ABSTRACT OF THE THESIS

Understanding Neonatal Cardiac Mitochondria Morphology and Structure and the Effect of Macrophage Phenotype on Cardiomyocyte Morphology

By

Jeoung Hyun Kawk

Master of Science in Biomedical Engineering

University of California, Irvine, 2017

Assistant Professor Anna Grosberg, Chair

Neonatal cardiomyocytes are not the most mature form of cardiomyocytes and may contain different structures and morphology compared to those of adult cardiomyocytes. Especially, mitochondrial morphology and structures has not been extensively studied in neonatal cardiomyocytes, leaving the criteria for normal neonatal mitochondrial morphology and structure unclear. Due to this uncertainty, the source of variability, whether it is physiology or external factors, in neonatal mitochondrial morphology and structure could not be accurately assessed. In this study, fixation and/or staining protocols were explored to test their influence on mitochondrial morphology and structure in neonatal cardiomyocyte culture. It was concluded that alterations in PFA concentration and/or incubation time and stain and primary antibody concentrations did not affect mitochondrial morphology and network. In the second part of the study, influence of cytokines in cardiac hypertrophy was investigated by analyzing changes in cell area occurred post introduction of specific cytokines. There are different macrophage populations, and they are often classified into fully polarized states of M1 and M2. M1 macrophages have pro-inflammatory characteristics whereas M2 macrophages have anti-inflammatory characteristics. It was shown that conditioned media obtained from M2 polarized macrophages that contain anti-inflammatory cytokines reduced the cardiomyocyte area, suggesting that cytokines released by M2 can protect and rescue the heart from cardiac hypertrophy. This finding substantiates the capacity of cytokines released from M2 polarized macrophages as possible treatment option for cardiac hypertrophy and adverse remodeling. Although the results regarding the stimulating factors and M1 polarized, or inflammatory macrophages, conditioned media on cardiomyocyte area were unconvincing and not fully understood, this study was successful in validating promising effect of anti-inflammatory cytokines in resolving cardiac hypertrophy.

Dendritic cells are crucial for the development of the adaptive immune response and have also been implicated in a number of inflammatory diseases. Much of what we know about dendritic cell function is limited to mouse models of disease, as human DCs remain difficult to identify consistently and samples can be difficult to acquire. We undertook a study to identify the prevalence of BDCA1+ DCs in a panel of healthy and diseased lung donors by carefully and comprehensively identifying DCs in parenchymal lung tissue. We found a dramatic increase in the prevalence of BDCA1+ DCs in the fibrotic lung diseases idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (HP) by flow cytometry. We also saw a significant increase in BDCA1+ DCs in Th2-high asthma by using the high-affinity IgE receptor, FcERI and MHCII as identification markers of DCs in biopsy sections. These increased numbers of BDCA1+ DCs could potentially contribute to disease and may represent a novel therapeutic target. Because of the potential role of FcERI in promoting Th2-mediated allergy via DCs, we also investigated regulation of this receptor on human DCs. Surprisingly, we found that human DCs and monocytes constitutively endocytose FcERI and IgE in the absence of crosslinking stimulus. Transgenic mice expressing human FcERIa showed similar FcERI regulation, which resulted in rapid human IgE endocytosis in blood DCs and monocytes in vivo. Serum IgE catabolism was accelerated in FcERI-transgenic mice and was significantly influenced by DC and monocyte numbers. DCs and monocytes may therefore contribute to serum IgE control through constitutive IgE endocytosis and could represent a novel therapeutic strategy for allergic conditions. Further work on the trafficking mechanism of FcERI regulation may provide a significant novel strategy for control of IgE-mediated allergies.

Antigen presenting cells (APCs), such as classical dendritic cells (DCs) and monocyte-derived DCs (moDCs), activate effector/memory Th2 cells at the airways to drive allergic asthma. We sought to better understand the identification and role of APCs in the lung. To distinguish between classical DCs and moDCs in allergic airway disease, it was shown that the MAR-1 monoclonal antibody, which recognizes mouse FcεRIα, stained moDCs but not classical DCs, suggesting that moDCs uniquely express FcεRI. Surprisingly, we found that moDCs stained with MAR-1 in FcεRIα–/– mice, indicating that MAR-1 binds other molecule(s). Ectopic expression of mouse Fc receptors in a human cell line revealed that MAR-1 antibody cross-reacts with FcγRI (CD64) and FcγRIV. We conclude that the MAR-1 staining on moDCs reflects the expression of FcγRI, rather than FcεRI, similar to tissue macrophages.

We also examined how antigen capture by APCs located near the airways in the steady state leads to the activation of Th2 inflammation upon allergen re-exposure. DCs were thought to be necessary for allergic airway recall responses, yet paradoxically, DCs did not appear to capture antigen at the conducting airways in microscopy studies. We tested whether DCs were necessary to induce allergic lung inflammation after sensitization, by selectively depleting classical DCs using Zbtb46-DTR mice. Unexpectedly, mice depleted of DCs during allergen re-exposure exhibited robust inflammation, indicating that alternative APCs are capable of driving local Th2 responses. Examination of airway proximal regions by microscopy showed that antigen was predominantly captured by CX3CR1-GFP+ cells that expressed MHC-II as well as macrophage markers. Because of their enrichment around the airways, we termed these cells Bronchus-Associated Macrophages (BAMs). BAMs were highly dendritic in morphology but were less motile than DCs and did not migrate to the lymph nodes, making them ideal candidates to serve as tissue-resident APCs at the airways. We found that BAMs presented antigen, activated T cells in culture, and interacted with T cells recruited to the airways in a model of allergic asthma. These observations establish BAMs as local APCs residing around the airways that are capable of promoting allergic airway inflammation.