Perinodal Adipose Tissue Participates in Immune Protection through a Lymphatic Vessel–Independent Route

Yujia Lin, Glory Leung, Dante Louie, Ania Bogoslowski, James Ross, Paul Kubes, Pierre-Yves von der Weid and Shan Liao
J Immunol July 1, 2018, 201 (1) 296-305; DOI: https://doi.org/10.4049/jimmunol.1800151

Abstract

Lymphatic vessels remove and transport excess interstitial fluid to lymph nodes (LNs) for fluid balance and immune protection. LNs are typically surrounded by perinodal adipose tissue (PAT). However, PAT is a blood vessel–rich but lymphatic-rare tissue; therefore, how excess fluid in PAT is removed remains unclear. Using C57BL/6 mice, fluorescent dye tracing and transmission electron microscopy results suggest that fluid in PAT can travel to the LN via collagen I+ channels (PAT-LN conduits), merge into a collagen-rich space between the PAT and LN capsule (PAT-LN sinus), and may enter the LN via the LN capsule–associated conduits. This newly identified route of fluid flow allows fluid to enter the draining LN even when the afferent lymphatic vessels are blocked, indicating that fluid trafficking in PAT-LN conduits is not dependent on functional lymphatic vessels. Similar to lymphatic vessels, PAT-LN conduits can deliver Ags to the LN for immune protection. Additionally, Staphylococcus aureus from intradermal or i.v. infection may use PAT-LN conduits to infect PAT and stimulate PAT immune protection. Our studies revealed a new route of material exchange between PAT and the LN. Ag accumulation and bacterial infection in PAT demonstrate that PAT not only provides energy and regulatory factors, but can also directly participate in immune protection, indicating a new immune function of PAT for host immunity.

This article is featured in In This Issue, p.3

Introduction

Afferent lymphatic vessels transport lymph and cells from tissue to the draining lymph nodes (LNs) for immune surveillance. Inside the LN, lymph flows around the subcapsular sinus (SCS) to medullary sinus (MS) or through the LN conduits (1, 2). LN conduits support fast delivery of fluid and small Ags into the T cell and B cell area to initiate immune responses (3, 4), as well as regulatory factors to regulate LN cell homeostasis (3, 5–7). Besides maintaining fluid homeostasis and immune surveillance, lymphatic vessels are known to be responsible for the uptake and transport of lipids (8–10). Anatomically, LNs are surrounded by the perinodal adipose tissue (PAT). A capsule composed of collagens surrounds the LN, which is generally considered as the barrier separating the LN from the surrounding PAT. Conventionally, the afferent lymphatic vessels are thought to be the only route that transports fluid or cells to the LNs. Although there may be more than one afferent lymphatic vessel to transport lymph to the LN, there is only one efferent lymphatic vessel to transport the filtered lymph out from the LN back into circulation (11–13). Because adipose tissue usually develops at the location where lymphatic vessels are rare (8), a long-term puzzle concerns how fluid and materials in PAT communicate with the LN.

The importance of the intimate communication between PAT and the LN is seen during LN development and immune responses (10, 14). LN development begins with the interaction between the lymphoid tissue organizer (LTo) cells and the lymphoid tissue inducer cells (15, 16). Adipocyte precursors in the developing PAT can differentiate into the LTo cells (17). Fat-soluble factors, such as vitamin A and its metabolite, retinoic acid, are known to regulate lymphoid organ development by inducing LTo cell differentiation and controlling the lymphoid tissue inducer cell maturation (18, 19). Vitamin A from diet is converted to functionally active retinoic acid in the gut by retinal dehydrogenase that is expressed by the intestinal epithelial cells and gut-associated dendritic cells (20–23). Recently, it has been demonstrated that transplantation of wild-type adipose tissue to the PAT of a vitamin A–deficient mouse restores peripheral LN cell homeostasis, suggesting that PAT directly participates in regulating LN development (24). Upon immune stimulation, PAT can provide energy, hormones, and cytokines to regulate immune responses in the LNs (25–29). Metabolic studies show that local immune stimulation increases PAT lipolysis and provides the energy for immune cell activation and proliferation in the LN (28, 30). However, what types of signals or materials from the circulation or local stimulation can access PAT, whether the activation of PAT is a secondary effect after LN activation, or whether PAT actively participates in immune protection remains poorly understood.

In this study, we found that fluid in PAT can enter the LN via collagen I+ PAT-LN conduits and PAT-LN sinus, from which fluid may enter the LN capsule–associated (LNC) conduits. PAT-LN conduits facilitate Ag delivery from the periphery to the LN even when afferent lymphatic vessels are blocked. Additionally, Staphylococcus aureus from intradermal or i.v. infection may use PAT-LN conduits to infect PAT. Our studies reveal a new mechanism of material exchange between PAT and the LN and a new function of PAT for immune protection.

Materials and Methods

Animals

C57BL/6 mice (purchased from The Jackson Laboratory) and Prox-mOrange mice (a gift from Dr. F. Kiefer) (31) were bred at the Health Sciences Animal Resource Center at the University of Calgary. All experiments were performed using 6- to 8-wk-old female mice. All animal protocols were reviewed and approved by the University of Calgary Animal Care and Ethics Committee and conformed to the guidelines established by the Canadian Council on Animal Care.

FITC skin sensitization

For inguinal LN (iLN) and the surrounding PAT, 200 μl of 2% FITC (Sigma-Aldrich) in 1:1 (v/v) acetone/dibutyl phthalate mixture was applied on shaved skin on flanks. For popliteal LN (pLN), 10 μl of 2% FITC was injected in the footpad or 50 μl of 2% FITC was applied on shaved skin of the leg.

Fluorescent-labeled OVA injection

Alexa Fluor 555 (Alexa555)–labeled OVA was diluted in saline and injected intradermally in the flanks at 20 μg in 50 μl of saline per site or 20 μg in 10 μl of saline at the footpad. To test Ag capture by APCs, Alexa555-OVA was injected with 20 ng of LPS. For tail vein injection, 20 μg was diluted in 200 μl of saline for injection.

pLN afferent lymphatic vessel blockade

Mice were anesthetized with ketamine/xylazine. Footpad was then injected with 5 μl of Evans blue to identify the pLN afferent lymphatic vessels. A small cut was made at the skin to expose the afferent lymphatic vessel to the pLN. Afferent lymphatic vessels were sutured at the site proximal to the PAT. The skin cut was then closed with suture. The sham leg received the same Evans blue injection, skin cut, and closure without suture to lymphatic vessels.

Time-lapse imaging

Mice were anesthetized with ketamine/xylazine. Skin and the adipose tissue around the pLN were carefully removed to expose pLN. Mice were placed on the heating pad and fixed on stage for time-lapse imaging under the multiphoton (MP) microscope. Images were taken as soon as 10 μl of FITC was injected in the footpad and continued for 20–30 min.

S. aureus infection

S. aureus (2.5 × 107) was suspended in 50 μl of saline and intradermally injected at the right side of flank. The draining PAT, draining iLN, and liver were collected to examine the S. aureus distribution 4 h after injection. For circulation infection, 5 × 107 S. aureus suspended in 200 μl of saline was injected via tail vein. iLN, inguinal PAT, liver, and gonadal adipose tissues were collected to examine S. aureus infection 4 h after the injection. Tissues were homogenized and cultured overnight to count the CFU.

Collection of tissues for imaging

Tissues of interest were collected from euthanized mice. Tissues were incubated in 4% formaldehyde for whole-mount staining or paraffin sections. For frozen sections, samples were mounted directly in OCT on dry ice.

Immunofluorescence and H&E staining

Standard protocols were used for immunofluorescent staining. Briefly, 10- to 20-μm frozen sections were blocked with 5% mouse serum for 1 h. Samples were incubated overnight with the primary Abs. After two to three washings in PBS, samples were incubated with secondary Abs for 60–90 min. The primary Abs were: rabbit anti-Lyve1 and rabbit anti–collagen I (Abcam) and rat anti-CD31 (BD Biosciences). The fluorescent-conjugated secondary Abs were purchased from Jackson ImmunoResearch Laboratories. H&E staining followed the manufacturer’s protocol (VWR) using 5-μm paraffin sections.

Second harmonic generation

We used a 900-nm MP laser to image FITC and the second harmonic generation (SHG) signal. To observe SHG signal, the MP laser power was set at 10%. However, to avoid FITC oversaturation, the MP laser was set at 2% when using higher magnification and longer time points for intravital imaging.

FACS analysis

To obtain single-cell suspensions, PAT was digested with collagenase I and DNase I for 30 min and pushed through the 70-μm cell strainer. LNs were directly pushed through the 40-μm cell strainer. RBCs were removed using ACK lysing buffer. Flow cytometry was done by using FACSCanto II equipment, and the results were analyzed with FlowJo.

Microscopy

Fluorescent images were taken with an SP8 multiphoton microscope (Leica). The objectives were ×20 in air, ×25 in water, and ×63 in oil for different studies. For the high-resolution images, the slice thickness was 0.2 μm with pinhole at 0.5 when using the ×63 oil objective. The resolution was ∼300 nm in this condition. The three-dimensional reconstruction and the diameter measurement were performed with Leica LAX software. Some of the analysis and image brightness adjustment were performed with ImageJ software.

Transmission electron microscopy

The tissue samples were prefixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for at least 2 h at 4°C. After washing three times with the same buffer, the samples were postfixed with cacodylate-buffered 1% osmium tetroxide for 2 h at room temperature and then dehydrated through a graded series of acetone and embedded in Epon resin. Ultrathin sections were cut on a Leica EM UC7 ultramicrotome using a diamond knife and collected on single-hole grids with Formvar supporting film. The sections were stained with 2% aqueous uranyl acetate and Reynolds’s lead citrate and observed in a Hitachi H7650 transmission electron microscope operated at 80 kV. The images were taken through an AMT16000 digital camera mounted on the microscope. Transmission electron microscopy (TEM) was performed by W.X. Dong in the Microscopy and Imaging Facility at the University of Calgary.

Results

Excessive fluid in PAT may flow through PAT-LN conduits to the LN

FITC is a commonly used fluorescent tracer to study lymph distribution in lymphatic vessels and LNs (32). To understand how fluid in PAT flows into the LN, we injected 20 μl of FITC solution intradermally at the flank or directly into PAT of the iLN. Two hours after injection, iLN and its surrounding PAT were collected to analyze FITC distribution. In contrast to the intradermal injection where FITC effectively highlighted PAT and iLN (including SCS, MS, and the conduits), FITC from the intra-PAT injection only highlighted PAT, iLN SCS, and, weakly, the MS (Fig. 1A, 1B). Because skin lymphatic vessels collect and transport fluid quickly into the draining iLN, the significantly reduced FITC accumulation in the iLN after intra-PAT injection suggests that fluid in PAT is less likely to flow through lymphatic vessels.

FIGURE 1.
FIGURE 1.

Fluid may flow through PAT-LN conduits to the LN besides lymphatic vessels. (A and B) FITC was injected intradermally at the flank, or directly into the PAT of iLNs. PAT and iLN were collected 2 h later. Cryosections show the FITC distribution. These experiments were performed with three mice per condition and repeated twice. (C) PAT and iLN were collected from Prox-mOrange reporter mice. Prox-mOrange+ lymphatic endothelial cells (red) and anti-Lyve1 staining (green) show only collecting lymphatic vessels exist in PAT; n = 3. (D–F) FITC sensitization on the skin of Prox-mOrange mice and samples were collected 2 h later. Cryosections show FITC distribution in dermis, s.c. tissue, PAT, and the iLN. Arrow shows one collecting lymphatic vessel in PAT with high density of FITC. Most of the FITC in PAT directly connects with LN capsule; n = 5. (G) In untreated mice, H&E staining shows that PAT connects to the LN capsule. (H) In untreated mice, anti–collagen I (blue) and DAPI show collagen I+ channels between the adipocytes (PAT-LN conduits, arrows). PAT-LN conduits open to a sinus between PAT and the LN capsule, the PAT-LN sinus (orange stars). (I–K). PAT and iLN were collected 2 h after FITC skin sensitization. FITC is located in the collagen I+ PAT-LN conduits (arrows) and accumulates in PAT-LN sinus (stars). Results are representative images from at least 10 iLN sections from 5 to 10 mice. (A–G) Original magnification, ×200; (H–K) original magnification, ×630.

To further characterize whether fluid in PAT flows through lymphatic vessels to the iLN, we used Prox-mOrange reporter mice (mOrange expression driven by the Prox1 promoter) (31), which highlights all of the lymphatic endothelial cells (both initial and collecting lymphatic vessels) to characterize lymphatic vessels in PAT. Anti-Lyve1 Ab staining (labels initial lymphatic vessels and some macrophages, but not collecting lymphatic vessels) in Prox-mOrange mice showed that only Prox-1+Lyve1− collecting lymphatic vessels exist in PAT (Fig. 1C, arrows). However, PAT lacks initial lymphatic vessels to collect interstitial fluid because no Prox-1+Lyve1+ initial lymphatic vessel was detected in PAT (Fig. 1C). Next, we sensitized FITC on the skin of Prox-mOrange mice to determine how FITC in PAT associated with lymphatic vessels. Prox-mOrange+ lymphatic vessels are concentrated in the dermis, s.c. tissue, and in the LN sinuses. Except the collecting lymphatic vessels (Fig. 1D, 1F, arrow), FITC in PAT was not associated with lymphatic vessels but appeared to connect directly with the LN capsule (Fig. 1D, 1F).

To understand how FITC in PAT connects with the LN capsule, we analyzed the architecture of the transition area between PAT and the iLN in untreated mice. H&E staining showed that PAT directly connects to the LN capsule (Fig. 1G). Anti–collagen I staining showed that many collagen I+ channels are present in the PAT interstitial space (Fig. 1H, arrows). We named these collagen I+ channels the PAT-LN conduits. The PAT-LN conduits open to a space between the PAT and the LN capsule–the PAT-LN sinus (Fig. 1H, orange stars). Two hours after FITC sensitization, FITC was colocalized with collagen I+ PAT-LN conduits (Fig. 1I–K, arrows) and accumulated at the PAT-LN sinus (Fig. 1I–K, stars). These results suggest that instead of traveling through lymphatic vessels, FITC in PAT may flow through PAT-LN conduits to the PAT-LN sinus, from where it enters the LN across the LN capsule.

PAT-LN conduits and PAT-LN sinus are collagen-rich channels

Previous reports showed that LN conduits are composed of collagen bundles surrounded by fibroblastic reticular cells and are able to mediate fluid flow in the LN (3, 6, 7). To understand whether PAT-LN conduits share the same structure as the LN conduits, we used TEM to characterize the ultrastructure of PAT-LN conduits and PAT-LN sinus. TEM images showed that PAT-LN conduits were quite different from the LN conduits. In PAT, collagen fibers lay along the adipocytes and formed the PAT-LN conduit channels between the adipocytes (Fig. 2A). The size of PAT-LN conduits varied from several hundred nanometers to several micrometers (Fig. 2A–C). Collagen bundles and extracellular vesicles were frequently observed in the PAT-LN conduits (Fig. 2B, 2C, arrows). PAT-LN conduits opened to the PAT-LN sinus (Fig. 2D, stars) between PAT and the LN capsule. The PAT-LN sinus appears as large PAT-LN conduits that were rich with collagen bundles (Fig. 2D, 2E, black arrows). Different from the LN conduits, collagen bundles in PAT-LN conduits were not surrounded with fibroblasts. Surprisingly, the LN capsule is not a simple collagen sheet to exclude the communication between PAT and the LN. Instead, it appeared that the LN capsule was quite permeable to fluid and small particles. Cells were positioned in the gaps between the collagen bundles at the LN capsule (Fig. 2D, 2E, white arrows). Small extracellular vesicles appeared to be able to transit across the LN capsule via the gaps between collagen bundles in the LN capsule (Fig. 2F, arrows). Thus, the ultrastructure of PAT-LN conduits and LN capsule suggest fluid and small particles may travel through PAT to the LN across the LN capsule.

FIGURE 2.
FIGURE 2.

Ultrastructure of PAT-LN conduits and PAT-LN sinus. TEM images show iLN and its surrounding PAT. (A) In PAT, collagen fibers lie along PAT-LN conduits between the adipocytes; original magnification, ×20,000. (B and C) Collagen bundles (black arrows) and extracellular vesicles (white arrows) in large PAT-LN conduits; original magnification, ×8000. (D) PAT-LN conduits (between the adipocytes) open to the collagen-rich PAT-LN sinus (stars). Black arrows point to collagen bundles; white arrows point to cells inside the LN capsule. Original magnification, ×1200. (E) Higher magnification of the PAT-LN sinus. Black arrows point to collagen bundle; white arrow points to cells in the gap between collagen fiber bundles in LN capsule. Original magnification, ×3000. (F) Extracellular vesicles (arrows) in gaps between collagen bundles in LN capsule. Stars indicate PAT-LN sinus. Original magnification, ×4000. TEM images are representative iLN from three mice.

Fluid flow through PAT-LN conduits to the LN is not dependent on the afferent lymphatic vessels

A previous study suggested that accumulation of fluid in PAT is due to the permeability of lymphatic vessels (33). It is also possible that fluid enters the LN sinus and then diffuses into PAT. To determine whether FITC accumulation in PAT is the secondary effect of lymphatic vessels’ permeability or fluid diffusion from the LN sinus, we sutured the afferent lymphatic vessels to the pLN, because these afferent lymphatic vessels are well defined (34, 35). The blockage of lymph flow in the afferent lymphatic vessels was confirmed with the following two observations. First, Evans blue dye, which filled the lymphatic vessels, could not pass the suture site after the surgery (Fig. 3A, arrows). Second, edema and significantly increased paw thickness were observed at the sutured, but not the sham, legs 2 h after suture (Fig. 3B).

FIGURE 3.
FIGURE 3.

Fluid flows through PAT-LN conduits to LN when afferent lymphatic vessels are blocked. (A) pLN afferent lymphatic vessel suture. Five microliters of Evans blue dye was injected in the footpad to show pLN afferent lymphatic vessels. A small skin cut was performed near the pLN and both afferent lymphatic vessels were sutured. The success of lymph flow blockade is indicated by Evans blue dye, which fills the afferent lymphatic vessels but cannot pass the suture site. (B) Evans blue dye and the paw thickness show the edema in the sutured leg, but not the sham leg. (C–E) FITC distribution in the draining pLNs and the downstream iLNs of the sham or sutured leg. *p < 0.01. (C) To prevent FITC from entering PAT through the surgical wound, FITC (10 μl) was injected at the footpad of the sham or the lymphatic sutured leg immediately after the surgery, and pLN and the surrounding PAT were collected 2 h later. FITC can highlight PAT and the pLN capsule when afferent lymphatic vessels are sutured. (D) FITC was injected in the footpad of sham mice or mice with sutured pLN afferent lymphatic vessels. pLNs were collected at 40 min, 2, 6, and 12 h after FITC injection. (E) The downstream iLNs were collected at different times after FITC injection. At 40 min after the FITC injection, FITC is restricted in the pLN and does not enter the iLN of the sham leg. Instead, FITC enters the iLN when pLN lymphatic vessels are sutured. The samples at different time points were prepared on the same day. The images were taken on the same day using exactly the same settings. (C–E) Original magnification, ×200. These experiments were performed with three mice per time point per condition and repeated twice.

To determine whether fluid can enter the pLN through PAT-LN conduits after lymphatic vessel blockage, FITC was injected in the footpad as soon as the surgery was completed. In the sham leg, FITC highlighted the whole pLN and the PAT surrounding pLN within 2 h (Fig. 3C, sham). In the lymphatic vessel sutured leg, albeit being significantly reduced, FITC managed to flow into the PAT and highlighted the capsule of pLN within 2 h (Fig. 3C). Thus, FITC flow through PAT-LN conduits and PAT-LN sinus is not dependent on functional afferent lymphatic vessels. To determine the FITC flow dynamics through PAT to the pLN after lymphatic blockage, pLNs were collected at 40 min, 2, 6, and 12 h after FITC injection in the footpad of the sham or sutured mice. When afferent lymphatic vessels were sutured, although FITC reached the LN capsule within 2 h, it took 6–12 h to highlight the whole pLN (Fig. 3D). FITC accumulation in the pLN after lymphatic suture suggests that FITC flow through PAT-LN conduits to the LN is independent of lymphatic vessels.

It is still possible that alternative lymphatic vessels may transport fluid to the pLN when lymphatic vessels are sutured. However, characterization of FITC flow dynamics clearly showed that afferent lymphatic vessels specifically transported fluid from the footpad into the pLN in the sham leg (Fig. 3D, 40 min). No FITC was detected in the downstream iLN by 40 min (Fig. 3E, sham iLN 40 min), but in the sutured leg, whereas FITC was substantially reduced in the draining pLN, FITC alternatively highlighted the downstream iLNs within 40 min (Fig. 3E, sutured iLN 40 min). These results suggest that when pLN afferent lymphatic vessels are sutured, the increased interstitial pressure may push lymphatic valves open to different collecting lymphatic vessels (36, 37), which helps fluid bypass the pLNs and enter the downstream iLNs. These results further demonstrate FITC flow through PAT to the pLN is independent of lymphatic vessels.

Fluid at the PAT-LN sinus may enter the LNC conduits

When lymphatic vessels were sutured, FITC highlighted PAT and the LN capsule first, and then the LN conduits (Fig. 3), and TEM images showed that the LN capsule is permeable (Fig. 2). Next, we aimed to understand whether fluid in PAT can enter the LN conduits. The kinetics of FITC distribution in the LN after skin sensitization showed that FITC signal was first detected at the outer edge of the LN capsule and in the collagen I+ LNC conduits within 20 min (Fig. 4A, arrows). FITC preferentially highlighted LNC conduits by 40 min and the whole LN conduits by 60 min (Fig. 4A). To avoid the possibility that the FITC flow dynamics along the LNC conduits are due to unexpected artifacts from the fixed samples, we performed intravital time-lapse imaging. Time-lapse video of three-dimensional–reconstructed images showed that lymph preferentially flowed along the LNC conduits (Fig. 4B, white arrows and Supplemental Video 1). The LNC conduits can be better visualized using higher magnification images (Fig. 4C, arrows, Supplemental Video 2). At 2 h after FITC sensitization, the whole-mount iLN image showed that FITC was mostly present along the LNC conduits (Fig. 4D). Finally, the three-dimensional–reconstructed image showed that FITC signal started from the outer edge of the collagen I+ LN capsule and entered the lumen of collagen I+ LNC conduits (Fig. 4E, 4F). Taken together, these results suggest that FITC traveling through PAT may flow into LNC conduits.

FIGURE 4.
FIGURE 4.

Fluid in PAT-LN sinus flows to LN capsule and LNC conduits. (A) PAT and iLN were collected at 5, 20, 40, and 60 min after FITC skin sensitization. Cryosections with anti–collagen I staining (red) show the dynamics of FITC distribution in the LN capsule and the LN conduits. Original magnification, top panel, ×200; bottom panel, ×630. (B and C) FITC flow dynamics visualized by intravital time-lapse imaging. (B) These images highlight critical steps in Supplemental Video 1. SHG signal shows the location of LN capsule, LNC conduits (white arrow), and the space around the pLN after surgical preparation. FITC first filled the space around the LN and specifically flows along the LNC conduits inside the pLN. MP laser power was set at 10% to show LN capsule with SHG signal. Original magnification, ×250. (C) Images that highlight the critical steps in Supplemental Video 2. Continued imaging of the pLN with higher magnification to focus on LNC conduits (white arrows) is shown. Owing to the extremely intense FITC signal, MP laser power was set at 2% to perform the time-lapse images without FITC signal oversaturation. Original magnification, ×750. These experiments were repeated in more than 10 mice. (D) LNs were collected 2 h after FITC sensitization. 3D-reconstructed image of whole mount LN show FITC is preferentially present in LNC conduits. Original magnification ×250. (E and F) anti-collagen I staining show FITC starts from the outer side of the collagen I+ LN capsule and locates in the lumen of collagen I+ LNC conduits (arrows). Original magnification ×1890. These experiments were repeated in >10 mice.

Ag delivery through PAT

Transportation of Ags to the draining LN is one of the major functions of lymph flow. To determine whether PAT-LN conduits can facilitate Ag delivery, we used Alexa555-OVA to trace Ag distribution in PAT. At 2 h postinjection, as expected, Alexa555-OVA was detected both in PAT and in the LN (Fig. 5A). To determine whether OVA simply flows through PAT or whether OVA can stimulate PAT-resident APCs, PAT was collected at 2 h after Alexa555-OVA (with LPS) injection. By FACS analysis, gated on the Alexa555+CD45+ cells, these cells were MHC class II+ APCs (Fig. 5B). Activation of PAT-resident APCs further demonstrated that OVA entered PAT.

FIGURE 5.
FIGURE 5.

PAT-LN conduits facilitate Ag delivery to the LN when lymphatic vessels are blocked. (A) Alexa555-OVA was injected intradermally at the flank. PAT and iLNs were collected 2 h later. Frozen sections show Alexa555-OVA distribution in PAT and the iLN. These experiments were performed with two mice per group and repeated three times. Original magnification, ×200. (B) PAT surrounding iLN was collected 2 h after Alexa555-OVA injection with LPS. Alexa555-OVA+ cells were analyzed with FACS analysis (n = 4 per group and the experiments were repeated twice). (C) Lymphatic vessels to the pLNs were sutured as described above. Alexa555-OVA (20 μg in 10 μl of saline with 20 ng of LPS) was injected in the footpad of sham or sutured leg. PLNs and iLNs were collected 6 h after the injection to exclude tissue migrating DCs. n = 3 per group and the experiments were repeated twice. ***p < 0.001.

To determine whether OVA can travel through PAT-LN conduits to the LN when the afferent lymphatic vessels are interrupted, we sutured the afferent lymphatic vessels to the pLN as described above and injected Alexa555-OVA with LPS. The draining pLNs and the downstream iLNs were collected 6 h later. In the sham leg, ∼1.2% of the pLN cells have captured OVA (Fig. 5C, white columns). When afferent lymphatic vessels were sutured, although significantly reduced, ∼0.4% of LN cells in the sutured pLN have captured OVA (Fig. 5C, black columns). These results suggest that PAT-LN conduits are able to transport Ag to the LN and stimulate LN-resident cells when afferent lymphatic vessels are sutured. Similar to FITC distribution, popliteal afferent lymphatic vessels specifically transported OVA to the draining pLN in sham leg. After lymphatic suture, the increased interstitial pressure likely pushes OVA to enter alternative functional lymphatic vessels, which bypasses the pLNs and enters the downstream iLNs (Fig. 5C). These results further demonstrate Ag delivery through PAT-LN conduits is lymphatic vessel–independent.

Circulating factors quickly enter PAT and may flow to the LN via PAT-LN conduits

Because lymphatic vessels can transport fluid much faster, it is likely that PAT-LN conduits play minor roles in transporting peripheral fluid or Ag to the LN under physiological conditions. Interstitial fluid comes from blood capillaries for oxygen and nutrient exchange. Because PAT is a blood vessel–rich tissue but lacks lymphatic vessels, we hypothesized that the physiological function of PAT-LN conduits is to remove excessive PAT interstitial fluid to the LN to maintain PAT fluid balance. To test this hypothesis, we i.v. injected Alexa555-OVA via the tail vein and collected iLN and the surrounding PAT 5 min and 2 h later. Alexa555-OVA was detected in PAT and LN within 5 min (data not shown). By 2 h, Alexa555-OVA was observed in PAT, LN blood vessels, and LN sinus (Fig. 6A). Some Alexa555-OVA in PAT appeared to feed into the LN (Fig. 6B, arrows). To exclude the possibility that Alexa555-OVA persists in the blood circulation, we perfused the mice with PBS to remove materials in blood vessels before collecting the samples. The Alexa555-OVA distribution pattern remained similar (data not shown). Thus, circulating OVA quickly enters and accumulates in PAT.

FIGURE 6.
FIGURE 6.

Circulating factors accumulate in PAT and likely enter the LN via PAT-LN sinus. (A–C) Alexa555-OVA was injected via the tail vein and PAT and iLN was collected 2 h later. (A) Alexa555-OVA accumulates in PAT and LN. Original magnification, ×200. (B) Alexa555-OVA in PAT appears to feed into the LN (white arrows). Original magnification, ×200. (C). Anti-CD31 (green) and anti–collagen I (blue) show that Alexa555-OVA in the PAT is colocalized with CD31+ blood vessels, and some of these vessels enter the PAT-LN sinus. Original magnification ×630. Images with separated colors are provided in Supplemental Fig. 1. (D) TEM images show a blood vessel in the PAT-LN sinus. Stars indicate the PAT-LN sinus. Original magnification, ×1200. (E and F) PAT and iLN were collected 2 h after FITC sensitization. Anti-CD31 and anti–collagen I staining show that FITC flows through collagen I+CD31− PAT-LN conduits and along the outside wall of collagen I+CD31+ vessels. (E) Original magnification, ×200; (F) original magnification, ×630. Images with separated colors are provided in Supplemental Fig. 1. (G) S. aureus was injected intradermally at the right side of the flank and the draining PAT, and iLN and liver were collected 4 h later. CFU were counted to determine bacteria infection. (H) Neutrophils (CD11b+Ly6G+) infiltrate both the draining LN and PAT 4 h after S. aureus intradermal injection. *p < 0.01. (I) S. aureus was injected via the tail vein and tissues were collected 4 h later to determine bacteria infection by counting CFU. A group of mice was perfused with PBS to remove remaining S. aureus in blood circulation before tissue collection. S. aureus CFU is similar in mice with or without PBS perfusion, indicating that S. aureus has left the circulation and infected PAT at this time point. (J) Schematic illustration of PAT-LN conduits, PAT-LN sinus, LN capsule, and LNC conduits. PAT-LN conduits include the collagen I+CD31− channels between the adipocytes and the outside wall of collagen I+CD31+ vessels. PAT-LN sinus is the collagen I–rich space between PAT and the LN capsule. Fluid from skin or PAT interstitial fluid may flow through PAT-LN conduits to PAT-LN sinus and then enter the LN across the LN capsule and/or along LNC conduits. In physiological conditions, PAT-LN conduits likely serve as the route to remove PAT excessive fluid to the LN for PAT fluid balance. Ags and S. aureus may use PAT-LN conduits and PAT-LN sinus to stimulate PAT immune protection.

To determine whether Alexa555-OVA travels through PAT-LN conduits to LN, we stained the PAT and LN with anti-CD31 (also named PECAM1) and anti–collagen I Abs. Alexa555-OVA was preferentially colocalized with CD31+collagen I+ vessels, and some of these vessels directly penetrated into the PAT-LN sinus (Fig. 6C). TEM images showed that blood vessels existed in the PAT-LN sinus (Fig. 6D). Thus, fluid exchange between the blood vessel and PAT may occur in the PAT-LN sinus.

To determine whether PAT-LN conduits are actually CD31+collagen I+ vessels, we used anti-CD31 and anti–collagen I Abs to stain PAT and iLN after FITC sensitization. FITC was located in all of the collagen I+ structure, within the collagen I+CD31− structure, and along the outside wall of the collagen I+CD31+ blood vessels (Fig. 6E, 6F; images with separate colors are provided in Supplemental Fig. 1A, 1B). Thus, PAT-LN conduits include both collagen I+CD31− interstitial space and the outside wall of collagen I+CD31+ vessel.

S. aureus enters PAT from either intradermal or i.v. infection

Previous reports show S. aureus skin infection causes dermal adipocyte expansion and antimicrobial peptide (cathelicidin) secretion (38). We hypothesized that S. aureus utilizes PAT-LN conduits to infect PAT and stimulate PAT immune protection. S. aureus was restricted in the draining PAT and iLN at 4 h after intradermal injection at the flank (Fig. 6G). Liver bacteria count suggests almost no bacteria entered the circulation and reached the liver at this time point (Fig. 6G). Next, we collected the draining PAT and iLN, as well as the contralateral PAT and iLN as control. At 4 h after S. aureus intradermal infection, neutrophils (CD11b+Ly6G+) were substantially recruited to the draining PAT and iLN (Fig. 6H), indicating that S. aureus activates immune responses in PAT.

Finally, to determine whether S. aureus can infect PAT via blood circulation, we injected 5 × 107 S. aureus via the tail vein and collected PAT, iLN, and liver 4 h later. S. aureus consistently accumulated in PATs and iLNs (Fig. 6I, nonperfusion). S. aureus counts were consistent between the nonperfused and the PBS-perfused mice, suggesting that S. aureus has left the circulation and infected PAT and iLN (Fig. 6I, perfusion). We also examined bacterial infection in gonadal adipose tissue (non–lymphoid-associated adipose tissue) and found that circulating S. aureus not only infects PAT but also other adipose tissue. These studies suggest that bacterial infection in PAT is unlikely dependent on lymphatic vessels or the secondary effect after LN activation, but more likely utilizes the PAT-LN conduits to actively participate in the immune protection.

Discussion

LNs are typically nestled in PAT, and the intimate communication between PAT and the LN is important for LN development and immune responses (10, 19, 24, 28, 39). The goal of this study was to understand how fluid and small molecules communicate between PAT and the LN. Although afferent lymphatic vessels contribute to most lymph flow to the LN, our study shows that peripheral fluid and Ags may flow from PAT-LN conduits to PAT-LN sinus, from which fluid may enter the LN across the LN capsule and/or along LNC conduits (Fig. 6J). Fluid flow through PAT is demonstrated by the fact that FITC and OVA can enter PAT and stimulate PAT-resident APCs, and that FITC and OVA can flow through PAT to the LN even after lymphatic vessels are sutured. FITC and OVA distribution patterns after the afferent lymphatic vessels are blocked indicate that PAT-LN conduits are not dependent on functional lymphatic vessels. The flow pattern characterized by the tracers from intradermal injection or i.v. injection suggests that PAT-LN conduits include collagen I+CD31− interstitial space and the outside wall of collagen I+CD31+ vessels in PAT (Fig. 6J). Interstitial fluid flow along the blood vessel wall has been reported in other organs, such as in the brain, and likely uses the passive force from the blood vessel pulsing (40). It is likely that PAT-LN conduits also adopt the passive force from the blood vessel pulsing for fluid transport to the LN.

Previous studies suggest that lymphatic vessel permeability causes fluid and Ag accumulation in PAT and that PAT-resident APCs can capture Ags draining from the periphery (33). Our studies suggest fluid may directly enter the PAT via PAT-LN conduits when afferent lymphatic vessels are blocked. As a consequence, Ags can stimulate PAT immune protection. These two mechanisms are not mutually exclusive. Previous reports show that dermal adipocytes can protect against S. aureus infection by secreting antimicrobial peptides (cathelicidin) (38). It has also been reported that adipose tissue may serve as a reservoir for chronic bacterial or viral infections (41, 42). Our studies showed that bacteria from either intradermal or i.v. infection might enter PAT and stimulate PAT immune protection. Thus, PAT can actively participate in immune protection, which unlikely is the secondary effect after LN activation. Our studies suggest a new immune function of PAT in host antimicrobial protection.

The significantly delayed and reduced FITC or OVA accumulation in the LN when lymphatic vessels are sutured suggests that lymphatic vessels are responsible for most lymph flow to the draining LN. It is likely that PAT-LN conduits play a minor role for peripheral fluid or Ag transport under physiological condition. How inflammation or other diseases, such as obesity, may impact PAT-LN conduits and Ag delivery are under investigation. During physiological conditions, the quick material exchange between blood circulation and PAT is demonstrated by Alexa555-OVA accumulation in PAT, which appears as early as 5 min after the tail vein injection (data not shown). Because PAT lacks initial lymphatic vessels, PAT-LN conduits probably serve as the route to bring excessive PAT fluid to the LN and join lymphatic circulation during physiological conditions. Although it appears that Alexa555-OVA may flow into the PAT-LN sinus, it is unlikely that PAT-LN conduits will serve as a critical route for the entrance of circulating factors to the LN in adult mice. Because the LN is highly vascularized, circulatory factors can directly communicate with LN via LN blood vessels, such as via high endothelial venules. Fluid and material exchange in PAT provides a mechanism to interpret why maternal or gut-derived molecular or cellular signals may access PAT and regulate LN development and cell homeostasis during embryogenesis (19, 24). However, whether this type of communication is important during early LN development remains to be investigated.

In summary, by exploring fluid exchange between PAT and the LN, our studies revealed that PAT might use PAT-LN conduits to facilitate Ag delivery and participate in immune protection, indicating a new function of immune communication between PAT and the LN.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Drs. Yan Shi and Peng Huang for critical input into this study and the manuscript preparation.

Footnotes

  • This work was supported by the University of Calgary start-up fund to S.L., provided by the Dianne and Irving Kipnes Foundation, the Canadian Institute of Health Research (to S.L.), and the Canada Foundation for Innovation (to S.L.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    Alexa555
    Alexa Fluor 555
    iLN
    inguinal LN
    LN
    lymph node
    LNC
    LN capsule–associated
    LTo
    lymphoid tissue organizer
    MP
    multiphoton
    MS
    medullary sinus
    PAT
    perinodal adipose tissue
    pLN
    popliteal LN
    SCS
    subcapsular sinus
    SHG
    second harmonic generation
    TEM
    transmission electron microscopy.

  • Received February 1, 2018.
  • Accepted April 27, 2018.

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