Obesity and Inflammation

Manindra SinghSigma Xi Student Research Showcase, 2013

Obesity and Inflammation

Overview

• Obesity - key facts and worldwide concern

• What is obesity?

• Obesity associated health risks and complications

• Inflammatory mechanisms for pathogenesis in obesity

• Adipokines in Obesity

• Why study obesity-associated inflammation?

• My research goals

• Preliminary results

• Conclusion

• Future studies and expected outcomes

• Worldwide obesity has doubled since 1980• In 2008, 35% of adults (>20 years) were overweight, and 11% were obese• In 2011, more than 40 million children below 5 years of age were obese• More than 10% of world’s adult population was obese in 2008

Obesity - key facts and worldwide concern

What is obesity?

Obesity is defined as an excess accumulation of adipose tissue (fat) when calorific intake exceeds energy expenditure

Factors contributing to obesity

Some genetic factorsExcess calorific intake

Inadequate physical activity

Obesity associated health risks and complications

A low-grade systemic inflammation associated with obesity plays a role in pathogenesis

Obesity is a metabolic disorder associated with high risk of cardiovascular diseases, insulin resistance, type II diabetes and some cancers

Inflammation in obese adipose tissue

• Obese adipose tissue secretes chemotactic factors which can attract immune cells from the circulation

• Infiltration by immune cells such as macrophages and their activation in adipose results in inflammation

Pharmacology & Therapeutics 130 (2011) 226–238

Inflammatory mechanisms in obesity-related pathogenesis

Macrophage recruitment and activation is the major cause of inflammation in obesity

Obese fat cells secrete pro-inflammatory adipokines , which induce classically activated M1 macrophages that release inflammatory molecules

Classically activated M1 macrophages cause chronic inflammation in obesity

Adipokines in Obesity

• Adipose tissue regulates metabolism and energy homeostasis by releasing factors known as adipokines

• Obesity decreases anti-inflammatory adipokines and increases proinflammatory adipokine secretion which

results in metabolic dysfunction and pathogenesis

• Adipokines can cross-talk with macrophage for their recruitment and activation in obese fat tissue

Why study obesity-associated inflammation?

• Low grade, chronic inflammation in obesity caused due to immunoinfiltration, leads to pathogenesis

• Inflammation reprograms the endocrine profile of adipose tissue to secrete factors leading to the development of insulin resistance, cardiovascular diseases, degenerative illness and cancers

• Metabolic dysfunction in obesity poses a challenge to health and cause increasing no. of deaths worldwide annually

Keeping above points in view, studying the underlying mechanisms for inflammation in obesity is important to understand the regulation of inflammatory pathways and devise novel therapeutics for the control of obesity-associated disease development

My research goals

Long term goals of my research includes:

• Understanding the signaling pathways and property of macrophage cell population in relation to adipokines

• Expression analysis of inflammatory proteins in adipose-derived macrophages

• The regulation of switch between inflammatory (M1) and anti-inflammatory (M2) macrophages under the effect of adipokines and growth factors in adipose

• Analysis of macrophage biology and activation under the effect of adipokines

Ongoing experiments:

Effect of adipokines and growth factors such as growth hormone (GH) for the surface expression of activation markers in cultured macrophage cell line

Experiment Outline

Collect the cells for the staining with fluorescent antibodies for surface markers

Culture mouse monocyte/ macrophage cell line in a suitable growth medium without serum

Treatment with adipokines and growth hormone for 72hr

Flow cytometry analysis for the expression of different surface markers of activation

RAW264.7 (Murine leukemic monocyte macrophage cell line) cells at 48h

Preliminary resultsFlow cytometry analysis for surface markers

A B

C D

Figure 1. FACS analysis of RAW264.7 cells after the treatment. Cells were collected and stained using fluorescence tag containing antibodies specific to different marker proteins. Gating strategy was used to isolate the CD11b +, F4/80+ macrophage cell population from the CD45+ leukocyte fraction. Cells treated with (A) medium only, (B) growth hormone, GH, (C) leptin, and (D) GH+leptin.

Flow cytometry analysis for surface markers

Figure 2. FACS analysis for surface markers expression in RAW 264.7 cells after the treatment with growth hormone (GH) at 100ng/ml for 72 hr. The surface expression was tested for (A) F4/80, (B) CD80, (C) MHCII and CD206. Red- isotype control, Blue – cells treated with medium only and Orange – GH treated.

Flow cytometry analysis for surface markers

Figure 3. FACS analysis for surface markers expression in RAW 264.7 cells after the treatment with adipokine Leptin, at 100ng/ml for 72 hr. The surface expression was tested for (A) F4/80, (B) CD80, (C) MHCII and CD206. Red- isotype control, Blue – cells treated with medium only and Orange – Leptin treated.

Flow cytometry analysis for surface markers

Figure 4. FACS analysis for surface markers expression in RAW 264.7 cells after the combined treatment with growth hormone (GH) and leptin (100ng/ml each) for 72 hr. The surface expression was tested for (A) F4/80, (B) CD80, (C) MHCII and CD206. Red- isotype control, Blue – cells treated with medium only and Orange – [Leptin + GH]treated.

Conclusions based on preliminary results

The effect of adipokine, leptin and growth hormone (GH) was tested on cultured mouse macrophage cell line.

• Flow cytometry analysis shows the high increase in expression of macrophage marker F4/80 and activation markers CD80 and MHCII in the cultured cells upon their treatment with both leptin and GH, compared to the medium only.

• Both GH and leptin treatment shows an enhanced expression of CD206 on the cell surface.CD206 is a marker for alternatively activated anti-inflammatory M2 macrophages.

The preliminary results so far go along with the idea that adipokines and growth factors in adipose tissue may cause activation of macrophages by up-regulating activation proteins and antigen presenting capabilities.

Also, the results show the relevance of adipokine and growth hormone treatment in macrophage phenotype determination.

Future studies and expected outcomes

• Results from cell culture studies will form the basis of in vivo analysis in mice models

• The study cytokine profile of macrophages in relevance to adipokines will help hypothesize the effector mechanism of these cells for inflammation

• Expression analysis of induced proteins at the transcript level in response to treatment would help understand the potential signal pathways of inflammation

• The final results will lead to the idea of novel therapeutic measures for the control of macrophage mediated inflammation

Thank You

Manindra SinghSigma Xi Student Research Showcase, 2013