Tài liệu Báo cáo khoa học: Bacterial-induced hepoxilin A3 secretion as a pro-inflammatory mediator pptx

MINIREVIEW
Bacterial-induced hepoxilin A
3
secretion as
a pro-inflammatory mediator
Beth A. McCormick
Department of Pediatric Gastroenterology, Massachusetts General Hospital, and Department of Microbiology and Molecular Genetics,
Harvard Medical School, Charlestown, MA, USA
Introduction
Recent studies suggest that 8S ⁄ R-hydroxy-11,12-
epoxyeicosa-5Z,9E,14Z-trienoic acid (hepoxilin A
3
;
HXA
3
) plays a central role in the directed migration of
neutrophils across mucosal surfaces infected with
pathogenic bacteria. This review will discuss recent
advances made in understanding the complex molecu-
lar events that orchestrate the directional movement of
neutrophils across the mucosal surface during bacterial
infection of the intestinal tract and lung and will, in
particular, emphasize the important role played by the
eicosanoid HXA
3
.
HXA
3
is secreted from epithelial cells
during bacterial infection and is a
potent neutrophil chemoattractant

Bacterial pathogens continually confront epithelial bar-
riers of the body, such as those of the gastrointestinal,
respiratory and reproductive tracts. Although mucosal
surfaces are generally impermeable to most foreign
entities, many microorganisms have developed sophis-
ticated strategies to breach or alter this barrier. In gen-
eral, microbial pathogens have evolved the capacity to
engage their host cells in very complex interactions
commonly involving the exchange of biochemical
Keywords
arachidonic acid; chemotaxis; eicosanoid;
hepoxolin A
3
; inflammation; intestine; lung;
neutrophils; Pseudomonas aeruginosa;
Salmonella typhimurium
Correspondence
B. A. McCormick, Department of Pediatric
Gastroenterology, Massachusetts General
Hospital, Harvard Medical School, CNY 114
16th Street (114–3503), Charlestown,
MA 02129, USA
Fax: +1 617 7264172
Tel: +1 617 7264168
E-mail:
(Received 13 Oct 2006, accepted 23 May
2007)
doi:10.1111/j.1742-4658.2007.05911.x
Bacterial infections at epithelial surfaces, such as those that line the gut
and the lung, stimulate the migration of neutrophils through the co-ordi-

nated actions of chemoattractants secreted from pathogen-stimulated epi-
thelial cells. One such factor involved in attracting polymorphonuclear
leukocytes across the epithelium and into the lumen has until recently
remained elusive. In 2004, we identified the eicosanoid, hepoxilin A
3
,tobe
selectively secreted from the apical surface of human intestinal or lung epi-
thelial cells stimulated with Salmonella enterica serotype Typhimurium or
Pseudomonas aeruginosa, respectively. In this role, the function of hepoxilin
A
3
is to guide neutrophils, via the establishment of a gradient, across the
epithelial tight junction complex. Interestingly, interruption of the synthetic
pathway of hepoxilin A
3
blocks the apical release of hepoxilin A
3
in vitro
and the transmigration of neutrophils induced by S. typhimurium both in
in vitro and in vivo models of inflammation. Such results have led to the
discovery of a completely novel pathway that is not only critical for
responses to bacterial pathogens but also has broad implications for
inflammatory responses affecting mucosal surfaces in general. Thus, the
objective of this review was to highlight the recent findings that implicate
hepoxilin A
3
as a key regulator of mucosal inflammation.
Abbreviations
AA, arachidonic acid; HpETE, hydroperoxy-eicosatetraenoic acid; HXA
3

,8S ⁄ R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid
(hepoxilin A
3
); IL-8, interleukin-8; LOX, lipoxygenase; PKC, protein kinase C; PLA
2
, phospholipase A
2
.
FEBS Journal 274 (2007) 3513–3518 ª 2007 The Author Journal compilation ª 2007 FEBS 3513
signals, the net result of which is often the triggering
of a host pro-inflammatory response. At the forefront
of this inflammatory response is the infiltration of neu-
trophils to the site of bacterial insult. Neutrophils rep-
resent a class of crucial white cells needed to defend
the host from such pathogenic injury, and thus the
accumulation of neutrophils at inflamed sites repre-
sents a characteristic feature of the innate host
response. However, the mechanisms by which neu-
trophils eradicate offending bacteria are nonspecific
and can lead to tissue damage, which, if excessive, con-
tributes to the pathology of the disease.
To reach inflamed sites, neutrophils traverse various
barriers, including the endothelium, basement mem-
brane (intestine) ⁄ interstitium (lung) and epithelium, in
response to localized inflammatory mediators. Overall,
such directed migration of neutrophils involves the
integrated actions of cytokines, adhesion molecules
with specificity for specific ligands, as well as highly
timed and compartmentalized secretion of various neu-
trophil-specific chemokines. Research from my laborat-

ory has begun to disclose the molecular and cellular
events underlying the directed infiltration of neutrophils
across epithelial mucosal surfaces during states of bac-
terial infection. This work has led to the current para-
digm that intestinal epithelial cells respond to luminal
pathogens, such as Salmonella typhimurium, by releas-
ing distinctive pro-inflammatory neutrophil chemo-
attractants that sequentially orchestrate neutrophil
movement across the intestinal epithelium [1–4]. More
specifically, S. typhimurium–intestinal epithelial cell
interactions induce the epithelial release of the potent
neutrophil chemokine, interleukin-8 (IL-8). Such baso-
lateral IL-8 release imprints subepithelial matrices with
long-lived haptotactic gradients that serve to guide neu-
trophils through the lamina propria to a subepithelial
position [2]. However, basolateral IL-8 release is insuffi-
cient to induce the migration of neutrophils across the
intestinal epithelium, suggesting that the production of
other inflammatory mediators, whose release would
probably be polarized apically, are important for the
execution of this step in the inflammatory pathway
[1,2]. In support of this contention, Kucharzik et al.
recently developed a double transgenic mouse model
with the ability to induce human IL-8 expression
restricted to the intestinal epithelium [5]. The results
from this transgenic model showed that although acute
induction of IL-8 in the intestinal epithelium is suffi-
cient to trigger neutrophil recruitment to the lamina
propria, additional signals are required for neutrophil
transepithelial migration and mucosal tissue injury.

Owing to the restrictive actions of the intestinal epi-
thelial tight junctions present at the neck of adjacent
epithelial cells, a distinct apical chemotactic factor
would be required for the continued migration of neu-
trophils across the epithelial tight junction. We recently
discovered that neutrophil transit through the epithelial
monolayer to the luminal surface is directed by the ap-
ically released eicosanoid, HXA
3
[4]. HXA
3
is a hyroxy
epoxide derivative formed from 12S-hydroperoxyei-
cosa-5Z,8Z,10E,14Z-tetraenoic acid (12S-HpETE), the
primary product of arachidonic acid (AA) formed by
12S-lipoxygenase (12S-LOX). Hepoxilins are documen-
ted to possess a wide range of biological activities, with
the A
3
form having been shown to potentiate glucose-
dependent insulin secretion [6], open S-type K
+
chan-
nels in Aplysia [7], modulate synaptic neurotransmission
in rat hippocampus [8], increase vascular permeability in
rat skin [9] and induce chemotaxis of neutrophils at con-
centrations as low as 30–40 nm [10]. Our findings repre-
sent the first demonstration that HXA
3
can be secreted

from epithelial cells, and that such secretion is regulated
by conditions that contribute to inflammation [4].
Neutrophil movement induced by HXA
3
HXA
3
directly stimulates neutrophils via a pertussis
toxin-sensitive receptor and elicits a Ca
2+
signal [11].
While these features are shared by most other chemo-
kines, analysis of HXA
3
-elicited neutrophil activation
reveals that, unlike other lipid- or peptide-based chemo-
attractants, HXA
3
, even at saturating concentrations,
elicits chemotactic activity in the absence of stimula-
tion of superoxide production and ⁄ or release of pri-
mary and ⁄ or secondary granules [3]. Thus, HXA
3
appears to function as a ‘pure’ neutrophil chemo-
attractant. Induction of polarized movement by neu-
trophils across the tight junction in response to HXA
3
is presumed to be achieved through its actions as a
Ca
2+
signaling molecule, and our earlier report of

intracellular Ca
2+
events following HXA
3
administra-
tion to isolated human neutrophils are consistent with
this hypothesis [3]. Most recent studies indicate that
the Ca
2+
signaling induced by HXA
3
appears to occur
through the activation of an intracellular receptor [12].
A previous elegant study by Mills et al. showed that
HXA
3
induces a reorganization of Ca
2+
within human
neutrophils from the endoplasmic reticulum into mito-
chondria [13]. In fact, HXA
3
has been shown to inhibit
subsequent Ca
2+
signaling events in cells where Ca
2+
signaling is normally induced by fMet-Leu-Phe, plate-
let-activating factor and leukotriene B
4

[14]. Further-
more, the binding of HXA
3
to a receptor in human
neutrophils shows clear specificity for this eicosanoid
compared with other compounds [15] in a manner sim-
ilar to our previously reported observations [4].
Bacterial-induced hepoxilin A
3
secretion B. A. McCormick
3514 FEBS Journal 274 (2007) 3513–3518 ª 2007 The Author Journal compilation ª 2007 FEBS
Functional consequences of HXA
3
release: the role of AA metabolism
Intestinal inflammation
The biological capacity of 12-LOX and its enzymatic
products, such as HXA
3
, is underappreciated com-
pared with the well-documented functional roles of
5-LOX and 15-LOX products, such as leukotriene B
4
and lipoxins, respectively. Nevertheless, key pieces of
work have not only demonstrated the formation of
hepoxilins through the 12-LOX pathway [10], but have
also uncovered the intriguing observation that epithe-
lial 12-LOX can be regulated at sites of mucosal
inflammation. Shannon et al. noticed that in the
healthy colonic mucosal epithelium, cells do not
express 12-LOX, whereas in tissue from patients with

inflammatory bowel disease, the colonic tissue is not
only actively involved with the disease, but also expres-
ses 12-LOX in mucosal epithelial cells and displays an
increase in 12-LOX enzymatic activity [16]. This study
was the first to demonstrate that 12-LOX participates
in colonic epithelial function. It also provides the first
in situ evidence for a selective increase in epithelial
12-LOX in inflammatory disease. Additionally, our
recent findings further demonstrate that inhibition of
the 12-LOX pathway, which is required for the synthe-
sis of HXA
3
, dramatically reduces neutrophil-mediated
tissue trauma associated with enteric infection [4].
Although these collective observations establish the
12-LOX pathway as yet another avenue for AA meta-
bolism involved in the events underlying inflammation,
such observations further underscore an emerging con-
cept suggesting that modulation of the 12-LOX path-
way during intestinal inflammation may be unique to
polarized epithelia and involved in host defense. In the
particular case of HXA
3
, its stimulated production and
release from the apical surface of infected intestinal
epithelial cells provides an unprecedented pathway of
regulated actions by a chemoattractant and, in addi-
tion, identifies a new scheme in innate immune
responses crucial for mediating neutrophil movement
through epithelial surfaces. Thus, while the epithelium

probably evolved to generate significant levels of
HXA
3
in response to colonization by pathogens, it is
certainly possible that HXA
3
generation is dysregulat-
ed under conditions such as inflammatory bowel dis-
ease because 12-LOX activity is induced at active sites
of this disease.
Because HXA
3
may play an important step underly-
ing the pathophysiology of inflammatory diseases, such
as inflammatory bowel disease, the investigation of
human 12-LOX genes at mucosal surfaces, and their
involvement with HXA
3
production, becomes an
important area of study. As this is an area of research
truly at its embryonic stage, at present, one can only
speculate as to the 12-LOX gene(s) responsible for the
synthesis of HXA
3
at mucosal surfaces. There are at
least four 12-LOX isoforms expressed in human tissue
[17,18]. These include platelet-type 12-LOX (p12-
LOX), epidermal-type 12-LOX (e12-LOX), 12R-LOX,
and 12 ⁄ 15-LOX (human 15-LOX-1). Of these four,
only three appear to be functional 12-LOXs [18];

although the human e12-lox transcript is expressed in
skin and hair follicles, it has been reported to be a
pseudogene, which lacks function [18], making it a less
likely candidate for the synthesis of HXA
3
. Platelet-
type 12-LOX is expressed in multiple tissues aside
from platelets, and can also be regulated at the
transcriptional level [17,18]. The enzymatic expression
of 12R-LOX forms 12R-hydroperoxy-eicosatetraenoic
acid (12R-HpETE) from AA with high specificity.
However, human 12R-LOX has very limited tissue dis-
tribution and, to date, only normal and psoriatic
human skin and tonsils have been found to express the
enzyme and convert exogenous AA to 12R-HETE [19].
Lastly, 15-LOX-1 produces primarily 15-HpETE, but
can also produce 12-HpETE [20,21]. Consequently,
this enzyme has been referred to as 12⁄ 15-LOX and
displays high homology (86.3%) at the protein level to
bovine leukocyte type 12-LOX [22]. Although the pro-
duction of 12-HpETE is a side reaction of 15-LOX-1,
it represents an intriguing candidate for the involve-
ment in HXA
3
production considering its expression in
both intestinal and airway epithelial cells [23].
Potential mechanisms underlying HXA
3
release
Identification of a factor such as HXA

3
, which is
responsible for the transmigration of neutrophils
across the mucosal barrier for entry into the intestinal
lumen, has addressed an important question of epithe-
lial pathobiology. Studies exploring the mechanism
underlying the release of HXA
3
during infection with
S. typhimurium revealed the involvement of the
S. typhimurium type III secreted effector protein, SipA
[24]. The Salmonella effector protein, SipA, promotes
a lipid signal transduction cascade that recruits an
ADP-ribosylation factor 6 guanine nucleotide exchange
factor (such as ARNO) to the apical plasma mem-
brane. ARNO facilitates ADP-ribosylation factor 6
activation at the apical membrane, which in turn
stimulates phospholipase D recruitment to and activity
at this site. The phospholipase D product, phosphati-
dic acid, is metabolized by a phosphohydrolase into
B. A. McCormick Bacterial-induced hepoxilin A
3
secretion
FEBS Journal 274 (2007) 3513–3518 ª 2007 The Author Journal compilation ª 2007 FEBS 3515
diacylglycerol, which recruits cytosolic protein kin-
ase C (PKC)-alpha to the apical membrane. Through
a process that is less understood, activated PKC-alpha
phosphorylates downstream targets that are respon-
sible for the production and apical release of HXA
3

,
which drives transepithelial neutrophil movement [25]
(Fig. 1).
Although immune cells recruited in response to
S. typhimurium, especially neutrophils, are thought to
be responsible for the clinical manifestations of this
infection, they probably play an important role in host
defense because nonimmunocompromised hosts gener-
ally clear this infection without medicinal intervention
(beyond hydration). Given that apically directed
migration of neutrophils is, by itself, thought to
contribute to epithelial cell dysfunction in a host of
mucosal diseases (i.e. cystic fibrosis and chronic
obstructive pulmonary disease of the lung, cirrhosis of
the skin, and urinary tract infections) [26], it is
conceivable that HXA
3
is produced by epithelial cells
at other mucosal surfaces. Indeed, we have shown that
lung epithelial cells produce HXA
3
in response to
Pseudomonas aeruginosa infection and HXA
3
, in turn,
appears to mediate neutrophils transmigration across
airway epithelial cells [27].
Lung inflammation
The inflammatory response mounted against bacterial
pathogens infecting the mucosal surface of the lung is

highly complex and multifaceted. Like the intestine,
one of the destructive consequences of an over-aggres-
sive inflammatory response is the accumulation of
activated neutrophils in the airway lumen that can
damage lung tissue. Mounting evidence reveals that
epithelial cells lining the luminal cavity, which separate
the lumenal contents from the underlying tissue, are
key players in orchestrating innate immune responses.
Fig. 1. Model of bacterial-induced signaling leading to the release of S ⁄ R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (hepoxilin A
3
;
HXA
3
). Interaction of bacterial pathogens of the intestine (S. typhimurium) and lung (P. aeruginosa) leads to the activation of a unique lipid
signal transduction cascade resulting in the up-regulation and activation of phosphorylated protein kinase C (pPKC), the signaling kinase
required for HXA
3
production. Whether pPKC directly or indirectly leads to the activation of phospholipase A
2
(PLA
2
) has yet to be deter-
mined, but this enzyme is responsible for the membrane release of arachidonic acid (AA), the precursor of HXA
3
. Once liberated within the
cytosol, AA is available as a substrate for 12-lipoxygenase (12-LOX), the enzyme responsible for the synthesis of HXA
3
. Through a mechan-
ism yet to be determined, HXA
3

is then released apically where it forms a concentration gradient through the epithelial cell tight junction,
resulting in the directed movement of neutrophils across the epithelial barrier. PMN, polymorphonuclear leukocyte.
Bacterial-induced hepoxilin A
3
secretion B. A. McCormick
3516 FEBS Journal 274 (2007) 3513–3518 ª 2007 The Author Journal compilation ª 2007 FEBS
Given the complexity of the route that neutrophils
must travel to reach the airway lumen (i.e. through the
endothelium), the basement membrane, the interstitial
space, the epithelial basement membrane and the epi-
thelial layer, it is likely that multiple neutrophil chemo-
attractants participate at discrete steps during this
recruitment process. In a scenario similar to the intes-
tine, during bacterial infection of the lung, such as
P. aeruginosa infection, IL-8 probably plays a major
role in recruiting neutrophils from the bloodstream to
the epithelium, whereas the production and apical
secretion of the eicosanoid HXA
3
is PKC dependent
and necessary for guiding neutrophils across the infec-
ted epithelium [27].
Current work from my laboratory is attempting to
define the mechanism(s) underlying HXA
3
production
and neutrophil transepithelial migration in response to
infection with P. aeruginosa. One probable means to
increase the production of HXA
3

is to increase the
availability of its precursor, AA. The liberation of AA
from phospholipid membranes is presumed to be the
rate-limiting step for the generation of eicosanoids,
with the idea being that the greater amount of free AA
to serve as substrate for the 12-lipoxygenase enzyme,
the greater potential to produce 12-HpETE and
HXA
3
. The major mechanism to generate free AA for
subsequent conversion to eicosanoids (via lipoxygenas-
es and cyclooxygenases) is by the action of phospho-
lipase A
2
(PLA
2
) [28]. PLA
2
represents a family of at
least 19 distinct proteins, which have been grouped
into three subfamilies. The sPLA
2
subfamily contains
small (14–19 kDa) enzymes that are secreted by cells
and act on the lumenal surface of cell membranes to
liberate AA [29]. Members of the cPLA
2
family are
distinguished by a dependency on calcium and are acti-
vated by phosphorylation. In addition, members of

this group are capable of shifting from the cytosol to
the perinuclear membrane where they interact with
phospholipids, resulting in the liberation of AA [29].
The third group is iPLA
2
, which resides in the cytosol
but its activation is independent of calcium [29].
Although isoforms of iPLA
2
have generally been
believed to participate in phospholipid remodeling,
recent studies have also documented the involvement
of iPLA
2
in mediating AA under certain circumstances
[30]. Indeed, our recent studies have shown that PLA
2
activity is required for P. aeruginosa-induced neutro-
phil transepithelial migration [31]. In addition, upon
infection, lung epithelial cells phosphorylate cPLA
2
and release significantly more AA from membrane
stores [31]. Based on these observations we have hypo-
thesized that increased PLA
2
activity, which mediates
AA release, is obligatory for the production of HXA
3
,
which in turn is required for orchestrating neutrophil

movement across lung epithelial monolayers (Fig. 1).
It is worth noting, however, that is it controversial as
to whether the activation of PLA
2
occurs by PKC.
Regardless, determination of the particular phospholi-
pase A
2
responsible for orchestrating neutrophil trans-
epithelial migration may lead to targeted therapies
designed to dampen inflammation in the lung.
Summary
Thus far it has been shown that pathogenic bacterial
interactions with either intestinal or airway epithelial
cells results in signal transduction cascades, which lead
to the production and secretion of HXA
3
, most prob-
ably through the action of the 12-LOX enzymatic
pathway. Therefore, this pathway may represent a con-
served innate immune mechanism for detection and
eradiation of pathogens interfacing with the host
mucosal surface. Of potential clinical significance,
signaling pathways leading to HXA
3
secretion may
provide an important new therapeutic target for the
treatment of acute and chronic diseases of intestinal,
lung and perhaps other mucosal surfaces.
Acknowledgements

I am indebted to the members of the McCormick
Laboratory, past and present, who have contributed to
this work. A special thank you is reserved for Dr
Randall J. Mrsny. This work was supported by the
National Institutes of Health (DK56754).
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