PMC

HDC⁺ myeloid cells promote intestinal adenomatous polyposis in APC^min/+ mice. (A) Representative fluorescence images showing accumulation of HDC⁺ (GFP⁺) cells in small intestine, colon, and spleen of APC^min/+;Hdc-GFP mice (n = 3). Scale bar = 50 µm (B) Experimental protocol for induction of HDC⁺ myeloid cell depletion in Hdc-CreERT2;APC^min/+;Rosa26-LSL-DTA mice by tamoxifen diet administration. (C) Number of intestinal polyps in 16-weeks-old Hdc-CreERT2;APC^min/+;Rosa26-LSL-DTA mice treated with tamoxifen diet, comparing genotyped DTA⁻ (n = 4) and DTA⁺ (n = 5) mice. (D) Representative H&E images of small intestine and colon histological sections from mice analyzed in (C). Scale bar = 50 µm. (E) Percentages of CD11b⁺Gr1⁺ myeloid cells in CD45⁺ cells from blood, spleen, and bone marrow, respectively, from mice analyzed in (C). Showing flow plots (left) and quantification (right) (F) Percentages of myeloid cells in digested intestinal tissue of mice analyzed in (C), with representative FACS plots (left), and quantification (right). *p < 0.05; ***p < 0.001, data are mean ± SEM, representing two to three independent experiments. Data were analyzed with two-tailed Student's t-test (E and F) and Mann–Whitney test (C).

HDC⁺ myeloid cells recruit Foxp3⁺ Tregs in colitis-associated colon carcinogenesis model. (A) Comparison of circulating chemokine levels in DTA⁻ (n = 5) and DTA⁺ (n = 6)AOM/DSS tumor-bearing mice sera. (B) Expression of Cxcl13 mRNA from sorted CD45⁺ cells in colon tumor tissue, comparing DTA⁻ and DTA⁺ (n = 3 per group). (C) Representative fluorescence staining showing Foxp3⁺ cells in colon tumor tissue in DTA⁻ and DTA⁺ AOM/DSS-treated mice (n = 3 per group). Scale bar = 50 µm. (D) Percentages of Foxp3-GFP⁺ cells in colon CD4⁺ cells in AOM/DSS-treated mice with (n = 3) or without (n = 4) rCxcl13 neutralization antibody. (E) Migration of Foxp3⁺ cells in response to Cxcl13. Showing quantitation of migrated Foxp3⁺ cell numbers by flow cytometry after co-culture of 5,000 splenic Foxp3-GFP⁺ cells (from spleens of AOM/DSS-treated Foxp3-GFP mice) in transwell plates with either HDC⁺ or HDC⁻ myeloid cells. N = 3 to 5 per group. *p < 0.05; **p < 0.01; ***p < 0.001, data are mean ± SEM, representing two to three independent experiments. Data were analyzed with two-tailed Student's t-test (A, B, and D) and Mann–Whitney test (E).

HDC marks a distinct granulocytic myeloid population. (A and B) Gene microarray comparison between HDC⁺ and HDC⁻ CD11b⁺Gr1⁺ bone marrow myeloid cells (n = 3 per group), showing differential expression of chemokine receptors and Pdl-1. (C) Proportion of G0 and Non-G0 cells from HDC⁺ or HDC⁻ bone marrow, spleen, and small intestine myeloid cells (n = 5 per group) as measured by flow cytometry using Ki-67 and Hoechst 33342 staining. Showing representative flow plots (left) and quantification (right). (D and E) Lineage tracing in Hdc-CreERT2;Rosa-tdTomato mice (n = 3) under homeostatic condition. FACS at different time points for tdTomato⁺ myeloid cells from spleen (D) and intestine (E) 6 d after tamoxifen withdrawal. (F and G) Lineage tracing in Hdc-CreERT2;Rosa-tdTomato mice (n = 5) in carcinogenic conditions in AOM/DSS-treated mice. FACS at different time points for tdTomato⁺ cells from spleen (F) and intestine (G) 6 d after tamoxifen withdrawal. *p < 0.05; **p < 0.01; ***p < 0.001, data are mean ± SEM, representing three independent experiments analyzed with two-tailed Student's t-test (C) and one-way analysis of variation (ANOVA) with Dunnett's pos-hoc test (D–G).

HDC⁺ myeloid cells promote colitis-associated colorectal carcinogenesis. (A) Experimental protocol for HDC⁺ myeloid depletion and rescue in Hdc-CreERT2;Rosa26-tdTomato;Rosa26-DTA mice subject to AOM/DSS colorectal carcinogenesis. (B) Macroscopic tumor images and tumor quantification from groups of AOM/DSS-treated mice as per protocol shown in (A), including DTA genotype negative mice (DTA⁻, n = 8), DTA genotype positive mice (DTA⁺, n = 10), and DTA⁺ mice treated with HDC⁺ myeloid cell adoptive transfer (HDC⁺ spleen myeloid cells from AOM/DSS-treated mice, n = 5). (C) Representative fluorescence images showing depletion of tdTomato⁺ cells in colon tumor frozen sections from mice analyzed in (B). Scale bar = 50 µm. (D) FACS plots and bar graphs showing the percentage of CD11b⁺Gr1⁺ myeloid cells in CD45⁺ bone marrow leukocytes of DTA⁻ or DTA⁺ AOM/DSS tumor mice analyzed in (B). (E) Macroscopic images of spleens and quantitation of splenic myeloid cells in DTA⁻ or DTA⁺ AOM/DSS-treated tumor mice analyzed in (B). (F) Percentage of CD11b⁺Gr1⁺ myeloid cells in circulating leukocytes in DTA⁺ and DTA⁻ mice analyzed in (B). (G) Representative FACS plots (left) and quantitation (right) of tumor-associated CD8⁺ T cells from mice analyzed in (B). *p < 0.05; **p < 0.01; ***p < 0.001, data are mean ± SEM, representing two independent experiments. Data were analyzed with Mann–Whitney test (B), two-tailed Student's t-test (D, E, and F), and one-way analysis of variation (ANOVA) with Dunnett's post-hoc test (H).

HDC⁺ myeloid-derived Cxcl13 augment Foxp3 expression through Stat3 phosphorylation. (A) Mouse experimental protocol. (B and C) Levels of phospho-Stat3 in Foxp3-GFP⁺ cells isolated by flow sorting from colon tumors. (B) Shows representative flow histogram graphs of phospho-Stat3, while (C) shows quantitation of phospho-Stat3⁺Foxp3⁺ cells (n = 4 DTA⁻, 5 DTA⁺, 4 DTA⁺ + HDC⁺ myeloid transfer, 5 DTA⁺ + rCxcl13). (D) Foxp3⁺ cell proliferation measured by BrdU incorporation assay. Foxp3-GFP⁺ cells cultured in vitro with either HDC⁺ or HDC⁻ myeloid cells (n = 5 to 8), with a knockdown in some groups of Cxcr5 or Cxcl13 by siRNA in Foxp3-GFP⁺ cells or HDC⁺ myeloid cells, respectively. rCxcl13 was added to Foxp3-GFP⁺ cells cultured without myeloid cells. Cells were isolated from spleens of AOM/DSS- and tamoxifen- treated Foxp3-GFP;Hdc-CreERT2;Rosa26-tdTomato mice. (E and F) Immunofluorescence images (E) and quantification (F) of CD4⁺ splenic T cells co-cultured with either HDC⁺ or HDC⁻ splenic myeloid cells. CD4⁺ cells were sorted from AOM/DSS-treated Foxp3-GFP mice and myeloid cells were isolated from AOM/DSS- and tamoxifen- treated Hdc-CreERT2;Rosa26-tdTomato mice. Cxcr5 or Cxcl13 were knocked down by siRNA before co-culture with CD4⁺ cells or HDC⁺ cells, respectively. Cells were stained with PE-conjugated antibody against phospho-Stat3 and (E) show representative staining of Foxp3-GFP and Phospho-Stat3-PE. The Foxp3-GFP⁺ and Phospho-Stat3⁺ cells were counted as shown in (F). (G) BrdU proliferation assay of cultured Foxp3-YFP⁺ splenic cells from AOM/DSS-treated Foxp3-YFP-Cre-Stat3+/+ or Foxp3-YFP-Cre-Stat3−/− mice, with or without Cxcl13 for 24 h (n = 5 to 9 per group). **p < 0.01; ***p < 0.001, data are mean ± SEM, representing two to three independent experiments. Data were analyzed with one-way analysis of variation (ANOVA) with Dunnett's post-hoc test (C, D, and G) and Mann–Whitney test (F).