Diet-induced obesity causes visceral, but not subcutaneous, lymph node hyperplasia via increases in specific immune cell populations

Abstract

Objectives: The spatial proximity of adipose depots to secondary lymph nodes allows a unique relation between the two systems. Obesity, predominately visceral adiposity, links to numerous diseases; hence, we postulate that secondary lymphatics within this region contributes to disease risk.

Material and methods: Male C57BL/6 mice were fed standard CHOW (18% kcal fat) or Western diet (45% kcal fat) for 7 weeks. Visceral and subcutaneous lymph nodes and associated adipose depots they occupy were excised. Lymph node morphology and resident immune cell populations were characterized via histopathology, immunofluorescence and flow cytometry. Adipose tissue immune cell populations were also characterized.

Results: Obesity caused lymph node expansion, increased viable cell number and deviations in immune cell populations. These alterations were exclusive to visceral lymph nodes. Notably, pro-inflammatory antigen presenting cells and regulatory T cells increased in number in the visceral lymph node. Obesity, however, reduced T regulatory cells in visceral lymph nodes. The visceral adipose depot also had greater reactivity towards HFD than subcutaneous, with a greater percent of macrophages, dendritic and CD8⁺ T cells. Immune cell number, in both the visceral and subcutaneous, however decreased as adipose depots enlarged.

Conclusion: Overall, HFD has a greater influence on visceral cavity than the subcutaneous. In the visceral lymph node, but not subcutaneous, HFD-induced obesity decreased cell populations that suppressed immune function while increasing those that regulate/activate immune response.

Figure 1

Average lymph node area (μm²). Visceral lymph node of (A) CHOW and (B) HFD mouse. Subcutaneous lymph node of (C) CHOW and (D) HFD mouse. Lymph node area for an individual sample was the sum of four sections sliced across the largest mid‐region portion of the lymph node. Lymph node area for an individual sample was the sum of four sections sliced across the largest mid‐region portion of the lymph node. E, HFD significantly increased mean lymph node size of visceral lymph nodes (*=.004) but not subcutaneous compared to CHOW controls

Figure 2

Lymph node immunocytochemistry (ICC) of B220⁺ (green) and CD3⁺ T cells (red). Visceral CHOW (A) and HFD (B) lymph node. Subcutaneous CHOW (C) and HFD (D) lymph node. HFD significantly increased the total area of the visceral lymph nodes (*=.011) (E) with specific enlargement in both the T‐cell zone (*=.017; CD3=red) (F) and B‐cell follicles (B220, green) (G) in the cortex

Figure 3

Immune cell percent in visceral and subcutaneous adipose tissue depots. In CHOW mice, the visceral adipose depot contained a significantly lower percent of CD11b⁺F4/80⁺, macrophages (*=.015) (A) and CD4⁺Foxp3⁺, regulatory T cells (*=.016) (C) compared with the subcutaneous depots from CHOW animals. In opposition, CD3⁺ CD4⁺ T helper cells (*=.008) (B) were significantly increased in the visceral adipose tissue depot of CHOW mice compared with respective subcutaneous adipose depots. HFD altered immune cell populations within lymph nodes. In the visceral lymph node, HFD significantly increased CD11b⁺F4/80⁺, macrophages (*=.0001) (A), CD3⁺ CD8⁺, cytotoxic T cells (*=.0001) (D) and CD11c⁺CD11b⁺, dendritic cells (*=.001) (E), but significantly decreased CD4⁺Foxp3⁺, regulatory T cells (*=.002) (C). HFD also significantly decreased regulatory T cells in the subcutaneous adipose depot (*=.002) (C); right column, respective scatter plots of significant diet differences

Figure 4

Total viable cells of CHOW and HFD animals in visceral and subcutaneous adipose tissue (cells/g) and visceral and subcutaneous lymph nodes. Total viable cell count was not different between CHOW visceral and subcutaneous adipose depots (A). Compared with controls, HFD caused a significant ~60% decrease in normalized (per gram adipose tissue) total viable cell number in both the visceral and subcutaneous adipose depots (*=.008). In the visceral only, HFD significantly increased lymph node viable cell number (*=.01) (B)

Figure 5

Total viable immune cell numbers in visceral and subcutaneous adipose tissue and lymph nodes (no. of cells per gram). Adipose tissue: In CHOW mice, the total number of CD3⁺ CD4⁺, T helper cells (*=.0001) (B) and CD3⁺ CD8⁺, cytotoxic T cells (*=.015) (D) within the visceral adipose depot were significantly greater than those in the subcutaneous. F4/80⁺ CD11b⁺, macrophages (*=.0001) (A) and CD4⁺Foxp3⁺, T regulatory cell (*=.0001) (C) number was significantly lower in the visceral adipose depot of CHOW mice compared with CHOW subcutaneous. HFD significantly reduced the viable number of CD3⁺ CD4⁺, T helper cells (*=.003) (B) in the visceral adipose depot and F4/80⁺ CD11b⁺, macrophages (*=.001) (A), CD3⁺ CD4⁺, T helper cells (*=.003) (B), CD3⁺ CD8⁺, cytotoxic T cells, (*=.004) (D) and CD11b⁺ CD11c⁺, dendritic cells (*=.045) (E) in the subcutaneous adipose depot relative to CHOW controls. Lymph nodes: HFD significantly increased viable number of visceral lymph node F4/80⁺ CD11b⁺, macrophages (*=.0001) (F), CD3⁺ CD4⁺, T helper cells (*=.003) (G) and CD11c⁺ CD11b⁺, dendritic cells (*=.23) (J) compared with respective CHOW controls. Visceral lymph node number of CD4⁺Foxp3⁺, T regulatory cells (*=.0001) (H) was significantly decreased in HFD mice compared with the CHOW controls. HFD did not change immune cell number in the subcutaneous lymph node.

Figure 6

Immune cell percent in visceral and subcutaneous lymph nodes. Visceral lymph nodes of CHOW mice contained a significantly greater percent of F4/80⁺ CD11b⁺ macrophages (*=.004) (A) and CD4⁺Foxp3⁺ regulatory T cells (*=.0001) (C) compared with CHOW subcutaneous lymph nodes. CD3⁺ CD4⁺ helper T‐cell percent (*=.012) (B), however, was significantly lower in the visceral lymph nodes of CHOW mice. HFD significantly decreased the percent of CD4⁺Foxp3⁺ regulatory T cells (*=.002) (C) in the visceral lymph node only. Figure C scatter plots ‐ Representative plot for CD4+Foxp3⁺ T cells from CHOW and HFD VLN respectively. (D) and (E) CD3⁺CD8⁺ and CD11c⁺CD11b⁺ immune cell percent was not different between lymph nodes and was not change by diet

Figure 7

Thymus area (μm²) of CHOW and HFD‐fed animals. Haematoxylin and eosin (H&E) stained CHOW and HFD (A) mouse thymus. HFD caused a significantly decrease, ~50%, in thymus area compared with CHOW (*=.014) (B). This reduction appeared secondary to generalized atrophy of both the cortex and medulla

Figure 8

Spleen area (μm²) of CHOW and HFD‐fed animals. Haematoxylin and eosin (H&E) stain of CHOW and HFD mouse spleen (A) HFD altered spleen architecture (A), without altering the total area of the spleen (B). HFD significantly decreased overall spleen lymphoid follicle number, ~50%, compared with CHOW (*=.002) (C). In HFD animals, the total follicle area was not changed compared with control (D), but secondary lymphoid follicles in HFD animals significantly increased approximately 2‐fold in number (*=.021) (E). Secondary follicle total area of HFD mice was also significantly increased ~2‐fold (*=.001) compared with CHOW animals (F)