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Identification and Characterization of Adipose Tissue Macrophage Populations in Cats During Development of Obesity Emily C. Graff, DVM, Dipl. ACVP (clinical pathology) Slide 2 U.S. Obesity Trends Slide 3 Obesity = Adipose Tissue Dysregulation Radin et al. Vet Clin Pathol 2009 Adipose tissue remodeling = altered adipokine regulation and chronic low grade inflammation Adipose tissue- cytology Slide 4 Macrophages (M) & Crown-Like Structures Cinti et al Journal of Lipid Research, 2005Weisberg et al. J Clin Invest. 2003 Adipose tissue macrophage numbers increase in obesity and participate in inflammatory pathways that are activated in adipose tissues of obese individuals. Weisberg et al. JCI 112 (2003) Slide 5 Macrophage Polarity Slide 6 Obesity-Associated Adipose Tissue Inflammation M2 > M1M1 > M2 Ouchi et al Nature Reviews immunology. Feb. 2011 Slide 7 Companion Animal Obesity Slide 8 The Cat as a Model for Human Obesity, Nutrition and Diabetes Similarities: Nutritional disorder Risk factor for T2DM Lifestyle Pathogenesis Amyloid deposition -cell loss GLUT-4 expression Differences: Obligate carnivores Do not develop atherosclerosis or hypertension Obrien Molecular and Cellular Endocrinology 2002 Slide 9 Tvarijonaviciute et al. Domestic Animal Endocrinology 2012 Study: Weight-loss in 37 client owned cats Cats: No change in serum amyloid A or serum haptaglobin People: Weight gain is associated with increased C-reactive protein Hoenig et al. Obesity 2013 Study: Longitudinal feline obesity model with 100% weight gain over 1 year Cats: Significant alterations in serum adipokines, but no significant increase in serum concentrations of IL-1, IL-6 or TNF- People: Weight gain is associated with marked adipokine dysregulation and significant increases in inflammatory cytokines Do Obese Cats Develop a Systemic Inflammatory Response? Possibly No! Slide 10 Miller et al. Journal of Nutrition 1998 Tumor Necrosis Factor- Levels in Adipose Tissue of Lean and Obese Cats Van de Velde et al. British Journal of Nutrition 2013 The cat as a model for human obesity: insights into depot- specific inflammation associated with feline obesity Increased adipocyte cell size, altered adipokine expression and increased pro-inflammtory cytokines in tissue Do Obese Cats Develop a Systemic Inflammatory Response? Possibly Yes! Slide 11 Identification of Depot Specific Inflammation Associated with Feline Obesity Slide 12 Gap in Knowledge What is actually contributing to the adipose tissue inflammation? Adipocytes Adipose tissue macrophages What, if any, are the systemic inflammatory patterns associated with obesity in cats? Biomarkers of inflammation (cytokines) Cellular markers of inflammation Slide 13 Initial Objective Preliminary Data Identify adipose tissue macrophages (ATM) and crown-like structures (CLS) in feline adipose tissue Two methods: 1.Immunohistochemistry of formalin fixed adipose tissue 2.Isolation of the stromal vascular fraction (SVF) followed by flow cytometric analysis Slide 14 Subcutaneous Adipose Tissue H&E 200XCD18 200X Lysozyme 200X Alpha-1 Antitrypsin Slide 15 Flow Cytometric Evaluation of SVF 4 g adipose tissue digested in collagenase Isolation of SVF via filtration and differential centrifugation Accuri C6 flow cytometer and Beckman Coulter MoFlo XDP for FACS to identify and sort cell populations PI stain to determine cell viability Prepared cytospin preparation of the cells for cytologic identification Slide 16 Flow Cytometry & Cytospin Results R1R2 Slide 17 Preliminary Data Results Immunohistochemistry: Lean cats do contain rare adipose tissue macrophages Isolation of SVF and flow cytometry: Concentration of adipose tissue macrophages Viable macrophages can be isolated from feline adipose tissue Slide 18 Identify and characterize feline macrophage populations in adipose tissue during the development of obesity Central hypothesis: During the development of obesity cats will demonstrate increased numbers of macrophages increased proportion of M1 type macrophages increased adipose tissue inflammation adipokine dysregulation systemic inflammation Slide 19 Specific Aims Specific Aim 1: Identify and enumerate macrophages in lean and obese feline adipose tissue Specific Aim 2: Confirm phenotype of the populations based on surface receptor expression and cytokine mRNA expression Specific Aim 3: Determine circulating feline adipokine profiles, specifically adiponectin, leptin, serum amyloid-A, TNF- and IL-6 in cats as changes in adipose inflammation occur Slide 20 Feline Obesity Model Baseline Lean Midpoint Overweight End of Study Obese 10% 50%20%60% 40% cat images from: http://www.healthyweightpet.com/uk/cathttp://www.healthyweightpet.com/uk/cat 30%70% Each cat will serve as their own control Cats will be fed same diet throughout the study CT scan to evaluate adipose tissue deposition (baseline and end of study) Samples collected Adipose tissue Visceral and subcutaneous (baseline and end of study) Serum ( at each 10% increase in weight gain) Slide 21 Sample Evaluation Perform IHC evaluation Adipose tissue macrophages Crown-like-structures Isolate SVF & sort cell populations Identify and quantify cell populations Evaluate systemic markers of Inflammation Adipokines Inflammatory cytokines Acute phase proteins Evaluate insulin sensitivity Slide 22 Expand Evaluation of SVF Evaluate M1 and M2 macrophage populations Apply specific macrophage surface markers RT-PCR for M1 and M2 cytokine profiles Classical - M1Alternative - M2General Surface markerCD11cCD163CD18 Cytokines expressed TNF, IL-6, IL-12IL-10, IL-1NA Induced byLPS (th1 cytokines)IL-10 (th2 cytokines)NA Slide 23 Cat Colony - Growth and Weight Neutering and recovery Slide 24 Expected Outcomes As obesity develops we expect to find: Increased numbers of adipose macrophages Increased percentage of M1 type macrophages Increased adipose tissue inflammation Altered systemic and tissue adipokine regulation Increased circulating inflammatory markers Slide 25 Summary Obesity and obesity-induced peripheral insulin resistance are epidemic in both people and companion animals Adipokine dysregulation and chronic inflammation are features of obesity-induced peripheral insulin resistance in humans Cats are naturally occurring model of human obesity and T2DM Further work is needed to evaluate feline obesity- associated inflammation Slide 26 Thank you Dr. Emily Graff was partially supported by the Charles and Sharron Capen Fellowship in Veterinary Pathology organized by the American College of Veterinary Pathologists and Society of Toxicologic Pathology Coalition for Veterinary Pathology Fellows. Dr. Robert L. Judd Dr. Desiree Wanders Olga Norris Ally Emmert Nathan Gray Dr. Elizabeth G. Welles Dr. Beth Spangler Dr. Robert Kemppainen Dr. Ellen Behrend Dr. Ray Dillon Dr. D.M. Tillson Sharron Barney Dr. R.C. Bird Ali Bird Dr. Pat Rynders Dr. Bettina Schemera Dr. Bob Cole