Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: DOCK2 maintains colonic IFN-γ-producing homeostatic T cells, impairs IL-22 and Reg3 AMP expression, and facilitates Verrucomicrobia colonization under physiological condition. ( A - G ) WT and Dock2 –/– mice were sacrificed under physiological conditions. Gross colonic appearance ( A ) and colon length measurements ( B ) were conducted. Following the isolation of leukocytes from the cLP, the proportions of CD4 + T cells (CD3ε + CD4 + ), CD8 + T cells (CD3ε + CD8α + ), γδT cells (CD3ε + TCRγδ + ), B cells (CD19 + ), NK cells (TCRγδ – NK1.1 + ), neutrophils (Ly6G + CD11b + ), monocytes (Ly6C + CD11b + ) and dendritic cells (MHC-II + CD11c + ) in CD45 + cells ( C ), the proportions of IFN-γ- or IL-17-producing cells in whole CD4 + and CD8 + T cells ( D ), and absolute numbers of IFN-γ- or IL-17-producing CD8 + T cells ( E ) were determined by FACS. The mRNA expression levels of genes encoding cytokines and T cell-associated transcription factors ( F ), and indicated AMPs and tight-junction proteins ( G ) in colon tissues were assessed by qPCR (n = 4/group) ( H , I ) mRNA expression levels of Muc2 (H) and Il18 ( I ) in colon tissues from WT and Dock2 –/– mice under normal or colitis conditions were examined by qPCR (n = 4/group). ( J ) Bacterial DNA was extracted from feces isolated from WT and Dock2 –/– mice under physiological conditions, and relative content of each indicated bacterial phylum or order was determined by qPCR using specific primers (WT, n = 11; Dock2 –/– , n = 5). ( K - M ) WT and Rorc –/– mice were sacrificed under physiological conditions. Following the isolation of leukocytes from the cLP, the proportions of IL-22/IL-17-producing cells in CD90.2 + Lineage – ILCs and in CD90.2 – Lineage + cells ( K ), absolute numbers of IL-22 + IL-17 + ILCs and IL-22 + IL-17 + Lineage + cells ( L ) were determined by FACS (n = 8/group). The mRNA expression levels of genes encoding IL-22, Reg3γ, Reg3β, and Mucin 2 in colon tissues were assessed by qPCR ( M ) (n = 6/group). ( N ) Bacterial DNA was extracted from feces isolated from WT and Rorc –/– mice under physiological conditions, and relative content of Akkermansia muciniphila was determined by qPCR using specific primers (n = 6/group). Data in ( A - N ) are from one of two independent experiments. Data in ( B - E , H - N ) are shown as mean ± SD and in (B-N) were analyzed using unpaired two-tailed Student’s t-test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Isolation, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: Dock2 signaling in T cells modifies IFN-γ- and IL-22-producing lymphocytes and protects mouse colitis in a commensal microbiota-dependent manner. ( A , B ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were sacrificed under physiological conditions (n = 4/group). Gross colonic appearance and colon length measurements ( A ) were conducted, and the mRNA expression of Reg3 family AMPs in colon tissues ( B ) were determined by qPCR. ( C - M ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were administered 2% DSS in drinking water for 7 days. Body weight loss ( C ) and disease activity index ( D ) were monitored daily during colitis induction (n = 3/group). On day 10 post-DSS treatment, mice were sacrificed, and gross colon observations, colon length measurements ( E , Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 7), and histological analyses of the distal colon (H&E staining) (F, G, n = 3/group) were conducted. Following leukocyte isolation from the cLP, the proportions of CD4 + and CD8 + T cells in leukocytes (H, Dock2 fl/fl n = 9, Cd4 -cre; Dock2 fl/fl n = 6), IFN-γ + in CD4 + and CD8 + T cells (I, Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 3) and IL-22 + and IL17 + populations in CD90.2 + Lineage – ILCs ( J ) and Lineage + lymphocytes ( K , Dock2 fl/fl n = 6, Cd4 -cre; Dock2 fl/fl n = 5) were quantified using FACS. The mRNA expression levels of genes encoding IL-22, RORγt and AHR in CD90.2 + Lineage – sorted ILCs ( L , Dock2 fl/fl n = 3, Cd4 -cre; Dock2 fl/fl n = 5) and the mRNA expression of Ifng , Il22 and Reg3 family AMPs in colon tissues ( M , Dock2 fl/fl n = 7, Cd4 -cre; Dock2 fl/fl n = 4) were determined by qPCR. ( N - S ) Dock2 fl/fl and Cd4 -cre; Dock2 fl/fl mice were co-housed for 4 weeks and then administered 2% DSS in drinking water for 7 days. Body weight loss ( N ) and disease activity index ( O ) were monitored daily during colitis induction. On day 10 post-DSS treatment, mice were sacrificed, and gross colon observations and colon length measurements ( P ), and histological analyses of the distal colon ( Q , H & E staining) were conducted (n = 3/group). Following leukocyte isolation from the cLP, the proportions of whole CD4 + and CD8 + T cells in CD45 + leukocytes (R) and IFN-γ + in CD4 + and CD8 + T cells (S) were quantified using FACS (n = 5/group) Data in ( A - B ) are from one of two, in ( C - M ) are from one of four, and in (N-S) are from one of two independent experiments. Data in ( B - D , F - S ) are shown as mean ± SD. Data in (B-D, F-S) were analyzed using unpaired two-tailed Student’s t-test. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Activity Assay, Staining, Isolation, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: Reg3 AMPs inhibit A. muciniphila expansion, and DOCK2-IFN-γ axis suppresses IL-22 by impairing Ahr transcription. ( A ) WT and Il22 –/– mice were sacrificed under physiological conditions, and the mRNA expression levels of genes encoding Reg3γ, Reg3β, and ZO1 in colon tissues were assessed by qPCR (WT, n = 5; Il22 –/– , n = 4). ( B ) A. muciniphila was cultured at 37 °C under anaerobic conditions with recombinant Reg3γ or Reg3β (5 μg/ml each peptide) added to the medium. Bacterial growth was measured spectrophotometrically at 3, 6, 9 and 12 h (n = 3 replicates/group). ( C ) WT mice were intrarectally administered recombinant Reg3γ or Reg3β for 5 h, followed by determination of A. muciniphila content in feces via qPCR (n = 4). ( D ) CD8 + or CD4 + T cells were isolated from splenocytes of WT mice, and were stimulated with anti-CD3/CD28 antibodies with the addition of DOCK2-Rac1 interaction inhibitor CPYPP. Four days after the stimulation, IFN-γ + in CD8 + or CD4 + T cells were quantified using FACS (n = 5 replicates/group). ( E ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AhR, and RORγt in ILCs were assessed by qPCR (n = 6 replicates/group). ( F ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ, AHR inhibitor BAY218 or AHR agonist ITE. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AHR, and RORγt in ILCs were assessed by qPCR (n = 3 replicates/group). ( G ) CD90.2 + ILCs were isolated from splenocytes of WT mice, and were stimulated with IL-1β with the addition of recombinant IFN-γ and STAT1 antagonist fludarabine. Twenty-four hours after the stimulation, the mRNA expression levels of genes encoding IL-22, AHR, and RORγt in ILCs were assessed by qPCR (n = 3 replicates/group). ( H ) Logo plot of the predicted binding motif of the transcription factor STAT1 to the AhR promoter. ( I ) The efficiency of STAT1 overexpression was confirmed by immunoblot analysis. ( J ) AHR mRNA levels in HT29 cells overexpressing STAT1 or empty vector control were quantified by qPCR (n = 3 replicates/group). ( K ) Schematic of AHR promoter regions (P1–P5) with predicted STAT1-binding sites. STAT1 binding was assessed by ChIP using anti-Flag antibody as a bait from paraformaldehyde cross-linked HT29 cells, followed by RT–PCR. ( L ) AHR mRNA levels in HT29 cells treated with IFN-γ (5, 20 or 50 ng/ml) for 48 h were assessed by RT-qPCR (n = 3 replicates/group). ( M ) Luciferase activity driven by AHR promoter was examined in HT29 cells co-transfected with Renilla and pGL3-AHR reporter plasmids, with or without 20 ng/ml IFN-γ (n = 3 replicates/group). ( N ) H3K27me3 protein levels in HT29 cells treated with LPS or IFN-γ were analyzed by immunoblot. ( O ) AHR mRNA levels in HT29 cells treated with IFN-γ in the presence or absence of GSK126 were quantified by qPCR (n = 3 replicates/group). ( P ) H3K27me3 protein levels in HT29 cells treated with increasing concentrations of GSK126 were analyzed by immunoblot. ( Q ) AHR mRNA levels in HT29 cells exposed to graded concentrations of GSK126 were assessed by qPCR (n = 3 replicates/group). ( R ) H3K27me3 enrichment at the AHR promoter following IFN-γ treatment for 24 h in HT29 cells were assessed by ChIP coupled with RT–qPCR (n = 3 replicates/group). ( S ) H3K27me3 enrichment at the AHR promoter following IFN-γ treatment with indicated doses in HT29 cells were assessed by ChIP coupled with RT–qPCR (n = 3 replicates/group). ( T - V ) WT mice were intraperitoneally administered AhR inhibitor BAY-218 (10 mg/kg) every other day for 3 times. On day 6 after the first administration, feces were harvested and A. muciniphila content was determined via qPCR ( T ) (n = 6). The mRNA expression levels of Ahr and Il22 in ILCs ( U ) (n = 3) and the expression of Reg3g , Reg3b , Ahr and Il22 in colon tissues ( V ) (non-treated n = 6, BAY-218 n = 4) were assessed by qPCR. Data in ( B ) are from one of three and in ( A , C - G , I - V ) are from one of two independent experiments. Data in ( A , B , D - G , J , L , M , O , Q – V ) are shown as mean ± SD. Data in ( A , J , M , T - V ) were analyzed using unpaired two-tailed Student’s t-test, in ( C ) using paired two-tailed Student’s t-test, and in ( B , D - G , L , O , Q - S ) using one-way ANOVA followed by Tukey’s multiple-comparisons test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Expressing, Cell Culture, Recombinant, Isolation, Binding Assay, Over Expression, Western Blot, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: Inflammation

Article Title: Dock2 Protects Colitis by Facilitating Akkermansia Colonization via Suppressing IL-22–Reg3 Activity

doi: 10.1007/s10753-026-02511-9

Figure Lengend Snippet: DOCK2-IFN-γ axis is also in the development of human IBD. ( A ) Colon tissues from one CD patient were obtained after resection, and immunohistochemical (IHC) staining for DOCK2 and REG3γ proteins of inflammatory colon tissues was performed. ( B ) Colon tissues from CD patients and healthy individuals were collected, and the relative mRNA expression of DOCK2 , IFNG , and REG3G in ileum and rectum tissues was determined by bulk RNA-seq analysis (HC, n = 44; CD, n = 46). ( C ) Point plots show the correlation of relative mRNA expression between DOCK2 and IFNG , and between AHR and IL22 by bulk RNA-seq analysis as described in ( B ). ( D ) CD3 + T cells were isolated from the inflammatory colon tissue after resection, and were stimulated with anti-CD3/CD28 antibodies with the addition of DOCK2-Rac1 interaction inhibitor CPYPP. Four days after the stimulation, the mRNA expression level of IFNG was assessed by qPCR (n = 4 replicates/group). ( E – G ) One public scRNA-seq dataset of colon cells obtained from IBD patients ( GSE150516 ) was reanalyzed. Heatmap shows the expression levels of top typical genes in each identified DOCK2 + cell cluster (subset) and Umap shows the cell types of DOCK2 + colonic cells ( E ). Violin plots display the expression of indicated genes in each identified cell cluster ( F ). The point plots show the correlation between the expression levels of DOCK2 and IFNG , and between AHR and IL22 in double-positive CD8 + or CD4 + T cells ( G ). ( H ) One public scRNA-seq dataset of colon cells obtained from IBD patients ( GSE214695 ) were reanalyzed, and the point plots show the correlation between the expression levels of DOCK2 and IFNG in double-positive CD8 + T cells in pooled colonic cells from 6 UC and 6 CD patients. ( I ) Point plots show the correlation between the expression levels of DOCK2 and CD8A , DOCK2 and IFNG , REG3G and IL22 , and AHR and IL22 in the colon tissues from all pooled human samples in the public RNA-seq dataset ( GSE117993 ). ( J ) The relative abundance of A. muciniphila in fecal bacteria from IBD patients and healthy individuals was determined by reanalyzing a public metagenomics sequencing dataset ( GSE111889 , HC n = 429, UC n = 459, CD n = 750). ( K ) The relative abundance of A. muciniphila in fecal bacteria from CD patients and healthy individuals was determined by 16S rDNA sequencing analysis (HC, n = 77; CD, n = 80). ( L ) The transcriptional level of the REG3G gene in colon tissues from non-IBD controls and IBD patients was determined by reanalyzing a public bulk RNA-seq dataset ( GSE117993 , Not IBD n = 55, IBD n = 135). ( M ) Point plots show the correlation between the relative abundance of A. muciniphila and the expression levels of REG3G , REG3A or IFNG in rectum tissues from the same individual UC patients in the public RNA-seq dataset ( GSE111889 ). Data in ( A , D ) are from one of two independent experiments. Data in ( B , J - L ) are shown as mean ± SEM, and in ( D ) are shown as mean ± SD. Data in ( B , D , J - L ) were analyzed using unpaired two-tailed Student’s t-test. Correlation significance in panels ( C , G - I , M ) was determined using Pearson’s correlation coefficient (r 2 ) test. Condition labels shown in the bottom panels apply to all graphs within each column. (ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: Akkermansia muciniphila was cultured on Brain Heart Infusion (BHI) medium in the presence of recombinant Reg3β (MCE, Cat# HY- P76003 ) or Reg3γ (MCE, Cat# HY- P71256 ) at 5 μg/mL for 3, 6, 9, or 12 h at 37 °C under anaerobic conditions.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, RNA Sequencing, Isolation, Bacteria, Metagenomics, Sequencing, Two Tailed Test

Journal: Molecular Metabolism

Article Title: METTL18 ensures pancreatic function by maintaining proper translation and proteostasis

doi: 10.1016/j.molmet.2026.102337

Figure Lengend Snippet: Proteomic alterations in the pancreas of Mettl18 -deficient mice. (A) Volcano plot of quantitative LC-MS/MS of the mouse pancreas. n = 3 per genotype. Proteins with |log2FC| > 0.5 and -Log 10 (P-value) > 4 are considered significantly changed. (B) Gene ontology (GO) analysis. A functional gene annotation of the 19 proteins (up) or 13 proteins (down) was performed with DAVID GO analysis (v6.8). Functional annotations against Biological Process, Molecular Function, and Cellular Component gene ontology with FDR less than 0.05 were shown. (C) Western blot of the pancreatic proteins from WT and KO mice. Top; anti-Reg1 antibody, bottom; anti-β-actin antibody. (D) Quantification of Reg1 protein in the pancreas. 16-week-old; n = 6; mean ± SEM. Student's t -test: p∗∗ = 0.00423. (E) qPCR quantification of mRNA expression of Reg1 in the pancreas. 16-week-old; n = 7; mean ± SEM. Student's t -test: n.s. >0.1. (F) Distribution of Reg1-FLAG. WT and two independent Mettl18 KO clones (KO1 and KO2) of 266-6 cells were transfected with Reg1-FLAG. After 48 h, cells were fractionated into soluble and insoluble fractions, and Reg1-FLAG was analyzed by Western blotting. n = 3; mean ± SEM. Dunnett's test: p∗ <0.05. See also . (G) WT and Mettl18 KO cells were transfected with the plasmid for Reg1-FLAG. 48 h after transfection, cells were fixed, and Reg1-FLAG aggregation was observed under a microscope. Arrowhead indicates aggregation foci. Scale bar: 10 μm. (H) Quantification of 266-6 cells with Reg1-FLAG aggregation. Mean value of three independent experiments was shown. Dunnett's test: p∗∗ = 0.00195, p∗∗∗ = 0.00054. (I) Distribution of Reg1-FLAG in rescued cells. WT and KO1 cells were transfected with Reg1-FLAG together with or without METTL18-WT-HA or its catalytic mutant, METTL18-mut-HA. Forty-eight hours after, cells were fractionated into soluble and insoluble fractions, and Reg1-FLAG was analyzed by Western blotting. n = 3; mean ± SEM. Statistical significance was assessed using Dunnett's test with the KO group as the reference. P∗∗ <0.01. See also . (J) WT and KO1 cells transfected with Reg1-FLAG together with or without METTL18-WT-HA or METTL18-mut-HA were fixed 48 h after transfection, and FLAG signals were visualized by fluorescence microscopy. Scale bar: 10 μm. (K) Quantification of the Reg1-FLAG aggregation. n = 3; mean ± SEM. Dunnett's test: p∗∗ = 0.00498, p∗∗∗<0.0001.

Article Snippet: Other antibodies used were as follows: anti-β-actin (clone 6D1, cat#M177-3; MBL); anti-phospho-PERK (Thr980) (clone G.305.4, cat#MA5-15033; Invitrogen); anti-RPL3 (cat#66130-1-Ig), anti-Reg1 (cat#15850-1-AP), and anti-PERK/EIF2AK3 (cat#24390-1-AP) (Proteintech Group); anti-IRE1 (cat#3294), anti-ATF4 (cat#11815), and anti-eIF5A (cat#20765) (Cell Signaling Technology); anti-phospho-IRE1 (cat#ab48187), anti-phospho-EIF2A (cat#ab32157), and anti-EIF2A (cat#ab5369) (Abcam); anti-IL-1β (cat#AF-401-NA; R&D Systems).

Techniques: Liquid Chromatography with Mass Spectroscopy, Functional Assay, Western Blot, Expressing, Clone Assay, Transfection, Plasmid Preparation, Microscopy, Mutagenesis, Fluorescence