15-epi-lipoxin A5 promotes neutrophil exit from exudates for clearance by splenic macrophages.

Specialized proresolving mediators (SPMs) promote local macrophage efferocytosis but excess leukocytes early in inflammation require additional leukocyte clearance mechanism for resolution. Here, neutrophil clearance mechanisms from localized acute inflammation were investigated in mouse dorsal air pouches. 15-HEPE (15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid) levels were increased in the exudates. Activated human neutrophils converted 15-HEPE to lipoxin A5 (5S,6R,15S-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), 15-epi-lipoxin A5 (5S,6R,15R-trihydroxy-7E,9E,11Z,13E,17Z-eicosapentaenoic acid), and resolvin E4 (RvE4; 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid). Exogenous 15-epi-lipoxin A5, 15-epi-lipoxin A4 and a structural lipoxin mimetic significantly decreased exudate neutrophils and increased local tissue macrophage efferocytosis, with comparison to naproxen. 15-epi-lipoxin A5 also cleared exudate neutrophils faster than the apparent local capacity for stimulated macrophage efferocytosis, so the fate of exudate neutrophils was tracked with CD45.1 variant neutrophils. 15-epi-lipoxin A5 augmented the exit of adoptively transferred neutrophils from the pouch exudate to the spleen, and significantly increased splenic SIRPa+ and MARCO+ macrophage efferocytosis. Together, these findings demonstrate new systemic resolution mechanisms for 15-epi-lipoxin A5 and RvE4 in localized tissue inflammation, which distally engage the spleen to activate macrophage efferocytosis for the clearance of tissue exudate neutrophils.

Lipoxin A4 ameliorates knee osteoarthritis progression in rats by antagonizing ferroptosis through activation of the ESR2/LPAR3/Nrf2 axis in synovial fibroblast-like synoviocytes.

BACKGROUND: Our previous studies have shown that lipoxin A4 (LXA4) can serve as a potential biomarker for assessing the efficacy of exercise therapy in knee osteoarthritis (KOA), and fibroblast-like synoviocytes (FLSs) may play a crucial role in KOA pain as well as in the progression of the pathology. OBJECTIVE: By analyzing the GSE29746 dataset and collecting synovial samples from patients with different Kellgren-Lawrence (KL) grades for validation, we focused on exploring the potential effect of LXA4 on ferroptosis in FLSs through the ESR2/LPAR3/Nrf2 axis to alleviate pain and pathological advancement in KOA. METHODS: The association between FLSs ferroptosis and chondrocyte matrix degradation was explored by cell co-culture. We overexpressed and knocked down LPAR3 in vitro to explore its potential mechanism in FLSs. A rat model of monosodium iodoacetate (MIA)-induced KOA was constructed and intervened with moderate-intensity treadmill exercise and intraperitoneal injection of PHTPP to investigate the effects of the LXA4 intracellular receptor ESR2 on exercise therapy. RESULTS: ESR2, LPAR3, and GPX4 levels in the synovium decreased with increasing KL grade. After LXA4 intervention in the co-culture system, GPX4, LPAR3, and ESR2 were upregulated in FLSs, collagen II was upregulated in chondrocytes, and MMP3 and ADAM9 were downregulated. LPAR3 overexpression upregulated the expression of GPX4, Nrf2, and SOD1 in FLSs, while downregulating the expression of MMP13 and MMP3; LPAR3 knockdown reversed these changes. Moderate-intensity platform training improved the behavioral manifestations of pain in KOA rats, whereas PHTPP treatment partially reversed the improvement in synovial and cartilage pathologies induced by platform training. CONCLUSION: LXA4 inhibited FLSs ferroptosis by activating the ESR2/LPAR3/Nrf2 axis, thereby alleviating the pain and pathological progression of KOA. This study brings a new target for the treatment of KOA and also leads to a deeper understanding of the potential mechanisms of exercise therapy for KOA.

Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk.

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

A signaling network map of Lipoxin (LXA4): an anti-inflammatory molecule.

Lipoxins (LXs) are a class of endogenous bioactive lipid mediators that are involved in the regulation of inflammation. They exert immunomodulatory effects by regulating the behaviour of various immune cells, including neutrophils, macrophages, and T and B cells, by promoting the clearance of apoptotic neutrophils. This helps to dampen inflammation and promote tissue repair. LXs regulate the expression of many inflammatory genes by modulating the levels of transcription factors, such as nuclear factor κB (NF-κB), activator protein-1 (AP-1), nerve growth factor-regulated factor 1A binding protein 1 (NGF), and peroxisome proliferator activated receptor γ (PPAR-γ), which are elevated in various diseases, such as respiratory tract diseases, renal diseases, cancer, neurodegenerative diseases, and viral infections. Lipoxin-mediated signaling is involved in chronic inflammation, cancer, diabetes-associated kidney disease, lung injury, liver injury, endometriosis, respiratory tract diseases, neurodegenerative diseases, chronic cerebral hypoperfusion, and retinal degeneration. In this study, we systematically investigated the intricate network of lipoxin signaling by analyzing the relevant literature. The resulting map comprised 467 molecules categorized as activation/inhibition, enzyme catalysis, gene and protein expression, molecular associations, and translocation events. This map serves as a valuable resource for understanding the complexity of lipoxin signaling and its impact on various cellular functions.

Association between arachidonate lipoxygenase 15,c.-292 C > T gene polymorphism and non-cystic fibrosis bronchiectasis in children: a pilot study on the effects on airway lipoxin A4 and disease phenotype.

BACKGROUND: Persistent airway inflammation is a central feature of bronchiectasis. Arachidonate 15-lipoxygenase (ALOX-15) controls production of endogenous lipid mediators, including lipoxins that regulate airway inflammation. Mutations at various positions in ALOX-15 gene can influence airway disease development. We investigated association between ALOX-15,c.-292 C > T gene polymorphism and bronchiectasis unrelated to cystic fibrosis in Egyptian children. Also, lipoxin A4 (LXA4) level in bronchoalveolar lavage (BAL) was studied in relation to polymorphism genotypes and disease phenotypes determined by clinical, pulmonary functions, and radiological severity parameters. METHODS: This was an exploratory study that included 60 participants. Thirty children with non-cystic fibrosis bronchiectasis (NCFB) were compared with 30 age and sex-matched controls. ALOX-15,c.-292 C > T polymorphism was genotyped using TaqMan-based Real-time PCR. LXA4 was measured in BAL using ELISA method. RESULTS: There was no significant difference between patients and controls regarding ALOX-15,c.-292 C > T polymorphism genotypes and alleles (OR = 1.75; 95% CI (0.53-5.7), P = 0.35) (OR = 1; 95% CI (0.48-2), p = 1). BAL LXA4 level was significantly lower in patients, median (IQR) of 576.9 (147.6-1510) ng/ml compared to controls, median (IQR) of 1675 (536.8-2542) (p = 0.002). Patients with severe bronchiectasis had a significantly lower LXA4 level (p < 0.001). There were significant correlations with exacerbations frequency (r=-0.54, p = 0.002) and FEV1% predicted (r = 0.64, p = 0.001). Heterozygous CT genotype carriers showed higher LXA4 levels compared to other genotypes(p = 0.005). CONCLUSIONS: Low airway LXA4 in children with NCFB is associated with severe disease phenotype and lung function deterioration. CT genotype of ALOX-15,c.-292 C > T polymorphism might be a protective genetic factor against bronchiectasis development and/or progression due to enhanced LXA4 production.

BML-111, the agonist of lipoxin A4, suppresses epithelial-mesenchymal transition and migration of MCF-7 cells via regulating the lipoxygenase pathway.

Introduction: Aberrant epithelial-mesenchymal transition (EMT) and migration frequently occur during tumour progression. BML-111, an analogue of lipoxin A4, has been implicated in inflammation in cancer research. Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, western blot, Reverse Transcription Polymerase Chain Reaction (RT-PCR), transwell assay, immunofluorescence, and immunohistochemistry were conducted in this study. Results: In vitro experiments revealed that BML-111 inhibited EMT and migration in CoCl2-stimulated MCF-7 cells. These effects were achieved by inhibiting MMP-2 and MMP-9, which are downregulated by 5-lipoxygenase (5-LOX). Moreover, BML-111 inhibited EMT and migration of breast cancer cells in BALB/c nude mice inoculated with MCF-7 cells. Conclusion: Our results suggest that BML-111 may be a potential therapeutic drug for breast cancer and that blocking the 5-LOX pathway could be a possible approach for mining effective drug targets.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Lipoxin A4 promotes antibiotic and monocyte bacterial killing in established Pseudomonas aeruginosa biofilm formed under hydrodynamic conditions.

Pseudomonas aeruginosa is a gram-negative, opportunistic bacteria commonly found in wounds and in lungs of immunocompromised patients. These bacteria commonly form biofilms which encapsulate the bacteria, making it difficult for antibiotics or immune cells to reach the bacterial cells. We previously reported that Lipoxin A4 (LxA4 ), a Specialized Pro-resolving Mediator, has direct effects on P. aeruginosa where it reduced biofilm formation and promoted ciprofloxacin antibiotic efficacy in a static biofilm-forming system. In the current studies, we examined the actions of LxA4 on established biofilms formed in a biofilm reactor under dynamic conditions with constant flow and shear stress. These conditions allow for biofilm growth with nutrient replenishment and for examination of bacteria within the biofilm structure. We show that LxA4 helped ciprofloxacin reduction of live/dead ratio of bacteria within the biofilm. THP-1 monocytes interacted with the biofilm to increase the number of viable bacteria within the biofilm as well as TNF-α production in the biofilm milieu, suggesting that monocyte interaction with bacterial biofilm exacerbates the inflammatory state. Pre-treatment of the THP-1 monocytes with LxA4 abolished the increase in biofilm bacteria and reduced TNF-α production. The effect of decreased biofilm bacteria was associated with increased LxA4 -induced monocyte adherence to biofilm but not increased bacteria killing suggesting that the mechanism for the reduced biofilm bacteria was due to LxA4 -mediated increase in adherence to biofilm. These results suggest that LxA4 can help antibiotic efficacy and promote monocyte activity against established P. aeruginosa biofilm formed under hydrodynamic conditions.

Lipoxin receptor agonist and inhibition of LTA4 hydrolase prevent tight junction disruption caused by P. aeruginosa filtrate in airway epithelial cells.

Airway diseases can disrupt tight junction proteins, compromising the epithelial barrier and making it more permeable to pathogens. In people with pulmonary disease who are prone to infection with Pseudomonas aeruginosa, pro-inflammatory leukotrienes are increased and anti-inflammatory lipoxins are decreased. Upregulation of lipoxins is effective in counteracting inflammation and infection. However, whether combining a lipoxin receptor agonist with a specific leukotriene A4 hydrolase (LTA4H) inhibitor could enhance these protective effects has not to our knowledge been investigated. Therefore, we explored the effect of lipoxin receptor agonist BML-111 and JNJ26993135 a specific LTA4H inhibitor that prevents the production of pro-inflammatory LTB4 on tight junction proteins disrupted by P. aeruginosa filtrate (PAF) in human airway epithelial cell lines H441 and 16HBE-14o. Pre-treatment with BML-111 prevented an increase in epithelial permeability induced by PAF and conserved ZO-1 and claudin-1 at the cell junctions. JNJ26993135 similarly prevented the increased permeability induced by PAF, restored ZO-1 and E-cadherin and reduced IL-8 but not IL-6. Cells pre-treated with BML-111 plus JNJ26993135 restored TEER and permeability, ZO-1 and claudin-1 to the cell junctions. Taken together, these data indicate that the combination of a lipoxin receptor agonist with a LTA4H inhibitor could provide a more potent therapy.

Effect of the Lipoxin Receptor Agonist BML-111 on Cigarette Smoke Extract-Induced Macrophage Polarization and Inflammation in RAW264.7 Cells.

Background: Macrophages are known to play a crucial role in the chronic inflammation associated with Chronic Obstructive Pulmonary Disease (COPD). BML-111, acting as a lipoxin A4 (LXA4) receptor agonist, has shown to be effective in protecting against COPD. However, the precise mechanism by which BML-111 exerts its protective effect remains unclear. Methods: In order to establish a cell model of inflammation, cigarette smoke extract (CSE) was used on the RAW264.7 cell line. Afterwards, an Enzyme-linked immunosorbent assay (ELISA) kit was employed to measure concentrations of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), interleukin-18 (IL-18), and interleukin-10 (IL-10) in the cell supernatants of the RAW264.7 cells.In this study, we examined the markers of macrophage polarization using two methods: quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, we detected the expression of Notch-1 and Hes-1 through Western blotting. Results: BML-111 effectively suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-18, as well as inflammasome factors NLRP3 and Caspase-1, while simultaneously up-regulating the expression of the anti-inflammatory cytokine IL-10 induced by CSE. Moreover, BML-111 reduced the expression of iNOS, which is associated with M1 macrophage polarization, and increased the expression of Arg-1, which is associated with M2 phenotype. Additionally, BML-111 downregulated the expression of Hes-1 and the ratio of activated Notch-1 to Notch-1 induced by CSE. The effect of BML-111 on inflammation and macrophage polarization was reversed upon administration of the Notch-1 signaling pathway agonist Jagged1. Conclusion: BML-111 has the potential to suppress inflammation and modulate M1/M2 macrophage polarization in RAW264.7 cells. The underlying mechanism may involve the Notch-1 signaling pathway.

Plasma and rectal mucosal oxylipin levels during aspirin and eicosapentaenoic acid treatment in the seAFOod polyp prevention trial.

BACKGROUND: Aspirin and eicosapentaenoic acid (EPA) have colorectal polyp prevention activity, alone and in combination. This study measured levels of plasma and rectal mucosal oxylipins in participants of the seAFOod 2 × 2 factorial, randomised, placebo-controlled trial, who received aspirin 300 mg daily and EPA 2000 mg free fatty acid, alone and in combination, for 12 months. METHODS: Resolvin (Rv) E1, 15-epi-lipoxin (LX) A4 and respective precursors 18-HEPE and 15-HETE (with chiral separation) were measured by ultra-high performance liquid chromatography-tandem mass spectrometry in plasma taken at baseline, 6 months and 12 months, as well as rectal mucosa obtained at trial exit colonoscopy at 12 months, in 401 trial participants. RESULTS: Despite detection of S- and R- enantiomers of 18-HEPE and 15-HETE in ng/ml concentrations, RvE1 or 15­epi-LXA4 were not detected above a limit of detection of 20 pg/ml in plasma or rectal mucosa, even in individuals randomised to both aspirin and EPA. We have confirmed in a large clinical trial cohort that prolonged (12 months) treatment with EPA is associated with increased plasma 18-HEPE concentrations (median [inter-quartile range] total 18-HEPE 0.51 [0.21-1.95] ng/ml at baseline versus 0.95 [0.46-4.06] ng/ml at 6 months [P<0.0001] in those randomised to EPA alone), which correlate strongly with respective rectal mucosal 18-HEPE levels (r = 0.82; P<0.001), but which do not predict polyp prevention efficacy by EPA or aspirin. CONCLUSION: Analysis of seAFOod trial plasma and rectal mucosal samples has not provided evidence of synthesis of the EPA-derived specialised pro-resolving mediator RvE1 or aspirin-trigged lipoxin 15­epi-LXA4. We cannot rule out degradation of individual oxylipins during sample collection and storage but readily measurable precursor oxylipins argues against widespread degradation.

Lipoxin A4 methyl ester attenuated ketamine-induced neurotoxicity in SH-SY5Y cells via regulating leptin pathway.

Ketamine, the widely used intravenous anesthetic, has been reported to cause neurotoxicity and disturbs normal neurogenesis. However, the efficacy of current treatment strategies targeting ketamine's neurotoxicity remains limited. Lipoxin A4 methyl ester (LXA4 ME) is relatively stable lipoxin analog, which serves an important role in protecting against early brain injury. The purpose of this study was to investigate the protective effect of LXA4 ME on ketamine-caused cytotoxicity in SH-SY5Y cells, as well as the underlying mechanisms. Cell viability, apoptosis and endoplasmic reticulum stress (ER stress) were detected by adopting experimental techniques including CCK-8 assay, flow cytometry, western blotting and transmission electron microscope. Furthermore, examining the expression of leptin and its receptor (LepRb), we also measured the levels of activation of the leptin signaling pathway. Our results showed that LXA4 ME intervention promoted the cell viability, inhibited cell apoptosis, and reduced the expression of ER stress related protein and morphological changes induced by ketamine. In addition, inhibition of leptin signaling pathway caused by ketamine could be reversed by LXA4 ME. However, as the specific inhibitor of leptin pathway, leptin antagonist triple mutant human recombinant (leptin tA) attenuated the cytoprotective effect of LXA4 ME against ketamine-induced neurotoxicity. In conclusion, our findings demonstrated LXA4 ME could exert a neuroprotective effect on ketamine-induced neuronal injury via activation of the leptin signaling pathway.

Exogenous Lipoxin A4 attenuates IL4-induced Mucin Expression in Human Airway Epithelial Cells.

Introduction: The proinflammatory cytokine interleukin-4 (IL-4) induces mucus hypersecretion by human airway epithelial cells and the MAP kinase signalling pathway may be important in terms of IL-4-induced MUC5AC gene expression. Lipoxin A4 (LXA4) is an arachidonic acid-derived mediator that promotes inflammation by binding to the anti-inflammatory receptors (ALXs) or the formyl-peptide receptor like-1 (FPRL1) protein expressed by airway epithelial cells. Here, we explore the effects of LXA4 on IL-4-induced mucin gene expression in, and secretion from, human airway epithelial cells. Methods: We co-treated cells with IL-4 (20 ng/mL) and LXA4 (1 nM) and measured the expression levels of mRNAs encoding MUC5AC and 5B via real-time polymerase chain reaction; protein expression levels were determined by Western blotting and immunocytofluorescence. The ability of IL-4 and LXA4 to suppress protein expression was determined by Western blotting. Results: IL-4 increased MUC5AC and 5B gene and protein expression. LXA4 suppressed IL-4-induced MUC5AC and 5B gene and protein expression by interacting with the IL4 receptor and mitogen-activated protein kinase (MAPK) pathway, including both phospho-p38 MAPK and phospho-extracellular signal-regulated kinase (phospho-ERK). IL-4 and LXA4 increased and decreased, respectively, the number of cells that stained with anti-MUC5AC and 5B antibodies. Conclusions: LXA4 may regulate mucus hypersecretion induced by IL4 in human airway epithelial cells.

Lipoxin A4 prevents high glucose-induced inflammatory response in cardiac fibroblast through FOXO1 inhibition.

Cardiac cells respond to various pathophysiological stimuli, synthesizing inflammatory molecules that allow tissue repair and proper functioning of the heart; however, perpetuation of the inflammatory response can lead to cardiac fibrosis and heart dysfunction. High concentration of glucose (HG) induces an inflammatory and fibrotic response in the heart. Cardiac fibroblasts (CFs) are resident cells of the heart that respond to deleterious stimuli, increasing the synthesis and secretion of both fibrotic and proinflammatory molecules. The molecular mechanisms that regulate inflammation in CFs are unknown, thus, it is important to find new targets that allow improving treatments for HG-induced cardiac dysfunction. NFκB is the master regulator of inflammation, while FoxO1 is a new participant in the inflammatory response, including inflammation induced by HG; however, its role in the inflammatory response of CFs is unknown. The inflammation resolution is essential for an effective tissue repair and recovery of the organ function. Lipoxin A4 (LXA4) is an anti-inflammatory agent with cytoprotective effects, while its cardioprotective effects have not been fully studied. Thus, in this study, we analyze the role of p65/NFκB, and FoxO1 in CFs inflammation induced by HG, evaluating the anti-inflammatory properties of LXA4. Our results demonstrated that HG induces the inflammatory response in CFs, using an in vitro and ex vivo model, while FoxO1 inhibition and silencing prevented HG effects. Additionally, LXA4 inhibited the activation of FoxO1 and p65/NFκB, and inflammation of CFs induced by HG. Therefore, our results suggest that FoxO1 and LXA4 could be novel drug targets for the treatment of HG-induced inflammatory and fibrotic disorders in the heart.

Lipoxin A4 depresses inflammation and promotes autophagy via AhR/mTOR/AKT pathway to suppress endometriosis.

BACKGROUND: Endometriosis is a benign gynecological disease with the feature of estrogen dependence and inflammation. The function of autophagy and the correlation with inflammation were not yet revealed. METHODS: Autophagosomes were detected by transmission electron microscopy. Gene Expression Omnibus (GEO) database was referred to analyze the expression of autophagy-related genes. Quantification of mRNA and protein expression was examined by qRT-PCR and Western Blot. Immunohistochemistry was performed to explore the expression of proteins in tissues. The mouse model of endometriosis was performed to analyze the autophagic activity and effect of LXA4. RESULTS: The expression of autophagy-related genes in endometriotic lesions were unusually changed. The number of autophagosomes and LC3B-II expression was diminished, and p62 was increased in ectopic lesions from both patients and mice. Interleukin 1ß (IL1ß) attenuated the expression of LC3B and promoted the level p62. The autophagy activator MG-132 upregulated the expression of LC3B and reduced IL1ß, IL6, and p62. LXA4 reversed the inhibitory effect of IL1ß on the expression of LC3B and p62, and blocking the receptor of LXA4 AhR (aryl hydrocarbon receptor) resulted in the incapacitation of LXA4 to influence the effect of IL1ß. LXA4 depressed the phosphorylation of AKT and mTOR to against IL1ß, and blocking AhR negatively regulated the effect of LXA4 on AKT/mTOR pathway. LXA4 reduced the ectopic lesions and the expression of IL1ß and p62, but enhanced LC3B-II in endometriotic mouse models. CONCLUSION: In endometriosis, increased inflammation of ectopic lesions prominently depresses autophagy. LXA4 could regulate autophagy by suppressing inflammatory response through AhR/AKT/mTOR pathway.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators.

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERß in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERß in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca2+ responses between sexes were found with carbachol only at 10-3 M, but not with histamine or leukotriene (LT) B4 at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10-7 M for 30 min significantly inhibited the LTB4-stimulated [Ca2+]i increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB4 response in females, while DHT and E2 significantly inhibited the LTB4 response in males. The SPM lipoxin A4 (LXA4) (10-9-10-8 M), but not the resolvins D1 or D2, induced an [Ca2+]i increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB4-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA4, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Dose-dependent ameliorating effect of lipoxin A4 on gentamicin-induced nephrotoxicity in rats: The role of TNFα, TGF-ß, ICAM-1, and JNK signaling.

Gentamicin (GEN) possesses a broad range of antimicrobial effects. However, it belongs to the aminoglycosides, and has the greatest potential for nephrotoxic effect above all other antibiotics from this group. This study aims to evaluate the possible protective effect of lipoxin A4 (LXA4) against GEN-induced nephrotoxicity in rats. Nephrotoxicity was induced in male Wistar rats by injection of GEN (80 mg/kg/day, i.p.) for 6 days starting from day 7 of the experiment. Rats were treated with either LXA4 (10 µg/kg/day, i.p.) or LXA4 (50 µg/kg/day, i.p.) for 14 days starting from day 1 of the experiment, along with GEN as the previous schedule. GEN resulted in a significantly elevated renal function in the form of higher serum creatinine and urea levels. Further, GEN induced abnormal renal histopathology including degenerated glomeruli and tubules, with perivascular inflammation and hemorrhage. All of these findings were significantly decreased by the LXA4 administration. Additionally, LXA4 remarkably reduced the increased serum lipid biomarkers. Moreover, LXA4 significantly inhibited the GEN-induced oxidative stress in the kidneys by decreasing the elevated levels of renal malondialdehyde (MDA) and total nitrite while raising the suppressed levels of renal superoxide dismutase (SOD) and serum total antioxidant capacity (TAC). LXA4 inhibited the up-regulated inflammatory mediators ICAM-1, TGFß 1 protein levels, and TNF-α protein expression. Finally, LXA4 suppressed the elevated apoptotic mediators; p-JNK and cleaved caspase-3 expression. Our results proved for the first time that LXA4 ameliorated GEN-induced nephrotoxicity through its antioxidant, anti-inflammatory and anti-apoptotic properties.

CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

Topical Aspirin Administration Improves Cutaneous Wound Healing in Diabetic Mice Through a Phenotypic Switch of Wound Macrophages Toward an Anti-inflammatory and Proresolutive Profile Characterized by LXA4 Release.

Patients with diabetes present a persistent inflammatory process, leading to impaired wound healing. Since nonhealing diabetic wound management shows limited results, the introduction of advanced therapies targeting and correcting the inflammatory status of macrophages in chronic wounds could be an effective therapeutic strategy to stop the sustained inflammation and to return to a healing state. In an excisional skin injury in a diet-induced diabetic murine model, we demonstrate that topical administration of low-dose aspirin (36 µg/wound/day) improves cutaneous wound healing by increasing wound closure through the promotion of the inflammation resolution program of macrophages. This treatment increased efferocytosis of wound macrophages from aspirin-treated diabetic mice compared with untreated diabetic mice. We also show that aspirin treatment of high-fat-fed mice oriented the phenotype of wound macrophages toward an anti-inflammatory and proresolutive profile characterized by a decrease of LTB4 production. The use of diabetic mice deficient for 5-LOX or 12/15-LOX demonstrated that these two enzymes of acid arachidonic metabolism are essential for the beneficial effect of aspirin on wound healing. Thus, aspirin treatment modified the balance between pro- and anti-inflammatory eicosanoids by promoting the synthesis of proresolving LXA4 through 5-LOX, LTA4, 12/15-LOX signaling. In conclusion, the restoration of an anti-inflammatory and proresolutive phenotype of wound macrophages by the topical administration of low-dose aspirin represents a promising therapeutic approach in chronic wounds.

Mechanical stress protects against chondrocyte pyroptosis through lipoxin A4 via synovial macrophage M2 subtype polarization in an osteoarthritis model.

Our previous study found that lipoxin A4 (LXA4) exerts therapeutic effects on osteoarthritis (OA). In this study, we evaluated the effects of LXA4 via synovial macrophage M1/M2 subtype polarization on chondrocyte pyroptosis and corresponding mechanisms during mechanical stimulation. Synovial macrophages were subjected to various LXA4 concentrations and cyclic tensile strain (CTS) conditions to determine optimal co-culture conditions. The effects of LXA4 on chondrocyte pyroptosis, as represented by macrophage M1/M2 subtype polarization, were detected by western blot, immunofluorescence, and flow cytometry analyses. Forty male Sprague-Dawley rats were randomly divided into four groups (n = 10): control (CG), OA (OAG), OA with moderate-intensity treadmill exercise (OAE), and OAE + BOC-2 (an LXA4 antagonist). All rats were evaluated using histology, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blot analyses. We found that with increasing Kellgren-Lawrence grade, LXA4 expression was downregulated in articular fluid and that CD86 and Arg1 expression was upregulated in the synovium of patients. In vitro, CTS and LXA4 both promoted M2 subtype polarization of synovial macrophages, which inhibited the nuclear translocation of NF-κB p65 and formation of NLRP3 in chondrocytes. In vivo, the OAE treatment exerted protective effects on articular cartilage and facilitated M2 polarization of synovial macrophages. These effects were suppressed by BOC-2 treatment. We concluded that moderate CTS enhances therapeutic effects of LXA4 by inhibiting the nuclear translocation of NF-κB p65 and NLRP3. Furthermore, the therapeutic effects of LXA4 during treadmill exercise in monoiodoacetate-induced OA were driven by promotion of synovial macrophage M2 subtype polarization.