Modulation of the IL-6 System and STAT-3 Activation in Lymphocytes of Lean and Obese Women

Modulation of the IL-6 System and STAT-3 Activation in Lymphocytes of Lean and Obese Women BY MARGARET M. SULLIVAN B.A., St. Mary s College, 1992 B.S., University of Illinois, Chicago, 1995 THESIS Submitted as partial fulfillment of the requirements for the degree of Master of Science in Human Nutrition in the Graduate College of the University of Illinois at Chicago, 2012 Chicago, Illinois Defense Committee: Giamila Fantuzzi, Chair and Advisor Carol Braunschweig Tracy Baynard

TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION... 1 A. Acute Inflammation... 2 B. Chronic Inflammation... 3 C. Chronic Inflammation in Obese Adipose Tissue... 4 D. The Interleukin-6 System... 7 E. The Interleukin-6 - Signal Transducer and Activator of Transcription -3 Pathway... 8 F. The Interleukin-6 System, Inflammation, and Obesity... 11 G. Leptin... 16 H. Adiponectin... 19 I. C-reactive Protein... 21 J. Inflammation, Obesity, and Cancer: Is Signal Transducer and Activator of Transcription -3 the Link?... 22 II. AIMS AND HYPOTHESES A. Rationale... 28 B. Specific Aims and Hypotheses... 28 III. METHODS A. Subjects... 30 B. Sample Collection... 30 C. Blood Analysis... 30 1. Enzyme-linked Immunosorbent Assay... 30 2. IL-6 Stimulation and Evaluation of Active Signal Transducer and Activator of Transcription -3-positive Cells... 31 D. Statistical Methods... 31 IV. RESULTS A. Subject Summary Characteristics... 34 B. Body Mass Index and the Interleukin-6 System... 37 C. Interleukin-6 System, Adipokines, and C-reactive Protein... 38 D. Percentage of Active Signal Transducer and Activator of Transcription - 3-positive Lymphocytes with and without Interleukin-6 Stimulation... 39 V. DISCUSSION... 43 VI. CONCLUSIONS... 51 CITED LITERATURE... 52 ii

LIST OF TABLES TABLE PAGE I. SUBJECT CHARACTERISTICS SUMMARY STATISTICS... 34 II. RESULTS WITHIN BMI GROUPS: IL-6, sil-6r, SGP130, LEPTIN, APN, AND CRP... 35 III. RESULTS WITHIN BMI GROUPS: SUBJECT % PSTAT-3 LYMPHOCYTES - WITH & WITHOUT IL-6 STIMULATION... 36 IV. SUBJECT ETHNICITY TOTALS WITHIN BMI GROUPS... 36 V. CORRELATION OF IL-6 SYSTEM COMPONENTS, LEPTIN, APN AND CRP WITH BMI... 37 VI. CORRELATION AMONGST IL-6 SYSTEM COMPONENTS, ADIPOKINES, AND CRP... 38 VII. CORRELATIONS OF % PSTAT-3 POSITIVE LYMPHOCYTES WITHOUT IL-6 STIMULATION... 40 VIII. COMPARISON OF % PSTAT-3-POSITIVE LYMPHOCYTES BETWEEN DIFFERENT IL-6 STIMULATION LEVELS... 41 IX. CORRELATION OF % PSTAT-3-POSITIVE LYMPHOCYTES STIMULATED WITH IL-6 WITH BMI... 41 iii

LIST OF FIGURES FIGURE PAGE 1. Interleukin-6: origins, target cells, and effectors... 8 2. IL-6 system activation... 9 iv

LIST OF ABBREVIATIONS ANOVA APN BMI Analysis of Variance Adiponectin Body Mass Index CD4 Cluster of differentiation 4 CD8 Cluster of differentiation 8 CRP ELISA HRT IFN-γ IL-6 IL-6R JAK MS NFƙB NK OB-R PCOS C-reactive protein Enzyme-linked immunosorbent assay Hormone replacement therapy Interferon gamma Interleukin-6 Interleukin-6 receptor Janus kinase Metabolic syndrome Nuclear factor kappab Natural Killer Leptin receptor Polycystic ovarian syndrome pstat-3 Active signal transducer and activator of transcription -3 sgp130 Soluble glycoprotein 130 sil-6r Soluble Interleukin-6 receptor SOCS-3 Suppressor of cytokine signals 3 v

LIST OF ABBREVIATIONS STAT Signal transducer and activator of transcription STAT-3 Signal transducer and activator of transcription -3 TH-1 TH-17 TNF-α VAT T helper 1 cells T helper 17 cells Tumor necrosis factor-alpha Visceral adipose tissue vi

SUMMARY In obesity, chronic inflammation develops in response to expanded adipose tissue. Chronic inflammation is a primary link in the etiology of many obesity-associated pathologies, including cancer. (1, 2, 3, 4, 5, 6) This relationship is particularly worrisome, given the increasing international obesity epidemic. (7, 8, 9) Inflammation becomes chronic in obesity as adipose tissue expands in response to nutrient excess. Obesity s chronic inflammatory environment is characterized by changes in production of a host of mediators produced by immune cells and adipocytes. (10, 11, 12, 13, 14) Among these are interleukin-6, the adipokines leptin and adiponectin, and signal transducer and activator of transcription -3, a pleiotropic transcription factor whose activators include interleukin-6 and leptin. The purpose of this study was to examine whether obesity is associated with alterations in constituents of the interleukin-6 system namely, interleukin-6, soluble interleukin-6 receptor, and soluble glycoprotein 130 and to investigate the association between obesity and the presence of active signal transducer and activator of transcription -3 in peripheral lymphocytes. Plasma from forty women across a body mass index spectrum were evaluated for IL-6 system constituents, adipokines, and C- reactive protein levels. Heparinized blood was stimulated with increasing amounts of interleukin-6 (0-10 ng/ml) and the presence of active signal transducer and activator of transcription -3 in lymphocytes was evaluated. Synopses of our three study aims and their results follow. Our first aim was to evaluate correlation of circulating levels of interleukin-6, soluble interleukin-6 receptor, and soluble glycoprotein 130 with increasing body mass vii

SUMMARY (continued) index, in order to test our first hypothesis that plasma levels of interleukin-6, soluble interleukin-6 receptor, and soluble glycoprotein 130 would increase as body mass index increased. Plasma levels of interleukin-6 did indeed increase as body mass index increased as predicted, but soluble interleukin-6 receptor and soluble glycoprotein 130 did not increase as body mass index increased, in disagreement with our hypothesis. Our second aim was to evaluate correlations of circulating levels of leptin, adiponectin, and C-reactive protein with levels of interleukin-6 system components in order to test our second hypothesis that leptin and C-reactive protein levels would increase as interleukin-6 system components increased, and that adiponectin would decrease as interleukin-6 system levels increased. As hypothesized, plasma levels of both leptin and C-reactive protein levels did increase as interleukin-6 increased; however, neither leptin nor C-reactive protein correlated with soluble glycoprotein 130 and soluble interleukin-6 receptor as we had predicted. Additionally, as opposed to what we hypothesized, adiponectin did not correlate with any interleukin-6 system components. Our third aim to was to compare the percentage of active signal transducer and activator of transcription -3-positive lymphocytes in subjects along a body mass index spectrum, with and without interleukin-6 stimulation, to test the hypothesis that the percentage of active signal transducer and activator of transcription -3 positive lymphocytes (without added interleukin-6) would increase as plasma interleukin-6 and body mass index increased, and that stimulation with interleukin-6 would result in relative increases of active signal transducer and activator of transcription -3-positive viii

SUMMARY (continued) cells. Our results did, as predicted, show that the percentage of active signal transducer and activator of transcription -3 positive lymphocytes without added interleukin-6 increased as plasma interleukin-6 and body mass index increased. Also, as we predicted, stimulation with interleukin-6 resulted in relative increases of active signal transducer and activator of transcription -3 positive cells, independent of body mass index increase. In sum, results of our investigation show that plasma interleukin-6 levels significantly increased with increasing body mass index. Increasing interleukin-6 levels correlated with both leptin and C-reactive protein. Also, the percentage of unstimulated and stimulated lymphocytes positive for active signal transducer and activator of transcription -3 progressively increased with increasing body mass index. These results suggest that constitutively active signal transducer and activator of transcription -3 via interleukin-6 and leptin may participate in determining the increased risk of pathology, including cancer, of obese individuals. ix

I. INTRODUCTION Obesity is now considered a global threat to health. Five hundred million people worldwide, or 1 out of every 10, is obese, a condition defined by the World Health Organization as having a body mass index (BMI) of 30kg/m2 or above. (8) In the United States alone, 68.8% of adults are overweight or obese and 35.7% are obese, resulting in an economic cost of approximately $270 billion per year due to medical care costs and economic productivity loss secondary to excess mortality and disability. (7, 9) Being obese contributes to increased risk of a myriad of other diseases as well. The links between obesity, diabetes, and cardiovascular diseases have long been recognized. (4, 5, 14, 15, 16, 17, 18, 19, 20) Additionally, epidemiological data suggest a significant association between increased BMI and an augmented frequency and poor prognosis of many cancers. (2, 6, 13, 21, 22, 23, 24, 27) Indeed, a meta-analysis review determined that individuals with a BMI > 40 were 52% (men) and 62% (women) more likely to die of cancer than individuals of normal weight. (13) Inflammation is one of the major mechanisms by which obesity increases cancer susceptibility. Chronic low grade inflammation in obesity is induced by enlarged and overabundant adipose tissue, producing pro-inflammatory factors that mediate and maintain an abnormal immune response to nutrient excess. Prolonged elevated levels of two such factors in obesity, interleukin-6 (IL-6) and signal transducer and activator of transcription -3 (STAT-3), may prove critical in linking obesity s chronic inflammation to cancer risk. Obesity is associated with increased levels of IL-6. How obesity may modulate components of the IL-6 system, how adipokines correlate with these components, 1

2 and lymphocyte STAT-3 response to IL-6 in the context of increasing BMI, has yet to be examined. In order to determine if obesity uniquely modulates these factors, plasma from women across a BMI spectrum were evaluated for IL-6 system constituents, adipokines, and presence of active STAT-3 in lymphocytes. A. Acute Inflammation Acute inflammation represents a critical component of the innate (non-specific) immune system, the aspect of immunity that responds in immediate but generic fashion during the first critical hours of an organism defense against infection or damaged self. Working in conjunction with the other parts of the innate immune response, namely anatomical barriers, the complement system, coagulation and neural factors, acute inflammation coordinates both first line defense and generation of the adaptive immune response. (28) It is initiated by specialized cells - resident macrophages, dendritic cells, histiocytes, Kupffer cells and mastocytes - present in all tissues, programmed to respond to harmful stimuli upon injury. (29, 30, 31) Recognition of pathogen-associated molecular patterns and damage-associated molecular patterns via pattern recognition receptors activates these specialized cells, which then initiate and promote a myriad of chemical responses that coordinate the local system of defense activity and attempted resolution. This vast array of molecules includes lipid and protein molecules (among which are prostaglandins and cytokines, respectively), chemoattractants (chemokines), cell adhesion proteins, and coagulation components. (31) These various molecules, along with components from the complement cascade, modulate the inflammatory response at the site of infection and beyond. Local blood

3 vessels dilate, allowing necessary proteins and cells to migrate to the site. Local endothelial cells concomitantly release cell adhesion proteins and chemokines that facilitate the migration of phagocytic leukocytes, at first neutrophils, to be followed by monocytes (circulatory macrophage precursors) and, finally, lymphocytes. Additionally, macrophages provide the first indicators to the system to convert its response from innate to adaptive immunity, which will address the specific pathogen directly in the future. Leukocytes continue to secrete cytokines to contain pathogen proliferation, coordinate injury resolution, and finally re-establish local and systemic homeostasis. (28) The resolution process is controlled by a range of tightly regulated biochemical and cellular mechanisms. (32, 33, 34) The acute inflammatory response is usually selflimiting and normally results in the return to tissue homeostasis. (32, 35, 36) As such, acute inflammation is the normal initial self-limiting response of the body to harmful stimuli, a finite process that ceases upon removal of the injurious agent. When acute inflammation becomes persistent or its resolution phase mechanisms dysregulated, it may evolve into chronic inflammation. B. Chronic Inflammation Chronic inflammation is an inflammatory abnormality that has been implicated as a causal, contributing, and aggravating factor in many diseases. (12, 36, 37, 38, 39, 40, 41) It primarily involves immune cells, and is characterized by a progressive shift in the type of cells (from primarily neutrophil to pro-inflammatory macrophages and lymphocytes) present at the site of inflammation, with simultaneous destruction and healing of the tissue from the inflammatory process. Onset, duration, mediators, and

4 outcomes negatively evolve into a perpetual state of potential harm, in contrast with the timely resolution to homeostasis normally found in acute inflammation. Abnormal inflammation contributes to autoimmune disorders including rheumatoid arthritis, multiple sclerosis, lupus, psoriasis, and inflammatory bowel disease. (28, 33, 34, 42, 43) It can also have etiological origins outside of the immune system. Chronic inflammatory processes have been identified in cancer, atherosclerosis, and ischemic heart disease as well as neurological disorders, including Parkinson s and Alzheimer s disease. (30, 37, 38, 39, 41, 44) What factors drive an organism to chronic inflammation remains a subject of ongoing investigation. Here we assess chronic inflammation in the context of obesity. C. Chronic Inflammation in Obese Adipose Tissue Adipose tissue is a loose connective tissue that may be viewed as a heterogeneous organ consisting of multiple cell types, whose main roles are to store energy, cushion, and insulate the body. Within adipose tissue is an adipocyte fraction, which contains lipid-laden adipocytes, and a stromal-vascular fraction, which includes pre-adipocytes, endothelial cells, macrophages and other immune cells. (2) Once thought to be inert, adipose tissue is now recognized as a metabolically active endocrine organ and a source of inflammatory modulators, producing and releasing a variety of pro-inflammatory and anti-inflammatory factors, including adipokines, cytokines and chemokines. (21, 45) Adipose tissue is subdivided mainly into the visceral and subcutaneous compartments. Visceral adipose tissue (VAT) surrounds internal organs and is located mainly beneath the abdominal wall, which encases the organs in

5 the abdominal cavity. (2) Excess VAT results in dysregulation of adipose tissue inflammatory factors, and has been linked to cardiovascular, metabolic, and inflammatory disease. (46, 48) Chronic inflammation in obesity is elicited by adipocytes as well as stromal and inflammatory cells in response to excess nutrients and energy. (2, 13, 49) The dynamic change occurring in the adipose tissue as a result of obesity, referred to as "adipose tissue remodeling," is characterized by adipocyte hypertrophy, followed by increased angiogenesis, immune cell infiltration, extracellular matrix overproduction, and increased production of pro-inflammatory factors, the end-result being low grade chronic inflammation. (12, 50) Adipocyte hypertrophy results in elevated production of several inflammatory mediators (13, 50, 51) that up-regulate pro-inflammatory mitogen-activated protein kinase pathways and reactive oxygen species production, promoting endoplasmic reticulum stress and adipokine dysregulation. (13, 50, 51) Increased expression of chemotactic factors, osteopontin, angiopoietin-like protein 2, and other mediators by such stressed adipocytes recruits additional monocytes to adipose tissue. (13, 49, 52, 53, 54) During such adipocyte-macrophage interaction, pro-inflammatory mediators such as saturated fatty acids (acting as danger-associated molecular patterns ligands for pattern recognition receptors) and interferon gamma (IFN-γ) promote the classically activated (pro-inflammatory) Type 1 macrophages over (anti-inflammatory) Type 2 macrophages. (12, 13, 49, 50, 55, 56, 57) Adaptive immune cells, including CD4 and CD8 T lymphocytes, also contribute in development and maintenance of inflamed adipose tissue in obesity. (50) In particular,

6 T helper 1 cells (TH-1) and T helper 17 (TH-17) cell lineages are primary inflammatory components present at elevated levels in obese adipose tissue. (58, 59, 60, 61, 62, 63, 64, 65, 66) Studies indicate that obesity selectively promotes expansion of TH-17 cells, inducing and exacerbating inflammation. (67) The contribution of CD4 lymphocytes to inflammation in obesity is not limited to their increased numbers. They also directly contribute to the dysregulation of other cells in the inflamed adipose tissue. Both rodent and human studies revealed proinflammatory T lymphocytes (mainly CD4 and to some extent CD8) present in obese VAT before the appearance of macrophages, indicating that T cells initiate (via the lymphocyte-derived TH-1-cytokine IFN-γ) activation of macrophage infiltration, and perpetuate continued recruitment of both cell types to the area. (60) Diet induced obesity in rodent models results in increased CD4 and CD8 cells, and macrophages, in VAT with a decrease in VAT T regulatory cells. (58) Lymphocyte-derived chemokines involved with T cell and macrophage recruitment are as well altered in inflamed adipose tissue. (61) Additionally, interleukin-17 inhibits adipocyte differentiation in human mesenchymal stem cells and promotes pro-inflammatory responses in adipocytes. (62, 63, 64, 65) Resulting ongoing accumulation of pro-inflammatory macrophages, adipocyte dysregulation, altered T cell activation, cell death, and the effects of numerous cytokines and chemokines characterize chronic inflammation in obesity. (4) Five of these components- the IL-6 system, leptin, adiponectin (APN), CRP, and the STAT-3 pathway and their relationships were examined in our study.

7 D. The Interleukin-6 System Interleukin-6 is a pleiotropic cytokine produced by both immune (including T cells, B cells, monocytes, macrophages,) and non-immune cells (including muscle cells, osteoblasts, fibroblasts, keratinocytes, endothelial cells, mesangial cells, adipocytes) in response to a variety of stimuli. (68, 69) It is, in turn, a pleiotropic effector on a variety of cells, including B and T lymphocytes, hematopoietic stem cells, hepatocytes, and adipocytes. Interleukin-6 works as an autocrine, paracrine, and hormonal communicator, depending on site of secretion and activation. In particular, typical interstitial IL-6 concentrations in adipose tissue are approximately 100 times higher than in plasma, denoting its critical autocrine and paracrine regulatory function in this tissue. (1).Upon release, IL-6 s biological roles include regulation of immune reactivity, the acute phase response, inflammation, angiogenesis, oncogenesis, and hematopoiesis. (68, 69) (Figure 1) Interleukin-6 is a member of the superfamily of IL-6 cytokines, whose common denominator is their interaction with the membrane-bound receptor subunit glycoprotein 130 (gp130), a trans-membrane protein that initiates signal transduction cascades following cytokine engagement. (68, 69) Membrane gp130 is ubiquitously expressed. (1, 68, 69) The pathway elicited with its stimulation is determined by the presence of additional receptor components specific to the individual IL-6 family member.

8 Figure 1. Interleukin-6: origins, target cells, and effectors. Interleukin-6 (IL-6) is a cytokine produced by T cells, endothelial cells, macrophages, fibroblasts, and other cells in response to a variety of stimuli. It, in turn, is a pleiotropic effector in a wide range of cells, serving as a vital communicator both within the immune system and between immune cells and other systems within the organism. It is a potent inducer of the acute-phase response and modulator of hematopoiesis, the immune response, and inflammation. E. The Interleukin-6 - Signal Transducer and Activator of Transcription -3 Pathway Interleukin-6 interacts with its target cells via the IL-6 System to elicit its effects. This 3-component system involves the interaction of IL-6, IL-6 receptor (IL-6R), its specific receptor component, and gp130. Activation of the IL-6 System can be via the classic method where the receptor is membrane-bound (mbil-6r), such as the case with hepatocytes and a subset of haemopoietic cells, or the trans method where a soluble form of the receptor, soluble IL-6R (sil-6r), existing in fluids, performs the IL- 6R function. Soluble IL-6R is believed to be derived from a membrane bound IL-6R via

9 the Zinc dependent metallopeptidase domain 17 (ADAM17) or by mrna splicing. (1, 68, 69) Trans signaling affords IL-6 the capacity to trigger responses in cell types that would remain unresponsive to IL-6 itself because they lack the mbil-6r. (66) A soluble version of gp130 (sgp130) also exists, and it has been shown to inhibit the trans method of IL-6 signaling. (1, 68, 69) (Figure 2) Figure 2. IL-6 system activation. IL-6 can signal via the classic method where the receptor is membrane bound (mbil-6r) such as the case within hepatocytes, macrophages, neutrophils, and some T cells, or via the trans method where a soluble IL-6R (sil-6r) found in various body fluids interacts with IL-6 and subsequently interacts with a membrane-bound gp130 which is ubiquitously expressed. In both methods, IL-6 first joins with its specific IL-6R subunit. This pair then engages the signal transducing unit, the membrane-bound gp130, leading to a stable trimer. The trimer then assembles with another IL-6/IL-6R/gp130 trimer to form a

10 hexameric complex, bringing gp130 cytoplasmic domains together, leading to intracellular Janus kinase (JAK)/gp130 activation, triggering either the mitogen-activated protein kinase pathway or the JAK/STAT pathway. (68, 69) In the JAK/STAT pathway, the pairing of gp130 cytoplasmic domains induces JAK self-phophorylation, leading to phosphorylation of the cytoplasmic domains of gp130 tyrosine residues, which in turn recruit STAT proteins to be phosphorylated by JAKs. Phosphorylated STAT proteins form hetero- or homodimers (STAT-3/STAT-1 or STAT-3/STAT-3) and subsequently translocate to the nucleus and activate the transcripton of genes, leading to a variety of events in target cells. (68, 69) STAT-3 is activated by numerous cytokines, including IL-6, oncostatin M, leukemia inhibitory factor, interleukin-10, and IFN-γ, as well as leptin. STAT-3- induced proteins play a variety of critical roles in regulating cell processes, including growth, differentiation, proliferation, and apoptosis. STAT-3 s role in adaptive immunity includes T cell development. (70) Once T cells have developed and exited the thymus, STAT-3 signals are needed to ensure correct subset development and function. STAT-3 has direct and essential roles in helper T cell development, lineage commitment, and function. Moreover, the balance between naïve CD4 T cell development into its mature derivatives, T helper and T regulatory cells, are believed to be regulated by STAT-3 and its cousins STAT-1 and STAT-5, a balance that is crucial to the homeostasis and function of the immune system. (66, 70) The IL-6/STAT-3 pathway is involved in both B and T cell growth and differentiation, liver and neuronal regeneration, embryonal development and fertility. (69, 71) Importantly, the IL-6/STAT-3 pathway is a primary signaling mechanism of the

11 pro-inflammatory CD4 TH-17 lineage, whose activity includes production of the cytokines IL-6 and interleukin-17 via a JAK/STAT-3 pathway. (70) Normally, the IL- 6/STAT-3 pathway is auto-regulatory. Suppressor of cytokine signals 3 (SOCS-3), one of the target genes of the JAK/STAT signaling pathway, binds with JAKs to inhibit the IL-6/STAT-3 pathway, and thus negatively regulates the signals to prevent overproduction. (72) F. The Interleukin-6 System, Inflammation, and Obesity Current knowledge on the role IL-6 and sil-6r in inflammatory disease is extensive, while information on sgp130 is more limited. Abnormally elevated IL-6-STAT- 3 signaling is believed to contribute to the pathogenesis of a variety of inflammatory diseases, including atherosclerosis, Crohn s disease, rheumatoid arthritis, and lupus. (1, 73, 74, 75) Increased levels of sil-6r are also present in several inflammatory diseases, where pro-inflammatory IL-6 trans-signaling is particularly relevant in stromal tissue cells. (1, 2, 13, 66, 76, 77, 78, 79, 80, 81) The role of sgp130 in inflammatory conditions is less clear. As an antagonist to IL-6 activity, logic would dictate that sgp130 levels would be reduced in inflammation; however, several studies document an increase in sgp130 levels in inflammatory diseases, possibly a mechanism to attempt to counteract chronic inflammation. (79, 82, 83) The potent role of sgp130 in reducing inflammation in vivo is demonstrated by the ability of a synthetic version of sgp130 (sgp130fc) to treat various inflammatory diseases. (75, 84) Thus, all components of the IL-6 system - IL-6, sil-6r and sgp130 - are elevated in patients with chronic inflammatory conditions.

12 As with inflammatory diseases, numerous studies have documented elevation of IL-6 in obesity and its associated chronic inflammation; however, there are minimal data on both sil-6r and sgp130 activity in obese subjects. Both tissue and serum IL-6 levels are abnormally elevated in obesity and contribute to development of chronic inflammation. (13, 48, 62, 63, 64, 65, 79, 80, 85) IL-6 levels are consistently found to be correlated with adiposity and other inflammatory factors, including activation of nuclear factor kappa- B (NFkB) and elevation of serum tumor necrosis factor-alpha (TNF-α), and CRP. In obesity, IL-6 is believed to exert pro-inflammatory activities via the JAK/STAT-3 pathway, inducing, among other responses, pro-inflammatory TH-17 lymphocytes (66) and dysfunctional myeloid-derived suppressor cells in obese adipose tissue. (1, 70, 80, 86, 87, 88, 89, 90, 91) IL-6/STAT-3 effects in obesity are present in numerous cell types, contributing to anomalies in development and communication between immune cells, adipocytes, and endothelial cells, amongst others. Elevated IL-6 in obesity may also contribute to modulation of glucose metabolism, although controversial data exist on the direction on this effect (86, 88). There are only few studies examining levels of sil-6r and sgp130 in the blood of obese populations, with and without comorbidities. Two of the three studies examining healthy obese subjects determined that there is a significant increase in serum levels of IL-6, but not sil-6r, with increasing BMI. (92, 93) The first study was an investigation on non-diabetic obese subjects, which measured IL-6 and sil-6r blood levels, before and after blood traveled through subcutaneous abdominal adipose tissue. Levels of IL-6 correlated significantly with both BMI and % body fat, but sil-6r did not. Additionally, levels of IL-6 were unrelated to those of sil-6r. (92) The second study assessed IL-6

13 and sil-6r in healthy, non-diabetic postmenopausal women 50 70 years old with a BMI >25 kg/m2. This study also found IL-6, but not sil-6r concentrations, correlating positively with BMI. Interestingly, with weight loss, IL-6 decreased, but concentrations of sil-6r did not change. (93) Authors of both studies therefore concluded that IL-6, but not sil-6r, relates to adiposity in non-diabetic obese subjects. (92, 93) Unfortunately, the two aforementioned studies on the IL-6 system and healthy obesity did not investigate levels of sgp130 in this setting. Conversely, a hormone replacement therapy (HRT) study evaluating systemic levels of IL-6 system components across three groups (premenopausal, postmenopausal-no HRT, postmenopausal-hrt users) reported no significant differences in IL-6 levels across groups; however, both sgp130 and sil-6r were higher in postmenopausal women compared to premenopausal women and lower in the HRTusing versus non-using controls. However, after adjusting for body fat amount, the differences were no longer significant, suggesting that both sgp130 and sil-6r are altered with increasing adiposity. (85) To expand upon the limited information we found regarding modulation of the IL- 6 system and obesity, we extended our review to incorporate four comorbidity obesity studies that included plasma IL-6 system evaluations. In these studies the relationship between obesity and IL-6 system components was more difficult to assess, due to the fact that obesity-il-6 system evaluations were usually secondary, indirect, or in combination with analysis of potential relationships between the IL-6 system and the comorbidities themselves.

14 The first comorbidity study investigating IL-6 and circulating sil-6r in type 1 diabetes found that neither IL-6 nor sil-6r correlated significantly with BMI in nondiabetic subjects. However, in diabetic subjects, BMI was found to correlate with plasma levels of both IL-6 and sil-6r. (94) The second study we reviewed was a crosssectional study investigating the relationship between metabolic syndrome (MS) and the IL-6 system in a population older than 65 years of age. (79) The presence of MS positively correlated with increase in BMI, IL-6, and sgp130 levels, with a trend toward higher levels of sil-6r; however, correlations between BMI and IL-6, sgp130 or sil-6r were not reported. Also, though IL-6 independently correlated with both waist circumference and insulin sensitivity, sgp130 and sil-6r correlations with insulin sensitivity in MS were independent of waist circumference. Authors therefore suggest that sgp130 and sil-6r correlations in the context of MS are mediated by insulin resistance, and not affected by BMI. Indeed, the association between high sgp130 levels and MS was no longer significant after adjustment for insulin resistance. (79) The third comorbidity study was designed to evaluate levels of sgp130 and degree of insulin sensitivity in lean and obese women with and without polycystic ovary syndrome (PCOS). Serum IL-6, sil-6r and sgp130, from PCOS and healthy women were analyzed. (83) PCOS women had higher serum sgp130 concentrations than the control group, and obese women had higher serum sgp130 concentrations than the lean women. The interaction between PCOS status and obesity was not significant, suggesting that an increase in sgp130 in PCOS was independent of obesity and an increase in sgp130 in obesity was independent of PCOS status. Serum sil-6r did not differ between lean and obese healthy women. Serum IL-6 increase correlated with

15 BMI, but not presence of PCOS. (83) Collectively, data from this study showed that healthy obese women (without PCOS) had significantly elevated IL-6 and sgp130 as compared to lean controls, whereas no difference in sil-6r was seen between obese and non-obese healthy subjects. Finally, an investigation on pro-inflammatory cytokines and cardiac abnormalities in uncomplicated obesity did correlate serum IL-6, sil-6-r, and sil-6-r/il-6 complex levels with increasing BMI. (48) Worth noting is that this study also examined whether levels of IL-6/sIL-6R correlate with cardiac abnormality indicators in this healthy obese population. Interestingly, echocardiographic parameters examined in the study all correlated with sil-6r, and sil-6r/il-6 complex levels as well. Furthermore, sil-6r, sil-6r/il-6 complex, and echocardiographic parameters were all higher in patients with a VAT area >130 cm2 than those with <130 cm2. These data therefore suggest that underlying cardiac dysfunction present in these obese subjects may have contributed to augmented IL-6 and sil-6r levels. (48) Thus, results of investigations evaluating the correlation of serum sil-6r and sgp130 with adiposity, with and without comorbidity, reported mixed results, with some studies demonstrating altered levels of these two components of the IL-6 system in obese individuals and others finding no difference between lean and obese subjects. Therefore, whether soluble IL-6 system components (sgp130 and sil-6r) and obesity correlate without comorbidity remains a question unanswered.

16 G. Leptin Leptin is a pleiotropic molecule which has roles in metabolism, endocrine and immune function. Produced and secreted by adipocytes, leptin signals through its leptin transmembrane receptor (OB-R) family. (95) Once leptin has bound to the long form of OB-R, it activates the STAT-3 pathway. Production of leptin is regulated by various factors, including food intake, the endocrine system, and the innate immune system. (95) As with IL-6, the negative feedback loop involving activation of the STAT-3 pathway by leptin itself induces expression of SOCS-3, which acts to inhibit leptin activity and induce leptin resistance. (96, 97) The major physiological site of leptin action is in the central nervous system as the central mediator of a feedback loop that regulates appetite and energy homeostasis. (2) Leptin initiates its specific OB-R/STAT-3 cascade in the hypothalamus to downregulate orexigenic molecules and stimulate release of anorexigenic peptides. (98, 96) This serves to inhibit anabolic and activate catabolic pathways, decreasing appetite while stimulating energy expenditure. (98, 99) In the periphery, leptin s other metabolic roles include increasing basal metabolism and modulating pancreatic cell function. (99) Leptin s role in immune development and function is well established. Its effects are elicited via STAT-3 activation by OB-R present on various immune cells, including neutrophils, macrophages, and T cells. (95) In innate immunity, leptin regulates the inflammatory response (95, 99) by stimulating the production of pro-inflammatory mediators including IL-6, IL-12, IFN-γ, and TNF-α. It also fosters NK cell development, activates neutrophil chemotaxis, as well as phagocytosis by monocytes/macrophages and chemokine secretion. (100) Leptin s effects in adaptive immunity include

17 proliferation of naïve CD4 T cells towards a pro-inflammatory TH-1-cell phenotype. (95) Leptin deficiency in ob/ob mice is associated with immunosuppression, with alterations in leukocyte production and function and thymic atrophy. (99) Absence of leptin leads to reduced sensitivity to T cell-activating stimuli, underscoring leptin s critical role in T cell development and differentiation. (101) Chronically elevated leptin levels are associated with obesity, overeating, and inflammation-related diseases including hypertension, MS, and cardiovascular disease. (102, 103) Leptin levels directly correlate with body fat mass, adipocyte size, and inflammatory state. (2, 96, 99,104, 105, 106) In obesity, excessive leptin may contribute to inflammation via mitogen-activated protein kinase, JNK and STAT-3 signaling, promotion of angiogenesis, and exacerbated production of IL-6, interleukin-1 beta, IFNγ, and TNF-α. (2, 107, 108) Data on elevation of leptin in obesity are extensive; however, information on the relationship of leptin with IL-6 system components in obesity is minimal. Several studies indicate that obesity is characterized by simultaneous elevation of both IL-6 and leptin. (109, 110) Furthermore, IL-6 and leptin share some biological activities. Leptin and IL-6 both signal via STAT-3, both induce SOCS-3, and both drive T lymphocytes towards a pro-inflammatory phenotype (TH-1 and TH-17, respectively), indicating that they may have redundancy in roles and effects. Moreover, leptin can induce production of IL-6, as demonstrated by a recent investigation showing that leptin, in a concentrationdependent manner, activates human peripheral blood B cells to induce secretion of IL-6, partly via activation of the JAK2/STAT-3 pathway. (111) This suggests that elevated levels of leptin in obesity may induce immune cells to secrete IL-6 at abnormally high

18 levels. Another study that correlated leptin to IL-6 production suggested that leptin may act as a key initiator of IL-6-related inflammatory effects in obesity. (108) Furthermore, another study showed leptin induced IL-6 production, signaling and subsequent STAT-3 activation as early events promoting the survival/proliferation of colon epithelial preneoplastic cells. (112) Though not evaluated in the context of obesity, this again shows leptin may directly contribute to IL-6 elevation. (108) Even with these limited data, it is worth considering that leptin and IL-6 not only independently correlate with obesity, but that leptin may also correlate with and contribute to IL-6 production in obesity. IL-6 leptin interactions in obesity may be aggravated by sil-6r and sgp130, but current information on these relationships is scarce. Leptin has indeed been shown to activate metalloprotease shedding to promote sil-6r and trans-signaling in inflammation. (112) Furthermore, adipocytes and preadipocytes actively produce leptin when stimulated by the IL-6/sIL-6R complex, but how these observations may translate to the obesity in vivo environment is undetermined. (113) In one study, levels of leptin and sil-6r in atherosclerotic coronary arteries positively correlated with one another. (103) Data is lacking on how leptin levels may relate to sgp130 in obesity, though leptin correlated with insulin resistance in a PCOS study, and sgp130 correlated with insulin resistance in a separate PCOS investigation, both independent of BMI. (83, 114) Thus, both sgp130 and sil-6r s relationship with leptin in the clinical setting remains under-investigated.

19 H. Adiponectin Adiponectin (APN) is an adipokine mainly produced by mature adipocytes, but it also can be found in skeletal muscle cells, cardiac myocytes and endothelial cells. (99) Adiponectin modulates a number of metabolic processes, including glucose regulation and fatty acid catabolism, primarily in the liver and muscle. (99) Adiponectin also regulates innate immunity, (99) acting mainly as an anti-inflammatory mediator. Adiponectin has anti-atherogenic, anti-inflammatory and insulin-sensitizing effects. (115) Studies have indicated that APN has anti-inflammatory effects on endothelial cells by inhibiting NF-kB activation and TNF-α-induced adhesion-molecule expression. (113) Adiponectin also induces the secretion of some anti-inflammatory cytokines, such as interleukin-10 and interleukin-1 receptor antagonist, by human monocytes, macrophages and dendritic cells and suppresses production of IFN-γ. Other studies have shown APN to inhibit production of IL-6, leptin, and CRP, while inducing production of SOCS-3. (116, 117) APN inhibits leptin-induced autocrine IL-6 production, sil-6r shedding, trans-il-6 signaling and subsequent STAT-3 phosphorylation in preneoplastic colon epithelial cells. (118) As with leptin, there is minimal information on how APN relates to the IL-6 system in obesity. In contrast to leptin, circulating levels of APN are negatively associated with obesity, BMI, visceral fat accumulation and insulin resistance. (99, 115, 116) Adipokine receptors reduction in obesity is correlated with reduced APN sensitivity. (118, 119) Pro-inflammatory mediators that are elevated in obesity, such as TNF-α and IL-6, inhibit APN gene expression and secretion from adipocytes. (113) Soluble IL-6R is believed to be a contributor to IL-6 s role in APN s attenuation in

20 obesity. In a study evaluating cytokine effects on APN secretion from human adipocytes, IL-6 was able to reduce (50%) APN production, but only in combination with exogenous sil-6r. (113) How sgp130 plays a role in APN levels in obesity remains unexplored. Investigating how these proposed mechanisms translate to clinical data unearthed studies evaluating both APN and IL-6 correlations to obesity, with only one relating APN to IL-6. In one study investigating the inflammatory profile and cardiovascular risk improvement in healthy obese women pre- and post-bariatric surgery, pre-surgery obese subjects had higher IL-6 and lower APN concentrations than lean controls. (120) However, preoperative levels of neither IL-6 nor APN significantly correlated with BMI in the obese. Moreover, though IL-6 decreased and APN increased significantly 12 months postop, only IL-6 (decrease) significantly correlated with BMI (decrease). (120) In contrast, another study evaluating obese women pre-and-postop gastric surgery, showed postop (17 months) APN increase, but not IL-6 decrease, associated significantly with decrease in BMI. (121) This study also showed that APN levels were negatively correlated with IL-6 preoperatively. Worth noting is that this study s patient population was (at baseline) evenly distributed along different stages of glucose tolerance. Impaired glucose tolerance may explain the discrepancy between the two studies. (121) Indeed, another study assessing patients with impaired glucose tolerance and type 2 diabetes after a physical training period, found that significant improvement in body fat was associated with significantly improved plasma concentrations of APN,

21 but not IL-6. Moreover, only changes in circulating APN, fasting plasma glucose and percentage body fat were determinants of changes in insulin sensitivity. (122) Indeed, whether APN and the IL-6 system relate in obesity, or whether APN itself truly relates to obesity is still unconfirmed. An exhaustive meta-analysis showed that for healthy lean-to-obese adults, no significant correlation between plasma APN and BMI or between leptin and APN exist, with authors concluding that obesity does not independently affect the plasma level of APN; rather, obesity-related changes in plasma APN levels may be a consequence of obesity-related metabolic disorders. (105) How these conclusions may translate to the IL-6 system s relationship with APN remains undetermined. I. C-reactive Protein Data have shown C-reactive protein (CRP) to be elevated in inflammatory diseases, obesity, and correlating with IL-6 and leptin, as well as being inversely related to APN; however, data evaluating the connection between sil-6r and sgp130 with CRP is limited. C-reactive protein is an acute-phase protein released into the blood stream by hepatocytes during the inflammatory response. (123) C-reactive protein is synthesized by the liver in response to factors, including cytokines (IL-6 and others), released by macrophages and adipocytes. (124,125) The physiological role of CRP is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system. (123) Concentrations of CRP are low in healthy individuals and elevated in infection and/or inflammation, correlating with disease progression. (74, 103, 126) Circulating levels of CRP are consistently higher in

22 the obese (46, 47, 95, 116, 120), and elevated CRP levels have been correlated with elevated IL-6 (116,124,125) in obesity and with leptin in inflammatory diseases. (103) Leptin, sil-6r, and CRP significantly positively correlate with one another in inflammatory disease states. (103) Clinical studies have shown CRP and APN inversely correlated in obesity. (116, 117, 121) Adiponectin reduces IL-6-induced CRP mrna levels in hepatocytes, and suppresses CRP synthesis and secretion from hepatic cells. (116) Therefore, the purported decrease of APN in obesity may contribute to CRP s elevation. Circulating levels of both CRP and sgp130 are elevated in PCOS, independent of obesity. (83,126) J. Inflammation, Obesity, and Cancer: Is Signal Transducer and Activator of Transcription -3 the Link? Chronic low-grade inflammation has emerged as a key pathogenic link between obesity and cancer. (2) When the inflammatory program is not resolved (as in the case with obesity), becoming chronic in nature, it creates an environment conducive to cancer development and progression. (42) Cancer risk in obesity is likely due to changes in adipocyte biology and in the non-adipose cells in the stromal-vascular fraction. (2) Factors connecting cancer to the adipose tissue micro-environment in obesity-driven inflammation include the IL-6 system components, leptin, and APN. (2, 13) A primary mechanism within the obese inflammatory milieu linking these factors to cancer development is the IL-6/STAT-3 pathway. (1, 13, 74, 80) Several transcription factors that are activated in inflamed tissues by cytokines or other inflammatory mediators are instrumental in cancer initiation and promotion.

23 STAT-3 is among the best characterized of these transcription factors. (13, 127, 128) STAT-3 s role in cancer derives from an imbalanced coordination of STAT-3 influence on immune cell production and response in the oncogenic setting. (127) Excessive STAT-3 signaling contributes to the recruitment of tumor-infiltrating inflammatory cells by controlling tumor cell production of chemotactic factors and the expression of chemokine receptors on hematopoietic cells. Persistent activation of STAT-3 is believed to mediate tumor-promoting inflammation and promotes pro-oncogenic inflammatory pathways, including NFkB and IL-6-gp130 JAK pathways, while opposing STAT-1 and NFkB-mediated TH-1 anti-tumour immune responses. (13, 127, 128) STAT-3 is believed to be involved in both the genesis and functionality of myeloid-derived suppressor cells, an immature population of myeloid cells that is present in most cancer patients, and is theorized to be the main player in the perturbation of myelopoiesis and hemopoiesis that compromises immune cell-mediated antitumor immunity and drives inflammation in cancer. (129) In addition, active STAT-3 induces vascular endothelial growth factor and metalloproteases, enhancing angiogenesis, tissue rearrangement, and metastasis formation. It also increases tumour cell proliferation and survival. (127, 128) STAT-3 has major roles in lymphocyte dysregulation in cancer. Elevated activation of STAT-3 negatively regulates the anti-tumor immune responses mediated by TH-1-type CD4 T cells and CD8 T cells, and promotes expansion of regulatory T cell function in the tumor microenvironment. (127) Active STAT-3 has been observed in several populations of malignant T cells (130, 131, 132), where it may directly induce expression of tumor-promoting activators. (133, 134) Thus, immune cell function (lymphocytes, in particular) in cancer is compromised, both as a result of and resulting

24 in STAT-3 s abnormal response in the oncogenic setting. Indeed, STAT-3 has been linked to lung, pancreatic, colon, and breast cancer amongst others. (127, 128, 135, 136, 137) The role of IL-6 in activating the STAT-3 pathways has been implicated in the modulation, growth, and metastases of several cancers, including breast, ovarian and prostate cancer, lymphoma, and melanoma. (137, 138, 143) The presence in the obese inflammatory environment of effects induced by the IL-6/STAT-3 pathways similar to those witnessed in oncogenesis suggests that elevated IL-6 in obesity may participate in increasing cancer risk. As in obesity s inflamed adipose tissue, IL-6 is one of the primary cytokines that activate STAT-3 in tumor and stromal cells. (61, 133, 144) In particular, IL-6 s role in inducing the JAK/STAT-3 pathway may modulate dysregulation of differentiation and activation of T lymphocytes in both oncogenesis and obesity. (58, 59, 60, 61, 76, 145) In both obese adipose tissue and tumors, ll-6/stat-3 also induces the population myeloid-derived suppressor cells. (86, 87, 91, 129, 146) Additionally, IL-6 promotes pro-inflammatory TH-17 cells and its own production via a feed-forward loop, particularly when produced at high levels, as witnessed in both obesity and cancer. (60, 61, 66, 147) Furthermore, crosstalk between proliferating inflammatory TH-17 cells and resident tissue cells - adipocytes in obesity, tumor cells in cancer - plays a decisive role in both inflammatory progression and maintenance in both conditions. (128, 148) Trans IL-6 signaling is linked to STAT-3 promotion of cancer. (149) Elevated levels of IL-6, sil-6, and sgp130 have been reported in cancer patients and correspond to disease severity and outcome (1, 73, 140, 142, 143, 149, 150, 151) As in obesity, sil-6r driven SOCS-3 dysfunction, immune cell infiltration, and crosstalk in cancer may

25 play roles. (2, 53, 59, 60, 136) For example, a rodent study on pancreatic ductal adenocarcinoma showed the involvement of IL-6 trans-signaling in aberrant activation of STAT-3 through deletion of SOCS-3 as accelerating cancer development in the pancreatic epithelium. (136) Furthermore, IL-6 trans-signaling induced abnormal infiltration of immune cells - primarily CD3 lymphocytes and macrophages - into the pancreatic epithelial tissue, the site of inflammation. Resulting subsequent IL-6 release by infiltrating macrophages promoted IL-6 trans-signaling crosstalk between immune cells and the local epithelial, stroma, and tumor cells to drive inflammation and tumor promotion. (53, 59, 60, 136) As in obese adipose tissue, such data support an IL- 6/STAT-3 positive-feedback inflammatory loop in the tumor environment, serving to promote the inflamed state. (13, 58, 59, 60, 61) Additionally, dysfunction associated with APN, leptin, and CRP production in the chronic inflammatory state of obesity may perpetuate oncogenesis. (115) High serum leptin is associated with increased risk of several cancers including colon, prostate, and breast. In addition, leptin levels are positively correlated with the likelihood of developing larger tumors. (137, 152) One study showed that leptin induces cell proliferation via STAT-3 signaling in a model of pre-neoplastic colon epithelial cells by inducing autocrine IL-6 production and trans-il-6 signaling. (152) Recalling that leptin also enhances the production of interleukin-1 beta and TNF-α leading to activation of NFkB and downstream effects that signal through the mammalian target of rapamycin, leptin s pro-angiogenic, pro-inflammatory and mitogenic signaling pathways may further contribute to oncogenesis. (2, 107) On the innate immunity level, one study showed that dramatic reduction of NK cells and Ob-R-expressing NK cells in the liver of obese