Dach1 transcription factor regulates the expression of peripheral node addressin and lymphocyte trafficking in lymph nodes

Abstract

Lymphocytes regulate the immune response by circulating between the vascular and lymphatic systems. High endothelial venules, HEVs, special blood vessels expressing selective adhesion molecules, such as PNAd and MAdCAM-1, mediate naïve lymphocyte migration from the vasculature into the lymph nodes and Peyer's patches. We have identified that DACH1 is abundantly expressed in developing HEV-type endothelial cells. DACH1 showed a restricted expression pattern in lymph node blood vessels during the late fetal and early neonatal periods, corresponding to HEV development. The proportion of MAdCAM-1⁺ and CD34⁺ endothelial cells is reduced in the lymph nodes of neonatal conventional and vascular-specific Dach1-deficient mice. Dach1-deficient lymph nodes in adult mice demonstrated a lower proportion of PNAd⁺ cells and lower recruitment of intravenously administered lymphocytes from GFP transgenic mice. These findings suggest that DACH1 promotes the expression of HEV-selective adhesion molecules and mediates lymphocyte trafficking across HEVs into lymph nodes.

Keywords: ECs, endothelial cells; Endothelial cell; HEV, High endothelial venules; ILNs, Inguinal lymph nodes; LNs, Lymph node; Lymph node; Lymphocyte; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MLNs, Mesenteric lymph nodes; PNAd, peripheral node addressin; PPs, Peyer's patches; Trafficking; Transcription factor; mAb, monoclonal antibody.

Graphical abstract

Fig. 1

The expression of DACH1 in the newborn mouse secondary lymphoid tissues. Mesenteric lymph nodes (MLNs), inguinal lymph nodes (ILN), Peyer's patches (PP) were harvested from newborn C57BL/6 mice. Immunohistochemistry was used to detect DACH1 (green, top left), MAdCAM-1 (red, bottom left), and CD34 (blue, top center) expression. Nuclei were stained with Hoechst33342 (bottom center). Top right: merged images of DACH1 and CD34. Bottom right: merged images of DACH1, CD34 and MAdCAM-1. Inset: merged images of DACH1, MAdCAM-1 and Hoechst33342. Data are representative of 7 experimental repeats with 7 mice in each group. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Time course of DACH1 expression pattern in fetal and postnatal MLNs. MLNs were collected from 16.5 dpc to two days after birth, and DACH1, MAdCAM-1, and CD34 expression were analyzed by immunohistochemistry. Scale bar: 100 μm. Data are representative of 3 experimental repeats with 3 mice in each group.

Fig. 3

The expression of HEV signature molecules in wild-type and the conventional Dach1-null peripheral LNs. Newborn ILNs of Dach1-null and the wild-type littermate control were serially cryosectioned and analyzed by immunohistochemistry. The images were digitally processed with a median filter (2.0 pixel radius) using ImageJ software. The numbers represent percentage of the mean fluorescent intensity (bottom) per LN area. Scale bar: 100 μm. Data are representative of 2 experimental repeats with 2 littermate mice in each group.

Fig. 4

Histological analysis of neonatal LNs in endothelial-specific Dach1-cKO mice. The percentage of MAdCAM-1⁺ and CD34⁺ in each tissue section of neonatal MLNs were quantified by ImageJ software. Data are representative of 6 tissue sections from littermate mice. Each plot in the scatter plot represents a single tissue section's value, and each line represents the median. The Mann-Whitney U test was used to analyze the data. *, P < 0.05. NS, not significant. Scale bar: 100 μm.

Fig. 5

Flow cytometric analysis of ECs in Dach1-cKO ILNs. (A) The percentage of HEV-ECs in 3-week-old ILNs were quantified by flow cytometry. CD34⁺ and PNAd⁺ cells in 7-AAD^- CD45^- stromal cell populations were analyzed. In each experiment, samples were prepared from the bilateral ILNs of three littermate mice per group and pooled. Each plot shows the value of each pooled sample in 6 experimental repeats. Mann-Whitney's U test was used as the significance test. The lines show the median value. *, P < 0.05, NS: not significant. (B) The percentage of CD31⁺podoplanin^- blood endothelial cells (BEC), CD31⁺podoplanin⁺ lymphatic endothelial cells (LEC), CD31^-podoplanin⁺ fibroblastic reticular cells (FRC) and CD31^-podoplanin^- double negative (DN) subsets in CD45^- non-myeloid cells in the bilateral ILNs of each mouse. Results are representative of two experimental repeats with three mice per group in each repeat. The Mann-Whitney U test was used to analyze the data. *, P < 0.05.

Fig. 6

Short-term lymphocyte migration in Dach1-cKO LNs. (A) The percentage of GFP⁺ lymphocyte in spleen, ILNs and MLNs of 3-week-old (control; n = 9, Dach1-cKO; n = 8) and 6–8 week-old mice (control; n = 8, Dach1-cKO; n = 7) were analyzed after injection of GFP⁺ cells. Each plot represents the ratio of LN GFP⁺ cells and splenic GFP⁺ cells of the same individual. The Mann-Whitney U test was used to analyze the data. *, P < 0.05, NS: not significant. (B) Whole-mount images of ILNs from control and Dach1-cKO mice intravenously injected with GFP⁺ lymphocytes. Representative images of 3 control and Dach1-cKO LNs are shown. Scale bar: 200 μm.