Fig 1: CD200R signaling regulates chemokine expression in TME(A) Differential gene expression analysis of the scRNA-seq data revealed significantly altered overall expression of chemokine genes in NB9464D tumors from CD200R−/− mice versus tumors from WT mice.(B) Violin plots for the expression of chemokine genes that show significant differences between tumors from WT and CD200R−/− mice. ∗∗∗P < 1e−100, ∗∗P < 1e−10 by Wilcoxon rank-sum test.(C and D) qPCR analyses for the expression of chemokine genes in NB9464D (C) and Yummer1.7 (D) tumors from WT and CD200R−/− mice. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-sided student t test.(E) ELISA assay for production of chemokines in NB9464D tumor lysates. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-sided student t test.(F) Featured plot data of scRNA-seq suggests that CCL24 and CCL8 were mainly expressed by TAMs, while CCL3, CXCL2 and CXCL3 were mainly expressed by neutrophils, but also by TAMs.(G) Differential gene expression analysis of the scRNA-seq data revealed altered expression of chemokine genes in TAMs and Neutrophils.(H) qPCR was used to quantify expression of chemokine genes in sorted CD11b+Ly6G− and CD11b+Ly6G+ cells from NB9464D tumors from WT and CD200R−/− mice.
Fig 2: Preventive exposure to FD leads to changes in MDM transcriptome that are associated with reduced CCL8 production and MHCII-mediated antigen presentation.(A) Volcano plot showing DEGs in OVA versus OVA + FD MDMs, analyzed by model-based analysis of single-cell transcriptomics (MAST). (B) Ccl8 gene expression in OVA versus OVA + FD. (C) Mouse serum CCL8 measured by enzyme-linked immunosorbent assay. (D) Serum CCL8 levels in human patients with asthma versus healthy controls. (E) WT mice were sensitized to OVA and treated daily with anti-CCL8 antibody or isotype 6 hours before OVA challenge. Representative BAL Giemsa cytospins and PAS-stained lung sections are shown. (F) Quantification of BAL eosinophils and (G) volume of mucus. (H) Lung inflammatory eosinophils (CD45+, Ly6gneg, CD125int, SiglecF+, CD11c+, and CD101+) quantified by flow cytometry. (I and J) CCL8 gene expression in murine BMDMs and human monocytes after 48 hours of FD exposure followed by 6 hours of LPS stimulation. (K) Average gene expression from MHCII-related genes (H2-Eb1, H2-Ab1, H2-Aa, and Ciita) were down-regulated in OVA + FD MDMs. (L) MHCII mean fluorescence intensity (MFI) on lung MDMs (CD45+, F4/80+, Ly6Gneg, CD11b+, and CCR2+). (M) HLA-DR MFI in human monocyte–derived APCs exposed to FD for 48 hours and LPS for 6 hours. (N) BMDMs pulsed with OVA323–339 peptide after FD and LPS stimulation and then cocultured with naive CD4+ T cells. T cell proliferation assessed by CellTrace™ Violet (CTV) MFI-based division index. (O) Proliferation-associated gene (Cenpe, Top2a, and Mki67) average expression within CD4+ T cell subset in OVA versus OVA + FD. Modules were clustered by KEGG/Reactome terms; antigen presentation node shows altered interaction strength. Results are plotted as means ± SEM, and difference between means were compared using one-way ANOVA followed by Tukey’s post hoc test (n = 4 to 19).
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