Manindra SinghSigma Xi Student Research Showcase, 2013
Obesity and InflammationOverview• 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
Obesity - key facts and worldwide concern• 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
What is obesity? Obesity is defined as an excess accumulation of adipose tissue (fat) when calorific intake exceeds energy expenditure Some genetic factorsExcess calorific intake Factors contributing to obesity Inadequate physical activity
Obesity associated health risks and complicationsObesity is a metabolic disorder associated with high risk of cardiovascular diseases, insulin resistance,type II diabetes and some cancersA low-grade systemic inflammation associated with obesity plays a role in pathogenesis
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 pathogenesisMacrophage recruitment and activation is the major cause of inflammation in obesityObese fat cells secrete pro-inflammatory adipokines , which induce classically activated M1macrophages 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?• Metabolic dysfunction in obesity poses a challenge to health and cause increasing no. of deaths worldwide annually• 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 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 goalsLong 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 OutlineCulture mouse monocyte/ macrophage cell line in a suitable growth medium withoutserum Treatment with adipokines and growth hormone for 72hr Collect the cells for the staining with fluorescent antibodies for surface markers 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 resultsThe 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 adiposetissue may cause activation of macrophages by up-regulating activation proteins and antigenpresenting capabilities.Also, the results show the relevance of adipokine and growth hormone treatment in macrophagephenotype 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
Manindra SinghSigma Xi Student Research Showcase, 2013