to iNOS. There was a significant increase in the
37
amygdala as a result of Hybrid treatment, which was attenuated by the antagonist. There was
no significant effects identified in the hippocampus.
Of the signaling molecules that I investigated, several molecules were identified that
were significantly different in both ethanol treated groups when compared to the control group,
including TLR2, NLRP3, and CXCR2 (Fig. 14). Notably, administration of the P2X7R antagonist
attenuated the Hybrid-induced increases in the signaling molecules, i.e. TLR2 and CXCR2 in
the hippocampus, and TLR2, CXCR2, and NLRP3 in the amygdala. TLR4 also showed a
similar trend as TLR2, though differences reported here were not significant.
Fig. 14. RT-qPCR of Neuroimmune response genes in hippocampus and amygdala. All
values reported as fold change compared to controls, mean ± SEM. * P < 0.05, ♦ P < 0.01
compared to control, except where noted (n=4)
38
5.2.3 Antagonist treatment model reduced liver steatosis in the Hybrid model
In my Aim 2 experiment, I recorded body weights throughout the study and observed
that the mice in the Dex+Veh increased in body weights overtime whereas those exposed to the
Hybrid treatment did not (Fig. 15 A). This change slowly progressed throughout the study with
significant difference in body weights beginning at day 20 and peaking at day 24. In addition to
change in body weight, I also found a significant difference in liver to body weight ratio between
the controls and Hybrid treated groups (Fig. 15 B). The antagonist treatment did not affect the
body weight or the liver/body ratio from those of the Hybrid exposure (Fig. 15).
H&E staining showed significant steatosis in the Hybrid exposure with and without the
antagonist, however the extent of the steatosis calculated as the adjusted threshold was
significantly lower in the EtOH+Ant group (Fig. 16).
Fig. 15. Body and liver weights of mice. A. Mice body weight throughout study. B. Post
mortem liver to body weights ratio. Mean ± SEM. # P < 0.001 compared to control, (n=10)
39
Fig. 16. Fat accumulation in liver with antagonist treatment. A. H&E staining of liver
tissues. Accumulation of fat after 4 weeks of the Hybrid exposure. B. Analysis of steatosis in
H&E slides. All values reported as fold change compared to control, mean ± SEM. * P < 0.05, #
P < 0.001 compared to control, except where noted (n=9)
5.2.4 Inflammatory changes in the liver are attenuated by P2X7R antagonist
Antagonist treatment suppressed the increases in TNF-α, MCP-1 and iNOS mRNA
levels caused by Hybrid exposure (Fig. 17). It also decreased the mRNA levels for IL-1β and IL-
6, though these were not elevated with the Hybrid exposure. Finally, the antagonist also
inhibited P2X7R mRNA levels despite that it was not changed as a result of Hybrid treatment.
40
Fig. 17. mRNA expression of inflammatory markers in liver. All values reported as fold
change compared to control, mean ± SEM. * P < 0.05, ** P < 0.005, # P < 0.001 compared to
control (n=9)
5.3 Discussion
Aim 2 studies tested the potential of the P2X7R antagonist A804598 to attenuate the
inflammatory responses in the brain and liver induced by 4 weeks of Hybrid exposure in
C57BL/6J mice. The first area investigated was the hippocampus for changes in astrocyte
morphology and proliferation. Similar to what was found in the Aim 1 study, there was a
significant increase in the number of astrocytes in the hippocampus after Hybrid treatment, as
evidenced by an increase in GFAP positive cells. Importantly, P2X7R antagonist treatment was
able to diminish these changes. The mechanism behind the activation of astrocytes is unknown,
but ROS and Ca
2+
mobilization are both required for GFAP expression on astrocytes (Gonzalez
et al., 2007). Activation of P2X7Rs leads to intracellular Ca
2+
increases and ROS generation.
The findings of my studies with the use of the P2X7R antagonist suggest that P2X7Rs
participate in astrocyte activation caused by Hybrid exposure.
The antagonist administration was effective in reducing the inflammatory response in the
brain as evidenced from Taqman gene expression RT-qPCR findings. Thus, the addition of the
41
P2X7R antagonist was able to suppress the Hybrid-induced increases in IL-1β and MCP-1 in
both hippocampus and amygdala regions. iNOS, encoded by the NOS2 gene, only saw an
increase in the hippocampus as a result of Hybrid exposure. Addition of the P2X7R antagonist
also attenuated this effect. Whereas Hybrid exposure caused an increase in TNF-α in the
hippocampus and amygdala, but only in the amygdala did the antagonist return mRNA levels
back to control. As described previously, IL-1β is one of the key cytokines believed to be directly
downstream of P2X7R. And previous studies have also reported a direct relationship between
P2X7R and IL-1β (Idzko et al., 2014; Solle et al., 2001; Takenouchi et al., 2009). Again, in both
brain regions, the antagonist was able to restore the level of MCP-1 back to that of controls.
Previous work reported that release of MCP-1 is regulated by NF-κB (Vetreno and Crews,
2014),my results suggest a role for P2X7R regulation of NF-κB, or perhaps through a novel
signaling route in the release of MCP-1. Lastly, P2X7R has also been implicated in regulating
TNF-α. Studies by Suzuki and colleagues suggested that mitogen-activated protein kinase
kinase and JNK are downstream of P2X7R and these kinases cause release of TNF-α as a
result of ATP stimulation (Suzuki et al., 2004). Activation of these kinases was inhibited by
treatment with a P2X7R antagonist. However, it was also shown that stimulation of P2X7R in
microglia suppressed glutamate-induced neurotoxicity, suggesting a protective role for P2X7R-
induced release of TNF-α.
The P2X7R antagonist was also effective in reducing the mRNA increases caused by
Hybrid exposure for signaling molecules such as TLR2, NLRP3, and CXCR2. The role of the
TLRs in ethanol induced neuroinflammation was briefly discussed earlier in the Introduction.
Similar to P2X7Rs, TLRs are found on glial cells and have been attributed to regulating NF-κB
signaling to induce inflammatory responses to ethanol (Szabo et al., 2007). Generally, TLR4
has received more attention as a key receptor in this mechanism. However, Pascual and
colleagues in comparing knock out mouse models of TLR4 with TLR2 found that both receptors
play similar roles in regulating ethanol induced cytokine release and behavioral changes
42
(Pascual et al., 2015). In my study, I observed some changes in TLR4 in the EtOH+Ant group,
but these changes were not as significant as TLR2. These results suggest a specific
relationship between P2X7R and TLR2 as compared to TLR4.
Alongside the change in TLR2 I observed a change in NLRP3, which belongs to the NLR
subfamily (believed to cooperate with TLRs to regulate inflammatory responses). NLRP3
specifically has been implicated in demyelination, metabolic disorders, and neurodegenerative
diseases such as multiple sclerosis and Alzheimer’s disease (AD) (Alfonso-Loeches et al.,
2014; De Nardo and Latz, 2011). Furthermore, Alfonso-Loeches and colleagues have shown
that ethanol can induce activation of astrocytic NLRP3s, driven by ROS generation (Alfonso-
Loeches et al., 2014). It has been suggested that P2X7R is upstream from NLRP3 and
regulates this mechanism (Surprenant and North, 2009). The results from my study support this
hypothesis in that I observed a decrease in ethanol induced NLRP3 mRNA expression following
inhibition of the P2X7R via addition of the antagonist.
The CXCR2 gene encodes for IL-8 receptor, which is a G protein-coupled receptor. This
receptor is known to be involved in polymorphonuclear neutrophil chemotaxis, which plays a
role in innate immune responses (Boppana et al., 2014). Furthermore, IL-8 receptors (CXCR1
and CXCR2) have been implicated in several inflammatory diseases such as rheumatoid
arthritis, inflammatory bowel disease, and AD. In AD, it was observed that astrocytes have
increased levels of IL-8 and the IL-8 receptor is believed to play a neuroprotective role (Liu et
al., 2014). Interestingly, recent studies suggest a new role for IL-8 and CXCR2 in the
development of alcoholic steatohepatitis (Wieser et al, Gut, 2016). In that no information is
available on the changes in brain IL-8 and CXCR2 in the studied experimental models. My
findings represent an important step forward showing changes in CXCR2 in the brain regions in
the Hybrid model. Overall, this latter work suggests that there is a functional link between
P2X7Rs and CXCR2 pathways.
43
In the liver, despite the P2X7R antagonist treatment not having a significant effect on the
Hybrid treatment in terms of liver weight, I did observe a decrease in fat accumulation. In
addition, the antagonist reduced the Hybrid-induced liver steatosis. Evidence of fat
accumulation in the liver is typically an indication of changes in metabolic function and
accompany liver damage (Han et al., 2016). Finally, the antagonist was able to suppress
increases in mRNAs of inflammatory TNF-α, MCP-1 and iNOS. And despite a slight decrease
due to Hybrid treatment, antagonist was still able to further decrease mRNA of IL-1β and IL-6.
Similar to the brain, these findings suggest a role for P2X7R in the inflammatory response
caused by Hybrid exposure.
Overall, findings from Aim 1 and 2 link Hybrid-induced changes in inflammatory markers
to P2X7R. However, there were several limitations of these findings. First, in contrast to the
Aim 1 study, I observed an increase in IL-1β in the hippocampus following Hybrid treatment. In
addition, IL-1β mRNA levels also increased in the amygdala, though this opposes the decrease
seen in Aim 1. MCP-1 only saw a slight increase in the hippocampus in the Aim 1 study.
Although this suggests some level of differences in the results, the primary findings of this effort
continue to illustrate the role of P2X7R in ethanol-induced inflammation in the brain and liver.
Furthermore, in the Aim 2 study I reported an increase in both amygdala and hippocampus in
the EtOH+Veh group. Again for TNF-α, the results of Aim 1 showed a decrease in the amygdala
whereas in Aim 2 I observed an increase. In the liver mRNA level of IL-1β increased in the Aim
1 study, but saw no change in the Aim 2 study. These inconsistencies may be due to
differences in the RT-qPCRs, i.e. conventional PCR used in Aim 1 vs Taqman custom multiplex
PCR employed for Aim 2 studies. This includes different primer sets for each marker being used
for the 2 studies and as a result, a difference in sensitivity. In addition, there are normally mouse
to mouse variability on how they respond to different exposures, resulting in some differences in
my results. Therefore, the best approach will be to increase the number of animals per each
experiment will help to overcome this issue.
44
Chapter 6
CONCLUSION
This thesis set out to investigate the role of P2X7Rs in ethanol-induced inflammatory
brain and liver damage. Collectively, my findings suggest that there is a functional role of
P2X7Rs and the neuroinflammatory response caused by exposure to chronic ethanol combined
with high-fat diet (Hybrid). Findings from my Aim 1 studies supported an early onset of the
neuroinflammatory response in the Hybrid model. Astrocyte proliferation and increases in pro-
inflammatory markers in the brain were found as early as 2 weeks after the exposure and were
further sustained through 4 weeks of treatment. Importantly, these changes were accompanied
by increases in the expression levels of P2X7Rs. In addition, changes found after ethanol
withdrawal indirectly supported the link between P2X7Rs and neuroinflammatory response.
Ethanol withdrawal caused aggravation of neuroinflammatory response at 2 weeks of exposure
which was accompanied by greater increases in P2X7R expression. Interestingly, the changes
in pro-inflammatory markers as well as P2X7R expression were less pronounced at 4 weeks of
exposure suggesting involvement of adaptive responses at that time period.
Findings of Aim 2 studies with the P2X7R antagonist administration further supported my
hypothesis on the functional link between P2X7Rs and neuroinflammatory responses. P2X7R
antagonist was able to attenuate astrocyte proliferation as well as increase pro-inflammatory
mediators in 2 different brain regions, hippocampus and amygdala.
My findings in the liver did not completely parallel my findings from the brain. For
example, the time differences identified in the changes in inflammatory markers suggested later
onset for the inflammatory response in the liver as compared to that in the brain. These changes
between time periods were also accompanied by a decrease P2X7R expression at 2 and 4
weeks of Hybrid exposure. Nonetheless, attenuation of several pro-inflammatory markers as
well as reduction in steatosis in the presence of antagonist support the notion that there is a
functional role of P2X7Rs in ethanol effects in the liver. Overall, my findings were mostly in
45
agreement with the previous reported data on the onset of inflammatory responses in liver in the
Hybrid model and suggest that longer exposure paradigm is required to effectively study the
involvement of P2X7Rs in liver effects.
In summary, my studies add support for an important role of P2X7Rs in chronic ethanol-
induced neuroinflammation and provide first insights into the involvements of P2X7Rs in
changes in liver pathology. Further research is necessary to establish a link between P2X7Rs
and ethanol’s damaging effects on the liver. However, my initial findings set the stage for
additional studies that will evaluate P2X7Rs as a novel therapeutic target against ethanol-
induced brain and liver damage.
46
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Abstract (if available)
Abstract
Alcohol use disorder (AUD) is a problem which afflicts millions of people throughout the world. Chronic consumption of alcohol leads to a myriad of health issues affecting several organs systems in the body. Two major organs where alcohol has a dramatic damaging impact are the brain and liver. Presently, there is a lack of pharmacological treatment options for prevention or reduction of AUD. By understanding the pathway(s) for ethanol action and its targets, especially in the liver and brain, we may hope to develop new approaches for treatment of AUD. Recent growing evidence has shown that inflammatory responses to alcohol exposure may be a mechanism for chronic alcohol-induced organ damage. Building evidence suggests that ATP-activated purinergic P2X7 receptors (P2X7Rs), primarily residing on immune cells throughout the body, play an important role in regulating inflammation and cell death. For example, P2X7Rs have been linked to neurological diseases including neurodegenerative pathologies and inflammatory conditions. Our laboratory is currently testing the hypothesis that P2X7Rs play a role in alcohol-induced neuroinflammation. Initial results from this work, using a model of intragastric (iG) ethanol exposure combined with high-fat diet (Hybrid) in C57BL/6J mice (over 8 weeks), identified upregulation of P2X7Rs in ethanol-sensitive brain regions. Importantly, these changes paralleled neuroinflammation and neurotoxicity in the same brain regions. These findings served the basis for my thesis that tested the hypothesis that there is a functional link between P2X7R-activated signaling events and ethanol-induced inflammatory processes. Two interrelated Aims were designed to test the hypothesis. Aim 1 tested the role of P2X7R in inflammatory response in the brain and liver in the early phases of ethanol exposure and withdrawal. Aim 2 explored the effects of a P2X7R antagonist on the expression of pro-inflammatory markers and mediators linked to P2X7R-signaling in the brain and liver. For Aim 1 studies, I used the Hybrid model at shorter time periods of exposure, i.e. 2 and 4 weeks. The effects of Hybrid exposure were investigated immediately after treatments as well as 24 hours after ethanol withdrawal. In Aim 2 studies, I used the 4 week Hybrid exposure to test the effects of a P2X7R antagonist. I evaluated the inflammatory response in different brain regions such as hippocampus and amygdala and in the liver using various techniques including immunofluorescence, Western blotting, and RT-qPCR. The results of these studies demonstrated that Hybrid treatment caused an inflammatory response after 2 and 4 weeks of exposure as evidenced by the activation of astrocytes, release of cytokines, and increase in P2X7R expression in the hippocampus and amygdala. In the liver, chronic alcohol consumption caused steatosis and an increase in cytokines, but unexpectedly, levels of P2X7R decreased. Furthermore, incorporation of a P2X7R antagonist into the study resulted in the attenuation of the changes caused by the Hybrid treatment. Collectively, results from my investigation supported the hypothesis that there is a functional link between P2X7Rs and the inflammatory responses that arise as a result of chronic ethanol exposure.
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Freire, Daniel
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Core Title
Role of purinergic P2X7 receptors in inflammatory responses in the brain and liver: a study using a mouse model of chronic ethanol and high-fat diet exposure
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School of Pharmacy
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Master of Science
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Molecular Pharmacology and Toxicology
Publication Date
07/21/2016
Defense Date
07/20/2016
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