ω3 fatty acid metabolite, 12-hydroxyeicosapentaenoic acid, alleviates contact hypersensitivity by downregulation of CXCL1 and CXCL2 gene expression in keratinocytes via retinoid X receptor α.

ω3 fatty acids show potent bioactivities via conversion into lipid mediators; therefore, metabolism of dietary lipids is a critical determinant in the properties of ω3 fatty acids in the control of allergic inflammatory diseases. However, metabolic progression of ω3 fatty acids in the skin and their roles in the regulation of skin inflammation remains to be clarified. In this study, we found that 12-hydroxyeicosapentaenoic acid (12-HEPE), which is a 12-lipoxygenase metabolite of eicosapentaenoic acid, was the prominent metabolite accumulated in the skin of mice fed ω3 fatty acid-rich linseed oil. Consistently, the gene expression levels of Alox12 and Alox12b, which encode proteins involved in the generation of 12-HEPE, were much higher in the skin than in the other tissues (eg, gut). We also found that the topical application of 12-HEPE inhibited the inflammation associated with contact hypersensitivity by inhibiting neutrophil infiltration into the skin. In human keratinocytes in vitro, 12-HEPE inhibited the expression of two genes encoding neutrophil chemoattractants, CXCL1 and CXCL2, via retinoid X receptor α. Together, the present results demonstrate that the metabolic progression of dietary ω3 fatty acids differs in different organs, and identify 12-HEPE as the dominant ω3 fatty acid metabolite in the skin.

Maternal ω3 docosapentaenoic acid inhibits infant allergic dermatitis through TRAIL-expressing plasmacytoid dendritic cells in mice.

BACKGROUND: Maternal dietary exposures are considered to influence the development of infant allergies through changes in the composition of breast milk. Cohort studies have shown that ω3 polyunsaturated fatty acids (PUFAs) in breast milk may have a beneficial effect on the preventing of allergies in infants; however, the underlying mechanisms remain to be investigated. We investigated how the maternal intake of dietary ω3 PUFAs affects fatty acid profiles in the breast milk and their pups and reduced the incidence of allergic diseases in the pups. METHODS: Contact hypersensitivity (CHS) induced by 2,4-dinitrofluorobenzene (DNFB) and fluorescein isothiocyanate was applied to the skin in pups reared by mother maintained with diets mainly containing ω3 or ω6 PUFAs. Skin inflammation, immune cell populations, and expression levels of immunomodulatory molecules in pups and/or human cell line were investigated by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. ω3 PUFA metabolites in breast milk and infant's serum were evaluated by lipidomics analysis using LC-MS/MS. RESULTS: We show that maternal intake of linseed oil, containing abundant ω3 α-linolenic acid, resulted in the increased levels of ω3 docosapentaenoic acid (DPA) and its 14-lipoxygenation products in the breast milk of mouse dams; these metabolites increased the expression of TNF-related apoptosis-inducing ligand (TRAIL) on plasmacytoid dendritic cells (pDCs) in their pups and thus inhibited infant CHS. Indeed, the administration of DPA-derived 14-lipoxygenation products to mouse pups ameliorated their DNFB CHS. CONCLUSION: These findings suggest that an inhibitory mechanism in infant skin allergy is induced through maternal metabolism of dietary ω3 PUFAs in mice.

Dietary coconut oil ameliorates skin contact hypersensitivity through mead acid production in mice.

Coconut oil is used as a dietary oil worldwide, and its healthy effects are recognized by the fact that coconut oil is easy to digest, helps in weight management, increases healthy cholesterol, and provides instant energy. Although topical application of coconut oil is known to reduce skin infection and inflammation, whether dietary coconut oil has any role in decreasing skin inflammation is unknown. In this study, we showed the impact of dietary coconut oil in allergic skin inflammation by using a mouse model of contact hypersensitivity (CHS). Mice maintained on coconut oil showed amelioration of skin inflammation and increased levels of cis-5, 8, 11-eicosatrienoic acid (mead acid) in serum. Intraperitoneal injection of mead acid inhibited CHS and reduced the number of neutrophils infiltrating to the skin. Detailed mechanistic studies unveiled that mead acid inhibited the directional migration of neutrophils by inhibiting the filamentous actin polymerization and leukotriene B4 production required for secondary recruitment of neutrophils. Our findings provide valuable insights into the preventive roles of coconut oil and mead acid against skin inflammation, thereby offering attractive therapeutic possibilities.

High accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man6-9GlcNAc2 structure (2.8%, 7.1%) and high abundance of the Man5GlcNAc2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man5GlcNAc2 structure, and Man I overexpression enhanced the conversion of Man6-9GlcNAc2 structures to the Man5GlcNAc2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins.

Integrative analysis of cancer dependency data and comprehensive phosphoproteomics data revealed the EPHA2-PARD3 axis as a cancer vulnerability in KRAS-mutant colorectal cancer.

Colorectal cancer (CRC), a common malignant tumour of the gastrointestinal tract, is a life-threatening cancer worldwide. Mutations in KRAS and BRAF, the major driver mutation subtypes in CRC, activate the RAS pathway, contribute to tumorigenesis in CRC and are being investigated as potential therapeutic targets. Despite recent advances in clinical trials targeting KRASG12C or RAS downstream signalling molecules for KRAS-mutant CRC, there is a lack of effective therapeutic interventions. Therefore, understanding the unique molecular characteristics of KRAS-mutant CRC is essential for identifying molecular targets and developing novel therapeutic interventions. We obtained in-depth proteomics and phosphoproteomics quantitative data for over 7900 proteins and 38 700 phosphorylation sites in cells from 35 CRC cell lines and performed informatic analyses, including proteomics-based coexpression analysis and correlation analysis between phosphoproteomics data and cancer dependency scores of the corresponding phosphoproteins. Our results revealed novel dysregulated protein-protein associations enriched specifically in KRAS-mutant cells. Our phosphoproteomics analysis revealed activation of EPHA2 kinase and downstream tight junction signalling in KRAS-mutant cells. Furthermore, the results implicate the phosphorylation site Y378 in the tight junction protein PARD3 as a cancer vulnerability in KRAS-mutant cells. Together, our large-scale phosphoproteomics and proteomics data across 35 steady-state CRC cell lines represent a valuable resource for understanding the molecular characteristics of oncogenic mutations. Our approach to predicting cancer dependency from phosphoproteomics data identified the EPHA2-PARD3 axis as a cancer vulnerability in KRAS-mutant CRC.

Comprehensive proteomic profiling of plasma and serum phosphatidylserine-positive extracellular vesicles reveals tissue-specific proteins.

Extracellular vesicles (EVs) are ubiquitously secreted by almost all tissues and carry many cargoes, including proteins, RNAs, and lipids, which are related to various biological processes. EVs are shed from tissues into the blood and expected to be used as biomarkers for diseases. Here, we isolated EVs from EDTA plasma and serum of six healthy subjects by an affinity capture isolation method, and a total of 4,079 proteins were successfully identified by comprehensive EV proteomics. Our reliable and detailed catalog of the differential expression profiles of EV proteins in plasma and serum between healthy individuals could be useful as a reference for biomarker discovery. Furthermore, tissue-specific protein groups co-regulated between blood EVs from healthy individuals were identified. These EV proteins are expected to be used for more specific and sensitive enrichment of tissue-specific EVs and for screening and monitoring of disease without diagnostic imaging in patient blood in the future.

Automated Proteomics Sample Preparation of Phosphatidylserine-Positive Extracellular Vesicles from Human Body Fluids.

Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and are present in all body fluids. Blood-derived EVs can be used as a promising source for biomarker monitoring in disease. EV proteomics is currently being analyzed in clinical specimens. However, their EV proteomics preparation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method using a magnetic particle processing robot for automated 96-well processing of magnetic particles for EV proteomics analysis that can be started with a low volume of multiple clinical samples. The automation of EV purification reduced the coefficient of variation of protein quantification from 3.5 to 2.2% compared with manual purification, enabling the quantification of 1120 proteins in 1 h of MS analysis. This automated proteomics EV sample preparation is attractive for processing large cohort samples for biomarker development, validation, and routine testing.

Temporal dynamics from phosphoproteomics using endoscopic biopsy specimens provides new therapeutic targets in stage IV gastric cancer.

Phosphoproteomic analysis expands our understanding of cancer biology. However, the feasibility of phosphoproteomic analysis using endoscopically collected tumor samples, especially with regards to dynamic changes upon drug treatment, remains unknown in stage IV gastric cancer. Here, we conducted a phosphoproteomic analysis using paired endoscopic biopsy specimens of pre- and post-treatment tumors (Ts) and non-tumor adjacent tissues (NATs) obtained from 4 HER2-positive gastric cancer patients who received trastuzumab-based treatment and from pre-treatment Ts and NATs of 4 HER2-negative gastric cancer patients. Our analysis identified 14,622 class 1 phosphosites with 12,749 quantified phosphosites and revealed molecular changes by HER2 positivity and treatment. An inhibitory signature of the ErbB signaling was observed in the post-treatment HER2-positive T group compared with the pre-treatment HER2-positive T group. Phosphoproteomic profiles obtained by a case-by-case review using paired pre- and post-treatment HER2-positive T could be utilized to discover predictive or resistant biomarkers. Furthermore, these data nominated therapeutic kinase targets which were exclusively activated in the patient unresponded to the treatment. The present study suggests that a phosphoproteomic analysis of endoscopic biopsy specimens provides information on dynamic molecular changes which can individually characterize biologic features upon drug treatment and identify therapeutic targets in stage IV gastric cancer.

Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis.

The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase-substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity. Thirty-seven ALK substrate candidates, including 34 phosphorylation sites not annotated in the PhosphoSitePlus database, were identified by integrated analysis of the phosphoproteome and crosslinking interactome of HEK 293 cells with doxycycline-induced ALK overexpression. Furthermore, KSRs of ALK were validated by an in vitro kinase assay. Finally, using phosphoproteomic data from ALK mutant cell lines and patient-derived cells treated with ALK inhibitors, we found that the prediction of ALK activity was improved when the KSRs identified in this study were used instead of the public KSR dataset. Our approach is applicable to other kinases, and future identification of KSRs will facilitate more accurate estimations of kinase activity and elucidation of phosphorylation signals.

Intestinal microbe-dependent ω3 lipid metabolite αKetoA prevents inflammatory diseases in mice and cynomolgus macaques.

Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.

12-Hydroxyeicosapentaenoic acid inhibits foam cell formation and ameliorates high-fat diet-induced pathology of atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease associated with macrophage aggregate and transformation into foam cells. In this study, we sought to investigate the impact of dietary intake of ω3 fatty acid on the development of atherosclerosis, and demonstrate the mechanism of action by identifying anti-inflammatory lipid metabolite. Mice were exposed to a high-fat diet (HFD) supplemented with either conventional soybean oil or α-linolenic acid-rich linseed oil. We found that as mice became obese they also showed increased pulsatility and resistive indexes in the common carotid artery. In sharp contrast, the addition of linseed oil to the HFD improved pulsatility and resistive indexes without affecting weight gain. Histological analysis revealed that dietary linseed oil inhibited foam cell formation in the aortic valve. Lipidomic analysis demonstrated a particularly marked increase in the eicosapentaenoic acid-derived metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) in the serum from mice fed with linseed oil. When we gave 12-HEPE to mice with HFD, the pulsatility and resistive indexes was improved. Indeed, 12-HEPE inhibited the foamy transformation of macrophages in a peroxisome proliferator-activated receptor (PPAR)γ-dependent manner. These results demonstrate that the 12-HEPE-PPARγ axis ameliorates the pathogenesis of atherosclerosis by inhibiting foam cell formation.

Vitamin B1 Supports the Differentiation of T Cells through TGF-ß Superfamily Production in Thymic Stromal Cells.

Homeostatic generation of T cells, which occurs in the thymus, is controlled at least in part by endogenous cytokines and ligands. In addition, nutritional factors are other key regulators for the homeostasis of host immunity, but whether and how nutrition affects the homeostatic generation of thymocytes remains to be established. Here, we showed that vitamin B1 deficiency resulted in a bias toward the maturation of γδ thymocytes accompanied by decreased differentiation into double-positive thymocytes during thymic involution. These events were mediated through the increased production of TGF-ß superfamily members due to the accumulation of branched-chain α-keto acids in thymic stromal cells. These findings revealed essential roles of vitamin B1 in the appropriate differentiation of T cells through the metabolism of thymic stromal cells.

Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens.

Rationale: Cancer phosphoproteomics can provide insights regarding kinases that can be targeted for therapeutic applications. Monitoring the phosphoproteomics in cancer is expected to play a key role in optimizing treatments with kinase inhibitors. Clinical phosphoproteomics in surgical tissues and patient-derived models has been studied intensively. However, the reported data may not accurately reflect the phosphosignaling status in patients due to the effect of ischemia occurring during surgery or changes in the characteristics of cancer cells when establishing the models. In contrast, endoscopic biopsies have an advantage for clinical phosphoproteomics because they can be rapidly cryo-preserved. We aimed to develop a highly sensitive method for phosphoproteomics in endoscopic biopsies of gastric cancer. Methods: Three tumor biopsies and three normal gastric biopsies were obtained by endoscopy at one time, and subjected to our optimized phosphoproteomics. Phosphopeptides were enriched with an immobilized metal affinity chromatography, and labeled with Tandem Mass Tag reagent. Quantified phosphosites were compared between the pairs of tumor/normal biopsies within same patient. Cancer-specific activated pathways and kinases were identified by pathway enrichment analysis and kinase-substrate enrichment analysis. Results: Our protocol enabled the identification of more than 10,000 class 1 phosphosites from endoscopic biopsies. A comparison between samples from cancer tissue and normal mucosa demonstrated differences in the phosphosignaling, including biomarkers of response to DNA damage. Finally, cancer-specific activation of DNA damage response signaling was validated by additional phosphoproteomics of other patients and western blotting of gastric cancer/normal cells. Conclusion: In summary, our pioneering approach will facilitate more accurate clinical phosphoproteomics in endoscopic biopsies, which can be applied to monitor the activities of therapeutic kinases and, ultimately, can be a useful tool to precision medicine.

Dietary Omega-3 Fatty Acid Dampens Allergic Rhinitis via Eosinophilic Production of the Anti-Allergic Lipid Mediator 15-Hydroxyeicosapentaenoic Acid in Mice.

The metabolism and generation of bioactive lipid mediators are key events in the exertion of the beneficial effects of dietary omega-3 fatty acids in the regulation of allergic inflammation. Here, we found that dietary linseed oil, which contains high amounts of alpha-linolenic acid (ALA) dampened allergic rhinitis through eosinophilic production of 15-hydroxyeicosapentaenoic acid (15-HEPE), a metabolite of eicosapentaenoic acid (EPA). Lipidomic analysis revealed that 15-HEPE was particularly accumulated in the nasal passage of linseed oil-fed mice after the development of allergic rhinitis with the increasing number of eosinophils. Indeed, the conversion of EPA to 15-HEPE was mediated by the 15-lipoxygenase activity of eosinophils. Intranasal injection of 15-HEPE dampened allergic symptoms by inhibiting mast cell degranulation, which was mediated by the action of peroxisome proliferator-activated receptor gamma. These findings identify 15-HEPE as a novel EPA-derived, and eosinophil-dependent anti-allergic metabolite, and provide a preventive and therapeutic strategy against allergic rhinitis.

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Many attempts have been made to reproduce the three-dimensional (3D) cancer behavior. For that purpose, Matrigel, an extracellular matrix from Engelbreth-Holm-Swarm mouse sarcoma cell, is widely used in 3D cancer models such as scaffold-based spheroids and patient-derived organoids. However, severe ion suppression caused by contaminants from Matrigel hampers large-scale phosphoproteomics. In the present study, we successfully performed global phosphoproteomics from Matrigel-embedded spheroids and organoids. Using acetone precipitations of tryptic peptides, we identified more than 20,000 class 1 phosphosites from HCT116 spheroids. Bioinformatic analysis revealed that phosphoproteomic status are significantly affected by the method used for the recovery from the Matrigel, i.e., Dispase or Cell Recovery Solution. Furthermore, we observed the activation of several phosphosignalings only in spheroids and not in adherent cells which are coincident with previous study using 3D culture. Finally, we demonstrated that our protocol enabled us to identify more than 20,000 and nearly 3,000 class 1 phosphosites from 1.4 mg and 150 µg of patient-derived organoid, respectively. Additionally, we were able to quantify phosphosites with high reproducibility (r = 0.93 to 0.95). Our phosphoproteomics protocol is useful for analyzing the phosphosignalings of 3D cancer behavior and would be applied for precision medicine with patient-derived organoids.

Deep Phospho- and Phosphotyrosine Proteomics Identified Active Kinases and Phosphorylation Networks in Colorectal Cancer Cell Lines Resistant to Cetuximab.

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.