The Transcription Factor ZEB2 Is Required to Maintain the Tissue-Specific Identities of Macrophages

Abstract

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.

Keywords: Clec4f-cre; Fcgr1-cre; Identity; LXRα; Macrophage; Transcription Factor; ZEB2.

Graphical abstract

Figure 1

ZEB2 Controls Mac Number and Surface Phenotype (A) Expression of CD64 and F4/80 by live CD45⁺Ly6G⁻Ly6C^- liver cells and Clec4F and Tim4 by total liver macs in Clec4f-crexZeb2^fl/fl and Zeb2^fl/fl mice. Absolute number of liver macs per gram of liver and % of total macs expressing Clec4F and Tim4. Data are pooled from four experiments with n = 11–13 per group. ^∗∗∗p < 0.001 Student’s t test. (B) Expression of SiglecF, F4/80, and CD11b by live CD45⁺CD64⁺CD11c⁺ Lung macs in Itgax-crexZeb2^fl/fl and Zeb2^fl/fl mice. AMs as a percentage of total live CD45⁺ cells, absolute number, and percentage of CD11b⁺ and CD11b⁻ AMs in Itgax-crexZeb2^fl/fl or Zeb2^fl/fl mice. Data are pooled from two experiments with n = 7–8 per group. ^∗p < 0.05, ^∗∗∗p < 0.001 Student’s t test. (C and D) t-SNE plot of SC-RNA-seq data of KCs from Clec4f-crexZeb2^fl/fl or Zeb2^fl/fl mice (C) or AMs from Itgax-crexZeb2^fl/fl or Zeb2^fl/fl mice (D), showing clusters, assigned groups, and CRE⁻ (Red) and CRE⁺ (Teal) overlay. (E) tSNE plots showing expression of Zeb2, Siglecf, and Ms4a1 in KCs. (F) tSNE plots showing expression of Zeb2, Epcam, and Cd101 in AMs. (G and H) Top 15 DE genes per group based on LogFC per group of KCs (G) or AMs (H). See also Figure S1.

Figure 2

Zeb2^−/− Macs Are Lost with Time (A) SiglecF and CD20 expression by Clec4F⁺ KCs at 6 and 12 weeks of age compared with Zeb2^fl/fl controls. Data are from one or two experiments with n = 7–10 per group. ^∗∗∗p < 0.001 one-way ANOVA with Bonferroni post-test. (B) Relative expression of Zeb2 mRNA normalized to β-actin as determined by qPCR of sorted SiglecF⁺ and SiglecF⁻ KCs compared with CRE⁻ controls. Data are pooled from one experiment with n = 5–7 per group. ^∗∗∗p <0.001 one-way ANOVA with Bonferroni post-test. (C) Expression and percentage of EpCam and CD101 by AMs at 6 and 12 weeks of age compared with Zeb2^fl/fl controls. Data are pooled from one or two experiments with n = 5–11 per group. (D) Relative expression of Zeb2 mRNA normalized to β-actin as determined by qPCR of sorted EpCam⁺ and EpCam⁻ AMs compared with CRE⁻ control AMs. Data are from one experiment with n = 5–7 per group. (E) Expression of Zeb2 mRNA and SiglecF in KCs from Zeb2^fl/fl and Clec4f-crexZeb2^fl/fl mice compared with label probe only control. Data are from one experiment with n = 5–6 per group. (F) Expression of Zeb2 mRNA and EpCam in AMs from Zeb2^fl/fl and Itgax-crexZeb2^fl/fl mice compared with label probe only control. Data are from one experiment with n = 4–5 per group. (G) Schematic of experimental set up. (H) Percentage SiglecF⁺CD20^int KCs amongst total CD45.2⁺ KCs and (I) percentage CD101⁺EpCam⁺ AMs amongst total CD45.2⁺ AMs at indicated time points (days) post the last dose of tamoxifen. Data are pooled from two experiments with n = 4–7 per time-point. ^∗∗∗p < 0.001, one-way ANOVA with Bonferroni post-test. In (H) and (I) each time point is compared to the previous time point and controls are pooled from donor macs from mice administered corn oil and host macs from mice administered tamoxifen. See also Figure S2.

Figure 3

ZEB2 Controls Tissue Identity of KCs and AMs (A) Schematic of experimental set up. (B) Expression of CD45.1 (donor) and Tim4 in total Clec4F⁺ KCs in Clec4f-crexZeb2^fl/fl chimeras. Percentage of total chimerism of KCs in Zeb2^fl/fl and Clec4f-crexZeb2^fl/fl mice. Data are pooled from two experiments with n = 6–10 per group.^∗∗∗p < 0.001 Student’s t-test. (C) Expression of CD45.1 (donor) and CD45.2 (host) in total lung AMs in Itgax-crexZeb2^fl/fl mice. Percentage of total chimerism of AMs in Zeb2^fl/fl and Itgax-crexZeb2^fl/fl mice. Data are pooled from two experiments with n = 5–8 per group. NS; non-significant. Student’s t-test. Percentage total chimerism calculated as ratio over the chimerism in blood Ly6C^hi monocytes in the same mouse. (D and E) Heatmap of expression of cell death-associated genes per group of KCs (D) or AMs (E) from SC-RNA-seq data. (F) Representative western blots (n = 2) for RIPK3, pMLKL, and Tubulin expression by total BAL cells isolated from CD45.1 mice that were irradiated (8 Gy) and reconstituted with Rosa26-cre^ert2xZeb2^fl/fl BM. 33 weeks post reconstitution, mice were fed 5 mg tamoxifen or corn oil as a control for 5 days. 27 days after the last dose, mice were sacrificed and BAL fluid isolated. BAL fluid from 3 or 4 mice was pooled per replicate and 150,000 cells were used per lane. Ratio of band intensity was calculated using ImageJ. Data are pooled from two experiments with n = 6–8 per group. Student’s t-test was used to calculate indicated p values. (G) Venn diagram showing DE genes specific to Zeb2^−/− KCs (group 3), Zeb2^−/− AMs (group 3), or shared between both mac populations. (H and I) Heatmap showing expression of top core genes across KC (H) or AM (I) groups from SC-RNA-seq data. Genes in red are significantly differentially expressed. See also Figure S3.

Figure 4

Loss of LXRα from KCs Recapitulates Main Features of Zeb2^−/− KCs (A) Expression of CD64 and F4/80 by live CD45⁺Ly6G⁻Ly6C⁻ liver cells in Clec4f-crexNr1h3^fl/fl and Nr1h3^fl/fl mice. Liver macs as a percentage of total live CD45⁺ cells and absolute number per gram of liver. (B) Expression of Clec4F and Tim4 by total liver macs in Clec4f-crexNr1h3^fl/fl and Nr1h3^fl/fl mice and percentage of total macs expressing Clec4F and Tim4. Data are pooled from two experiments with n = 12 per group. ^∗∗∗p < 0.001 Student’s t test. (C) Percentage total chimerism of total Clec4F⁺ KCs in Nr1h3^fl/fl and Clec4f-crexNr1h3^fl/fl mice. Data are pooled from two experiments with n = 6–8 per group. ^∗∗∗p < 0.001; Student’s t-test. (D) t-SNE plot of SC-RNA-Seq data from KCs from Clec4f-crexNr1h3^fl/fl and Nr1h3^fl/fl mice, showing clusters, assigned groups and CRE⁻ (Red) and CRE⁺ (Teal) overlay. (E) Heatmaps showing top DE genes (15 downregulated, 15 upregulated) based on LogFC in KCs with loss of LXRα and expression of the same genes by the indicated groups of KCs from Zeb2^fl/fl and Clec4f-crexZeb2^fl/fl mice. (F and G) Histogram and MFI of CD55 expression in (F) Zeb2^+/+ (Zeb2^fl/fl), SiglecF⁺Zeb2^−/− KCs, and SiglecF⁻Zeb2^+/− KCs from Clec4f-crexZeb2^fl/fl mice and (G) Nr1h3^+/+ (Nr1h3^fl/fl) and Nr1h3^−/− KCs from Clec4f-crexNr1h3^fl/fl mice. (H) Venn diagram showing DE genes specific to Zeb2^−/− KCs, Nr1h3^−/− KCs, or shared between both mac populations. See also Figure S4.

Figure 5

Loss of ZEB2 Affects Mac Phenotype and/or Number across Tissues (A) Expression of CD64, F4/80 and CD11b by live CD45⁺Ly6G^-CD64⁺Ly6C⁻MHCII⁻ SMs in Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice. SMs as a percentage of total live CD45⁺ cells, absolute number and percentage of CD11b⁺ and CD11b⁻ SMs in Fcgr1-crexZeb2^fl/fl or Zeb2^fl/fl mice. (B) Expression of CD64, F4/80, CD11c, and CD11b by live CD45^int microglia in Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice. Microglia as a percentage of total live CD45⁺ cells, absolute number and percentage of CD11c⁺ and CD11c⁻ microglia in Fcgr1-crexZeb2^fl/fl or Zeb2^fl/fl mice. (C) Expression of Ly6C and MHCII (monocyte waterfall) by live CD45⁺CD11b⁺Ly6G⁻SiglecF⁻ non cDCs in Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice. Percentage of live CD45⁺ and absolute number of Ly6C⁺MHCII⁻, Ly6C⁺MHCII⁺, and Ly6C⁻ Macs in Fcgr1-crexZeb2^fl/fl or Zeb2^fl/fl mice. Data are pooled from two experiments with n = 8–11 per group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 Student’s t test. See also Figure S5.

Figure 6

Loss of Zeb2 Results in Altered Transcriptome across Mac Lineage (A and B) t-SNE plot of SC-RNA-seq data from SMs (A) and microglia (B) sorted from Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice, showing clusters of macs, assigned groups and CRE⁻ (Red) and CRE⁺ (Teal) overlay and expression of indicated genes. (C) t-SNE plot of SC-RNA-seq data from total CMs from Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice, showing clusters of macs and Cd74, Itgax, H2-Aa, and H2-Eb1 expression. (D) t-SNE plot of SC-RNA-seq data from Cd74^hi CMs from Fcgr1-crexZeb2^fl/fl and Zeb2^fl/fl mice, showing clusters, assigned groups and CRE⁻ (Red) and CRE⁺ (Teal) overlay and expression of indicated genes. See also Figure S6.

Figure 7

Loss of ZEB2 Results in Loss of Mac Tissue-Specific Identity across Tissues (A–C) Heatmap showing expression of top core SM (A), microglia (B), or Cd74^hi CM (C) genes across indicated groups from SC-RNA-Seq analysis. Genes in red are significantly differentially expressed. (D) t-SNE showing all macs sequenced by SC-RNA-seq from the indicated five tissues. Zeb2^−/− macs are shown in bold color, Zeb2^+/+ or Zeb2^+/− macs are shown in faded color. Open circles represent cells arising from CRE⁻ mice while filled circles are those isolated from CRE⁺ mice (Fcgr1-cre, Itgax-cre, or Clec4f-cre). (E) Venn Diagram detailing overlap of DE genes between all five tissue mac populations in the absence of Zeb2. (F) Heatmap showing 32 DE genes conserved across all five tissue mac populations. See also Figure S7.