Aus dem Institut für Anatomie und Zellbiologie der Justus-Liebig-Universität Gießen Leiter: Prof. Dr. Eveline Baumgart-Vogt Peroxisomal functions in the lung and their role in the pathogenesis of lung diseases Habilitationsschrift zur Erlangung der Venia legendi des Fachbereichs Medizin der Justus-Liebig-Universität Gießen vorgelegt von Srikanth Karnati Gießen 2018 Die nachfolgende Arbeit nimmt Bezug auf folgende Originalarbeiten: 1. Karnati S*, Graulich T, Oruqaj G, Pfreimer S, Seimetz M, Stamme C, Mariani TJ, Weissmann N, Mühlfeld C, Baumgart-Vogt E (2016). Postnatal development of the secretory cells of the distal airways, the bronchiolar club cells in the mouse lung: stereological and molecular biological studies. Cell and Tissue Research. Jun;364(3):543- 57. 2. Karnati S, Baumgart-Vogt E (2009) PeroXisomes in airway epithelia and future prospects of these organelles for pulmonary cell biology. Histochem Cell Biol. Apr: 131(4):447-54. 3. Karnati S, Lüers G, Pfreimer S and Baumgart-Vogt E (2013) Manganese SuperoXide dismutase 2 (MnSOD) is localized to mitochondria but not in peroXisomes. Histochemistry and Cell Biology, Aug:140(2):105-17 4. Karnati S, Palaniswamy S, Alam MR, Oruqaj G, Stamme C, Baumgart-Vogt E (2015) C22- bronchial and T7-alveolar epithelial cell lines of the immortomouse are eXcellent murine cell culture model systems to study pulmonary peroXisome biology and metabolism. Histochemistry and Cell Biology Mar;145(3):287-304. 5. Oruqaj G§, Karnati S§, Vijayan V, Kotarkonda LK, Boateng E, Zhang W, Ruppert C, Günther A, Shi W, Baumgart-Vogt E (2015) Compromised peroXisomes in idiopathic pulmonary fibrosis, a vicious cycle inducing a higher fibrotic response via TGF-β signaling. Proc Natl Acad Sci. 112(16):E2048-57. § Both authors contributed equally to the study. 6. Vijayan V, Tumpara S, Karnati S, Garikapati V, Linke M, Kamalyan L, Mali SR, Sudan K, Kollas A, Schmid T, Schulz S, Spengler B, Weichhart T, Immenschuh S, Baumgart-Vogt E (2017) A new immunomodulatory role for peroXisomes in macrophages activated by the TLR-4 ligand LPS. Journal of Immunology Mar 15;198(6):2414-2425. doi: 10.4049/jimmunol.1601596 Übersichtsarbeiten 7. Boateng E, El-Merhie N, Trompak O, Tumpara S, Seimetz M, Pilatz A, Baumgart-Vogt E, Karnati S* (2016) Targeting PPARγ in Lung fibroblasts: Prospects of therapeutic treatment for IPF. PVRI Chronicle vol 3, issue 2, p33-37 I Tables and contents 1 INTRODUCTION ............................................................................................................................................... 1 1.1 ANATOMY OF THE LUNG ......................................................................................................................... 1 1.2 RESPIRATORY TRACT .............................................................................................................................. 1 1.3 TERMINAL BRONCHIOLES AND RESPIRATORY BRONCHIOLES .............................................................................. 3 1.4 WHY ARE CLUB CELLS AND CLUB CELL STEREOLOGY IMPORTANT? ....................................................................... 4 1.5 LUNG PARENCHYMA .............................................................................................................................. 5 1.6 IDIOPATHIC PULMONARY FIBROSIS ............................................................................................................. 6 1.7 THE PATHOGENESIS OF IDIOPATHIC PULMONARY FIBROSIS ................................................................................ 7 1.8 ROLE OF TGFΒ1 AND ROS GENERATION IN THE PATHOGENESIS OF IPF .............................................................. 8 1.9 ROLE OF PEROXISOMAL METABOLISM AND PPAR-MEDIATED PEROXISOME PROLIFERATION IN PULMONARY FIBROSIS .... 11 1.10 DISCOVERY OF PEROXISOMES AND PEROXISOMAL ENZYMES .......................................................................... 14 1.11 PEROXISOMAL BIOGENESIS AND METABOLIC FUNCTIONS ............................................................................. 15 1.12 PEROXISOMAL FUNCTIONS IN SCAVENGING ROS AND RNS .......................................................................... 17 1.13 DO PEROXISOMES CONTAIN SOD2? ...................................................................................................... 18 1.14 PEROXISOMES IN THE LUNG: WHERE WE STAND ........................................................................................ 19 1.15 PULMONARY PEROXISOMES AND THEIR IMPORTANCE IN THE LUNG METABOLISM ............................................... 22 2 AIMS OF THE WORK ....................................................................................................................................... 24 3 APPLIED METHODOLOGY FOR THE ANALYSIS OF AIMS OF THE WORK ........................................................... 26 4 RESULTS AND DISCUSSION ............................................................................................................................. 28 4.1 CLUB CELL DIFFERENTIATION IS A POSTNATAL PHENOMENON .......................................................................... 28 4.2 ABUNDANCE OF PEROXISOMES IN THE BRONCHIOLAR AND ALVEOLAR EPITHELIAL CELLS OF THE DEVELOPING LUNG ....... 32 4.3 SOD2 IS NOT LOCALIZED IN PEROXISOMES ................................................................................................ 37 4.4 C22 AND T7 ARE THE BEST CELL LINES TO STUDY THE FUNCTIONS OF PEROXISOMES IN THE CLUB AND AECII CELLS ....... 42 4.5 PPAR-DEPENDENT PEROXISOME SIGNALING AS A TREATMENT STRATEGY FOR IPF ............................................... 49 4.6 PPARS AND THEIR ROLE IN PREVENTING THE IDIOPATHIC PULMONARY FIBROSIS .................................................. 56 4.7 PPAR-INDEPENDENT PEROXISOME SIGNALING IN CHRONIC INFLAMMATORY LUNG DISORDERS ................................ 58 5 SUMMARY ..................................................................................................................................................... 60 6 ZUSAMMENFASSUNG .................................................................................................................................... 62 7 OUTLOOK ....................................................................................................................................................... 64 COMPARTMENTALIZATION OF PEROXISOMAL MEMBRANE PROTEINS BY SUPER- RESOLUTION MICROSCOPY .................... 64 8 REFERENCES ................................................................................................................................................... 67 9 ACKNOWLEDGMENTS .................................................................................................................................... 84 II Introduction 1 Introduction Fresh air entering the mouth and nose is brought to the blood-gas barrier in the lungs by a repetitively branching network of airways. This airway tree achieves an enormous amplification in cross-sectional area from the trachea to the terminal bronchioles. 1.1 AnatomY of the lung The lung is a very complex heterogeneous air-filled organ specially adapted for gas exchange. The lung parenchyma is divided into lobes and segments. Each lobe having its own blood supply originating from the bronchial arteries. The lungs are positioned in the pleural cavity within the thoracic cavity and are surrounded by the visceral pleural membrane which is directly adherent to the lung surface tissue, and the parietal pleural membrane which lines the chest wall. In between these pleural membranes, approximately 100–300 ml of serous fluid acting as a lubricating agent was observed in interpleural space (Noppen et al., 2000). Contraction of the intercostal muscles, movement of the ribs and the diaphragm increases the intrathoracic volume. In consequence, during inspiration, the intrapleural pressure decreases to approximately -6mm Hg, and the lungs expand, causing the pressure in the airways to become slightly negative and leading to air flow into the lungs. During expiration, the diaphragma and intercostales external muscles relax causing the thorax and lungs to recoil. The air pressure within the lungs increases to above the pressure of the atmosphere, causing air to be forced out of the lungs. 1.2 RespiratorY tract The two functionally and structurally distinct regions of the respiratory system are the upper and lower respiratory tracts. The upper respiratory tract consists of nasal cavity, pharynx, and larynx (collectively called as nasopharyngeal region), while the lower respiratory tract consists of trachea, bronchi, bronchioles until terminal bronchioles and alveolar region. The 1 Introduction respiratory system includes two zones: the proximal conducting zone and the distal respiratory zone. In the proximal conducting zone (nose, pharynx, larynx, trachea, bronchi, bronchioles, and terminal bronchioles), the inhaled air is filtered, warmed, and humidified in the conducting airways, thus protecting the distinct lining epithelia of the lower tract from damage, while in the distal respiratory zone (respiratory bronchioles, alveolar ducts, and alveoli), the gas exchange occurs (Duncker, 2008). In the human, the dichotomous bifurcation of the branching morphogenesis of the trachea gives rise to two daughter bronchi with a similar smaller diameter than the mother branch. In the human lung, these proximal airways undergo divisions 23 times (13 times in the mouse) to generate distal airways down to the respiratory