DISSERTATION THE PATHOGENESIS OF DIABETES-TUBERCULOSIS COMORBIDITY Submitted by Brendan K. Podell Department of Microbiology, Immunology and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2014 Doctoral Committee: Advisor: Randall J. Basaraba Steven Dow Joel Rovnak Mary Jackson Douglas Ishii Copyright by Brendan K Podell 2014 All Rights Reserved ABSTRACT THE PATHOGENESIS OF DIABETES-TUBERCULOSIS COMORBIDITY Exposure to the bacterium, Mycobacterium tuberculosis, only leads to the active form of tuberculosis disease (TB) in 5-10% of infected individuals. The development of active TB, at any stage of infection, is often the result of a known TB risk factor, either intrinsic to the individual or acquired as a communicable or non-communicable disease. An association between diabetes and TB has long been recognized, but only recently was diabetes confirmed to increase the risk of developing active TB disease. The convergence of a growing diabetes epidemic on regions with endemic TB has positioned diabetes as an emerging global threat to TB control. Of particular importance is the rapidly growing incidence of type 2 diabetes, which accounts for up to 95% of the global diabetic population. Since the potential impact of this growing comorbidity has only been recently emphasized, little is known regarding the mechanisms of dysregulated immune function and metabolism by which diabetes predisposes to active TB disease. The current understanding of this comorbidity is further limited by the lack of appropriate animal models that replicate the pathogenesis of both human type 2 diabetes and TB. The guinea pig is a well-established model of TB that replicates human pathology and disease progression. This species was emphasized in this series of studies with the goal of better understanding the impact of type 2 diabetes on TB progression and the mechanisms that may change the host response to M. tuberculosis infection. In Chapter 2, we investigated the impact of hyperglycemia alone, induced as post- prandial hyperglycemia through daily administration of sucrose, on TB disease progression in non-diabetic guinea pigs. Guinea pigs receiving daily sucrose developed both higher bacterial burdens in pulmonary and extrapulmonary tissue and also more severe pathology by day 60 of ii infection. This exacerbated disease manifestation was accompanied by the accumulation of advanced glycation end-products, which are inflammatory by-products of chronic hyperglycemia with known involvement in the development of diabetes-related complications. Interestingly, by monitoring glucose and lipid metabolism in these guinea pigs, we learned that TB alone leads to severe metabolic disturbances, manifesting as hyperglycemia and accumulation of circulating total free fatty acids. From this study, we were able to conclude that not only does mild post- prandial hyperglycemia worsen the course of TB disease in guinea pigs, but also, infection with M. tuberculosis alone induces metabolic disease resembling diabetes, similar to what has been previously reported in human TB. These conclusions rationalize the investigation of novel adjunctive therapies to restore metabolic homeostasis, which may improve the host response to infection, limit bacterial growth, and increase the efficacy of frontline antimicrobial drugs. In Chapter 3, we developed a novel model of type 2 diabetes in the guinea pig to be used in future investigations of type 2 diabetes-TB comorbidity. Previously, the guinea pig as a diabetic model has been described only in the context of β-cell cytotoxicity with the drug, streptozotocin (STZ), but with variable efficacy. In this study, we initially optimized the dose response and STZ preparation to achieve an induction of hyperglycemia that was uniform with limited mortality. This hyperglycemic response was transient but could be stabilized through continued β-cell stress, in the form of a high fat, high sugar diet. Feeding of this modified diet led to impaired glucose tolerance and a compensatory β-cell response that could be abrogated with the use of a single optimized dose of STZ. This novel model of type 2 diabetes develops both insulin resistance and β-cell failure, which replicate the typical progression of type 2 diabetes in humans, all within a reasonable experimental timeframe. From this study, two models emerged, a type 2 diabetic guinea pig as well as a model of impaired glucose tolerance, or prediabetes, that would be used to investigate the mechanisms of diabetes-TB comorbidity. iii In Chapter 4, the newly developed guinea pig models were used to investigate the overall impact of type 2 diabetes and impaired glucose tolerance on TB progression and the host immune response to M. tuberculosis infection. Although impaired glucose tolerance alone had limited impact on TB progression with exacerbation of disease only at chronic end points, M. tuberculosis infected type 2 diabetic guinea pigs closely resembled the reported manifestations of human diabetes-TB comorbidity including more severe TB disease, higher bacterial burdens, and a robust innate and cell-mediated immune response. Despite evidence of strong Th1 cell-mediated immunity, which is known to be critical for limiting bacterial growth and disease progression, diabetic guinea pigs were unable to control bacterial growth and developed damaging neutrophilic inflammation. To better understand the immune mechanisms leading to uncontrolled bacterial growth and severe disease, in Chapter 5, we investigated the innate and adaptive immune response over the course of early infection in type 2 diabetic guinea pigs. Diabetic guinea pigs were slow to develop early lesions with delayed bacterial transport to the lung draining lymph node, and a corresponding delay in antigen-specific Th1 immunity. Early alterations in cytokine expression were identified that may explain the delayed development of cell-mediated immunity and allow for substantial growth of M. tuberculosis in the lung of infected diabetic guinea pigs. These data indicate that not only does type 2 diabetes increase the severity of TB but also that the chronic inflammatory process associated with TB itself may worsen diabetes. This has important implications worthy of further investigation revolving around the diagnostic criteria for diabetes when associated with TB, the impact of active TB on medical management of diabetes, and the investigation of novel therapeutic targets, both metabolic and immunological, to enhance the host immune response to infection and limit TB disease severity in diabetics. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Randall Basaraba, for providing me with the opportunity to pursue advanced research training and to work jointly in an area of research that is highly innovative and is certain to have a significant impact on my developing research career. Members of the Basaraba laboratory were instrumental in completing this project and I would especially like to thank Forrest Ackart for his assistance. I would also like to thank my committee members, Drs. Mary Jackson, Joel Rovnak, Steven Dow, and Douglas Ishii for their insight and assistance along the way. Additionally, I would like to extend my gratitude to Dr. Edward Hoover, as head of the biomedical research training program for veterinarians, and the T32 National Research Service Award committee for providing the opportunity to pursue my research training under the funding of a National Research Service Award fellowship. Lastly, I would like to acknowledge the contributions provided by Colorado State University collaborators throughout this project: Drs. Christopher Bell and Adam Chicco, Department of Health and Exercise Science (Chapters 2 and 3); and Drs. Diane Ordway, Andres Obregon-Henao, Marcela Henao-Tamayo, and Ian Orme, Department of Microbiology, Immunology and Pathology (Chapter 4). v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................................. v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x Chapter 1 - Review of the Literature .............................................................................................. 1 Tuberculosis - Pathophysiology, Disease Diagnosis, and Treatment ........................................... 1 The Immunopathogenesis of Tuberculosis .................................................................................... 6 Diabetes Mellitus - Diagnosis and Pathogenesis ........................................................................ 14 The Comorbidity of Tuberculosis and Diabetes - Current Knowledge ......................................... 30 The Guinea Pig Model of Tuberculosis ....................................................................................... 38 References .................................................................................................................................. 55 Chapter 2 - Non-diabetic hyperglycemia exacerbates disease severity in Mycobacterium tuberculosis infected guinea pigs ...............................................................................................