Journal: Science Advances
Article Title: Type 2 lymphocytes restrict type 3 lymphocytes during liver fibrosis and colocalize in fibroblast niches
doi: 10.1126/sciadv.aea6805
Figure Lengend Snippet: ( A and B ) Thick section confocal imaging from livers of RORγtGFP; Il5tdtomato- Cre/+; Rosa26 RFP/+ mice at the steady state (A) or after 4-week CCl 4 treatment or 14 days post-BDL (B). Higher magnification shows RORγtGFP + cells near IL-5 + lymphocytes (yellow arrows). PV (zone 1). Images represent two experiments; n = 7 or 8 mice per group. ( C ) Quantification of RORγt + cells <60 μm from Col1; n ≥ 3 mice per group. ( D ) Confocal imaging of control and 4-week CCl 4 –treated IL-17A lineage tracker mice (Il17atdtomato- Cre/+; Rosa26 RFP/+ ). n ≥ 4 mice per group. ( E ) IL-17 + lymphocytes per tissue volume. n = 4 or 5 mice per group. ( F ) Flow gating scheme for liver RORγtGFP + cells from reporter mice. ( G ) Quantification of RORγtGFP + subsets among CD45 + leukocytes in vehicle- or CCl 4 -treated mice. n ≥ 4 mice per group. ( H ) Percent RORytGFP + liver cells from reporter mice ± 4-week CCl 4 treatment; pooled from two experiments; n ≥ 9 mice per group. ( I ) PDGFRα + Sca1 + lung AFs cultured for 6 to 7 days with lung ILC2s and γδ T cells; TGF-β added to induce MF differentiation. n = 3 experiments. ( J ) Tbx21-ZsGreen “Tbet” reporter schematic, relevant to (J) to (M), and representative liver image after 4-week CCl 4 treatment or vehicle. Two mice per group, two sections each. ( K ) Perivascular IL-5 + , IL-17A + , and Tbet + lymphocytes: total; periportal <60 μm from α-SMA + , Col1a low or Col1a − , GS − ; and pericentral <60 μm from α-SMA low , Col1a + , GS + . n = 4 to 6 mice per group. ( L ) Percent Tbet + cells <60 μm from Col1. n = 2 to 4 mice per group. ( M ) Flow gating scheme for liver ZsGreen expression in Tbet-ZsGreen mice after 4-week CCl 4 treatment. All scale bars, 200 μm. Bar graphs indicate the means (±SE), Student’s t test [(E), (H), and (L)] or one-way ANOVA with Tukey post test [(C) and (K)]. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.
Article Snippet: For the therapeutic intervention, mice were placed on CCl 4 [0.5 ml/kg, intraperitoneally (ip), three times per week] for 4 weeks and treated with 250 μg of mouse anti–IL-17a (clone 17F3, BioXcell) antibody intraperitoneally in a volume of 200 μl (diluted in PBS) at the beginning of CCl 4 application and throughout the experiment or with the RORγt antagonist GSK805 (Sigma-Aldrich, cat. no. 5313690001) from weeks 2 to 4 daily at 10 mg/kg ip in corn oil.
Techniques: Imaging, Control, Cell Culture, Expressing
![( A and B ) Thick section confocal imaging from livers of RORγtGFP; Il5tdtomato- Cre/+; Rosa26 RFP/+ mice at the steady state (A) or after 4-week CCl 4 treatment or 14 days post-BDL (B). Higher magnification shows RORγtGFP + cells near IL-5 + lymphocytes (yellow arrows). PV (zone 1). Images represent two experiments; n = 7 or 8 mice per group. ( C ) Quantification of RORγt + cells <60 μm from Col1; n ≥ 3 mice per group. ( D ) Confocal imaging of control and 4-week CCl 4 –treated <t>IL-17A</t> lineage tracker mice (Il17atdtomato- Cre/+; Rosa26 RFP/+ ). n ≥ 4 mice per group. ( E ) IL-17 + lymphocytes per tissue volume. n = 4 or 5 mice per group. ( F ) Flow gating scheme for liver RORγtGFP + cells from reporter mice. ( G ) Quantification of RORγtGFP + subsets among CD45 + leukocytes in vehicle- or CCl 4 -treated mice. n ≥ 4 mice per group. ( H ) Percent RORytGFP + liver cells from reporter mice ± 4-week CCl 4 treatment; pooled from two experiments; n ≥ 9 mice per group. ( I ) PDGFRα + Sca1 + lung AFs cultured for 6 to 7 days with lung ILC2s and γδ T cells; TGF-β added to induce MF differentiation. n = 3 experiments. ( J ) Tbx21-ZsGreen “Tbet” reporter schematic, relevant to (J) to (M), and representative liver image after 4-week CCl 4 treatment or vehicle. Two mice per group, two sections each. ( K ) Perivascular IL-5 + , IL-17A + , and Tbet + lymphocytes: total; periportal <60 μm from α-SMA + , Col1a low or Col1a − , GS − ; and pericentral <60 μm from α-SMA low , Col1a + , GS + . n = 4 to 6 mice per group. ( L ) Percent Tbet + cells <60 μm from Col1. n = 2 to 4 mice per group. ( M ) Flow gating scheme for liver ZsGreen expression in Tbet-ZsGreen mice after 4-week CCl 4 treatment. All scale bars, 200 μm. Bar graphs indicate the means (±SE), Student’s t test [(E), (H), and (L)] or one-way ANOVA with Tukey post test [(C) and (K)]. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8224/pmc12978224/pmc12978224__sciadv.aea6805-f4.jpg)
