Spatially resolved heterogeneity of lymphatic vasculature in the adult lung

Abstract

Background.

Lung lymphatic drainage of interstitial fluid is essential for preventing pulmonary edema, a condition characterized by the accumulation of extravascular fluid that impedes respiratory gas exchange and can be fatal. Beyond fluid regulation, lung lymphatics also serve as conduits for immune cell trafficking, supporting both surveillance and mounting of host immune responses during lung infection. Despite their essential roles, the cellular and functional heterogeneity of lung lymphatic endothelial cells (LECs) remains poorly defined.

Methods.

Leveraging single nucleus sequencing technology, we investigated LEC heterogeneity in the homeostatic adult mouse lung.

Results.

We identified Stabilin-2 (Stab2), a Class H scavenger receptor, as a unique marker of a distinct subset of LECs in lymphatic vessels adjacent to pulmonary veins and in alveolar regions. A separate distinct subset of LECs is defined by expression of Polycystic Kidney and Hepatic Disease 1-Like-1 (Pkhd1l1), a component of stereocilia, and these lymphatic vessels are localized to the bronchovascular bundle.

Conclusions.

Our findings suggest that lung LECs are not a uniform population but instead compromise molecularly distinct and spatially organized lymphatic vessels, possibly with specialized functions. These results lay the groundwork for further studies geared towards understanding the roles of cellular origin in lung LEC heterogeneity, whether lymphatics in other organs have analogous spatial organization, and whether these disparate cells and vessels have functional differences in fluid homeostasis and immune regulation during pulmonary disease pathogenesis.

Graphical Abstract

To the editor:

Lung lymphatics consist of an interconnected network of vessels which play crucial roles in removing excess interstitial fluid and in facilitating immune cell trafficking to lymph nodes, essential for maintaining lung homeostasis. Lymphatic vessels are lined by specialized lymphatic endothelial cells (LECs), but lung LECs remain understudied compared to LECs of other organs and non-lymphatic endothelial cells of the lung. Recent work revealed significant heterogeneity within endothelial cells of the pulmonary capillaries, identifying at least two transcriptionally and functionally distinct cell populations with unique proliferative properties and microanatomic location.¹ The discovery of pulmonary capillary endothelial cell heterogeneity has substantially reshaped our understanding of alveolar biology.² In contrast, lung LEC heterogeneity has not yet been demonstrated.

We leveraged single nucleus RNA sequencing (snRNAseq) to investigate LEC heterogeneity in the healthy adult mouse lung. To perform RNA sequencing of lung LEC nuclei, we coupled a nucleus lineage labeling strategy with fluorescence-activated nuclei sorting (FANS).^3,4 For this, Prox1-CreER^T2/CAG-Sun1/sfGFP mice were used for GFP tagging of nuclear membranes in Prox1-expressing cells. Prox1 is expressed in only two cell types in the lung, LECs and pulmonary neuroendocrine cells (PNECs). GFP⁽⁺⁾ nuclei were isolated by FANS after detergent-dependent tissue lysis followed by cDNA preparation, library construction and RNA sequencing. After clustering of single nuclei data with Celda^5–7, the LEC nuclei were readily distinguished from all other clusters by co-expression of Prox1 and GFP transcripts and the absence of Epcam expression (present in PNECs⁸). When LECs were re-clustered in isolation from all other nuclei, two distinct subpopulations of similar size were identified, computationally delineated as Cluster 1 and 2 (Fig 1A). Differential expression analysis revealed 29 genes that were upregulated in Cluster 1 LECs and 22 genes that were upregulated in Cluster 2 LECs (FDR<0.05, Fig 1B). Among the top DEGs that define Cluster 1 LECs (Fig 1C), stabilin 2 (Stab2) is a Class H scavenger receptor with roles in clearance of cellular debris and immune cell movement across endothelial surfaces. In contrast, polycystic kidney and hepatic disease 1-like-1 (Pkhd1l1), a component of stereocilia, was expressed more highly in Cluster 2 LECs (Fig 1C).

Figure 1. Nuclear transcriptional profiling of murine lung LECs reveals two distinct gene signatures, and top DEGs from each cluster are also expressed in human lung and human gut LEC subsets.

snRNAseq of healthy adult Prox1CreER^T2/CAG-Sun1-sfGFP mouse lungs revealed two clusters of LECs with significant transcriptional differences (A). The volcano plot shows DEGs up- and downregulated in Cluster 1 as compared to Cluster 2 (B). Stab2 and Pkhd1l1 are among the top three DEGs that define either Cluster 1 or 2 LECs (C). In one human single-nuclei data set in LungMAP (D), the expression pattern of each of the top three DEGs in Clusters 1 and 2 are shown with the lymphatic cluster circled in yellow, emphasizing the comparatively more restricted expression pattern of Stab2 and Pkhd1l1 in human lung (E-F). Using the gene coexpression tool in ShinyCell to assess Stab2 and Pkhd1l1 expression patterns in human lung LECs specifically, separate subpopulations were identified that expressed only Pkhd1l1, only Stab2, both genes and neither gene (G). Gene coexpression data in each of these subpopulations is shown as number of LECs in the lymphatic cluster as well as percent of all LECs. The lymphatic cluster within a human gut single-cell atlas was also explored for Stab2 and Pkhd1l1 expression patterns, showing non-overlapping expression of these two genes in LECs in healthy human intestinal tissue (H).

To further explore the significance of our findings in human lung LECs, we utilized a LungMAP human single-nuclei data set to investigate the expression patterns for the top DEGs defining our murine LEC clusters (Fig 1D).^9,10 Of the top 3 DEGs defining Cluster 1 and the top 3 DEGs defining Cluster 2 (Fig 1E–F), only Stab2 and Pkhd1l1 had a comparatively restricted cellular expression pattern in human lung, whereas genes such as Nav3 were also expressed in many other cell types such as lung fibroblasts, smooth muscle, blood endothelium, and alveolar type 1 cells. Furthermore, using the gene coexpression tool in ShinyCell to assess Stab2 and Pkhd1l1 expression patterns in human lung LECs specifically, separate subpopulations were identified that expressed only Pkhd1l1 (56.9% of healthy human lung LECs), only Stab2 (4.2%), both genes (30.1%) or neither gene (8.8%) (Fig 1G). In addition to human lung, we investigated available single-cell data sets for other human organs including an openly available gut cell atlas, with intestinal cells collected from a wide age range of healthy patients.¹¹ Consistent with mouse and human lung LEC data, the gut LEC cluster had non-overlapping expression of Stab2 and Pkhd1l1 in two similar-sized subclusters of LECs (Fig 1H). For subsequent validation of our snRNAseq data set, we therefore focused on Stab2 and Pkhd1l1 as markers for unique subtypes of LECs.

Using in situ hybridization in healthy adult C57BL/6 mouse lung tissue, we localized and quantified expression of Stab2 and Pkhd1l1 in lung LECs. Immunofluorescent costaining of vascular endothelial growth factor receptor-3 (VEGFR3) was employed to delineate lymphatic vessels in lung sections.¹² We evaluated distinct microanatomic regions specific to the lung including ‘pulmonary vein’, ‘alveolar space’ and ‘bronchovascular bundle’, the latter of which is a conserved compartment of structures in the mammalian lung including airways (bronchi or bronchioles), pulmonary arteries, and their interstitium in close proximity. Stab2^(High) LECs localized primarily to pre-collector lymphatic vessels adjacent to pulmonary veins and in smaller initial lymphatic vessels in alveolar spaces (Fig 2A). A distinct and non-overlapping subset of LECs was identified by relatively high expression of Pkhd1l1, and these lymphatic vessels were localized primarily in the bronchovascular bundle (Fig 2B). These data reveal that at least two types of lung LECs with distinct transcriptomes segregate to spatially distinct lymphatic vessels in the mouse lung. LEC subtypes populated lymphatic vessels of similar size next to either bronchovascular bundles with airways and arteries (Pkdh1l1^(High)) or pulmonary veins (Stab2^(High)), as well as smaller initial lymphatics in the distal lung (Stab2^(High)). We also investigated lymphatics in human lung tissue, and consistent with the higher prevalence of Pkhd1l1-expressing LECs in human single cell and single nuclei data sets (Fig 1G), we identified Pkhd1l1^(High)/Stab2⁽⁻⁾ LECs adjacent to airways in the bronchovascular bundle region (Fig 2C). This underscores the translational relevance of our findings of distinct lung LEC subsets defined by molecular and spatial characteristics, possibly with specialized functions.

Figure 2. Lung LEC subtypes are patterned in at least two anatomically distinct gene signatures.

In situ hybridization for mRNA targets was performed in healthy adult C57BL/6J mouse lung tissue, costaining for VEGFR3 by immunofluorescence (A-B). Stab2 mRNA was found predominantly in perivenous and alveolar space lymphatic vessels (A), while Pkhd1l1 mRNA was found predominantly in lymphatic vessels adjacent to bronchovascular bundles (B). Briefly, three regions of interest were randomly assigned per left lung section per mouse (including 3 female and 3 male C57BL/6 mice), a sample size chosen a priori based on historical variability for this endpoint in our lab and consistent with prior quantitative histological studies.¹² Every lymphatic vessel therein was assigned an anatomic location (bronchovascular, pulmonary vein, alveolar space). The number of mRNA puncta were counted and the lymphatic perimeter measured using the polygon annotation tool in QuPath. The number of Stab2 or Pkhd1l1 mRNA puncta per μm of lymphatic vessel was averaged for each anatomic location. Each dot represents one mouse, and 3 equal-sized regions of interest were analyzed per mouse. In (A) and (B), data are shown as mean and standard deviation. For normally distributed data, one-way repeated-measures ANOVA was used with anatomic location as a within-subjects factor, followed by Tukey’s multiple comparisons test performed to determine RNA expression significance between anatomic regions, and adjusted p-values are shown. All significance levels (α) were set to 0.05. AF, autofluorescence. For Stab2 in (A): bronchovascular vs. pulmonary vein, mean difference −0.036, CI −0.060 to −0.012; bronchovascular vs. alveolar space, mean difference −0.056, CI −0.080 to −0.031; pulmonary vein vs. alveolar space, mean difference −0.019, CI −0.044 to 0.005. For Pkhd1l1 in (B): bronchovascular vs. pulmonary vein, mean difference 0.013, CI 0.009 to 0.017; bronchovascular vs. alveolar space, mean difference 0.017, CI 0.013 to 0.021; pulmonary vein vs. alveolar space, mean difference 0.004, CI 0.0004 to 0.008. We next investigated lymphatics in a human lung tissue sample, and identified multiple Pkhd1l1^(High) LECs adjacent to airways in the bronchovascular bundle region shown, with white arrow heads denoting Prox1⁽⁺⁾/Pkhd1l1⁽⁺⁾ lymphatic vessels (C). These lymphatics were Stab2⁽⁻⁾ (not shown). All scale bars represent 50 um in (C).

Although it is established that lymphatic vessels exhibit a hierarchical organization of specialized subtypes, including collecting and pre-collecting vessel LECs, valvular LECs and initial LECs,^13–15 our current findings reveal a novel form of LEC heterogeneity in the lung that extends beyond this known classification. Lymphatics are found throughout the lung including in each of the defined microanatomic regions we studied, with larger pre-collector lymphatic vessels located adjacent to pulmonary veins and bronchovascular bundles, and smaller and more rare initial lymphatics located in the distal lung.^16,17 One interesting aspect of murine lung collector and pre-collector lymphatic vessels that set them apart from similarly sized lymphatic vessels of other organs is that they lack smooth muscle coverage, and lymph flow is instead thought to be primarily facilitated by constant, cyclic respiratory motion.^18,19 This makes staining with smooth muscle markers less useful in delineating the hierarchical division among lung lymphatic vessels by histology. The population of LECs we identified in distal airspaces is most consistent with initial LECs based on small vessel size, however the larger Stab2^(High) lymphatic vessels observed near pulmonary veins and the Pkhd1l1^(High) lymphatic vessels near the bronchovascular bundle were observed to be of similar size range. This suggests that these two LEC subtypes likely reside in distinct pre-collector lymphatic vessel subtypes found in different anatomic regions of the lung.

This discovery raises unanticipated new questions such as whether lungs have separate lymphatic vessels derived from different cellular origins, whether distinct lymphatic subtypes coalesce proximally into a common collecting system, whether lymphatics in other organs such as the gut have analogous spatial organization (perhaps with regard to adjacent structures such as arteries or veins), and whether these unique cells and vessels have functional differences. In the lung, there are important differences between disease processes that are localized primarily to bronchovascular bundle regions, pulmonary vein-adjacent regions, and alveolar spaces. For example, pulmonary edema associated with acute heart failure is primarily localized to perivenous regions and distal alveolar spaces and may suggest a role for subspecialized lymphatics at these sites. Airway-centric diseases such as asthma, COPD and bronchiectasis may also suggest a role for specialized lymphatics during pathogenesis or to facilitate resilience in these anatomic locations. Another interesting question raised here is whether LEC and/or lymphatic vessel subsets may have unique immune functions in the lung. Stabilin receptors are scavenger receptors with wide-ranging roles in hyaluronic acid homeostasis, tumor metastasis, and atherosclerosis, but also LPS, clearance, immune cell adhesion and transcytosis, any or all of which may have significance in lung homeostasis and disease pathogenesis.^20–23 On the other hand, Pkhd1l1 has a known role as a critical stereocilia component in hair cells of the inner ear²⁴, and its presence in specific lung LEC subtypes supports emerging evidence for the role of cilia in lymphatic biology.^25,26

In sum, we report the existence of transcriptionally distinct and spatially organized lung LEC subtypes. These findings have implications for future studies investigating lung fluid homeostasis, regional immune cell regulation, and pulmonary disease pathogenesis, and there is also potential for Stab2 and Pkhd1l1 to play novel functional roles in lymphatics that have yet to be uncovered.

Supplementary Material

• Expanded Methods

• Major Resources Table

What are the Clinical Implications?

The discovery of unique lymphatic vessels within bronchovascular bundles as compared to lymphatic vessels adjacent to pulmonary veins and in distal airspaces raises new clinically relevant questions. For example, the lung may contain lymphatic vessels derived from different cellular origins during development, which may have implications for developmental lung disease. It is also possible that unique lung lymphatic vessels have functional differences that contribute to resilience or disease pathogenesis localized within separate anatomic compartments of the lung. For example, pulmonary edema associated with acute heart failure is primarily localized to perivenous regions and distal alveolar spaces and may suggest a role for subspecialized lymphatics at these sites. Airway-centric diseases such as asthma, COPD and bronchiectasis may also suggest a role for specialized lymphatics during pathogenesis or to facilitate resilience in these anatomic locations. Another interesting question raised here is whether LEC and/or lymphatic vessel subsets may have unique immune functions in the lung, which would have clinical implications for myriad inflammatory and infectious diseases of the lung.

Abbreviations and Acronyms:

LEC

Lymphatic endothelial cell

FANS

Fluorescence-activated nuclei sorting

Stab2

Stabilin-2

PNEC

Pulmonary neuroendocrine cell

Pkhd1l1

Polycystic kidney and hepatic disease 1-like-1

VEGFR3

Vascular endothelial growth factor receptor-3