Journal: Science Advances

Article Title: Pathogenic GM-CSF drives functional diversification of inflammatory macrophages in autoimmune arthritis

doi: 10.1126/sciadv.aec0986

Figure Lengend Snippet: ( A and B ) Synovial tissue from BM chimeras (CD45.2 > CD45.1 SKG) was analyzed 6 weeks post–mannan injection. Mice without mannan injection were analyzed as healthy controls. Flow cytometry analysis of the CD45.1 + recipient– or CD45.2 + donor–derived cells among synovial myeloid cells. ( C ) Experimental design for the adoptive transfer of Ly6C hi monocytes. W, weeks. ( D ) Expression of Ly6C, MHCII, CD11c, and CCR2 on synovial CD11b + Ly6G − CD45.1 + donor–derived cells at days 1 and 3 posttransfer. Day 0 shows pretransfer Ly6C hi monocytes. ( E ) Arthritis scores of WT ( n = 6) and Csf2rb −/− SKG mice ( n = 9) 12 weeks post–mannan injection. ( F ) Experimental design of mixed BM chimera reconstituted with a 1:1 mixture of BM cells from Ccr2 −/− and WT SKG mice ( n = 6) or from Ccr2 −/− and Csf2rb −/− SKG mice ( n = 7). ( G ) Arthritis scores of the mixed BM chimeras shown in (F). ( H ) Flow cytometry analysis of myeloid cells in the synovium of the mixed BM chimera of Ccr2 −/− and Csf2rb −/− SKG mice. ( I ) Contribution of CD45.1 + CD45.2 + Ccr2 −/− cells to each synovial subset, normalized to BM CD45 + cell chimerism. ( J and K ) Intracellular IL-17A and GM-CSF staining of CD4 + T cells in the synovium of the mixed BM chimeras ( n = 6 each). ** P < 0.01. Statistical analyses were performed using multiple t tests comparing arthritic and healthy control mice (B), Student’s t test (E and K), two-stage step-up method of Benjamini, Krieger, and Yekutieliun (G), and one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test (I). Error bars denote the SD in panels. Data are pooled from two (B, G, I, and K) or three (E) independent experiments; data in (A), (D), (H), and (J) are the representatives of two independent experiments.

Article Snippet: Ccr2 −/− (B6.129S4- Ccr2 tm1Ifc /J), Il17a eGFP (C57BL/6- Il17a tm1Bcgen /J), Csf2rb −/− (B6.129S1- Csf2rb tm1Cgb /J), and CD45.1 congenic (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from the Jackson Laboratory.

Techniques: Injection, Flow Cytometry, Derivative Assay, Adoptive Transfer Assay, Expressing, Staining, Control, Comparison

Journal: Science Advances

Article Title: Pathogenic GM-CSF drives functional diversification of inflammatory macrophages in autoimmune arthritis

doi: 10.1126/sciadv.aec0986

Figure Lengend Snippet: ( A ) UMAP visualization of pooled MNPs isolated from the inflamed synovium of mixed BM chimeras generated from CD45.1 WT SKG and CD45.2 Csf2rb −/− SKG mice (see experimental schematic in fig. S4A; gating strategy provided in fig. S4B), analyzed by scRNA-seq. Data represent merged results from four independent samples. ( B ) Relative frequency of each transcriptionally defined cluster derived from CD45.1 + WT and CD45.2 + Csf2rb −/− MNPs. ( C and D ) Dot plots (C) and UMAP (D) displaying the expression of selected signature genes across MNP clusters. Ly6c1 hi mono, Ly6c1 hi monocytes; MΦ, macrophages. ( E and F ) Flow cytometry analysis of neutrophils, Ly6C hi monocytes, macrophages, OCPs, and cDCs from CD45.1 + WT and CD45.2 + Csf2rb −/− synovial cells in mixed BM chimeras. (F) Percentages of Ly6C hi monocytes, macrophages, OCPs, and cDCs among synovial CD45.1 + WT and CD45.2 + Csf2rb −/− CD11b + cells ( n = 6). ( G to I ) Flow cytometry analysis of EpCAM + macrophages and intracellular staining of Arg1 and components of complement 1q (C1q), gated on CD45.1 + WT and CD45.2 + Csf2rb −/− CD11b + Ly6G − Ly6C lo CD26 − synovial macrophages from mixed BM chimeras. (H) Percentages of EpCAM + macrophages within CD45.1 + WT and CD45.2 + Csf2rb −/− CD11b + Ly6G − Ly6C lo CD26 − synovial macrophages ( n = 8). (I) Frequencies of cells among the indicated quadrants (Q1 to Q4) shown in (G) ( n = 8). * P < 0.05 and ** P < 0.01. Statistical analyses were performed using paired t test (F and H) and multiple t tests comparing CD45.1 + WT and CD45.2 + Csf2rb −/− cells (I). Error bars denote the SD in panels. Data in (F), (H), and (I) are pooled from two independent experiments. Data in (E) and (G) are the representatives of two independent experiments.

Article Snippet: Ccr2 −/− (B6.129S4- Ccr2 tm1Ifc /J), Il17a eGFP (C57BL/6- Il17a tm1Bcgen /J), Csf2rb −/− (B6.129S1- Csf2rb tm1Cgb /J), and CD45.1 congenic (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from the Jackson Laboratory.

Techniques: Isolation, Generated, Derivative Assay, Expressing, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Pathogenic GM-CSF drives functional diversification of inflammatory macrophages in autoimmune arthritis

doi: 10.1126/sciadv.aec0986

Figure Lengend Snippet: UMAP visualization showing the expression patterns of ( A ) key proinflammatory cytokines and ( B ) indicated genes across synovial MNPs. ( C ) Flow cytometry analysis of Arg1 and C1q expression, along with surface markers EpCAM and CD11c, within CD11b + Ly6G − Ly6C lo CD26 − synovial MNPs from control (healthy) and arthritic SKG mice. Arthritic SKG mice were analyzed 4 weeks after mannan injection. ( D ) Total numbers of Arg1 + and EpCAM + macrophages in the synovium of control and arthritic SKG mice ( n = 6 each). ( E and F ) Intracellular staining of TNF-α, IL-6, and pro–IL-1β in CD11b + Ly6G − Ly6C lo CD26 − CD200 − synovial macrophages derived from CD45.1 + WT and CD45.2 + Csf2rb −/− cells in mixed BM chimeric mice, 4 weeks post–mannan injection. (F) Percentages and total numbers of TNF-α + , IL-6 + , and pro–IL-1β + macrophages, as shown in (E) ( n = 6). ( G ) UMAP displaying the expression levels of Ccl17 , Irf4 , and Pdcd1lg2 . ( H ) Gating strategy for sorting synovial cell subsets from arthritic SKG mice, including cDCs, Ly6C hi monocytes, EpCAM + macrophages, CD11c lo macrophages, and CD11c hi EpCAM − macrophages from arthritic SKG mice. ( I ) Relative mRNA expression of Ccl17 in the indicated sorted subsets ( n = 4). Expression levels were normalized to those of Hprt . ** P < 0.01. Statistical analyses were performed using Student’s t test (D), paired t test (F), and one-way ANOVA, followed by Tukey’s multiple-comparison test (I). Error bars denote the SD in panels. Data in (C), (E), (H), and (I) are the representatives of two independent experiments; data in (D) and (F) are pooled from two independent experiments.

Article Snippet: Ccr2 −/− (B6.129S4- Ccr2 tm1Ifc /J), Il17a eGFP (C57BL/6- Il17a tm1Bcgen /J), Csf2rb −/− (B6.129S1- Csf2rb tm1Cgb /J), and CD45.1 congenic (B6.SJL- Ptprc a Pepc b /BoyJ) mice were purchased from the Jackson Laboratory.

Techniques: Expressing, Flow Cytometry, Control, Injection, Staining, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Multidimensional characterization of cellular ecosystems in Hodgkin lymphoma

doi: 10.1101/2025.03.18.643177

Figure Lengend Snippet: (A) Proportion of mutation types across recurrently mutated genes. (B) Lollipop diagram depicting the distribution of CSF2RB mutations in our in-house cohort. (C) WB analysis of CSF2RB expression and phosphorylation status of STAT5 in the L-428 model. Representative images are shown (left). Densitometry analysis was carried out by Image Lab (right). Data are presented as mean ± SEM. Statistical analyses were conducted using one-way ANOVA followed by Dunnett’s multiple comparisons test. (D) Top pathways from LymphoChip upregulated in E788* cells compared to WT cells in the L-428 model. (E) Heatmap showing transcript levels of genes in the L-428 model. Genes with consistent expression changes (absolute fold change ≥ 1.4) across all three experimental models (L-428, U-HO1 + IL-3, U-HO1 + IL-5) are shown. (F) TARC and IL-13 concentrations in supernatant of L-428 model at baseline. Data are presented as mean ± SEM. Statistical analyses were conducted using one-way ANOVA followed by Dunnett’s multiple comparisons test (TARC), and a negative binomial regression model to account for overdispersion in the data (IL-13). (G) TARC and IL-13 concentrations in supernatant of L-428 (left) model treated with pacritinib (0.5 μM) for 48 hours, and U-HO1 (right) model in the presence of IL-3 (1 ng/mL) or IL-5 (1 ng/mL) treated with or without pacritinib (0.5 μM) for 48 hours. Data are presented as mean ± SEM. Pre- and post-treatment comparisons were conducted using an unpaired, two-sided t -test for each cell type. (H) CCR4+CD4+ T cell migration towards L-428 CSF2RB WT or E788* supernatant in a transwell migration assay, showing the percentage of migrated cells relative to the input control. A paired, two-sided t -test was used to compare the two conditions. Data are presented as mean ± SEM (n=4). (I) Spleen tissues from humanized mouse model, sacrificed at 12 weeks after CB injection, were stained with hematoxylin and eosin (H&E), CD30, CD4, PAX5, and FOXP3. The image is presented at a magnification of 20X. (J) Percentage of CD4+CD25+FOXP3+ Tregs identified in spleen of humanized CNW mice engrafted with L-428 CSF2RB WT or E788* cells by FCM. An unpaired, two-sided t -test was used to compare the two conditions, and data are shown as the mean ± SEM (n=6 for WT and n=5 for E788*). (K) Bar graph depicts serum TARC concentration from CNW mice engrafted L-428 WT (n=6) and E788* (n=5) cells, sacrificed at 12 weeks after CB injection. An unpaired, two-sided t -test was used to compare the two conditions. The data are shown as mean ± SEM.

Article Snippet: Immunoblotting was performed as previously described( Alig et al ., 2023 ) using the following primary antibodies: STAT1 Rabbit mAb (D1K9Y), STAT3 Mouse mAb (124H6), STAT6 Rabbit mAb (D3H4), AKT(pan) Rabbit mAb (C67E7), Phospho-STAT1 (Y701) Rabbit mAb (58D6), Phospho-STAT3 (Y705) Rabbit mAb (D3A7), Phospho-STAT5 (Y694) Rabbit mAb (C11C5), Phospho-STAT6 (Y641) Rabbit mAb (C11A12), Phospho-AKT (S473) Rabbit mAb (193H12), GAPDH Rabbit mAb (14C10) (all from Cell Signaling Technology, dilution 1:1000), STAT5 Rabbit polyclonalAb (C-17) (dilution 1:5000), CSF2RB (IL-3/IL-5/GM-CSFRβ Mouse mAb (A-3) (dilution 1:100) (Santa Cruz Biotechnology), followed by secondary staining using anti-Mouse (dilution 1:10000; Promega, catalog no. W4021) or Rabbit IgG (H+L) HRP conjugate (dilution 1:5000; Promega, catalog no. W4011), and detected using Amersham ECL Detection Reagents (Cytiva, catalog no. RPN3004).

Techniques: Mutagenesis, Expressing, Migration, Transwell Migration Assay, Control, Injection, Staining, Concentration Assay