Loyola University Chicago
Loyola eCommons
Dissertations
Theses and Dissertations
2015
The Gut-Liver Axis Regulates Pulmonary
Inflammation After Intoxication and Burn Injury
Michael M. Chen
Loyola University Chicago, [email protected]
Recommended Citation
Chen, Michael M., "The Gut-Liver Axis Regulates Pulmonary Inflammation After Intoxication and Burn Injury" (2015). Dissertations.
Paper 1633.
http://ecommons.luc.edu/luc_diss/1633
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Copyright © 2015 Michael M. Chen
LOYOLA UNIVERSITY CHICAGO
THE GUT-LIVER AXIS REGULATES PULMONARY INFLAMMATION AFTER
INTOXICATION AND BURN INJURY
A DISSERTATION SUBMITTED TO
THE FACULTY OF THE GRADUATE SCHOOL
IN CANDIDACY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
PROGRAM IN INTEGRATIVE CELL BIOLOGY
BY
MICHAEL M. CHEN
CHICAGO, ILLINOIS
AUGUST 2015
ACKNOWLEDGEMENTS
I would like to thank my family for their love and my fellow lab mates for making
me look forward to work each day. Together with my mentor, Liz, they have made this
part of my training a truly memorable and enjoyable experience. There are not enough
pages in this document that would be needed to express my gratitude to Liz for her
selfless mentorship, coffee, food, life advice, and support. I will remember and cherish
her guidance and friendship for many years to come. Finally, I would also like to thank
my committee for investing in my training and education as well as acknowledge the
National Institutes of Health, the American Medical Association Foundation, and the
Stritch School of Medicine MD/PhD program for funding.
iii
TABLE OF CONTENTS
ACKNOLWEDGEMENTS
iii
LIST OF TABLES
vi
LIST OF FIGURES
vii
LIST OF ABBREVIATIONS
ix
CHAPTER 1: INTRODUCTION
1
CHAPTER 2: REVIEW OF THE RELATED LITERATURE: ALCOHOL
MODULATION OF THE POST BURN HEPATIC RESPONSE
Socioeconomic and Clinical Consequences
Extrahepatic Events
Intrahepatic Mechanisms
Conclusions
4
6
10
17
CHAPTER 3: AN ALTERATION IN THE GUT-LIVER AXIS DRIVES
PULMONARY INFLAMMATION AFTER INTOXICATION AND BURN
Abstract
Introduction
Materials and Methods
Results
Summary
18
19
20
25
32
CHAPTER 4: KUPFFER CELL P38 MAPK SIGNALING DRIVES POST BURN
HEPATIC DAMAGE AND PULMONARY INFLAMMATION WHEN
ALCOHOL INTOXICATION PRECEDES INJURY
Abstract
Introduction
Materials and Methods
Results
Summary
33
34
36
41
48
CHAPTER 5: ALCOHOL POTENTIATES POST BURN REMOTE ORGAN
DAMAGE THROUGH SHIFTS IN FLUID COMPARTMENTS MEDIATED BY
BRADYKININ
Abstract
Introduction
Materials and Methods
Results
49
50
52
54
iv
Summary
60
CHAPTER 6: INTOXICATION BY INTRAPERITONEAL INJECTION OR ORAL
GAVAGE EQUALLY POTENTIATE POST BURN ORGAN DAMAGE AND
INFLAMMATION
Abstract
Introduction
Materials and Methods
Results
Summary
61
62
64
67
77
CHAPTER 7: DISCUSSION
Role of the Intestinal Barrier
Kupffer Cell Regulation of the Hepatic Response
Influence of Fluid Shifts
Ethanol Administration in our Animal Model
Final Conclusions
78
82
86
89
92
APPENDIX: DETAILED METHODS DESCRIPTIONS
Intoxication and Burn Injury Protocol
Kupffer Cell Isolation
Tail Vein Injection
ImageJ Analysis of Lung Histology
Mesenteric Lymph Node Plating for Bacterial Translocation
96
97
99
101
102
102
REFERENCES
104
VITA
122
v
LIST OF TABLES
Table 1. Interventions attempted in the setting of intoxication and burn
vi
93
LIST OF FIGURES
Figure
Page
1. The gut-liver axis after burn injury
10
2. Potential signaling pathway alterations in Kupffer cells after burn injury
and/or alcohol intoxication
14
3. Mechanisms of steatosis development after burn injury and/or alcohol
intoxication
16
4. Decreasing bacterial translocation attenuated hepatosteatosis
26
5. Liver damage 24 hours after intoxication and burn is diminished when
Bacterial translocation is inhibited.
28
6. Interleukin-6 production is decreased with PIK or antibiotic treatment
29
7. Pulmonary inflammation 24 hours after intoxication and burn is
attenuated by restoration of the gut-liver axis
31
8. Kupffer cell p38 activation is increased when intoxication precedes burn
42
9. The absence of Kupffer cells or p38 inhibition equally attenuate hepatic
damage
43
10. Interleukin-6 production is decreased with clodronate liposomes or
p38 inhibition after intoxication and burn
45
11. Pulmonary inflammation after intoxication and burn is attenuated by
Kupffer cell depletion or p38 inhibition.
46
12. In vivo Kupffer cell depletion and p38 inhibition
47
13. Fluid status 24 hours after injury
55
14. Remote organ damage 24 hours after injury
57
vii
15. HOE140 restores fluid balance 24 hours after intoxication and burn
58
16. HOE140 attenuates remote organ damage 24 hours after intoxication
and burn
59
17. Blood alcohol concentration (BAC)
68
18. Circulating blood granulocytes 24 hours after injury
69
19. Serum IL-6 at 24 hours after injury
69
20. Histologic state of the ileum 24 hours after intoxication and burn
71
21. Intestinal damage 24 hours after injury
72
22. Hepatic damage 24 hours after injury
73
23. Liver weight to body weight ratio 24 hours after injury
74
24. Histologic state of the lungs 24 hours after injury
75
25. Quantification of pulmonary congestion 24 hours after injury
76
26. Pulmonary inflammation after intoxication and burn
77
27. Overlapping responses to burn injury and alcohol exposure alter
the gut-liver axis
94
viii
LIST OF ABBREVIATIONS
Abx
antibiotics
ALDH
aldehyde dehydrogenase
α2A-AR
alpha-2a adrenoceptor
ALT
alanine aminotransferase
ANOVA
one-way analysis of variance
AP-1
activator protein-1
APR
acute phase responses
ARDS
acute respiratory distress syndrome
AST
aspartate aminotransferase
BAC
blood alcohol content
BUN
blood urea nitrogen
CD
cluster of differentiation
CFU
colony forming unit
CLO
clodronate
CoA
coenzyme A
ELISA
enzyme-linked immunosorbent assay
ER
endoplasmic reticulum
H&E
hematoxylin and eosin
ix
ICAM-1
intracellular adhesion molecule-1
IκB
inhibitor of kappa B
IKK
IκB kinase
IL
interleukin
i.p.
intraperitoneal
IRAK
interleukin-1 receptor-associated kinase
LBP
LPS binding protein
LPS
lipopolysaccharide
LW:BW
liver weight to total body weight ratio
MAPK
mitogen-activated protein kinase
MLCK
myosin light chain kinase
MLN
mesenteric lymph node
NAD
nicotinamide adenine dinucleotide
NADPH
nicotinamide adenine dinucleotide phosphate
NF-κB
nuclear factor kappa B
OCT
optimal cutting temperature
P
phosphorylated
PMN
neutrophil
p38i
p38 inhibitor SB203580
PIK
permeant inhibitor of MLCK
qRT-PCR
quantitative real-time polymerase chain reaction
ROS
reactive oxygen species
x
SEM
standard error of the mean
SIRS
Systemic Inflammatory Response Syndrome
TBSA
total body surface area
TG
triglyceride
TLR
Toll-like receptor
TNF
tumor necrosis factor
VLDL
very low-density lipoprotein
PI3K
phosphoinositide 3-kinase
ROS
reactive oxygen species
TBSA
total body surface area
TNFα
tumor necrosis factor alpha
ZO-1
zonula occludens
xi
CHAPTER 1
INTRODUCTION
The widespread and rapidly increasing trend of binge drinking is accompanied by
a concomitant rise in the prevalence of trauma patients under the influence of alcohol at
the time of their injury. Epidemiologic evidence suggests up to half of all adult burn
patients are intoxicated at the time of admission and the presence of alcohol is an
independent risk factor for death in the early stages post burn. The major site of alcohol
metabolism and toxicity, the liver is crucial to post burn outcome and experimental
evidence implies an injury threshold exists beyond which burn-induced hepatic
derangement is observed. Alcohol may lower this threshold for post burn hepatic damage
through a variety of mechanisms including modulation of extrahepatic events, alteration
of the gut-liver axis, and changes in signaling pathways. The direct and indirect effects
of alcohol may prime the liver for the second-hit of many overlapping physiologic
responses to burn injury. In an effort to gain a deeper understanding of how alcohol
potentiates post burn hepatic damage we proposed studies to identify the mechanism of
how ethanol intoxication prior to burn injury drives the aberrant inflammatory response
relative to either ethanol or burn alone.
The resident macrophages in the liver, known as Kupffer cells, coordinate the
hepatic response after injury and are a producer of systemic cytokines such as IL-6.
Cytokine production by Kupffer cells can be induced by gut-derived lipopolysaccharide
1
2
(LPS), which can be found in the portal system after the combined injury due to intestinal
barrier breakdown. Furthermore, ethanol can sensitize Kupffer cells to LPS through
downstream signaling proteins such as mitogen-activated protein kinases (MAPK)
leading to even greater IL-6 production after encountering LPS. This is important, as we
have previously demonstrated that increased IL-6 after this combined injury drives
pulmonary inflammation. With this knowledge, we hypothesize that binge ethanol
intoxication exacerbates the hepatic response to burn injury through a feed-forward loop
involving increased intestinal permeability, ethanol-induced sensitization of Kupffer cells
to LPS via MAPK signaling, and hepatic IL-6 production. To test this hypothesis, three
aims are proposed: 1) to determine the effects of alcohol intoxication on the post burn
hepatic response, 2) if restoring gut barrier function or reducing intestinal microbial
burden attenuates hepatic damage and IL-6 production, and 3) identify the role of Kupffer
cells and any specific MAPK isoforms responsible for LPS-induced production of IL-6.
Furthermore, the effects on pulmonary inflammation will be assessed for each aim.
Hepatic damage will be evaluated through serum markers and histologic analysis.
Cytokine production will be measured at the mRNA and protein levels. We will limit
bacterial translocation by restoring intestinal barrier function after injury with permeant
inhibitor of kinase (PIK) and via prophylactic sterilization of the gut with oral antibiotics.
The role of Kupffer cells will be determined by selectively depleting them before injury
with clodronate liposomes. Kupffer cells will be isolated and assessed for MAPK
phosphorylation and IL-6 production. Finally, MAPK isoforms identified as important in
the Kupffer cell response to the combined injury will be inhibited in vivo. These studies
3
will help gain an understanding of the mechanisms by which alcohol worsens post burn
outcome and are aimed at identifying novel therapeutic targets in the clinically relevant
setting of burn injury preceded by ethanol intoxication. As ethanol is a risk factor for
many types of injury, a benefit may also apply to other forms of trauma.
CHAPTER 2
REVIEW OF RELATED LITERATURE:
ALCOHOL MODULATION OF THE POST BURN HEPATIC RESPONSE
Socioeconomic and Clinical Consequences
Alcohol is the most abused substance in the US and the third leading cause of
preventable death [1], much of which is associated with unintentional injury [2,3]. Binge
drinking, defined as consuming five or more drinks on one occasion, is an increasingly
prevalent form of intoxication [4] and is the characteristic pattern of drinking among
patients presenting with traumatic injury, including burns [5]. Nearly 50% of patients
admitted for burns have a positive blood alcohol content (BAC) and these patients have
worse clinical outcomes than individuals who sustain similar injuries not under the
influence [6,7,8]. Specifically, they are twice as likely to acquire an infection, require
more surgical procedures and have a longer duration of stay in the ICU than their
nonintoxicated counterparts [6,8,9]. As a result of these complications, burn patients with
prior alcohol exposure have been identified as a patient population at high risk for death
in the early stages post burn [10]. With 1 million burn injuries in the US each year,
resulting in 40,000 hospitalizations [11], this represents a substantial burden of morbidity,
mortality and socioeconomic cost that is worsened by alcohol abuse. Despite the
established consequences, there are currently few differences in the treatment and
management of burn patients with and without prior alcohol exposure. The increased
4
5
morbidity exhibited by patients with pre-injury alcohol exposure may be due to an early
increased inflammatory response and subsequent immune suppression [12,13], though
the inciting cells and mechanisms responsible for these phenomena remain largely
unknown.
As a responsive organ with metabolic, hormonal and immune functions, the liver
is central to the physiologic response to burn injury and plays a pivotal role in post burn
outcome [14]. This is highlighted by the close correlation between hepatic function and
post burn prognosis as seen in a national sample of 31,338 adult burn patients [15].
Correspondingly, hepatic cirrhosis, often caused by chronic alcoholism, is a high
premorbid contributor to death in the setting of severe burn (3). Given the comorbidities
associated with chronic alcoholism, it is not surprising that clinical and animal studies
demonstrate amplified liver damage when chronic alcohol exposure precedes thermal
injury. In recent years, however, the prevalence of binge drinking has increased
precipitously and now represents the majority of alcohol consumed in the US [4]. Due to
its high association with injury in an otherwise healthy population, binge drinking may be
the more clinically relevant mode of alcohol abuse in burn patients today [5,16]. Indeed,
more recent studies suggest the majority of intoxicated burn patients are now binge
drinkers not chronic alcoholics [6]. Animal studies also confirm using various doses and
timing before injury that the presence of intoxication at the time of injury, even at a
single dose, is the key factor in the ability of alcohol to worsen post burn outcome.
Therefore we focus this review on the potential mechanisms of how alcohol, consumed
acutely as a binge, affects the post burn hepatic response.
6
The total body surface area (TBSA) affected by a burn is a major factor in
determining the severity of injury, with increased TBSA correlating to increased systemic
inflammation [17]. In an animal model, substantial hepatic derangements were present
after a 40% TBSA burn that were absent when reduced to 20% TBSA [18]. This may
suggest an injury threshold beyond which an aberrant hepatic response occurs. Work
from our lab and others demonstrate significant hepatic damage in a mouse model of 1520% TBSA burn only when intoxication precedes the injury [19,20,21,22,23]. This may
indicate that alcohol lowers the threshold for an impaired post burn hepatic response.
Indeed, alcohol exposure and burn injury have many overlapping physiologic effects that
work in a synergistic manner to cause hepatic derangement. While burn-induced liver
dysfunction may last for years after the initial injury [24], we center this review on the
short term response in hopes of better characterizing the early events that may predispose
to the later hypermetabolic state. We summarize below potential mechanisms of
alcohol’s ability to worsen post burn outcome through extrahepatic events, direct effects
on the liver and alterations in signaling pathways before discussing existing treatment
regimens.
Extrahepatic Events
Intestinal Ischemia
Animal studies demonstrate catecholamine-mediated vasoconstriction of the
splanchnic vasculature makes the intestines particularly susceptible to ischemic injury
after burn [25]. Decreased cardiac output in the early phases post burn [26] worsens
circulating blood flow, leading to intestinal damage and a loss of barrier function [27,28]
7
in rodents. A single intoxication event with alcohol prior to a burn injury in mice
enhances intestinal damage leading to greater dissociation of tight junction complexes in
the intestinal epithelium [29,30,31] and resultant permeability [32,33] compared to either
insult alone. The mechanism for alcohol’s ability to exacerbate post burn intestinal
damage may stem from its action on the post burn hemodynamic response and tissue
oxygen requirements. We recently reported that alcohol given as a single oral gavage
(1.1 g/kg) potentiates post burn bradykinin signaling in mice, leading to greater
extravasation of fluid from the vascular compartment compared to a burn alone [34].
This adds a degree of hypovolemia into a scenario in which organs supplied by the
splanchnic vasculature are already at risk for ischemic injury [28]. Likewise, clinical
data demonstrate intoxicated burn patients require more aggressive fluid resuscitation
than do their non-intoxicated counterparts [6], also indicating a shift in fluid
compartments. Choudhry et al. demonstrated that a BAC of 100 mg/dl before burn injury
in rats decreases blood flow and oxygen delivery to the intestines and liver [35].
Additionally, animal studies indicate that within 90 minutes of drinking, a single dose of
alcohol increases oxygen demand in the liver by 48% [36] with oxygen consumption rate
doubling in the liver after 2.5 hours of alcohol exposure [37]. While this does not rule
out the influence of other effects of alcohol in post burn remote organ ischemia, it points
towards the ability of alcohol to enhance post burn third spacing of fluid while creating a
hypermetabolic state, widening the disparity between oxygen delivery and requirement in
the intestines and liver.
8
The Gut-Liver Axis
The high baseline bacterial content of the intestines represents an enormous
potential reservoir for systemic infections and sepsis after injury [38,39,40]. Trauma,
such as a burn, causes a disruption in normal peristalsis leading to bacterial overgrowth in
the intestine and this bacterial load is even greater when alcohol ingestion precedes the
injury in rats [33]. While the exact mechanism by which alcohol enhances post burn
bacterial growth in the intestine remains unknown, experimental evidence points to
modulation of hormonal control [41], shifts in intestinal flora population [42], and the
promotion of adynamic ileus [43] as possible causes, as reviewed elsewhere [44].
Animal models report that after disruption of intestinal barrier integrity, bacteria and
bacterial products such as lipopolysaccharide (LPS) translocate from the lumen of the
intestine into the lymphatic [45,46] and portal [47] systems. The number of bacteria
found in the mesenteric lymph nodes is directly proportional to the degree of increased
bacterial growth in the intestines after injury [33], suggesting important roles for both the
luminal bacterial burden and the degree of intestinal permeability. As both bioload and
permeability are exacerbated with the combined injury, it is befitting that many animal
studies report up to 10 times greater amounts of bacterial translocation when alcohol
precedes a burn than after a burn alone [19,29,30,32,34,48]. Much of the translocated
bacteria and LPS will pass through the liver, which receives 75% of its blood supply from
the portal system [14] and is therefore sensitive to portal system hemodynamic changes
and content. As the largest reticular endothelial system organ and the first to encounter
9
portal blood, the liver plays an important role in clearing live intestinal bacteria and LPS.
The relationship between intestinal microbiota and the liver is known as the “gut-liver
axis” and is proposed to regulate a myriad of human diseases, such as nonalcoholic fatty
liver disease, primary sclerosing cholangitis, celiac disease, and others [49,50,51,52,53].
This interaction is prominent after burn injury as seen in autopsies of burn patients where
the amount of bacterial translocation correlates with the degree of steatosis [54]. We
have previously reported that in mice, antecedent ethanol exposure (single dose of 1.1
g/kg) potentiates post-burn intestinal damage [19,29,48], hepatic steatosis [23,55] and IL6 production [19,21,48,56] compared to either intoxication or a burn alone. Furthermore,
limiting crosstalk between the gut and liver after injury, either through restoration of tight
junction complexes or prophylactic sterilization of the gut, attenuated hepatic
derangements to the level of a burn alone [20]. This suggests that the increased hepatic
damage seen when intoxication precedes a burn stems from the ability of alcohol to alter
the gut-liver axis (Figure 1). In addition to increasing the delivery of LPS into the portal
system after burn, alcohol also modifies the post burn hepatic response through its
influence on Kupffer cells in the liver.
10
Figure 1. The gut-liver axis after burn injury. Burn injury leads to intestinal damage,
allowing LPS to enter the portal system and illicit a hepatic response which can
contribute to pulmonary inflammation.
Intrahepatic Mechanisms
Kupffer Cell Sensitization to LPS
Representing the body’s largest tissue-fixed macrophage population, Kupffer cells
continuously sample portal blood for foreign antigen and orchestrate the hepatic response
to injury [57]. Kupffer cells can become activated when LPS binds to Toll-like receptor
4 (TLR4), an interaction dependent on its co-receptor, CD14 [58] and enhanced in the
presence of LPS binding protein (LBP) [59]. TLR4 signaling can result in a variety of
downstream signaling pathways, many of which result in pro-inflammatory cytokine
production [60,61]. At low levels, cytokines, such as interleukin-6 (IL-6), are beneficial
to hepatocyte survival, but exposure to levels above an acceptable threshold or for a
prolonged duration can be detrimental, as seen in a mouse model [62]. Kupffer cells
protect themselves from overstimulation by expressing low levels of CD14 relative to
11
other monocytes [63]. After burn injury, Kupffer cells fulfil the role of clearing gutderived LPS from the portal system while avoiding over-stimulation. The importance of
maintaining homeostatic Kupffer cell function in the injury setting was nicely
demonstrated in a model of systemic inflammation where Kupffer cell depletion
increased gut-origin septicemia but attenuated toxic symptoms associated with
macrophage hyperactivity [64]. Animals studies have shown that hyperactive Kupffer
cells can cause hepatic damage after many types of injury, including hemorrhagic
pancreatitis [65], trauma and sepsis [66], as well as burns [67]. Burn injury causes a
transient induction of CD14 in Kupffer cells, leading to enhanced TLR4 signaling and
cytokine production in mice [68]. This is supported by work with CD14 knockout mice
and TLR4 defective animals implicating LPS as the initial inducing agent associated with
acute phase responses (APR) in the liver after burn [69]. As a part of the APR,
hepatocytes increase production of LBP leading to even greater LPS binding to Kupffer
cell TLR4 in rats [70]. Over-stimulated Kupffer cells contribute not only to hepatic
damage and steatosis [71], but also to systemic levels of potentially damaging proinflammatory cytokines. It is of great clinical importance, therefore, that Kupffer cell
sensitivity to post-burn LPS is significantly increased when alcohol intoxication precedes
burn injury as seen in a mouse model (Chen, in preparation).
Alcohol has a well described and dynamic influence on the sensitivity of
macrophages to LPS, with paradoxical effects depending on the duration of exposure
[72]. After a single dose of alcohol (4g/kg), rat Kupffer cells display a brief initial
decrease in LPS sensitivity that is reciprocally increased by 24 hours, correlating to a 5-
12
fold increase in CD14 expression [73]. This is supported by studies using a mouse model
of acute intoxication where the activity of interleukin-1 receptor-associated kinase
(IRAK), a protein downstream of TLR4, was increased 21 hours after a single dose of
alcohol [74]. In contrast to chronic alcoholics, binge drinkers experience only a minor
and transient spike of LPS in portal blood [36] and therefore increased Kupffer cell CD14
has limited clinical significance. In the context of a burn however, alcohol-exposed
Kupffer cells have abundant and prolonged LPS exposure leading to aberrant increases in
both the co-receptor and ligand for TLR4 signaling.
Recent work from our laboratory demonstrated that a single dose of alcohol (1.1
g/kg) potentiated post-burn hepatic damage via Kupffer cell derangement in mice (Chen
et al, in preparation). Specifically, Kupffer cells isolated from mice exposed to both
alcohol and burn were sensitized to LPS and produced significantly greater amounts of
IL-6 in a p38-dependent manner. A member of the mitogen activated protein kinase
(MAPK) family, p38 has a well-established role in post burn remote organ damage
[75,76,77,78,79], is altered by intoxication [80,81] and plays a role in enhanced TLR4
reactivity following injury [82]. Antecedent depletion of Kupffer cells or global p38
inhibition after intoxication and burn injury in mice attenuated hepatic and pulmonary
damage to a similar extent suggesting a prominent role for Kupffer cell p38 (Chen et al,
in preparation). Furthermore, both the absence of Kupffer cells and p38 inhibition
decreased hepatic damage to a level seen after a burn alone, again pointing to alcohol’s
ability to alter the gut-liver axis as a key mechanism for worsening post burn outcome.
13
Cytokine Production
A paramount consequence of hyperactive Kupffer cells is the excessive release of
cytokines. While beneficial at appropriate levels, aberrant cytokine release is a hallmark
of the Systemic Inflammatory Response Syndrome (SIRS) and a common complication
after burn [83]. The multifunctional cytokine IL-6 is particularly elevated in thermally
injured patients [84,85] and correlates to mortality risk after injury [23,84,86,87]. In
animal models of intoxication and burn, increased systemic levels of IL-6 are found
relative to either alcohol or burn alone [19,21,23] with the highest concentrations found
in the liver [21]. Furthermore, systemic IL-6 decreases in the absence of Kupffer cells
after intoxication and burn (Chen et al, in preparation) and other models of trauma
support the idea that Kupffer cells may be the systemic source of IL-6 after injury [88].
Identifying the systemic source of IL-6 may be crucial to successful treatment of
intoxicated burn patients, as recent evidence in animal models revealed excessive IL-6 is
responsible for amplified pulmonary inflammation [89] and gut permeability [29,48] in
this combined injury setting. These findings do not rule out the influence of other
cytokines and signaling pathways which are altered by intoxication and burn, some of
which are depicted in Figure 2. However, they implicate alterations in the gut-liver axis,
specifically in Kupffer cell IL-6 production, as key mechanisms for the ability of alcohol
to worsen post burn hepatic damage and overall outcome.
14
Figure 2: Potential signaling pathway alterations in Kupffer cells after burn injury and/or
alcohol intoxication. activator protein-1 (AP-1), alpha-2a adrenoceptor (α2A-AR), cluster
of differentiation 14 (CD14), inhibitor of kappa B (IκB), interleukin-1 receptor-associated
kinase (IRAK), interleukin-6 (IL-6), endoplasmic reticulum (ER), lipopolysaccharide
(LPS), LPS binding protein (LBP), nicotinamide adenine dinucleotide phosphate-oxidase
(NADPH oxidase), nuclear factor kappa B (NF-κB), Toll-like Receptor 4 (TLR4), tumor
necrosis factor (TNF). Numbers in figure 2 denote associated references. *Chen et al, in
preparation.
Oxidative Stress
Another factor contributing to hepatic cytokine production after alcohol or burn is
oxidative stress. Hepatic aldehyde dehydrogenase (ALDH) activity is reduced after a
burn injury in rats allowing for the accumulation of toxic aldehydes produced by lipid
peroxidation [90]. The generated reactive oxygen species (ROS) can directly damage
intracellular proteins and lead to activation of multiple inflammatory signaling pathways
including MAPKs and NF-κB [91,92]. In addition, antioxidant enzymes in rat livers are
15
down regulated within 24 hours after a burn [93], increasing the susceptibility of the liver
to oxidative stress. Alcohol directly stimulates the release of ROS in Kupffer cells
within 3 hours of intoxication as seen in a study of a rat model [94]. Other animal studies
implicate this ethanol-induced oxidative stress as a key regulator of Kupffer cell LPS
sensitivity after a single dose of alcohol (5g/kg) [95,96]. Furthermore, a byproduct of
alcohol metabolism, acetaldehyde, is an example of the aforementioned toxic aldehydes
normally cleared by ALDH [90]. Not surprisingly, animal studies revealed alcohol
increased post-burn hepatic oxidative stress and IL-6 production in a dose dependent
manner [23].
Steatosis
The accumulation of triglycerides within hepatocytes, known as steatosis, is a
common finding after burn injury with the clinical significance depending on the etiology
and severity [97,98]. Animal and clinical studies demonstrate that post burn β-adrenergic
peripheral lipolysis increases the delivery of free fatty acids to the liver [99,100,101],
which has concomitantly decreased ability for very low-density lipoprotein (VLDL)triglyceride export [101]. The resulting hepatic fatty infiltration is associated with
increased incidence of sepsis and mortality in pediatric burn patients [54], highlighting
the importance of organ integrity. Steatosis is also commonly caused by alcohol
consumption [102] and the degree of steatosis is substantially increased in mice with the
combination of alcohol and burn over either insult alone [20,22]. Animal studies show
that alcohol metabolism increases the ratio of NADH:NAD+ which then inhibits βoxidation of fatty acids while increasing the rate of their esterification [103], leading to
16
excessive triglyceride storage. Uncontrolled hyperglycemia [104], oxidative stress [105],
LPS signaling [106] and Kupffer cell cytokine secretion [71] can also contribute to
steatosis development. As these factors are all influenced by both alcohol and burn
(Figure 3), they may represent potential mechanisms by which intoxication potentiates
post burn steatosis.
Figure 3: Mechanisms of steatosis development after burn injury and/or alcohol
intoxication. Acetyl coenzyme A (acetyl CoA), glycerol 3-phosphate (glycerol 3-p),
nicotinamide adenine dinucleotide (NAD), triglyceride (TG), very low-density
lipoprotein (VLDL)
17
Conclusions
Intoxication plays a detrimental role in traumatic injury as both a causative agent
and complicating factor in recovery. This is dramatically observed in burns where high
prevalence and established consequences warrant further investigation into the
mechanisms by which alcohol worsens the post burn response. Furthermore, the very
presence of alcohol, regardless of dependence or pre-existing hepatic sequelae,
potentiates post burn hepatic damage. Clinical and animal studies suggest alcohol alters
the post burn gut-liver axis in the early phases after injury which may lower the threshold
for longer term hepatic derangement and hypermetabolism. As our understanding of this
combined injury grows, more targeted therapeutic approaches may be possible.
CHAPTER 3
AN ALTERATION IN THE GUT-LIVER AXIS DRIVES
PULMONARY INFLAMMATION AFTER INTOXICATION AND BURN
Abstract
Approximately half of all adult burn patients are intoxicated at the time of their
injury and have worsened clinical outcomes compared to those without prior alcohol
exposure. This study tested the hypothesis that intoxication alters the gut-liver axis
leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver.
To this end, C57BL/6 mice were given 1.2 g/kg ethanol 30 minutes prior to a 15% total
body surface area burn. To restore gut barrier function, a specific myosin light chain
kinase inhibitor [membrane-permeant inhibitor of kinase (PIK)] was administered 30
minutes after injury which we have demonstrated to reduce bacterial translocation from
the gut. Limiting bacterial translocation with PIK attenuated hepatic damage as measured
by a 47% reduction in serum alanine aminotransferase (p<0.05) as well as a 33%
reduction in hepatic IL-6 mRNA expression (p<0.05) compared to intoxicated burned
mice without PIK. This mitigation of hepatic damage was associated with a 49% decline
in pulmonary neutrophil infiltration (p<0.05) and decreased alveolar wall thickening
compared to matched controls. These results were reproduced by prophylactically
18
19
reducing the bacterial load in the intestines with oral antibiotics before intoxication and
burn. Overall these data suggest the gut-liver axis is deranged when intoxication precedes
burn and that limiting bacterial translocation in this setting attenuates hepatic damage and
pulmonary inflammation.
Introduction
As the most frequently abused substance in the United States, alcohol has a high
association with unintentional injuries [2,3] and is the third leading cause of preventable
death [1]. The practice of consuming enough alcohol to bring the blood alcohol
concentration (BAC) to 0.08% or above, known as binge drinking, is an increasingly
prevalent form of intoxication [4] and the characteristic consumption pattern among
patients presenting with traumatic injury, including burns [5]. Approximately half of
adult burn patients are intoxicated at the time of admission and these patients have worse
clinical outcomes than individuals who sustain similar injuries not under the influence
[6,7,8]. Specifically, they are twice as likely to acquire an infection, undergo 60% more
surgical procedures, require more days on mechanical ventilation, and stay longer in the
intensive care unit [6,9]. As a result of these complications, burn patients with prior
alcohol exposure have been identified as a patient population at high risk for death in the
early stages post burn [10]. With 1 million burn injuries in the US each year, resulting in
40,000 hospitalizations [11], this represents a substantial burden of morbidity, mortality
and socioeconomic cost that is worsened by alcohol abuse. Despite the prevalence and
known repercussions of being intoxicated at the time of a burn injury, there is little
difference in the treatment and management of burn patients with and without prior
20
alcohol exposure. In fact, relatively little is known about the mechanisms by which
alcohol affects the post burn response. The complicated natural history of burn injuries as
well as the complex and duration-dependent effects of alcohol impact nearly every major
organ system [21]. Therefore, the detrimental response to intoxication and burn likely
encompasses aberrations, and potentially crosstalk, between multiple organs.
The studies reported herein examine the role of the gut-liver axis after
intoxication and burn by limiting bacterial translocation through pharmacologic
restoration of intestinal barrier function after injury. This was accomplished by
administration of an inhibitor of myosin light chain kinase (MLCK) after a burn, which
we have previously demonstrated decreases intestinal barrier damage and subsequent
bacterial translocation in this setting [29]. In a separate group of animals, the initial
bacterial burden within the intestines was decreased before injury by prophylactic oral
antibiotics. Finally, systemic effects of manipulating the gut-liver axis were investigated
by examining pulmonary inflammation and interleukin-6 levels.
Materials & Methods
Animals
Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor,
ME) and housed in sterile micro-isolator cages under specific pathogen-free conditions in
the Loyola University Chicago Comparative Medicine Facility. All experiments were
conducted with approval of and strict accordance to the Loyola Institutional Animal Care
and Use Committee. All experiments were performed with mice weighing between 23-25
21
g and during the hours of 8-10 AM to avoid confounding factors related to circadian
rhythms.
Murine Model of Intoxication and Burn Injury
A murine model of a single binge ethanol exposure and burn injury was employed
as described previously [107] with minor modifications. Briefly, mice were given a single
intraperitoneal (i.p.) dose of ethanol (1.12 g/kg) that resulted in a blood ethanol
concentration of 150–180 mg/dl at 30 minutes or saline vehicle. The mice were then
anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine), their dorsum shaved, and
placed in a plastic template exposing 15% of the total body surface area and subjected to
a scald injury in a 90-92ºC water bath or a sham injury in room-temperature water. The
scald injury resulted in an insensate, full-thickness burn [108]. The mice were then
resuscitated with 1.0 ml saline and allowed to recover on warming pads. The intoxicated
and burn injured mice were split into two groups and at 30 minutes after injury, given the
specific long MLCK inhibitor PIK (membrane-permeant inhibitor of kinase,100μl i.p. of
50 μM) or saline control as described previously [29]. A separate group of mice received
prophylactic antibiotics (neomycin 100 mg/kg/day and polymyxin E (60mg/kg/day) via
drinking water for 10 days before intoxication and burn. This resulted in a 3.67 log
reduction in culturable aerobic fecal bacteria at the time of injury. Mice were sacrificed
by CO2 narcosis followed by exsanguination 24 hours after injury. The control arms of
sham vehicle, sham ethanol, and burn vehicle are presented herein to demonstrate
baseline results and provide injury context for the assessed parameters. The data from
these control groups can be found in the manuscript figures and are consistent with
22
previously reported results in the intestines [29], liver [23], and lungs[89]. As this study
examines a therapeutic intervention for the established consequences of intoxication and
burn injury in this animal model, the discussion of this study will focus on the animals
given the combined injury of ethanol and burn, with and without treatments.
Histopathologic Examination of the Ileum.
At 6 and 24 hours post-injury, mice were euthanized by CO2 narcosis and the
ileum was harvested and fixed overnight in 10% formalin. Samples were then embedded
in paraffin, sectioned at 5 microns, and stained with hematoxylin and eosin (H&E).
Images were taken at 200x.
Immunofluorescent Staining in the Ileum
Immunofluorescent staining was done as previously described [29] with minor
modifications. Briefly, a small section of ileum (5 mm) was embedded in optimal cutting
temperature (OCT) media and frozen at -80ºC. The ileum was sectioned (8 μm) and
stained with rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA) followed by goat anti-rabbit
Alexa Fluor 488 (Invitrogen, Carlsbad, CA). Sections were further stained with
fluorescent-conjugated phalloidin (actin) and Hoechst nuclear stain (Invitrogen Carlsbad,
CA). Images were taken at 400x magnification.
Bacterial Translocation
Bacterial translocation was assessed as previously described [29,48]. Briefly, 3–5
mesenteric lymph nodes (MLN) per mouse were removed and placed in cold RPMI, and
kept on ice. MLN were separated from connective tissue and homogenized in RPMI
23
using frosted glass slides. Homogenates were plated in triplicate on tryptic soy agar and
placed in a 37°C incubator overnight.
Histopathologic Examination of the Liver
The whole liver was removed at the time of sacrifice, weighed and embedded in
OCT media or snap frozen in liquid nitrogen. Frozen sections were cut at 7 μm, stained
with Oil Red O, and then examined for the presence of fat droplets as described
previously [22]. Briefly, sectioned tissue was rinsed in isopropyl alcohol and then placed
in a filtered working solution of Oil Red O for 20 minutes. The slides were then rinsed in
isopropyl alcohol, followed by tap water and counterstained with hematoxylin for 1
minute. The slides were then serially rinsed in tap water, ammonia water (lithium
carbonate), and finally tap water. Representative images were taken at 400x
magnification.
Liver Cytokines and Triglyceride Quantification
Liver tissue was homogenized in 1 ml of BioPlex cell lysis buffer (BioRad,
Hercules, CA) and analyzed for cytokine production using an enzyme-linked
immunosorbent assay (ELISA) for interleukin-6 (IL-6; BD Biosciences, Franklin Lakes,
NJ). The results were normalized to total protein using the BioRad protein assay
(BioRad, Hercules, CA). A portion of the frozen liver was used in a Triglyceride
Quantification Kit according to manufacturer instruction (Abcam, Cambridge, MA).
Hepatic IL-6 mRNA Expression
RNA was isolated from liver homogenate using RNeasy Mini Kit (Qiagen,
Valencia, CA) and cDNA was produced using iScript cDNA synthesis kit (Bio-Rad,
24
Hercules, CA). Quantitative real-time polymerase chain reaction (qRT-PCR) was
performed using the StepOne Plus Real-Time PCR Systems (Applied Bio-Systems,
Carlsbad, CA) and data analyzed by the 2-ddCT relative quantification method [109].
Serum Measurements
At 24 hours post-injury mice were euthanized, blood was collected via cardiac
puncture and harvested for serum by centrifugation after clotting. Serum aliquots were
used to measure IL-6 by ELISA (BD Biosciences, Franklin Lakes, NJ) or liver
transaminase levels using a DRI-CHEM 7000 (HESKA, Loveland, CO) as described
previously [19].
Histopathologic Examination of the Lungs
The upper right lobe of the lung was inflated with 10% formalin and fixed overnight as
described previously [110]. The lung was then embedded in paraffin, sectioned at 5 µm,
and stained with hematoxylin and eosin (H&E). The sections were analyzed
microscopically in a blinded fashion for the number of neutrophils in 10 high power
fields as a marker of inflammation. Representative images were taken at 400x
magnification. Ten high power fields (400X) per animal were analyzed using the Javabased imaging program ImageJ (National Institutes of Health, Bethesda, MD). The
images were converted to binary to differentiate lung tissue from air space and then
analyzed for the percent area covered by lung tissue in each field of view as described
previously [89].
25
KC Analysis of Lung Homogenates
The right middle lung lobe was snap-frozen in liquid nitrogen. The tissues were
then homogenized in 1 ml of BioPlex cell lysis buffer according to manufacturer’s
instructions (BioRad, Hercules, CA). The homogenates were filtered and analyzed using
an ELISA for KC (BD Biosciences, Franklin Lakes, NJ), a neutrophil chemokine and
murine analog for human IL-8. The results were normalized to total protein using the
BioRad protein assay (BioRad, Hercules, CA).
Statistical Analysis
Statistical comparisons (GraphPad Instat) were made between intoxicated burned
mice given saline and intoxicated burned mice given a treatment (PIK or antibiotics)
using a Student's t-test and Tukey's post hoc test (p<0.05). Data are reported as mean
values ± the standard error of the mean (SEM).
Results
Limiting Bacterial Translocation Attenuates Hepatosteatosis
after Intoxication and Burn
Twenty-four hours after intoxication and burn, intestinal villi demonstrated a loss
of tight junction integrity (Figure 4A), a 62-fold increase in bacterial translocation
(Figure 4B), and a 14-fold elevation in hepatic triglycerides (Figure 4C) compared to
sham injured animals. PIK treatment after intoxication and burn restored tight junction
complexes as demonstrated by colocalization of ZO-1 and actin (Figure 4A). This
corresponded to a 71% decrease (p<0.05) in bacterial translocation (Figure 4B), and a
75% reduction (p<0.05) in liver triglycerides (Figure 4C) compared to saline-treated
26
Figure 4. Decreasing bacterial translocation attenuated hepatosteatosis. (A) Tight junction
protein localization (400x). zonula occludens protein-1 (ZO-1; green), phalloidin (red)
and nuclei (blue). (B) Bacterial translocation. colony forming units (CFU). (C) Liver
sections with ORO (400x). (D) Hepatic triglycerides. *p<0.05 compared to PIK and Abx
by Student’s T test. N=4-6 animals per group.
represents sham vehicle,
represents sham ethanol, and
represents burn vehicle.
27
animals undergoing the same injury. Antibiotic prophylaxis failed to restore tight junction
complexes in the terminal ileum (Figure 4A) but did reduce bacterial translocation by
93% (p<0.05) (Figure 4B) which corresponded to an 84% decrease (p<0.05) in liver
triglycerides (Figure 4C) relative to intoxicated and burn injured mice given saline. The
attenuation of steatosis after intoxication and burn in PIK or antibiotic-treated animals
was evident upon histologic examination in Oil Red O-stained images (Figure 4D).
PIK or Antibiotic Treatment Reduces Hepatic Damage
After Intoxication and Burn
In comparison to sham injured animals, mice given ethanol and burn were found
to have a >25-fold increase in serum AST (Figure 5A) and ALT (Figure 5B) as well as
1.3-fold increase in liver weight to body weight ratio (Figure 5C). PIK treatment after the
combined insult alleviated hepatic damage as measured by a 49% reduction (p<0.05) in
serum AST, a 47% decrease (p<0.05) in serum ALT and a 24% lower (p<0.05) liver
weight to body weight ratio compared to intoxicated and burned animals given saline
(Figure 2 A-C). Analogous to PIK treatment, antibiotic prophylaxis reduced serum AST
by 44% (p<0.05), ALT by 43% (p<0.05) and liver weight to body weight ratio by 23%
(p<0.05), relative to animals given saline after ethanol and burn (Figure 5A-C).
Hepatic and Systemic Levels of IL-6 are Reduced by Limiting Bacterial
Translocation after Intoxication and Burn
Twenty-four hours after intoxication and burn, an approximate 60-fold increase in
hepatic mRNA expression (Figure 6B) corresponded to a 10-fold increase in IL-6 protein
28
Figure 5. Liver damage 24 hours after intoxication and burn is diminished when bacterial
translocation is inhibited. (A) Serum levels of aspartate aminotransferase (AST) after
treatment with saline vehicle, MLCK inhibition (PIK), or prophylactic antibiotics (Abx).
(B) Serum levels of alanine aminotransferase (ALT). (C) Liver weight to total body
weight ratio. *p<0.05 compared to PIK and Abx treatments by Student’s T test. N=4-6
animals per group.
represents sham vehicle,
represents sham ethanol, and
represents burn vehicle.
29
levels in the liver (Figure 6A) and serum (Figure 6C) compared to sham-injured mice.
After the combined insult of intoxication and burn, PIK treatment led to a 33% reduction
(p<0.05) in hepatic mRNA expression (Figure 6B), a 71% decrease (p<0.05) in IL-6
protein levels in the liver (Figure 6A) and 70% lower (p<0.05) serum IL-6 (Figure 6C).
Similarly, IL-6 levels in mice that received antibiotic prophylaxis reduced hepatic IL-6
by 51% (p<0.05), IL-6 mRNA expression by 45% (p<0.05), and serum IL-6 by 68%
(p<0.05) compared to mice undergoing intoxication and burn given saline.
Figure 6. Interleukin-6 production is decreased with PIK or antibiotic treatment. (A)
Hepatic IL-6. (B) IL-6 mRNA expression levels in liver. (C) Serum IL-6 levels. *p<0.05
vs PIK and Abx by Student’s T test. N=4-6 animals per group.
represents sham
vehicle,
represents sham ethanol, and
represents burn vehicle.
30
Pulmonary Inflammation after Intoxication and Burn is Attenuated
with PIK or Antibiotics
Intoxication and burn substantially increased alveolar wall thickness by visual
examination (Figure 7A) compared to sham injured animals. Imaging software was
employed to quantify the amount of lung tissue versus air space as a measure of alveolar
wall thickness as described previously [19]. A 3-fold increase in lung tissue area was
observed in intoxicated and burned mice compared to those receiving a sham injury
(Figure 7B) which was accompanied by a >10-fold increase in neutrophil infiltration
(Figure 7C) and pulmonary KC levels (Figure 7D). PIK treatment as well as antibiotics
lessened pulmonary congestion and cellularity upon visual examination (Figure 7A).
Consistent with visual findings, PIK treatment decreased pulmonary congestion by 23%
(p<0.05) compared to matched controls and similarly, antibiotic prophylaxis lessened
congestion by 29% (p<0.05) (Figure 7B). Neutrophil accumulation was reduced by 49%
(p<0.05) with PIK treatment and 43% (p<0.05) with antibiotics compared to intoxicated
burned mice given saline (Figure 7C). Finally, levels of the neutrophil chemokine, KC,
were measured in lung homogenates and were reduced by 48% (p<0.05) and 41% in PIK
and antibiotic treated animals respectively compared to intoxicated and burn injured mice
given saline after injury (Figure 7D).
31
Figure 7. Pulmonary inflammation 24 hours after intoxication and burn is attenuated by
restoration of the gut-liver axis. (A) Lung histology (hematoxylin and eosin at 400x) of
intoxicated and burn-injured mice receiving PIK treatment or prophylactic antibiotics
(Abx). (B) Pulmonary congestion quantified using imaging software to calculate the lung
tissue area in 10 fields of view. (C) Neutrophil (PMN) quantification in 10 high power
fields of view of intoxicated and burn-injured mice. (D) Pulmonary KC levels. *p<0.05
compared to PIK and antibiotic (Abx) treatments by Student’s T test. N=4-6 animals per
group.
represents sham vehicle,
represents sham ethanol, and
represents burn vehicle.
32
Summary
The studies above demonstrate a clear and reversible interaction between bacterial
translocation from the intestines and the hepatic response to burn injury and ethanol
exposure. Interestingly, both PIK and antibiotic treatments reduced the amount of
bacterial translocation to the extent observed after a burn alone (Figure 4B, [19,29,48])
and the subsequent responses seen in the liver and lungs matched the degree observed in
post burn injuries without ethanol exposure (Figures 5 & 7, [19,89,111]). These results
may suggest that intoxication worsens post burn outcome through alterations in the gutliver axis since the additional damage observed with antecedent ethanol exposure can be
reversed by restoring gut-liver crosstalk to levels seen after a burn alone. Clinically, there
are no accurate predictors for increased burn risk amongst alcohol abusers and
indiscriminate antibiotic prophylaxis in this population may raise numerous issues with
antimicrobial stewardship, drug compliance, altering endogenous flora, drug interactions
and cost. Therefore, an ideal therapeutic in this setting could be administered after the
injury. PIK is still an experimental research drug and unfortunately there are currently no
licensed drugs that act on MLCK inhibition to our knowledge. However we believe our
data demonstrates a proof of concept that can hopefully be taken into human trials as
drugs become available.
CHAPTER 4
KUPFFER CELL P38 MAPK SIGNALING DRIVES POST BURN HEPATIC
DAMAGE AND PULMONARY INFLAMMATION WHEN ALCOHOL
INTOXICATION PRECEDES BURN INJURY
Abstract
The widespread and increasing trend of binge drinking is accompanied by a
concomitant rise in the prevalence of trauma patients intoxicated at the time of injury.
Nearly half of adult burn patients are intoxicated at the time of admission and the
presence of alcohol is an independent risk factor for death in the early stages post burn.
We recently reported hepatic IL-6 drives pulmonary inflammation in this setting and we
now seek to determine the role of Kupffer cells in this aberrant response. To this end,
mice were given ethanol (1.2g/kg) by oral gavage 30 minutes prior to a 15% burn injury.
Kupffer cells were isolated 24 hours after injury and analyzed for p38 activity and IL-6
production. In separate experiments, Kupffer cell depletion with clodronate liposomes or
p38 inhibition was achieved via intraperitoneal injection of SB203580. Kupffer cells of
intoxicated burned mice had a 2-fold (p<0.05) elevation of p38 activation relative to burn
alone and this corresponded to a 43% (p<0.05) increase in IL-6 production. Kupffer cell
depletion attenuated hepatic damage as seen by decreases of 53% (p<0.05) in serum ALT
and 74% (p<0.05) in hepatic triglycerides, as well as a 77% reduction (p<0.05) in serum
IL-6 levels compared to matched controls.
33
This mitigation of hepatic damage was
34
associated with a 54% decrease (p<0.05) in pulmonary neutrophil infiltration and reduced
alveolar wall thickening by 45% p<0.05), suggesting a crucial role for Kupffer cells in
both hepatic and pulmonary damage in this setting. In vivo p38 inhibition conferred
nearly identical hepatic and pulmonary protection after the combined injury as mice
depleted of Kupffer cells, highlighting the role of Kupffer cell p38 in causing injury.
Taken together, these data suggest intoxication exacerbates post burn hepatic damage
through p38-dependent IL-6 production in Kupffer cells which may serve as a potential
therapeutic target in this common clinical scenario.
Introduction
Severe burns are a devastating injury affecting every major organ system, with the
degree of systemic inflammation correlating to the size of the burn [17]. Advances in
wound care, fluid resuscitation and infection control have dramatically increased survival
rates [112], though hepatic derangements and a hypermetabolic state can persist for
years after the initial injury [24]. The physiologic response to a burn depends on a
variety of factors including the age of the patient, the size and severity of the burn, and
the presence of alcohol [10]. Nearly 50% of patients admitted for burns have a positive
blood alcohol content at presentation and these patients have worse clinical outcomes
than individuals who sustain similar injuries not under the influence of alcohol [6,7,8].
Specifically, they are twice as likely to acquire an infection, require more surgical
procedures and have a longer duration of stay in the intensive care unit than their
nonintoxicated counterparts [6,9]. As a result of these complications, burn patients with
prior alcohol exposure have been identified as a patient population at high risk for death
35
in the early stages post burn [10]. However, few differences currently exist in their
treatment and management despite the established consequences. With 1 million burn
injuries in the US each year, resulting in 40,000 hospitalizations [11], this represents a
substantial burden of morbidity, mortality and socioeconomic cost that is worsened by
alcohol abuse. Binge drinking in particular is an increasingly prevalent form of
intoxication [4] and is the characteristic pattern of drinking among patients presenting
with traumatic injury, including burns [5]. Recent studies suggests the majority of
intoxicated burn patients are binge drinkers without evidence of chronic alcoholism [5],
consistent with the majority of alcohol consumption in the US [4]. The increasing
prevalence of binge drinking and its high association with traumatic injury in an
otherwise healthy population warrant further investigation into the mechanism by which
alcohol worsens the post burn response.
As a vital organ with metabolic, hormonal and immune functions, the liver is
central in the response to burn injury and plays a pivotal role in post-burn outcome [14].
This is highlighted by the close correlation between hepatic function and prognosis after
burn [15]. The liver is also the major site of alcohol metabolism and toxicity and we
have previously demonstrated that intoxication potentiates post burn hepatic damage
[19,20,22,23]. Furthermore, we reported that increased hepatic interleukin-6 (IL-6)
drives pulmonary inflammation in the setting of this combined injury [20,89], making it
essential to understand the source and underlying mechanisms of its production. Animal
studies demonstrate that post burn intestinal permeability is exacerbated when
intoxication precedes the injury [32,33], leading to greater translocation of bacteria and
36
bacterial products such as lipopolysaccharide (LPS) into the lymphatic [45,46,48] and
portal [47] systems. Within the liver, Kupffer cells line the hepatic sinusoids and sample
the portal blood for foreign antigen. Upon activation by LPS, Kupffer cells release proinflammatory cytokines which orchestrate an appropriate hepatic response [60]. In an
injury context, however, Kupffer cells can become hyperactive and release excessive
levels of cytokines, including IL-6, causing hepatic and systemic damage [64,67] .
Independently, both alcohol [73,96] and burn [67,113] are known to increase Kupffer cell
sensitivity to LPS through Toll-like receptor 4 (TLR4) signaling and downstream p38
mitogen activated protein kinase (MAPK). We therefore sought to examine the role of
Kupffer cells in the response to intoxication and burn. We describe within findings that
demonstrate intoxication augments Kupffer cell IL-6 production through increased p38
activation after burn.
Materials & Methods
Animals
Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor,
ME) and housed in sterile micro-isolator cages under specific pathogen-free conditions in
the Loyola University Chicago Comparative Medicine Facility. All experiments were
conducted with approval of and strict accordance to the Loyola Institutional Animal Care
and Use Committee. All experiments were performed with mice weighing between 2325 g and during the hours of 8-10 AM to avoid confounding factors related to circadian
rhythms.
37
Murine Model of Intoxication and Burn Injury
A murine model of a single binge ethanol exposure and burn injury was employed
as described previously [107] with minor modifications. Briefly, mice were given a
single dose of ethanol (1.12 g/kg) by oral gavage that resulted in a blood alcohol
concentration of approximately 180 mg/dl at 30 minutes [19]. The mice were then
anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine), their dorsum shaved and they
were placed in a plastic template exposing 15% of the total body surface area (TBSA)
and subjected to a scald injury in a 92ºC water bath or a sham injury in room-temperature
water. The scald injury resulted in an insensate, full-thickness burn [108]. The mice
were then resuscitated with 1.0 ml of 0.9% normal saline and placed on warming pads
until recovered from anesthesia. Mice were euthanized by carbon dioxide narcosis
followed by exsanguination 24 hours after injury. For experiments involving in vivo p38
inhibition, the intoxicated and burn injured mice were split into two groups and at 30
minutes after injury, given an intraperitoneal injection of the selective p38 MAPK
inhibitor, SB203580 (InvivoGen, 10mg/kg) or vehicle control (saline). SB203580 at this
dose has been previously demonstrated to specifically inhibit Kupffer cell p38 MAP
kinase 24 hours after burn injury ([113], Fig. S1). For experiments involving antecedent
Kupffer cell depletion, clodronate liposomes (Encapsula NanoSciences, 0.5mg/kg) or
vehicle control (empty liposomes) were administered via tail vein injection. This method
of administration has been shown to cause Kupffer cell depletion while limiting the
effects on other macrophage populations ([114], Fig. S1). The control arms of sham
vehicle, sham ethanol, and burn vehicle are presented herein for the clodronate and p38i
38
experiments to demonstrate baseline results and provide injury context for the assessed
parameters. The data from these control groups can be found in the manuscript figures
and are consistent with previously reported results in the liver [19,20,23], and lungs
[19,34,89]. As this study examines the role of Kupffer cells in the established
consequences of intoxication and burn injury in this animal model, the discussion of the
in vivo studies will focus on the animals given the combined injury of ethanol and burn,
with and without treatments. Furthermore, as the treatment vehicle controls given to
intoxicated and burn injured mice (saline and empty liposomes for p38 inhibition and
clodronate, respectively) did not differ from intoxication and burn without vehicle
controls, only one set of results are shown for clarity.
Blood and Serum Measurements
At 24 hours post injury, mice were euthanized, blood was collected via cardiac
puncture, an aliquot was placed into a microcapillary tube, and read for a complete blood
count with differential by Hemavet (Drew Scientific, Dallas, TX). The remaining blood
was harvested for serum by centrifugation after clotting. Serum aliquots were used to
measure IL-6 by ELISA (BD Biosciences, Franklin Lakes, NJ) or liver transaminase
levels using a DRI-CHEM 7000 (HESKA, Loveland, CO) as described previously [19].
Histologic Examination of the Liver
The whole liver was removed at the time of sacrifice, weighed and a portion was
embedded in OCT media or snap frozen in liquid nitrogen. Frozen sections were cut at 7
μm, stained with Oil Red O, and then examined for the presence of fat droplets as
described previously [22]. Briefly, sectioned tissue was rinsed in isopropyl alcohol and
39
then placed in a filtered working solution of Oil Red O for 20 minutes. The slides were
then rinsed in isopropyl alcohol, followed by tap water and counterstained with
hematoxylin for 1 minute. The slides were then serially rinsed in tap water, ammonia
water (lithium carbonate), and finally tap water. Representative images were taken at
400x magnification.
Assessment of Liver Cytokines and Triglyceride Quantification
Liver tissue was homogenized in 1 ml of BioPlex cell lysis buffer according to
manufacturer’s instructions (BioRad, Hercules, CA) and analyzed for cytokine
production using an ELISA for interleukin-6 (IL-6; BD Biosciences, Franklin Lakes, NJ).
The results were normalized to total protein using the BioRad protein assay (BioRad,
Hercules, CA). A portion of the frozen liver was used in a Triglyceride Quantification
Kit according to manufacturer instruction (Abcam, Cambridge, MA).
Isolation of Kupffer Cells
Kupffer cells were isolated 24 hour after injury as described by Kuriakose et al.
[115] with minor modifications. Briefly, rinsed liver tissue was incubated at 37°C for 30
minutes in collagenase IV (5000 CDU/mL) and run through an automated gentleMACS
Dissociator (Miltenyi Biotec, Auburn, CA) before being passed through a sterile 100um
strainer into phosphate-buffered saline (Miltenyi Biotec, Auburn, CA) containing 0.5%
bovine serum albumin. The hepatocytes were then removed by low-speed centrifugation
at 20xg for 4 minutes, and the residual cell suspension was centrifuged twice at 300xg for
10 minutes at 4°C with the cell pellet washed with Red Blood Cell Lysis Solution
between centrifugations. CD11b+ cells were then enriched by positive selection using
40
AUTOMACS (Miltenyi Biotec Auburn, CA). Enriched liver CD11b cells isolate by this
+
method have been shown to be greater than 96% positive for F4/80 expression as
assessed by flow cytometry.
Cell Culture
Isolated Kupffer cells were cultured at a concentration of 3x105 per well in RPMI
medium supplemented with 10% fetal bovine serum and 50µg/ml gentamycin for 2
hours. Cells were pretreated with SB203580 (10uM) for 1 hour before stimulation with
LPS (100ng/mL) followed by incubation at 37°C with 5% CO2 and 95% humidity for 18
hours. At the concentration used, SB203580 is highly selective for p38 [116].
Supernatants were removed and analyzed for cytokine production using an enzymelinked immunosorbent assay (ELISA) for interleukin-6 (IL-6; BD Biosciences, Franklin
Lakes, NJ). Activation of p38 was determined by cell-based ELISA (RayBiotech Inc,
Norcross, GA) using primary antibodies against p38 and phospho-p38 (Thr180/Tyr182)
as specified per manufacturer instructions.
Histologic Examination of the Lungs
The upper right lobe of the lung was inflated with 10% formalin and fixed
overnight as described previously [110]. The lung was then embedded in paraffin,
sectioned at 5 µm, and stained with hematoxylin and eosin (H&E). The sections were
analyzed microscopically in a blinded fashion for the number of neutrophils in 10 high
power fields as a marker of inflammation. Representative images were taken at 400x
magnification. Ten high power fields (400X) per animal were analyzed using the Javabased imaging program ImageJ (National Institutes of Health, Bethesda, MD). The
41
images were converted to binary to differentiate lung tissue from air space and then
analyzed for the percent area covered by lung tissue as described previously [89].
Statistical analysis
Statistical comparisons (GraphPad Instat) were made between the sham vehicle,
sham ethanol, burn vehicle, and burn ethanol animals, resulting in four total groups
analyzed. One-way analysis of variance (ANOVA) was used to determine differences
between groups, and Tukey's post hoc test once significance was achieved (p<0.05).
Statistical comparisons made between intoxicated burned mice given saline and
intoxicated burned mice given a treatment (clodronate or SB203580) were performed
using a Student's t-test (p<0.05). Data are reported as mean values ± the standard error of
the mean (SEM).
Results
Kupffer Cell p38 Activation and IL-6 Production
Total p38 was detectable in all samples and did not change between treatment
groups. Ethanol intoxication or burn injury alone increased Kupffer cell p38 activation
approximately 2-fold (p<0.05) over nonintoxicated sham injured animals (Figure 8A),
corresponding to an approximate 2-fold increase in IL-6 production (Figure 8B) after
LPS stimulation. The combination of ethanol and burn injury led to a 4-fold (p<0.05)
increase in p38 activation relative to sham, which was 2-fold (p<0.05) greater than either
insult alone. IL-6 levels paralleled p38 activation with the combined injury
demonstrating a 3-fold (p<0.05) and 1.5-fold (p<0.05) increase over sham injury or
isolated intoxication or burn, respectively (Figure 8B). Ex vivo p38 inhibition abrogated
42
the increases in LPS-induced p38 activation (Figure 8A) or IL-6 production (Figure 8B)
in Kupffer cells from any injury group.
Figure 8. Kupffer cell p38 activation is increased when intoxication precedes burn.
Kupffer cells were isolated 24 hours after injury and assessed for (A) p38 activation and
(B) IL-6 production after LPS-stimulation with and without the presence of the p38
inhibitor SB203580 (p38i). *p<0.05 compared to all groups given LPS without inhibitor.
#p<0.05 compared to Sham Vehicle given LPS without inhibitor. N=4-6 animals per
group.
Kupffer Cell Depletion or p38 Inhibition Attenuates Hepatic Damage
after Intoxication and Burn
In comparison to sham injured animals, mice given ethanol and burn were found
to have a >15-fold increase in serum AST (Figure 9A) and ALT (Figure 9B) as well as
1.4-fold increase in liver weight to body weight ratio (Figure 9C) which corresponded to
12-fold (p<0.05) greater triglyceride accumulation (Figure 9D). Inhibition of p38 after
the combined insult alleviated hepatic damage as measured by a 35% reduction (p<0.05)
in serum AST, a 58% decrease (p<0.05) in serum ALT and a 22% lower (p<0.05) liver
weight to body weight ratio compared to intoxicated and burned animals given saline
43
Figure 9. The absence of Kupffer cells or p38 inhibition equally attenuate hepatic
damage. (A) Serum levels of aspartate aminotransferase (AST) after treatment with
vehicle, clodronate liposomes (CLO) or SB203580 (p38i). (B) Serum levels of alanine
aminotransferase (ALT). (C) Liver weight to total body weight ratio (LW:BW). (D)
Hepatic triglycerides. (E) Liver sections stained with Oil Red O at 400x magnification.
*p<0.05 compared to CLO and p38i by Student’s T test. N=4-6 animals per group.
represents Sham Vehicle,
represents Sham Ethanol, and
represents
Burn Vehicle.
44
(Figure 9A-C). Analogous to SB203580 treatment, antecedent Kupffer cell depletion
reduced serum AST by 44% (p<0.05), serum ALT by 53% (p<0.05) and liver weight to
body weight ratio by 28% (p<0.05), relative to animals given saline after ethanol and
burn (Figure 9A-C). The decreased hepatic damage after p38 inhibition or the absence of
Kupffer cells paralleled decreases in hepatic triglycerides of 59% (p<0.05) and 74%
(p<0.05), respectively (Figure 9D). The attenuation of steatosis after intoxication and
burn in SB203580 or clodronate-treated animals was evident upon histologic examination
in Oil Red O-stained images (Figure 9E).
Hepatic and Systemic Levels of IL-6 are Reduced by Limiting p38 Signaling
in Kupffer Cells after Intoxication and Burn
Twenty-four hours after intoxication and burn, a 20-fold increase in IL-6 protein
levels in the liver (Figure 10A) and serum (Figure 10C) were observed compared to
sham-injured mice. After the combined insult of intoxication and burn, p38 inhibition led
to a 57% reduction (p<0.05) in hepatic IL-6 levels (Figure 10A) which corresponded to a
68% decrease (p<0.05) in serum IL-6 (Figure 10C). Similarly, IL-6 levels in mice that
lacked Kupffer cells at the time of injury had reduced hepatic IL-6 by 65% (p<0.05) and
serum IL-6 by 77% (p<0.05) compared to mice undergoing intoxication and burn with
intact Kupffer cells.
45
Figure 10. Interleukin-6 production is decreased with clodronate liposomes (CLO) or
SB203580 (p38i) after intoxication and burn. (A) Interleukin-6 (IL-6) protein levels in
liver homogenates. (B) Serum IL-6 levels. *p<0.05 compared to CLO and p38i by
Student’s T test. N=4-6 animals per group.
represents Sham Vehicle,
represents Sham Ethanol, and
represents Burn Vehicle.
Pulmonary Inflammation after Intoxication and Burn is Attenuated with
SB203580 or Clodronate
Intoxication and burn substantially increased alveolar wall thickness by visual
examination (Figure 11A) compared to sham injured animals. Imaging software
(ImageJ) was employed to quantify the amount of lung tissue versus air space as a
measure of alveolar wall thickness as described previously [19]. A 3-fold increase in
lung tissue area was observed in intoxicated and burned mice compared to those
receiving a sham injury (Figure 11B) which was accompanied by an 8-fold increase in
neutrophil infiltration (Figure 11C). Clodronate or inhibition of p38 lessened pulmonary
congestion and cellularity upon visual examination (Figure 11A). Consistent with visual
findings, p38 inhibition decreased pulmonary congestion by 43% (p<0.05) compared to
matched controls and similarly, Kupffer cell depletion lessened congestion by 45%
(p<0.05) (Figure 11B). Neutrophil accumulation was reduced by 58% (p<0.05) with
46
SB203580 treatment and 54% (p<0.05) with clodronate compared to matched controls
(Figure 11C).
Figure 11. Pulmonary inflammation after intoxication and burn is attenuated by Kupffer
cell depletion or p38 inhibition. (A) Lung histology (hematoxylin and eosin at 400x) of
intoxicated and burn injured mice receiving clodronate liposomes (CLO) or SB203580
(p38i). (B) Neutrophil (PMN) quantification in 10 high power fields of view of
intoxicated and burn-injured mice. (C) Pulmonary congestion quantified using imaging
software to calculate the lung tissue area in 10 fields of view. *p<0.05 compared to
CLO and p38i by Student’s T test. N=4-6 animals per group.
represents Sham
Vehicle,
represents Sham Ethanol, and
represents Burn Vehicle.
In Vivo Treatment Efficacy
Liver sections of mice administered clodronate liposomes (Figure 12B) exhibited
depletion of Kupffer cells as demonstrated by the low CD68 staining compared to the
livers of mice administered empty liposomes (Figure 12A). In contrast no differences
were found in numbers of circulating monocytes (Figure 12C) or alveolar macrophages
(Figure 12D-E). In vivo SB203580 treatment abrogated LPS-induced p38 activation in
47
Kupffer cells isolated from intoxicated and burn injured mice (Figure 12F). Despite an in
vitro study reporting SB203580 specifically inhibits p38 activity but not p38 activation
[117], inhibition of p38 phosphorylation after SB203580 administration is widely
reported and used as a mechanism to verify in vivo efficacy [75,78,113,118,119].
Inhibition of p38 was confirmed through parallel decreases in LPS-induced IL-6
production (Figure 12G).
Figure 12. In vivo Kupffer cell depletion and p38 inhibition. (A) Liver sections stained
for CD68 from mice receiving empty liposomes or (B) clodronate liposomes. (C)
Peripheral blood monocytes. (D) Lung sections stained for alveolar macrophage marker
SiglecF in mice receiving empty liposomes or (E) clodronate liposomes. (F) p38
activation in isolated Kupffer. (G) IL-6 production in isolated Kupffer cells. *p<0.05
compared to all groups by one-way ANOVA. N=3-6 animals per group.
48
Summary
The studies above delineate a clear and reversible role for Kupffer cell p38
MAPK in the hepatic response to intoxication and burn. Kupffer cells isolated after the
combined injury demonstrated increased p38 activation and IL-6 production,
corresponding to worsened hepatic damage and pulmonary inflammation. Furthermore,
antecedent depletion of Kupffer cells or p38 inhibition after injury attenuated damage in
the liver and lungs to the degree observed in post burn injuries without alcohol exposure
(Figures 2 & 4, [19,89,111]). We recently reported near identical hepatic and pulmonary
protection from limiting bacterial translocation after intoxication and burn as the current
study where the ability of Kupffer cells to respond to LPS was impaired. Taken together,
these results may suggest that intoxication worsens post burn outcome through
sensitizing Kupffer cells while increasing the delivery of LPS to the liver. Of clinical
importance is the finding that alcohol mediates this post burn response through the action
of Kupffer cell p38 MAPK, perhaps revealing a much needed therapeutic target for this at
risk patient population.
CHAPTER 5
ALCOHOL POTENTIATES POST BURN REMOTE ORGAN DAMAGE THROUGH
SHIFTS IN FLUID COMPARTMENTS MEDIATED BY BRADYKININ
Abstract
Of the 450,000 burn patients each year, 50% have a positive blood alcohol
content and this predisposes them to worsened clinical outcomes. Despite high
prevalence and established consequences, the mechanisms responsible for alcoholmediated complications of post burn remote organ damage are currently unknown. To
this end, mice received a single dose of alcohol (1.12 g/kg) or water by oral gavage and
were subjected to a 15% total body surface area burn. Animals with a burn alone lost
~5% of their body weight in 24 hours whereas intoxicated and burned mice lost only 1%
body weight (p<0.05) despite a 17% increase in hematocrit (p<0.05) and a 57% increase
in serum creatinine (p<0.05) over burn injury alone. This retention of water weight
despite increased dehydration suggests that intoxication at the time of a burn causes a
shift in fluid compartments that may exacerbate end organ ischemia and damage as
evidenced by a 3-fold increase in intestinal bacterial translocation (p<0.05), a 30%
increase (p<0.05) in liver weight to body weight ratio, and an increase in alveolar wall
thickness over a burn alone. Furthermore, administration of the bradykinin antagonist
HOE140 30 minutes after intoxication and burn restored fluid balance and alleviated end
49
50
organ damage. These findings suggest that alcohol potentiates post burn remote organ
damage through shifts in fluid compartments mediated by bradykinin.
Introduction
Remote organ damage after burns can be caused by aberrations in the
inflammatory response, known as the systemic inflammatory response syndrome (SIRS),
though the exact mechanisms are currently unknown. The physiologic response to a burn
is dependent on the age of the patient, the size and severity of the injury, and patientspecific risk factors such as alcohol use. Alcohol is the most abused substance in the US
and the third leading cause of preventable death [1], much of which is associated with
unintentional injuries [120]. Binge drinking in particular is an increasingly prevalent form
of intoxication [4] and is the characteristic pattern of drinking among patients presenting
with traumatic injury, including burns [5]. Nearly 50% of patients admitted for burns
have a positive blood alcohol content (BAC) at the time of admission and this
predisposes them to worsened clinical outcomes compared to patients with similar
injuries not under the influence of alcohol [121]. Specifically, intoxicated patients were
found to be twice as likely to acquire an infection, required 60% more surgical
procedures, had longer durations of stay in the intensive care unit, and generated more
cost than their non-intoxicated counterparts [6,9]. With nearly 450,000 burns requiring
medical attention each year in the American healthcare system [11], alcohol greatly
contributes to the socioeconomic burden of this destructive injury as both a causative
agent and complicating factor in recovery. Despite the high prevalence and established
consequences of being intoxicated at the time of a burn injury, there are currently few
51
differences in the treatment and management of burn patients with and without prior
alcohol exposure. This may be due in part to the dynamic natural history of burns as well
as the complex and duration dependent effects of alcohol. In order to develop much
needed targeted therapies, the effects of intoxication on the physiologic response to burn
injury need to be studied and manipulated under controlled conditions.
Animal models have provided meaningful insight into the pathophysiology of
burn injuries preceded by intoxication. Previous studies in our laboratory and others have
demonstrated that relative to either insult alone, the combined injury of intoxication and
burn results in elevated levels of neutrophil infiltration and edema in the lung [61], which
occurs in an interleukin-6 (IL-6) dependent manner [89] . Furthermore, this pulmonary
inflammation is accompanied by an increased susceptibility to pulmonary bacterial
infections and decreased oxygen saturation compared to non-intoxicated controls [122].
This correlates with clinical data linking elevated serum IL-6 in trauma patients to
mortality risk [123] as well as findings that burn patients with a positive BAC acquire
more pulmonary infections [124]. Despite this knowledge, there remains a need to
address the currently unknown mechanism by which alcohol intoxication worsens the
post burn response and outcome. The need for such understanding is highlighted by the
failure of single cytokine therapies in animal and clinical studies of similar injuries
leading to SIRS [125]. Clinical data demonstrates intoxicated burn patients require more
aggressive fluid resuscitation than their non-intoxicated counterparts [6] which may
indicate some degree of hypovolemia. This is of significant concern as early ischemiareperfusion injury may be the inciting event that initiates SIRS, which is linked to
52
pulmonary failure and ARDS [126]. One mechanism by which systemic capillary
leakiness in this setting may ensue is through the vasoactive mediator, bradykinin. No
one to date has investigated the role of bradykinin signaling in the setting of intoxication
and burn but alcohol is known to potentiate the action of bradykinin [127] while burn
injuries cause systemic bradykinin production [26]. To this end, the studies outlined
herein examined a possible mechanism by which alcohol exacerbates post burn remote
organ damage through potentiating third spacing of fluid and enhancing ischemic
damage.
Materials and Methods
Animals
Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor,
ME) and sacrificed when 8-10 weeks old. Mice were housed in sterile micro-isolator
cages under specific pathogen-free conditions in the Loyola University Chicago
Comparative Medicine Facility. All experiments were conducted with approval of and
strict accordance to the Loyola Institutional Animal Care and Use Committee
Murine Model of Intoxication and Burn Injury
A murine model of a single binge ethanol exposure and burn injury was employed
as described previously [107] with minor modifications. Briefly, mice were given a single
dose of ethanol (1.12 g/kg) by oral gavage that results in a blood alcohol concentration of
approximately 180 mg/dl at 30 minutes [19]. The mice were then anesthetized (100
mg/kg ketamine and 10 mg/kg xylazine), their dorsum shaved, weighed, and placed in a
plastic template exposing 15% of the total body surface area and subjected to a scald
53
injury in a 92ºC water bath or a sham injury in room-temperature water. The scald injury
resulted in an insensate, full-thickness burn [108]. The mice were then resuscitated with
1.0 ml of 0.9% normal saline and placed on warming pads until recovered from
anesthesia. At 30 minutes after burn injury, a separate group of mice was given the
selective bradykinin receptor antagonist HOE 140 or saline control. HOE 140 stock
solution (0.083 mg/mL) was prepared in saline and administered as a 150 µL
intraperitoneal injection to achieve a dose of approximately 0.5 mg/kg. Mice were
sacrificed by carbon dioxide narcosis followed by exsanguination 24 hours after injury.
All experiments were performed a minimum of three times with one representative data
set shown.
Blood and Serum Measurements
Blood was collected via cardiac puncture and an aliquot was placed into a
microcapillary tube then measured for hematocrit by Hemavet (Drew Scientific, Dallas,
TX). The remaining blood was harvested for serum by centrifugation after clotting.
Serum aliquots were used to measure blood urea nitrogen and creatinine levels using a
DRI-CHEM 7000 (HESKA, Loveland, CO).
Bacterial Translocation
Bacterial translocation was assessed as previously described [29,48]. Briefly, 3–5
mesenteric lymph nodes (MLN) per mouse were removed, placed in cold RPMI, and kept
on ice. MLN were separated from connective tissue and mechanically homogenized in
RPMI using frosted glass slides. Homogenates were plated in triplicate on tryptic soy
54
agar and placed in a 37°C incubator overnight. Colonies were counted the following day,
averaged, and divided by the total number of lymph nodes harvested.
Histopathologic Examination of the Liver and Lungs
The whole liver was removed at the time of sacrifice, weighed, and normalized to
the total body weight. The upper right lobe of the lung was inflated with 10% formalin
and fixed overnight as described previously [110]. The lung was then embedded in
paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E).
Representative images were taken at 400x magnification.
Statistical Analysis
Statistical comparisons (GraphPad Instat) were made between the sham vehicle,
sham ethanol, burn vehicle, and burn ethanol treatment groups, resulting in four total
groups analyzed. One-way analysis of variance (ANOVA) was used to determine
differences between treatment responses, and Tukey's post hoc test once significance was
achieved (p<0.05). Statistical comparisons made between the intoxicated burned mice
with saline or HOE140 treatment were performed using a Student's t-test and Tukey's
post hoc test (p<0.05). Data are reported as mean values ± the standard error of the mean
(SEM).
Results
Intoxication Increases Post Burn Dehydration
Despite Fluid Retention
Sham injured animals with and without intoxication lost approximately 6% of
their total body weight 24 hours after injury, presumably due to effects of anesthesia and
55
Figure 13. Fluid status 24 hours after injury. (A) Total body weight loss; (B) hematocrit,
(C) creatinine, and (D) blood urea nitrogen (BUN) levels. *p<0.05 compared to all
groups, #p<0.05 compared to sham groups by one-way ANOVA. N=4-6 animals per
group.
stress of physical manipulation (Figure 13A). Similarly, mice receiving a burn alone lost
>5% weight. When intoxication preceded the burn injury, however, mice lost 78%
(p<0.05) less body weight than sham injured animals and 73% (p<0.05) less than mice
56
receiving a burn alone (Figure 13A). Despite this apparent retention of water weight,
mice undergoing the combined injury of intoxication and burn had an approximate 30%
increase (p<0.05) in hematocrit compared to sham injured animals regardless of alcohol
exposure and a 17% increase (p<0.05) over a burn alone (Figure 13B). Similarly,
intoxication and burn raised serum creatinine levels 3-fold (p<0.05) over sham injury and
1.5-fold (p<0.05) over burn alone (Figure 13C). A 2.3-fold increase (p<0.05) in serum
blood urea nitrogen (BUN) was found after a burn alone compared to sham injury which
was raised an additional 1.3-fold when preceded by intoxication (Figure 13D).
Remote Organ Damage is Increased when
Intoxication Precedes Burn
Bacterial translocation to the mesenteric lymph nodes was increased >15-fold
(p<0.05) after a burn injury compared to sham injured animals with and without alcohol
exposure, and increased an additional 3-fold (p<0.05) when ethanol preceded the burn
(Figure 14A). The increase in bacterial translocation with the combined injury was
accompanied by a 40% (p<0.05) and 30% (p<0.05) increase in the relative size of the
liver compared to sham injury and burn alone, respectively (Figure 14B). Histologic
images of the lungs 24 hours after injury demonstrate an increase in alveolar wall
thickness and cellularity with a burn alone which is exacerbated with antecedent
intoxication (Figure 14C).
57
Figure 14. Remote organ damage 24 hours after injury. (A) bacterial translocation
reported as the number of colony forming units (CFU) per mesenteric lymph node, (B)
liver weight to total body weight ratio, (C) Lung histology by H&E staining.
Representative images shown at 400x magnification. *p<0.05 compared to all groups by
one-way ANOVA. N=4-6 animals per group.
Bradykinin Antagonism Partially Restores Fluid
Balance after Intoxication and Burn
HOE140 treatment 30 minutes after intoxication and burn partially normalized
weight loss at 24 hours injury as evidenced by a 10-fold (p<0.05) increase in weight loss
compared to mice given saline vehicle (Figure 3A). Bradykinin antagonism with
58
HOE140 also alleviated dehydration after the combined injury as seen by a 12% decrease
(p<0.05) in hematocrit (Figure 3B), 20% reduction (p<0.05) in serum creatinine (Figure
3C) and 20% lower (p<0.05) BUN (Figure 3D).
Figure 15. HOE140 restores fluid balance 24 hours after intoxication and burn. (A) Total
body weight loss; (B) hematocrit, (C) creatinine, and (D) blood urea nitrogen (BUN)
levels. *p<0.05 by Student’s T test. N=4-6 animals per group.
59
Bradykinin Antagonism Attenuates End Organ
Damage after Intoxication and Burn
Early treatment with HOE140 after intoxication and burn injury led to a 47%
reduction (p<0.05) in bacterial translocation to the mesenteric lymph nodes (Figure 4A),
a 20% decrease (p<0.05) in liver weight to body weight ratio (Figure 4B) and attenuated
pulmonary congestion as seen by a diminution of alveolar wall thickness and cellularity
(Figure 4C).
Figure 16. HOE140 attenuates remote organ damage 24 hours after intoxication and burn.
(A) Bacterial translocation reported as the number of colony forming units (CFU) per
mesenteric lymph node, (B) liver weight to total body weight ratio, (C) lung histology by
H&E staining. Representative images shown at 400x magnification. *p<0.05 by
Student’s T test. N=4-6 animals per group.
60
Summary
The findings of this study suggest that alcohol potentiates post burn third spacing
of fluid through the action of bradykinin, perhaps leading to greater intestinal ischemia
and initial injury itself. We chose to examine a 24 hour time point because this allows for
the greatest differences between treatment groups and avoids any confounding factors
related to circadian rhythms. However, we have shown that there is increased intestinal
damage as soon as 2 hours after injury when intoxication precedes burn [29].
Furthermore, because bradykinin antagonism partially restored fluid balance and
attenuated intestinal damage we feel it can be concluded that bradykinin plays an
important early role in this scenario, though more direct evidence of bradykinin
involvement would be needed to be certain. This does not rule out the significance of
other vasoactive agents after burn, such as histamine and serotonin, but given the
potentiating effect of ethanol on bradykinin, B2 antagonism may be particularly suitable
therapeutic target in this combined injury.
CHAPTER 6
INTOXICATION BY INTRAPERITONEAL INJECTION OR ORAL GAVAGE
EQUALLY POTENTIATE POST BURN ORGAN DAMAGE AND INFLAMMATION
Abstract
The increasing prevalence of binge drinking and its association with trauma
necessitate accurate animal models to examine the impact of intoxication on the response
and outcome to injuries such as burn. While much research has focused on the effect of
alcohol dose and duration on the subsequent inflammatory parameters following burn,
little evidence exists on the effect of the route of alcohol administration. We examined
the degree to which intoxication before burn injury causes systemic inflammation when
ethanol is given by intraperitoneal (i.p.) injection or oral gavage. We found that
intoxication potentiates post burn damage in the ileum, liver and lungs of mice to an
equivalent extent when either ethanol administration route is used. We also found a
similar hematologic response and levels of circulating interleukin-6 (IL-6) when either
ethanol paradigm achieved intoxication before burn. Furthermore, both i.p. and gavage
resulted in similar blood alcohol concentrations at all time points tested. Overall, our
data show an equal inflammatory response to burn injury when intoxication is achieved
by either i.p. injection or oral gavage, suggesting findings from studies using either
ethanol paradigm are directly comparable.
61
62
Introduction
Ethanol is the most commonly abused substance in the United States and the third
leading cause of preventable death [128], many of which are associated with
unintentional injuries [2]. Binge drinking, defined as reaching a blood alcohol content of
0.08 [129], in particular, is an increasingly prevalent form of intoxication[4] and the
characteristic drinking pattern of trauma patients [5]. As a central nervous system
depressant, alcohol likely plays a causative role in many accidents but the diverse cellular
effects of alcohol and its metabolites can also negatively alter the physiologic response to
the injuries it helped cause [130]. As a small neutral compound capable of freely
traversing lipid membranes, alcohol can influence nearly every cell in the body with
effects dependent on the amount and duration of exposure [131]. Even a single dose of
alcohol in animals has been shown to worsen systemic inflammation after injuries, such
as burns [21,111]. Burns are a devastating injury with a complex natural history and high
association with alcohol [132]. Nearly half of adult burn patients have a positive blood
alcohol concentration (BAC) at the time of admission and this predisposes them to
worsened clinical outcomes compared to patients with similar injuries not under the
influence [120]. Specifically intoxicated patients were found to be twice as likely to
acquire an infection, required more surgical procedures, had longer durations of stay in
the intensive care unit and generated more cost than their non-intoxicated counterparts
[6]. Interestingly, these patients are not typically chronic alcoholics but considered binge
drinkers [133], consistent with the majority of alcohol consumption in the US [4]. With
nearly 450,000 burns requiring medical attention each year in the American healthcare
63
system [11], alcohol greatly contributes to the socioeconomic burden of this destructive
injury as both a causative agent and complicating factor in recovery. Despite the high
prevalence and established consequences of binge intoxication at the time of burn injury,
there are currently few differences in the treatment and management of burn patients with
and without prior alcohol exposure. This may be due in part to the aforementioned
dynamic natural history of burns as well as the complex and duration dependent effects of
alcohol. In order to develop much needed targeted therapies, the effects of intoxication
on the physiologic response to burn injury need to be studied and manipulated under
controlled conditions. To this end, mouse models of binge ethanol exposure and burn
have been in use for nearly 20 years and yielded insightful information into the
mechanisms by which ethanol exacerbates the response to burn. Of note is the finding
that ethanol can potentiate burn-induced damage in the intestine [33,44], liver [22,23] and
lungs [61,134] with increased serum interleukin-6 playing an important role in
inflammation of these organs [48,89]. The majority of these studies administer ethanol
by gavage or i.p. injection to reach a desired BAC in mice. While presumably the
presence and level of intoxication are the most important factors in these models, no one
to date has investigated the impact of the route of ethanol administration in the context of
burn. It is important to establish if results from historical experiments using i.p. injection
and gavage are directly comparable as well as to be aware of any unintentional
confounding factors in future studies. Herein we examine the effects of ethanol, given by
gavage or i.p. injection, on post-burn inflammation and damage in the intestines, liver
and lungs of mice.
64
Materials and Methods
Animals
Male wild-type (C57BL/6) mice were purchased from Jackson Laboratories (Bar
Harbor, ME) and sacrificed at 8-10 weeks old. Mice were housed in sterile micro-isolator
cages under specific pathogen-free conditions in the Loyola University Medical Center
Comparative Medicine facility. All experiments were conducted in accordance with the
Institutional Animal Care and Use Committee.
Murine Model of Ethanol and Burn Injury
A murine model of a single binge ethanol intoxication and burn injury was
employed using either i.p. injection or oral gavage as described previously [107,135].
Briefly, i.p. mice were given a single i.p. dose of 150 μl of 20% (v/v) ethanol solution
(1.12 g/kg) or saline control. Gavaged mice were given a single dose of 300 μl of 10%
(v/v) ethanol solution (1.12 g/kg) or water control. The mice were then anesthetized (100
mg/kg ketamine and 10 mg/kg xylazine), their dorsum shaved, placed in a plastic
template exposing 15% of the total body surface area, and subjected to a scald injury in a
92-95° C water bath or a sham injury in room-temperature water. The scald injury results
in an insensate, full-thickness burn [108]. The mice were then resuscitated with 1.0 ml
saline and allowed to recover on warming pads. All experiments were performed between
8 and 9 am to avoid confounding factors related to circadian rhythms.
Blood Alcohol Concentration (BAC)
Mice were sacrificed at 30 minutes, 1 hour or 4 hours after a single dose of
ethanol (1.12 g/kg) administered by either i.p. injection or gavage. Whole blood was
65
collected via cardiac puncture, incubated at room temperature for 20 minutes and then
centrifuged at 3000 rpm at 4° C for 20 minutes. Serum was isolated and BAC was
measured using the GM7 Micro-Stat Analyzer (Analox, Lunenburg, MA).
Blood and Serum Measurements
At 24 hours post-injury mice were euthanized, blood was collected via cardiac
puncture, an aliquot was placed into a microcapillary tube, and read for a complete blood
count with differential by Hemavet (Drew Scientific, Dallas, TX). The remaining blood
was harvested for serum as described above and stored at -80°C. Serum aliquots were
used to measure IL-6 by enzyme linked immunosorbent assay (ELISA) (BD Biosciences,
Franklin Lakes, NJ) or liver transaminase levels using a DRI-CHEM 7000 (HESKA,
Loveland, CO).
Histopathologic Examination of the Ileum and liver
At 24 hours post-injury mice were euthanized and the ileum, liver, and lungs were
harvested. The ileum was fixed overnight in 10% formalin, embedded in paraffin,
sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). The length of 5
individual villi in 5 fields of view (100X) were measured for a total of 25 measurements
per animal. The average was considered representative of the villus length in the ileum
and demonstrative images are presented herein. The whole liver was removed at the time
of sacrifice, weighed and normalized to total body weight.
Bacterial Translocation
Bacterial translocation was assessed as previously described [29]. Briefly, 3–5
mesenteric lymph nodes per mouse were removed and placed in cold RPMI, and kept on
66
ice. Nodes were separated from connective tissue and homogenized with frosted glass
slides. Homogenates were plated on tryptic soy agar and incubated at 37°C overnight.
Histopathologic Examination of the Lung
The upper right lobe of the lung was inflated with 10% formalin and fixed
overnight as described previously [110], embedded in paraffin, sectioned at 5 μm, and
stained with hematoxylin and eosin (H&E). Photographs were taken in a blinded fashion
of 10 high power fields (400X) per animal and analyzed using the Java-based imaging
program ImageJ (National Institutes of Health, Bethesda, MD). The images were
converted to binary to differentiate lung tissue from air space and then analyzed for the
percent area covered by lung tissue in each field of view as described previously [89].
Neutrophils were counted in a blinded fashion in 10 high power fields (400X).
KC Analysis of Lung Homogenates
The right middle lung lobe was snap-frozen in liquid nitrogen. The tissues were
then homogenized in 1 ml of BioPlex cell lysis buffer according to manufacturer’s
instructions (BioRad, Hercules, CA). The homogenates were filtered and analyzed for
cytokine production using an ELISA for KC (BD Biosciences, Franklin Lakes, NJ). The
results were normalized to total protein using the BioRad protein assay (BioRad,
Hercules, CA).
Statistical Analysis
Statistical comparisons were made between i.p. and gavage animals in the sham
vehicle, sham ethanol, burn vehicle, and burn ethanol treatment groups, resulting in 8
total groups analyzed. One-way analysis of variance was used to determine differences
67
between treatment responses, and Tukey’s post-hoc test once significance was achieved
(p<0.05). Data are reported as mean values ± the standard error of the mean.
Results
Blood Alcohol Concentration is Equal
after I.P. Injection or Gavage
To determine if the route of ethanol administration impacted the kinetics of its
absorption and clearance, the blood alcohol concentration (BAC) of mice was determined
at 30 minutes, 1 hour and 4 hours after a single dose of 1.12 g/kg ethanol by i.p. injection
or gavage. Mice receiving ethanol by i.p. injection were found to have a BAC of 143
mg/dL at 30 minutes, which was reduced 33% by 1 hour and 69% by 4 hours (Figure 17).
Similarly, mice receiving ethanol by gavage demonstrated a BAC of 141mg/dL at 30
minutes, which by 1 hour was decreased by 41% and by 77% at 4 hours (Figure 17). No
significant difference between BAC in mice receiving ethanol via i.p. injection or gavage
at each time point was found, suggesting that equivalent amounts of ethanol absorb into
the bloodstream and are cleared at similar rates.
Intoxication by I.P. Injection or Gavage Increases
Peripheral Blood Granulocytes after Burn
To examine if administration route effected the hematologic response to
intoxication and burn, the number of circulating granulocytes was enumerated by an
automated counter after burn or sham injury when preceded by ethanol given by i.p.
injection or gavage. In gavaged mice, there was a 2-fold increase (p<0.05) in blood
granulocytes in intoxicated burned mice relative to sham injured mice regardless of prior
68
Figure 17. Blood alcohol concentration (BAC). Mice were administered 1.12g/kg
ethanol and the subsequent BAC measured at various time points. Data are presented at
mean values ± SEM. N=6-9 animals per group
intoxication status (Figure 18). Likewise in mice given an i.p. ethanol injection before
burn, a 3-fold increase (p<0.05) in blood granulocytes was found when compared to
sham injured mice with and without prior intoxication (Figure 18). No significant
differences were found between i.p. injected and gavaged mice within treatment groups
suggesting both routes of ethanol administration induce an equal neutrophilic
leukocytosis after burn injury.
Serum IL-6 is Elevated When Intoxication Precedes Burn
Injury Regardless of Administration Route
Circulating IL-6 levels were quantified by ELISA in all treatments groups of i.p.
injected and gavaged mice. Burn injury alone increased the amount of serum IL-6 by
greater than 25-fold above sham injured animals in both i.p. and gavage mice (Figure 19).
When intoxication preceded the burn, a further 3 to 4-fold increase (p<0.05) above burn
69
Figure 18. Circulating blood granulocytes 24 hours after injury. *p<0.05 compared to
Sham groups. Data are presented at mean values ± SEM. N=3-6 animals per group.
*
*
Figure 19. Serum IL-6 at 24 hours after injury. *p<0.05 compared to Sham groups.
Data are presented at mean values ± SEM. N=4-8 animals per group.
70
alone was observed, regardless of the route of ethanol administration (Figure 19).
No significant differences were found between i.p. and gavage mice within treatment
groups suggesting that intoxication before burn injury increases serum IL-6 irrespective
of the ethanol paradigm used.
Villus Blunting is Similar in I.P. and Gavage
Intoxicated Mice After Burn
We previously reported that intoxication by i.p. injection furthers the diminution
of ileal villi after burn [29]. Consistent with our earlier observations, at 24 hours after
burn (Figure 20, E-F), villi in the ileum were shortened in comparison to sham injured
animals regardless of intoxication status (Figure 20, A-D). Furthermore when mice were
intoxicated by i.p. injection (Figure 20, G) or gavage (Figure 20 H), villus blunting was
pronounced beyond burn alone.
The average villus length in the ileum of burn injured mice was blunted by greater
than 20% (p<0.05) compared to sham injured animals regardless of intoxication status or
administration method (Figure 21A). Antecedent intoxication by i.p. injection or gavage
caused a further ~20% reduction (p<0.05) compared to burn alone (Figure 21A),
demonstrating this increased intestinal damage is present to a similar extent whether
intoxication is achieved by i.p. injection or gavage. This villus blunting corresponded to
an increase in bacterial translocation to the mesenteric lymph nodes where intoxication
increased the number of colony forming units by >400-fold over sham animals and 5-fold
over burn alone (Figure 21B). No significant differences between i.p. and gavage mice
71
were found within treatment groups suggesting both routes of ethanol administration
effect intestinal damage after burn injury in a similar manner.
Figure 20. Histologic state of the ileum 24 hours after intoxication and burn injury. The
first row is representative of villi in the ileum of sham injured mice given vehicle by i.p.
injection (A) or gavage (B). The second row show villi from sham injured mice given
ethanol by i.p. injection (C) or gavage (D). The third row villi appear rounded and
widened as seen in burned mice given vehicle by i.p. injection (E) or gavage (F). The
final row demonstrate the villus blunting observed in burn injured mice administered
ethanol by i.p. injection (G) or gavage (H).
72
A
B
Figure 21. Intestinal damage 24 hours after injury. Villus length in the ileum (A) and
bacterial load in per mesenteric lymph node (B) 24 hours after injury. *p<0.05 compared
to Sham groups. Data are presented at mean values ± SEM. N=4-6 animals per group.
I.P. or Gavage Intoxication Equally Exacerbates
Hepatic Damage After Burn
Hepatic damage was measured by levels of serum alanine aminotransferase
(ALT) and aspartate aminotransferase (AST), and by the liver weight to total body weight
ratio. Burn alone increased serum ALT levels by greater than 6-fold compared to sham
injured animals (Figure 22A). This increase was found irrespective of the presence or
absence of ethanol or route of administration before sham injury. When i.p. or gavage
mice were intoxicated at the time of burn however, a greater than 12-fold elevation
(p<0.05) was observed over sham injured animals which corresponded to a ~2-fold
increase (p<0.05) above burn alone (Figure 22A). A similar pattern was found for serum
AST with burn alone causing a greater than 5-fold increase relative to sham injured
groups in both i.p. injected and gavaged mice with and without ethanol (Figure 22B).
73
Intoxication in both i.p. and gavage mice at the time of burn increased serum AST an
additional 2-fold (p<0.05) which is a greater than 20-fold elevation (p<0.05) over sham
injured mice (Figure 22B).
B
A
*
*
*
*
Figure 22. Hepatic damage 24 hours after injury. Serum alanine aminotransferase (ALT)
(A) and serum aspartate aminotransferase (AST) (B) 24 hours after injury. *p<0.05
compared to Sham and Burn Vehicle groups. Data are presented at mean values ± SEM.
N=3-5 animals per group.
Finally, the liver weight to total body weight ratio (LW:BW) was recorded as a
measure of hepatic edema. No significant changes in LW:BW were found between sham
groups regardless of ethanol intoxication or administration route (Figure 23). Similarly,
burn alone did not cause a significant change in LW:BW relative to sham injured mice.
However when mice received ethanol by i.p. injection or gavage before burn, a ~47%
increase (p<0.05) above all other groups was observed. Taken together, the serum
transaminase and LW:BW suggest that ethanol potentiates liver damage after burn injury
irrespective of the route intoxication was achieved.
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*
*
Figure 23. Liver weight to body weight ratio 24 hours after injury. *p<0.05 compared to
Sham and Burn Vehicle groups. Data are presented at mean values ± SEM. N=4-6
animals per group.
I.P. or Gavage Administration of Ethanol Enhances
Alveolar Wall Thickness After Burn
At 24 hours after intoxication and burn injury, there is a marked increase in the
thickness of the alveolar wall and increased cellularity, which is more pronounced than
after burn alone (Figures 24 and 25). The alveolar wall thickness and cellularity was
quantified using imaging software to measure the area of lung tissue in 10 high power
fields per animal which is reported as a percentage of the entire field of view. A
significant increase in tissue area, corresponding to a relative decrease in air space, was
found after burn injury, compared to sham animals (p<0.05). Intoxication increased the
tissue area after burn regardless of how it was achieved (p<0.05), indicating a greater
level of pulmonary congestion.
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Figure 24. Histologic state of the lungs 24 hours after injury. The first row is
representative of the lungs of sham injured mice given vehicle by i.p. injection (A) or
gavage (B). The second row show lungs from sham injured mice given ethanol by i.p.
injection (C) or gavage (D). The third row displays an increase in alveolar wall thickness
as seen in burned mice given vehicle by i.p. injection (E) or gavage (F). The final row
demonstrate the amplified alveolar wall thickness and cellularity in burn injured mice
administered ethanol by i.p. injection (G) or gavage (H).
76
Neutrophil Accumulation and Pulmonary KC Levels are Amplified a
fter I.P. or Gavage Intoxication and Burn
Similar to previous studies [61,110,134], following the combined insult of i.p.
ethanol injection and burn, there was a 20-fold increase in pulmonary neutrophils
compared to sham animals (p<0.05) and a 2-fold increase over burn alone (p<0.05)
(Figure 26A). This neutrophil accumulation in i.p. intoxicated animals corresponded to a
6-fold increase in KC compared to sham animals (p<0.05) and a 2-fold elevation
compared to burn alone (Figure 26B). When an equal amount of ethanol was given by
gavage, similar results were observed with intoxicated burned mice having neutrophil
numbers and KC levels that were 15 and 6-fold above sham animals (p<0.05) and 2.5 and
2-fold above burn alone, respectively (Figure 26). No significant differences between i.p.
and gavage mice within treatment groups were found suggesting that intoxication
enhances post-burn pulmonary neutrophil accumulation and KC regardless of
administration route.
Figure 25. Quantification of pulmonary congestion 24 hours after injury. *p<0.05
compared to Sham and Burn Vehicle groups. #p<0.05 compared to Sham groups. Data
are presented at mean values ± SEM. N=4-6 animals per group.
77
A
B
Figure 26. Pulmonary inflammation after intoxication and burn. Pulmonary neutrophils
in 10 high power (400x) fields of view (A) and pulmonary KC levels (B) 24 hours after
injury. #p<0.05 compared to Sham groups. *p<0.05 compared to Burn Vehicle groups.
Data are presented at mean values ± SEM. N=4-8 animals per group.
Summary
Animal studies using mice offer controlled conditions, manipulatable genomes,
and pharmacologic interventions not available in humans. An important variable is the
level of intoxication achieved before burn and while historically animal studies have
administered known amounts of ethanol by i.p. injection, oral gavage is considered a
more physiologic method of intoxication. We now describe that post burn inflammation
and damage in the ileum, liver and lungs of mice are exacerbated to an equal extent when
preceded by intoxication achieved by i.p. injection or gavage. Furthermore, the
administration route had no impact on the hematologic changes observed when
intoxication precedes burn. Taken together our data suggest that either i.p. injection or
gavage are appropriate for studying the effects of ethanol on post burn inflammation and
response.
CHAPTER 7
DISCUSSION
As a small neutral compound with the ability to cross lipid membranes, alcohol
can affect nearly any cell in the body with varying consequences depending on the
context, amount and duration of exposure [131]. Burns are a devastating and dynamic
injury involving potential complications in every major organ system including cardiac
impairment [136], renal dysfunction [137], intestinal damage [138], hepatic
derangements [14] and pulmonary failure [139]. It is unlikely, given the permeating
effects of both burns and alcohol, that there is a single cell type or molecule solely
responsible for the multi-system sequelae observed in burn patients who were intoxicated
at the time of their injury. Rather, the involvement of several organs, each influencing the
response of others seems plausible and supported by findings reported herein.
The Role of the Intestinal Barrier
Emerging evidence points to a strong relationship between intestinal microbiota
and the liver which is proposed to regulate a myriad of human diseases [49,50,51,52,53].
Independently, burn injury [23] and alcohol [27,53] have also been shown to alter aspects
of the gut-liver axis but the combination of the two insults is relatively unexplored
despite its prevalence in the trauma population [4,5]. We have previously reported that in
mice, antecedent ethanol exposure potentiates post burn intestinal damage [19,29,48],
hepatosteatosis [23,55] and IL-6 production [19,21,48,56], as well as exacerbates
78
79
pulmonary inflammation [19,61,89,134] compared to either intoxication or a burn alone.
In Chapter 3, we demonstrate through the use of two separate approaches that gut-derived
bacteria, or bacterial products, may play a causative yet reversible role in the wellestablished ability of ethanol to worsen post burn systemic inflammation.
The intestinal barrier is normally maintained by multiple types of junction
complexes including the tight junction proteins ZO-1 and occludin. In our current study,
we chose to investigate only ZO-1 localization as a representation of tight junction
integrity but have verified similar results with occludin in this animal model elsewhere
[96,140]. In agreement with these studies, PIK treatment restored intestinal barrier
function after injury (Figure 4A) and we now report the benefits of this treatment on the
hepatic and pulmonary responses to intoxication and burn. The finding that PIK treatment
dampened steatosis development after intoxication and burn (Figure 4) suggests that the
aberrant hepatic response observed in this setting is caused, at least in part, by the
translocation of intestinal bacteria. Furthermore, decreasing intestinal bacterial load
before injury alleviated these parameters to an equal extent. These findings do not rule
out the possible influence of other etiologies of steatosis (see Figure 3), such as oxidative
stress [141] or changes in fat metabolism [14] but are supported by data from autopsies of
burn patients which reveal an association between fatty liver infiltration and bacterial
translocation from the intestines [54]. Similarly, in a hemorrhagic shock model, gut
sterilization of rats was found to confer hepatic protection [142].
80
The degree of steatosis in our intoxicated burned mice paralleled other markers of
hepatic damage as measured by serum AST, ALT and relative size of the liver after
injury (Figures 5A-C). Severe derangement of AST and ALT in burn patients are a
harbinger for complications and death [15] and the liver can increase its size by 225%
after a burn alone [143]. The liver is also the major site of ethanol metabolism and
toxicity which is known to sensitize the liver to gut-derived LPS [74,95,96]. Ethanol
priming of the liver to post burn intestinal LPS [73] may help explain our findings that
intoxication potentiated post burn hepatic damage which was attenuated with either PIK
or antibiotic treatment (Figure 5). The etiology of acute liver injury in the setting of
intoxication and burn is of significant clinical concern as the liver is a central player in
regulating the inflammatory response that is thought to be responsible for the worsened
prognosis [144].
An important player in any inflammatory response, IL-6 is particularly elevated in
thermally injured patients [84,85] and correlates to increased mortality risk after injury
[86]. Moreover we and others have reported that antecedent intoxication significantly
raises IL-6 levels of burn injured mice [19,21,23] and reducing IL-6 attenuates
pulmonary inflammation [89] and gut permeability [48], possibly serving as a link
between the gut, liver and lung in this common clinical scenario. The studies of Chapter 3
demonstrate that hepatic IL-6 production at the mRNA and protein level can be
manipulated through limiting hepatic exposure to intestinal bacteria (Figure 6A&B).
Furthermore, this correlates to IL-6 levels in the serum (Figure 6C) indicating the liver
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could be the source of systemic IL-6 when burn is preceded by ethanol exposure. This
agrees with studies of other trauma models where the liver is considered the source of
systemic IL-6 after injury [88]. Additionally, Li et al [21] found elevated IL-6 in the
plasma, ileum, liver and lungs of mice receiving ethanol and burn injury with the highest
concentration of IL-6 in the liver.
As discussed in Chapter 2, IL-6 production after intoxication and burn in the liver
may be initiated through the interaction between gut-derived LPS and TLR4 on Kupffer
cells. Residing in the sinusoids of the liver, Kupffer cells are tissue-fixed macrophages
that survey the blood for circulating antigen and mobilize an immune response. After
burn there is an increase in circulating LPS [27] which, through binding to TLR4,
stimulates Kupffer cells to express inflammatory genes and produce cytokines such as IL6 [60]. Ethanol exposure alters TLR4 signaling in a variety of ways including changes to
CD14 expression [73], lipid rafts [145], and mitogen-activated protein kinases
(MAPKs)[146,147], which can increase IL-6 production after exposure to LPS (See
Figure 2). Furthermore, the absence of TLR4 in the setting of intoxication and burn
significantly decreases circulating IL-6 [61]. The experiments of Chapter 4 investigate
the role of Kupffer cells in this setting but evidence from other models of trauma support
the idea that Kupffer cells may be the systemic source of IL-6 after injury [88].
Increased serum IL-6 is linked to poor survival in patients with acute respiratory
distress syndrome (ARDS) [148], though whether IL-6 plays a causative role or is simply
a marker of mortality risk remains to be conclusively demonstrated. Likewise, alcohol
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abuse is also associated with a worsened ARDS prognosis [149] and is a common
complication after a thermal injury [150]. ARDS is characterized by inflammation and
edema in the lung parenchyma leading to impaired gas exchange. Similarly, Figure 7A
demonstrates an increase in alveolar wall thickness in the combined injury and is
consistent with previously reported results [19,61,89,122,134]. The degree of reduction in
pulmonary inflammation observed with PIK treatment (Figure 7) matches the level of
reduction found in previous studies of IL-6 deficient mice undergoing the same combined
injury [89], suggesting the pulmonary benefit in PIK-treated animals may be related to
decreased IL-6 levels. The parallel results in antibiotic-treated mice imply the pulmonary
benefits of PIK treatment stem from the limitation of bacterial translocation and not a
direct action of PIK on the lungs.
Kupffer Cell Regulation of the Hepatic Response
After a severe burn injury, hepatic dysfunction and associated hypermetabolism
can persist for years despite clinical intervention [24]. Animal studies suggest the
occurrence of hepatic derangement is dependent on the size of the burn [18]. In a direct
comparison, substantial hepatic derangements were observed in a 40% TBSA burn that
were absent when reduced to 20% [18]. The authors concluded from this work that
increasing the severity of injury does not lead to a simple dose-dependent response but
rather qualitatively different responses, though this may also suggest an injury threshold
beyond which an aberrant hepatic response occurs. Work from our lab and others
demonstrate significant hepatic damage in a mouse model of 15% TBSA burn only when
83
intoxication precedes the injury [19,20,21,22,23]. This may indicate that alcohol lowers
the threshold for an aberrant post burn hepatic response. Many overlapping physiologic
effects of both alcohol and burn converge on Kupffer cell activity (See Figure 2) that may
work in a synergistic fashion to lower this threshold for hepatic damage and
derangement, as supported by the findings contained herein.
We demonstrate that a single intoxication event increases post burn sensitivity of
Kupffer cells to LPS to a greater extent than after a burn alone (Figure 8). This is of
clinical importance as Kupffer cell hyperactivity is known to cause hepatic damage after
many types of injury including hemorrhagic pancreatitis [65], trauma and sepsis [66], as
well as burns [67]. Kupffer cell sensitization to LPS has been shown to occur in a rat
models of 30% TBSA burn [67,113], which agrees with the results of Kupffer cells
isolated from burn-injured animals in Figure 8 and is supported by work with TLR-4
defective animals [69]. Additionally, alcohol, even after a single dose, also increases
Kupffer cell sensitivity to LPS [73], a finding recapitulated in Figure 8 and supported by
work demonstrating the role of Kupffer cells in mediating alcohol-induced liver damage
[151]. While burn injury or alcohol may sensitize Kupffer cells via a variety of
mechanisms including changes in CD14 expression [73], lipid rafts [145], and mitogenactivated protein kinases (MAPKs)[146,147], the functional consequence is increased
TLR4 signaling and cytokine production. Accordingly, we have previously demonstrated
that the absence of TLR4 attenuates systemic IL-6 and remote organ damage 24 hours
after intoxication and burn [61]. However, animal models of systemic inflammation
84
suggest overall outcome may require a balance of maintaining Kupffer cell function
without over-stimulation [64], highlighting the need to understand the underlying
mechanisms of Kupffer cell hyperactivity. Of the many downstream signaling proteins
of TLR4, p38 MAPK may have particular importance in the setting of intoxication and
burn.
A member of the MAPK family, p38 has a well-established role in post burn
remote organ damage [75,76,77,78,79], is altered by alcohol exposure [80] and enhances
TLR4 reactivity following injury [82]. We found increased p38 activity in Kupffer cells
isolated from burn-injured animals (Figure 8A), a finding that has also been reported after
trauma-hemorrhage [152]. In the combined injury, significantly increased Kupffer cell
p38 activation (Figure 8A) and IL-6 production (Figure 8B) was found over either insult
alone, mirroring the degree of damage at the organ level (Figures 9&11). These
observations from isolated Kupffer cells demonstrate that ethanol augments p38
activation in Kupffer cells after burn, which corresponds to heightened IL-6 production.
Furthermore, ex vivo inhibition of p38 (Figure 8A) abrogated LPS-induced IL-6
production (Figure 8B) suggesting that the increased IL-6 after intoxication and burn is
produced in a p38-dependent manner.
In order to further examine the role of Kupffer cell p38 in the hepatic response to
burns preceded by alcohol exposure, mice were antecedently depleted of Kupffer cells
with clodronate liposomes or given a p38 inhibitor after injury. The clodronate dose and
administration route used is a well-established method to achieve relatively selective
85
depletion of Kupffer cells ([114], Figures 12A&B), while leaving other macrophage
populations such as the circulating monocytes (Figure 12C) and alveolar macrophages
(Figures 12D&E) intact. The dosage of p38i used has previously been shown to inhibit
p38 activation in Kupffer cells 24 hours after burn [113] which we confirm through
decreased p38 phosphorylation (Figure 12F) and IL-6 production (Figure 12G) after LPS
stimulation.
Similar to the results of Chapter 3, the absence of Kupffer cells conferred hepatic
protection from intoxication and burn as measured by decreases in serum AST, serum
ALT, and the liver to body weight ratio. It is interesting therefore that global p38
inhibition attenuated hepatic damage to an extent equal to that of antecedent Kupffer cell
depletion, suggesting that p38 specifically within Kupffer cells may be responsible for
the increased hepatic damage. Furthermore, both treatments reduced hepatic damage to
the extent seen after a burn alone, implying that alcohol may potentiate post burn hepatic
damage through Kupffer cell p38 activity. Similarly, steatosis was reduced with either
treatment to the degree of a burn alone, implying a prominent role for Kupffer cell p38 in
hepatic triglyceride accumulation after intoxication and burn. While these findings do
not rule out the possible influence of other etiologies of steatosis (see Figure 3), they are
supported by recent evidence which indicates Kupffer cells can promote steatosis via
increased cytokine production [71].
Chapter 3 demonstrated that the increased hepatic IL-6 production when
intoxication precedes burn is dependent on gut-derived LPS. The present studies
86
demonstrate that hepatic IL-6 production can also be manipulated through p38 inhibition
or Kupffer cell depletion (Figure 10). This may be important as IL-6 at low levels is
beneficial to hepatocyte survival but above a threshold of excessive amount or prolonged
duration can be detrimental [62]. IL-6 levels in the serum (Figure 10B) paralleled hepatic
IL-6 reinforcing that the liver could be the source of systemic IL-6 when burn is preceded
by ethanol exposure.
The degree of reduction in pulmonary inflammation observed with p38i treatment
(Figure 11) matches the level of reduction found in previous studies of IL-6 deficient
mice undergoing the same combined injury [89], suggesting the pulmonary benefit in
p38i-treated animals may be related to decreased IL-6 levels. This matches the studies of
Chapter 3 where decreased IL-6 attenuated neutrophil accumulation in the lung (Figures
6&7). The parallel results in clodronate-treated mice imply the pulmonary benefits of
p38i treatment stem from its effect on Kupffer cells and not a direct action of p38
inhibition in the lungs. While more studies would be needed to confirm the precise
action of Kupffer cell p38 on pulmonary inflammation after intoxication and burn, our
findings are supported by work demonstrating a direct role of Kupffer cells in lung injury
during endotoxic shock [153].
The Influence of Fluid Shifts
Chapters 3 and 4 demonstrate that the gut-liver axis is paramount to the post burn
response and can be manipulated through blocking the interaction of gut-derived LPS and
Kupffer cells after injury. With this in mind we sought to examine possible causes for
87
the increased intestinal damage and permeability when intoxication precedes burn. Fullthickness burns can induce excessive vascular permeability both at the site of injury and
in remote organs, leading to detrimental hypodynamic changes in circulation [26]. Burn
patients who were intoxicated at the time of their injury require greater amounts of fluid
resuscitation [6], perhaps indicating a greater degree of fluid extravasation. This is
supported by findings contained in Chapter 5 where mice given ethanol and burn injury
have increased hematocrit, creatinine, and BUN compared to mice given either ethanol or
burn alone (Figures 13B-D). Interestingly, the combination of intoxication and burn led
to weight retention 24 hours after injury (Figure 13A). This apparent retention of fluid
despite increased dehydration suggests that intoxication at the time of a burn causes a
shift in fluid compartments that may exacerbate end organ ischemia and damage in the
intestines (Figure 14A). Catecholamine-mediated vasoconstriction of the splanchnic
vasculature makes the intestines particularly susceptible to hypovolemia [25], made
worse by third spacing of fluid as evidenced by edema in the liver (Figure 14B) and lungs
(Figure 14C). One mechanism by which systemic capillary leakiness in this setting may
ensue is through the action of the vasoactive agent, bradykinin.
Bradykinin is a potent vasoactive agent produced systemically after a burn with
effects of vasodilation and vascular permeability [26]. The effects of bradykinin are
mediated by two cellular receptors classified as B1 and B2. B1 receptors are present only
at low levels in healthy tissues while B2 receptors are constitutively expressed in most
issues. Acute inflammatory responses such edema formation are mediated through B2
88
receptors whereas chronic inflammation is associated with B1 activity [154]. Accordingly,
most experimental and clinical studies examining bradykinin antagonism in an acute
inflammatory setting have focused on B2 receptor blockade. HOE140 is a potent,
selective, and long-acting B2 receptor antagonist and has been used to treat experimental
sepsis [155], acute pancreatitis [156], and local inflammation [157] or microvascular
changes [158] after thermal injury though with varying degrees of success. B2 receptor
antagonism may be particularly suited to treat the common clinical scenario of
intoxication and burn injury because burn injuries cause systemic bradykinin production
[26], while its action is known to be potentiated in the presence of alcohol [127], perhaps
affording bradykinin undue influence on vascular permeability after this combined injury.
In agreement with this, we found that B2 blockade early after intoxication and burn
restores fluid balance (Figure 15) and attenuates end organ damage (Figure 16). This
highlights the early important role of bradykinin after burn with antecedent alcohol
exposure and also suggests third spacing is at least partially responsible for the increased
remote organ damage in this setting.
The pathophysiology of both binge intoxication and burn injury converge on
bradykinin signaling and could explain numerous observations in animal models and
patients as described above. The exacerbated injury in the intestines, liver, and lungs of
mice when ethanol precedes burn injury is well documented and agrees with and expands
upon the findings of Figure 14. Therapeutic interventions in animals to date have focused
on mitigating events downstream of the initial injury, such as manipulating levels of IL-6
89
[89], IL-18 [159], intracellular adhesion molecule-1 (ICAM-1) [160], and Toll-like
receptor 4 (TLR4) [61] While some of these interventions have proven efficacious in
providing protection in various organs, as a whole they focus on restoring aberrations that
follow the combination of intoxication and burn instead of addressing the underlying
cause of the exacerbated injury.
Ethanol Administration in our Animal Model
In order to more accurately represent physiologic drinking in our animal model,
the route of ethanol administration was changed from being given via i.p. injection
(Chapter 3) to oral gavage (Chapters 4&5). The studies in Chapter 6 indicate that in the
context of burn injury in mice, intoxication at an equivalent BAC and duration
exacerbates organ inflammation and damage to a similar extent whether given by oral
gavage or i.p. injection. Equal BAC’s at the time of injury resulting in comparable
amounts of organ damage is consistent with findings that suggest ethanol acts through
worsening ischemic damage [161], altering cytokine networks [162,163], and impairing
immune responses [144] after burn injury and perhaps not through interactions at the site
of absorption. The near infinite water solubility of ethanol allows for a quick distribution
throughout the blood and we observed peak BACs near 30 minutes when ethanol was
administered by oral gavage or i.p. injection. As seen in Figure 17, identical doses of
ethanol by both paradigms resulted in nearly the same BAC at 30 minutes, 1 hour and 4
hours after administration. The absorption time and BAC of the i.p. mice agree with our
previously published studies [23,29,163] but the equivalency of BAC profiles between
90
gavage and i.p. injection are in contrast with work by Livy et al [164] who concluded that
in mice, ethanol given by gavage resulted in a lower BAC than an equivalent amount of
ethanol given by i.p. injection. They proposed this discrepancy may be due to
metabolism by gastric alcohol dehydrogenase, which only occurs when ethanol traverses
the gastrointestinal tract. While the reasons for the contrasting results are unclear, several
possibilities, including differences in ethanol amount (3.8g/kg vs 1.12g/kg), the volume
of ethanol administered (up to 0.41 ml vs 0.15 ml (i.p.)), and discrepancy between the
vehicle used in i.p. injections (water vs saline) may have been contributing factors.
Nevertheless, the mice used in our studies, which were given equal doses of ethanol by
gavage and i.p. injection, demonstrated equivalent BAC profiles and an over exuberant
inflammatory response after burn. A further discussion regarding considerations of i.p.
and gavage ethanol administration can be found elsewhere as reviewed by D’Souza ElGuindy et. al. [165].
A neutrophilic leukocytosis is seen in a variety of illnesses and conditions and is
widely regarded an indicator of infection or inflammation. Trauma can also induce a
leukocytosis where it is considered an acute phase marker and is clinically associated
with increased morbidity and mortality risk [166]. We observed, in Figure 18, that
intoxication by either paradigm induced a similar granulocytic leukocytosis at 24 hours
after burn. The sequestration of these circulating neutrophils in end organs after injury is
proposed as a major mechanism in the pathogenesis of multiple organ failure [167]. We
and others have shown that intoxication at the time of burn leads to increased neutrophil
91
infiltration into the gut, liver and lungs of mice within 24 hours [21,111,168].
Furthermore, prevention of neutrophil transmigration using ICAM knockout mice in this
setting decreased pulmonary inflammation [134], highlighting the important role of
neutrophil infiltration in this setting.
Circulating neutrophils migrate from the blood into tissues along a density
gradient of chemoattractants, which in the mouse include KC. In mice, a burn injury
increases pulmonary KC and ethanol has been shown to amplify this accumulation both
in the absence [61,89,134] and presence of an intratracheal infection with Pseudomonas
aeruginosa [122,163]. We observed that both ethanol paradigms increase pulmonary KC
equally after burn (Figure 26B) and this corresponded to increased neutrophil numbers in
the lung (Figure 26A). The leukocytosis after intoxication and burn, together with an
increase in neutrophil chemoattractants, likely plays a key role in the subsequent
pulmonary inflammation and appears to be independent of the method of ethanol
administration.
As discussed above elevated levels of circulating IL-6 also correlate with
mortality risk in trauma patients [169] and are further increased when intoxication
precedes burn injury [23,56,131]. We confirm our previous findings that burn alone
increases serum IL-6 levels in mice and intoxication at the time of injury raises
circulating IL-6 even further. We now report that this amplified IL-6 level when
intoxication precedes burn injury is not affected by the route of ethanol administration
(Figure 19).
92
We report in Chapter 6 that gavage or i.p. intoxication potentiated post burn
intestinal damage as demonstrated by histology (Figure 20) and villus length (Figure
21A). Furthermore, intestinal damage corresponded to an increase in bacterial
translocation (Figure 21B) and hepatic damage as assessed by serum transaminase levels
(Figure 22) and hepatic weight (Figure 23). These findings were independent of the
ethanol administration route in our model and support the ability to directly compare
results between studies using either administration route.
When examined by histology (Figure 24), the lungs of mice from both gavage and
i.p. paradigms appear congested relative to all other treatment groups. When the amount
of tissue relative to air space was quantified (Figure 25), the alveolar wall thickening and
increased cellularity seen visually was found to be increased after burn and further
increased with prior intoxication. This finding agrees with our previously reported work
with i.p. injected mice [89] and is unaffected by the route of administration.
Final Conclusions
The increasing prevalence of binge drinking and its high association with
traumatic injury warrant further investigation into the mechanisms underlying the
worsened clinical outcomes associated with this combined injury. The existing literature
reviewed in Chapter 2 suggests that the liver is at the center of the post burn systemic
response with crosstalk between the intestinal microbiome and the liver playing an
important role after injury. There is a paucity in the literature, however, regarding the
role of the gut-liver axis in the common clinical scenario of burns preceded by alcohol
93
intoxication. Previous studies in our laboratory demonstrated intoxication potentiates
post burn hepatic damage and IL-6 production in a dose-dependent manner. In Chapter 3
we show through two separate approaches that interruption of gut-liver crosstalk, through
restoration of intestinal barrier function or prophylactic sterilization of the gut, attenuates
hepatic damage and IL-6 production after the combined injury, highlighting the role of
intestinal damage and subsequent bacterial translocation. In Chapter 4 we demonstrate,
through antecedent Kupffer cell depletion, that Kupffer cells play a critical role in
mediating the injurious hepatic response to intoxication and burn. Furthermore we show
that alcohol increases post burn Kupffer cell sensitivity to LPS through increased p38
MAPK signaling, providing a potential mechanism for the increased hepatic and systemic
levels of IL-6 found in the combined injury. Accordingly, global p38 inhibition also
attenuated hepatic damage and IL-6 production in alcohol-exposed and burn injured
Consequence of
intoxication and
burn
Intestinal
permeability
Experimental
Intervention
Restoration of tight
junction complexes
Mechanism of
Intervention
Post injury effect
↓ LPS delivery to
liver
↓ Hepatic damage
Antibiotics
p38 inhibition
Hepatic damage
Depletion of
Kupffer cells
Fluid extravasation
Bradykinin
antagonism
↓ Kupffer cell
hyperactivity
↓ Pulmonary
inflammation
↓IL-6
↓ intestinal damage
Table 1. Interventions attempted in the setting of intoxication and burn.
94
mice. Taken together, the studies of Chapter 4 suggest that specifically targeting Kupffer
cell p38 in the setting of intoxication and burn may have therapeutic benefit. This was
supported by the decreased pulmonary inflammation associated with lowered IL-6 levels
in all experimental interventions used (Table 1). In Chapter 5 we found that alcohol
increased intestinal damage through potentiating bradykinin signaling, leading to greater
fluid extravasation from the vascular compartment. The subsequent intestinal barrier
breakdown may be the inciting event that alters the gut-liver axis observed in Chapter 3
(Figure 27).
Figure 27: Overlapping responses to burn injury and alcohol exposure alter the gut-liver
axis. interleukin-6 (IL-6), Kupffer cells (KC), lipopolysaccharide (LPS), tumor necrosis
factor (TNF)
95
In summation, the studies contained herein reveal that the gut-liver axis drives
pulmonary inflammation after intoxication and burn injury. We demonstrate multiple
mechanisms by which alcohol modulates the post burn hepatic response and provide
preliminary evidence for potential therapeutic targets for this common clinical scenario.
APPENDIX
DETAILED METHODS
[96]
[97]
Intoxication and Burn Injury Protocol
Materials
Hot plate
Water basins
Animal clippers with #40 surgical clip comb
Thermometer
1cc syringes with 27 gauge needle
3cc syringes with 20 gauge oral gavage needle
0.9% normal saline (sterile)
Burn injury template (check for proper size according to weight of mice)
Ketamine
Xylazine
Ethanol
Procedure
1. Fill two water basins with deionized water and heat one until 92-95°C, leaving the
other at room temperature.
2. Tail mark and weigh the mice.
3. Gavage mice with ethanol at the desired dose with 150uL of ethanol mixed in water
or water alone. Allow 2-3 minutes between cages so there is time to shave and burn
the mice at their scheduled time.
[98]
4. Thirty (30) min. after ethanol administration, anesthetize the mice by injecting a
mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) in sterile saline by i.p.
injection.
5.
As soon as they are asleep (approx. 5 min. after anesthesia injection), thoroughly
shave the backs of the mice (Oster animal clippers with #40 comb).
6. One at a time, place animals in appropriate burning template to give a 14-16% total
body surface area scald injury according to their weight (see chart in procedure
room), and proceed with the burn injury (or sham – dunking mice in room temp
water). Make sure the shaved part of the back is “sealed” on the burning template by
gently pressing the mouse down into the template.
7. Hold the template containing the animal in the water for 7 seconds. Quickly blot the
mouse’s back on paper towels. Removing as much hot water as possible from the
animal prevents further scalding. Put the mouse back in a clean cage.
8.
As soon as possible after the injury, resuscitate each mouse with 1.0 ml of 0.9%
sterile saline (i.p.), pre-warmed in 37C water bath, or on warming pads.
9. Keep cages on heating pads for the next 3-4 hours.to help the mice recover from
procedure. Mice can be returned to the mouse room once they are walking and fairly
alert.
10. Place some food pellets on the bottom of each cage; the mice can’t stretch their necks
as easily to reach for food after burn injury. Replace water bottles.
[99]
Kupffer Cell Isolation
Materials
100µm mesh filters
50mL conical tubes
gentleMACS Dissociator
gentleMACS C Tubes
Krebs-Ringer-Buffer (KRB): 154mM NaCl, 5.6mM KCl, 5.5mM Glucose, 20.1mM
HEPES, 25mM NaHCO3, adust to pH 7.4 with NaOH.
PEB buffer: 0.5% bovine serum albumin and 2mM EDTA in phosphate-buffered saline.
Diultue 1:20 in autoMACS Rinising Solution.
DNase I solution: 30,000 U/mL DNase I in KRB
Collagenase IV solution: 5,000 Collagenase Digestion Units (CDU)/mL in KRB
0.5M CaCl2 solution
0.2M MgCl2 solution
Red Blood Cell Lysis Solution
Centrifuge
Procedure
1. For each sample, mix 4.4mL KRB into a gentleMACS C tube and add 20uL CaCl2
solution, 50ul MgCl2 solution, 500uL Collagenase IV solution and 25uL DNase I
solution. Incubate at 37°C for 30 minutes.
2. Remove whole liver and carefully separate the gallbladder. Mince liver tissue with
razor blades.
[100]
3. Incubate minced tissue in a prewarmed collagenase IV solution for 30 minutes at
37°C.
4. Run C tube on gentleMACS Dissociator program “m_liver_01”.
5. Incubate samples for 30 minutes at 37°C under slow continuous rotation.
6. Run C tube on gentleMACS Dissociator program “m_liver_02”.
7. Pass contents of the C tube through a 100µm mesh filter that has been rinsed with
PEB buffer. Collect in a 50uL conical tube. Rinse C tube with 5mL PEB buffer and
pass through filter again. Wash the filter with 10 mL of PEB buffer collecting all
rinses in the 50mL tube. Add 10mL more of PEB buffer.
8. Centrifuge suspension at 17xg for 4 minutes at 4°C to remove hepatocytes.
9. Collect supernatant and transfer to new 50mL tube that has been prerinsed with PEB
buffer.
10. Centrifuge suspension at 300xg for 10 minutes at 4°C. Aspirate supernatant
11. Resuspend cell pellet in 1mL PEB buffer and add 10mL Red Blood Cell Lysis
Solution.
12. Incubate at room tempterature for 5 minutes.
13. Add 30mL of PEB buffer and centrifuge at 300xg for 10 minutes at 4°C. Aspirate
supernatant.
14. Resuspend cell pellet in 2mL of PEB buffer and adjust volume with PEB buffer to a
final volume of 30mL.
15. Centrifuge at 300xg for 10 minutes at 4°C. Aspirate supernatant.
16. Resuspend in 90uL of PEB buffer per 107 cells and add 50uL of CD11b MicroBeads.
[101]
17. Mix well and incubate for 15 minutes at 4°C.
18. Wash cells by adding 10mL of PEB buffer and centrifuge at 300xg for 10 minutes at
4°C. Aspirate supernatant.
19. Resuspend in 500uL of PEB buffer.
20. Prime autoMACS and exchange necessary reagents or magnetic columns.
21. Place tube containing magnetically labeled cells in the autoMACS separator and run
on “Possel” for positive selection.
Tail Vein Injection
Materials
Hollow Styrofoam housing with a small hole in the side
27 gauge needle with syringe
Heating Pad
Ethanol (70% v/v)
Procedure
1. Place animal cage on warming pad.
2. Bend needle to a 120° angle with the syringe.
3. Place mouse under Styrofoam housing, pulling tail through the side hole.
4. Wet the tail with ethanol and rub proximal portion of the tail until a lateral tail vein is
visible.
5. Insert needle into lateral vein and slowly inject drug or saline control.
[102]
ImageJ Analysis of Lung Tissue Histology
Materials
Lung H&E slides
Microscope with camera
ImageJ program
Procedure
1. Save 10 high power images per animal as jpeg files in a separate folder for each
animal.
2. Import folder as an “Image Sequence” in the ImageJ program.
3. Convert to 8-bit and adjust contrast and threshold if necessary. Must apply the same
settings to all sets of images.
4. Convert to binary.
5. Set measurements to be captured with threshold as area fraction.
6. Select “measure” and record.
Mesenteric Lymph Node Plating for Bacterial Translocation
Materials
1.5 mL tubes
RPMI with 5% FBS
Frosted glass slides
Small petri dishes
Tryptic soy agar plates
Cell spreaders
[103]
Procedure
1. Collect lymph nodes in a 1.5 mL tube with 0.5 mL RPMI (without antibiotic and with
5% FBS), add RPMI sterilely to tubes on day of sac, keep on ice.
2. Separate lymph nodes from connective tissue and transfer to a new 1.5 mL tube
containing 0.5 mL RPMI. (Lymph nodes should sink in RPMI.)
3. Transfer nodes (cut tip from a 1 mL pipette tip to allow transfer of nodes and RPMI)
to the frosted side of one glass slide. Homogenize by crushing lymph nodes between
the frosted sides of 2 glass slides over a small petri dish to collect the homogenate.
Wash slides with 200 uL of RPMI and transfer collected homogenate from small petri
dish to 1.5 mL epi tube.
4. Vortex homogenates. Pipette 100 uL of homogenate onto a Tryptic Soy Agar (TSA)
plate. Plate in triplicate.
5. Leave plates overnight in 37C incubator.
6. Count colonies at 24 hour.
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VITA
Michael M Chen attended the University of Wisconsin – Madison for his
undergraduate where he obtained a Bachelor’s of Science in Medical Microbiology and
Immunology. After graduation Michael spent four years at Kimberly-Clark Corporation
as a scientist and product developer. In 2010 Michael matriculated into the Loyola
University Chicago Stritch School of Medicine MD/PhD program. After completing two
years of medical school, he began his graduate education in the Integrative Cell Biology
Program under the mentorship of Dr. Elizabeth J Kovacs.
Michael’s dissertation work on alcohol’s modulation of the post burn hepatic
response was supported by a predoctoral Ruth L. Kirchstein National Research Service
Award from the National Institute of Health and a Research Seed Grant from the
American Medical Association Foundation. Upon completion of his graduate studies,
Michael will return to finish medical school.
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