Anti-inflammatory Lipid Mediators Research Articles

Effect of BAL Lipoxin A4 Level in Children with Bronchiectasis on Disease Severity

Abstract Background A central feature of most cases of bronchiectasis is airway inflammation. The elevated neutrophil count has been strongly correlated with impaired lung function, bronchiectasis severity, and disease duration. There is a failure of resolution of inflammation in bronchiectasis, leading to irreversible damage and dilatation of the bronchial airways with loss of mucociliary function. Lipoxins were the first agents to be identified as anti-inflammatory endogenous lipid mediators involved in the resolution of inflammation. Aim of the Work This study aimed to measure Lipoxin A4 level in bronchoalveolar lavage (BAL) of children with Cystic Fibrosis bronchiectasis (CF) and Non-CF bronchiectasis as a prognostic marker for clinical and radiological severity in these children as regard Bronchiectasis severity score. Methods A cross-sectional study was conducted at Children’s Hospital, Ain Shams University from December 2020 to December 2021, on children following up at the Pediatric Pulmonology Clinic. The study included 15patients with cystic fibrosis (CF) bronchiectasis, 15 patients with non-CF bronchiectasis, and 30 control cases,without breathing difficulty or signs of RDS. Human Lipoxin A4 was measured in BAL by ELISA. Results There was a significant difference between patients and controls as regards Lipoxin A4 level (p = 0.002),being higher in control group, but no significant difference in its level between CF and non-CF bronchiectasischildren(p = 0.663). Conclusion Severe bronchiectasis is associated with decreased airway Lipoxin A4 levels as lowest level of lipoxinA4 was found in severe cases by bronchiectasis severity score.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

The Effect of Acute Knee Injuries and Related Knee Surgery on Serum Levels of Pro- and Anti-inflammatory Lipid Mediators and Their Associations With Knee Symptoms

Background: Despite an acute knee injury being a major risk factor for osteoarthritis, the factors that initiate and maintain this risk of longer-term knee symptoms are poorly understood. Bioactive lipids derived from omega-3 and -6 polyunsaturated fatty acids have key roles in the regulation of the inflammatory response and have been linked to joint damage and osteoarthritis pain in translational models. Hypothesis: There would be associations between systemic levels of bioactive lipids and knee symptoms longitudinally after an acute knee injury and related knee surgery. Study Design: Controlled laboratory study. Methods: This study analyzed a subset of young, active adults who had sustained an acute knee injury (recruited via a surgical care pathway) and healthy age- and sex-matched controls. Surgery, if performed, was conducted after the baseline serum sample was taken and before the 3-month and 2-year visits. Liquid chromatography–tandem mass spectrometry of 41 bioactive lipids was carried out in sera of (1) 47 injured participants (median age, 28 years) collected at baseline (median, 24 days after injury), 3 months, and 2 years, along with the Knee injury and Osteoarthritis Outcome Score, and (2) age- and sex-matched controls. Results: Levels of the omega-3 polyunsaturated fatty acids eicosapentaenoic acid (P≤ .0001) and docosahexaenoic acid (P≤ .0001) and the pro-resolving lipid mediators 17– and 14–hydroxydocosahexaenoic acid, and 18-hydroxyeicosapentaenoic acid were all significantly greater at baseline in injured participants compared with the later time points and also higher than in healthy controls (P = .0019 and P≤ .0001, respectively). Levels of pro-inflammatory prostaglandins E2 and D2, leukotriene B4, and thromboxane B2 were significantly lower at baseline compared with the later time points. Higher levels of 8,9–, 11,12–, and 14,15–dihydroxyeicosatrienoic acid (DHET) were cross-sectionally associated with more severe knee pain/symptoms according to the Knee injury and Osteoarthritis Outcome Score at 2 years (P = .0004, R2 = 0.251; P = .0002, R2 = 0.278; and P = .0012, R2 = 0.214, respectively). Conclusion: The profile of pro-resolving versus pro-inflammatory lipids at baseline suggests an initial activation of pro-resolution pathways, followed by the later activation of pro-inflammatory pathways. Clinical Relevance: In this largely surgically managed cohort, the association of soluble epoxide hydrolase metabolites, the DHETs, with more severe knee symptoms at 2 years provides a rationale for further investigation into the role of this pathway in persisting knee symptoms in this population, including potential therapeutic strategies.

Lipidomic analysis of microfat and nanofat reveal different lipid mediator compositions.

Microfat and nanofat are two commonly used techniques in various surgical procedures from skin rejuvenation to scar correction that are known to contribute to tissue regeneration. While microfat mainly contains adipocytes and is well suited for tissue augmentation, nanofat is rich in lipids, adipose derived stem cells, microvascular fragments, and growth factors, making it attractive for esthetic use. We have previously demonstrated that the mechanical processing of microfat into nanofat significantly changes its proteomic profile. Considering that mechanical fractionation leads to adipocyte disruption and lipid release, we aimed to analyze the lipidomic profile for its regenerative properties. Microfat and nanofat samples were isolated from fourteen healthy patients. Lipidomic profiling was performed by liquid chromatography tandem mass spectrometry. Resulting data was compared against the Human Metabolome and LIPID MAPS® Structure Database. Metaboanalyst was used to analyze metabolic pathways and lipids of interest. From 2,388 mass-to-charge ratio features, metabolic pathway enrichment analysis between microfat and nanofat samples revealed 109 pathways that were significantly enriched. While microfat samples revealed higher intensity levels of sphingosines, different eicosanoids and fat-soluble vitamins, increased levels of coumaric acids and prostacyclin were found in nanofat. This is the first study that has analyzed the lipidomic profile of micro- and nanofat, providing evidence that mechanical emulsification of microfat into nanofat leads to changes in their lipid profile. From 109 biological pathways, anti-inflammatory, anti-fibrotic and anti-melanogenic lipid mediators were particularly enriched in nanofat samples when compared to microfat. Although further studies are necessary to have a deeper understanding on the composition of these specific lipid mediators in nanofat samples, we propose that they might contribute to its regenerative effects on tissue.

Silymarin and Inflammation: Food for Thoughts.

Inflammation is a vital defense mechanism, creating hostile conditions for pathogens, preventing the spread of tissue infection and repairing damaged tissues in humans and animals. However, when inflammation resolution is delayed or compromised as a result of its misregulation, the process proceeds from the acute phase to chronic inflammation, leading to the development of various chronic illnesses. It is proven that redox balance disturbances and oxidative stress are among major factors inducing NF-κB and leading to over-inflammation. Therefore, the anti-inflammatory properties of various natural antioxidants have been widely tested in various in vitro and in vivo systems. Accumulating evidence indicates that silymarin (SM) and its main constituent silibinin/silybin (SB) have great potential as an anti-inflammation agent. The main anti-inflammatory mechanism of SM/SB action is attributed to the inhibition of TLR4/NF-κB-mediated signaling pathways and the downregulated expression of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-12, IL-23, CCL4, CXCL10, etc. Of note, in the same model systems, SM/SB was able to upregulate anti-inflammatory cytokines (IL-4, IL-10, IL-13, TGF-β, etc.) and lipid mediators involved in the resolution of inflammation. The inflammatory properties of SM/SB were clearly demonstrated in model systems based on immune (macrophages and monocytes) and non-immune (epithelial, skin, bone, connective tissue and cancer) cells. At the same time, the anti-inflammatory action of SM/SB was confirmed in a number of in vivo models, including toxicity models, nonalcoholic fatty liver disease, ischemia/reperfusion models, stress-induced injuries, ageing and exercising models, wound healing and many other relevant model systems. It seems likely that the anti-inflammatory activities of SM/SB are key elements on the health-promoting properties of these phytochemicals.

Bioactive Lipid Mediators Predict Platelet Function in Transfusion Recipients: A Phase 1 Randomized Clinical Trial in Healthy Humans

Background: Platelets are stored at room temperature for 5-7 days (RSP) and are transfused to patients who are bleeding or at risk of bleeding. Due to frequent and severe shortages, the FDA recently approved cold-stored platelets in plasma (CSP) for up to 14 days despite a lack of post-transfusion data. It is also unclear which donors are best suited to provide platelets for either RSP or CSP and which in vitro markers accurately predict post-transfusion RSP or CSP platelet function. Objective: To evaluate the post-transfusion function and predictors of post-transfusion function of platelets stored for the maximum approved storage times (7-day RSP, 14-day CSP) in healthy volunteers on acetylsalicylic acid (ASA). Methods: We conducted a randomized cross-over study in ten healthy humans. Subjects on ASA were randomized to receive either autologous RSP or CSP first. We obtained blood from recipients before and after transfusion for platelet function testing and samples from the storage bag before and after storage for lipid metabolite analysis. The first round was followed by a second round with transfusion of the alternative product and the same testing sequence. Results: RSP reversed platelet inhibition significantly better in αIIbβ3 integrin activation-dependent assays. In contrast, CSP led to significantly more thrombin generation in recipients. Overall, RSP contained more pro-inflammatory lipid mediators while CSP had more anti-inflammatory lipid mediators. In particular, LPC species including LPC-O species (formerly known as Lyso-platelet activating factor [LPAF]), LPE, and LPI were significantly more abundant in RSP than in CSP, while LPS was more abundant in CSP than in RSP. Similarly, the relative increase of LPC, LPE, and LPI, and linoleic acid-dependent oxylipins, such as diHOMEs and HODEs were higher in RSP recipients than in CSP recipients. Linoleic acid-dependent oxylipins correlated positively with platelet aggregation in units and recipients of RSP and CSP. Polyunsaturated fatty acids (PUFAs) showed a negative correlation with platelet aggregation in both RSP and CSP units and recipients. Most importantly, LPC-O (LPAF) species showed a strong negative correlation with endogenous thrombin generation potential in CSP units after collection (pre-storage), post-storage, and in transfusion recipients. Conclusion: Results from our trial provide the first efficacy data of autologous and extended-stored CSP in plasma. We report the first correlational data between unit and recipient lipid mediators and post-transfusion platelet function. Taken together, we show that the main mechanism of post-transfusion CSP function appears to be based on procoagulant properties. In the future, testing platelet donors for LPC-O (LPAF) species could identify donors whose platelets are better suited for cold storage than room-temperature storage.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

Promising Anti-Inflammatory Tools: Biomedical Efficacy of Lipoxins and Their Synthetic Pathways.

Lipoxins (LXs) have attracted widespread attention as a class of anti-inflammatory lipid mediators that are produced endogenously by the organism. LXs are arachidonic acid (ARA) derivatives that include four different structures: lipoxin A4 (LXA4), lipoxin B4 (LXB4), and the aspirin-induced differential isomers 15-epi-LXA4 and 15-epi-LXB4. Because of their unique biological activity of reducing inflammation in the body, LXs have great potential for neuroprotection, anti-inflammatory treatment of COVID-19, and other related diseases. The synthesis of LXs in vivo is achieved through the action of lipoxygenase (LO). As a kind of important enzyme, LO plays a major role in the physiological processes of living organisms in mammals and functions in some bacteria and fungi. This suggests new options for the synthesis of LXs in vitro. Meanwhile, there are other chemical and biochemical methods to synthesize LXs. In this review, the recent progress on physiological activity and synthetic pathways of LXs is summarized, and new insights into the synthesis of LXs in vitro are provided.

Novel thiazolopyridine derivatives of diflapolin as dual sEH/FLAP inhibitors with improved solubility

Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.

Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane.

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators-palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)-in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3-5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis invitro and invivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators invivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

The relationship between unique gut microbiome-derived lipid metabolites and subsequent revascularization in patients who underwent percutaneous coronary intervention

Background and aimsStudies have recently revealed the linoleic acid metabolic pathway of Lactobacillus plantarum, the representative gut bacterium in human gastrointestinal tract, and the anti-inflammatory effects of metabolites in this pathway. However, no clinical trials have evaluated the association between these metabolites and revascularization in patients who underwent percutaneous coronary intervention (PCI). MethodsWe retrospectively reviewed patients who underwent PCI with subsequent revascularization or coronary angiography (CAG) without revascularization. Patients with frozen blood samples at the index PCI and revascularization or follow-up CAG were enrolled. ResultsAmong 701 consecutive patients who underwent PCI, we enrolled 53 patients who underwent subsequent revascularization and 161 patients who underwent follow-up CAG without revascularization. Patients who underwent revascularization showed significantly lower plasma 10-oxo-octadecanoic acid (KetoB) levels (720.5 [551.6–876.5] vs. 818.4 [641.1–1103.6 pg/mL]; p = 0.01) at index PCI. Multivariate logistic regression analysis revealed that decreased plasma KetoB levels at the index PCI were independently associated with subsequent revascularization after PCI (odds ratio; 0.90 per 100 pg/mL increase, 95% confidence interval; 0.82–0.98). Additionally, in vitro experiments showed that the addition of purified KetoB suppressed the mRNA levels of IL-6 and IL-1β in macrophages and IL-1β mRNA in neutrophils. ConclusionsPlasma KetoB level at index PCI was independently associated with subsequent revascularization after PCI, and KetoB could act as an anti-inflammatory lipid mediator in macrophages and neutrophils. The assessment of gut microbiome-derived metabolites may help predict revascularization after PCI.

Linking platelet lipidome and inflammatory response in septic patients

Sepsis is characterized by major endothelial dysfunction, microvascular alterations, and coagulopathy. Platelets are important players in sepsis as they promote thrombo-inflammation and generate lipidic mediators of inflammation. Previously, we observed that platelet acetyl-CoA carboxylase 1 (ACC1), responsible for de novo lipid synthesis, is phosphorylated and thus inhibited by AMP-activated protein kinase (AMPK) upon thrombin stimulation, leading to platelet lipidome changes. The role of the AMPK-ACC signaling in the regulation of platelet lipidome during sepsis, and its potential impact on inflammation, has never been investigated. To investigate whether changes in ACC phosphorylation can influence the platelet lipidome and the inflammatory response of patients during sepsis. Platelets and plasmas were isolated from 48 septic and 48 control patients. In parallel, WT and DKI mice injected intraperitoneally with lipopolysaccharides were used as a model of endotoxemia. Platelets ACC1 phosphorylation/expression levels were measured by western blot. Lipidomics analysis was carried out by untargeted liquid chromatography–mass spectrometry or on the commercial Lipidyzer platform. Plasma factors were measured by Enzyme-linked immunosorbent assays (ELISA). Platelet ACC1 expression and phosphorylation were increased both in septic patients and in septic mice. Lipidomics analysis highlighted significant changes of lipids in human and murine platelets during sepsis, with a substantial reduction in phosphatidylcholines and phosphatidylethanolamines containing long polyunsaturated fatty acid chains (ω3 and ω6), which are key phospholipids for the generation of pro- and anti-inflammatory lipid mediators. Our preliminary data obtained in humans and mice, suggest that elevated ACC phosphorylation in sepsis is associated with a higher content of these phospholipids as well as a plasma reduction in myeloid inflammatory parameters. Our data reveal that critical changes in the platelet lipidome occur during sepsis and may contribute to the pathophysiology of the disease. Our results also suggest that ACC can influence the platelet lipid content and/or composition.

Serum lipid mediator profiles in COVID-19 patients and lung disease severity: a pilot study

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is highly heterogeneous, ranging from asymptomatic to severe and fatal cases. COVID-19 has been characterized by an increase of serum pro-inflammatory cytokine levels which seems to be associated with fatal cases. By contrast, the role of pro-resolving lipid mediators (SPMs), involved in the attenuation of inflammatory responses, has been scarcely investigated, so further studies are needed to understand SPMs metabolism in COVID-19 and other infectious diseases. Our aim was to analyse the lipid mediator metabolome, quantifying pro- and anti-inflammatory serum bioactive lipids by LC–MS/MS in 7 non-infected subjects and 24 COVID-19 patients divided into mild, moderate, and severe groups according to the pulmonary involvement, to better understand the disease outcome and the severity of the pulmonary manifestations. Statistical analysis was performed with the R programming language (R Foundation for Statistical Computing, Vienna, Austria). All COVID-19 patients had increased levels of Prostaglandin E2. Severe patients showed a significant increase versus controls, mild- and moderate-affected patients, expressed as median (interquartile range), in resolvin E1 [112.6 (502.7) vs 0.0 (0.0) pg/ml in the other groups], as well as in maresin 2 [14.5 (7.0) vs 8.1 (4.2), 5.5 (4.3), and 3.0 (4.0) pg/ml, respectively]. Moreover, 14-hydroxy docosahexaenoic acid (14-HDHA) levels were also increased in severe vs control and mild-affected patients [24.7 (38.2) vs 2.4 (2.2) and 3.7 (6.4) ng/mL, respectively]. Resolvin D5 was also significantly elevated in both moderate [15.0 (22.4) pg/ml] and severe patients [24.0 (24.1) pg/ml] versus controls [0.0 (0.0) pg/ml]. These results were confirmed by sparse partial least squares discriminant analysis which highlighted the contribution of these mediators to the separation between each of the groups. In conclusion, the potent inflammatory response to SARS-CoV-2 infection involves not only pro- but also anti-inflammatory lipid mediators that can be quantified in easily accessible serum samples, suggesting the need to perform future research on their generation pathways that will help us to discover new therapeutic targets.

Specialized pro-resolving lipid mediators regulate inflammatory macrophages: A paradigm shift from antibiotics to immunotherapy for mitigating COVID-19 pandemic.

The most severe clinical manifestations of the horrifying COVID-19 disease, that claimed millions of lives during the pandemic time, were Acute respiratory distress syndrome (ARDS), Coagulopathies, septic shock leading eventually to death. ARDS was a consequence of Cytokine storm. The viral SARS-COV2infection lead to avalanche of cytokines and eicosanoids causing "cytokine storm" and "eicosanoid storm." Cytokine storm is one of the macrophage-derived inflammatory responses triggered by binding of virus particles to ACE2 receptors of alveolar macrophages, arise mainly due to over production of various pro-inflammatory mediators like cytokines, e.g., interleukin (IL)-1, IL-2, and tumor necrosis factor (TNF)- α, causing pulmonary edema, acute respiratory distress, and multi-organ failure. Cytokine storm was regarded as the predictor of severity of the disease and was deemed one of the causes of the high mortality rates due to the COVID-19. The basis of cytokine storm is imbalanced switching between an inflammation increasing - pro-inflammatory (M1) and an inflammation regulating-anti-inflammatory (M2) forms of alveolar macrophages which further deteriorates if opportunistic secondary bacterial infections prevail in the lungs. Lack of sufficient knowledge regarding the virus and its influence on co-morbidities, clinical treatment of the diseases included exorbitant use of antibiotics to mitigate secondary bacterial infections, which led to the unwarranted development of multidrug resistance (MDR) among the population across the globe. Antimicrobial resistance (AMR) needs to be addressed from various perspectives as it may deprive future generations of the basic health immunity. Specialized pro-resolving mediators (SPMs) are generated from the stereoselective enzymatic conversions of essential fatty acids that serve as immune resolvents in controlling acute inflammatory responses. SPMs facilitate the clearance of injured tissue and cell debris, the removal of pathogens, and augment the concentration of anti-inflammatory lipid mediators. The SPMs, e.g., lipoxins, protectins, and resolvins have been implicated in exerting inhibitory influence on with cytokine storm. Experimental evidence suggests that SPMS lower antibiotic requirement. Therefore, in this review potential roles of SPMs in enhancing macrophage polarization, triggering immunological functions, hastening inflammation resolution, subsiding cytokine storm and decreasing antibiotic requirement that can reduce AMR load are discussed.

P068 Polyunsaturated fatty acid excess fuels pro-inflammatory transcriptional networks in GPX4 deficient intestinal epithelial cells

Abstract Background There is increasing evidence that the rise in Crohn’s disease (CD) incidence is partially driven by constituents of a Western-style diet such as polyunsaturated fatty acids (PUFA). Recently we could demonstrate that PUFA excess induces a CD-like enteritis in mice lacking antioxidant GPX4 in their intestinal epithelial cells (IECs) and that CD patients clinical disease activity positively correlates with PUFA intake. In this project we investigated the role of epithelial lipid composition and lipid mediators during PUFA induced metabolic gut inflammation. Furthermore, we transcriptionally profiled GPX4 deficient small intestinal cells after stimulation with ω3- and ω6-PUFA. Methods Mice lacking one allele of Gpx4 in their IECs (GPX4+/-IEC) and wild-type controls (WT) were fed a PUFA (10% fish oil) enriched Western diet (PUFA-WD) for 12 weeks. Intestinal inflammation was assessed by histopathology. IECs were harvested by scraping of small intestinal sections and lipid profiles and mediators were analysed with LC/MS. GPX4 in murine small intestinal cells (MODE-K) was knocked down by siRNA (siGpx4) scrambled siRNA served as control (siCtrl). MODE-K were stimulated with ω3-, ω6-PUFA, 5,6- and 8,9-DHET or vehicle. Cytokine expression in cell culture supernatant was analysed by ELISA and transcriptome was analysed by bulk RNA sequencing. Results Histopathology revealed gut inflammation in GPX4+/-IEC but not WT mice after 12 weeks PUFA-WD feeding. LC/MS analysis of IECs derived from GPX4+/-IEC and WT mice fed the PUFA-WD depicted a significant increase in eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6) containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species (Fig 1a). All other PE and PC species showed a reduced amount, confirming the enrichment of the specific PUFAs within the IECs by the PUFA-WD. Lipid mediator analysis further revealed an increase in 5,6-DHET and 8,9-DHET only in IECs derived from GPX4+/-IEC but not WT mice (Fig 1b). When stimulated with PUFA, siGpx4 MODE-K produced high amounts of neutrophil attracting chemokine CXCL-1 which is attenuated by the supplementation of anti-inflammatory lipid mediators 5,6-DHET and 8,9-DHET (Fig 2). Transcriptome analysis revealed the upregulation of pro-inflammatory pathways including TNFa, MAPK and NF-κB in siGpx4 vs. siCtrl MODE-K after PUFA stimulation (Fig 3). Conclusion By integrating lipidomic and transcriptomic data of a reductionist cell culture system and a model organism, we could show that PUFA excess in a GPX4 deficient individual fuels a pro-inflammatory transcriptional network which outweighs anti-inflammatory ω3-PUFA metabolites influence culminating in gut inflammation.

Protectin Conjugates in Tissue Regeneration 1 Inhibits Macrophage Pyroptosis by Restricting NLRP3 Inflammasome Assembly to Mitigate Sepsis via the cAMP-PKA Pathway

Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel anti-inflammatory and proresolving lipid mediator biosynthesized from docosahexaenoic acid. Excessive activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and consequent pyroptosis are involved in diverse inflammatory diseases. However, how PCTR1 affects NLRP3 inflammasome activation and pyroptosis are still unclear. Here, we demonstrated that PCTR1 inhibited NLRP3 inflammasome activation and pyroptosis. These results show that PCTR1 dose-dependently inhibited gasdermin D cleavage in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin stimulation. Additionally, PCTR1 treatment after LPS priming inhibited caspase-1 activation and subsequent mature interleukin-1β release independent of the nuclear factor-kappa B pathway. PCTR1 exerted its inhibitory effects by blocking NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC) interaction and ASC oligomerization, thereby restricting NLRP3 inflammasome assembly. However, the inhibitory effect of PCTR1 could be reversed by KH7 and H89, which are the inhibitors of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Moreover, PCTR1 treatment alleviated lung tissue damage and improved mouse survival in LPS-induced sepsis. Our study unveils the molecular mechanism of negative regulation of NLRP3 inflammasome activation and pyroptosis by a novel lipid mediator and suggests that PCTR1 may serve as a potential treatment option for NLRP3-inflammasome driven diseases.

Lipase-catalyzed synthesis of 1,3-diacylglycerols containing stearidonic, γ-linolenic and α-linolenic acids in a solvent-free system

1,3-Diacylglycerols (1,3-DAG) are minor components in edible oils with potential for the management of overweight and obesity. In this work, 1,3-DAG containing stearidonic acid (SDA), γ-linolenic acid (GLA), and α-linolenic acid (ALA), were synthesized by lipase-mediated catalysis following a Taguchi experimental design. SDA, GLA, and ALA are polyunsaturated fatty acids (PUFA) able to act as precursors of anti-inflammatory lipid mediators in the human body and may help with prevention or relief of inflammatory diseases. Solvent-free synthesis of 1,3-DAG was successfully performed by esterifying glycerol with free fatty acids (FFA) obtained from Echium plantagineum seed oil. Crude reaction products were further purified by saponification. Two lipases (Lipozyme® RM-IM and Lipozyme® 435) and three variables (substrate molar ratio, lipase load and reaction time) were used to optimize the reaction conditions. At the optimal conditions, 88.7 mol% of DAG was produced (68.0 mol% 1,3-DAG and 20.7 mol% 1,2-DAG) using Lipozyme® 435 at 60 °C, a glycerol:FFA molar ratio of 1:3, a lipase load of 2.0 wt%, and 4 h reaction time. Proportions of SDA, GLA and ALA in the 1,3-DAG were 32.7, 32.3 and 22.0% respectively. The synthesized 1,3-DAG have potential as functional ingredients in foods for weight and obesity management.

LipoxinA4 analog BML-111 protects podocytes cultured in high-glucose medium against oxidative injury via activating Nrf2 pathway

Numerous studies have shown that the activation of the Nrf2 pathway alleviates oxidative stress and podocyte damage. Emerging evidence indicates that the dual anti-inflammatory and pro-resolution lipid mediator, lipoxin A4 (LXA4), has antioxidant activity. The aim of the present study was to confirm that BML-111, an analog of LXA4, prevents oxidative injury in diabetic podocytes via the regulation of the Nrf2 pathway. Here, we found that BML-111 inhibited high glucose (HG)-induced oxidative injury in the podocyte cell line, MPC5, in vitro, through activating Nrf2. Mechanistically, BML-111 significantly activated Nrf2 and its phase II enzymes, including Nqo1 and Ho-1. Moreover, BML-111 suppressed the migration of MPC5 cells. Additionally, BML-111 decreased the expression of Vcam, Icam and inflammatory cytokines (Il-1α, Il-6, and Tnf) in MPC5 cells. Importantly, BML-111 ameliorated blood glucose levels (approximately 75% of that in the SMZ group) and kidney damage by activating Nrf2, and its phase II enzymes, in diabetic mice. These effects are mainly mediated by Fpr2, a specific LXA4 receptor. Our findings demonstrate that BML-111 alleviates the injury of diabetic podocytes and kidneys by regulating the Nrf2 pathway.

A dose-response study with omega-3 rich canola oil as a novel source of docosahexaenoic acid (DHA) in feed for Atlantic salmon (Salmo salar) in seawater; effects on performance, tissue fatty acid composition, and fillet quality

Long-chain omega-3 rich oils from crops genetically modified with algal genes are a promising new source of the omega-3 fatty acid DHA (docosahexaenoic acid; 22:6n-3) in Atlantic salmon diets. In this study, the fish oil and fish meal content were held constant across diets, and conventional canola oil was replaced with DHA canola (DHA-CA) oil in a step-wise manner at 0%, 25%, 50%, and 100% replacement, respectively. Growth of salmon, reared in seawater, from approximately 465 g to 1.5 kg was equal across all dietary treatments. Survival was >98% in all cases, with no difference between the dietary groups. Digestibility of lipids was high, approximately 93% in all dietary groups. Fatty acid retention in whole body was also high, with greater retention of α-linolenic acid (ALA, 18:3n-3) and eicosapentaenoic acid (EPA; 20:5n-3) in groups containing higher proportions of DHA-CA oil. Fillet, liver, and intestinal fatty acid compositions were influenced by the different fatty acid contents of the diets, with progressively higher inclusion of ALA, EPA, and DHA and a higher n-3:n-6 ratio in fish fed the 25%, 50%, and 100% DHA-CA diets. Increased dietary inclusion levels of DHA reduced the conversion to 1-14C labelled ALA to 1-14C EPA in liver cells, but some conversion of ALA to 1-14C EPA was maintained even at high dietary DHA levels. There was also a significant increase in anti-inflammatory pro-resolving lipid mediators, resolvins, with increased dietary DHA-CA. The visual colour impression of both muscle and skin was improved at higher DHA-CA inclusion levels in diets.Overall, our findings demonstrates that DHA-CA oil is an effective ingredient in Atlantic salmon diets in seawater, and that fatty acids (FA) from this oil are incorporated into the fish tissues in a manner equivalent to FA from conventional sources, with positive effects on fillet quality.