Journal: bioRxiv

Article Title: The CCR4/CCL17 axis drives intestinal acute Graft versus Host disease after allogeneic bone marrow transplantation

doi: 10.1101/2024.03.02.583093

Figure Lengend Snippet: (A+B) Transwell migration assay of splenic T cells from either C57BL6/N wild-type mice or CCR4-/-animals was measured towards a CCL17 (800 ng/ml) gradient. Cells were counted via FACS for 90 seconds. WT CD3+ T cells ( p=0.0046 ) and CD4+ T cells ( p=0.0035 ) showed significant migration towards CCL17. (C) Naïve CD8+ T cells were isolated from the spleen of C57BL6/N mice. These cells were co-cultured with αCD3/CD28 beads and CCL17 (800ng/ml) when indicated. Proliferation was measured by CFSE distribution 60 hours later. (p p=0.8986 ) (C) Naïve CD4+ T cells were differentiated into Th1, Treg or Th2 cells in the presence (red) or absence of CCL17 (blue). Representative plots are shown. (D) Survival of BALB/c recipient mice after alloHSCT. Survival is not affected by CCL17 of donor bonemarrow or T cells; p=0.11 . Schematic figure created using biorender.com. (E) CCL17-/-recipient mice showed prolonged survival compared to CCL17 +/+ recipients, p=0.0069 . Schematic figure created using biorender.com. The data are pooled from 3 independent experiments with at least 3 mice per group. (F) Samples from gut biopsies of patients with the suspicion of intestinal aGvHD were collected. An experienced pathologist performed GvHD grading and samples were stained for CCL17 expression. Patients with proven aGvHD (n=12) show increased CCL17 positive area in representative areas compared to patients without GvHD (n=9). Representative samples of each group are shown.

Article Snippet: 600 μl of RPMI 1640 medium supplemented with 10% FBS, 1% P/S containing CCL17 (800ng/ml, R&D Systems, #529-TR), when indicated, was added to a well of a 24 well plate and a 6.5 mm Transwell® Polycarbonate Membrane insert with a 5.0 μm pore (Costar #3421/Sigma # CLS3421-48EA) was added to each well.

Techniques: Transwell Migration Assay, Migration, Isolation, Cell Culture, Staining, Expressing

Journal: bioRxiv

Article Title: The CCR4/CCL17 axis drives intestinal acute Graft versus Host disease after allogeneic bone marrow transplantation

doi: 10.1101/2024.03.02.583093

Figure Lengend Snippet: (A) CCL17egfp/+ recipient mice underwent alloHSCT. Mice receiving only bone marrow cells (no aGvHD induction) were compared to those receiving additional T cells for induction of aGvHD. Quantification of the CCL17 positive area was higher in the GvHD group p=0.027 . The experiment was performed twice. Representative data from one experiment is shown. Schematic figure created using biorender.com. Representative images of the small intestine stained for CD11c (DC marker) and DAPI are shown. Note the co-localization of CCL17(eGFP) and CD11c(PE). (B) Irradiated BALB/c mice received bone marrow cells and additional T cells from C57BL6/N donors. Mice were fed with ruxolitinib until day 3. Mice were sacrificed and CCL5 ( p=0.3267 ), CXCL10 ( p=0.0622 ) and CCL17 ( p=0.0045 ) expression was determined via qPCR using the ΔΔCT method. Pooled data from 2 independent experiments. Schematic figure created using biorender.com. (C) Splenic DCs were treated with IL-33 for 18 hours, which induced CCL17 secretion ( p<0.0001). (D) The presence of ruxolitinib (1µM) dampend CCL17 levels ( p=0.023 ). Schematic figure created using

Article Snippet: 600 μl of RPMI 1640 medium supplemented with 10% FBS, 1% P/S containing CCL17 (800ng/ml, R&D Systems, #529-TR), when indicated, was added to a well of a 24 well plate and a 6.5 mm Transwell® Polycarbonate Membrane insert with a 5.0 μm pore (Costar #3421/Sigma # CLS3421-48EA) was added to each well.

Techniques: Staining, Marker, Irradiation, Expressing

Journal: Scientific Reports

Article Title: Targeting fibroblast CD248 attenuates CCL17-expressing macrophages and tissue fibrosis

doi: 10.1038/s41598-020-73194-x

Figure Lengend Snippet: Cd248 disruption reduced infiltration and pro-fibrotic phenotype switching of kidney macrophages during renal fibrosis. ( a ) Representative images of F4/80 immunostaining for macrophages in control and D14 UUO kidneys. F4/80 + staining was brown. Original magnification, × 100. Scale bar, 100 μm. ( b ) Quantification of F4/80 + areas on LPF images of kidney sections taken at 100 × magnification n = 10. ( c ). Representative images of green fluorescent protein (GFP) + circulation-derived cells and F4/80 + (red) macrophages in control and day 3 (D3) UUO-kidneys of WT and Cd248 –/– parabionts joined surgically to transgenic GFP ( GFP Tg ) mice. Arrowhead indicates an F4/80 + GFP + macrophage. Original magnification, × 100. Scale bar, 100 μm. ( d ) Quantification of GFP + , F4/80 + and F4/80 + GFP + cells on LPF images of control (upper panel) and D3 UUO (lower panel) kidneys of WT and Cd248 –/– parabionts. n = 3. ( e ) qPCR of genes encoding cytokines and enzymes in macrophages isolated from D7 UUO kidneys. n = 4. ( f ) qPCR of Nos2 , Arg1 and Ccl17 in lipopolysaccharide (LPS) and interferon γ (IFNγ)–primed RAW264.7 macrophages co-cultured with D7 UUO-kidney myofibroblasts isolated from WT or Cd248 –/– mice in the Transwell system. RAW264.7 macrophages co-cultured with medium were used as control. n = 4. ( g ) qPCR of Ccl17 in WT bone marrow–derived macrophages (BMDMs, Mϕ) co-cultured with medium only (control) or with WT or Cd248 –/– UUO-kidney myofibroblasts (MF) in the same dish. Recombinant CD248 (rCD248) was included in the culture as indicated. n = 5. Data are expressed as means ± standard errors of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with post hoc Tukey’s multiple comparisons test in ( b , g ) and unpaired t-test in ( d , e , f ).

Article Snippet: Lower chambers of Costar Transwell plates (5 μm pores size, 3421; Corning, NY) were filled with 0.6 ml of starvation media with or without supplementation of 500 ng/ml recombinant murine CCL17 (529-TR/CF; R&D Systems).

Techniques: Disruption, Immunostaining, Control, Staining, Derivative Assay, Transgenic Assay, Isolation, Cell Culture, Recombinant

Journal: Scientific Reports

Article Title: Targeting fibroblast CD248 attenuates CCL17-expressing macrophages and tissue fibrosis

doi: 10.1038/s41598-020-73194-x

Figure Lengend Snippet: Anti-CCL17 antibody administration attenuated murine obstructive renal fibrosis. ( a ) Representative images of picrosirius red staining in control and UUO kidneys on day 10 after surgery in mice administered with anti-CCL17 antibody or control IgG. ( b ) Quantification of Sirius red + collagen fibrils on LPF images of kidney sections. (c) Representative images of F4/80 immunostaining for macrophages in control and UUO kidneys on day 10 after surgery. ( d ) Quantification of F4/80 + areas on LPF images of kidney sections. ( e ) Representative images of αSMA immunostaining for myofibroblasts in control and UUO kidneys on day 10 after surgery. ( f ) Quantification of αSMA + areas on LPF images of kidney sections. The original magnification of representative images was × 100 in ( a , c , e ) and the LPF images of kidney sections were taken at 100 × magnification in ( b , d , f ). Scale bar, 100 μm. Data are expressed as means ± standard errors of the mean. n = 6. * P < 0.05, ** P < 0.01 and *** P < 0.001 by one-way ANOVA with post hoc Tukey’s multiple comparisons test.

Article Snippet: Lower chambers of Costar Transwell plates (5 μm pores size, 3421; Corning, NY) were filled with 0.6 ml of starvation media with or without supplementation of 500 ng/ml recombinant murine CCL17 (529-TR/CF; R&D Systems).

Techniques: Staining, Control, Immunostaining

Journal: Scientific Reports

Article Title: Targeting fibroblast CD248 attenuates CCL17-expressing macrophages and tissue fibrosis

doi: 10.1038/s41598-020-73194-x

Figure Lengend Snippet: CD248 interacted with galectin-3 to induce CCL17-expressing pro-fibrotic macrophages. ( a ) qPCR of genes encoding cytokines and chemokines in macrophages isolated from D7 UUO kidneys of WT and Lgals3 knockout ( Lgals3 –/– ) mice. n = 5. ( b,c ) HEK293T cells were transfected with and without plasmid DNA expressing galectin-3 or CD248-DDK separately. CD248-DDK (DDK-IP) or galectin-3 (Gal3-IP) was immunoprecipitated from the cell lysates, and then immunoblot analyses of galectin-3 and CD248-DDK were performed. ( d ) Flow cytometry of the binding of CD248 extracellular domain (CD248ECD-EGFP) to WT and Lgals3 –/– BMDMs. Blue and red lines indicate results for WT and Lgals3 –/– BMDMs, respectively, with CD248ECD-EGFP. Black and gray lines indicate results for WT and Lgals3 –/– BMDMs, respectively, with control medium. ( e ) Flow cytometry of CD248ECD-EGFP binding to WT BMDMs in the presence of 25 mM lactose (red) or sucrose (blue). Conditioned medium from HEK293T cells in the presence of lactose (gray) or sucrose (black) was used as control. ( f ) qPCR of Ccl17 in WT and Lgals3 –/– BMDMs in the absence (control) or presence of 200 ng/ml rCD248 for 48 h. n = 6. ( g ) Gel plots of PCR for Ccr4 and Gapdh in D7 UUO-kidney myofibroblasts isolated from WT and Cd248 –/– mice. The reaction cycles for Ccr4 and Gapdh were 30 and 15, respectively. ( h ) qPCR for genes Col1a1 and Acta2 in isolated D7 UUO-kidney myofibroblasts after incubation with vehicle (Con), TGF-β1, and CCL17 for 24 h. Data are expressed as means ± standard errors of the mean. * P < 0.05, ** P < 0.01 and *** P < 0.001 by unpaired t-test in ( a ) and one-way ANOVA with post hoc Tukey’s multiple comparisons test in ( f , h ).

Article Snippet: Lower chambers of Costar Transwell plates (5 μm pores size, 3421; Corning, NY) were filled with 0.6 ml of starvation media with or without supplementation of 500 ng/ml recombinant murine CCL17 (529-TR/CF; R&D Systems).

Techniques: Expressing, Isolation, Knock-Out, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Flow Cytometry, Binding Assay, Control, Incubation