B cell-derived GABA elicits IL-10⁺ macrophages to limit anti-tumour immunity

GABAを標的とする抗腫瘍免疫機構 --代謝産物を介した免疫細胞間制御の一端を解明--. 京都大学プレスリリース. 2021-11-10.Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8⁺ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses

Genetic influences on human blood metabolites in the Japanese population

Summary: An increase in ethnic diversity in genetic studies has the potential to provide unprecedented insights into how genetic variations influence human phenotypes. In this study, we conducted a quantitative trait locus (QTL) analysis of 121 metabolites measured using gas chromatography-mass spectrometry with plasma samples from 4,888 Japanese individuals. We found 60 metabolite-gene associations, of which 13 have not been previously reported. Meta-analyses with another Japanese and a European study identified six and two additional unreported loci, respectively. Genetic variants influencing metabolite levels were more enriched in protein-coding regions than in the regulatory regions while being associated with the risk of various diseases. Finally, we identified a signature of strong negative selection for uric acid (Sˆ = −1.53, p = 6.2 × 10−18). Our study expanded the knowledge of genetic influences on human blood metabolites, providing valuable insights into their physiological, pathological, and selective properties

A phenome-wide association study (PheWAS) to identify the health impacts of 4-cresol sulfate in the Nagahama Study

Abstract Gut-microbiota derived metabolites are important regulators of host biology and metabolism. To understand the impacts of the microbial metabolite 4-cresol sulfate (4-CS) on four chronic diseases [type 2 diabetes mellitus, metabolic syndrome (MetS), non-alcoholic fatty liver disease, and chronic kidney disease (CKD)], we conducted association analyses of plasma 4-CS quantified by liquid chromatography coupled to mass spectrometry (LC–MS) in 3641 participants of the Nagahama study. Our results validated the elevation of 4-CS in CKD and identified a reducing trend in MetS. To delineate the holistic effects of 4-CS, we performed a phenome-wide association analysis (PheWAS) with 937 intermediate biological and behavioral traits. We detected associations between 4-CS and 39 phenotypes related to blood pressure regulation, hepatic and renal functions, hematology, sleep quality, intraocular pressure, ion regulation, ketone and fatty acid metabolisms, disease history and dietary habits. Among them, 19 PheWAS significant traits, including fatty acids and 14 blood pressure indices, were correlated with MetS, suggesting that 4-CS is a potential biomarker for MetS. Consistent associations of this gut microbial-derived metabolite on multiple endophenotypes underlying distinct etiopathogenesis support its role in the overall host health, with prospects of probiotic-based therapeutic solutions in chronic diseases

Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms

Construction of a Ca²⁺-Gated Artificial Channel by Fusing Alamethicin with a Calmodulin-Derived Extramembrane Segment

Using native chemical ligation, we constructed a Ca²⁺-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca²⁺, this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca²⁺ significantly lengthened the specific channel open state and increased the mean channel current, while Mg²⁺ produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca²⁺-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein

Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

Macropinocytosis is among ubiquitous cellular uptake mechanisms of peptide-based intracellular delivery. Due to its capability of engulfing large macromolecules, macropinocytosis shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. We previously reported SN21, a peptide derived from the N-terminus of stromal cell-derived growth factor 1α (SDF-1α), as a potent macropinocytosis inducer. In this work, we obtained the 8-residue analog P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A with membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins

Impact of anticancer drugs on the therapeutic efficacy and side effects of hepatic arterial embolization for hepatocellular carcinoma

Abstract Background and Aim Transcatheter arterial chemoembolization (TACE) using various anticancer drugs is often performed to treat hepatocellular carcinoma (HCC). We aimed to compare the therapeutic efficacy and side effects of TACE with anticancer drugs versus transcatheter arterial embolization (TAE) without anticancer drugs for HCC. Methods Patients with HCC were randomized to either the TACE or TAE group. Up to five target nodules were treated in each patient. Lipiodol (Lp; 10 mL), contrast media (CM; 10 mL), epirubicin (40 mg), mitomycin C (10 mg), miliplatin (70 mg), and 1–2‐mm 2‐day soluble gelatin sponge particles (2D‐SGS) were injected into the TACE group, whereas Lp (10 mL), CM (10 mL), and 2D‐SGS were injected into the TAE group. Treatment effect (TE) of the target nodules was graded (TE1–TE4) and patient responses were assessed. Three months after treatment, blood tests were performed to compare tumor markers and adverse events. Results Fifty‐four patients and 161 target nodules were included; 75 nodules in 28 patients were treated by TACE, and 86 nodules in 26 patients were treated by TAE. The number of nodules graded TE1, TE2, TE3, and TE4 was 1, 28, 7, and 39, respectively, in the TACE group and 2, 25, 7, and 52, respectively, in the TAE group. The response rates were 89% (25/28) and 73% (19/26) in the TACE and TAE groups, respectively. There were no significant differences in TE, response rates, or blood test results between the two groups. Conclusion In hepatic arterial embolization for HCC, anticancer drugs did not have any impact on the therapeutic efficacy or side effects at 3 months after embolization

Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

Macropinocytosis is among ubiquitous cellular uptake mechanisms of peptide-based intracellular delivery. Due to its capability of engulfing large macromolecules, macropinocytosis shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. We previously reported SN21, a peptide derived from the N-terminus of stromal cell-derived growth factor 1α (SDF-1α), as a potent macropinocytosis inducer. In this work, we obtained the 8-residue analog P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A with membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins

The Interplay Between Metabolites and MicroRNAs in Aqueous Humor to Coordinate Corneal Endothelium Integrity

Purpose: The purpose of the study was to clarify the interplay between metabolites and microRNAs (miRs) in the aqueous humor (AqH) of bullous keratopathy (BK) patients to retain human corneal endothelium (HCE) integrity. Design: Prospective, comparative, observational study. Participants: A total of 55 patients with BK and 31 patients with cataract (Cat) as control. Methods: A biostatic analysis of miRs and metabolites in the AqH, hierarchical clustering, and a least absolute shrinkage and selection operator (Lasso) analysis were employed. The miR levels in AqH of BK (n = 18) and Cat (n = 8) patients were determined using 3D-Gene human miR chips. Hierarchical clusters of metabolites detected by liquid chromatography–mass spectrometry or gas chromatography–mass spectrometry in AqH specimens from 2 disease groups, BK (total n = 55) and Cat (total n = 31), were analyzed twice to confirm the reproducibility. The analytical procedure applied for investigating the association between metabolites and miRs in AqH was the exploratory data analysis of biostatistics to avoid any kind of prejudice. This research procedure includes a heat-map, cluster analysis, feature extraction techniques by principal component analysis, and a regression analysis method by Lasso. The cellular and released miR levels were validated using reverse transcription polymerase chain reaction and mitochondria membrane potential was assessed to determine the functional features of the released miRs. Main Outcome Measures: Identification of interacting metabolites and miRs in AqH attenuating HCE degeneration. Results: The metabolites that decreased in the AqH of BK patients revealed that 3-hydroxyisobutyric acid (HIB), 2-aminobutyric acid (AB) and branched-chain amino acids, and serine were categorized into the same cluster by hierarchical clustering of metabolites. The positive association of HIB with miR-34a-5p was confirmed (P = 0.018), and the Lasso analysis identified the interplay between miR-34a-5p and HIB, between miR-24-3p and AB, and between miR-34c-5p and serine (P = 0.041, 0.027, and 0.009, respectively). 3-hydroxyisobutyric acid upregulated the cellular miR-34a expression, mitochondrial membrane potential, and release of miR-184 in dedifferentiated cultured HCE cells. Conclusions: Metabolites and miRs in AqH may synchronize in ensuring the integrity of the HCE to maintain efficient dehydration from the stroma. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references