Obesity Impairs Lymphatic Fluid Transport and Dendritic Cell Migration to Lymph Nodes

<div><p>Introduction</p><p>Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system.</p><p>Methods</p><p>Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8–10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up.</p><p>Results</p><p>High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages.</p><p>Conclusions</p><p>Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.</p></div

Obesity impairs dendritic cell migration in wild-type but not RAG mice.

<p><b>A.</b> Representative flow diagram (top) and quantification (bottom) of migrated DCs (MHCII^high/CD11c⁺/GFP⁺) in control and obese wild-type and RAG mice. <b>B., C.</b> Correlation between migrated DCs and weight in wild-type (<b>B</b>) and RAG (<b>C</b>) mice.</p

Obesity decreases lymph node uptake.

<p><b>A., B.</b> Tc⁹⁹ lymphoscintigraphy in wild-type (<b>A</b>) and RAG (<b>B</b>) mice. Yellow arrow denotes spleen. White arrow is the lymph node; arrow head denotes Tc⁹⁹ injection site. <b>C., D.</b> Mean Tc⁹⁹ uptake in wild-type (C) and RAG (B) control and obese mice. <b>E.</b> Mean rate of Tc⁹⁹ uptake in control and obese wild-type and RAG mice.</p

Obese mice have smaller lymph nodes with fewer lymphatic vessels.

<p><b>A.</b> Representative immunofluorescent staining (LYVE-1 =  red; DAPI  =  blue) of inguinal lymph nodes (2× magnification) harvested from control (left panels) and obese (right panels) wild-type and RAG mice. <b>B.</b> Mean lymph node area of inguinal lymph nodes harvested from control/obese wild-type and RAG mice. <b>C.</b> Mean number of lymphatic vessels per unit area in inguinal lymph nodes of control/obese wild-type and RAG mice. <b>D.</b> Mean lymphatic vessel area in the inguinal lymph nodes of control/obese wild-type and RAG mice.</p

High fat diet results in obesity and subcutaneous tissue inflammation.

<p><b>A.</b> Body weight of obese and control mice. <b>B.</b> Gross photograph of subcutaneous tissue adipose deposition in control and obese mice (top). Representative photomicrograph of full-thickness back punch biopsy specimens in control and obese mice (20× magnification). Brackets indicate subcutaneous fat deposition. <b>C.</b> Mean subcutaneous fat thickness in back punch biopsy of control and obese mice. <b>D.</b> Serum cholesterol, triglycerides, and glucose levels in 12 hour fasting blood samples from control and obese mice. <b>E., F.</b> Representative low (<b>E;</b> 10x) and high (<b>F;</b> 40x) power photomicrographs of control and obese mice back punch (upper) and tail (lower) histological sections stained for CD45 (brown) and LYVE-1 (red). Black box in figure E is magnified in F. <b>G.</b> Mean number of CD45⁺ cells/hpf (40x) in control and obese mice. <b>H.</b> Mean number of peri-lymphatic CD45⁺ cells/hpf (40x) in control and obese mice.</p

Obesity causes changes in the architecture and cellular composition of lymph nodes.

<p><b>A.</b> Representative florescent micrograph (2.5× magnification) of inguinal lymph nodes harvested from control and obese mice and stained for B cells (B220; purple) and T cells (CD3; green). Note loss of follicular pattern and disorganization in obese mice. <b>B.</b> Representative florescent micrographs (2.5x) of inguinal lymph nodes harvested from control (left panels) and obese (right panels) wild-type mice stained for CCL21 (red) and DAPI (blue). CCL21 expression is shown on top panels with DAPI overlay on the bottom. Dotted circled represent follicular regions. Note loss of CCL21 expression gradients in obese animals. <b>C.</b> Representative whole mount immunofluorescent staining (Green  =  LYVE-1; Red  =  CCL21; Blue  =  DAPI) of ear sections from control (top) and obese (bottom). Sections are shown at 63× magnification. LYVE-1 staining is shown on the left; CCL21 is the middle panel; overlay is shown on the right panels. Dashed lines in the middle panel is the region of LYVE-1 vessel. <b>D.</b> Mean area of LYVE-1/CCL21 co-localization (presented as a percentage of LYVE-1 area overall) in control and obese mice. <b>E., F. G., H.</b> Flow cytometry analysis of T-helper cells (CD45+/CD3+/CD4+; <b>E</b>), cytotoxic T cells (CD45⁺/CD3⁺/CD8⁺; <b>F</b>); B cells (CD45⁺/B220⁺/CD19⁺; <b>G</b>), and macrophages (CD45^+//B220⁺/CD11b⁺; <b>H</b>). Mean percentage of cells from 4–5 animals per group is presented.</p

Obesity impairs lymphatic flow.

<p><b>A., B.</b> Representative florescent photomicrograph of microlymphangiography (<b>A</b>) and quantification of florescence intensity at a fixed distance from the injection site (black box on photograph; <b>B</b>) in control and obese mice. The microlymphangiography photographs depict the uptake of florescent labeled macromolecule in the dermal lymphatics of the tail at various time points following injection in the distal tail. Note rapid uptake (10 minutes) and increased florescence intensity in control mice as compared with obese mice. This difference is quantified in figure <b>B</b> in 5 animals per group. <b>C.</b> Representative low power (20x; upper) and higher power (40x; lower) photomicrographs of back punch tissue sections from control and obese mice stained for LYVE-1 (brown). Boxes in upper panel represent area magnified in high power views. Brackets denote diameter of the lymphatic vessels. <b>D.</b> Mean lymphatic vessel area in control and obese mice. <b>E.</b> Mean number of LYVE-1⁺ lymphatic vessels/hpf in back punch biopsies of control and obese mice. <b>F., G.</b> Representative PET-CT of control and obese mice demonstrating lymphatic chain after distal tail injection of 18F-FDG. The lymphatic chain is also shown separately for greater detail. Arrow denotes location of para-aortic lymph nodes. Mean normalized sacral node uptake ± SEM (gray shading) of 18F-FDG over time in control and obese mice.</p