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: mccormic@helix.mgh.harvard.edu (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. 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Biochem Pharmacol 63, 1969–1977. 31 Hurley BP, Williams NL & McCormick BA (2006) Involvement of phospholipase A2 in Pseudomonas aeru- ginosa mediated PMN trans-epithelial migration. Am J Physiol Lung Cell Mol Physiol 290, 703–9. Bacterial-induced hepoxilin A 3 secretion B. A. McCormick 3518 FEBS Journal 274 (2007) 3513–3518 ª 2007 The Author Journal compilation ª 2007 FEBS . nucleotide exchange factor (such as ARNO) to the apical plasma mem- brane. ARNO facilitates ADP-ribosylation factor 6 activation at the apical membrane, which. that implicate hepoxilin A 3 as a key regulator of mucosal inflammation. Abbreviations AA, arachidonic acid; HpETE, hydroperoxy-eicosatetraenoic acid; HXA 3 ,8S