Lipid Mediators in Inflammation

Lipids are potent signaling molecules that regulate a multitude of cellular responses, including cell growth and death and inflammation/infection, via receptor-mediated pathways. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. This diversity arises from their synthesis, which occurs via discrete enzymatic pathways and because they elicit responses via different receptors. This review will collate the bioactive lipid research to date and summarize the major pathways involved in their biosynthesis and role in inflammation. Specifically, lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins, and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins, and maresins) will be discussed herein

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances. Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a 'sepsis-like' state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations

A Comparison of Human Neutrophils Acquired from Four Experimental Models of Inflammation

Defects in neutrophil function have been implicated in a wide spectrum of clinical conditions. Several models are employed to study activated human neutrophils akin to those found at a site of inflammation. These include whole blood (WB) ex vivo stimulation with lipopolysaccharide (LPS) and in vivo techniques: cantharidin blister, skin windows and intra-dermal injection of UV-killed E.coli (UVKEc). Neutrophils obtained from these have never been compared. We compared the activation status of neutrophils from each technique in order to inform the optimal model for use in human studies. Healthy male volunteers were randomised to undergo one of the four techniques (n = 5/group). LPS: WB stimulated with 1ng/ml of LPS for 4 hours. Cantharidin: 12.5μl of 0.1% cantharidin elicited a single blister, aspirated at 24 hours. Skin windows: four 6mm mechanical-suction blisters created, de-roofed and an exudate-collection chamber placed over the windows for 4 hours before aspiration. UVKEc: 1.5 x 107 UVKEc injected intra-dermally. A single 10mm mechanical-suction blister formed and aspirated at 4 hours. Unstimulated WB used as the control. Flow cytometry was used to determine activation status using CD16, CD11b, CD54, CD62L and CD88. Functional status was assessed with a phagocytosis assay. The pattern of neutrophil activation was similar in all models. Neutrophil CD11b was elevated in all models, most markedly in UVKEc (p<0.0001), and CD54 was also elevated but only significant in the LPS model (p = 0.001). CD62L was significantly reduced in all 4 models (p<0.0001) and CD88 was also suppressed in all. There were no changes in CD16 in any model, neither was there any significant difference in the phagocytic capacity of the neutrophils. In summary, there are no significant differences in activation marker expression or phagocytic capacity in the neutrophils obtained from each technique. Therefore we believe whole blood stimulation is the best model in experimentally challenging inpatient populations

Lipid mediators in immune dysfunction after severe inflammation.

Sepsis, trauma, burns, and major surgical procedures activate common systemic inflammatory pathways. Nosocomial infection, organ failure, and mortality in this patient population are associated with a quantitatively different reprioritization of the circulating leukocyte transcriptome to the initial inflammatory insult, greater in both magnitude and duration, and secondary to multiple observed defects in innate and adaptive immune function. Dysregulation of inflammatory resolution processes and associated bioactive lipid mediators (LMs) mechanistically contribute to this phenotype. Recent data indicate the potential efficacy of therapeutic interventions that either reduce immunosuppressive prostaglandins (PGs) or increase specialized proresolving LMs. Here, we reassess the potential for pharmacological manipulation of these LMs as therapeutic approaches for the treatment of critical illness (CI)

Novel translational model of resolving inflammation triggered by UV-killed E. coli

Whilst numerous studies investigating the aetiology of inflammatory diseases have been performed in rodents, the applicability of these data to human pathophysiology is frequently debated. Regardless of the strengths and weaknesses of rodent models in biomedical research, there is a need to develop models of experimental inflammation in humans. Here, we describe a self-resolving acute inflammatory response triggered by the intradermal injection of UV-killed Escherichia coli into the forearm of healthy volunteers. Cells and exudates were harvested from onset to resolution by applying negative pressure over the inflamed site. Onset was characterized by high blood flow, neutrophilia and peak levels of pro-inflammatory cytokines, whilst resolution showed a decline in blood blow, reduction in neutrophils, increase in monocytes/macrophages and waning of classic pro-inflammatory cytokine levels. An anti-inflammatory effect, defined as suppression of onset phase events, was demonstrated by administering naproxen, a conventional non-steroidal anti-inflammatory drug. In summary, this model of resolving acute inflammation is minimally invasive, highly tractable and allows simultaneous investigation of the vascular response, cellular trafficking and chemical mediator profile of onset and resolution phases of acute inflammation in humans. It can serve as a translational platform to provide mechanistic insights and to test the clinical efficacy of novel anti-inflammatory and pro-resolving drugs, and also as a tool in patients to explore inherent defects in resolution pathways

Potent anti-inflammatory effects of an H2 S-releasing naproxen (ATB-346) in a human model of inflammation

ATB-346 is a hydrogen sulfide-releasing non-steroidal anti-inflammatory drug (H2 S-NSAID) derived from naproxen, which in preclinical studies has been shown to have markedly reduced gastrointestinal adverse effects. However, its anti-inflammatory properties in humans compared to naproxen are yet to be confirmed. To test this, we used a dermal model of acute inflammation in healthy, human volunteers, triggered by ultraviolet-killed Escherichia coli. This robust model allows quantification of the cardinal signs of inflammation along with cellular and humoral factors accumulating within the inflamed skin. ATB-346 was non-inferior to naproxen in terms of its inhibition of cyclooxygenase activity as well as pain and tenderness. ATB-346 significantly inhibited neutrophil infiltration at the site of inflammation at 4 h, compared to untreated controls. Subjects treated with ATB-346 also experienced significantly reduced pain and tenderness compared to healthy controls. Furthermore, both classical and intermediate monocyte subsets infiltrating the site of inflammation at 48 h expressed significantly lower levels of CD14 compared to untreated controls, demonstrating a shift toward an anti-inflammatory phenotype. Collectively, we have shown for the first time in humans that ATB-346 is potently anti-inflammatory and propose that ATB-346 represents the next generation of H2 S-NSAIDs, as a viable alternative to conventional NSAIDs, with reduced adverse effects profile

Monocyte dysfunction in decompensated cirrhosis is mediated by the prostaglandin E2-EP4 pathway

Background & Aims: Infection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction. Methods: Using samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE2 antagonists in whole blood using polychromatic flow cytometry and cytokine production. Results: We show that hepatic production of PGE2 via the cyclo-oxygenase 1–microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)–DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE2 mediated this dysfunction via its EP4 receptor. Conclusions: PGE2 mediates monocyte dysfunction in decompensated cirrhosis via its EP4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission. Lay summary: Patients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E2, which is present at elevated levels, via its EP4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation