Obesity Alters the Immune Response to Influenza Virus Infection- A Mechanism for Immune Modulation Alexia Genese Smith A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Nutrition, School of Public Health. Chapel Hill 2007 Approved by: Advisor: Melinda Beck Reader: Jean Handy Reader: Tal Lewin Reader: Terry Combs Reader: Kay Lund © 2007 Alexia Genese Smith ALL RIGHTS RESERVED ii ABSTRACT Alexia Genese Smith Obesity Affects the Immune Response to Influenza Virus Infection - A Mechanism for Immune Modulation (Under the direction of Melinda A. Beck) The incidence of obesity worldwide has reached epidemic proportions. This has had a significant economic and social impact due to the increase in co-morbidities associated with obesity (1). Obesity leads to changes in immune parameters under basal conditions (2), suggesting that obesity may result in impaired immune responses during an infection. Influenza virus infection is a leading cause of morbidity and mortality worldwide, and proper immune regulation is necessary for clearance of virus with minimal damage to the host. Given that obesity leads to impairment in immune function, we hypothesized that obesity would result in an impaired immune response following infection with influenza virus. Using a mouse model of diet-induced obesity, we found that influenza infected mice had minimal and delayed activation of the innate immune response, as well as a mortality rate that was eight times greater than normal weight mice. The lack of response was partially due to a reduction in mononuclear cell infiltration into the lungs during the early stages of infection. Obese mice also had impaired dendritic cell function, which is a critical link between the innate and cell-mediated immune responses, suggesting that cell-mediated responses during influenza infection were also affected by obesity. Indeed, obese mice had iii lower induction of cytokines that promote anti-viral T cell responses, impaired proliferation and cytokine production by T cells, and delayed infiltration of T cells to the infected lung. What was the cause of the immune dysfunction? Once possibility was the high circulating leptin levels found in obese mice. Leptin is a hormone that plays a role in both innate and cell-mediated immunity through phosphorylation of Signal transducer and activator of transcription 3 (STAT3) subsequent to binding to its receptor. Despite high circulating leptin, we found that obese mice had reduced STAT3 phosphorylation in immune cells during infection, indicating leptin signaling was impaired and may have mediated some of the immune changes that occurred in obese mice. When we analyzed the impact of direct leptin on immune cell function, we found that direct leptin signaling was not required for normal function. This was shown by correction of immune cell function when a functional neuro-specific leptin receptor was expressed in leptin receptor-deficient, db/db mice. Therefore, the impairment in immune responses that occur in obese mice is, in part, likely due to a lack of centrally mediated effects of leptin. Overall, this work implicates obesity as a risk factor for impaired immune responses during influenza virus infection and, furthermore, suggests that responses to other infections may be affected by obesity. Thus, the rapidly growing obese population in this country and other may at substantial risk for increased morbidity and mortality during the next influenza pandemic. iv Dedication First and foremost I want to thank my advisor, Melinda Beck, for being the person she is. Melinda allows her students the freedom to imagine possibilities, while patiently instilling the values that are necessary for succeeding in the reality of science. Secondly, I want to thank Patricia Sheridan, a great friend on whose shoulders I have stood throughout my time in graduate school. Her influence on me as a person and scientist is more than I would have ever expected, and for which I am eternally grateful. Finally I want to thank my mom, my sister, and my husband for their unwaivering love and support. v TABLE OF CONTENTS Page TABLE OF CONTENTS.........................................................................................................vi LIST OF TABLES....................................................................................................................x LIST OF FIGURES.................................................................................................................xi LIST OF ABBREVIATIONS................................................................................................xiii CHAPTER I. BACKGROUND AND SIGNIFICANCE.............................................................................1 A. Specific Aims................................................................................................................1 B. Obesity: A Public Health Perspective...........................................................................2 C. Animal Models of Obesity.............................................................................................2 D. Obesity as a Modulator of Immune Function................................................................3 1. Adipokine release from adipose tissue.....................................................................6 2. Leptin.......................................................................................................................6 2.1 Leptin Signaling................................................................................................7 2.2 Leptin Resistance............................................................................................12 2.3 Leptin and Immune Function..........................................................................13 2.3.1 Innate Immune Response.......................................................................13 vi 2.3.2 Cell-Mediated Immune Response..........................................................14 E. Influenza A: A Public Health Perspective....................................................................16 F. Influenza A: Life Cycle................................................................................................16 G. Immune Response to Influenza Infection....................................................................19 1. Innate Immune Response........................................................................................19 1.1 Respiratory Epithelial Cells............................................................................19 1.2 Macrophage (MΦ)..........................................................................................20 1.3 Dendritic Cells (DC).......................................................................................20 1.4 Natural Killer (NK) Cells................................................................................21 2. Cell-Mediated Immune Response...........................................................................21 2.1 Major Histocompatibility Complex ...............................................................22 2.2 T cells..............................................................................................................22 2.2.1 CD4+ T cells ..........................................................................................23 2.2.2 CD8+ T cells...........................................................................................24 3. Virus Induced Cytokine Signaling..........................................................................26 3.1 Viral Recogition..............................................................................................26 3.2 Type I Interferon ............................................................................................26 3.3 Type II Interferon............................................................................................28 3.4 IL-12 ..............................................................................................................29 3.5 IL-6.................................................................................................................29 3.6 IL-10 ..............................................................................................................30 3.7 Signaling Deactivation....................................................................................32 vii II. INCREASED MORTALITY AND ALTERED IMMUNE FUNCTION IN DIET INDUCED OBESE MICE INFECTED WITH INFLUENZA VIRUS A. Abstract........................................................................................................................34 B. Introduction..................................................................................................................35 C. Materials and Methods.................................................................................................37 D. Results .........................................................................................................................41 E. Discussion.....................................................................................................................45 III. SELECTIVE IMPAIRMENT IN DENDRITIC CELL FUNCTION AND ALTERED ANTIGEN-SPECIFIC CD8+ T CELL RESPONSES IN DIET-INDUCED OBESE MICE A. Abstract........................................................................................................................58 B. Introduction..................................................................................................................59 C. Materials and Methods.................................................................................................60 D. Results .........................................................................................................................65 E. Discussion.....................................................................................................................71 IV. CENTRAL LEPTIN RECEPTOR SIGNALING RESCUES DEFECT IN NATURAL KILLER CELL CYTOXICITY IN db/db MICE DURING INFLUENZA INFECTION A. Abstract........................................................................................................................84 B. Introduction..................................................................................................................85 C. Methods........................................................................................................................87 D. Results .........................................................................................................................92 E. Discussion.....................................................................................................................95 V. SYNTHESIS A. Overview of Research Findings.................................................................................108 B. Increased mortality and altered immune response in obese mice infected with influenza virus.................................................................................................................109 viii C. Selective impairment in dendritic cell function and altered antigen-specific CD8+ T cell responses in diet-induced obese mice infected with influenza virus...........................................................................................111 D. Central leptin receptor signaling rescues defect in natural killer cell cytoxicity in db/db mice during influenza infection.................................................115 E. Future Studies.............................................................................................................117 1. Catecholamine/Neuropeptide-mediated changes in immune function during obesity.............................................................................................117 2. Central versus periperal mediation of leptin's effects on other immune responses............................................................................................117 3. Memory response to influenza infection...............................................................118 4. Different dietary fatty acid sources on the immune response to influenza infection...............................................................................................118 F. Public Health Significance.........................................................................................118 REFERENCES.....................................................................................................................120 ix LIST OF TABLES Page Table 1.1 Factors that may link obesity and immune function………………………... 5 Table 1.2 Cytokines and chemokines expressed during influenza virus infection.…… 31 Table 1.3 SOCS proteins and their target molecules………………………………….. 33 Table 3.1 Total number of cells in lung during influenza infection…………………....78 Table 4.1 Phenotype of Nse-Rb db/db transgenic mice……………………………….. 101 Table 4.2 Serum glucose, insulin, and leptin in Nse-Rb db/db mice..……………….... 102 Table 4.3 Effects of influenza infection on db/db, Nse-Rb db/db, and Nse-Rb +/+ mice………………………………………………………. 106 x