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Type 2 cytokines promote epithelial changes that help to expel worms during intestinal helminth infection. Oyesola et al. show that prostaglandin D2 and its receptor CRTH2 are novel negative regulators of this response, dampening the Type 2 cytokine–mediated program., Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine–mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2–CRTH2 pathway negatively regulates the Type 2 cytokine–driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.

In the last twenty years an impressive body of evidence in diverse inflammatory animal disease models and human tissues, has established polyunsaturated fatty acids (PUFA) derived specialized-pro-resolving mediators (SPM), as essential mediators for controlling acute inflammation, immune responses and wound healing and for resolving acute inflammation in many non-ocular tissues. SPM pathways and receptors are highly expressed in the ocular surface where they regulate wound healing, nerve regeneration, innate immunity and sex-specific regulation of auto-immune responses. Recent evidence indicates that in the eye these resident SPM networks are important for maintaining ocular surface health and immune homeostasis. Here, we will review and discuss evidence for SPMs and other PUFA-derived mediators as important endogenous regulators and biomarkers of ocular surface health and disease and their therapeutic potential.

Resolution of inflammation is defective after spinal cord injury (SCI), which impairs tissue integrity and remodeling and leads to functional deficits. Effective pharmacological treatments for SCI are not currently available. Maresin 1 (MaR1) is a highly conserved specialized proresolving mediator (SPM) hosting potent anti-inflammatory and proresolving properties with potent tissue regenerative actions. Here, we provide evidence that the inappropriate biosynthesis of SPM in the lesioned spinal cord hampers the resolution of inflammation and leads to deleterious consequences on neurological outcome in adult female mice. We report that, after spinal cord contusion injury in adult female mice, the biosynthesis of SPM is not induced in the lesion site up to 2 weeks after injury. Exogenous administration of MaR1, a highly conserved SPM, propagated inflammatory resolution after SCI, as revealed by accelerated clearance of neutrophils and a reduction in macrophage accumulation at the lesion site. In the search of mechanisms underlying the proresolving actions of MaR1 in SCI, we found that this SPM facilitated several hallmarks of resolution of inflammation, including reduction of proinflammatory cytokines (CXCL1, CXCL2, CCL3, CCL4, IL6, and CSF3), silencing of major inflammatory intracellular signaling cascades (STAT1, STAT3, STAT5, p38, and ERK1/2), redirection of macrophage activation toward a prorepair phenotype, and increase of the phagocytic engulfment of neutrophils by macrophages. Interestingly, MaR1 administration improved locomotor recovery significantly and mitigated secondary injury progression in a clinical relevant model of SCI. These findings suggest that proresolution, immunoresolvent therapies constitute a novel approach to improving neurological recovery after acute SCI.SIGNIFICANCE STATEMENTInflammation is a protective response to injury or infection. To result in tissue homeostasis, inflammation has to resolve over time. Incomplete or delayed resolution leads to detrimental effects, including propagated tissue damage and impaired wound healing, as occurs after spinal cord injury (SCI). We report that inflammation after SCI is dysregulated in part due to inappropriate synthesis of proresolving lipid mediators. We demonstrate that the administration of the resolution agonist referred to as maresin 1 (MaR1) after SCI actively propagates resolution processes at the lesion site and improves neurological outcome. MaR1 is identified as an interventional candidate to attenuate dysregulated lesional inflammation and to restore functional recovery after SCI.

Neutrophils are generally the first immune cells recruited during the development of sterile or microbial inflammation. As these cells express many innate immune receptors with the potential to directly recognize microbial or endogenous signals, we set out to assess whether their functions are locally influenced by the signals present at the onset of inflammation. Using a mouse model of peritonitis, we demonstrate that neutrophils elicited in the presence of C-type lectin receptor ligands have an increased ability to produce cytokines, chemokines, and lipid mediators in response to subsequent TLR stimulation. Importantly, we found that licensing of cytokine production was mediated by paracrine TNF-α-TNFR1 signaling rather than direct ligand sensing, suggesting a form of quorum sensing among neutrophils. Mechanistically, licensing was largely imparted by changes in the posttranscriptional regulation of inflammatory cytokines, whereas production of IL-10 was regulated at the transcriptional level. Altogether, our data suggest that neutrophils rapidly adapt their functions to the local inflammatory milieu. These phenotypic changes may promote rapid neutrophil recruitment in the presence of pathogens but limit inflammation in their absence.

Chronic itch remains a highly prevalent disorder with limited treatment options. Most chronic itch diseases are thought to be driven by both the nervous and immune systems, but the fundamental molecular and cellular interactions that trigger the development of itch and the acute-to-chronic itch transition remain unknown. Here, we show that skin-infiltrating neutrophils are key initiators of itch in atopic dermatitis, the most prevalent chronic itch disorder. Neutrophil depletion significantly attenuated itch-evoked scratching in a mouse model of atopic dermatitis. Neutrophils were also required for several key hallmarks of chronic itch, including skin hyperinnervation, enhanced expression of itch signaling molecules, and upregulation of inflammatory cytokines, activity-induced genes, and markers of neuropathic itch. Finally, we demonstrate that neutrophils are required for induction of CXCL10, a ligand of the CXCR3 receptor that promotes itch via activation of sensory neurons, and we find that that CXCR3 antagonism attenuates chronic itch.

Atopic dermatitis (AD) is the most common skin disease in children. It is characterized by relapsing inflammation, skin-barrier defects, and intractable itch. However, the pathophysiology of itch in AD remains enigmatic. Here, we examine the contribution of Tmem79, an orphan transmembrane protein linked to AD in both mice and humans. We show that Tmem79 is expressed by both keratinocytes and sensory neurons, but that loss of keratinocytic Tmem79 is sufficient to elicit robust scratching. Tmem79 -/- mice demonstrate an accumulation of dermal mast cells, which are diminished following chronic treatment with cyclooxygenase inhibitors and an EP3 receptor antagonist. In Tmem79 -/- mice, mast cell degranulation produces histaminergic itch in a histamine receptor 1/histamine receptor 4 (H4R/H1R)-dependent manner that may involve activation of TRPV1- afferents. TMEM79 has limited sequence homology to a family of microsomal glutathione transferases and confers protection from cellular accumulation of damaging reactive species, and may thus play a role in regulating oxidative stress. In any case, mechanistic insights from this model suggest that therapeutics targeting PGE2 and/or H1R/H4R histaminergic signaling pathways may represent useful avenues to treat Tmem79-associated AD itch. Our findings suggest that individuals with mutations in Tmem79 develop AD due to the loss of protection from oxidative stress.

Recently identified regulatory PMN control immune-driven dry eye disease (DED) in females by producing the arachidonic acid (ω-6)-derived specialized pro-resolving mediator (SPM), LXA4, in lymph nodes. Dietary ω-3 docosahexaenoic acid (DHA) is protective in DED but mechanisms of action remain elusive. DHA is converted to ω-3 SPMs by PMN via the same lipoxygenases (LOX) that generate LXA4. We investigated if dietary DHA amplifies SPM formation and affects T effector cell function and/or regulatory PMN in DED. DED was induced in mice on a DHA-enriched or ω-3-deficient diet. DHA deficiency amplified DED with marked sex-specific differences. Dietary DHA protection against dry eye disease correlated with increased PMN levels in lymph nodes, ocular tissues, and unexpectedly, selective amplification of LXA4 tissue levels. Dietary DHA increased 12/15-LOX and decreased 5-LOX expression in lymph nodes and isolated lymph node PMN, which correlated with amplified LXA4 formation. Acute DHA treatment rescued DHA-deficient females from exaggerated DED by amplifying lymph node LXA4 formation, increasing Treg and decreasing TH1 and TH17 effector cells. Our results identify DHA regulation of LXA4 producing PMN in ocular tissues and lymph nodes in health and immune disease as novel mechanism and determinant for T-cell responses to routine ocular injury or stress signals.

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

Astrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs), and a risk of systemic spread of the bacterium. We investigated host mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-LOX in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12-LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-LOX exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high-level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX–dependent HXA3 production and subsequent PMN transepithelial migration.

The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are precursors to immune regulatory and specialized pro-resolving mediators (SPM) of inflammation termed resolvins, maresins, and protectins. Evidence for lipid mediator formation in vivo can be gained through evaluation of their 5-lipoxygenase (LOX) and 15-LOX metabolic pathway precursors and downstream metabolites. We performed a secondary blood sample analysis from 60 participants in the Mothers, Omega-3, and Mental Health study to determine whether SPM and SPM precursors are augmented by dietary EPA- and DHA-rich fish oil supplementation compared to soy oil placebo. We also aimed to study whether SPM and their precursors differ in early and late pregnancy or between maternal and umbilical cord blood. We found that compared to placebo supplementation, EPA- and DHA-rich fish oil supplementation increased SPM precursor 17-hydroxy docosahexaenoic acid (17-HDHA) concentrations in maternal and umbilical cord blood (P = 0.02). We found that the D-series resolvin pathway marker 17-HDHA increased significantly between enrollment and late pregnancy (P = 0.049). Levels of both 14-HDHA, a maresin pathway marker, and 17-HDHA were significantly greater in umbilical cord blood than in maternal blood (P < 0.001, both).

Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity but did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1-/-Casp8-/- mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology.

Lipid droplet (LD) formation occurs during infection of macrophages with numerous intracellular pathogens, including Mycobacterium tuberculosis. It is believed that M. tuberculosis and other bacteria specifically provoke LD formation as a pathogenic strategy in order to create a depot of host lipids for use as a carbon source to fuel intracellular growth. Here we show that LD formation is not a bacterially driven process during M. tuberculosis infection, but rather occurs as a result of immune activation of macrophages as part of a host defense mechanism. We show that an IFN-γ driven, HIF-1α dependent signaling pathway, previously implicated in host defense, redistributes macrophage lipids into LDs. Furthermore, we show that M. tuberculosis is able to acquire host lipids in the absence of LDs, but not in the presence of IFN-γ induced LDs. This result uncouples macrophage LD formation from bacterial acquisition of host lipids. In addition, we show that IFN-γ driven LD formation supports the production of host protective eicosanoids including PGE2 and LXB4. Finally, we demonstrate that HIF-1α and its target gene Hig2 are required for the majority of LD formation in the lungs of mice infected with M. tuberculosis, thus demonstrating that immune activation provides the primary stimulus for LD formation in vivo. Taken together our data demonstrate that macrophage LD formation is a host-driven component of the adaptive immune response to M. tuberculosis, and suggest that macrophage LDs are not an important source of nutrients for M. tuberculosis., Author summary Mycobacterium tuberculosis, the causative agent of the disease tuberculosis, causes more deaths annually than any other single bacterial pathogen. M. tuberculosis primarily lives in macrophages, immune cells which specialize in phagocytosing and killing pathogens. In order to survive this inhospitable environment, M. tuberculosis must be both resistant to the bactericidal activities of macrophages and able to exploit macrophages as a replicative niche. Intracellular bacterial pathogens acquire all of their nutrients from the host cell, and M. tuberculosis relies on lipids as a key carbon source during infection. It has been proposed that lipid droplets, organelles that store neutral lipids and are observed in macrophages during M. tuberculosis infection, are an accessible nutrient source for M. tuberculosis. Furthermore, it is thought that M. tuberculosis provokes macrophages into making LDs as a pathogenic strategy. Here, we present evidence that LD formation is actually a programmed macrophage immune response, and part of the broader program of IFN-γ activation of macrophages. Furthermore, we present data uncoupling bacterial acquisition of host lipids from the presence of LDs in macrophages. Thus, we find that in addition to supporting immune functions, LDs are also unlikely to be an important nutrient source for M. tuberculosis in macrophages.

Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A(4) receptors (ALX) constitute a LXA(4) circuit that is a key feature of inflammatory resolution. LXA(4) analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA(4) circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA(4), respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA(4), but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA(4) circuit as an endogenous regulator of pathological angiogenesis.

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A3, an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A3 is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A3 is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A3 synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A3 synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.

Salmonella spp. and Shigella spp. are frequently implicated in outbreaks of enteric disease due to the consumption of contaminated food. In the United States alone, the number of prevalent cases of salmonellosis is estimated to range from 80,000 to 3,700,000 annually (7). Moreover, Shigella sp. infections are responsible for ∼163 million cases of dysentery and 1 million deaths each year worldwide (27). While the majority of infections occur in third-world countries, where contaminated food and drinking water are common (27), even developed nations cannot escape several outbreaks in a given year (65). Therefore, studying the molecular pathogenesis of these gram-negative enteric pathogens is of paramount importance, especially taking into consideration emerging antibiotic resistance and the lack of appropriate vaccines. Salmonella enterica serotype Typhimurium and Shigella flexneri interact with the intestinal epithelium to induce intestinal inflammation characterized by the transepithelial migration of neutrophils (2, 12, 33, 41, 49). Symptoms of salmonellosis manifest within 48 h of the ingestion of contaminated food or water and include nausea, vomiting, and acute, mostly self-limiting diarrhea. Following its ingestion, this motile pathogen colonizes and penetrates the intestinal epithelium and gains access to systemic sites, such as the liver and spleen, through lymphatic and blood circulation (70). The ability of S. enterica serovar Typhimurium to cause disease in humans is related to the acquisition of virulence genes termed pathogenicity islands. Located on a 40-kb segment of the bacterial chromosome termed SPI-1 (Salmonella pathogenicity island 1) are 25 virulence genes that encode structural components and secreted substrates of a type III secretion system (TTSS) (11, 21). This TTSS is essential for the ability of S. enterica serovar Typhimurium to invade and induce proinflammatory responses (21). Like S. enterica serovar Typhimurium, Shigella flexneri enters the human host by being ingested via contaminated food or water, passaging through the stomach, and infecting the large intestine. Infection of the colonic mucosa by Shigella results in bacillary dysentery (shigellosis), an acute inflammatory disease characterized by abdominal cramps, fever, and severe diarrhea often containing blood and mucus (66). In contrast to what is the case for Salmonella, humans (and certain higher primates) serve as the only natural hosts and reservoirs for Shigella (66). Thus, this organism has evolved to be highly host specific as well as efficient, with a particularly low infectious dose in comparison to other enteric pathogens (77). Also distinct from Salmonella, Shigella has a unique mode of entry requiring access to the basolateral surface. It has been established that Shigella alters the tight junctional complex, thereby allowing the pathogen to traverse the paracellular space and access the basolateral surface, an event that also decreases barrier function (64). Once at the basolateral surface, Shigella rapidly invades and disseminates through the epithelium, a consequence of the TTSS and additional proteins encoded in a 31-kb region (the mxi-spa locus) of the large 220-kb virulence plasmid (5, 69). While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in the massive transepithelial migration of neutrophils across the intestinal mucosa (2, 12, 33, 41, 49). Both S. enterica serovar Typhimurium and S. flexneri induce epithelial cells to secrete a repertoire of chemokines that play an active role in recruiting polymorphonuclear leukocytes (PMNs) from the peripheral circulation and directing them across the epithelium to the intestinal lumen (15, 25, 43, 44, 61, 63, 68, 74). In the case of S. enterica serovar Typhimurium, the effector protein SipA is necessary to initiate the cellular events that lead to PMN transepithelial migration during acute states of active intestinal inflammation (10, 35, 73). Regarding the molecular mechanism underlying these cellular events, it has been revealed so far that SipA activates a novel ADP ribosylation factor 6- and phospholipase D-dependent lipid signaling cascade (10) that in turn activates protein kinase C α (PKC α) (73), events that ultimately lead to the apical secretion of a potent PMN chemoattractant, eicosanoid hepoxilin A3 (HXA3; 8-hydroxy-11,12-epoxy-eicosatetraenoic acid) (51). HXA3, an endogenous 12/15-lipoxygenase (12/15-LOX) product, forms a chemotactic gradient across the epithelial tight junctional complex that directs PMNs across the intestinal epithelium to the luminal surface (51). The mechanisms underlying Shigella-induced PMN transepithelial migration are less defined. Studies addressing PMN movement across model intestinal epithelia indicate that PMN transmigration does occur as a result of Shigella infection and that this event further facilitates bacterial invasion (48, 62). Furthermore, the induction of PMN transepithelial migration is dependent not only on a functional TTSS and invasive ability (24, 67) but also on intercellular spread (16). Recently, lipopolysaccharide and the S. flexneri Osp proteins have been shown to be involved in the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway (26, 85, 86). In turn, ERK1/2 phosphorylation and its nuclear localization have been shown to play a role in PMN transepithelial migration (26). The purpose of this study was to determine if S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce PMN transepithelial migration (PKC and ERK1/2, respectively). We found that S. flexneri, similarly to S. enterica serovar Typhimurium, induced the apical secretion of the potent PMN chemoattractant HXA3 from model intestinal epithelia. In addition, PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA3. Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A2 (PLA2), a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA3.

Host defense is essential to all vertebrates, and programs of inflammation and wound healing must be highly integrated in tissue and organ structures, such as the skin, cornea, and mucosa, that provide crucial barriers and interfaces with the external environment. Certain aspects of inflammation and wound healing have posed a conundrum for biologists, especially to the extent that the two programs may appear to operate in opposition to each other. The recruitment of neutrophils to injured tissue, for example, is essential to inflammation and defense against infection, but can at the same time impair wound healing. One mechanism for regulating this duality is provided by lipid autacoids, which act to restrain leukocyte activation and to promote the resolution of inflammation. Emerging evidence indicates that lipid autacoids also have a central role in wound healing and in fact mediate a privileged injury response, as is observed in the cornea, characterized by rapid healing as well as effective host defense.

A key feature in the pathophysiology of acute renal injury and glomerulonephritis is exacerbated or non-resolving inflammation. A rapidly evolving field has identified lipoxins and novel ω-3 lipid autacoids as important mediators of inflammatory resolution. These renal lipid autacoid circuits counterbalance proinflammatory cascades, are antifibrotic and maintain renal function. Therapeutic or genetic amplification of protective lipid autacoid circuits heralds a new strategy to halt progressive renal disease and acute renal injury.

Immune-driven dry eye disease primarily affects women; the cause for this sex-specific prevalence is unknown. Polymorphonuclear neutrophils (PMN) have distinct phenotypes that drive inflammation but also regulate lymphocytes and are the rate-limiting cell for generating anti-inflammatory lipoxin A4 (LXA4). Estrogen regulates the LXA4 circuit to induce delayed female-specific wound healing in the cornea. However, the role of PMNs in dry eye disease remains unexplored. We discovered an LXA4-producing tissue PMN population in the corneal limbus, lacrimal glands, and cervical lymph nodes of healthy male and female mice. These tissue PMNs, unlike inflammatory PMNs, expressed a highly amplified LXA4 circuit and were sex-specifically regulated during immune-driven dry eye disease. Desiccating stress in females, unlike in males, triggered a remarkable decrease in lymph node PMN and LXA4 formation that remained depressed during dry eye disease. Depressed lymph node PMN and LXA4 in females correlated with an increase in effector T cells (Th1 and Th17), a decrease in regulatory T cells (Treg), and increased dry eye pathogenesis. Ab depletion of tissue PMN abrogated LXA4 formation in lymph nodes, as well as caused a marked increase in Th1 and Th17 cells and a decrease in Tregs. To establish an immune-regulatory role for PMN-derived LXA4 in dry eye, females were treated with LXA4. LXA4 treatment markedly inhibited Th1 and Th17 and amplified Treg in draining lymph nodes, while reducing dry eye pathogenesis. These results identify female-specific regulation of LXA4-producing tissue PMN as a potential key factor in aberrant effector T cell activation and initiation of immune-driven dry eye disease.

Heme oxygenase (HO) represents an intrinsic anti-inflammatory system based on its ability to regulate leukocyte function and inhibit expression of proinflammatory cytokines. This anti-inflammatory function is linked to the inducible isoform HO-1; the role of the constitutive isoform HO-2 is unknown. The current study was undertaken to investigate the role of HO-2 in the regulation of the acute inflammatory and reparative response by using HO-2-null mice and well-established animal models of epithelial injury and antigen-induced peritonitis. Here we show that in vivo deletion of HO-2 disables execution of the acute inflammatory and reparative response after epithelial injury and leads to an exaggerated inflammatory response in antigen-induced peritonitis. HO-2 deletion was associated with impaired HO-1 induction, indicating that HO-2 is critical for HO-1 expression and that the subsequent failure to up-regulate the HO system may contribute to unresolved inflammation and the development of chronic inflammatory conditions. Indeed, supplementation with the HO bioactive product, biliverdin, rescued the acute inflammatory and reparative response in HO-2-null mice. Thus, HO-2 sets in place a basal tone of anti-inflammatory signals that may be a prerequisite for the ordered execution of an inflammatory and reparative response.

1. Lipoxins (LX) and aspirin-triggered 15-epi-lipoxins (ATL) exert potent anti-inflammatory actions. In the present study, we determined the anti-inflammatory efficacy of endogenous LXA(4) and LXB(4), the stable ATL analog ATLa2, and a series of novel 3-oxa-ATL analogs (ZK-996, ZK-990, ZK-994, and ZK-142) after intravenous, oral, and topical administration in mice. 2. LXA(4), LXB(4), ATLa2, and ZK-994 were orally active, exhibiting potent systemic inhibition of zymosan A-induced peritonitis at very low doses (50 ng kg(-1)-50 microg kg(-1)). 3. Intravenous ZK-994 and ZK-142 (500 microg kg(-1)) potently attenuated hind limb ischemia/reperfusion-induced lung injury, with 32+/-12 and 53+/-5% inhibition (P

Signaling pathways instrumental in the temporal and spatial progression of acute inflammation toward resolution are of wide interest. Here a transgenic mouse with myeloid-selective expression of human lipoxin A4 receptor (hALX) was prepared and used to evaluate in vivo the effect of hALX expression. hALX-transfected HEK293 cells transmitted LXA4 signals that inhibit TNFalpha-induced NFkappaB activation. Transgenic FvB mice were generated by DNA injections of a 3.8 kb transgene consisting of the full-length hALX cDNA driven by a fragment of the hCD11b promoter. When topically challenged via dermal ear skin, hALX transgenic mice gave attenuated neutrophil infiltration (approximately 80% reduction) in response to leukotriene B4 (LTB4) plus prostaglandin E2 (PGE2) as well as approximately 50% reduction in PMN infiltrates (P0.02) to receptor-bypass inflammation evoked by phorbol ester. The hALX transgenic mice gave markedly decreased PMN infiltrates to the peritoneum with zymosan and altered the dynamics of this response. Transgenic hALX mice displayed increased sensitivity with50% reduction in PMN infiltrates to suboptimal doses (10 ng/mouse) of the ligand lipoxin A4 stable analog compared with10% reduction of PMN in nontransgenic littermates. Soluble mediators generated within the local inflammatory milieu of hALX mice showed diminished ability to activate the proinflammatory transcription factor NFkappaB. Analyses of the lipid-derived mediators from exudates using LC-MS tandem mass spectroscopy indicated an altered profile in hALX transgenic mice that included lower levels of LTB4 and increased amounts of lipoxin A4 compared with nontransgenic littermates. Together these results demonstrate a gain-of-function with hALX transgenic mouse and indicate that ALX is a key receptor and sensor in formation of acute exudates and their resolution.

Aspirin (ASA) is unique among current therapies because it acetylates cyclooxygenase (COX)-2 enabling the biosynthesis of R-containing precursors of endogenous antiinflammatory mediators. Here, we report that lipidomic analysis of exudates obtained in the resolution phase from mice treated with ASA and docosahexaenoic acid (DHA) (C22:6) produce a novel family of bioactive 17R-hydroxy-containing di- and tri-hydroxy-docosanoids termed resolvins. Murine brain treated with aspirin produced endogenous 17R-hydroxydocosahexaenoic acid as did human microglial cells. Human COX-2 converted DHA to 13-hydroxy-DHA that switched with ASA to 17R-HDHA that also proved a major route in hypoxic endothelial cells. Human neutrophils transformed COX-2-ASA–derived 17R-hydroxy-DHA into two sets of novel di- and trihydroxy products; one initiated via oxygenation at carbon 7 and the other at carbon 4. These compounds inhibited (IC50 ∼50 pM) microglial cell cytokine expression and in vivo dermal inflammation and peritonitis at ng doses, reducing 40–80% leukocytic exudates. These results indicate that exudates, vascular, leukocytes and neural cells treated with aspirin convert DHA to novel 17R-hydroxy series of docosanoids that are potent regulators. These biosynthetic pathways utilize omega-3 DHA and EPA during multicellular events in resolution to produce a family of protective compounds, i.e., resolvins, that enhance proresolution status.

Lipoxin (LX) A4 and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA4 receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific 3H-LXA4 binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA4 recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B4 receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA4 recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Parenteral nutrition (PN)-associated liver disease (PNALD) is a life-threatening complication of the administration of PN. The development of PNALD may be partly due to the composition of the lipid emulsion administered with PN: soybean oil-based lipid emulsions (SOLE) are associated with liver disease, while fish oil-based lipid emulsions (FOLE) are associated with prevention and improvement of liver disease. The objective of this study was to determine how the choice of lipid emulsion modified the production of bioactive lipid mediators (LMs). We utilized a mouse model of steatosis to study the differential effect of FOLE and SOLE. We subsequently validated these results in serum samples from a small cohort of human infants transitioning from SOLE to FOLE. In mice, FOLE was associated with production of anti-inflammatory, proresolving LMs; SOLE was associated with increased production of inflammatory LMs. In human infants, the transition from SOLE to FOLE was associated with a shift toward a proresolving lipidome. Together, these results demonstrate that the composition of the lipid emulsion directly modifies inflammatory homeostasis.

Background The severity and longevity of inflammation is controlled by endogenous counter-regulatory signals. Among them are long-chain polyunsaturated fatty acid (PUFA)-derived lipid mediators, which promote the resolution of inflammation, an active process for returning to tissue homeostasis. Objective To determine whether endogenous production of lipid-derived resolution agonists is regulated differentially in patients with highly active and less active multiple sclerosis (MS). Design Matched-pairs study in University hospital Neurology department. Patients Based on clinical (relapse frequency) and paraclinical (MRI lesions, contrast enhancement) criteria, 10 pairs of age- and sex-matched patients with relapsing-remitting MS were assigned either to a group with highly active or less active MS. Lipid mediators were quantified in serum and cerebrospinal fluid using LC-MS/MS-based lipidomics. Results Levels of the key arachidonic (ω-6) and docosahexaenoic acid (ω-6)-derived mediators prostaglandins (PG), leukotrienes, hydroxyeicosatetraenoic acids (HETE) and resolution agonists lipoxin A4 (LXA4), resolvin D1 (RvD1) and neuroprotectin D1 (NPD1) were quantified. In the patient group with highly active MS, 15-HETE and PGE2 were increased, which are products of the 15-lipoxygenase and cyclooxygenase pathways. The proresolution mediator RvD1 was significantly upregulated and NPD1 was detected in the highly active group only. LXA4 levels were not increased in patients with highly active MS. Conclusions Lipid mediator pathways are regulated differentially in the cerebrospinal fluid of MS patients, depending on disease severity. Non-exhaustive or possibly ‘delayed’ resolution pathways may suggest a defective resolution program in patients with highly active MS. Longitudinal analyses are required to hetero-typify this differential resolution capacity, which may be associated with disease progression, longevity and eventual termination.

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A3 is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A3 in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A3, including phospholipase A2 and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A3 precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A3 are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A3 and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A3 serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A3 are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A3 and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A3 is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Acute inflammation is a frequent, essential and beneficial response to maintain normal tissue function. PMN are the primary effector cells of acute inflammatory responses and their timely resolution by macrophages from an injured, stressed or infected tissues are required for the successful execution of this routine tissue response. Dysregulation of this fundamental program is a major factor in the global disease burden and contributes to many ocular diseases. Counter-regulatory signals are critical to the controlled activation of innate and adaptive immune responses in the eye and recent studies have identified two circuits in the cornea, uvea and/or retina, namely 15-lipoxygenase and heme-oxygenase, which control inflammation, promote resolution of PMN and afford neuroprotection. The role of these counter-regulator and pro-resolution circuits may provide insight into ocular inflammatory diseases and opportunities to restore stressed ocular tissue to a pre-inflammatory state, namely homeostasis, rather than limiting therapeutic options to palliative inhibition of pro-inflammatory circuits.

Exacerbated inflammation plays an important role in the pathogenesis of ischemic renal injury (IRI), which is the major cause of intrinsic acute renal failure. Clinical studies suggest that long-term treatment with ω-3 polyunsaturated fatty acids (PUFA) improves renal function and lowers the risk of death or end-stage renal disease. Docosahexaenoic acid, a principle ω-3 PUFA of fish oils, is of particular interest as it is found in most human tissues and is converted to protectin D1 (PD1), which exhibits antiinflammatory and proresolving bioactions. We set out to investigate the impact of acute dietary modulation of ω-3 or ω-6 PUFA on IRI and renal lipid autacoid circuits, using an established mouse model and liquid chromatography-mass spectroscopy/mass spectroscopy-based lipidomics. Thirty minutes of renal ischemia significantly elevated serum creatinine in the ω-6 diet group while renal function remained normal in the matched ω-3 diet group. Notably, extending ischemia to 45 min caused 100% mortality in the ω-6 group, in sharp contrast to 0% mortality in the ω-3 group. Protection against IRI in the ω-3 group correlated with decreased polymorphonuclear leukocyte recruitment, chemokine and cytokine levels, abrogated formation of lipoxygenase- and cyclooxygenase-derived eicosanoids, and increased renal levels of PD1. Systemic treatment with PD1 reduced kidney polymorphonuclear leukocyte influx and, more importantly, amplified renoprotective heme-oxygenase-1 protein and mRNA expression in injured and uninjured kidneys. These findings suggest therapeutic or dietary amplification of PD1 circuits restrains acute renal injury and that short-term changes in dietary ω-3 and ω-6 PUFA dramatically impacts renal lipid autacoid formation and outcome of IRI.

The eye must contain highly evolved programs to limit inflammation and promote wound healing as an errant response can lead to blindness. However, pathways that protect the delicate visual axis and account for its atypical inflammatory responses remain to be clearly defined. Hence, research efforts have been initiated to elucidate the role of the anti-inflammatory LXA 4 circuits in the eye. LXA 4 is formed in healthy and injured corneas and both its receptor and 12/15-lipoxygenase are predominately expressed in epithelial cells. An essential role for LXA 4 in preserving ocular function is supported by 12/15-LOX deficient mice that exhibit a phenotype of impaired wound healing and LXA 4 formation. A novel epithelial bioaction role for LXA 4 has been uncovered in the cornea as topical LXA 4 promotes wound healing and limits the sequelae of injury. These emerging studies indicate that the LXA 4 circuit may hold a fundamental role in maintaining an ocular environment that actively restricts inflammation while promoting wound healing.

The surface of the eye actively suppresses inflammation while maintaining a remarkable capacity for epithelial wound repair. Our understanding of mechanisms that balance inflammatory/reparative responses to provide effective host defense while preserving tissue function is limited, in particular, in the cornea. Lipoxin A(4) (LXA(4)) and docosahexaenoic acid-derived neuroprotectin D1 (NPD1) are lipid autacoids formed by 12/15-lipoxygenase (LOX) pathways that exhibit anti-inflammatory and neuroprotective properties. Here, we demonstrate that mouse corneas generate endogenous LXA(4) and NPD1. 12/15-LOX (Alox15) and LXA(4) receptor mRNA expression as well as LXA(4) formation were abrogated by epithelial removal and restored during wound healing. Amplification of these pathways by topical treatment with LXA(4) or NPD1 (1 microg) increased the rate of re-epithelialization (65-90%, n = 6-10, p0.03) and attenuated the sequelae of thermal injury. In contrast, the proinflammatory eicosanoids, LTB(4) and 12R-hydroxyeicosatrienoic acid, had no impact on corneal re-epithelialization. Epithelial removal induced a temporally defined influx of neutrophils into the stroma as well as formation of the proinflammatory chemokine KC. Topical treatment with LXA(4) and NPD1 significantly increased PMNs in the cornea while abrogating KC formation by 60%. More importantly, Alox15-deficient mice exhibited a defect in both corneal re-epithelialization and neutrophil recruitment that correlated with a 43% reduction in endogenous LXA(4) formation. Collectively, these results identify a novel action for the mouse 12/15-LOX (Alox15) and its products, LXA(4) and NPD1, in wound healing that is distinct from their well established anti-inflammatory properties.

Host defense mechanisms are impaired in patients with congenital neutrophil (polymorphonuclear neutrophils (PMN)) defects. Impaired PMN chemotaxis is observed in localized aggressive periodontitis (LAP), a familial disorder characterized by destruction of the supporting structures of dentition. In the present studies, we sought evidence for molecular events underlying this aberrant human PMN phenotype. To this end, PMN transendothelial migration and superoxide anion generation were assessed with LAP patients and asymptomatic family members, as well as patients with other chronic mucosal inflammation. PMN from LAP patients showed decreased transmigration across vascular endothelial monolayers (18 ± 12% of control, n = 4) and increased superoxide anion generation (358 ± 37%, p = 0.003). Gene expression was analyzed using oligonucleotide microarrays and fluorescence-based kinetic PCR. cDNA microarray and kinetic-PCR analysis revealed diminished RNA expression of leukocyte-type diacylglycerol (DAG) kinase α in PMN from LAP patients (4.6 ± 1.7 relative units, n = 6, p = 0.007) compared with asymptomatic individuals (51 ± 27 relative units, n = 7). DAG kinase activity was monitored by DAG phosphorylation and individual DAG molecular species were quantified using liquid chromatography and tandem mass spectrometry-based lipidomics. DAG kinase activity was also significantly decreased (73 ± 2%, p = 0.007) and correlated with increased accumulation of 1,2-diacyl-sn-3-glycerol substrates (p = 0.01). These results implicate defects in both PMN transendothelial migration and PMN DAG kinase α signaling as disordered functions in LAP. Moreover, they identify a potential molecular lesion in PMN signal transduction that may account for their aberrant responses and tissue destruction in this disease.

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFkappaB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-beta-induced NFkappaB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion.

Leukotrienes (LTs) and prostaglandins (PGs) amplify acute inflammation, whereas lipoxins (LXs) have unique anti-inflammatory actions. Temporal analyses of these eicosanoids in clinical and experimental exudates showed early coordinate appearance of LT and PG with polymorphonuclear neutrophil (PMN) recruitment. This was followed by LX biosynthesis, which was concurrent with spontaneous resolution. Human peripheral blood PMNs exposed to PGE2 (as in exudates) switched eicosanoid biosynthesis from predominantly LTB4 and 5-lipoxygenase (5-LO)-initiated pathways to LXA4, a 15-LO product that "stopped" PMN infiltration. These results indicate that first-phase eicosanoids promote a shift to anti-inflammatory lipids: functionally distinct lipid-mediator profiles switch during acute exudate formation to "reprogram" the exudate PMNs to promote resolution.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with ω-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 ω-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX5 and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as ω-5 and ω-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2–nonsteroidal antiinflammatory drug–dependent oxygenations and cell–cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of ω-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Asthma is characterized by chronic airway inflammation resulting from overproduction of pro-inflammatory mediators, such as leukotrienes (LT). The authors questioned the biosynthetic capacity of asthmatic patients for lipoxins (LX) and 15-epimer lipoxins (15-epi-LX), endogenous regulators of inflammatory responses that inhibit pro-inflammatory events. Levels of LXA4, 15-epi-LXA4 and LTC4 were determined in 14 clinically characterized aspirin-intolerant asthmatics (AIA), 11 aspirin-tolerant asthmatics (ATA) and eight healthy volunteers using a stimulated whole blood protocol. Both LXA4 and 15-epi-LXA4 were generated in whole blood activated by the divalent cation ionophore, A23187. Higher levels of LXA4 were produced in ATA than either AIA or healthy volunteers. Exposure of AIA whole blood to interleukin-3 prior to A23187 did not elevate their reduced capacity to generate LXA4. Generation of a bronchoconstrictor, LTC4, was similar in both AIA and ATA. Consequently, the ratio of LXA4:LTC4 quantitatively favoured the bronchoconstrictor for AIA and differed from both ATA and healthy subjects. In addition, the capacity for 15-epi-LXA4 generation was also diminished in AIA, since whole blood stimulated in the presence of aspirin gave increased levels only in samples from ATA. The present results indicate that asthmatics possess the capacity to generate both lipoxins and 15-epimer-lipoxins, but aspirin-intolerant asthmatics display a lower biosynthetic capacity than aspirin-tolerant asthmatics for these potentially protective lipid mediators. This previously unappreciated, diminished capacity for lipoxin formation by aspirin-intolerant asthmatic patients may contribute to their more severe clinical phenotype, and represents a novel paradigm for the development of chronic inflammatory disorders.

Neutrophil-dependent vascular injury gives rise to increased vascular permeability, edema, and further release of chemoattractants. Leukotriene B 4 (LTB 4 ) is among the most potent neutrophil stimuli and thus participates in tissue injury via recruiting neutrophils (PMNs, polymorphonuclear leukocytes) in pathophysiologic scenarios (1). Lipoxins are trihydroxytetraene-containing eicosanoids that are, among other in vivo sites, also generated within vascular lumen primarily by platelet-leukocyte interactions by pathways (Figure 1) that are activated during multicellular responses such as inflammation, atherosclerosis, and thrombosis (for a review, see Reference 2). In addition, aspirin has been found to trigger the transcellular biosynthesis of a new group of compounds termed 15-epi-lipoxins (15-epi-LXs) or aspirin-triggered lipoxins, which are likely to contribute to some of the beneficial actions ascribed to aspirin by serving as local endogenous antiinflammatory lipid mediators (2). Thus, these LX branches illustrated in Figure 1 involve cell-cell interactions that appear to produce endogenous stop signals or antiinflammatory compounds, while the 5-lipoxygenase pathway generates leukotrienes that evoke cellular events that are considered to be proinflammatory.

Multicellular host responses to infection, injury or inflammatory stimuli lead to the formation of lipid mediators (LMs) by the host (Cotran et al. 1994). The host response depends on a complex set of events that initiate inflammation and appear to be aimed at resolution.

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators, inhibiting prostanoid production and initiating 15-epi-lipoxin generation through the acetylation of cyclooxygenase II. These aspirin-triggered lipoxins (ATL) may mediate some of ASA’s beneficial actions and therefore are of interest in the search for novel antiinflammatories that could manifest fewer unwanted side effects. Here, we report that design modifications to native ATL structure prolong its biostabilityin vivo. In mouse whole blood, ATL analogs protected at carbon 15 [15(R/S)-methyl-lipoxin A4(ATLa1)] and the omega end [15-epi-16-(para-fluoro)-phenoxy-LXA4(ATLa2)] were recoverable to ≈90 and 100% at 3 hr, respectively, compared with a ≈40% loss of native lipoxin A4. ATLa2retains bioactivity and, at levels as low as ≈24 nmol/mouse, potently inhibited tumor necrosis factor-α-induced leukocyte recruitment into the dorsal air pouch. Inhibition was evident by either local intra-air pouch delivery (≈77% inhibition) or systemic delivery by intravenous injection (≈85% inhibition) and proved more potent than local delivery of ASA. Rank order for inhibiting polymorphonuclear leukocyte infiltration was: ATLa2(10 μg, i.v.) ≈ATLa2(10 μg, local) ≈dexamethasone (10 μg, local) >ASA (1.0 mg, local). Applied topically to mouse ear skin, ATLa2also inhibited polymorphonuclear leukocyte infiltration induced by leukotriene B4(≈78% inhibition) or phorbol ester (≈49% inhibition), which initiates endogenous chemokine production. These results indicate that this fluorinated analog of natural aspirin-triggered lipoxin A4is bioavailable by either local or systemic delivery routes and is a more potent and precise inhibitor of neutrophil accumulation than is ASA.

Neutrophil (PMN) activation is critical in inflammation and reperfusion injury, suggesting that PMN-directed therapies may be of clinical use. Here, leukotriene B4 (LTB4)-induced PMN influx in ear skin was equivalent between 5-lipoxygenase knockout and wild-type mice. To explore actions of lipoxin (LX) in PMN-mediated tissue injury, we prepared several novel LX stable analogues, including analogues of LXA4 and aspirin-triggered 15-epi-LXA4 as well as LXB4, and examined their impact in PMN infiltration and vascular permeability. Each applied topically to mouse ears inhibited dramatically PMN-mediated increases in vascular permeability (IC50 range of 13-26 nmol) with a rank order of 15(R/S)-methyl-LXA4 > 16-para-fluoro-phenoxy-LXA4 approximately 5(S)-methyl-LXB4 >/= 16-phenoxy-LXA4 > 5(R)-methyl-LXB4. These LX mimetics were as potent as an LTB4 receptor antagonist, yet results from microphysiometry with mouse leukocytes indicated that they do not act as LTB4 receptor level antagonists. In addition, within 24 h of delivery, > 90% were cleared from ear biopsies. Neither IL-8, FMLP, C5a, LTD4, nor platelet-activating factor act topically to promote PMN influx. When applied with LTB4, PGE2 enhanced sharply both infiltration and vascular permeability, which were inhibited by a fluorinated stable analogue of aspirin-triggered LX. These results indicate that mimetics of LXs and aspirin-triggered 15-epi-LXA4 are topically active in this model and are potent inhibitors of both PMN infiltration and PMN-mediated vascular injury.

Detection of microbial products by host inflammasomes is critical for innate immune surveillance, while eicosanoids, signaling lipids that include the prostaglandins and leukotrienes, are rapid triggers of inflammation. Here we report for the first time a link between inflammasome activation and eicosanoid synthesis. Using a novel protein toxin, we show that systemic cytosolic delivery of bacterial flagellin in vivo leads to loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (

The hallmark of pneumonia caused by Streptococcus pneumoniae (pneumococcus) is massive recruitment of polymorphonuclear cells (PMNs) from the circulation into lung alveolar spaces, causing considerable tissue damage. Moreover, pneumococcal translocation across lung epithelium into blood can lead to potentially lethal septicemia. The PMN influx caused by the bacterial insult, might lead to the disruption of the barrier function of the respiratory epithelium, thus contributing to the transepithelial migration of S. pneumoniae into the bloodstream. Our results show that S. pneumoniae triggered PMN migration from basolateral to apical side of polarized monolayers of lung epithelial cell line H292, and this PMN migration was associated with elevated levels of bacterial migration in the reverse direction. HPLC/MS studies of infected H292 culture supernatants showed increased levels of hepoxilin A3 (HXA3), a 12-LOX-derived eicosanoid that can act as a PMN chemoattractant. Administration of a 12-LOX inhibitor significantly reduced both pneumococcus-induced PMN migration and translocation of bacteria itself. Interestingly, 12-LOX knockout mice, or wild type mice treated with a 12-LOX inhibitor, showed diminished lung inflammation and bacteremia, and showed 100% survival in an otherwise lethal lung challenge. Our results indicate that during S. pneumoniae lung infection, HXA3 plays important roles in both pulmonary inflammation and bacterial invasion of the bloodstream.

The successful execution of an acute inflammatory response is nowhere as critical and evolved as in the delicate visual axis because uncontrolled ocular inflammation impairs vision and leads to blindness. In particular, the transparent, avascular, and immune-privileged cornea has a tightly regulated and remarkable inflammatory/reparative response that is highly dependent on the generation and release of specific and temporally defined arrays of mediators.1–4 In this regard, lipid autacoids such as arachidonic acid (AA)-derived cytochrome P450 metabolites, lipoxins, and hydroxyeicosatetraenoic acids (HETEs) have emerged as key mediators of corneal inflammation and pathologic angiogenesis and the resolution of these responses.3,5–7 Lipid autacoids are formed in most tissues and are essential regulators of inflammatory and immune responses.1,8–11 Recent findings have uncovered a critical role for resident protective lipid circuits, namely 12/15-lipoxygenase (12/15-LOX; Alox15), in the cornea. 12/15 LOX is highly expressed in mouse and human corneas12,13 and generates AA- and ω-3 polyunsaturated fatty acid-derived mediators (i.e., lipoxins, resolvins, and protectins) that inhibit inflammation and pathologic angiogenesis and promote wound healing.7,13–15 This emerging field underscores the importance of resident endogenous lipid autacoid circuits in the eye to maintain and execute essential acute inflammatory responses. Cyclooxygenase (COX)-derived prostaglandins (PG) are of primary interest because they are critical and early response mediators that initiate or amplify inflammation. In particular, PGE2 has traditionally been identified as a prevalent inflammatory mediator in many tissues and inflammatory diseases.10,16 More recently, it has been implicated in tumor-driven angiogenesis.17 Not surprisingly, it is a primary target of several important classes of clinically used drugs (i.e., nonsteroidal anti-inflammatory drugs [NSAIDs] and corticosteroids) for inflammation, pain, and colon cancer. PGE2 is produced by many cells of the body, including fibroblasts, macrophages/monocytes, dendritic cells, and some types of malignant cells, and it exerts its effects through binding to G-protein–coupled receptors (EP). Four EP receptor subtypes have been identified—EP-1, EP-2, EP-3, and EP-4—which are coupled to different intracellular signal transduction pathways.18,19 EP-2 and EP-4 receptors are coupled to Gs and mediate increases in cyclic adenosine monophosphate (cAMP) concentrations, whereas activation of EP-3 inhibits adenylate cyclase through Gi; EP-1 stimulates intracellular Ca2+ mobilization through Gq. Hence, the distribution and relative expression of these four receptor subtypes provide an elegant system that can account for the ability of PGE2 to evoke pleiotropic and sometimes opposing bioactions that are tissue- and cell type–specific.18–20 Although PGE2 induces reactions that give rise to the initial cardinal signs of inflammation (i.e., edema),21 it can also inhibit inflammation by downregulating host responses. PGE2 is a negative regulator of neutrophil, monocyte, and lymphocyte function.10,11,18 Specifically, PGE2 has been shown to negatively regulate allergic reactions, including contact hypersensitivity, conjunctivitis, and asthma, through EP-3 in several murine experimental models, and, through EP-4, to maintain intestinal homeostasis by downregulation of the immune response.22–25 Moreover, PGE2 decreases macrophage production of proinflammatory cytokines such as TNF-α26 and stimulates anti-inflammatory cytokine production,27 mechanisms that both contribute to the resolution of inflammation. PGE2 can also promote the resolution of inflammation through the induction of key enzymes, namely 12/15-LOX, in polymorphonuclear leukocytes (PMNs), and the formation of the anti-inflammatory and pro-resolving lipoxin A4.28 Hence, the role of PGE2 in inflammatory and immune responses is complex; it is temporally defined and largely dependent on EP receptor expression. The capacity of ocular tissues to synthesize PGE2 was initially determined on the basis of 14C-AA conversion in vitro and in vivo.29–31 Subsequently, studies that used NSAIDs, PGE2, and PGE2 analogs, as well as EP receptor knockout mice, have demonstrated that in the eye, this prostanoid causes miosis, vasodilation, disrupts the integrity of the blood-aqueous barrier, and, through regulating the dynamics of aqueous humor flow, can increase or reduce intraocular pressure.32–35 Importantly, NSAIDs are a primary treatment option for ocular indications including the inhibition of intraoperative miosis and the management of postoperative inflammation and pain. However, in contrast to other tissues and despite reports of COX-2 upregulation and PG formation in models of ocular inflammation,3 the endogenous role of this prominent prostanoid and primary therapeutic target in the corneal inflammatory/reparative response, surprisingly, remains to be fully defined. To this end, we assessed the role of the PGE2 circuit in mediating inflammatory responses in the cornea. Here, we report that endogenous PGE2 in the cornea is not a general inflammatory mediator. Increased PGE2 formation and upregulation of EP-2 and EP-4 receptor expression were key features of a severe and chronic injury that was associated with inflammatory neovascularization but not a feature of acute self-resolving inflammation. Moreover, topical PGE2 treatment selectively amplified leukocyte recruitment and pathologic angiogenesis in response to chronic inflammation but did not affect wound healing or leukocyte recruitment during acute epithelial injury, providing evidence for a selective role of the PGE2 circuit in mediating corneal inflammatory responses.