Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: LTH-sEV promotes LSEC capillarization in HFD mice and aggravates the progression of MASLD. ( A ) NTA analysis of LTH-sEV; ( B ) TEM images of LTH-sEV, scale bar = 100 nm; ( C ) Western blot analysis of CD9, CD63, Alix, TSG101, and calnexin protein expression in hepatocyte (HepG2) and LTH-sEV; ( D ) Schematic diagram of LTH-sEV injection into mice on HFD and NCD diet; ( E ) In vivo imaging detection of DiR-labeled LTH-sEV in mice; ( F ) SEM images and porosity of hepatic sinusoidal in NCD-fed mice treated with PBS, LTH-sEV, HFD-fed mice treated with PBS, LTH-sEV, n = 6, scale bar = 500 nm; ( G ) Immunohistochemistry images of CD31, Ang-2 in each group, scale bar = 50 μm, n = 6; ( H ) ELISA analysis of serum Ang-2 in each group, n = 6; ( I ) Western blot analysis of Ang-2 expression in each group, n = 3; ( J ) H&E staining, immunohistochemistry images of α-SMA, Sirius red staining in each group, scale bar = 50 μm, n = 6; ( K ) Serum ALT, AST expression in each group, n = 6; ( L ) qRT-PCR analysis of IL-1β, IL-6, and TNFα expression in each group, n = 6; ( M ) ELISA analysis of IL-1β, IL-6, and TNFα level in mice liver or serum, n = 6. Compared with HFD or NCD group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ns, no significance.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Western Blot, Expressing, Injection, In Vivo Imaging, Labeling, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: OGT level in serum and serum sEV is positively associated with MASLD. ( A ) ELISA analysis of serum OGT expression in healthy individuals and patients with MASLD, n = 50; ( B ) ROC curve of serum OGT, AUC = .6832, P = .0016, Compared with Healthy group, ∗∗ P < .01; ( C ) Correlation analysis of serum OGT and ALT, r = .2154, P = .0438; ( D ) Correlation analysis of serum OGT and AST, r = .2493, P = .0171; ( E ) Correlation analysis of serum OGT and TG, r = .2541, P = .0197; ( F ) Immunohistochemical images of OGT expression in liver of healthy individuals and patients with MASLD; ( G ) Schematic diagram of the collection of Healthy-sEV and MASLD-sEV; ( H–I ) NTA and TEM detection of Healthy-sEV and MASLD-sEV, scale bar = 100 nm; ( J ) Western blot analysis of CD9, CD63, Alix and TSG101 protein expression in Healthy-sEV and MASLD-sEV; ( K ) Western blot analysis of OGT protein expression in Healthy-sEV and MASLD-sEV, n = 3; ( L ) ELISA analysis of of OGT in Healthy-sEV and MASLD-sEV, n = 50, Compared with Healthy-sEV group, ∗∗ P < .01; ( M ) ROC curve of OGT in serum sEV, AUC = .7228, P = .0001; ( N ) Correlation analysis of sEV-OGT and serum ALT, r = .2395, P = .0246; ( O ) Correlation analysis of sEV-OGT and serum AST, r = .2493, P = .0171; ( P ) Correlation analysis of sEV-OGT and Fib-4 score (Fib-4 = age × AST/PLT × √ALT), r = .3100, P = .0113.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemical staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs

doi: 10.1172/JCI196788

Figure Lengend Snippet: ( A ) Fecal microbiota from Clec4n –/– mice were cultured on MRS medium supplemented with neomycin for 3 days. Single colonies were isolated and identified by PCR amplification and sequencing of bacterial 16S rDNA. ( B – F ) L.j . WXY strain was isolated from mouse feces. WT mice were treated with antibiotics (ABX), followed by oral transfer of the indicated bacterial strains twice over 7 days. Whole fecal microbiota from normal WT mice were then transferred back for 3 days, followed by DSS treatment for 7 days and sacrifice on day 10. Body weight loss ( B ), DAI ( C ), colon length measurement ( D ), distal colon histology ( E ), and cLP neutrophil and Treg frequencies ( F ) ( B – D , and F , PBS, n = 7; L . j . WXY, n = 7; Lactobacillus mixture, n = 8; E . coli , n = 7; E , n = 3 /group). ( G ) Correlation between fecal Lactobacillus abundance and colonic S100a8 / S100a9 mRNA expression in C57BL/6J mice under steady-state conditions ( n = 18). ( H ) L.j . WXY was cultured anaerobically at 37°C with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each). Bacterial growth was quantified spectrophotometrically at 6 and 24 hours ( n = 6 technical replicates/group). ( I ) Clec4n –/– mice received intrarectal administration of recombinant S100A8 + S100A9 for 5 hours, followed by quantification of fecal L.j . abundance by qPCR ( n = 10). ( J and K ) WT and Clec4n –/– recipient mice were lethally irradiated and reconstituted with BM cells from WT or Clec4n –/– donors. After 30 days, colonic expression of S100a8 and S100a9 was assessed by qPCR ( J ), and fecal Lactobacillus abundance was measured by qPCR on days 10, 20, and 30 after transfer ( K ) (WT→WT, n = 8; WT→ Clec4n –/– , n = 7; Clec4n –/– →WT, n = 8; Clec4n –/– → Clec4n –/– , n = 8.). ( L – N ) WT and Clec4n –/– mice were treated intraperitoneally with anti-Ly6G neutralizing Ab or isotype control IgG (100 μg/mouse) every other day for 5 doses. Two days after the final injection, colonic tissues and feces were collected. S100A8 and S100A9 protein levels in colon lysates were measured by ELISA ( L ), fecal L.j . abundance was determined by qPCR ( M ), and colonic Il6 and Tnf expression was assessed by qPCR ( N ) (control [con] IgG, n = 3–4; anti-Ly6G, n = 3–4). ( O – S ) WT mice were orally administered culture supernatant (sup.) from L.j . WXY or heat-killed (hk) bacteria daily for 3 days before and throughout 7 days of DSS treatment ( n = 10 total administrations). Body weight loss ( O ), DAI ( P ), colon length measurement at sacrifice on day 9 ( Q ), distal colon histology ( R ), and cLP Treg frequencies ( S ) (PBS n = 8; WXY-sup. n = 8; WXY-hk n = 9). ( T ) CD11b + and CD11c + cells isolated from WT cLP were stimulated in vitro with L.j . WXY, Lactobacillus mixture, or E . coli . After 12 hours, Il10 and Tgfb1 mRNA expression was quantified by qPCR ( n = 4 technical replicates/group). ( U ) CD62L + naive CD4 + T cells isolated from WT spleen and lymph nodes were polarized toward Treg differentiation in the presence of L.j . WXY culture supernatant. After 6 days, Il10 and Tgfb1 expression was measured by qPCR ( n = 9 technical replicates from 3 biological replicates/group). ( V and W ) ABX-treated WT mice received oral L.j . WXY or PBS, followed by fecal microbiota transplantation from normal WT mice and subsequent DSS treatment. Cecal contents were collected on day 10 for untargeted metabolomic analysis by liquid chromatography. Histogram ( V ) shows the top 20 enriched metabolites, and volcano plot ( W ) highlights significantly altered metabolites in WXY-treated mice. ( X ) Targeted metabolomic analysis of l -glutamic acid levels in cecal contents from 8-week-old WT and Clec4n –/– mice under physiological conditions ( n = 3/group). ( Y ) l -Glutamic acid concentrations in culture supernatants of L.j . WXY, Lactobacillus mixture, or E . coli measured by targeted metabolomics ( n = 3 replicates/group). Data in B – F , H – K , and O – S are pooled from 2 independent experiments. Data in B – F , J , L – U , X , and Y are presented as mean ± SD. Statistical analysis: 1-way ANOVA with Bonferroni’s multiple-comparison test ( B , C , K , O , and P ); 1-way ANOVA with Tukey’s multiple-comparison test ( D – F , J , L – N , Q – T , and Y ); Spearman’s correlation test ( G ); 2-way ANOVA test with repeated measures ( H ); paired Student’s t test ( I ); or 2-tailed unpaired Student’s t test ( U and X ).

Article Snippet: Lactobacillus cultures were incubated in MRS medium containing a 1:1 mixture of recombinant S100A8 and S100A9 peptides (5 μg/mL each; HY-P71076, MCE) for 6 or 24 hours under anaerobic conditions.

Techniques: Cell Culture, Isolation, Amplification, Sequencing, Expressing, Recombinant, Irradiation, Control, Injection, Enzyme-linked Immunosorbent Assay, Bacteria, In Vitro, Transplantation Assay, Metabolomic, Liquid Chromatography, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii –derived glutamic acid–maintained Tregs

doi: 10.1172/JCI196788

Figure Lengend Snippet: ( A ) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. ( B ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR ( Engyodontium : HC, n = 59; CD, n = 62. Lacutobacillus : HC, n = 62; CD, n = 63). ( C ) Relative contents of Engyodontium sp. in fecal fungi and L.j . in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). ( D ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in B ( n = 97 [including HC, n = 48 and CD, n = 49]). ( E ) Correlation between Ct value of L.j . and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). ( F ) C57BL/6J SPF mice were orally colonized with Engyodontium sp. ( Engyod. ) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j . was quantified by qPCR ( n = 10 /group). ( G ) Relative expression of CLEC6A , S100A8 , and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). ( H ) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G . ( I ) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR ( n = 25). ( J ) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. ( K ) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J . ( L ) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset ( GSE117993 ) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). ( M ) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b + cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR ( n = 7). ( N ) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR ( n = 7 technical replicates from 2 biological replicates/group). ( O ) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( P ) Correlation between fecal L.j . abundance and colonic expression of CLEC6A , S100A8 , or S100A9 in patients with UC ( n = 25). ( Q ) CD11b + cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression ( n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. ( R ) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates ( n = 3 replicates/group). ( S ) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). ( T ) Correlation between 1/Ct value of L.j . and fecal glutamic acid levels in patients with CD ( n = 8). ( U ) Correlation between 1/Ct value of L.j . and glutamic acid concentration in colon tissues from patients with UC ( n = 25). ( V ) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l -glutamic acid. After 5 days, the proportion of Foxp3 + CD4 + among CD45 + leukocytes was analyzed by flow cytometry ( n = 6 technical replicates from 2 biological replicates/group). Data in F , M , N , Q , and V are pooled from 2 independent experiments. Data in B , C , G , and L are presented as mean ± SEM, and in F , N , Q , R , S , and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test ( B , C , and G ), 2-tailed unpaired Student’s t test ( F , N , Q – S , and V ), 1-way ANOVA with Kruskal-Wallis and Dunn’s test ( L ), Spearman’s correlation test ( D , E , H , I , O , P , T , and U ), or paired Student’s t test ( M ).

Article Snippet: Lactobacillus cultures were incubated in MRS medium containing a 1:1 mixture of recombinant S100A8 and S100A9 peptides (5 μg/mL each; HY-P71076, MCE) for 6 or 24 hours under anaerobic conditions.

Techniques: Isolation, Bacteria, Expressing, Control, RNA Sequencing, Immunohistochemistry, In Vitro, Cell Culture, Recombinant, Liquid Chromatography, Concentration Assay, Flow Cytometry, MANN-WHITNEY