Th2 Cytokines IL-4, IL-13, and IL-10 Promote Differentiation of Pro-Lymphatic Progenitors Derived from Bone Marrow Myeloid Precursors

Abstract

Myeloid-lymphatic endothelial cell progenitors (M-LECP) are a subset of bone marrow (BM)-derived cells characterized by expression of M2-type macrophage markers. We previously showed significant contribution of M-LECP to tumor lymphatic formation and metastasis in human clinical breast tumors and corresponding mouse models. Since M2 type is induced in macrophages by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10, we hypothesized that these factors might promote pro-lymphatic specification of M-LECP during their differentiation from BM myeloid precursors. To test this hypothesis, we analyzed expression of Th2 cytokines and their receptors in mouse BM cells under conditions leading to M-LECP differentiation, namely, CSF-1 treatment followed by activation of TLR4. We found that under these conditions, all three Th2 receptors were strongly upregulated in >95% of the cells that also secrete endogenous IL-10, but not IL-4 or IL-13 ligands. However, addition of any of the Th2 factors to CSF-1 primed cells significantly increased generation of myeloid-lymphatic progenitors as indicated by co-induction of lymphatic-specific (eg, Lyve-1, integrin-a9, collectin-12, and stabilin-1) and M2-type markers (eg, CD163, CD204, CD206, and PD-L1). Antibody-mediated blockade of either IL-10 receptor (IL-10R) or IL-10 ligand significantly reduced both immunosuppressive and lymphatic phenotypes. Moreover, tumor-recruited Lyve-1⁺ lymphatic progenitors in vivo expressed all Th2 receptors as well as corresponding ligands, including IL-4 and IL-13, which were absent in BM cells. This study presents original evidence for the significant role of Th2 cytokines in co-development of immunosuppressive and lymphatic phenotypes in tumor-recruited M2-type myeloid cells. Progenitor-mediated increase in lymphatic vessels can enhance immunosuppression by physical removal of stimulatory immune cells. Thus, targeting Th2 pathways might simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that ultimately promote tumor spread.

Keywords: Th2 cytokines; lymphangiogenesis; lymphatic endothelial progenitors; myeloid cell differentiation.

FIG. 1.

Upregulation of IL-4R, IL-13R, and IL-10R during CSF-1/LPS induced differentiation of M-LECP. Mouse BM cells were differentiated with CSF-1 followed by LPS as described under Materials and Methods section. Transcripts and protein levels of IL-4R, IL-13R, and IL-10R were determined daily by qPCR (A) and flow cytometry (B–D), respectively. Quantitative PCR data in (A) are presented by fold increase of target transcripts in differentiated M-LECP compared with ex vivo cells. Values represent the mean ± SD of triplicate or duplicate for qPCR and flow cytometry analyses, respectively. (B, C) Mean percent of positive cells and MFI for each target were determined daily. Note that some SD bars are not visible due to their smaller size compared with the symbol. (D) Representative histograms of IL-4R, IL-13R, and IL-10R protein expression in ex vivo (blue lines) and differentiated (red lines) cells. The black line represents cells stained with secondary antibody alone. Percent of positive ex vivo and differentiated cells are indicated for each target. All analyses were reproduced three times. BM, bone marrow; LPS, lipopolysaccharide; M-LECP, myeloid-lymphatic endothelial cell progenitor; MFI, mean fluorescent intensity.

FIG. 2.

Expression of IL-4, IL-13, and IL-10 cytokines in BM cells differentiating into M-LECP. BM cells were differentiated with CSF-1 followed by LPS as described under Materials and Methods section. (A) Fold change in mRNA expression of the indicated cytokines was calculated based on cDNA concentration-normalized Ct values for qPCR. (B) Conditioned medium of differentiating BM cells collected on days 3 and 6 was analyzed for secreted IL-4, IL-13, and IL-10 proteins using ELISA. All values represent mean ± SD of duplicate of experiments that were reproduced three times. ELISA, enzyme-linked immunosorbent assay.

FIG. 3.

Th2 cytokines upregulate immunosuppressive M2 markers in BM myeloid precursors. CSF-1-primed BM cells were stimulated with 10 ng/mL of IL-4 (A), IL-13 (B), or IL-10 (C) instead of LPS. Expression of M2 surface markers CD163, CD204, CD206, and PD-L1 was determined by flow cytometry on day 6 of differentiation. Representative histograms of Th2 cytokine-induced M2 targets are presented. Numbers indicate percent of positive cells for each target. (D) Mean percent of positive cells ± SD and (E) mean MFI ( × 10³) ± SD for each M2 marker induced by IL-4, IL-13, or IL-10 in CSF-1-primed cells, as indicated by symbols. All analyses were reproduced three times.

FIG. 4.

Th2 cytokines promote the lymphatic identity in CSF-1-primed myeloid precursors. BM cells were differentiated with CSF-1 and either (A) IL-4 or (B) IL-10. Flow cytometry dot plots of cells stained for M2 markers PD-L1 or IL-10R and lymphatic specific proteins Lyve-1, podoplanin (Pdpn), integrin-a9 (Itga9), collectin-12 (Colec12), or stabilin-1 (Stab1). Percentage of double-positive cells is highlighted in red font. (C) The mean percent of positive cells and (D) MFI ( × 10³) ± SD for each LEC target. Statistical significance between cells differentiated with Th2 cytokines and CSF-1 alone determined by a Student's t-test with P values ≤0.05 and it is indicated by *. (E–H) Standard differentiation of BM cells by CSF-1/LPS was performed in the presence of control or blocking antibodies to IL-10R or IL-10 ligand. On day 6, cells were stained for CD11b in combination with anti-Lyve-1 (E) or anti-stabilin-1 (F) antibodies. Percent of double-positive cells for each dot plot is identified in red font. (G) The mean percent of positive cells ± SD and (H) mean MFI ( × 10³) ± SD for each LEC marker expressed in control and IL-10 pathway blocking antibodies. Statistical significance between marker expression in differentiated cells in the presence of control and blocking antibodies was determined by a Student's t-test with P values ≤0.05 and it is indicated by *. ND (not done) indicates markers that were not analyzed in some assays. Each assay was performed in duplicate and reproduced in three independent experiments. LEC, lymphatic endothelial cell.

FIG. 5.

Blockade of IL-10 inhibits immunosuppressive M2 phenotype as well as pro-vascular and lymphatic endothelial differentiation. Total mRNA was isolated from BM cells differentiated with CSF-1 and LPS in the presence of a control and IL-10R blocking antibody. Relative expression of (A) M1 markers, (B) M2 markers, (C) Th2 pathway cytokines and receptors, (D) LEC markers, (E) Vegf family members, (F) NF-κB pathway regulators, (G) selected transcription factors, and (H) selected miscellaneous markers was determined by qPCR. The mean fold increase or decrease ± SD caused by an anti-IL-10R antibody relative to control IgG were calculated based on normalized Ct values. P values determined by Student's t-test are indicated by * ≤ 0.05, ** ≤ 0.01, and *** ≤ 0.001 for differences between expression levels in the presence of anti-IL-10R and control antibodies. All assays were performed in triplicate and reproduced twice.

FIG. 6.

Tumor microenvironment contains IL-4, IL-13, and IL-10, which can activate Th2 receptor-positive myeloid-lymphatic progenitors. BALB/c and C57BL/6 mice were orthotopically implanted in the mammary fat pad with mouse breast cancer lines EMT6 and MMTV-PyMT, respectively. Tumors were harvested when the size reached 500 mm³. (A–C) Tumor lysates collected from four to five mice were used for measurement of IL-4, IL-13, and IL-10 by ELISA. The mean concentrations ± SD for each cytokine were determined from triplicate readings. (D–F) Tumors sections obtained five mice per group were co-stained for a lymphatic marker Lyve-1 and Th2 receptors (D) IL-4R, (E) IL-13R, or (F) IL-10R. Nuclei were visualized by Hoechst's stain. Scale bars are 20 μm. Representative images are shown. All images were acquired at 400 × magnification.