Abstract
BackgroundMyeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M-LECP differentiation are currently unclear. We previously showed that activation of Toll-like receptor-4 (TLR4) induces myeloid-lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro-lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process.Methodology/Principal findingsHuman and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic-specific markers vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel-specific markers; (3) transient expression of VEGFR-3, but sustained increase of vascular endothelial growth factor-C (VEGF-C) and a variety of inflammatory cytokines; (4) dependency of VEGFR-3 upregulation and other LEC genes on NF-κB; and (5) novel expression of lymphatic-specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M-LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models.Conclusions/SignificanceWe established a novel TLR4-dependent protocol for in vitro production of functionally competent M-LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.
Highlights
The lymphatic system plays a key role in physiology to ensure tissue homeostasis, lipid metabolism, and immune defense [1,2]
After 4 days of differentiation, transcripts from Toll-like receptor-4 (TLR4)-activated cells were compared with those from cells treated by Colony stimulating factor 1 (CSF1) alone using RT-qPCR and an inhouse constructed array of genes commonly expressed in endothelial, stem/progenitor and activated immune cells (S3 Table; Gene Expression Omnibus (GEO) datasets GSE75518 & GSE78162)
The main findings of this study are: 1) TLR4 activation causes myeloid-lymphatic transition (MLT) of adult human and mouse myeloid cells; 2) MLT reprogramming is initiated and regulated by NF-κB pathway; 3) MLT is associated with upregulation of multiple inflammatory cytokines and receptors possibly required to promote the functions of M-lymphatic endothelial cell progenitors (LECP) in autocrine, paracrine and chemotactic manners; 4) this process occurs for human and mouse myeloid cells and is independent of T and B lymphocytes; and 5) Monocyte-derived Lymphatic Endothelial Cell Progenitors (M-LECP) produced in vitro by culturing with TLR4 ligands are capable of increasing the density and the function of new lymphatics in mouse models in vivo
Summary
The lymphatic system plays a key role in physiology to ensure tissue homeostasis, lipid metabolism, and immune defense [1,2]. Previous studies established that postnatal lymphangiogenesis is induced by chronic inflammation, tissue injury or cancer [3,5]. Whether this process requires lymphatic endothelial cell progenitors (LECP) remains a subject of debate [6,7]. Clarification of this question would advance our current understanding of lymphatic biology and promote the rational design of therapies intending to control lymphatic formation under pathological conditions. Myeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. The goals were to assess the potential of different TLR4 ligands to induce prolymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process
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