As a pediatric pulmonologist, my long-term career goal is to become a physician-scientist who is an expert in the immunologic control of microbial infections in cystic fibrosis (CF). My short-term educational and research plans for this K08 award are focused on gaining scientific independence through a detailed understanding of the macrophage control of Burkholderia infections in CF. My resident and fellowship research training has allowed me to discover deficiencies in macrophage autophagy in both murine and human CF models that play a key role in Burkholderia virulence in CF. Newly acquired data suggest that Burkholderia species affect the CF macrophage oxidative burst, but it is unknown how this ties in with autophagy signaling in CF. The pursuit of these potentially intertwined pathways will generate the necessary fundamental knowledge for developing R01 grant proposals with the ultimate goal of future clinical trials of novel host-directed therapies for improving treatment of acute and chronic infections in CF. The K08 mechanism will allow me to focus at least 75% of my efforts on developing an independent translational research career. My career development plan will utilize a structured approach with specific objectives to allow me to transition to an independent research career. My training objectives include: 1) Gaining expertise in the study of autophagy and NADPH oxidase-mediated pathways relating to pathogen clearance; 2) Training in the manipulation of macrophage signaling responses to pathogens; and 3) Enhancing translational research design skills for the development of pre-clinical and clinical trials. I will achieve these objectives through hands-on scientific training ia my research mentors and mentoring advisory committee, relevant coursework, participation in a Master's in Public Health for Professionals program, and scientific seminars. Through this approach I will both broadly and specifically enhance my research skills in order to fully develop my independent research lab and research program to facilitate my achievement of independent funding by the completion of a K08 award. The research plan utilizes a human macrophage model of Burkholderia infection in CF to study evasion of host immune responses. My preliminary data suggests that Burkholderia are able to suppress an already dysfunctional oxidative burst in CF to avoid host killing. This also leads to a downstream reduction in autophagosome formation, further decreasing pathogen killing. We hypothesize that Burkholderia survival in CF is predicated upon both the disruption of the NADPH oxidase for generation of reactive oxygen species and the deficient assembly of autophagosomes for targeted intracellular bacterial killing. We will analyze both the assembly and generation of the oxidative burst, as well as signaling interactions between ROS and autophagy initiation. The anticipated outcome of this work is that it will advance our understanding of potential antibiotic- independent targets for drug delivery and/or discovery for antibiotic-resistant Burkholderia infections in CF. The significance of these studies is reflected in our current lack of a gold standard treatment for these deadly infections in CF. The environments of the Center for Microbial Pathogenesis at the Research Institute at Nationwide Children's Hospital (RINCH) and the Center for Microbial Interface Biology at The Ohio State University are outstanding and ideally suited for the achievement of this research plan and training objectives. I will utilize a skilled mentoring team to provide comprehensive research guidance and training. Dr. Larry Schlesinger is a world leader in macrophage biology and macrophage-pathogen interactions with a long and distinguished training record. His leadership is coupled with co-mentor Dr. Amal Amer's expertise in Burkholderia pathogenesis, including novel mechanisms of autophagy deficiency. Additionally, my mentor advisory committee features further expertise in clinical, translational, and basic CF research in addition to CF-specific bacterial pathogenesis. I also have the unique benefit of working in two state-of-the-art facilities and receiving support from experts in both clinical and basic science research cores. Institutional support from the RINCH will allow me to continue to devote 75% of my time towards the proposed training and research. Through this interdisciplinary mentoring approach I will greatly enhance my scientific progression and successful competition for independent R01 level funding during the course of this K08 award.

Public Health Relevance

Burkholderia cenocepacia and B. multivorans are highly transmissible pathogens that cause significant harm in cystic fibrosis (CF) and immunocompromised patients. There are limited treatment options due to high antibiotic resistance and the ability of the bacteria to replicate in host macrophages and avoid natural host immune defenses. In this study we will determine key deficiencies in clearing Burkholderia from CF macrophages. This will provide evidence for novel therapeutics to more effectively clear Burkholderia infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Clinical Investigator Award (CIA) (K08)
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Microbiology and Infectious Diseases B Subcommittee (MID)
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Lu, Kristina
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Nationwide Children's Hospital
United States
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Zhang, Shuzhong; Shrestha, Chandra L; Kopp, Benjamin T (2018) Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have differential effects on cystic fibrosis macrophage function. Sci Rep 8:17066
Robledo-Avila, Frank H; Ruiz-Rosado, Juan de Dios; Brockman, Kenneth L et al. (2018) Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses. J Immunol 201:2016-2027
Krause, Kathrin; Kopp, Benjamin T; Tazi, Mia F et al. (2018) The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients. J Cyst Fibros 17:454-461
Shrestha, Chandra L; Assani, Kaivon D; Rinehardt, Hannah et al. (2017) Cysteamine-mediated clearance of antibiotic-resistant pathogens in human cystic fibrosis macrophages. PLoS One 12:e0186169
Assani, Kaivon; Shrestha, Chandra L; Robledo-Avila, Frank et al. (2017) Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia. J Immunol 198:1985-1994