Single-cell transcriptomics of popliteal lymphatic vessels and peripheral veins reveals altered lymphatic muscle and immune cell populations in the TNF-Tg arthritis model

Abstract

Background Lymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls. Methods Single-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations. Results Fluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrb expression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod. Conclusions Single-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy

“…In this work, we found that direct LMC damage may not be responsible for the reduced lymphatic function and onset of severe inflammatory-erosive arthritis in TNF-Tg mice. Alternative mechanisms, such as chemical inhibition of lymphatic contractility through the accumulation of peri-lymphatic inflammatory immune cells 22 or interactions with adjacent LECs 11 , ought to be investigated in future studies. Of particular relevance, NOS inhibition is effective in recovering lymphatic contractility in some studies 8 , 11 , 33 ,while recent investigation of global genetic iNOS ablation demonstrated limited effects on lymphatic function and arthritis progression in TNF-Tg mice 29 .…”

“…myosin heavy chain 11 (Myh11)) 36 or transcriptional changes (i.e. Zbtb16 ) 22 may have recovered during the treatment period associated with amelioration of arthritis. In addition, based on our unsuccessful prospero homeobox 1 (Prox1) staining (as previously performed 26 ) and lack of bona fide valve associated markers (i.e., integrin-α9 37 ), we were limited in our capacity to analyze specific PLV regions (i.e., valve vs non-valve).…”

“…After the 6-weeks of treatment at 9.5-months-old, the mice were euthanized for PLV harvest and immunostaining, as previously described 22 , 26 . Briefly, mice were administered a lethal dose of ketamine/xylazine cocktail (intraperitoneal), and Evan’s Blue dye (Millipore Sigma Cat# E2129) was injected intradermal into both hindpaws to visualize the PLVs.…”

“…Despite the well-described relationship between reduced lymphatic contractility and arthritis progression, the mechanisms associated with the lymphatic dysfunction and joint inflammation remain largely unknown. Several reports indicate that inflammatory factors (i.e., inducible nitric oxide synthase (iNOS)) produced by accumulated peri-lymphatic immune cells may underlie the reduced lymphatic contractility 8 , 11 , 22 , while others posit that direct LMC tissue damage is a pivotal event 9 , 23 . For instance, ultrastructural imaging revealed a notable loss of LMC structure and integrity associated with reduced PLV contraction frequency and lymphatic clearance in vivo by near-infrared imaging of indocyanine green (NIR-ICG) 9 .…”

Multi-omics Analysis Identifies IgG2b Class-Switching with ALCAM-CD6 Co-Stimulation in Lymph Nodes During Advanced Inflammatory-Erosive Arthritis

“…However, with the onset of Advanced arthritis and lymphatic dysfunction, these activated macrophages stagnate within the popliteal lymphatic vessels and PLN, and mediate tissue destruction and immune cell activation (Li et al, 2013). This lymphatic stagnation is associated with LEC (Bouta et al, 2017) and lymphatic muscle cell (LMC) (Jiao et al, 2022; Kenney et al, 2020; Kenney et al, 2022c; Liang et al, 2021) apoptosis and efferocytosis by joint-draining macrophages leading to intracellular iron accumulation (Fig. 6D).…”