Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A) Gene ontology enrichment of genes that are significantly upregulated in A20 ZF7 versus WT intestines by bulk RNAseq. Red bars highlight categories related to TH17 differentiation. Blue bars highlight categories related to cellular proliferation. (B) qPCR analyses of Il17a and Il22 expression from intact small intestine (relative to Actb ). (C) Volcano plot of all annotated UCSC Refseq genes from bulk RNAseq analyses of A20 ZF7 versus WT small intestines. Horizontal dashed line indicates adjusted p-value (FDR) of 0.01. (D) Representative immunohistochemical analyses of CD4 expression in WT and A20 ZF7 small intestines. Data are representative of 3 mice from each genotype. Bar, 100 microns. (E) Flow cytometry of small intestinal lamina propria cells from WT and A20 ZF7 mice. Data shown as mean + SEM. Statistics calculated using unpaired two-tailed Student t-test with Welch correction. *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, Immunohistochemical staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A, B) UMAP clusters of scRNAseq analyses of SILP from WT and A20 ZF7 mice. (C) Relative proportions of cell subsets in WT and A20 ZF7 SILP. TH17 subset includes both proliferative (mustard yellow) and non-proliferative (sky blue) compartments. (D) Projection of Il17a and Il22 expression onto UMAP clusters shown in panels A and B. (E) Violin plots of Il17a and Il22 expression in TH17 cells from indicated genotypes of mice. Statistics calculated using unpaired two-tailed Wilcoxon rank sum test. *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A20’s Linear Ubiquitin Binding Motif Restrains Pathogenic Activation of TH17/22 cells and IL-22 Driven Enteritis

doi: 10.1101/2024.12.31.630926

Figure Lengend Snippet: (A) Flow cytometric analysis of RORγt expression in in vitro differentiated TH17 cells from WT and A20 ZF7 mice. (B) qPCR analyses of Il17a and Il22 expression in cells generated as in (A) above. “TH17” indicates TH17 differentiation conditions. “FICZ” indicates treatment with the AHR agonist FICZ. Note increased IL-22 expression in absence of supplementary treatment with PMA/ionomycin. (C) ELISA of IL-22 secretion from cells generated as in (A). (D) ATAC-seq of genomic loci at/near the Il22 locus in cells generated as in (A) above. Note increased DNA accessibility across Il22 gene in enlarged plot (locus g). (E) Chromatin IP of acetylated H3K27 at indicated Il22 loci (a,b,c in (D) above) in cells generated as in (A) above. Note increased H3K27 acetylation at locus b in A20 ZF7 cells, coinciding with increased Il22 transcription. (F) Flow cytometric analyses of RORγt expression in CRISPR/Cas9-edited primary human T cells differentiated in vitro using TH17 conditions. (G) qPCR analyses of expression of indicated genes in paired isogenic human TH17 cells that were engineered with CRISPR/Cas9 and either A20 ZF7 targeted or non-targeting guide RNAs. Note increased expression of TNFAIP3 , IL17A , and IL22 in A20 ZF7 ablated TH17 cells. Three pairs of isogenic samples from two healthy donors are shown. Data shown as mean + SEM. Statistics calculated using unpaired two-tailed Student t-test with Welch correction (B, C, E) or paired ratio t-test (G). *p<0.05, ** p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Cells were subsequently differentiated into TH17 cells in plates pre-coated with 5 ug/mL anti-CD3 in TH17 medium (Immunocult XF serum-free medium with 50 uM beta-mercaptoethanol, 30 ng/mL recombinant human IL-6 (Proteintech HZ-1019), 2.5 ng/mL recombinant human TGFb1 (PeproTech 100-21), 10 ng/mL recombinant human IL-1β (Proteintech HZ-1164), 10 ng/mL recombinant human IL-23 (Proteintech HZ-1254), 10 ug/mL anti-IL4 (BioXCell BE0240, clone MP4-25D2), and 10 ug/mL anti-IFNγ (BioXCell BE0235, clone B133.5)) at a density of 1 million cells per mL.

Techniques: Expressing, In Vitro, Generated, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, CRISPR, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Itaconate and its derivatives ameliorate autoimmunity by suppressing Th17 cells via regulating mitophagy

doi: 10.1186/s12964-025-02621-1

Figure Lengend Snippet: Itaconate and its derivatives 4-OI and DMI inhibit Th17 cells via regulating mitophagy. A Structures of ITA and the ITA photo-affinity probe named x-ITA which was synthesized based on the structure of ITA. B Visualization of ITA-interacting proteins in Th17 cells by in-gel fluorescence, CBB was used for protein staining and quantitative analysis. C Principal component analysis of ITA-interacting proteins in Th17 cells ( n = 3). D Volcano plot of ITA-interacting proteins quantified in Th17 cells. E and F Biological analysis of ITA-interacting proteins in Th17 cells. ( G ) Biological process analysis of the ITA-interacting proteins quantification in Th17 cells. The enriched processes relatied to mitochondria are marked in red. H Transmission electron microscopy (TEM) showed mitochondria morphology in presence or absence of ITA. The scale bar is 1.0 μm in the normal TEM micrograph (left) and 500 nM in the enlarged (right). Green arrows showed normal mitochondria in Veh group, and red arrows showed the swollen mitochondria. I Flow cytometric analysis of MitoTracker Green fluorescence in the presence or absence of ITA ( n = 5). (J) Flow cytometric analysis of MitoSOX fluorescence in the presence or absence of ITA ( n = 5). (K) Flow cytometric analysis of JC-1 staining in the presence or absence of ITA ( n = 5). L Statistic analysis of flow cytometry data of Th17 cells for mitophagy with and without 48 h ITA treatment and/or CCCP ( n = 5). M Immunoblotting for mitophagy-related proteins in the presence or absence of ITA in Th17 cells. β-actin is shown as a loading control. Graphs show the relative abundance of the protein calculated as the density of the protein band divided by the density of the β-actin band. N Flow cytometric analysis of MitoTracker Green fluorescence in the presence or absence of ITA with or without PMI ( n = 5). O Flow cytometric analysis of IL17A frequency in the presence or absence of ITA with or without PMI ( n = 5). P ELISA analysis of IL17A secretion in the presence or absence of ITA with or without PMI ( n = 7). P -values were calculated by two-tailed Student’s t -tests ( I - K and M ) or one-way ANOVA with Tukey’s test ( L and N - P ). Data are representative of mean ± SD. Differences were considered statistically significant at * p ≤ 0.05. ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: Th17 cells were loaded in polarization medium with 100 nM mitophagy Dye (Mitophagy Detection Kit, Dojindo Molecular Technologies, Japan) after 3 days of polarization.

Techniques: Synthesized, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test