Fig 1: TKO Tregs Display Marked Defects in Mobilization In Vitro and In Vivo(A) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were treated with anti-CD3 antibodies and TGF-β1 for differentiation into in vitro differentiated Tregs (iTregs). Percentage of WT and TKO CD4+CD25+Foxp3+ Tregs were measured by flow cytometry at the indicated time points (n = 6 per group). (B and C) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were activated by anti-CD3 antibodies for 24 h and subjected to qRT-PCR analysis (B) or ELISA from supernatants (C) for the indicated cytokines, chemokines, and growth factors (n = 5–9 per group).(D and E) Transwell migration study of CD4+CD25+ Tregs isolated from WT and TKO mice after 12 weeks of HFD. Cells were assessed for migration in the presence or absence of CCL19 (D) or CCL20 (E) (n = 3 per group).(F and G) Flow cytometry for CCR7 (F) or CCR6 (G) expression in WT and TKO Tregs (n = 6 per group).(H) Schematic of PKH26-labeled HFD WT and TKO Tregs adoptively transferred to HFD C57BL/6 mice. Flow cytometry shows percentage of PKH26-expressed cells in liver, VAT, and SAT of recipient mice (n = 6 per group).(I and J) Schematic of glucose uptake study of differentiated 3T3-L1 cells co-cultured with HFD WT and TKO iTreg supernatant (supe) (I). (J) Fluorescence intensity of 2-Deoxy-D-glucose (2-DG) uptake by differentiated 3T3-L1 cells co-cultured with supernatants of WT and TKO CD4+ Tregs in the presence or absence of insulin stimulation (n = 4 per group).(K and L) Schematic of glucose production study of mouse primary hepatocytes co-cultured with HFD WT and TKO iTreg supernatants (K). (L) Glucose production by mouse primary hepatocytes co-cultured with supernatants of HFD WT and TKO CD4+ Tregs (n = 6 per group).Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM.Related to Figures S3 and S4.
Fig 2: Optn deficiency hinders CII-induced DC migration. (A) qRT-PCR analyses of Optn in WT and Optn KO BMDCs. n = 3. (B) Scheme (left) and quantification (right) for Transwell analysis of CCL19/21-triggered migration of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h. n = 3. (C) Scheme (left), representative images (middle) and bar graph (right) for the in vivo WT and Optn KO BMDC migration after labeling with CFSE and treating with CII (100 μg/mL) for 24 h. n = 3. (D) PCA analysis of transcriptome profiles of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h. n = 3. (E) Volcano plot of transcriptome profiles of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h (P < 0.05, fold-change >1.5). n = 3. (F) GSEA analyses of genes enriched in CII-pulsed WT or Optn KO BMDCs. n = 3. (G) Heatmap analyses of representative genes involved in cell migration in CII-pulsed WT or Optn KO BMDCs. n = 3. (H) qRT-PCR analyses of indicated genes in WT and Optn KO BMDCs after CII (100 μg/mL) treated for 24 h. n = 3. (I) Immunostaining of F-Actin (green) and membrane MHC-II (red) in WT and Optn KO BMDCs after CII (100 μg/mL) treatment for 24 h. Scale bar: 3 μm. n = 3. Data are presented as mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
Fig 3: CII establishes a characteristic gene profile to activate DC migration. (A) Scheme of the experimental approach for Affymetrix microarray gene chip analysis from the EMBL-EBI database (accession no. E-MTAB-2904). (B) Volcano plot of genes differentially expressed in human MoDCs after LPS or CII stimulation for 20 h (fold change >2; P < 0.05). n = 6. (C) Venn diagram of differentially up-regulated genes after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (D) Heatmap and clustering analysis of differentially up-regulated genes after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (E) Bar plots of gene ontology analysis of the up-regulated genes in different clusters of (D). n = 6. (F) Gene tree analysis based on gene expression levels after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (G, H) GSEA enrichment scores for indicated gene sets in CII (G) or LPS (H) stimulated human MoDCs. n = 6. (I) Transwell analysis of CCL19/21-triggered migration of BMDCs upon LPS (50 ng/mL) or CII (100 μg/mL) treatment for 20 h. n = 3. Data are presented as mean ± SD; ∗∗∗P < 0.001.
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