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Emily C. Graff, DVM, Dipl. ACVP (clinical pathology)

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1 Emily C. Graff, DVM, Dipl. ACVP (clinical pathology)
Identification and Characterization of Adipose Tissue Macrophage Populations in Cats During Development of Obesity Emily C. Graff, DVM, Dipl. ACVP (clinical pathology)

2 U.S. Obesity Trends During the past 20 years, there has been a dramatic increase in obesity in the United States and rates remain high. In 2010, no state had a prevalence of obesity less than 20%. Thirty-six states had a prevalence of 25% or more. Obesity is a form of adipose tissue dysregulation and a serious endocrine disorder.

3 Obesity = Adipose Tissue Dysregulation
Adipose tissue is a complex endocrine organ comprised of numerous tissue types. Normal adipose tissue is made up of adipocytes, pre-adipocytes, macrophages, ECM and blood vessels. In obesity there is dramatic remodeling of many of the components of normal adipose anatomy. The concept of adipose tissue remodeling refers to the turnover of cells within adipose tissue and the renovation of the ECM in response to requirements for growth and expansion, changes in hormones, aging or disease. There is not only tissue and organ dysregulation with obesity, but also cellular dysfunction within each adipocyte. Many of these changes alter adipokine production. Adipokines are proteins and signaling molecules produced specifically by the adipocytes. The classic change described in obese adipocytes is an increase in Leptin (an inflammatory adipokine) and a decrease in Adiponectin (an anti-inflammatory adipokine) The change that we have been focusing on currently in our lab is the Chronic Low Grade Inflammation that is associated with not only the adipose tissue. Adipose tissue- cytology Radin et al. Vet Clin Pathol 2009 Adipose tissue remodeling = altered adipokine regulation and chronic low grade inflammation

4 Macrophages (MΦ) & Crown-Like Structures
Weisberg et al. J Clin Invest. 2003 Cinti et al Journal of Lipid Research, 2005 “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) While macrophages are present in normal adipose tissue and participate in normal remodeling there is a change in macrophage behavior and phenotype in obesity. Crown like structures which were first described in 2003, consist of macrophages surrounding dead adipocytes Molecules that are broken down from dying adipose tissue activate Toll-like receptors (specifically FFA with TLR-4) activating pro-inflammatory cytokine and chemokine response. High fat diet (>60% lard) for over16 weeks result in nearly complete remodeling of epididymal fat depot of mice. With time the macrophages phagocytose the lipid and other cellular constituents and can assume the appearance of lipid laden cells. Whether the macrophage responds to or directly contributes to the adipocyte death is not completely clear; However, it is clear that inflammation appears to increase in the adipose tissue as adiposity increases.

5 Macrophage Polarity M1 M2
In the tissues, macrophages are stimulated by the adipocytes to differentiate into various phenotypes. In lean individuals macrophages are polarized to the M2 state by IL-4 and IL-13. M2 macrophages up-regulate scavenger receptors and arginase-1 (reduces the iNOS reaction) as well as secreting the anti-inflammatory cytokine IL-10. In obese states macrophages are polarized to the M1 state by interferon γ and other inducers of inducers of TNF. In turn M1 macrophages produce pro-inflammatory cytokines TNFα, IL-6 and IL-12 resulting increased ROS, and nitrogen intermediates. M1 M2

6 Obesity-Associated Adipose Tissue Inflammation
With increased obesity adipose tissue macrophages are polarized from anti-inflammatory macrophages (M2) to predominance of inflammatory type macrophages (M1). Both the number of M1 macrophages and M1:M2 ratio are associated with systemic inflammation and induction of insulin resistance. These results represent findings in various rodent models. There is some concern that this may not completely reflect what is seen in humans Recent data suggest that there may be a distinct difference between rodent and human macrophage infiltration in adipose tissue. This difference was noted not only in number, but also polarity. In addition, mouse obesity models often represent acute changes in weight (weeks on a specifically enriched HFD) rather than chronic changes associated with a sedentary lifestyle and increased but varied caloric intake. These potential differences highlight the need for a good intermediate model of obesity. Ouchi et al Nature Reviews immunology. Feb. 2011 M2 > M1 M1 > M2 INFLAMMATION

7 Companion Animal Obesity
Like obesity in people, companion animal obesity has become a significant epidemic. Obese horses are prone to development of lamminits and obesity associated shortened life-span has been reported in Labrador retrievers. Since the 1970’s obesity in cats has risen to ~35% with a concurrent rise in type 2 diabetes. In cats obesity is also associated with an increased risk of interstitial cystitis, osteoarthritis and certain cancers (specifically mammary cancer and squamous cell carcinoma) The cat has also been proposed as a model for human nutrition and disease

8 The Cat as a Model for Human Obesity, Nutrition and Diabetes
Similarities: Nutritional disorder Risk factor for T2DM Lifestyle Pathogenesis Amyloid deposition β-cell loss GLUT-4 expression Differences: Obligate carnivores Do not develop atherosclerosis or hypertension Like people, obesity is the most common nutritional disorder of cats and is a risk factor for diabetes. Thus, The cat has been implicated as a naturally occurring animal model of type 2 DM and a model of human obesity and nutritional disorders. As a companion animal it mimics the lifestyle of its human counterparts with excessive caloric intake paired with a sedentary lifestyle. Similar to developments in obese people, obese cats show peripheral tissue insulin resistance and demonstrate glucose intolerance. These clinical findings represent a similar pathogenesis between cats and people with obesity being directly associated with increased secretion of islet amyloid polypeptide, the formation of islet amyloid and the progressive loss of Beta-cell mass. In addition, cats have similar changes in GLUT-4 transporter expression as seen in obese humans There are also some significant differences between cats and people most notably, cats are considered obligate carnivores that persistently undergo gluconeogenesis and lack expression of hepatic glucokinase. Clinically the most unique differences between cats and humans is the lack of clinical development of atherosclerosis and hypertension which are often the main causes of mortality in people with metabolic syndrome. Does this make cats and inappropriate model for obesity, or does it make them an important comparative species? In human medicine, metabolic syndrome and obesity are associated with chronic low grade inflammation. O’brien Molecular and Cellular Endocrinology 2002

9 Do Obese Cats Develop a Systemic Inflammatory Response? – Possibly No!
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-α People: Weight gain is associated with marked adipokine dysregulation and significant increases in inflammatory cytokines Do cats develop a systemic inflammatory response associated with Obesity? Since 2012 there have been a few of papers that by a variety of different methods evaluate circulating inflammatory biomarkers. A paper from the lab of Alexander German evaluated weight-loss from 37 client owned cats. In this study the authors noted no change in the major acute phase proteins SAA or haptoglobin in cats. This is in contrast to what is reported in people who gain weight. In February of 2013, Dr. Hoenig published results from a longitudinal study of a feline obesity model where she was able to achieve 100% weight gain within one year. As the cats became obese they noted significant changes in the serum adipokine profiles, but no changes in serum IL-1, IL-6 or TNF-alpha. This is in stark contrast to finding in both obese children and adults who exhibit marked changes in both adipokines and circulating inflammatory cytokines.

10 Do Obese Cats Develop a Systemic Inflammatory Response? – Possibly Yes!
Miller et al. Journal of Nutrition 1998 Tumor Necrosis Factor-α 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 In contrast to the previous papers, there are others who suggest that inflammation at least at the level of the tissue is present in obese cats. As early as 1998, a paper by Miller et al noted an almost 10 fold increase in TNF-alpha protein in the adipose tissue of obese cats compared to lean. These findings are consistent with what is seen in human and rodent models of obesity. In May of this year, Van de Velde et al suggested that cats may still be an excellent model for human obesity and demonstrated that obese cats have increased adipocyte cell size, altered adipokine gene expression and increased pro-inflammatory cytokines and chemokines in tissues. Together these findings suggest that cats likely develop adipose tissue inflammation associated with obesity, but unlike people, they do not develop a strong systemic inflammatory response.

11 Identification of Depot Specific Inflammation Associated with Feline Obesity
In addition, Van de Velde evaluated various adipose tissue depots for changes in adipocyte cell size and infiltration of inflammatory cells. Using immunohistochemistry they evaluated the number of t-lymphocytes, b-lymphocytes and macrophages in the various adipose tissue depots in populations of lean and obese cats. Van de Velde noted that obesity associated changes are more predominant in the subcutaneous tissue of cats.

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 It appears that even if there is no clear evidence of systemic inflammation, tissue inflammation is occurring. But what is actually contributing to the adipose tissue inflammation? Is it associated with the adipocytes directly? What is the role of the adipose tissue macrophages and other infiltrating and resident immune cells? If tissue inflammation is present, what if any, are the systemic inflammatory markers associated with obesity in cats? There do not appear to be consistent serum biomarkers of inflammation, but what about cellular markers such as monocytes?

13 Initial Objective – Preliminary Data
Identify adipose tissue macrophages (ATM) and crown-like structures (CLS) in feline adipose tissue Two methods: Immunohistochemistry of formalin fixed adipose tissue Isolation of the stromal vascular fraction (SVF) followed by flow cytometric analysis Before we could address these questions, we needed to determine if adipose tissue macrophages are present in cats and develop methods to evaluate the adipose tissue based on methods that are currently used in human and rodent models. We approached this through two separate methods. We collected subcutaneous adipose tissue from lean cats. Part of the fat was placed in formalin and IHC was performed utilizing general macrophage. With the remainder of the adipose tissue we wanted to isolate the stromal vascular fraction and identify various macrophage populations using flow cytometric analysis and FACS.

14 Subcutaneous Adipose Tissue
H&E 200X CD18 200X Lysozyme 200X H&E stains and IHC markers including CD18, Lysozyme and Aplha-1 Atitrypsin all demonstrated similar findings with rare CLS and rare macrophages present in lean cats. Alpha-1 Antitrypsin

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 In order to preform flow cytometric analysis 4 grams of SQ adipose tissue was digested in a collagenase solution. The SVF was isolated using filtration and differential centrifugation. Isolated cells were then evaluated on the Beckman Coulter MoFlo flow cytometer for identification and sorting of cells. The isolated population of macrophages was stained with propidium idodide to determine cell viability and cytospin preparations of the cells were prepared to confirm cell identity.

16 Flow Cytometry & Cytospin Results
Here are two representative tracing from FACS of the stromal vascular fraction. The two gated populations represent adipose tissue macrophages and the larger lipid laden cells. The upper panels show viability based on lack of PI stain uptake and Cytospin preparations identified a fairly homogenous population of mononuclear cells.

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 Based on the findings of our preliminary data we are able to conclude that lean cats do contain rare adipose tissue macrophages and we are able to demonstrate a measurable concentration of viable adipose tissue macrophage populations in the SVF via flow cytometric analysis.

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 This leads us to our current goal and the purpose of this project: To identify and characterize feline adipose tissue macrophage populations during the development of obesity. We believe that with the development of obesity there will be increased number of macrophages, an increased proportion of M1 type macrophages and increased adipose tissue inflammation. In addition we expect to confirm the presence of tissue and systemic adipokine dysregulation and possible identify concurrent systemic inflammation.

19 Specific Aims Specific Aim 1: Specific Aim 2: Specific Aim 3:
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-α and IL-6 in cats as changes in adipose inflammation occur

20 Feline Obesity Model Each cat will serve as their own control
10% 20% 30% 40% 50% 60% 70% Baseline Lean Midpoint Overweight End of Study Obese 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) In order to address these three specific aims we have acquired a colony of 12 male cats. In this study each cat will serve as their own control cats will be fed the same diet throughout the entire study. Therefore, any weight gain will be due to increased caloric intake. Obesity and adipose tissue deposition will be evaluated via CT scans performed at the baseline and conclusion of the study. In addition adipose tissue samples will be also be collected at the baseline and at the end of the study from the inguinal subcutaneous fat pad and from the falciform visceral fat pad. At each time point during the weight gain progression of a 10%, 20% and 30% so of weight gain serum and whole blood will also be collected to evaluate circulating inflammation as well as determine a HOM-IR to asses insulin sensitivity. cat images from:

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

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 - M1 Alternative - M2 General Surface marker CD11c CD163 CD18 Cytokines expressed TNFα, IL-6, IL-12 IL-10, IL-1 NA Induced by LPS (th1 cytokines) IL-10 (th2 cytokines)

23 Cat Colony - Growth and Weight
In March of this year, we acquired 12, 6-month old intact male cats. Since their arrival the animals have been neutered and placed on an appropriate diet to maintain weight for their growth. All animals are now within one kg. There is only a slight variation in individual cat weights between each week Baseline data will be collected once the cats reach a year of age this August Neutering and recovery

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

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

26 Thank you 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 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.

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