Comparative Transcriptomic Analysis Identifies a Range of Immunologically Related Functional Elaborations of Lymph Node Associated Lymphatic and Blood Endothelial Cells

Abstract

Lymphatic and blood vessels are formed by specialized lymphatic endothelial cells (LEC) and blood endothelial cells (BEC), respectively. These endothelial populations not only form peripheral tissue vessels, but also critical supporting structures in secondary lymphoid organs, particularly the lymph node (LN). Lymph node LEC (LN-LEC) also have been shown to have important immunological functions that are not observed in LEC from tissue lymphatics. LN-LEC can maintain peripheral tolerance through direct presentation of self-antigen via MHC-I, leading to CD8 T cell deletion; and through transfer of self-antigen to dendritic cells for presentation via MHC-II, resulting in CD4 T cell anergy. LN-LEC also can capture and archive foreign antigens, transferring them to dendritic cells for maintenance of memory CD8 T cells. The molecular basis for these functional elaborations in LN-LEC remain largely unexplored, and it is also unclear whether blood endothelial cells in LN (LN-BEC) might express similar enhanced immunologic functionality. Here, we used RNA-Seq to compare the transcriptomic profiles of freshly isolated murine LEC and BEC from LN with one another and with freshly isolated LEC from the periphery (diaphragm). We show that LN-LEC, LN-BEC, and diaphragm LEC (D-LEC) are transcriptionally distinct from one another, demonstrating both lineage and tissue-specific functional specializations. Surprisingly, tissue microenvironment differences in gene expression profiles were more numerous than those determined by endothelial cell lineage specification. In this regard, both LN-localized endothelial cell populations show a variety of functional elaborations that suggest how they may function as antigen presenting cells, and also point to as yet unexplored roles in both positive and negative regulation of innate and adaptive immune responses. The present work has defined in depth gene expression differences that point to functional specializations of endothelial cell populations in different anatomical locations, but especially the LN. Beyond the analyses provided here, these data are a resource for future work to uncover mechanisms of endothelial cell functionality.

Keywords: RNA-Seq; antigen presentation; chemokines; cytokines and receptors; endothelial cell; lymph node; lymphatic; scavenger receptors.

Figure 1

Differential gene analysis and hierarchical clustering of differentially expressed genes in LN-LEC, LN-BEC, and D-LEC revealed distinct and shared subsets by at least two populations. (A) Venn diagrams showing pairwise comparisons of LN-LEC, LN-BEC, and D-LEC. (B) Total number of unique and cell type specific 5X-DEG. (C) Hierarchical clustering of distinct and shared subsets of 5X-DEG. Complete lists of 5X-DEGs in each subset are listed in Table S2.

Figure 2

5X-DEG in all five subsets expressed distinct and shared members of the ECM constituents, remodeling enzymes, cell adhesion molecules. Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods).

Figure 3

5X-DEG in D-LEC only subset showed enriched representation of different cytoskeletal protein groups compared to other 5X-DEG subsets. Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods).

Figure 4

5X-DEG revealed immunomodulatory role of LEC and BEC via cytokines and innate effector molecules production. Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods).

Figure 5

5X-DEG revealed immunosensory role LEC and BEC via cytokines and pathogen sensing. Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods).

Figure 6

5X-DEG in LN-LEC+LN-LBEC shared subset revealed overrepresentation of molecules involved in antigen processing and presentation via MHC-I and MHC-II pathways. Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods).

Figure 7

5X-DEG in LN-LEC only subset revealed enriched overrepresentation of molecules involved in receptor-mediated endocytosis and scavenger receptor activity. (A) Heatmap analysis based on Z-score values of average log2 FPKM for replicates in each cell type (details in Methods). (B) Flow cytometry analyses of LN stromal cell populations for expression of Marco and Msr-1. (C) Immunofluorescent (IF) staining in adjacent tissue sections of LN for detection of Marco and Msr-1 co-expression with Lyve-1 (LEC marker).