Elucidating the role of tumor-associated ALOX5+ mast cells with transformative function in cervical cancer progression via single-cell RNA sequencing.

Background: Cervical cancer (CC) is the fourth most common malignancy among women globally and serves as the main cause of cancer-related deaths among women in developing countries. The early symptoms of CC are often not apparent, with diagnoses typically made at advanced stages, which lead to poor clinical prognoses. In recent years, numerous studies have shown that there is a close relationship between mast cells (MCs) and tumor development. However, research on the role MCs played in CC is still very limited at that time. Thus, the study conducted a single-cell multi-omics analysis on human CC cells, aiming to explore the mechanisms by which MCs interact with the tumor microenvironment in CC. The goal was to provide a scientific basis for the prevention, diagnosis, and treatment of CC, with the hope of improving patients' prognoses and quality of life. Method: The present study acquired single-cell RNA sequencing data from ten CC tumor samples in the ArrayExpress database. Slingshot and AUCcell were utilized to infer and assess the differentiation trajectory and cell plasticity of MCs subpopulations. Differential expression analysis of MCs subpopulations in CC was performed, employing Gene Ontology, gene set enrichment analysis, and gene set variation analysis. CellChat software package was applied to predict cell communication between MCs subpopulations and CC cells. Cellular functional experiments validated the functionality of TNFRSF12A in HeLa and Caski cell lines. Additionally, a risk scoring model was constructed to evaluate the differences in clinical features, prognosis, immune infiltration, immune checkpoint, and functional enrichment across various risk scores. Copy number variation levels were computed using inference of copy number variations. Result: The obtained 93,524 high-quality cells were classified into ten cell types, including T_NK cells, endothelial cells, fibroblasts, smooth muscle cells, epithelial cells, B cells, plasma cells, MCs, neutrophils, and myeloid cells. Furthermore, a total of 1,392 MCs were subdivided into seven subpopulations: C0 CTSG+ MCs, C1 CALR+ MCs, C2 ALOX5+ MCs, C3 ANXA2+ MCs, C4 MGP+ MCs, C5 IL32+ MCs, and C6 ADGRL4+ MCs. Notably, the C2 subpopulation showed close associations with tumor-related MCs, with Slingshot results indicating that C2 subpopulation resided at the intermediate-to-late stage of differentiation, potentially representing a crucial transition point in the benign-to-malignant transformation of CC. CNVscore and bulk analysis results further confirmed the transforming state of the C2 subpopulation. CellChat analysis revealed TNFRSF12A as a key receptor involved in the actions of C2 ALOX5+ MCs. Moreover, in vitro experiments indicated that downregulating the TNFRSF12A gene may partially inhibit the development of CC. Additionally, a prognosis model and immune infiltration analysis based on the marker genes of the C2 subpopulation provided valuable guidance for patient prognosis and clinical intervention strategies. Conclusions: We first identified the transformative tumor-associated MCs subpopulation C2 ALOX5+ MCs within CC, which was at a critical stage of tumor differentiation and impacted the progression of CC. In vitro experiments confirmed the inhibitory effect of knocking down the TNFRSF12A gene on the development of CC. The prognostic model constructed based on the C2 ALOX5+MCs subset demonstrated excellent predictive value. These findings offer a fresh perspective for clinical decision-making in CC.

Multiomics integrated analysis and experimental validation identify TLR4 and ALOX5 as oxidative stress-related biomarkers in intracranial aneurysms.

BACKGROUND: Intracranial aneurysm (IA) is a severe cerebrovascular disease, and effective gene therapy and drug interventions for its treatment are still lacking. Oxidative stress (OS) is closely associated with the IA, but the key regulatory genes involved are still unclear. Through multiomics analysis and experimental validation, we identified two diagnostic markers for IA associated with OS. METHODS: In this study, we first analyzed the IA dataset GSE75436 and conducted a joint analysis of oxidative stress-related genes (ORGs). Differential analysis, functional enrichment analysis, immune infiltration, WGCNA, PPI, LASSO, and other methods were used to identify IA diagnostic markers related to OS. Next, the functions of TLR4 and ALOX5 expression in IA and their potential targeted therapeutic drugs were analyzed. We also performed single-cell sequencing of patient IA and control (superficial temporal artery, STA) tissues. 23,342 cells were captured from 2 IA and 3 STA samples obtained from our center. Cell clustering and annotation were conducted using R software to observe the distribution of TLR4 and ALOX5 expression in IAs. Finally, the expression of TLR4 and ALOX5 were validated in IA patients and in an elastase-induced mouse IA model using experiments such as WB and immunofluorescence. RESULTS: Through bioinformatics analysis, we identified 16 key ORGs associated with IA pathogenesis. Further screening revealed that ALOX5 and TLR4 were highly expressed to activate a series of inflammatory responses and reduce the production of myocytes. Methotrexate (MTX) may be a potential targeted drug. Single-cell analysis revealed a notable increase in immune cells in the IA group, with ALOX5 and TLR4 primarily localized to monocytes/macrophages. Validation through patient samples and mouse models confirmed high expression of ALOX5 and TLR4 in IAs. CONCLUSIONS: Bioinformatics analysis indicated that ALOX5 and TLR4 are the most significant ORGs associated with the pathogenesis of IA. Single-cell sequencing and experiments revealed that the high expression of ALOX5 and TLR4 are closely related to IA. These two genes are promising new targets for IA therapy.

Integrating Lipidomics, Metabolomics, and Network Pharmacology to Reveal the Mechanism of Cannabidiol against Inflammation in High-Fat, High-Cholesterol Diet-Induced Mice.

Inflammation plays a critical role in the development of numerous diseases. Cannabidiol (CBD), found in hemp, exhibits significant pharmacological activities. Accumulating evidence suggests that CBD has anti-inflammatory and cardiovascular protection effects, but the potential mechanisms require further exploration. In this study, we aimed to reveal the mechanisms of CBD against high-fat, high-cholesterol (HFC) diet-induced inflammation combining metabolomics with network pharmacology. First, plasma lipidomics results indicated that oxidized lipids could serve as potential biomarkers for HFC diet-induced inflammation, and CBD reversed the elevated levels of oxidized lipids. The HFC diet was also found to enhance intestinal permeability, facilitating the entry of lipopolysaccharides (LPSs) into the circulatory system and subsequently increasing systemic inflammation. Additionally, cell metabolomic results indicated that CBD could reverse 10 important differential metabolites in LPS-induced RAW 264.7 cells. Using network pharmacology, we identified 49 core targets, and enrichment analysis revealed that arachidonic acid was the most significantly affected by CBD, which was closely associated with inflammation. Further integrated analysis focused on three key targets, including PTGS2, ALOX5, and ALOX15. Molecular docking showed high affinities between key targets and CBD, and qPCR further demonstrated that CBD could reverse the mRNA expression of these key targets in RAW 264.7 cells. Collectively, this finding integrates lipidomics and metabolomics with network pharmacology to elucidate the anti-inflammatory effects of CBD and validates key therapeutic targets.

Elucidating the role of S100A10 in CD8+ T cell exhaustion and HCC immune escape via the cPLA2 and 5-LOX axis.

Hepatocellular carcinoma (HCC) is a common malignant tumor with a complex immune evasion mechanism posing a challenge to treatment. The role of the S100A10 gene in various cancers has garnered significant attention. This study aims to elucidate the impact of S100A10 on CD8+ T cell exhaustion via the cPLA2 and 5-LOX axis, thereby elucidating its role in immune evasion in HCC. By analyzing the HCC-related data from the GEO and TCGA databases, we identified differentially expressed genes associated with lipid metabolism and developed a prognostic risk model. Subsequently, through RNA-seq and PPI analyses, we determined vital lipid metabolism genes and downstream factors S100A10, ACOT7, and SMS, which were significantly correlated with CD8+ T cell infiltration. Given the most significant expression differences, we selected S100A10 for further investigation. Both in vitro and in vivo experiments were conducted, including co-culture experiments of CD8+ T cells with MHCC97-L cells, Co-IP experiments, and validation in an HCC mouse model. S100A10 was significantly overexpressed in HCC tissues and potentially regulates CD8+ T cell exhaustion and lipid metabolism reprogramming through the cPLA2 and 5-LOX axis. Silencing S100A10 could inhibit CD8+ T cell exhaustion, further suppressing immune evasion in HCC. S100A10 may activate the cPLA2 and 5-LOX axis, initiating lipid metabolism reprogramming and upregulating LTB4 levels, thus promoting CD8+ T cell exhaustion in HCC tissues, facilitating immune evasion by HCC cells, ultimately impacting the growth and migration of HCC cells. This research highlights the critical role of S100A10 via the cPLA2 and 5-LOX axis in immune evasion in HCC, providing new theoretical foundations and potential targets for diagnosing and treating HCC.

CRISPR/Cas9-mediated neuronal deletion of 5-lipoxygenase alleviates deficits in mouse models of epilepsy.

INTRODUCTION: Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. OBJECTIVES: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. METHODS: Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. RESULTS: Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn ISer603. CONCLUSION: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.

Inhibition of host 5-lipoxygenase reduces overexuberant inflammatory responses and mortality associated with Cryptococcus meningoencephalitis.

Cryptococcosis, caused by fungi of the genus Cryptococcus, manifests in a broad range of clinical presentations, including severe pneumonia and disease of the central nervous system (CNS) and other tissues (bone and skin). Immune deficiency or development of overexuberant inflammatory responses can result in increased susceptibility or host damage, respectively, during fungal encounters. Leukotrienes help regulate inflammatory responses against fungal infections. Nevertheless, studies showed that Cryptococcus exploits host 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, to facilitate transmigration across the brain-blood barrier. To investigate the impact of host 5-LO on the development of protective host immune responses and mortality during cryptococcosis, wild-type (C57BL/6) and 5-lipoxygenase-deficient (5-LO-/-) mice were given experimental pulmonary and systemic Cryptococcus sp., infections. Our results showed that 5-LO-/- mice exhibited reduced pathology and better disease outcomes (i.e., no mortality or signs associated with cryptococcal meningoencephalitis) following pulmonary infection with C. deneoformans, despite having detectable yeast in the brain tissues. In contrast, C57BL/6 mice exhibited classical signs associated with cryptococcal meningoencephalitis. Additionally, brain tissues of 5-LO-/- mice exhibited lower levels of cytokines (CCL2 and CCL3) clinically associated with Cryptococcus-related immune reconstitution inflammatory syndrome (C-IRIS). In a systemic mouse model of cryptococcosis, 5-LO-/- mice and those treated with a Federal Drug Administration (FDA)-approved 5-LO synthesis inhibitor, zileuton, displayed significantly reduced mortality compared to C57BL/6 infected mice. These results suggest that therapeutics designed to inhibit host 5-LO signaling could reduce disease pathology and mortality associated with cryptococcal meningoencephalitis. IMPORTANCE: Cryptococcosis is a mycosis with worldwide distribution and has a broad range of clinical manifestations, including diseases of the CNS. Globally, there is an estimated 179,000 cases of cryptococcal meningitis, resulting in approximately 112,000 fatalities per annum and 19% of AIDS-related deaths. Understanding how host immune responses are modulated during cryptococcosis is central to mitigating the morbidity and mortality associated with cryptococcosis. Leukotrienes (LTs) have been shown to modulate inflammatory responses during infection. In this study, we show that mice deficient in 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, exhibit reduced pathology, disease, and neurological signs associated with cryptococcal meningitis. Additionally, mice given an experimental cryptococcal infection and subsequently treated with an FDA-approved 5-LO synthesis inhibitor exhibited significantly reduced mortality rates. These results suggest that therapeutics designed to inhibit host 5-LO activity could significantly reduce pathology and mortality rates associated with cryptococcal meningitis.

Quercetin Alleviates LPS-Stimulated Myocardial Injury through Regulating ALOX5/PI3K/AKT Pathway in Sepsis.

Quercetin (QUE) has been found to inhibit the progression of sepsis-related diseases, including sepsis-induced cardiomyopathy (SIC). More information about the role and mechanism of QUE in SIC progression deserves further exploration. Human cardiomyocytes (AC16) were induced with LPS to mimic SIC cell models. Cell proliferation and apoptosis were determined using CCK8 assay, EdU assay, and flow cytometry. Cell inflammation and ferroptosis were evaluated by detecting IL-1ß, TNF-α, Fe2+, ROS, GSH, and GPX4 levels. 5-lipoxygenase (ALOX5) expression was examined by quantitative real-time PCR and western blot. LPS treatment reduced AC16 cell proliferation, while enhanced apoptosis, inflammation, and ferroptosis. QUE repressed LPS-induced AC16 cell apoptosis, inflammation, and ferroptosis. ALOX5 was upregulated in SIC patients, and its expression was reduced by QUE. ALOX5 knockdown restrained LPS-induced apoptosis, inflammation, and ferroptosis in AC16 cells. The inhibitory effect of QUE on LPS-induced myocardial injury could be reversed by ALOX5 overexpression. QUE promoted the activity of PI3K/AKT pathway by reducing ALOX5 expression. QUE could alleviate LPS-induced myocardial injury by regulating ALOX5/PI3K/AKT pathway, suggesting that QUE might be used for treating SIC.

Artemisia argyi essential oil alleviates asthma by regulating 5-LOX-CysLTs and IDO-1-KYN pathways: Insights from metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia argyi essential oil (AAEO) is a traditional herbal remedy for asthma. However, the potential effect of AAEO on asthma has not been elucidated. AIM OF THE STUDY: To investigate the protective properties of AAEO upon asthma and elucidate its mechanism. MATERIALS AND METHODS: The effects of AAEO in asthma were assessed by histology and biochemical analysis. Then, we integrated real-time reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry and metabolomics analysis to reveal its mechanism. RESULTS: In vivo, AAEO reduced the counts of white blood cells (WBCs) and cytokines in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of OVA-sIgE and muc5ac. Metabolomics results showed that AAEO can exert therapeutic effects on asthmatic mice by regulating disordered arachidonic acid metabolism and tryptophan metabolism. Further studies shown that AAEO inhibited the expression of 5-LOX and reduced the accumulation of CysLTs in mice. Meanwhile, AAEO promoted the activity of IDO-1, facilitated the conversion of tryptophan to kynurenine, and regulated the imbalance of Treg/Th17 immunity. Immunohistochemical results showed that AAEO promoted the expression of IDO-1. RT-qPCR results showed that AAEO promoted the expression of IL-10 and Foxp3 mRNA, and inhibited the expression of IL-17A and RORγt mRNA, thus regulated the imbalance of Treg/Th17 immunity and exerted its therapeutic effects. CONCLUSION: AAEO treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating lung tissue metabolism. The anti-asthmatic activity of AAEO may be achieved by reprogramming 5-LOX-CysLTs and IDO-1-KYN pathways.

Expression characteristics of lipid metabolism-related genes and correlative immune infiltration landscape in acute myocardial infarction.

Lipid metabolism is an important part of the heart's energy supply. The expression pattern and molecular mechanism of lipid metabolism-related genes (LMRGs) in acute myocardial infarction (AMI) are still unclear, and the link between lipid metabolism and immunity is far from being elucidated. In this study, 23 Common differentially expressed LMRGs were discovered in the AMI-related mRNA microarray datasets GSE61144 and GSE60993. These genes were mainly related to "leukotriene production involved in inflammatory response", "lipoxygenase pathway", "metabolic pathways", and "regulation of lipolysis in adipocytes" pathways. 12 LMRGs (ACSL1, ADCY4, ALOX5, ALOX5AP, CCL5, CEBPB, CEBPD, CREB5, GAB2, PISD, RARRES3, and ZNF467) were significantly differentially expressed in the validation dataset GSE62646 with their AUC > 0.7 except for ALOX5AP (AUC = 0.699). Immune infiltration analysis and Pearson correlation analysis explored the immune characteristics of AMI, as well as the relationship between these identified LMRGs and immune response. Lastly, the up-regulation of ACSL1, ALOX5AP, CEBPB, and GAB2 was confirmed in the mouse AMI model. Taken together, LMRGs ACSL1, ALOX5AP, CEBPB, and GAB2 are significantly upregulated in AMI patients' blood, peripheral blood of AMI mice, myocardial tissue of AMI mice, and therefore might be new potential biomarkers for AMI.

Osteomesopyknosis associated with a novel ALOX5 variant that impacts the RANKL pathway.

BACKGROUND: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. METHODS: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments. RESULTS: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. CONCLUSION: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.

The role of human 5-Lipoxygenase (5-LO) in carcinogenesis - a question of canonical and non-canonical functions.

5-Lipoxygenase (5-LO), a fatty acid oxygenase, is the central enzyme in leukotriene (LT) biosynthesis, potent arachidonic acid-derived lipid mediators released by innate immune cells, that control inflammatory and allergic responses. In addition, through interaction with 12- and 15-lipoxgenases, the enzyme is involved in the formation of omega-3 fatty acid-based oxylipins, which are thought to be involved in the resolution of inflammation. The expression of 5-LO is frequently deregulated in solid and liquid tumors, and there is strong evidence that the enzyme plays an important role in carcinogenesis. However, global inhibition of LT formation and signaling has not yet shown the desired success in clinical trials. Curiously, the release of 5-LO-derived lipid mediators from tumor cells is often low, and the exact mechanism by which 5-LO influences tumor cell function is poorly understood. Recent data now show that in addition to releasing oxylipins, 5-LO can also influence gene expression in a lipid mediator-independent manner. These non-canonical functions, including modulation of miRNA processing and transcription factor shuttling, most likely influence cancer cell function and the tumor microenvironment and might explain the low clinical efficacy of pharmacological strategies that previously only targeted oxylipin formation and signaling by 5-LO. This review summarizes the canonical and non-canonical functions of 5-LO with a particular focus on tumorigenesis, highlights unresolved issues, and suggests future research directions.

In Vitro Inhibition of Colorectal Cancer Gene Targets by Withania somnifera L. Methanolic Extracts: A Focus on Specific Genome Regulation.

An approach that shows promise for quickening the evolution of innovative anticancer drugs is the assessment of natural biomass sources. Our study sought to assess the effect of W. somnifera L. (WS) methanolic root and stem extracts on the expression of five targeted genes (cyclooxygenase-2, caspase-9, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2) in colon cancer cell lines (Caco-2 cell lines). Plant extracts were prepared for bioassay by dissolving them in dimethyl sulfoxide. Caco-2 cell lines were exposed to various concentrations of plant extracts, followed by RNA extraction for analysis. By explicitly relating phytoconstituents of WS to the dose-dependent overexpression of caspase-9 genes and the inhibition of cyclooxygenase-2, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2 genes, our novel findings characterize WS as a promising natural inhibitor of colorectal cancer (CRC) growth. Nonetheless, we recommend additional in vitro research to verify the current findings. With significant clinical benefits hypothesized, we offer WS methanolic root and stem extracts as potential organic antagonists for colorectal carcinogenesis and suggest further in vivo and clinical investigations, following successful in vitro trials. We recommend more investigation into the specific phytoconstituents in WS that contribute to the regulatory mechanisms that inhibit the growth of colon cancer cells.

Synovial 5-Lipoxygenase-Derived Oxylipins Define a Lympho-Myeloid-Enriched Synovium.

OBJECTIVE: Oxylipins are bioactive lipids derived from polyunsaturated fatty acids (PUFAs) that modulate inflammation and may remain overexpressed in refractory synovitis. In plasma, they could also be biomarkers of synovial pathology. The aim of this study is to determine if synovial oxylipins in inflamed joints correlate with plasma oxylipins and with synovial histologic patterns. METHODS: Patients with established rheumatoid or psoriatic arthritis with active disease despite treatment were recruited, and paired synovial tissue (ST) and plasma were collected. Oxylipins were determined by liquid chromatography with tandem mass spectrometry and were classified into groups according to their PUFA precursor and enzyme. The expression of CD20, CD68, CD3, and CD138 was obtained to describe synovial histology. Cell-specific expression of oxylipin-related genes was identified by examining available synovial single-cell RNA sequencing data. RESULTS: We included a total of 32 ST and 26 paired-plasma samples. A total of 71 oxylipins were identified in ST, but only 24 were identified in plasma. Only levels of 9,10-dihydroxyoctadecenoic acid and tetranor-Prostaglandin FM had a significant positive correlation between plasma and ST. Several oxylipins and oxylipin-related genes were differentially expressed among synovial phenotypes. Specifically, several 5-lipoxygenase (LOX)-derived oxylipins were statistically elevated in the lympho-myeloid phenotype and associated with B cell expression in rheumatoid arthritis samples. CONCLUSION: The lack of correlation between ST and plasma oxylipins suggests that ST lipid profiling better characterizes active pathways in treated joints. Synovial 5-LOX-derived oxylipins were highly expressed in lympho-myeloid-enriched synovium. Combination therapy with 5-LOX inhibitors to improve refractory inflammation may be needed in patients with this histologic group.

ALOX5 drives the pyroptosis of CD4+ T cells and tissue inflammation in rheumatoid arthritis.

Pyroptosis, an inflammatory form of programmed cell death, is linked to the pathology of rheumatoid arthritis (RA). Here, we investigated the molecular mechanism underlying pyroptosis in T cells isolated from patients with RA. Compared with healthy individuals, patients with RA had more pyroptotic CD4+ T cells in blood and synovia, which correlated with clinical measures of disease activity. Moreover, the mRNA expression and protein abundance of arachidonate 5-lipoxygenase (ALOX5), which converts arachidonic acid to leukotriene A4 (LTA4), were increased in CD4+ T cells from patients with RA and, among patients with RA, were lowest in those in clinical remission. Knockdown or pharmacological inhibition of ALOX5 suppressed CD4+ T cell pyroptosis and improved symptoms in two rodent models of RA. Mechanistically, the increase in ALOX5 activity in RA CD4+ T cells enhanced the production of the LTA4 derivative LTB4, which stimulated Ca2+ influx through ORAI3 channels, leading to the activation of NLRP3 inflammasomes and pyroptosis. Our findings reveal a role for ALOX5 in RA and provide a molecular basis for further exploring the clinical utility of ALOX5 inhibition in RA and for using ALOX5 as a biomarker to distinguish active disease and remission in RA.

Magnolin alleviated DSS-induced colitis by inhibiting ALOX5-mediated ferroptosis.

Inflammatory bowel disease (IBD) is a chronic and incurable disorder associated with higher cancer risk and currently faces unsatisfactory treatment outcomes. Ferroptotic cells secrete damage-associated molecular patterns (DAMPs) that recruit and activate immune cells, particularly macrophages. Magnolin has excellent antioxidant and anti-inflammatory properties, but its effect on IBD has not yet been clearly understood. This study aimed to investigate the therapeutic effects and mechanism of magnolin in IBD. For this purpose, in vivo and in vitro colitis models were established using dextran sulfate sodium (DSS), followed by optimization of magnolin concentration 2.5 µg/mL in vitro and 5 mg/kg in vivo. Bioinformatics analysis identified potential magnolin target sites and evaluated ferroptosis-associated gene expressions. Body weight, food intake, disease activity index (DAI), pathological changes, and inflammation levels were assessed. The effect of magnolin on ferroptosis and macrophages was evaluated using quantitative real time-polymerase chain reaction (qRT-PCR), immunofluorescent staining, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blotting. Results indicated that magnolin at a lower dose (5 mg/kg) alleviated DSS-induced colitis symptoms and reduced inflammation in mice. The bioinformatics analysis showed arachidonate 5-lipoxygenase (ALOX5) as a potential magnolin target. Furthermore, magnolin inhibited the expression of ALOX5 with no effect on GPX4. Moreover, magnolin regulated macrophage differentiation into the M2 phenotype and suppressed pro-inflammatory factors, that is, interleukin-6 and tumor necrosis factor-α (IL-6 and TNFα). These results suggested that magnolin possesses significant therapeutic potential in treating IBD by suppressing ALOX5-mediated ferroptosis, inhibiting M1 while promoting M2 macrophages, which is envisaged to provide novel strategies for treating IBD.

Dysregulated Leukotriene Metabolism in Patients with COVID-19.

This study aimed to examine the leukotriene metabolism during COVID-19. In total, 180 participants were included in this study, of which 60 were healthy controls, 60 required intensive care units (ICU), and 60 did not require intensive care (non-ICU). The serum levels of 5-lipoxygenase (5-LO), 5-LO activating protein (ALOX5AP), and cysteinyl leukotriene (CYSLT) were measured, and the mRNA expression levels of 5-LO, ALOX5AP, and cysteinyl leukotriene receptor 1 (CYSLTR1) were investigated. Compared with the control group, both the non-ICU and ICU groups had lower levels of 5-LO and mRNA expression. ICU patients had lower levels of 5-LO and mRNA expression than non-ICU patients. CYSLTR1 mRNA expression was highest in the ICU group, followed by the non-ICU group, and healthy controls had the lowest mRNA expression levels. CYSLT levels were higher in the control group than in the non-ICU and ICU groups. CYSLTR1 expression was higher in patients than in controls; therefore, selective leukotriene receptor blockers can be used as treatment options. CYSLTR1 expression was higher in the ICU group than in the non-ICU group. Furthermore, CYSLTR1 mRNA expression may be a promising biomarker of COVID-19 severity.

ALOX5 deficiency contributes to bladder cancer progression by mediating ferroptosis escape.

Ferroptosis is an iron-dependent form of regulated cell death driven by the lethal lipid peroxides. Previous studies have demonstrated that inducing ferroptosis holds great potential in cancer therapy, especially for patients with traditional therapy failure. However, cancer cells can acquire ferroptosis evasion during progression. To date, the therapeutic potential of inducing ferroptosis in bladder cancer (BCa) remains unclear, and whether a ferroptosis escape mechanism exists in BCa needs further investigation. This study verified that low pathological stage BCa cells were highly sensitive to RSL3-induced ferroptosis, whereas high pathological stage BCa cells exhibited obviously ferroptosis resistance. RNA-seq, RNAi-mediated loss-of-function, and CRISPR/Cas9 experiments demonstrated that ALOX5 deficiency was the crucial factor of BCa resistance to ferroptosis in vitro and in vivo. Mechanistically, we found that ALOX5 deficiency was regulated by EGR1 at the transcriptional level. Clinically, ALOX5 expression was decreased in BCa tissues, and its low expression was associated with poor survival. Collectively, this study uncovers a novel mechanism for BCa ferroptosis escape and proposes that ALOX5 may be a valuable therapeutic target and prognostic biomarker in BCa treatment.

Oncogenic PIK3CA recruits myeloid-derived suppressor cells to shape the immunosuppressive tumour microenvironment in luminal breast cancer through the 5-lipoxygenase-dependent arachidonic acid pathway.

BACKGROUND: Oncogenic PIK3CA mutations (PIK3CAmut ) frequently occur in a higher proportion in luminal breast cancer (LBC), especially in refractory advanced cases, and are associated with changes in tumour cellular metabolism. Nevertheless, its effect on the progression of the immune microenvironment (TIME) within tumours and vital molecular events remains veiled. METHODS: Multiplex immunohistochemistry (mIHC) and single-cell mass cytometry (CyTOF) was used to describe the landscape of TIME in PIK3CAmut LBC. The PIK3CA mutant cell lines were established using CRISPER/Cas9 system. The gene expression levels, protein secretion and activity of signaling pathways were measured by real-time RT-PCR, ELISA, immunofluorescence staining or western blotting. GSEA analysis, transwell chemotaxis assay, live cell imaging, flow cytometry metabolite analysis targeting arachidonic acid, Dual-luciferase reporter assay, and Chromatin immunoprecipitation assay were used to investigate the underlying function and mechanism of the PI3K/5-LOX/LTB4 axis. RESULTS: PIK3CAmut LBC cells can induce an immunosuppressive TIME by recruiting myeloid-derived suppressor cells (MDSCs) and excluding cytotoxic T cells via the arachidonic acid (AA) metabolism pathway. Mechanistically, PIK3CAmut activates the transcription of 5-lipoxygenase (5-LOX) in a STAT3-dependent manner, which in turn directly results in high LTB4 production, binding to BLT2 on MDSCs and promoting their infiltration. Since a suppressive TIME is a critical barrier for the success of cancer immunotherapy, the strategies that can convert "cold" tumours into "hot" tumours were compared. Targeted therapy against the PI3K/5-LOX/LTB4 axis synergizing with immune checkpoint blockade (ICB) therapy achieved dramatic shrinkage in vivo. CONCLUSIONS: The results emphasize that PIK3CAmut can induce immune evasion by recruiting MDSCs through the 5-LOX-dependent AA pathway, and combination targeted therapy with ICB may provide a promising treatment option for refractory advanced LBC patients.

Human cytomegalovirus infection enhances 5­lipoxygenase and cycloxygenase­2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro­inflammatory enzymes 5­lipoxygenase (5­LO) and cyclooxygenase­2 (COX­2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin­embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki­67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5­LO and COX­2. The CRC cell lines Caco­2 and LS­174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti­inflammatory drug celecoxib (CCX) and analyzed by reverse transcription­quantitative PCR and immunofluorescence for 5­LO, COX­2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX­2, 5­LO and KI­67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5­LO and COX­2 transcript and proteins in both Caco­2 and LS­174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX­2, 5­LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

Association between a single nucleotide polymorphism of the ALOX5 gene and susceptibility to multisystem tuberculosis in a Chinese Han population.

BACKGROUND: Host genetic single nucleotide polymorphisms can exert an influence susceptibility to tuberculosis infection. Previous investigations have demonstrated an association between the polymorphism in the ALOX5 gene and a range of diseases, encompassing not only noninfectious conditions like asthma, acute myocardial infarction, and cerebral infarction but also infections caused by various pathogens. However, the relationship between ALOX5 gene polymorphism and susceptibility to tuberculosis has received limited research attention. The ALOX5 gene encodes arachidonic acid 5-lipoxygenase(5-LO), which serves as the initiating catalyst in the generation of the inflammatory mediator leukotriene. Leukotrienes, products derived from the 5-LO pathway, are potent proinflammatory lipid mediators that assume a pivotal role in tuberculosis infections.Consequently, ALOX5 gene variants may be intricately associated with the pathogenesis of tuberculosis. In instances where the host exhibits immunocompromisation, infection with Mycobacterium tuberculosis can impact multiple systems. The involvement of multiple systems significantly augments the complexity of treatment and escalates patient mortality rates. Regrettably, the underlying mechanisms driving multisystem tuberculosis pathogenesis remain enigmatic, with clinicians paying scant attention to this aspect. Although the protein encoded by the ALOX5 gene represents a pivotal enzyme that catalyzes the metabolism of arachidonic acid into LXA4, and thereby plays a significant role in the inflammatory response during tuberculosis infection, studies investigating ALOX5 gene polymorphism and its association with susceptibility to multisystem tuberculosis in the Chinese Han population are exceptionally scarce. Therefore, the primary objective of this study is to comprehensively examine the correlation between ALOX5 gene polymorphisms and susceptibility to tuberculosis within the Chinese Han population, with particular emphasis on multisystemic tuberculosis. METHODS: A case‒control study design was employed, encompassing 382 individuals with pulmonary tuberculosis and 367 individuals with multisystemic tuberculosis as the case groups, along with 577 healthy controls.Whole blood DNA was extracted from all patients and healthy controls. Subsequently, three tag polymorphisms (rs2029253, rs7896431, rs2115819) within the ALOX5 gene were selectively identified and genotyped. RESULTS: After adjusting for age and sex, the presence of allele A at rs2029253 exhibited a pronounced association with an elevated risk of TB susceptibility when compared to the tuberculosis group and healthy control group. (ORa: 2.174, 95% CI: 1.827-2.587; Pa<0.001, respectively). Notably, the rs2029253 AG genotype and AA genotype displayed a significantly increased susceptibility to tuberculosis (ORa: 2.236, 95% CI: 1.769-2.825; Pa <0.001 and ORa: 4.577, 95% CI: 2.950-7.100; Pa <0.001, respectively) compared to the GG genotype. Moreover, in the analysis utilizing genetic models, rs2029253 also exhibited a markedly heightened susceptibility to tuberculosis in additive models, dominant models, and recessive models (Pa <0.001). Conversely, no significant association was observed between rs7896431, rs2115819, and tuberculosis. In the subgroup analysis, when comparing the pulmonary tuberculosis group with the healthy control group, we observed no significant disparities in the distribution frequencies of alleles, genotypes, and gene models (additive model, dominant model, and recessive model) for the three tag SNPs, with P-values were >0.05 after adjusting for age and sex. Additionally, we noted that the presence of allele A at rs2029253 was linked to an increased susceptibility to tuberculosis in the multisystemic tuberculosis group relative to the healthy control group (ORa: 2.292, 95% CI: 1.870-2.810; Pa<0.001). Similarly, the rs2029253 AG genotype, AA genotype, and gene models, including the additive model, dominant model, and recessive model, demonstrated a significantly elevated risk of tuberculosis susceptibility. CONCLUSIONS: The polymorphism in the ALOX5 gene is associated with susceptibility to multisystemic tuberculosis in the Chinese Han population.