Adipose tissue extrinsic factor: Obesity-induced inflammation and the role of the visceral lymph node

Abstract

Obesity-related adverse health consequences occur predominately in individuals with upper body fat distribution commonly associated with increased central adiposity. Visceral adipose tissue accumulation is described to be the greatest driver of obesity-induced inflammation, however evidence also supports that the intestines fundamentally contribute to the development of obesity-induced metabolic disease. The visceral adipose depot shares the same vasculature and lymph drainage as the small intestine. We hypothesize that the visceral lymph node, which drains adipose tissue and the gastrointestinal tract, is central to the exacerbation of systemic pro-inflammation. Male C57BL/6 mice were fed CHOW or high fat diet (HFD) for 7 weeks. At termination the mesenteric depot, visceral lymph node and ileum, jejunum and Peyer's patches were collected. Cytokine concentration was determined in adipose tissue whereas immune cell populations where investigated in the visceral lymph node and intestinal segments by flow cytometry. Visceral adipose tissue and the gastrointestinal tract mutually influence immune cells enclosed within the visceral lymph node. HFD increased visceral lymph node immune cell number. This likely resulted from 1.) an increase in immune cells migration from the small intestines likely from activated dendritic cells that travel to the lymph node and 2.) cytokine effluent from visceral adipose tissue that promoted expansion, survival and retention of pro-inflammatory immune cells. Overall, the visceral lymph node, the immune nexus of visceral adipose tissue and the small intestines, likely plays a fundamental role in exacerbation of systemic pro-inflammation by HFD-induced obesity. The research of Tim Bartness greatly enhanced the understanding of adipose tissue regulation. Studies from his laboratory significantly contributed to our awareness of extrinsic factors that influence body fatness levels. Specifically, the work he produced eloquently demonstrated that adipose tissue was more complex than an insulating storage center; it was connected to our brains via the sympathetic and sensory nervous system. Mapping studies demonstrated that adipose tissue both receives and sends information to the brain. Further, his lab demonstrated that nervous system connections contributed to lipolysis, thermogenesis and adipocyte proliferation and growth. The work of Tim Bartness will continue to influence adipose tissue research. As such, Tim Bartness directly inspired the following research. Adipose tissue extrinsic factors are not limited to the peripheral nervous system. The lymphatic system is an additional extrinsic factor that cross talks with adipose tissue, however its role in this context is under emphasized. Here we begin to elucidate how the lymphatic system may contribute to the comorbidities associated with visceral adipose tissue accumulation.

Keywords: Central obesity; Lymphatics; Metabolic disease; Pro-inflammation; Visceral adiposity.

Figure 1. Visceral adipose tissue cytokine concentrations (pg/ml)

Compared with CHOW, HFD increased the concentration of pro-inflammatory interleukins IL-1b A.) and IL-6 (* = 0.003) C.). The anti-inflammatory IL-5 B.) was also significantly increased (* = 0.05) whereas the other IL-13 was decreased D.). Additionally pro-inflammatory cytokines that were significantly increased include KC GRO (* = 0.018) E.) and TNFα (* = 0.019) F.).

Figure 2. Visceral lymph node immune cell number and frequency

A.) Total visceral lymph node viable cells number - HFD significantly increased total number of immunes cells within the visceral lymph node compared with CHOW (* = 0.02). B.) Percent frequencies (%) of individual immune cell populations – CD4⁺FoxP3⁺ cells (regulatory T) were the only immune cells to be significantly decreased in HFD fed mice compared with CHOW (* = 0.039). The inset represents scatter plots of the significant population enclosed within the circle. C.) Viable immune cells for distinct populations - F4/80⁺CD11b⁺macrophages (* = 0.022), CD11b⁺CD11c⁺ dendritic cells (* = 0.017), CD3⁺CD4⁺helper T cells (* = 0.024), CD3⁺ pan T cell (* = 0.02), and B220⁺ B cells (* = 0.002) were increased in the visceral lymph node of HFD fed mice compared with CHOW. CD4⁺FoxP3⁺ regulatory T cells, however, were decreased in HFD mice (** = 0.011)

Figure 3. Immune cell frequency (%) for specific populations within the ileum

A.) CD4⁺FoxP3⁺ regulatory T (* = 0.011), NKG2D⁺ (* = 0.002) and NKG2D⁺CD4⁺ (* = 0.014) immune cells were significantly decreased in HFD fed mice compared with CHOW, whereas CD11c⁺MHCII⁺dendritic cells (* = 0.014) were significantly increased. B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. Circles indicate double positive populations while the square represents all cells positive for a single marker, NKG2D.

Figure 4. Immune cell frequency (%) for specific populations within the jejunum

A.) CD4⁺FoxP3⁺ regulatory T (* = 0.015), NKG2D⁺CD4⁺ (* = 0.034), and CD3⁺CD4⁺ helper T cells (* = 0.011) were significantly decreased in HFD fed mice compared with CHOW, whereas CD11c⁺MHCII⁺dendritic cells (* = 0.008) were significantly increased. B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. All populations are double positive and indicated by circles.

Figure 5. Immune cell frequency (%) for specific populations within the Peyer’s patches collected along the Ileum and jejunum

A.) Significant decreases occurred in CD4⁺FoxP3⁺ regulatory T (* = 0.011), NKG2D⁺ (*= 0.002), NKG2D⁺CD4⁺ (p= 0.014) and CD11b⁺Gr1⁺ Myeloid suppressor (* = 0.042) immune cells of HFD mice compared with CHOW. The frequency of CD11b⁺MHCII⁺ dendritic cells, however, were significantly increased in HFD mice (* = 0.005). B.) Representative scatter plots of immune cell frequency for populations significantly altered by HFD. The circles indicate double positive populations while the square indicates all cells positive for a single marker NKG2D.