Donor-Host Lymphatic Anastomosis after Murine Lung Transplantation

Abstract

Background.

Establishing lung lymphatic drainage is thought to be important for successful lung transplantation. To date, there has been a complete absence of knowledge of how lymphatic connections are reestablished after lung transplant, despite evidence suggesting that this does indeed occur. The present study aimed to elucidate whether and how lymphatic anastomosis occurs after lung transplant.

Methods.

An orthotopic murine model of lung transplant using lymphatic reporter mice and whole mount immunohistochemistry was used to evaluate the lymphatic vasculature and donor-host connections after lung transplantation.

Results.

Immunohistochemistry of transplanted lungs demonstrated robust lymphatic vessels, and functional assays demonstrated lymphatic drainage in the transplanted lung that was comparable to that in native lungs. Lymphatic vessels in the donor lung exhibited active sprouting toward the host at the anastomosis within the first 3 days after lung transplantation, with more numerous and complex lymphatic sprouting developing thereafter. Donor lymphatic vessels were numerous at the site of anastomosis by day 14 after lung transplantation and formed physical connections with host lymphatic vessels, demonstrating a mechanism by which lymphatic drainage is reestablished in the transplanted lung.

Conclusions.

Lymphatic drainage after lung transplantation is established by active sprouting of donor lymphatic vessels towards the host and the formation of donor-host lymphatic connections at the level of the transplant anastomosis.

Introduction

The pulmonary lymphatics transport fluid and traffic immune cells from the lungs to the draining lymph nodes and are critically important for lung homeostasis. Establishing lung lymphatic drainage is thought to be important for successful lung transplantation, but precisely how the transplanted lung accomplishes this is not understood. Elegant work in canine and mouse models has shown that lymphatic function is restored 2-4 weeks after lung transplantation, and that stimulation of lymphangiogenesis has beneficial effects for graft survival in the setting of rejection.^1,2 Given the known role of lymphatics for fluid transport and immune surveillance, interruption of lymphatic vessels in the transplanted lung has also been hypothesized as a possible contributor to primary graft dysfunction in humans, though evidence for this is scant.^3,4 Although one study found an association between increased lymphatic density and acute rejection after human lung transplant,⁵ others have shown either no correlation between lymphatic density and rejection,^1,6 that decreased lymphatic function is associated with rejection,² or a positive effect of stimulation of lymphangiogenesis on graft function.¹ Data in humans and animal models of kidney and heart transplantation suggest that increased lymphatic density is associated with better outcomes and decreased susceptibility to rejection in these organs.^7–9 Thus, accumulating evidence suggests that lymphatic vessels may be beneficial for graft survival, though this remains a controversial topic, and one with limited data.

Lymphatic vessels can be visualized throughout the transplanted lung,³ but direct imaging demonstrating interactions of donor and recipient lymphatics has not been reported. Thus, the contribution of donor and host lymphatic vessels to the transplanted lung and how lymphatic drainage from the transplanted lung is established remains unknown. To address this question, we examined the behavior of donor and host lymphatic vessels at the site of anastomosis after lung transplantation in an orthotopic murine model. Using lymphatic reporters that fluorescently label the lymphatic endothelium and immunostaining, we were able to distinguish donor versus host lymphatic vessels and analyze their relative contribution at the transplant anastomosis.

Materials and Methods

Mice

Prox1-EGFP BAC transgenic mice, previously described by our lab¹⁰, and Prox1-tdtTomato mice were obtained from The Jackson Laboratory. All mice were strictly maintained on a backcrossed C57BL/6 background.

Lung transplantation

Prox1-EGFP mice were used as donors to either wild-type C57Bl/6 or Prox1-tdtTomato recipient mice. Details of the transplantation procedure were previously described.¹⁰ Briefly, the heart-lung block was retrieved and flushed with heparin and saline, and the left lung isolated. The recipient mouse was anesthetized with isoflurane, intubated, and connected to a ventilator. The mouse was then placed in the supine position and an incision made at the left chest between the 3rd and 4th ribs to expose the left pulmonary artery and vein. After a micro-serrefine clamp (FST 18055) was placed at the left hilum, transverse incision was made on the left bronchus. A running stitch of 11-0 suture (Ethilon 2881G) was used for end-to-end anastomosis of donor to recipient bronchus. The donor pulmonary artery and vein were connected to recipient pulmonary artery and vein respectively end-to-side using 11-0 sutures. The native left lung was removed after implantation of the donor lung.

Immunohistochemistry

Mice were sacrificed and tissue was perfused with PBS prior to lung retrieval and fixation with 4% paraformaldehyde overnight. Slides from paraffin-embedded sections were immunostained with antibodies for LYVE1 (R&D Systems, AF2125), VEGFR3 (R&D Systems, AF743), and PROX1 (Abcam, ab76696) overnight. Slides where then washed and stained with Alexa Fluor conjugated secondary antibodies (ThermoFisher).

Dextran Assay

50ul of 5 mg/ml dextran-568 (10 000 kD MW, ThermoFisher) was administered to anesthetized, intubated mice via endotracheal catheter. Fifty minutes after administration, the mice were sacrificed for procurement of mediastinal lymph nodes. Lymph nodes were imaged using an Olympus SZX16 dissecting microscope. Quantification of fluorescence intensity was performed using ImageJ.

Whole mount staining

Lung tissue was fixed overnight in 4% PFA at 4°C. For lungs, thick coronal sections were made using a scalpel. Tissue was permeabilized in 0.1% BSA + 0.3% Triton-X in PBS. Incubations with anti-LYVE1 primary antibody (R&D AF2125) and AlexaFluor 594-conjugated secondary antibody (ThermoFisher) were performed in 0.3% Triton-X in PBS. The tissue was mounted with Vectashield (Vector Labs) and imaged using a Leica TCS SP8 confocal microscope.

Statistics

Images shown are representative of at least 3 independent experiments. Quantitative data are expressed as mean ± SEM, and number of samples per condition are indicated in figure legends. Statistical significance was determined by unpaired two-tailed Student’s t test using Prism statistical software. P values less than 0.05 were considered statistically significant.

All studies were performed with approval of the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia.

Results

Immunohistochemistry of transplanted lungs in mice demonstrated robust lymphatic vessels, as seen by staining for the lymphatic markers VEGFR3 (Figure 1A, B) and PROX1/LYVE1 (Figure 1C, D). We did not observe any difference in the appearance of vessels between the transplanted and native lungs. Functionally, there was no significant difference between native and transplanted lungs in lymphatic drainage of dextran from the lung parenchyma to the mediastinal lymph nodes by day 25 posttransplant (Figure 1E, F). These data are similar to previous reports^1,2 and confirm restoration of efficient lymphatic drainage in the transplanted lung.

Figure 1: Restoration of lymphatic drainage after lung transplant is associated with sprouting of donor lymphatic vessels.

A-D: Immunohistochemistry for lymphatic vessels using the markers VEGFR3 (A, B) and PROX1/LYVE1 (C, D) in control lung tissue and lung transplants at Day 25 posttransplant. E: Drainage of dextran-568 (red) to mediastinal lymph nodes (mLN, E) in native (top) and transplanted lungs (bottom) 50 minutes after endotracheal administration. F: Quantification of dextran drainage from native (n = 4) and transplanted (n = 4) lungs to mediastinal lymph nodes, as quantified by mean fluorescence. G-I: Whole mount microscopy of the area of anastomosis after lung transplant from donor mice expressing the lymphatic reporter Prox1-EGFP (green). Dotted lines indicate areas of sutures between donor and host tissue. J: Whole mount microscopy of donor lung from Prox1-EGFP mouse at day 14 posttransplant. K, L: Higher magnification images of the donor lymphatic sprouting shown in J. Scale bars = 25 μm. n.s. = nonsignificant. Immunohistochemistry data shown is representative of at least 5 independent transplant experiments. Whole mount staining is representative of 3 independent mice that received lung transplants.

Since major collecting lymphatic vessels are severed and not surgically reconnected during lung transplantation procedures, the anatomical basis for restoration of lymphatic function after lung transplantation is unclear. We hypothesized that lymphatic drainage is reestablished by sprouting of donor and/or host lung lymphatics and the creation of new donor-host lymphatic anastomoses around the site of where the donor lung is sutured to the host. To enable visualization of the lymphatics, we performed lung transplantation using lymphatic reporter mice (Prox1-EGFP), in which lymphatic endothelial cells from the donor lung are marked by GFP expression, as donors to wild type recipients. Whole mount imaging of the area of anastomosis showed sprouting of donor lung lymphatics towards the host at days 3-14 posttransplant, which became more numerous and complex over time (Figure 1G–I). While sprouting of donor lung lymphatics was generally towards the host, we also found evidence of donor lymphatics sprouting towards other donor lymphatic vessels (Figure 1J–L).

The finding of donor lung lymphatic sprouting toward the host after lung transplant suggested development of anastomoses of these lymphatics with host lymphatic vessels. To address this possibility and visualize both donor and host lymphatics, we first performed whole mount immunohistochemistry using staining for the lymphatic endothelial marker LYVE1 in lung transplants from Prox1-EGFP donor mice into wild type recipients. We found that donor lymphatics physically connected with host LYVE1⁺ vessels at the transplant anastomosis by day 14 posttransplant (Figure 2A–C).

Figure 2: Direct connection of donor and host lymphatic vessels after lung transplantation.

A-C: Whole mount immunostaining of lymphatics in lung transplants from Prox1-EGFP (green) donor mice to wild type recipients using the marker LYVE1 (red). Higher magnification of boxed area in A is shown in B. An additional higher magnification image of a direct donor-host lymphatic connection is shown in C. D-F: Whole mount imaging of lymphatics in lung transplants from Prox1-EGFP (green) donor mice to Prox1-tdTomato (red) recipient mice. Higher magnification of boxed area in D is shown in E. Images in D and F represent 2 independent transplant experiments. G: Quantification of the number of lymphatic anastomoses observed per mm² at the area of connection between donor and host tissue. Scale bars = 25 μm. Data shown is representative of 3 independent mice that received lung transplants.

To more definitely determine whether donor and host lymphatic vessels anastomose after lung transplant, we used Prox1-EGFP mice as donors for lung transplant to Prox1-tdTomato recipients, such that both donor (GFP⁺) and recipient (tdTomato⁺) lymphatic endothelial cells are labeled by reporter expression. Whole mount imaging at day 14 posttransplant revealed an abundance of both donor and host-derived lymphatic vessels at the transplant anastomosis (Figure 2D–F). We saw clear connections between donor and host lymphatic vessels at the area of transplant anastomosis at this time point (Figure 2E,G). The appearance of these donor-host lymphatic anastomoses by day 14 correlated with our functional data for reestablishment of lymphatic drainage in the mouse (Figure 1E, F), and with reported canine models,² and for the first time provides a mechanism for the reestablishment of lymphatic drainage in the lung after transplant.

Discussion

Reestablishment of lymphatic function is proposed to be a critical event after lung transplant that enables normal immune cell surveillance and drainage of lung tissue protein and interstitial fluid.^1,3 Here, we show in an orthotopic mouse model that the mechanism for reestablishment of lymphatic drainage in the transplanted lung is sprouting of donor lymphatics towards the host and direct connections of donor and host lung lymphatics at the site of transplant anastomosis. This is the first evidence of donor-host lymphatic vessel anastomosis after solid organ transplant, and answers the long sought-after question of how lymphatic drainage is achieved despite the lack of surgical reconnection of these vessels.

Though sprouting lymphangiogenesis can be induced in the lung by VEGFC in multiple models,^1,10–12 the factors that contribute to proper anastomosis of donor and host lymphatic vessels after lung transplant are unknown. The data shown here clearly demonstrate directional sprouting of lymphatic vessels at the transplant anastomosis, suggesting the presence of guidance cues for the lymphatic endothelial cells. It is feasible that multiple factors including anatomic issues of the donor or host, surgical technique, or presence of rejection or inflammation may affect these cues and therefore compromise the formation of donor-host lymphatic connections. Furthermore, it is unknown whether donor-host lymphatic connections occur in the setting of other solid organ transplants in the same manner as in the lung.

The importance of lymphatic function in the lung has been described in a variety of settings.¹³ We have recently showed that mice with impaired pulmonary lymphatic flow form tertiary lymphoid organs (TLOs) in the lung parenchyma and develop alveolar damage that resembles emphysema.¹⁰ Interestingly, we also found that lymphatic deletion in lung transplants leads to the formation of TLOs within the graft.¹⁰ The connection between lymphatic dysfunction and TLO formation is notable in the setting of lung transplantation, given that TLOs may play an important role in local immunity in the graft.^14–16 Proper establishment of lymphatic connection after lung transplant surgery may therefore be a critical early event in the survival of the graft, perhaps in part due to the role of lymphatic dysfunction towards the development of TLOs.

In this study, we have shown that donor-host lymphatics form anastomoses to reestablish lymphatic drainage after lung transplant in mice. These data add another layer of complexity for understanding graft survival, and warrant further investigations of how to optimize or accelerate lymphatic anastomoses.

Abbreviations

PROX1

Prospero homeobox 1

LYVE1

Lymphatic vessel endothelial hyaluronan receptor 1

VEGF3

Vascular endothelial growth factor receptor 3

GFP

Green fluorescent protein

VEGFC

Vascular endothelial growth factor C

TLO

Tertiary lymphoid organ