Journal: Hepatology Communications

Article Title: Arrb2 in hepatocytes promotes M2 macrophage polarization, ameliorates hepatic ischemia–reperfusion injury through upregulating metabolite 6-ketoLCA

doi: 10.1097/HC9.0000000000000916

Figure Lengend Snippet: 6-ketoLCA upregulated by Arrb2 in hepatocytes promotes M2 macrophage polarization, alleviating hepatic IRI. (A–E) Liver and serum samples from liver-specific Arrb2-CKO and WT mice with or without 6-ketoLCA treatment after I/R (n=6). (A) HE and TUNEL staining were analyzed microscopically. Scale bar: 200 μm; Suzuki score and necrosis area were examined. (B) Quantitative analysis of the bile acid metabolite 6-ketoLCA in liver tissue. (C) Liver function levels (ALT, AST, TBA, GGT) were analyzed between groups. (D) qRT-PCR analysis of mouse liver tissue mRNA expression for inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (E) iNOS, CD86, CD206, and CD163 expression in PMMs was assessed by WB analysis. (F) The CCK8 assay measured the viability of RAW264.7 cells exposed to varying concentrations and durations of 6-ketoLCA (n=3). (G, I) Co-culture of RAW264.7 and AML12 cells. (G) ELISA for culture medium supernatant inflammatory factors (IL-6, TNF-α, IL-10, TGF-β). (I) BCL2, BAX, and GAPDH expression in AML12 cells was assessed by WB analysis. (H, J) Flow cytometry detected the proportions of M1 (CD86, PE) and M2 (CD206, APC) macrophages treated with IL-4 and 6-ketoLCA. The ratio of M1/M2 macrophages was measured. All results are presented as mean ± SD, and statistical significance was assessed. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CKO, conditional knockout; GGT, gamma-glutamyl transferase; HE, hematoxylin and eosin; I/R, ischemia/reperfusion; IL-6, interleukin 6; IL-10, interleukin 10; IRI, ischemia–reperfusion injury; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PMMs, primary mouse macrophages; TBA, total bile acids; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; WB, western blotting; WT, wild type.

Article Snippet: RAW264.7 were cultured in 10% FBS RPMI-1640 medium with 6-ketoLCA (100 ng/mL) and IL-4 (20 ng/mL) to induce polarization to M2-like macrophage. shRNA targeting TGR5 (GeneCopoeia, Rockville, MD, USA) was transfected into RAW264.7 cells utilizing Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol to achieve TGR5 knockdown.

Techniques: TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, CCK-8 Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Knock-Out, Reverse Transcription, Polymerase Chain Reaction, End Labeling, Western Blot

Journal: Frontiers in Immunology

Article Title: CRAC Channel Controls the Differentiation of Pathogenic B Cells in Lupus Nephritis

doi: 10.3389/fimmu.2021.779560

Figure Lengend Snippet: CRAC channel inhibition suppressed B cell differentiation. (A) Blimp-1 expression in B cells from patients with lupus nephritis (LN) or healthy controls (HC) was measured by Western blot and representative bands of six independent samples. (B, C) Naive B cells from LN or HC were cultured in the presence of anti-IgM (5 μg/ml), anti-CD40 (1 μg/ml), IL-2 (20 ng/ml), IL-4 (20 ng/ml), and IL-21 (50 ng/ml) for 8 days. Percentages of CD19 low CD138 + plasma cells were determined by flow cytometry. Representative counter plots are shown and data from five samples. Data are mean ± SEM. *p < 0.05 by t-test. (D) GSEA plot for the gene set of B-cell differentiation showing the enrichment scores for YM-58483 or vehicle-treated B cells (n = 3). (E) Blimp-1 expression in B cells treated with YM58483 or vehicle was measured by Western blot and representative bands of six independent samples. (F) Blimp-1, ORAI1 expression in B cells treated with ORAI1 siRNA or scramble siRNA was measured by Western blot. Representative bands of six samples are shown. (G, H) CD138 and IgG expression in B cells treated with YM-58483 or vehicle. Representative counter plots are shown, and the percentages of CD138 + or CD138 + IgG + cells are summarized in dot plot with bar plot (n = 5). (I, J) CD138 and IgG expression in B cells treated with ORAI1 siRNA or scramble siRNA. Representative counter plots are shown, and the percentage of CD138 + or CD138 + IgG + cells are summarized in dot plot with bar plot (n = 5). For the Western blot data, relative expression values to GAPDH are indicated above each lane. *p < 0.05, **p < 0.01 by paired t-test.

Article Snippet: Cells were stimulated with antihuman IgM (5 μg/ml, Sigma, #10759) and antihuman CD40 (1 μg/ml, Bioxcell, #BE0189) antibodies in the presence of interleukin (IL)-2 (20 ng/ml, PeproTech), IL-4 (25 ng/ml, Sino Biological) and IL-21 (50 ng/ml, Sino Biological) for 8 days.

Techniques: Inhibition, Cell Differentiation, Expressing, Western Blot, Cell Culture, Flow Cytometry

Journal: Theranostics

Article Title: Grafted human ESC-derived astroglia repair spinal cord injury via activation of host anti-inflammatory microglia in the lesion area

doi: 10.7150/thno.70929

Figure Lengend Snippet: Grafted astroglia activate anti-inflammatory microglia via IL-4 signaling. (A-C) Representative images showing CD68 (red) and IL-4R (green) staining in the lesion center at 4 weeks post-transplantation. A1-C1 are higher magnification images of boxed areas in A-C ; A1'-C1' are three-dimensional models of the microglia in A1-C1 . Scale bars: 30 μm (A-C) , 5 μm (A1-C1, A1'-C1') . (D) Quantification of IL-4R + CD68 + cells relative to all CD68 + cells ( n = 5 per group). Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test. Data are mean ± SD; ** p < 0.01, *** p < 0.001. (E) Western blotting for IL-4, p-STAT6, and Arg1 expression in the lesion area at 3 to 28 days after transplantation. (F-H) Quantification of IL-4, p-STAT6 and Arg1 expression at 3 to 28 days ( n = 3 per group). One-way ANOVA followed by Tukey's multiple comparisons test for each time point; once one-way ANOVA failed, Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparisons test or unpaired t test with Welch's correction. Data are mean ± SD; * p < 0.05, ** p < 0.01, n.s., not significant. (I) Diagram depicting the model for anti-inflammatory polarization of microglia to promote repair of the spinal cord lesion after astroglial transplantation.

Article Snippet: Primary antibodies used for WB included: monoclonal anti-PDGFR-β (Abcam cat. #ab32570; RRID: AB_2262874; 1:5000), monoclonal anti-fibronectin (Abcam cat. #ab2413; RRID: AB_777165; 1:5000), monoclonal anti-IL-4 (Sino Biological cat. #11846-R404; 1:1000), monoclonal anti-phospho-STAT6 (CST cat. #56554; RRID: AB_2799514; 1:500), polyclonal anti-Arg1 (GeneTex cat. #GTX109242; RRID: AB_2036264; 1:1000), polyclonal anti-Iba1 (Abcam cat. #ab5076; RRID: AB_2224402; 1:1000).

Techniques: Staining, Transplantation Assay, Western Blot, Expressing