Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
DSpace
UBCV
Pawluk, Megan
Master of Science - MSc
University of British Columbia
Radiation dermatitis (RD) is observed in up to 90% of patients receiving radiation therapy. Symptoms of RD include redness, scaling, and crusted wounds. Severe symptoms can prevent future radiation treatments. Current treatments are not effective and may cause severe side effects. Granzyme B (GzmB) was originally characterized as a serine protease secreted by cytotoxic lymphocytes to promote target cell apoptosis. It is now recognized that GzmB can be secreted by other immune cell and non-immune cell populations. GzmB is elevated in a number of inflammatory skin disorders, such as atopic dermatitis, autoimmune blistering, impaired wound healing and scarring. Previous studies suggest that extracellular GzmB cleaves E-cadherin, filaggrin, and decorin leading to reduced epithelial barrier function and scarring. Based on these findings, I hypothesized that GzmB contributes to increased RD severity and delayed healing through the cleavage of epithelial barrier proteins and/or matrix proteins. GzmB expression and cell source, E-cadherin, filaggrin, and decorin levels were assessed using immunohistochemical analysis of biopsies from RD patients. The role of GzmB was investigated in an established RD murine model, comparing GzmB knockout (GzmB-/-) to wild type (WT) mice. The dorsal skin was exposed to a single 40 gray dose of radiation. RD severity was blindly scored. Tissues were harvested at days 4, 14, and 46 post-radiation exposure. Tissues were examined for GzmB, immune cell infiltrate, scarring, and decorin. Macrophages and mast cells expressed GzmB while E-cadherin, filaggrin, and decorin were reduced in human RD skin compared to healthy skin. GzmB-/- mice exhibited a decrease in RD severity compared to WT mice from day 4 to day 12. CD3+ T-cell levels were reduced and neutrophil infiltration was observed at day 14. GzmB was expressed by resident mast cells in WT mice. GzmB-/- mice exhibited less scar area and more decorin than WT mice at day 46. In summary, GzmB may contribute to RD severity through E-cadherin, filaggrin, and decorin cleavage. The RD murine model suggests that depletion of GzmB attenuates severity of RD and could mitigate scarring. These studies provide a rationale for pursuing GzmB as a novel therapeutic target for the treatment of RD.
GRANZYME B: A NOVEL THERAPEUTIC TARGET FOR RADIATION DERMATITIS by Megan Pawluk B.Sc., The University of Victoria, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2023 © Megan Pawluk, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Granzyme B: A Novel Therapeutic Target For Radiation Dermatitis submitted by Megan Pawluk in partial fulfilment of the requirements for the degree of Master of Science in Pathology and Laboratory Medicine Examining Committee: Dr. David Granville, Professor, Pathology and Laboratory Medicine, UBC Supervisor Dr. Tillie Hackett, Professor, Anesthesiology, Pharmacology and Therapeutics, UBC Supervisory Committee Member Dr. Christopher Carlsten, Professor, Medicine, UBC Additional Examiner Dr. Honglin Luo, Professor, Pathology and Laboratory Medicine, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Kevin Bennewith, Professor, Pathology and Laboratory Medicine, UBC Supervisory Committee Member iii Abstract Radiation dermatitis (RD) is observed in up to 90% of patients receiving radiation therapy. Symptoms of RD include redness, scaling, and crusted wounds. Severe symptoms can prevent future radiation treatments. Current treatments are not effective and may cause severe side effects. Granzyme B (GzmB) was originally characterized as a serine protease secreted by cytotoxic lymphocytes to promote target cell apoptosis. It is now recognized that GzmB can be secreted by other immune cell and non-immune cell populations. GzmB is elevated in a number of inflammatory skin disorders, such as atopic dermatitis, autoimmune blistering, impaired wound healing and scarring. Previous studies suggest that extracellular GzmB cleaves E-cadherin, filaggrin, and decorin leading to reduced epithelial barrier function and scarring. Based on these findings, I hypothesized that GzmB contributes to increased RD severity and delayed healing through the cleavage of epithelial barrier proteins and/or matrix proteins. GzmB expression and cell source, E-cadherin, filaggrin, and decorin levels were assessed using immunohistochemical analysis of biopsies from RD patients. The role of GzmB was investigated in an established RD murine model, comparing GzmB knockout (GzmB-/-) to wild type (WT) mice. The dorsal skin was exposed to a single 40 gray dose of radiation. RD severity was blindly scored. Tissues were harvested at days 4, 14, and 46 post-radiation exposure. Tissues were examined for GzmB, immune cell infiltrate, scarring, and decorin. Macrophages and mast cells expressed GzmB while E-cadherin, filaggrin, and decorin were reduced in human RD skin compared to healthy skin. GzmB-/- mice exhibited a decrease in RD severity compared to WT mice from day 4 to day 12. CD3+ T-cell levels were reduced and neutrophil infiltration was observed at day 14. GzmB was expressed by resident mast cells in WT mice. GzmB-/- mice exhibited less scar area and more decorin than WT mice at day 46. In summary, GzmB may iv contribute to RD severity through E-cadherin, filaggrin, and decorin cleavage. The RD murine model suggests that depletion of GzmB attenuates severity of RD and could mitigate scarring. These studies provide a rationale for pursuing GzmB as a novel therapeutic target for the treatment of RD. v Lay Summary Radiation dermatitis is an inflammatory skin condition and the most common side effect of radiation therapy. Up to 90% of patients who receive radiation therapy develop radiation dermatitis. Radiation dermatitis is characterized by skin redness, itchiness, pain, and scaling, which, if severe, can prevent further cancer treatments. Current treatments are ineffective or cause severe side effects. Thus, new research avenues to identify novel therapeutic targets are needed. We have shown that a protein-degrading enzyme, granzyme B, plays a key role in other inflammatory skin diseases by degrading key structural components of the skin leading to inflammation and loss of function. My work examined the contribution of granzyme B in radiation dermatitis. I assessed human radiation dermatitis skin samples as well as experimental models of radiation dermatitis to investigate the role of granzyme B. The results of this study may provide support for a novel treatment option for this devastating condition. vi Preface  All histology, experiments, and analysis were performed by Megan Pawluk with the assistance of Layla Nabai, under the supervision of Dr. David Granville.  All thesis writing was done by Megan Pawluk under the direction of Dr. David Granville  The animal model used was performed by Dr. Sho Hiroyasu in collaboration with the lab of Dr. Kevin Bennewith. Dr. Brennan Wadsworth performed all the murine irradiation.  Blinded RD severity analysis in the RD mouse model was performed by Dr. Sho Hiroyasu who is a postdoctoral fellow and dermatologist.  Experimental design of in vitro work was completed by Megan Pawluk under the consultation of Dr. David Granville.  Figure 11 is the work done by Dr. Sho Hiroyasu in Dr. David Granville’s lab with collaboration with Dr. Kevin Bennewith’s lab, specifically Dr. Brennen Wadsworth. Dr. Sho Hiroyasu monitored and imaged all the mice, he scored the severity of RD and ran the analysis on the RD severity scores. This work is presented with permission from Dr. Sho Hiroyasu.  All experimental procedures using human samples were approved by the University of British Columbia (UBC) Review Ethics Board (H16-02161, H15-03345 and H12-00540). All animal procedures were performed in accordance with the guidelines for animal experimentation approved by the Animal Experimentation Committee of University of British Columbia, Canada (A17-0333). vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Acronyms and Abbreviations ....................................................................................... xiii Acknowledgements .................................................................................................................... xiv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................1 1.1 Radiation Dermatitis Background and Significance of Study ........................................ 1 1.2 Skin Structure and Function............................................................................................ 2 1.2.1 Epithelial Barrier and Barrier Proteins in the Epidermis ............................................ 3 1.2.2 Dermis and the Extracellular Matrix........................................................................... 6 1.3 Skin Inflammation Response with Radiation Exposure ................................................. 7 1.3.1 Acute Inflammation Response with Radiation Exposure ........................................... 8 1.3.2 Chronic Inflammation Response with Radiation Exposure ...................................... 10 1.4 Skin Structure with Radiation Exposure ....................................................................... 11 1.4.1 Epithelial Barrier and Barrier Proteins and the Epidermis with RD ......................... 11 1.4.2 Fibrosis and Scarring with RD .................................................................................. 13 1.5 Granzymes .................................................................................................................... 14 viii 1.6 Granzyme B Mediated Apoptosis ................................................................................. 16 1.7 Extracellular Granzyme B in the Skin .......................................................................... 17 1.8 Granzyme B in Skin Diseases and Inflammation ......................................................... 22 1.9 Rationale, Hypothesis and Aims ................................................................................... 25 Chapter 2: Materials and Methods ............................................................................................27 2.1 Human Samples ............................................................................................................ 27 2.2 Hematoxylin and Eosin on Paraffin Embedded Human Skin Samples ........................ 28 2.3 Immunohistochemistry on Paraffin Embedded Human Skin Samples ......................... 28 2.4 Immunofluorescence Double Staining on Paraffin Embedded Human Skin Samples . 30 2.5 Masson’s Trichrome Staining on Paraffin Embedded Human Skin Samples .............. 32 2.6 Laboratory Mice............................................................................................................ 33 2.7 Animal Models.............................................................................................................. 34 2.8 Immunohistochemistry on Mice Paraffin Embedded Skin Samples ............................ 35 2.9 Masson’s Trichrome Staining on Paraffin Embedded Mice Skin Samples .................. 36 2.10 Toluidine Blue O on Paraffin Embedded Mice Skin Samples ..................................... 37 2.11 Statistics ........................................................................................................................ 38 Chapter 3: Results........................................................................................................................39 3.1 GzmB Expression is Increased in Human RD Skin Samples ....................................... 39 3.2 Infiltration of CD68 and Mast Cell Tryptase Expressing Cells is Increased in Human RD Skin Samples ...................................................................................................................... 40 3.3 GzmB Staining Co-localizes to CD68 and Mast Cell Tryptase Staining in Human RD Skin Samples ............................................................................................................................. 42 3.4 Infiltration of Neutrophil Cells is not Increased in Human RD Skin Samples ............. 43 ix 3.5 Epidermal Barrier Proteins, E-cadherin and Filaggrin, are Reduced in Human RD Skin Samples ..................................................................................................................................... 44 3.6 Extracellular Matrix Protein, Decorin, is Reduced in Human RD Skin Samples ......... 46 3.7 GzmB-/- Mice Exhibit Reduced RD Severity Compared to WT Mice after Radiation Exposure ................................................................................................................................... 47 3.8 GzmB Levels are Elevated in Mice Skin Post-radiation Exposure .............................. 48 3.9 GzmB Staining Overlays with TBO Staining in WT Mice Skin Post-radiation Exposure ................................................................................................................................... 50 3.10 Mast Cells are Present in WT and GzmB-/- Mice 4 and 14 Days Post-radiation Exposure ................................................................................................................................... 51 3.11 WT and GzmB-/- Mice Skin May Exhibit a Decrease of CD3+ T-cell Presence in the Skin Post-radiation Exposure .................................................................................................... 54 3.12 WT and GzmB-/- Mice may Exhibit an Infiltration of Neutrophils in the Skin 14 Days Post-radiation Exposure ............................................................................................................ 56 3.13 GzmB-/- Mice Exhibit Increased Decorin Levels and Reduced Scarring in the Skin 46 Days Post-radiation Exposure. .................................................................................................. 58 3.14 Neutrophil Infiltration is not Observed in WT and GzmB-/- Mice at Day 46 ............... 60 Chapter 4: Discussion ..................................................................................................................62 Chapter 5: Conclusion and Future Directions ..........................................................................79 References .....................................................................................................................................81 x List of Tables Table 1. GzmB extracellular substrates. ....................................................................................... 19 Table 2. Table of RD patient information. .................................................................................... 27 Table 3. Table of antibodies used for immunohistochemistry and immunofluorescence. ........... 30 xi List of Figures Figure 1. Structures of the epidermis and dermis in the skin.......................................................... 3 Figure 2. Acute to chronic RD. ....................................................................................................... 8 Figure 3. Extracellular GzmB in the skin. .................................................................................... 25 Figure 4. Outline of animal model. ............................................................................................... 35 Figure 5. GzmB expression is increased in human RD skin samples. .......................................... 39 Figure 6. Infiltration of CD68 and Mast Cell Tryptase expressing cells is increased in human RD skin. ............................................................................................................................................... 41 Figure 7. GzmB staining co-localizes to CD68 and Mast Cell Tryptase in human RD skin........ 43 Figure 8. Infiltration of neutrophil cells is not increased in human RD skin................................ 44 Figure 9. Epidermal barrier proteins, E-cadherin and filaggrin, are reduced in human RD skin. 45 Figure 10. Extracellular matrix protein, decorin, is reduced in human RD skin samples. ........... 46 Figure 11. GzmB-/- mice exhibit reduced RD severity compared to WT mice skin after radiation exposure. ....................................................................................................................................... 48 Figure 12. GzmB levels are elevated in mice skin post-radiation exposure. ................................ 49 Figure 13. GzmB staining overlays with TBO (mast cell) staining in WT mice skin post-radiation exposure. ....................................................................................................................................... 51 Figure 14. Mast cells are present in WT and GzmB-/- mice skin post-radiation exposure. .......... 53 Figure 15. WT and GzmB-/- mice skin may exhibit a decrease in CD3+ T-cell presence post-radiation exposure. ........................................................................................................................ 55 Figure 16. WT and GzmB-/- mice may exhibit an infiltration of neutrophils in the skin 14 days post-radiation exposure. ................................................................................................................ 57 xii Figure 17. GzmB-/- mice exhibit smaller scar area and more decorin in the skin 46 days post-radiation exposure. ........................................................................................................................ 59 Figure 18. Neutrophil infiltration is not observed in WT or GzmB-/- mice at day 46. ................. 61 Figure 19. Summary of findings. .................................................................................................. 80 xiii List of Acronyms and Abbreviations CTL Cytotoxic T lymphocytes DNCB 2, 4-dinitro-chlorobenzene GzmB Granzyme B GzmB-/- Gy Granzyme B knockout gray IL Interleukin MCP-1 Monocyte chemoattractant protein-1 MIP-1 Macrophage inflammatory protein-1 NK PAR Natural killer Protease-activated receptor PI-9 Protease inhibitor-9 RD Radiation dermatitis TGF-β Transforming growth factor-beta TNF-α Tumor necrosis factor-alpha TBO Toluidine blue O WT Wild type xiv Acknowledgements I would like to thank the Cancer Research Society for generously funding this thesis project. I would also like to thank the Faculty of medicine for granting me the KOA team fund in 2021. I would like to thank all the past and present lab members in the Granville lab for supporting me throughout my graduate education. My supervisor, Dr. David Granville, who took a chance on me and hired me as a graduate student. Dr. Granville has educated, guided, and supported me through these past years. I have also had the privilege to have worked with many postdoctoral fellows in Dr. Granville’s lab who has helped me trouble shoot, provided guidance, and have been great additional mentors on countless occasions. Dr. Sho Hiroyasu, Dr. Layla Nabai, Dr. Matt Zeglinski, Dr. Alexandre Aubert, and Dr. Karen Jung, a very large thank you to you all for all of your support. I also would like to thank my fellow Granville lab graduate student, Katlyn Richardson, for guiding me through the Master’s program and being my rock every single day. I would also like to thank the amazing ICORD community which I have grown close to these past few years as well as all the members on the ITC committee who have all become great friends. I would like to extend my thanks to my committee members: Dr. David Huntsman, Dr. Kevin Bennewith, and Dr. Tillie Hackett (as well as Dr. Sarah Hedtrich for her input in the first year) for their academic support and helping me perfect and offer guidance with my project. Your insight and encouragement has helped me get this thesis to the finish line. Lastly, my family and friends who have supported me far before I had even started my Master’s program. My friends, you are all so patient with me as I complete my education and always offering me your time and support. Thank you Ryan Mullin for joining my life as I began this graduate student journey and sticking with me as I navigate my way through it. Thank you xv for all the endless support, your consistent belief in my abilities, and keeping me laughing. Thank you to my siblings, Rachel and Jacob Pawluk, who are my forever cheerleaders. Thank you to my parents, Ken and Kendel Pawluk, who have supported me throughout my entire life and always guided me towards a life with purpose and joy. Each individual mentioned above has had an integral part of my life in the lab and I wouldn’t be where I am today without each and every one of them. xvi Dedication I dedicate my thesis to my parents, Ken and Kendel Pawluk, who have been by my side from day one and have always encouraged me to work hard and believe in myself. Thank you for offering me the tools needed to explore my interests and discover my love for science. Without their constant and unconditional support I would not have been able to gain the skill and reached the success I have today. I will always be grateful for their endless support and encouragement, and constant time, patience, and advice. From the bottom of my heart, thank you!1 Chapter 1: Introduction 1.1 Radiation Dermatitis Background and Significance of Study Radiation therapy is an important therapeutic approach for treating various types of cancer. At present, greater than 50% of patients receive radiation therapy for many types of cancers including breast, rectal, and non-small cell lung cancers in the United States1. Radiation dermatitis (RD) is the result of both the radiation injury and the inflammatory response. During radiation therapy, up to 90 % of patients develop moderate to severe acute skin reactions including erythema, dry desquamation, moist desquamation, edema, ulceration, and necrosis2–5. Severe RD especially occurs in patients with breast, head and neck, skin, and lung cancer, due to the higher radiation dose to the skin as well as increased radiosensitivity in these areas.6,7 There are several other factors that can contribute to an increased risk of severe RD which includes, concurrent chemotherapy, concurrent targeted therapy like epidermal growth factor receptor inhibitors, genetic factors which include DNA repair-deficiency disorders, age, smoking, obesity, and sun exposure.8 One of the downfalls in assessing RD in the clinic is that there is no universally agreed upon metrics for severity scoring. However, one of the most commonly used scoring systems is the U.S. National Cancer Institute Common Terminology Criteria for Adverse Events. This grading system measures RD severity from grade 1-to-5. Grade 1 is when there is mild erythema (redness of the skin, indicating inflammation and increased blood flow to the skin), and dry desquamation (characterized as peeling of dry, scaly skin). At grade two there is moderate erythema and moist desquamation (sloughing of the epidermis) but all confined to the skin folds. Grade 3 is when the moist desquamation has gone beyond the skin folds and there may be bleeding due to trauma. Grade 4 is characterized by skin necrosis or ulceration of the full 2 thickness of the dermis. There can be spontaneous bleeding and the lesions can lead to life-threatening consequences. Grade 5 is when there is death due to dermatitis alone, however this is a very rare event.9,10 In most cases, the skin reaction is mild or moderate, but approximately 20 to 45 percent of patients experience higher grade dermatitis with moist desquamation and ulceration.6,11 RD not only affects quality of life but often results in interruption of primary cancer treatment.12 There are currently no known effective, targeted treatments for RD. Corticosteroids are the most commonly recommended treatment for RD symptoms since it has anti-inflammatory effects and reduces the surge of RD induced cytokines. However, it is a nonspecific treatment and there are adverse effects. Other forms of treatment for RD have been explored such as Aloe vera, chamomile, hyaluronic acid, sucralfate, silver sulfadiazine, and silver nylon dressings. However, there is little evidence supporting that these treatments prevent or reduce the severity of RD.12–14 Since prevention and/or treatment of RD is essential, there is a need to elucidate the mechanisms of RD to find more effective, targeted treatment strategies. 1.2 Skin Structure and Function The skin is one of the largest organs of the human body and is one of the organs with immediate access to the environment. The skin is the first line of defense against external insults. The skin is made up of many layers of diverse, specialized cells, which play specific roles to allow for the skin to protect the internal organs from the environment, thermoregulation, water and pH balance, as well as the detection of external stimuli. There are three main layers of the skin: the first layer closest to the environment is the epidermis, the second is the dermis, and the third is the hypodermis as outlined in Figure 1. 3 Figure 1. Structures of the epidermis and dermis in the skin. Skin is comprised of three main layers, the hypodermis, the dermis, and the epidermis. The epidermis is comprised of four sublayers, the basal layer, the stratum spinosum, the stratum granulosum, and the stratum corneum. The epidermis is made up of keratinocyte cells. Each sublayer of the epidermis indicates a different level of differentiation of the keratinocytes. E-cadherin can be found in all layers of the epidermis. Filaggrin can be found in the stratum granulosum and the stratum corneum. The hemidesmosomes within the dermal-epidermal junction anchor the epidermis to the dermis. In the dermis, Type I collagen is present and is organized in a basket weaving pattern assisted by decorin binding to the collagen. Note: Image created based on Jung, K., Pawluk, M. et al. 2022.15 1.2.1 Epithelial Barrier and Barrier Proteins in the Epidermis The epidermis has a major role in maintaining skin barrier function such as reducing trans-epidermal water loss, preventing entry of foreign particles/chemicals/allergens/pathogens, regulation of inflammation, and other functions. The epidermis is divided into sublayers which have a strata of keratinocytes at different stages of differentiation.15, 16 The differentiation process can be followed by observing different differentiation markers of the keratinocytes. The inner most layer of the epidermis is the stratum basal which is made up of basal keratinocytes which are the highest proliferating and least differentiated group of keratinocytes. In the stratum basal layer, the basal keratinocytes will exit the cell cycle at the mitosis stage and differentiate 4 across the epidermis until they are in the stratum corneum as corneocytes (flattened and denucleated keratinocytes). Keratins KRT5 and KRT14 are expressed in the basal keratinocytes and are the basal layer markers. The next layer is the stratum spinosum which is responsible for the strength and flexibility of the skin. Early differentiation markers, keratin KRT1 and KRT10, are expressed in keratinocytes in the stratum spinosum. Other cornified envelop precursors are found in the stratum spinosum which include involucrin and transglutaminase-1. The next layer is the stratum granulosum where granular keratinocytes produce filaggrin as precursor profilaggrin. Profilaggrin eventually transitions to the stratum corneum once mature. Late differentiation markers are cornified envelope protein loricrin and keratin cross-linking protein filaggrin which are expressed in the stratum granulosum. The outer most layer is the stratum corneum which is made up of corneocytes (flattened and denucleated keratinocytes) and plays a predominant role in barrier function.15–17 The skin functions as a barrier between the internal organs and external environments. The epidermis of the skin contributes to the epithelial barrier function. Filaggrin (Filament aggregating protein) is a protein which is crucial for the function of the stratum corneum and is produced from keratinocytes as a precursor form, profilaggrin, in the stratum granulosum. 15–18 Profilaggrin in primarily located within the keratohyalin granules which are found within the cytoplasm of the keratinocytes of the stratum granulosum. Keratohyalin granules also contain keratin intermediate filaments which contribute to the cytoskeletal network of the keratinocytes. While the keratinocytes differentiate into the stratum corneum, profilaggrin is cleaved to generate positively charged filaggrin monomers. The positively charged filaggrin monomers can now bind with keratin filaments which promotes the collapse of the keratinocyte resulting in a corneocyte and forms the corneocyte fibrous matrix. Once in the outer layer of the stratum 5 corneum, peptidylarginine deiminases can further modify the positively charged filaggrin monomers into a neutrally charged filaggrin monomer. The neutrally charged filaggrin monomer can disassociate from keratins and degrade into free amino acids, which contribute to the natural moisturizing factor of the skin. This is essential for regulation of skin hydration, pH and immune regulation.17, 19 Filaggrin has been implicated in skin diseases specifically those with barrier disorders, including atopic dermatitis, atopic eczema, and allergies. Treatments targeting filaggrin loss are a large area of interest for future therapeutics for cutaneous disease.19 Adherens junctions, including E-cadherin (epidermal cadherin), are key cell adhesion structures for maintaining a strong interaction between keratinocytes within the epidermis which contributes to the epidermal barrier function of the skin20. E-cadherin can be found in all layers of the epidermis and regulate the organization of other intercellular junctions including desmosomes and tight-junctions.15 Adherens junctions are responsible for initiation and continuous cell-cell adhesion, intracellular signalling, and actin cytoskeleton regulation. E-cadherin is a single pass transmembrane glycoprotein which binds to another E-cadherin protein on other cells allowing for cell adhesion. E-cadherin is a Ca2+ dependent adhesion protein since the binding of Ca2+ to E-cadherin is required for proper organization of E-cadherin’s extracellular matrix domain. The extracellular matrix domain is the portion of E-cadherin which interacts and binds to adhesion proteins on opposing cells. The cytoplasmic domain of E-cadherin binds to many proteins which regulated E-cadherin recycling, regeneration, control of actin cytoskeleton, and intracellular signaling. The intracellular matrix domain of E-cadherin can make contact with intracellular proteins causing E-cadherin clusters to grow laterally. This lateral growth of E-cadherin clusters strengthens the contact to an opposing cells adhesion proteins, thus making the adhesion interaction between the two cells stronger.21 The loss of E-6 cadherin has been detected in epithelial barrier dysfunction such as atopic dermatitis and eczema dermatitis.22, 23 Tight junctions play a role in barrier function though regulation of passing ions and small molecules between cells. Although the loss of E-cadherin does not completely disassemble tight junctions, E-cadherin loss prevents assembly of key contributors to barrier function and thus contributes to decreased barrier function.24 The loss of E-cadherin is observed in diseases where barrier function plays a role in disease progression and severity, for example atopic dermatitis and eczema dermatitis.22,23,25,26 1.2.2 Dermis and the Extracellular Matrix The dermis has fewer cells than the epidermis and predominantly consists of the extracellular matrix as well as blood vessels, lymphatics, nerves, and sweat glands. The dermis is split into two sections: the outer papillary dermis which is located between the appendages from the epidermis, and the inner reticular dermis where the connective tissue is much denser.27 The dermis is mainly made up of extracellular matrix which functions as a crucial structural component. The extracellular matrix plays a role in cell adhesion, migration, proliferation, differentiation, and cell fate determination. One key component of the extracellular matrix is decorin. Decorin is a small leucine-rich proteoglycan, found in all parts of the dermis, and is produced and secreted by fibroblasts. Decorin binds to a large extracellular matrix protein, type I collagen and regulates collagen fibrillogenesis.28,29 Type 1 collagen forms the macrostructure of the skin and is normally observed in histology as fibrils in a jumbled basket weave pattern in healthy skin.29,30 In scar tissue, the collagen fibrils are seen in histology arranged parallel to one another while hair follicles and glands are absent.31,32 Decorin also is an anti-fibrotic protein as it is known to bind to transforming growth factor-beta (TGF-β), a pro-fibrotic factor, and neutralize 7 and repress TGF- β activity. Thus, the loss of decorin and increasing TGF-β bioavailability has been proposed to contribute to scar tissue formation.28,33–35 Degradation of decorin has been found in many skin conditions including ultraviolet radiation, cutaneous burns, and aging.34–36 1.3 Skin Inflammation Response with Radiation Exposure RD occurs in two pathological stages: acute - where inflammation and barrier disruption occurs, and chronic - where fibrosis is established.12 Inflammation cytokines can be seen at both acute and chronic RD. The severity of inflammation and the cytokines produced can indicate development of acute and chronic RD. The mechanism of RD inflammation is not completely understood; however, keratinocytes, fibroblasts, and endothelial cells stimulate skin resident immune cells as well as promote infiltration of other immune cells. Radiation can result in a dose dependent accumulation of diverse immune cells such as macrophages, mast cells, and neutrophils.37 Many inflammatory mediators and cytokines have been found to be induced with radiation including monocyte chemoattractant protein-1(MCP-1), Interleukin(IL)-1, IL-6, tumor necrosis factor-alpha (TNF-α).37 TGF-β is a profibrotic factor which is elevated in RD and can result in scarring in chronic RD.38 Cytokines and inflammation contribute to both acute and chronic RD and can determine the severity of RD as outlined in Figure 2. 8 Figure 2. Acute to chronic RD. RD has two stages: Acute RD and chronic RD. Acute RD is the early stages of RD where inflammation can increase due to an increase in inflammatory cytokines. The inflammatory cytokines will recruit infiltrated immune cells and mast cells will degranulate which will cause more inflammation and tissue damage. Erythema and desquamation are clinical signs of acute RD and barrier disruption can be determined by measuring trans-epidermal water loss. In chronic RD fibrosis inducing cytokines are present in the skin. These cytokines can cause fibroblasts to differentiate into myofibroblasts which can contribute to collagen disorganization, fibrosis, and scarring. RD fibrosis and scarring can appear as skin stiffness, soft tissue atrophy, and alopecia in the clinic. 1.3.1 Acute Inflammation Response with Radiation Exposure Ionizing radiation induces many changes to keratinocytes, which are the first layer of exposed cells. In acute RD inflammatory cytokines are involved in the damage response.20 Acute RD is correlated to an increase in various cytokines and chemokines, in particular, IL-1α, IL-1β, TNF-α, IL-6, IL-8, macrophage inflammatory protein-1 (MIP-1), and MCP-1.39–42 Keratinocytes secrete MIP-1, IL-6, IL-8, and MCP when irradiated with ultraviolet and other forms of radiation such as γ-radiation.43–45 MIP-1 is increased in thoracic RD and has a critical role in radiation induced inflammation through binding its chemokine receptor, CC chemokine receptor 1, causing an increase in immune cell infiltration and later fibrosis, in the lungs.46 IL-6 and IL-8 are induced in irradiated keratinocytes.43 IL-6 is a major player in acute inflammation by mediating a wide variety of immune responses and is necessary for wound healing. IL-6 can further promote proinflammatory and profibrotic responses in differentiated macrophages.47 IL-8 9 is responsible for activating and attracting neutrophils to the inflammatory site.46–48 MCP-1 plays a role in acute RD by recruiting monocytes to the irradiated site and in chronic RD through its involvement in tissue fibrosis. MCP-1 and IL-1 are robust chemo-attractants for monocytes and can also activate the macrophages to produce more cytokines that will increase the inflammation response. In a mouse study of RD, they found that MCP-1 levels were about seven times higher in their RD treated mice then the control mice.41 Trans-endothelial migration of immune cells, such as neutrophils, macrophages, and leukocytes, from circulation to irradiated skin is considered a ‘‘hallmark’’ of radiation-induced skin injury.40,49 After radiation exposure, cytokines from irradiated skin cells recruit immune cells and neutrophils are the first immune infiltrated cells to reach the site of infection and release more pro-inflammatory cytokines.12,50 Monocytes are recruited, which differentiate into macrophages once they reach the irradiated tissue.51 In addition, ionizing radiation induces degranulation of mast cells in the dermis.52 Mast cells release a variety of proteolytic enzymes after radiation exposure which contribute to connective tissue changes.52, 53 7 days after a 17.9 gray (Gy) dose of radiation was exposed to mice, macrophages and some lymphocytes were evident in the skin. However, at day 14 macrophage inflammation was prevalent with fewer lymphocytes.54 A study looking at CD3+ T-cell levels in the blood of patients after radiation exposure to the Chernobyl disaster, showed less CD3+ T-cells in the blood of the patients exposed to radiation compared to the controls.55 Another study had taken different immune cells and exposed the cells to radiation in vitro. The results showed that T-cells were the most radio-sensitive cells in the blood population and would start apoptosis soon after radiation exposure. B-cells were also much more radiosensitive than the least radiosensitive group, monocytes, and macrophages. The study also included an in vivo model where peripheral blood was taken from 10 patients who had received total-body irradiation. They observed that the number of T-cells declined after the first day of radiation exposure and remained low. The monocytes did not decline as extensively as the T-cells.56 1.3.2 Chronic Inflammation Response with Radiation Exposure Fibrosis is a hallmark for chronic RD and involves skin thickening hardening, tension, and scarring which can be caused by prolong exposure to injury or inflammation. Chronic RD typically presents months to years after radiation exposure, however, the events which can lead to fibrosis can begin within acute RD.51 Neutrophils arrive at the irradiated site and are stimulated by pro-inflammatory proteins such as TNF-α, IL-1, and IL-6 early on in RD development. Stimulated neutrophils can cause more inflammation by producing and secreting more pro-inflammatory cytokines, such as IL-1α and TNF-α. IL-1α and TNF-α act as signals to activate local fibroblasts to facilitate wound healing.48 Neutrophils can also sustain the chemokine response which can lead to scarring found in chronic RD.57 Macrophages as well as fibroblasts can secrete TGF-β, a profibrotic factor, which can be found early in radiation damage and is involved in the development of radiation fibrosis pathogenesis. An increase in TGF-β expression from fibroblasts in the skin has been seen to occur as early as 6 hours after exposure to γ-irradiation at both protein and RNA level from a single dose of radiation ranging from 16-64 Gy.38 TGF-β levels have been observed to be elevated in all phases of fibrosis development in an in vivo pig RD skin model.38 TGF-β regulates and controls cell proliferation, differentiation, wound healing , and synthesis of the extracellular matrix by activating fibroblasts through the TGF-β/Smad signaling pathway.38,58,59 Skin fibrosis through the TGF-β pathway involves the phosphorylation of the Smad-2 and Smad-3 proteins which lead to nucleus translocation, 11 activation of transcription factors, and triggers fibrosis through fibroblast migration, fibroblast differentiation into myofibroblasts, increase in extracellular matrix protein expression, and profibrotic growth factors.28, 38, 59 The TGF-β signaling pathway has been targeted for radiation fibrosis. For example pentoxifylline, a competitive nonselective phosphodiesterase inhibitor, has been studied for its ability to inhibit TGF-β expression, however, more studies need to be done, since some studies find pentoxifylline used alone is not effective at reducing fibrosis.12, 38 1.4 Skin Structure with Radiation Exposure The loss of barrier proteins reduce the epithelial barrier function, resulting in dryness, itchiness, and inflammation of the skin and have been correlated with increased severity of acute RD.39 Acute RD can present as dry desquamation which is thickened, scaly, peeling, and dry skin. In more severe acute RD there can be moist desquamation, which can occur due to the loss of integrity of the epithelial barrier.39 Chronic RD can be clinically presented through skin stiffness, soft tissue atrophy, and alopecia. Cutaneous fibrosis can change the form and function of the skin and has been an area of focus for treatment and prevention.51 1.4.1 Epithelial Barrier and Barrier Proteins and the Epidermis with RD Early acute RD can progress 10 to 14 days after radiation exposure and is characterized by the clinical presentation of desquamation as well as apoptotic keratinocytes, vacuolization of the basal layer, and epidermal edema. Epidermal barrier disruption is a hallmark for acute RD.20 Epithelial barrier disruption can cause loss of epidermis, thus re-epithelialisation is needed for skin repair. Re-epithelization, through migration and proliferation of basal keratinocytes, can begin within 10 days of radiation exposure. This especially occurs in the absence of severe 12 infection.60 The loss of epithelial barrier function can allow for penetration of foreign antigens, pathogens, bacterial infections, and trans-epidermal water loss. There is a greater chance for bacterial infections when more epithelial barrier is lost. Higher radiation doses can cause greater barrier disruption leading to more severe symptoms and prolonged healing. In severe RD, re-epithelization may be greater than 10 days.61 This correlates with the clinical presentations of RD severity. Dry desquamation observed at grade 1 is clinically characterized as peeling of dry, scaly skin and indicates barrier disruption. With an increase in radiation dose, moist desquamation can be seen and is characterized as epidermal necrosis, blister formation, and sloughing of the epidermis.39 The major role of E-cadherin is to adhere the cells within the epidermis together so that more cell-cell interactions can occur and an epithelial barrier can be established. Cleaved fragments of E-cadherin have been found in serum from patients with many inflammatory skin diseases such as Dariers disease, psoriasis vulgaris, pemphigus vulgaris, eczema dermatitis, and atopic dermatitis.22, 23, 62 The loss of E-cadherin staining have been detected in epithelial barrier dysfunction disease skin tissues such as atopic dermatitis and eczema dermatitis. In atopic dermatitis lesion human skin, levels of E-cadherin are reduced when compared to the controls.22 Human eczematous dermatitis lesion samples showed less E-cadherin when compared to control skin.23 The loss of E-cadherin, resulting in less cell-cell contact, has been detected in a mouse model of RD.20 However, more studies on the cause for E-cadherin loss and E-cadherin levels in human RD are needed. Filaggrin is a protein which is crucial for the function of the stratum corneum.18 The stratum corneum plays a role in osmotic balance and when the stratum corneum is damaged there is an increase in trans-epidermal water loss. The loss of filaggrin is correlated to increase trans-13 epidermal water loss.15, 63 2, 4-dinitro-chlorobenzene (DNCB) was used to induce dermatitis like lesions in mice skin. The levels of trans-epidermal water loss was increased where filaggrin expression was decreased in mice skin treated with DCNB when compared to non-treated mice.64 Dermatitis like inflammatory skin reaction was induced in an oxazolone treated mouse model. In this model the trans-epidermal water loss was increased and filaggrin protein levels were decreased in oxazolone treated mice when compared to non-treated mice.22 The loss of the epidermal barrier function in RD patients has been shown through increased trans-epidermal water loss which is a known parameter used in the clinic to indicate damage to the stratum corneum.65 Thus the loss of filaggrin may be prevalent in RD. 1.4.2 Fibrosis and Scarring with RD Chronic RD involves multiple stages and consists of inflammation, proliferation, and remodeling. An increase in connective tissue causes fibrosis and can lead to organ dysfunction.12 Fibrosis can been found in chronic RD and is characterized by dermal thickening and hardening, scarring, retraction, hyper- and hypopigmentation, atrophy, hair loss, and alopecia. TGF-β is a pro-fibrotic factor found elevated in many stages of RD and is a contributor to chronic RD fibrosis.38 TGF-β binds to its receptor found on fibroblasts and activates the signaling pathway which causes fibroblasts to differentiate into myofibroblasts. Myofibroblasts have been shown to secrete excessive extracellular matrix proteins, including collagen. An increase of the extracellular matrix proteins can cause the stiffness seen clinically in radiation induced fibrosis.51 The initial loss of hair follicles and sweat glands may be due to cell apoptosis. As explained above, in chronic wound healing the extracellular matrix does not return to its original state. This can cause the extracellular matrix to be unable to promote proper communication between 14 keratinocytes and fibroblasts. Communication between keratinocytes and fibroblasts, through the role of the extracellular matrix, is responsible for the formation and maintenance of hair follicles and glands in the skin. Thus, the abnormal extracellular matrix found in fibrosis renders the hair follicle and glands to be compromised and appear absent in fibrosis.66,67 Histologically, scarring is characterized by increase in collagen, collagen lining up parallel to one another, and absence of pilosebaceous units (hair follicle, hair shaft, and sebaceous glands) which has been seen in chronic RD.8, 31, 32 Decorin is a dermatan sulfate glycosaminoglycan proteoglycan that is associated with type I collagen which allows decorin to play a role as a stabilizer of the extracellular matrix tissue. Decorin is an anti-fibrotic factor since it can regulate the bioavailability of TGF-β. Decorin binds to TGF-β, sequestering the isoforms to the extracellular matrix, which inhibits TGF-β’s activation.28 When Decorin is cleaved TGF-β is released and becomes profibrotic.33A study looking at an in vitro model of human breast stromal fibroblasts, which were exposed to γ-irradiation, showed a down-regulation of both the mRNA and protein levels of decorin.68 However, the role of decorin in RD needs to be further explored. Decorin is decreased in diseases showing fibrosis, including hypertrophic scars, burns, and ultraviolet light exposure.34, 35, 69 Further studies are required to be able to understand more about the mechanism focusing on barrier protein loss and decorin levels in RD. 1.5 Granzymes Granzymes (granule-secreted enzymes) are a family of serine protease with a role in immune mediated apoptosis. The human genome includes five granzymes: granzyme A, granzyme B (GzmB), granzyme H, granzyme K, and granzyme M. However, the mouse genome 15 includes eleven granzymes (granzyme A, B, C, D, E, F, G, K, L, M, and N) which could have resulted due to evolution and different immune chllanges.70,71 Evolution and different immune challenges, could be the cause for the different number of granzymes among species.72, 73 Granzymes are classically known to be stored in granules of cytotoxic T lymphocytes (CTL) and natural killer cells (NK). Perforin, a pore forming protein, is secreted along with granzymes to allow for pore formation and entry of granzymes into the target cell, activating targeted cell death. In recent years, diverse and specific functions for each granzyme has been discovered. Each granzyme contains a similar His-Asp-Ser catalytic triad which enables their proteolytic activity; however, granzymes exhibit unique preferred amino acid cleavage sites.72,74,75 Granzyme A is a tryptase and cleaves basic residues of arginine and lysine. Granzyme A originally was known to play a role in caspase independent cell death but has now been discovered to play a role in extracellular degradation, protease-activated receptor (PAR) activation, proinflammation, and cytokine release. GzmB is an Asp-ase and cleaves acidic residues on amino acids aspartic acid and glutamic acid. GzmB was most commonly known for its role in apoptosis. However, GzmB has been found to play a role in autoimmunity, barrier dysfunction, basement membrane disruption, extracellular matrix degradation, and impaired remodeling.71 Granzyme H is a chymase which cleaves aromatic residues of amino acids phenylalanine, tryptophan, and tyrosine. Granzyme H was originally known for its role in cell death but more recently has been known for its role in antiviral immunity. Granzyme K is a tryptase and cleaves basic residues on arginine and lysine. Granzyme K was most commonly known for its role in necrosis but has been discovered to be involved in endothelial activation, PAR activation, proinflammation, and cytokine release.71 Granzyme M is a metase and cleaves 16 aliphatic residues on leucine and methionine amino acids. Granzyme M is largely understudied and is only currently emerging with a role in innate immunity.71 Granzymes are synthesized and targeted to the endoplasmic reticulum where the target signal is then removed producing an inactive precursor granzyme. The precursor granzyme will then move to the Golgi apparatus and is shuttled into a lysosome which will become the secretory granule. In the granule, the inhibitory N-terminal dipeptide of the precursor granzyme will be cleaved by cathepsin C. The granzyme is then stored in a scaffold of chondroitin sulfate proteoglycan serglycin which concentrates the granzymes. The storage of the granzyme in the scaffold and the acidic environment mitigates the enzymatic activity of the granzymes within the secretory vesicle. Once granzymes are released to the cytoplasm, typically at a neutral pH environment, the granzymes are then optimally active.76, 77 1.6 Granzyme B Mediated Apoptosis GzmB, an aspase-like serine protease, is the most studied granzyme and was originally appreciated for its cytotoxic role in CTL/NK-mediated target cell apoptosis.78 The target cell is most commonly a virus-infected cell, cell with intracellular pathogen, or tumor cell. GzmB is secreted into the synaptic cleft at the binding site where the CTL T-cell receptor binds with the target cells major histocompatibility complex-peptide.76 GzmB is secreted along with a pore forming protein known as perforin. Perforin facilitates GzmB entry into target cells, allowing activation of the apoptotic pathway.76, 77 GzmB preferentially cleaves proteins directly after an aspartic acid residue. GzmB can also cleave adjacent to glutamic acid residues, however with less preference.72,75,79,80 In the target cell, GzmB activates the apoptotic pathway by cleaving many caspases, especially caspase 3. GzmB has also been shown to cleave pro-caspase-8 into 17 active caspase-8 which is a major initiator of the apoptotic pathway. The cleavage of pro-caspase-8 still involves caspase-3 activation since activated caspase-8 leads to caspase-3 activation.72,75 A caspase independent pathway can also be activated by GzmB through cleavage of Bid, a pro-apoptotic protein. When Bid is cleaved by GzmB it becomes a granzyme-truncated Bid which can translocate to the mitochondria and releases other apoptotic factors including cytochrome C. Downstream of cytochrome C, the procaspase-9-activating heptameric protein complex (apoptosome) is formed and apoptosis of the target cell is activated.72,75,79,80 The only known human endogenous inhibitor for GzmB is an intracellular inhibitor called protease inhibitor-9 (PI-9). PI-9, a serine protease inhibitor, is used by immune cells as a defense against accidental exposure to active GzmB within its cytosol. PI-9 inhibits GzmB through binding GzmB and forming inactive, irreversible complexes.81 However, there is no known endogenous extracellular inhibitor in humans.15 1.7 Extracellular Granzyme B in the Skin GzmB can be expressed in many different immune cells within the skin including macrophages, T-cells, natural killer cells, basophils, and mast cells, as well as non-inflammatory cells, including keratinocytes.77,82–85 Approximately one third of secreted GzmB, is secreted into the extracellular space during NK/CTL-mediated target cell apoptosis.33 In addition, mast cells and keratinocytes may secrete GzmB without perforin, further contributing to extracellular GzmB accumulation.86 GzmB is detected in the extracellular milieu and bodily fluids in inflammatory diseases, such as synovial fluid of rheumatoid arthritis patients, cerebrospinal fluid of multiple sclerosis, and bronchoalveolar lavage in chronic obstructive pulmonary disease and 18 lung inflammation.87 The proteolytic activity of GzmB is retained in the extracellular environment because there are no endogenous extracellular GzmB inhibitors identified.15,87 Extracellular GzmB is elevated in a number of skin disorders, including atopic dermatitis, autoimmune blistering, chronic wound healing, aging, and scarring.22, 33, 34, 88–90 Extracellular GzmB cleaves numerous substrates that contribute to reduced epithelial barrier function, blistering, impaired matrix remodeling, and scarring (Table 1). Some of the GzmB substrates identified include fibronectin, decorin, laminin, vitronectin, collagen VII, collagen XVII, α6/β4 integrin, E-cadherin, filaggrin, and occludin as in Table 1.22, 33, 88, 91–95 19 Table 1. GzmB extracellular substrates. GzmB substrate Location Result of cleavage Reference α6/β4 integrin Dermal-epidermal junction Blistering 88 Collagen VII Dermal-epidermal junction Blistering 88 Collagen XVII Dermal-epidermal junction Blistering 88 Decorin Dermis Scarring 33 E-cadherin Epidermis Epithelial barrier disruption 22 Fibronectin Dermal-epidermal junction, Dermis Impaired cell adhesion and migration 92 Filaggrin Epidermis Epithelial barrier disruption 22 IL-1α Epidermis, Dermis Inflammation 95 JAM-A Epidermis Epithelial barrier disruption 22 Laminin Dermal-epidermal junction Impaired cell adhesion and migration 92 Occludin Epidermis Epithelial barrier disruption 93 pro-IL-18 Epidermis, Dermis Inflammation 94 Vitronectin Dermis Impaired cell adhesion and migration 92 ZO-1 Epidermis Epithelial barrier disruption 22 E-cadherin, a cell-cell junction protein, and filaggrin, an important structural protein with a primary role in epidermal barrier integrity, have both been shown to be cleaved by GzmB.22 In 20 vitro studies and an ex vivo human skin model have shown GzmB can cleave both E-cadherin and filaggrin in a dose dependent manner. E-cadherin and filaggrin cleavage and loss is inhibited when a GzmB inhibitor is incubated with GzmB prior to exposure to the substrates. With the loss of both E-cadherin and filaggrin, epithelial barrier integrity can be lost and an increase in disease severity can occur. GzmB was increased and filaggrin was lost in atopic dermatitis human lesional skin when compared to healthy, indicating GzmB may play a role in the loss of filaggrin in atopic dermatitis which was confirmed in a mouse model of induced dermatitis. In atopic dermatitis human lesions, mast cells were upregulated and expressed GzmB, indicating mast cells as a potential GzmB source in atopic dermatitis. In a dermatitis inflammation mouse model, WT and GzmB-/- mice were treated with oxazolone to induce a dermatitis like skin inflammation reaction. The mice model showed the GzmB-/- mice had less filaggrin loss when compared to the WT mice. The loss of filaggrin was also prevented when a GzmB inhibitor was used to treat the WT mice which were induced with oxazolone. Filaggrin plays a key role in maintenance of water content in the stratum corneum and trans-epidermal water loss has been seen in atopic dermatitis human lesions compared to the healthy human skin. A mouse model treated with oxazolone to induce a dermatitis like skin inflammation response, showed an increase in trans-epidermal water loss which was reduced in GzmB-/- mice. This further indicates the role GzmB plays in barrier function disruption. E-cadherin, which is found in all epidermal cell layers, is important for cell adhesion as well as regulating the organization of other types of intracellular junctions and tight junctions which contribute to epidermal barrier function. The same atopic dermatitis human lesional skin samples, which showed an increase in GzmB, also showed a decrease in E-cadherin when compared to the healthy skin samples. This indicates GzmB may play a role in the loss of E-cadherin in lesional atopic dermatitis samples which was confirmed in 21 a mouse model of dermatitis. In a dermatitis inflammation mouse model, WT and GzmB-/- mice were treated with oxazolone to induce a dermatitis like skin inflammation reaction. The mice model showed the GzmB-/- mice had less E-cadherin loss when compared to the WT mice. The loss of E-cadherin was also prevented when a GzmB inhibitor was used to treat the WT mice which were induced with oxazolone.22 However the role of GzmB in epithelial barrier dysfunction in RD needs to be explored. Decorin is an anti-fibrotic protein which is located in the extracellular matrix in the dermis of the skin. The loss of decorin can play a role in skin fibrosis.22 An in vitro model has shown GzmB can cleave decorin in a dose dependent manner.33 GzmB was increased and decorin was reduced in atopic dermatitis human lesional skin when compared to healthy, indicating GzmB may play a role in the loss of decorin in atopic dermatitis human lesional skin. This was confirmed in a mouse model of dermatitis. WT and GzmB-/- mice were treated with oxazolone to induce a dermatitis like skin inflammation reaction. The mice model showed the GzmB-/- mice had less decorin loss when compared to the WT mice treated with oxazolone.22 In a UV radiation model where WT mice were exposed to radiation for several weeks, GzmB was elevated in the skin of the WT mice with mast cells indicating a potential GzmB source. In the same UV radiation model where both WT and GzmB-/- mice were exposed to UV radiation, the levels of decorin were shown to be decreased in the WT UV treated mice and the decorin levels were rescued in the GzmB-/- UV treated mice.34 In a burn model where WT mice were treated with a GzmB inhibitor or a sham treatment, the levels of decorin were rescued in the GzmB inhibitor treated burns than the non-treated burns. The mice treated with the GzmB inhibitor showed a smaller scar area and a faster wound closer in the burn wound model.35 Cleavage of decorin allows for bioavailability of TGF-β. A study has shown that GzmB cleavage of decorin 22 leads to the release of active TGF-β. Extracellular matrix proteoglycans supplemented with TGF-β were exposed to GzmB. TGF-β was released from the extracellular matrix and found in the supernatant. The supernatants, containing TFG-β released from the extracellular matrix by GzmB, were then incubated with human coronary artery smooth muscle cells and found the phosphorylation and activation of Smad-3, indicating the TGF-β released from the extracellular matrix by GzmB is active.33 Active TGF-β can lead to fibrosis and scarring. Although a study has already looked at decorin levels in irradiated human breast stromal fibroblasts, decorin, and its contribution to fibrosis, needs to be further studied in RD.68 1.8 Granzyme B in Skin Diseases and Inflammation Extracellular GzmB can cleave a variety of substrates which can be found within the skin. Cleavage of these substrates can result in a variety of different physiological functions. Recently, GzmB-mediated cleavage of E-cadherin and filaggrin was observed in a mouse oxazolone-induced model of dermatitis. In this study both wild type (WT) and GzmB knockout (GzmB-/-) mice were subjected to a model of dermatitis. A significant reduction of E-cadherin and filaggrin was found in the WT mice, whereas the GzmB-/- mice exhibited less E-cadherin and filaggrin degradation.22 In psoriasis and atopic dermatitis, there is a significant higher concentration of GzmB then in control skin. However more studies need to explore the role of GzmB in psoriasis.96 GzmB also cleaves tight junctions ZO-1, JAM-A, and occludin in retinal pigment epithelial cells in vitro which suggests extracellular GzmB may contribute to blood-retinal barrier function.93 Cleavage of barrier proteins by GzmB can result in furthering disease severity and prolong inflammation and healing. 23 GzmB has been detected at high levels in cellular infiltrate and blister fluids of patients with autoimmune blistering disease and severe adverse drug reactions.97, 98 The dermal-epidermal junction is a basement membrane structure in the skin which anchors the epidermis to the dermis. The dermal-epidermal junction contains hemidesmosomes which include α6/β4 integrin and collagen XVII.99 In a study looking at GzmB in blistering diseases, GzmB was found elevated at the dermal-epidermal junction in tissue samples from patients with different blistering diseases, including epidermolysis bullosa acquisita, bullous pemphigoid, and dermatitis herpetiformis. Collagen XVII, collagen VII, and α6/β4 integrin were diminished in the different blistering tissue samples. This may have been due to the GzmB present in the blister samples since cleavage of α6/β4 integrin, collagen XVII and collagen VII by GzmB has been shown.88 In another study using WT and GzmB-/- mice in a epidermolysis bullosa acquisita blistering model, the GzmB-/- mice exhibited less blistering, and mitigated the loss of both type XVII collagen and α6 integrin.89 GzmB has also been found to be elevated in mice model of alopecia areata, which is an autoimmune, non-scarring, hair loss disease. The complete role of GzmB in alopecia areata needs to be further explored, however the cleavage of the extracellular matrix and autoantigen may contribute to the impaired hair follicle function in alopecia areata.100–102 The presence of GzmB in autoimmune and blistering diseases increases disease severity through contribution to the destruction of epidermal-dermal junctions and potentially the extracellular matrix, thus contributing to the disease progression. The cleavage of decorin has been observed in many chronic skin inflammatory conditions. A model of skin aging where levels of decorin were compared in WT and GzmB-/- mice found that GzmB was associated with decorin loss.36 In a mouse model of chronic UV exposure to WT and GzmB-/- mice showed that GzmB degrades decorin. 34 A mouse model of 24 delayed wound healing used both WT, APO-E knockout mice on a high fat diet (accelerates mice aging), and GzmB and APO-E double knockout mice on a high fat diet. The GzmB and APO-E double knockout mice exhibited faster wound closures then the APO-E knockout mice (accelerated aging model). The APO-E knockout mice displayed more degraded fibronectin, vitronectin, and decorin when compared to the GzmB and APO-E double knock out mice.90 In a diabetic mice wound model, the diabetic mice showed higher levels of GzmB when compared to WT mice. When the mouse GzmB inhibitor serpina3n was added to the excisional wounds in the diabetic mice, the wound closure, including re-epithelialization and contraction, was significantly accelerated.103 GzmB has also been identified in hypertrophic scars and keloids and decorin has been reported to be reduced with in these scars. The role of mast cells as the source of GzmB within hypertrophic scars and keloids has been hypothesized; however, more of the role of GzmB in hypertrophic scars and keloids needs to be studied.104–107 GzmB can cleave proIL-18 to the active proinflammatory cytokine IL-18 form.94,108 IL-18 increases production of IL-4 and IL-13 and enhances the Th2 response.109 IL-1α has also been shown to be a substrate of GzmB, and when cleaved, can have a higher proinflammatory potency.95 IL-8 is important for the activation and recruitment of neutrophils to the site of injury.89 In a mouse model of epidermolysis bullosa acquisita, extracellular GzmB mediated secretion of macrophages inflammatory protein-2, a murine homologue of IL-8. Since neutrophils are common in many inflammatory diseases, extracellular GzmB may contribute to increase in inflammation through neutrophil infiltration and activation through the ability of GzmB to activate secretion of IL-8. The increase in active TGF-β, through the cleavage of decorin, can contribute to further inflammatory response including IL-31, a cytokine which plays a major role in chronic itch.110 GzmB may contribute to inflammation also through activation of 25 IL-8 secretion, cleavage of IL-1α, and TGF- β activation through decorin cleavage. A summary of a few effects of extracellular GzmB in the skin can be found in Figure 3. Figure 3. Extracellular GzmB in the skin. Extracellular GzmB can be secreted by lymphocytes, natural killer cells (NK cells), macrophages, and mast cells. Extracellular GzmB can also: a) activate IL-8 which will recruit neutrophils to the skin and increase inflammation, b) activate IL-1α which can increase inflammation, c) cleave pro-IL-18 to active IL-18 which can increase inflammation, d) cleave filaggrin and E-cadherin which can cause an increase in barrier disruption. GzmB can cleave Epidermal-dermal junction proteins which can cause blistering as well as decorin which can result in collagen disorganization which can lead to fibrosis and scarring. Note: Image created based on Jung, K., Pawluk, M. et al. 2022.15 1.9 Rationale, Hypothesis and Aims RD is a common side effect of radiation therapy that is triggered by inflammation following exposure to ionizing radiation. The immune response in RD is characterized by an early neutrophil infiltration response, increase in macrophages and degranulation of mast cells.12,50,53 GzmB can be produced by macrophages and mast cells and is involved in other skin reactions including atopic dermatitis and ultraviolet radiation exposure.22,34,82 Inhibition of GzmB in murine models of inflammatory skin reactions such as dermatitis and ultraviolet 26 radiation exposure reduces severity, suggesting that targeting GzmB in skin diseases may be a potential therapeutic option. GzmB is also involved in the loss of both epidermal barrier proteins, E-cadherin and filaggrin, and an anti-fibrotic factor decorin in many skin diseases models including dermatitis and ultraviolet radiation. The loss of barrier function and increase in fibrosis are hallmarks for RD severity and thus GzmB may be a contributor to the loss of barrier function, through cleavage of barrier proteins, and through increase of fibrosis, through cleavage of anti-fibrotic proteins, in RD.22, 33, 34 In this thesis, the impact of GzmB is assessed in both human RD and a murine model of RD. This study will observe GzmB presence and expression from macrophages and mast cells in RD, the contribution to epithelial barrier dysfunction, and the reduction of RD severity through genetic deficiency of GzmB. A murine model with a single radiation dose of 40 Gy to the dorsal skin of GzmB-/- mice, observed over 4, 14, and 46 days was chosen to look at effects of RD severity, inflammation, and GzmB substrates based on previous murine RD models.111–114 I hypothesized that GzmB contributes to increased RD severity and delayed healing through the cleavage of epithelial barrier proteins and/or matrix proteins. My specific aims were: 1) To assess the location, level, source, and role of GzmB expression in human RD. 2) To evaluate the RD severity, immune cell infiltration, and decorin expression in WT and GzmB-/- mice induced with a model of RD 27 Chapter 2: Materials and Methods 2.1 Human Samples In accordance with Vancouver General Hospital (Vancouver, BC, Canada) research guidelines and policies, we obtained written informed consent from each participant in the study. All experimental procedures using human samples were approved by the University of British Columbia (UBC) Review Ethics Board. Skin from patients with RD was obtained from St. Paul’s Hospital, British Columbia, Canada, and Vancouver General Hospital Departments of Pathology and Laboratory Medicine, British Columbia, Canada (see Table 2 for RD patient information) with approval (H16-02161). Healthy skin was collected from a study where both healthy skin and burn skin was obtained from the University of British Columbia, Canada, and Vancouver General Hospital Burns Clinic, British Columbia, Canada, with approval (H15-03345 and H12-00540). Table 2. Table of RD patient information. Patient # VS14-06328 A3 VS15-16838 A4 VS16-39997 A4 11-27145 A1 VS14-05297 B1 12-35593 A1X2 Age (years) 75 79 75 84 50 44 Sex Male Female Male Male Female Female Location Scalp Neck Back Scalp Calf Shoulder RD stage Late acute/ early chronic Chronic Chronic Chronic Chronic Chronic 28 2.2 Hematoxylin and Eosin on Paraffin Embedded Human Skin Samples Five-micron sections of each skin tissue sample was deparaffinized through two washes of 100% xylene for 10 minutes. The slides where then incubated in absolute Isopropyl alcohol, 95% Isopropyl alcohol, and 70% Isopropyl alcohol for two minutes each. The slides were then washed in water for one minute before incubating in Hematoxylin for five minutes. The slides were washed in water for one minute before they were quickly dipped in 1% acid alcohol for differentiation. The slides were washed in water for 30 seconds before the slides were quickly dipped in lithium carbonate. The slides were then washed in water for one minute and checked under the microscope to make sure the nucleus of the cells were adequately stained. The slides were washed in water for another minute before being incubated in 70% Isopropyl alcohol for 30 seconds. The slides were then quickly dipped in 1% Eosin in 80% alcohol. The excess eosin is drained away and the slides are then incubated in 80% Isopropyl and 90% Isopropyl for 10 minutes, then finally incubated in absolute Isopropyl for one minute twice. The slides were then air dried and mounted with a coverslip then assessed by a PhD/MD dermatologist to determine the stage of the RD, either chronic or acute. 2.3 Immunohistochemistry on Paraffin Embedded Human Skin Samples Five-micron sections of each skin tissue sample was deparaffinized and rehydrated through three washes of 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of tris-buffered saline. Slides for immunohistochemistry were prepared by heating them for 15 mins in 80˚C citrate buffer (pH 6.0) and then cooled at room temperature for 30 minutes. The slides were blocked for one hour, at room temperature, in 10% goat, rabbit, or horse serum depending on the host of the secondary antibody used. Then the slides were incubated with the designated primary 29 antibody prepared in 10% of either goat, rabbit, or horse serum overnight at 4˚C. The antibodies used were rabbit anti human GzmB polyclonal antibody (Abcam, ab4059), mouse anti human CD68 (monocytes) monoclonal antibody (DAKO, Clone PG-M1), mouse anti human Mast Cell Tryptase monoclonal antibody (Bio Rad, MCA1438), rat anti human Neutrophil Marker NIMP-R14 monoclonal antibody (Santa Cruz, sc59338), goat anti human E-cadherin polyclonal antibody (R&D, AF748), rabbit anti human filaggrin polyclonal (Abcam, ab81468), and goat anti human Decorin polyclonal antibody (R&D, AF143) on human skin tissue samples. The next day the slides were incubated with biotinylated secondary antibody raised in the same animal species as the animal serum used for blocking, and in the primary antibody mix, and targeting the host of the first antibody. The secondary antibodies that were used were biotinylated goat anti rabbit secondary antibody (1:350-1:400,Vector Laboratories), rabbit anti goat secondary antibody (1:350-1:400, Vector Laboratories), goat anti rat secondary antibody (1:350-1:400, Vector Laboratories), and horse anti mouse secondary antibody (1:350-1:400, Vector laboratories) prepared in 5% of animal serum from the host of the secondary antibody, and incubated for 30 minutes at room temperature. The slides are then washed three times in tris-buffered saline and incubated with Vectastain Elite ABC reagent (Vector Laboratories) for 30 minutes. Then the slides are washed again in tris-buffered saline three times for five minutes and incubated with NovaRED substrate (Vector laboratories) until the desired staining intensity is obtained. Only one section sample from each tissue was stained for each immunohistochemistry stain. The slides were then counterstained with hematoxylin for 15-30 seconds and quantified using Aperio slide scanner and Aperio ImageScope (v12.4.3.5008) Positive Pixel Count V9 algorithm. All primary antibodies used are outlined in Table 3. 30 Table 3. Table of antibodies used for immunohistochemistry and immunofluorescence. Antibody Species Final concentration Source and catalog # GzmB Human Human Mouse 1/100 (IF) 1/300 (IHC) 1/200 (IHC) Abcam, ab4059 Abcam, ab4059 Abcam, ab4059 CD68 Human Human 1/200 (IHC) 1/100 (IF) DAKO, Clone PG-M1 DAKO, Clone PG-M1 Mast Cell Tryptase Human 1/1000 (IHC+IF) Bio Rad, MCA1438 Neutrophil marker Human Mouse 1/250 (IHC) 1/250 (IHC) Santa Cruz, sc-59338 Santa Cruz, sc-59338 CD3 Mouse 1/150 (IHC) Abcam, ab16669 E-cadherin Human 1/200 (IHC) R&D, AF748 Filaggrin Human 1/400 (IHC) Abcam, ab81468 Decorin Human Mouse 1/1000 (IHC) 1/2000 (IHC) R&D, AF143 R&D, AF1060 2.4 Immunofluorescence Double Staining on Paraffin Embedded Human Skin Samples Immunofluorescence was performed on the human RD skin tissues and control tissues mentioned above. Five-micron sections of each skin tissue sample was deparaffinized and rehydrated through three washes of 100% xylene, and a single wash of 100% ethanol, 90% ethanol, 70% ethanol, and two washes of tris-buffered saline. The slides were heated for 15 mins in 80˚C citrate buffer (pH 6.0) and then cooled at room temperature for 30 minutes. The slides were blocked for one hour, at room temperature, in 10% goat, rabbit, or horse serum depending on the host of the secondary antibody used. Then the slides were incubated with the designated primary 31 antibody prepared in 10% of either goat, rabbit, or horse serum overnight at 4˚C. The primary antibodies used was rabbit anti human GzmB polyclonal antibody (Abcam, ab4059). The next day the slides were incubated with biotinylated secondary antibody raised in the same animal species as the animal serum used for blocking, and in the primary antibody mix, and targeting the host of the first antibody. The secondary antibodies that were used were biotinylated goat anti rabbit secondary antibody (1:350-1:400, Vector Laboratories) prepared in 5% of animal serum from the host of the secondary antibody, and incubated for 30 minutes at room temperature. The slides are then washed three times in tris-buffered saline and incubated with Vectastain Elite ABC reagent (Vector Laboratories) for 30 minutes. Then added 1:50 dilute fluorescein Amplification Reagent (green fluorescent, 488nm absorbance) mixed in tris-buffered saline to the samples and incubated at room temperature for 5 minutes. The samples were then washed three times in tris-buffered saline. Prior to adding the next primary antibody, the samples were incubated with Avidin solution for 15 mins, washed with tris-buffered saline two times for two minutes, and then incubated with Biotin solution for 15 minutes. This step prevents the interaction with the second set of labeling reagents with the first set of labeling reagents. The samples were then washed again with tris-buffered saline two times for two minutes. The slides were blocked for one hour, at room temperature, in 10% goat, rabbit, or horse serum depending on the host of the secondary antibody used. Then the slides were incubated with the designated primary antibody prepared in 10% of either goat, rabbit, or horse serum overnight at 4˚C. The primary antibodies used for this second step were mouse anti human CD68 (monocytes) monoclonal antibody (DAKO, Clone PG-M1) and mouse anti human Mast Cell Tryptase monoclonal antibody (Bio Rad, MCA1438). The next day the slides were incubated with biotinylated secondary antibody raised in the same animal species as the animal serum used for blocking, and in the primary antibody mix, and targeting the 32 host of the first antibody. The secondary antibody that was used was horse anti mouse secondary antibody (1:350-1:400, Vector laboratories) prepared in 5% of animal serum from the host of the secondary antibody, and incubated for 30 minutes at room temperature. The slides are then washed three times in tris-buffered saline and incubated with Vectastain Elite ABC reagent (Vector Laboratories) for 30 minutes. Then added 1:50 dilute fluorescein Amplification Reagent (red fluorescent, 595nm absorbance) mixed in tris-buffered saline to the samples and incubated at room temperature for five minutes. The samples were then washed in tris-buffered saline for five minutes three times. DAPI (1:15000) was then applied to the samples for two minutes and 30 seconds. Finally, sections were rinsed, mounted and visualized using confocal imaging on Zeiss Axio Observer Z.1 laser scanning confocal microscope and images were captured using the Zen Software. All primary antibodies used are outlined in Table 3. 2.5 Masson’s Trichrome Staining on Paraffin Embedded Human Skin Samples Paraffin embedded human tissue sections were processed for Masson’s Trichrome staining at the UBC Center for Heart Lung Innovation (Providence Research, Vancouver, BC). Five-micron sections of each skin tissue samples was deparaffinized and rehydrated through three washes of 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of tris-buffered saline. The slides were washed in distilled water for one minute then left staining with Mordant in Bouin solution overnight. The slides were then washed under running tap water for one minute then washed in distilled water for one minute. The slides were then incubated in Weigert’s Hematoxylin working solution for 15 minutes. The slides were then washed under running tap water for 10 minutes then washed in distilled water for one minute. The slides were then incubated in Biebrich scarlet-acid fuchsin solution for one minute. The slides were then washed in distilled water for 33 three dips then incubated in phosphomolybdic, phosphotungstic acid solution, and distilled water (1:1:2 parts) for 10 minutes three times. The slides were then incubated in Aniline Blue solution for 10 minutes, then washed in distilled water for one quick dip. The slides were then incubated in 1% Acetic acid solution for four minutes. The slides were then incubated in 90% ethanol for one minute twice then incubated in 100% ethanol for one minute twice. The slides were then incubated in Xylene 100% for three minutes twice then mounted with cytoseal before imaging with Aperio slide scanner. 2.6 Laboratory Mice C57Bl/6 (WT) and GzmB knock out mice (GzmB-/-) mice with C57Bl/6 background were obtained from Jackson Laboratory (Bar Harbor, ME, USA). GzmB-/- mice were maintained as homozygous colonies or by continuous backcross to C57Bl/6 mice. WT and GzmB-/- mice were bred and housed in a 12h light/dark cycle with controlled room temperature at 20-26˚C and 40-60% relative humidity at the ICORD vivarium, UBC or the Genetic Engineered Models facility, St. Paul’s Hospital, UBC. All procedures were performed in accordance with the guidelines for animal experimentation approved by the Animal Experimentation Committee of University of British Columbia, Canada (#A17-0333). All radiation exposures took place at BC Cancer Research Center with all approvals for animal experiments and radiation safety training completed there. The animal model used was performed by Dr. Sho Hiroyasu in collaboration with the lab of Dr. Kevin Bennewith. Dr. Brennan Wadsworth performed all the murine irradiation 34 2.7 Animal Models Mice were subjected to a single 40 Gy radiation dose at about 20 weeks of age. The timeline for the animal model is outlined in Figure 4. Dorsal skin of both WT and GzmB-/- mice was shaved two days prior to irradiation. Shaved dorsal skin was stretched under anesthesia. The non-irradiation area was shielded with a lead shield and a radiation collimator which was used to irradiate a 0.5-1cm half circle of the skin. The dorsal skin was then irradiated with x-rays from a 300keV X-ray source (X-RAD 320). The mice were exposed to a single 40 Gy dose of radiation given with a rate of 5 Gy/min. WT and GzmB-/- mice are then monitored and scored every other day following a clinical scoring system adapted for mice from Douglas and Fowler (1976)115 , and described elsewhere.40 The mice were then euthanized at day 4 (N=4 per group), 14 (N=6 for WT, N=7 for GzmB-/-), and for a preliminary chronic study at day 46 (N=7 per group). The skin samples were collected for analysis. One mouse was euthanized two days early in the WT day 14 group at the veterinarian’s request. Based on preliminary results, approximately 20-30% of animals may not develop dermatitis.50 In order to perform a statistical analysis on the severity of dermatitis, 4 animals were needed per group. To get statistical significance 7 mice were irradiated per group for day 14 and 46, and 4 mice were irradiated per group for day 4. 35 Figure 4. Outline of animal model. Representative image of the timeline of the RD model and endpoints for the mouse study. 2.8 Immunohistochemistry on Mice Paraffin Embedded Skin Samples Five-micron sections of each skin tissue samples was deparaffinized and rehydrated through three washes of 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of tris-buffered saline. Slides for immunohistochemistry were prepared by heating them for 15 mins in 80˚C citrate buffer (pH 6.0) and then cooled at room temperature for 30 minutes. The slides were blocked for one hour, at room temperature, in 10% goat, rabbit, or horse serum depending on the host of the secondary antibody used. Then the slides were incubated with the designated primary antibody prepared in 10% of either goat, rabbit, or horse serum overnight at 4˚C. The antibodies used were rabbit anti mouse GzmB polyclonal antibody (Abcam, ab4059), rabbit anti mouse CD3 monoclonal antibody (Abcam, ab16669), rat anti mouse Neutrophil Marker NIMP-R14 monoclonal antibody (Santa Cruz, sc59338), and goat anti mouse decorin polyclonal antibody (R&D, AF1060) on mice skin tissue samples. The next day the slides were incubated with biotinylated secondary antibody raised in the same animal species as the animal serum used 36 for blocking, and in the primary antibody mix, and targeting the host of the first antibody. The secondary antibodies that were used were biotinylated goat anti rabbit secondary antibody (1:350-1:400, Vector Laboratories), rabbit anti goat secondary antibody (1:350-1:400, Vector Laboratories), and goat anti rat secondary antibody (1:350-1:400, Vector laboratories) prepared in 5% of animal serum from the host of the secondary antibody, and incubated for 30 minutes at room temperature. The slides are then washed three times in tris-buffered saline and incubated with Vectastain Elite ABC reagent (Vector Laboratories) for 30 minutes. Then the slides are washed again in tris-buffered saline three times for five minutes and incubated with NovaRED substrate (Vector laboratories) until the desired staining intensity is obtained. Only one section sample from each tissue was stained for each immunohistochemistry stain. The slides were then counterstained with hematoxylin for 15-30 seconds and quantified using Aperio slide scanner and Aperio ImageScope (v12.4.3.5008) Positive Pixel Count V9 algorithm. All primary antibodies used are outlined in Table 3. 2.9 Masson’s Trichrome Staining on Paraffin Embedded Mice Skin Samples Paraffin embedded mice tissue sections were processed for Masson’s Trichrome staining at the UBC Center for Heart Lung Innovation (Providence Research, Vancouver, BC). Five-micron sections of each skin tissue samples was deparaffinized and rehydrated through three washes of 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of tris-buffered saline. The slides were washed in distilled water for one minute then left staining with Mordant in Bouin solution overnight. The slides were then washed under running tap water for one minute then washed in distilled water for one minute. The slides were then incubated in Weigert’s Hematoxylin working solution for 15 minutes. The slides were then washed under running tap water for 10 37 minutes then washed in distilled water for one minute. The slides were then incubated in Biebrich scarlet-acid fuchsin solution for one minute. The slides were then washed in distilled water for three dips then incubated in Phosphomolybdic, Phosphotungstic acid solution, and distilled water (1:1:2 parts) for 10 minutes three times. The slides were then incubated in Aniline Blue solution for 10 minutes, then washed in distilled water for one quick dip. The slides were then incubated in 1% Acetic acid solution for four minutes. The slides were then incubated in 90% ethanol for one minute twice then incubated in 100% ethanol for one minute twice. The slides were then incubated in Xylene 100% for three minutes twice then mounted with cytoseal before imaging with Aperio slide scanner. 2.10 Toluidine Blue O on Paraffin Embedded Mice Skin Samples Five-micron sections of each skin tissue samples was deparaffinized and rehydrated through three washes of 100% xylene, 100% ethanol, 90% ethanol, 70% ethanol, and two washes of distilled water. The slides were then stained with Toluidine Blue O solution (pH 2.0-2.5) for two to three minutes. The slides were washed in water for five minutes three times. The slides were then air dried for two hours. The slides were then incubated in 100% ethanol for one minute two times. The slides were then incubated in xylene 100% and mounted with a cover slip. The slides were scanned using Aperio slide scanner. The slides were then left in Xylene 100% to allow the coverslips to be removed. Then the slides were used for GzmB immunohistochemistry staining as described above. The amount of TBO cells were manually counted on the Aperio ImageScope (v12.4.3.5008) and the area which was acquired in Aperio ImageScope (v12.4.3.5008). 38 2.11 Statistics All histology data were analyzed with nonparametric unpaired Wilcoxon test. The data is presented with median ± interquartile range with each individual score represented on the graph. The data is presented as a whisker and box plot which indicates all individual scores. The box plots indicate median ± interquartile range of the intensity levels of the immunohistochemistry stains in the epidermis for E-cadherin and filaggrin levels, epidermis and dermis for all cell levels, and dermis for decorin levels which is defined as the total intensity of staining/total area which was analyzed. The results are presented as relative to the median score of the healthy human skin samples or the wild type control samples. For the TBO slides, the data is presented as a whisker and box plot which indicates all individual scores. The box plots indicate median ± interquartile range of the number of cells in the epidermis and dermis defined as total number of cells/total area which was analyzed. The results are presented as relative to the median score of the wild type control samples. For the area of scar tissue in the mice skin, the data is presented as a whisker and box plot which indicates all individual scores. The box plots indicate median ± interquartile range of the total area which was analyzed as scar area within the dermis. The results are presented as relative to the median score of the wild type control samples. All severity scores were analyzed with Welch two sample t-test. P-values are represented as follows: p < 0.05 is denoted by *. p< 0.05 was considered significant in all experiments. All statistical analyses were performed in JMP Pro version 16 (JMP Statistical Discoveries Software, Cary, North Carolina) or analyzed using R (R Foundation for Statistical computing, Vienna, Austria). 39 Chapter 3: Results 3.1 GzmB Expression is Increased in Human RD Skin Samples Formalin fixed, paraffin embedded healthy human skin specimens were used to compare to the formalin fixed and paraffin embedded human RD skin specimens. An immunohistochemistry antibody specific for GzmB was used to determine GzmB levels. Significantly elevated GzmB levels (p<0.0051) were found in human RD skin tissues when compared to healthy human skin tissues (Figure 5a and 5b). GzmB positive cells were predominantly located within the dermis; however, a small cell population was located near the basal keratinocytes found within the epidermis at the dermal-epidermal junction. Figure 5. GzmB expression is increased in human RD skin samples. a) Representative GzmB immunohistochemistry image of healthy and RD human skin. GzmB positive cells are labelled with black arrows. Scale bars indicate 200 µm, N=6 per group. b) Quantification of GzmB positive intensity in healthy and RD human skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of GzmB in the epidermis and dermis defined as (total intensity of GzmB in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. 40 3.2 Infiltration of CD68 and Mast Cell Tryptase Expressing Cells is Increased in Human RD Skin Samples Macrophage levels were assessed using an anti-CD68 antibody. A significant increase in CD68 (p<0.0131) staining was observed in human RD skin samples compared to healthy skin samples (Figure 6a and 6b). Mast cells were assessed using an anti-Mast Cell Tryptase antibody. A significant increase was also observed in Mast Cell Tryptase (p<0.0306) staining in human RD skin samples compared to healthy skin samples (Figure 6c and 6d). The increase in both macrophages and mast cells indicated an influx in inflammatory cell recruitment in RD compared to controls. 41 Figure 6. Infiltration of CD68 and Mast Cell Tryptase expressing cells is increased in human RD skin. a) Representative CD68 (macrophage marker) immunohistochemistry image of healthy and RD human skin. CD68 positive cells are labelled with black arrows. Scale bars indicate 200 µm, N=6 per group. b) Quantification of CD68 positive intensity in healthy and RD human skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of CD68 in the epidermis and dermis defined as (total intensity of CD68 in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. c) Representative Mast Cell Tryptase (mast cell marker) immunohistochemistry image of healthy and RD human skin. Mast Cell Tryptase positive cells are labelled with black arrows. Scale bars indicate 200 µm, N=6 per group. b) Quantification of Mast Cell Tryptase positive intensity in healthy and RD human skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of Mast Cell Tryptase in the epidermis and dermis defined as (total intensity of Mast Cell Tryptase in epidermis and dermis)/(total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. 42 3.3 GzmB Staining Co-localizes to CD68 and Mast Cell Tryptase Staining in Human RD Skin Samples Macrophages and mast cells can express and secrete GzmB into the extracellular milieu.82, 83 Macrophages and mast cells are increased in human RD skin samples which also have an increase in GzmB staining (Figure 6a to 6d). To assess if the GzmB expressing cells were also CD68 (macrophage marker) or Mast Cell Tryptase (mast cell marker) expressing cells, in human RD, we used immunofluorescence and confocal microscopy to see if the GzmB staining co-localizes to the CD68 and Mast Cell Tryptase staining. Co-localization of GzmB with CD68 in human RD tissues was detected (Figure 7a), suggesting that macrophages are an important source of GzmB in human RD. Co-localization of GzmB with Mast Cell Tryptase was also detected and suggests that mast cells are an important source of GzmB in RD (Figure 7b). CD68 positive macrophages that co-localize to GzmB were found throughout the dermis. Many were located near the epidermis with a few in the basal layer of the epidermis. The majority of the GzmB positive cells were of macrophage (CD68) and of mast cell (Mast Cell Tryptase) origin. 43 Figure 7. GzmB staining co-localizes to CD68 and Mast Cell Tryptase in human RD skin. a) Representative confocal image of GzmB immunofluorescence (green) and CD68 immunofluorescence (red) overlaid in human RD skin at 40X magnification with a scale bar showing 50 µm, Blue = DAPI (nuclear stain) b) Representative confocal image of GzmB immunofluorescence (green) and Mast Cell Tryptase immunofluorescence (red) overlaid in human RD skin at 40X magnification with a scale bar showing 50 µm, Blue = DAPI (nuclear stain). 3.4 Infiltration of Neutrophil Cells is not Increased in Human RD Skin Samples Neutrophils are the first infiltrated immune cell to arrive to the irradiated site. Neutrophils and mast cells have a close relationship in wound healing and progressing the immune response.48 Since mast cells are present in RD, we investigated the levels of neutrophils in our human RD skin samples compared to our healthy human skin samples. The neutrophil levels were analyzed using immunohistochemistry. There was no significant increase in neutrophil staining in the human RD skin samples when compared to the healthy skin samples (Figure 8a and 8b). The lack of increase in neutrophils could hint that the human RD skin samples are showing a later stage of inflammation response since we see macrophages and mast cells elevated with a lack of neutrophils. 44 Figure 8. Infiltration of neutrophil cells is not increased in human RD skin. a) Representative neutrophil immunohistochemistry image of healthy and RD human skin. Neutrophil cells are labelled within the black box. Scale bars indicate 200 µm, N=6 per group. b) Quantification of Neutrophil staining intensity in healthy and RD human skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of neutrophils in the epidermis and dermis defined as (total intensity of neutrophil staining in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. 3.5 Epidermal Barrier Proteins, E-cadherin and Filaggrin, are Reduced in Human RD Skin Samples E-cadherin and filaggrin play a role in barrier function, and are substrates of GzmB. Thus, the levels of epidermal barrier proteins, E-cadherin and filaggrin, were explored in human RD skin tissue samples. A significant reduction of E-cadherin (p<0.0202) was found in human RD skin tissues when compared to healthy human skin tissue (Figure 9a and 9b). There was also a significant reduction in filaggrin (p<0.0453) in human RD skin tissues when compared to healthy human skin tissues (Figure 9c and 9d). 45 Figure 9. Epidermal barrier proteins, E-cadherin and filaggrin, are reduced in human RD skin. a) Representative E-cadherin immunohistochemistry image of healthy and RD human skin. Positive E-cadherin staining is labelled with black arrows. Scale bars indicate 200 µm, N=6 per group. b) Quantification of E-cadherin positive intensity presented as a dot plot which indicates all individual scores and box plots indicates median ± interquartile range of the intensity levels of E-cadherin in the epidermis defined as (total intensity of E-cadherin in epidermis)/ (total area of examined epidermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. c) Representative filaggrin immunohistochemistry image of healthy and RD human skin. Positive filaggrin staining is labelled with black arrows. Scale bars indicate 200 µm, N=6 per group. d) Quantification of filaggrin positive intensity presented as a dot plot which indicates all individual scores and box plots indicates median ± interquartile range of the intensity levels of filaggrin in the stratum corneum and stratum granulosum defined as (total intensity of filaggrin in stratum corneum and stratum granulosum)/ (total area of examined stratum corneum and stratum granulosum) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. 46 3.6 Extracellular Matrix Protein, Decorin, is Reduced in Human RD Skin Samples Since decorin is an established GzmB substrate and scarring is a major side effect of radiation exposure, the levels of decorin were explored in human RD skin tissue samples. There was a significant difference (p=0.0051) in the levels of decorin in human RD skin tissues when compared to healthy human skin tissues (Figure 10a and 10b). Figure 10. Extracellular matrix protein, decorin, is reduced in human RD skin samples. a) Representative decorin immunohistochemistry image and Masson’s trichrome staining image of healthy and RD human skin. Scale bars indicate 200 µm, N=6 per group. b) Quantification of decorin positive intensity presented as a dot plot which indicates all individual scores and box plots indicates median ± interquartile range of the intensity levels of decorin in the dermis defined as (total intensity of decorin in the dermis)/ (total area of examined dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the healthy human skin. Wilcoxon test presented, *p<0.05, N=6 per group. 47 3.7 GzmB-/- Mice Exhibit Reduced RD Severity Compared to WT Mice after Radiation Exposure To determine if removing GzmB would affect the severity of RD in a murine model, both WT and GzmB-/- mice dorsal skin were exposed to a single 40 Gy dose of radiation. The severity of the RD was scored blindly every other day by a MD/PhD dermatologist. Representative images of the dorsal skin of WT and GzmB-/- mice over days 2 to 14 post-radiation exposure show decreased RD severity in the GzmB-/- mice at days 4, 6, 8, 10, and 12 post-radiation exposure when compared to WT mice (Figure 11a). There is a statistical significant decrease in RD severity in the GzmB-/- mice at day 4 (p<0.03339), day 6 (p<0.01596), day 8 (p<0.007776), and day 12 (p<0.01408) when compared to WT mice (Figure 11b). 48 Figure 11. GzmB-/- mice exhibit reduced RD severity compared to WT mice skin after radiation exposure. a) Representative images of the dorsal skin of WT and GzmB-/- mice at day 2, 4, 6, 8, 10, 12, and 14 post exposure to a single 40 Gy radiation dose. Severity scores for each mouse at each time point is indicated in the top left corner b) Quantification of RD severity in WT and GzmB-/- mice scored every other day from days 0-14 post a single 40 Gy radiation exposure. Individual WT scores are indicated with a blue marker and individual GzmB-/- scores are indicated with a yellow marker. N=18 from days 0-4 for both groups, N=14 for both groups from days 5-10, then N=14 for GzmB-/- group on day 12-14 and N=13 for WT group for days 12-14. Welch two sample t-test was applied and *p-value<0.05. 3.8 GzmB Levels are Elevated in Mice Skin Post-radiation Exposure Since GzmB was elevated in human RD, the first step was to evaluate the presence of GzmB in mice RD skin at days 4 and 14 post-radiation exposure. Formalin fixed, paraffin embedded WT mice dorsal skin 4 days and 14 days post exposure to a single 40 Gy radiation dose was used to compare to the formalin fixed, paraffin embedded, non-irradiated mice dorsal skin. An immunohistochemistry antibody specific for GzmB was used to determine GzmB levels 49 and a significant elevation of GzmB (p<0.0081) was found in WT mice skin 14 days post-radiation exposure when compared to non-irradiated mice skin (Figure 12a and 12b). Most of the GzmB positive cells were located within the dermis. A small population GzmB positive cells were detected near the basal keratinocytes found at the dermal-epidermal junction. The location of GzmB positive cells in the irradiated mouse skin corresponded to the location of GzmB in the human RD skin samples (Figure 5a). Figure 12. GzmB levels are elevated in mice skin post-radiation exposure. a) Representative GzmB immunohistochemistry image of WT control skin, WT 4 days post-radiation exposure skin, and WT 14 days post-radiation exposure skin. GzmB positive cells are labelled with black arrows. Scale bars indicate 200 µm. b) Quantification of GzmB positive intensity in WT control, WT day 4, and WT day 14 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of GzmB in the epidermis and dermis defined as (total intensity of GzmB in epidermis and dermis)/(total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the WT control skin. Wilcoxon test presented, *p<0.05, WT control N=5, WT day 4 N=4, and WT day 14 N=6. 50 3.9 GzmB Staining Overlays with TBO Staining in WT Mice Skin Post-radiation Exposure Since mast cells are abundant and co-localize to GzmB in human RD, we analyzed if the GzmB positive cells were also TBO positive cells (mast cells) by staining TBO then GzmB on the same tissue sample for mice RD skin at days 4 and 14 post-radiation exposure. Formalin fixed, paraffin embedded WT mouse dorsal skin 4 days and 14 days post exposure to a single 40 Gy radiation dose was used to compare to the formalin fixed, paraffin embedded, control, non-irradiated mice skin. An immunohistochemistry antibody specific for GzmB was used to determine GzmB expression and TBO was used to determine mast cell localization. Comparing the two staining techniques, there is an overlap of both GzmB and TBO staining in the WT mice skin 14 days post-radiation exposure (Figure 13). GzmB positive and TBO positive cells were predominantly located within the dermis of the mice skin. 51 Figure 13. GzmB staining overlays with TBO (mast cell) staining in WT mice skin post-radiation exposure. a) Representative sequential GzmB immunohistochemistry and toluidine blue O (TBO) stained image of WT control skin, WT 4 days post-radiation exposure skin, and WT 14 days post-radiation exposure skin. TBO stains mast cells dark blue to purple. Cells stained with TBO are labelled with black arrows and the red boxes indicate overlapping GzmB and TBO staining. Scale bars indicate 200 µm. 3.10 Mast Cells are Present in WT and GzmB-/- Mice 4 and 14 Days Post-radiation Exposure To assess the infiltration of mast cells, WT and GzmB-/- mice skin 4 days and 14 days post exposure to a single 40 Gy radiation dose was used to compare to non-irradiated WT and GzmB-/- mice skin. TBO staining for mast cells was used to determine mast cell levels and showed no significant increase in infiltration of mast cells in WT or GzmB-/- mice skin 4 days and 14 days post-radiation exposure when compared to non-irradiated mice skin (Figure 14a and 14b). However, there was a trend (p=0.0741) of fewer mast cells in the GzmB-/- mice skin when 52 compared to the WT mice skin at day 14. The lack of an increase in mast cell infiltration in this mouse model is not similar to the infiltration of mast cells observed in human RD (Figure 6c and 6d), however the mice samples represent acute RD at an early time point and the human samples represent chronic RD at later time points and this may indicate why there is a difference in mast cell infiltration. 53 Figure 14. Mast cells are present in WT and GzmB-/- mice skin post-radiation exposure. a) Representative toluidine blue O (TBO) stained images of WT and GzmB-/- control skin, WT and GzmB-/- skin 4 days post-radiation exposure, and WT and GzmB-/- skin 14 days post-radiation exposure. TBO stains mast cells dark blue to purple. Cells stained with TBO are labelled with black arrows. Scale bars indicate 200 µm. b) Quantification of TBO positive cells in WT and GzmB-/- mice control skin, WT and GzmB-/- day 4 mice skin, and WT and GzmB-/- day 14 mice skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the cell counts of TBO in the epidermis and dermis defined as (total cells of TBO in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from TBO images and presented as relative to the median score of the WT control skin. Wilcoxon test presented, *p<0.05, WT control N=5, GzmB-/- control N=3, WT and GzmB-/- day 4 N=4, and WT day 14 N=6, GzmB-/- day 14 N=7. 54 3.11 WT and GzmB-/- Mice Skin May Exhibit a Decrease of CD3+ T-cell Presence in the Skin Post-radiation Exposure CD3+ T-cells are known to produce GzmB and are an infiltrated immune cell in many acute wound healing models. Thus, we wanted to analyze the levels of CD3+ T-cells in mice RD skin at days 4 and 14 post-radiation exposure. WT and GzmB-/- mice skin 4 days and 14 days post exposure was used to compare to non-irradiated WT and GzmB-/- mice skin. An immunohistochemistry antibody specific for CD3+ T-cells was used to determine CD3+ T-cell levels. A significant reduction of CD3+ T-cells in WT mice dorsal skin at 4 days (p=0.0373) and 14 days (p=0.0225) post-radiation exposure was observed when compared to non-irradiated mice skin. A significant reduction of CD3+ T-cells in GzmB-/- mice dorsal skin at 4 days (p=0.0304) post-radiation exposure was observed when compared to non-irradiated mice skin (Figure 15a and 15b). However, there was only a trend (p=0.0682) of less CD3+ T-cells in the GzmB-/- mice skin at day 14 when compared to the non-irradiated GzmB-/- mice skin. 55 Figure 15. WT and GzmB-/- mice skin may exhibit a decrease in CD3+ T-cell presence post-radiation exposure. a) Representative CD3 (T-cell marker) immunohistochemistry staining images of WT and GzmB-/- control skin, WT and GzmB-/- 4 days post-radiation exposure skin, and WT and GzmB-/- 14 days post-radiation exposure skin. CD3-positive cells are labelled with black arrows. Scale bars indicate 200 µm. b) Quantification of CD3-positive intensity in WT and GzmB-/- control, WT and GzmB-/- day 4, and WT and GzmB-/- day 14 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of CD3 in the epidermis and dermis defined as (total intensity of CD3 in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the WT control skin. Wilcoxon test presented, *p<0.05, WT control N=5, GzmB-/- control N=3, WT and GzmB-/- day 4 N=4, and WT day 14 N=6, GzmB-/- day 14 N=7. 56 3.12 WT and GzmB-/- Mice may Exhibit an Infiltration of Neutrophils in the Skin 14 Days Post-radiation Exposure Since neutrophils are a hallmark of RD and is the first infiltrated cell to the irradiated site, we wanted to analyze the levels of neutrophil cells in mice RD skin at days 4 and 14 post-radiation exposure when compared to non-irradiated mice skin.12 Formalin fixed, paraffin embedded WT and GzmB-/- mice skin 4 days and 14 days post exposure to a single 40 Gy radiation dose was used to compare to non-irradiated WT and GzmB-/- mice skin. Neutrophil immunohistochemistry staining was used to determine neutrophil levels and showed a significant elevation of neutrophils in WT and GzmB-/- mice skin 14 days post-radiation exposure (p=0.0081 and p=0.0227) when compared to non-irradiated mice skin. Additionally, a significant elevation of neutrophils were found in WT mice skin 4 days (p=0.0200) post-radiation exposure when compared to non-irradiated mice skin (Figure 16a and 16b). However there was a trend (p=0.0741) of fewer neutrophils in the GzmB-/- mice skin when compared to the WT mice skin at day 14. 57 Figure 16. WT and GzmB-/- mice may exhibit an infiltration of neutrophils in the skin 14 days post-radiation exposure. a) Representative neutrophil immunohistochemistry staining images of WT and GzmB-/- control skin, WT and GzmB-/- 4 days post-radiation exposure skin, and WT and GzmB-/- 14 days post-radiation exposure skin. Neutrophil cells are labelled with black arrows. Scale bars indicate 200 µm. b) Quantification of neutrophil staining intensity in WT and GzmB-/- control, WT and GzmB-/- day 4, and WT and GzmB-/- day 14 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of neutrophil staining in the epidermis and dermis defined as (total intensity of neutrophil staining in epidermis and dermis)/(total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the WT control skin. Wilcoxon test presented, *p<0.05, WT control N=5, GzmB-/- control N=3, WT and GzmB-/- day 4 N=4, and WT day 14 N=6, GzmB-/- day 14 N=7. 58 3.13 GzmB-/- Mice Exhibit Increased Decorin Levels and Reduced Scarring in the Skin 46 Days Post-radiation Exposure. Since fibrosis and scarring is a hallmark of chronic RD, we wanted to analyze scarring in a preliminary RD mice model which was brought out to day 46. WT mice skin 46 days post exposure to a single 40 Gy radiation dose was used to compare to the GzmB-/- mice skin 46 days post exposure to a single 40 Gy radiation dose. Scar area was determined in the dermis of the skin by looking for an increase in collagen fibers lined up parallel to one another indicating collagen disorganization, a potential increase in the thickness of the epidermis indicting injury, and a lack of hair follicles and glands indicating scar tissue. These features were observed using Masson’s trichrome staining and the scarring area in the dermis was outlined and measured. There was a significantly smaller (p=0.0073) scar area in the GzmB-/- mice skin 46 days post-radiation exposure when compared to WT mice skin 46 days post-radiation exposure (Figure 17a and 17b). Since decorin loss can lead to scarring and is a GzmB substrate, we wanted to analyze the levels of decorin in GzmB-/- mice skin 46 days post-radiation exposure when compared to WT mice skin at 46 days post-radiation exposure. Immunohistochemistry methods was used to determine decorin levels and showed that the GzmB-/- mice skin 46 days post-radiation exposure had significantly increased (p=0.0409) decorin staining then the WT mice skin 46 days post-radiation exposure (Figure 17a and 17c). 59 Figure 17. GzmB-/- mice exhibit smaller scar area and more decorin in the skin 46 days post-radiation exposure. a) Representative decorin immunohistochemistry staining and Masson’s trichrome staining images of WT and GzmB-/- skin 46 days post-radiation exposure. Area of scar is outlined by a black dotted line. Scale bars for images at 1X indicate 3mm and scale bars for images at 20X indicate 200 µm. b) Quantification of the scar area in WT and GzmB-/- day 46 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the scar area in the dermis defined as (total scar area in the dermis) which were quantified from immunohistochemistry and Masson’s trichrome staining images and presented as relative to the median score of the WT skin. Wilcoxon test presented, *p<0.05, WT day 46 N=7, GzmB-/- day 46 N=7. c) Quantification of decorin staining intensity in WT and GzmB-/- day 46 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of decorin staining in the dermis defined as (total intensity of decorin staining in dermis)/ (total area of examined dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the WT skin. Wilcoxon test presented, *p<0.05, WT day 46 N=7, GzmB-/- day 46 N=7. 60 3.14 Neutrophil Infiltration is not Observed in WT and GzmB-/- Mice at Day 46 Neutrophil levels were assessed in WT and GzmB-/- skin 46 days post-radiation exposure and compared the levels to the WT and GzmB-/- control skin. There was no significant increase in neutrophil staining in the WT and GzmB-/- skin 46 days post-radiation exposure when compared to the WT and GzmB-/- control skin (Figure 18a and 18b). The lack of increase in neutrophils hints that the inflammation in the WT and GzmB-/- day 46 skin is at a later stage. This is similar to the human RD results. 61 Figure 18. Neutrophil infiltration is not observed in WT or GzmB-/- mice at day 46. a) Representative neutrophil immunohistochemistry staining images of WT and GzmB-/- control skin and WT and GzmB-/- skin 46 days post-radiation exposure. Scale bars indicate 200 µm b) Quantification of neutrophil staining intensity in WT and GzmB-/- control and WT and GzmB-/- day 46 skin presented as a dot plot which indicates all individual scores and box plots indicate median ± interquartile range of the intensity levels of neutrophils in the epidermis and dermis defined as (total intensity of neutrophil staining in epidermis and dermis)/ (total area of examined epidermis and dermis) which were quantified from immunohistochemistry images and presented as relative to the median score of the WT control skin. Wilcoxon test presented, *p<0.05, WT control N=5, GzmB-/- control N=3, WT and GzmB-/- day 46 N=7 each. 62 Chapter 4: Discussion RD is a common side effect of radiation therapy and can limit a patient’s ability to continue receiving radiation therapy if the RD gets too severe. Since there is a lack of current universally agreed upon therapy for reducing the severity of RD, a therapy which will help mitigate the severity of RD is crucial.2, 13 GzmB was previously investigated in numerous skin diseases as a protease affecting barrier function and wound healing through the cleavage of E-cadherin, filaggrin and decorin. The loss of barrier proteins increase trans-epidermal water loss while the absence of decorin may contribute to scarring, both of which may contribute to RD disease severity.12,22,33,65 However, there has been no current research to assess GzmB as a potential therapeutic target in RD. In this thesis, the levels, location, source, and role of GzmB are explored in human RD skin samples. A RD mouse model, where murine dorsal skin was exposed to a single large dose of radiation, was selected to investigate the benefit of GzmB deletion in mice. Detecting the presence of GzmB in human RD skin is an important first step. Elevated GzmB levels were present in human RD skin when compared to healthy human skin controls. Some of the GzmB staining has been observed amongst the basal cells of the epidermis, however most of the GzmB staining is found in the dermis. The location and elevation of GzmB is similar in atopic dermatitis lesional skin where GzmB positive cells were found throughout the dermis, especially in regions of inflammatory infiltrated cells.22 In human skin blistering samples of bullous pemphigoid, dermatitis herpetiformis, and epidermolysis bullosa acquisita, the level and location of GzmB was observed. GzmB was elevated in the upper dermis, at the dermal epidermal junction in areas of increased neutrophils, and a few GzmB positive cells were found in the epidermis.88 In human skin pressure injury, positive GzmB cells were found distributed 63 throughout the dermis with a few GzmB positive cells found in the epidermis.116 This finding is similar to the location we observed in our human RD skin samples. However, it appears that in different diseases the location where the majority of GzmB is observed may change. In the blistering diseases GzmB was found in the upper dermis, within the blisters, and at the epidermal-dermal junction. In RD skin the GzmB positive cells were found evenly distributed among the dermis.88 This could be caused by the cell types expressing GzmB or due to the site of injury within the skin and will require future investigation. After radiation exposure, monocytes are recruited to the site of injury and are then differentiated into macrophages once they reach the irradiated tissue.51 Mast cells release a variety of proteolytic enzymes, after radiation exposure, which contribute to tissue damage and RD.52, 53 This suggests mast cells may be a large contributor to RD severity through tissue damage from their degranulated products. Since macrophages and mast cells are elevated in RD and contribute to GzmB production, the levels of macrophages and mast cells in human RD were assessed. There was a significant increase in CD68 and Mast Cell Tryptase staining in human RD skin tissues when compared to healthy human skin tissues. The increase in macrophages and mast cells indicates an increase in inflammatory cell recruitment. The mast cells and macrophages appear to be found all over the dermis, there are even some macrophages and mast cells in the dermis by the dermal-epidermal junction. In a previous study, the human mast cell line (Human leukemic mast cell line, HMC-1) was exposed to a 5 Gy dose of radiation and showed an increase in tryptase release from the mast cell granules.53 In an ex vivo human skin model of RD, where normal human skin was exposed to a 5 Gy dose of radiation, an increase in mast cell degranulation was observed in the dermis.53 This demonstrates how radiation exposure can affect mast cells and cause mast cell degranulation which could increase inflammation and 64 contribute to RD development. Mast cells were also found elevated in pressure injuries and were located within the dermis, similar to our human RD skin samples. The human pressure injury samples also showed a subpopulation of CD68 positive cells located in the dermis, close to the epidermis, which is similar to what is found in the human RD samples.116 However, more CD68 positive cells were observed to be spread throughout the dermis in RD tissues than the pressure injury tissues. This indicates a potential difference in location of recruited cells which could be based on the type of injury to the skin or the time point since the initial injury. These findings show that macrophages and mast cells are present in human RD tissue samples and are located within all parts of the dermis. We have shown that macrophages are increased in human RD skin tissues and some studies have shown that GzmB can be expressed from macrophages as discussed previously.82 Thus, we investigated if the macrophages found in human RD skin tissues also express GzmB. A proportion of the GzmB positive cells co-localized to the CD68 marker, indicating macrophages are a potential source of GzmB in human RD. The majority of the GzmB positive cells that were observed were CD68 positive. CD68 positive macrophages that co-localize to GzmB were identified throughout the dermis. The majority were located near the epidermis with a few within the basal layer of the epidermis. The co-localization of CD68 to GzmB was also shown in human pressure injury skin.116 The human pressure injury samples showed a subpopulation of cells which was CD68 positive that co-localized with GzmB, however in contrast to the RD results, majority of the CD68 positive cells were not expressing GzmB in the pressure injury samples.116 We have also shown that mast cells are increased in our human RD skin tissues. Thus, we wanted to investigate if the mast cells found in the human RD skin tissues also express GzmB. Some of the GzmB positive cells co-localized to the Mast Cell Tryptase marker, indicating mast 65 cells are a potential source of GzmB in human RD. Mast Cell Tryptase positive cells that co-localized to GzmB were found throughout the dermis. Many were located near the epidermis. Many of the GzmB positive cells were positive for Mast Cell Tryptase with only a few mast cells not expressing GzmB suggesting there may be different subsets of mast cells present. The findings share similarities with human pressure injuries and lesional human atopic dermatitis skin where all of the mast cells expressed GzmB and were the main source of GzmB.22,116 The location of the GzmB positive mast cells in our RD human skin samples is comparable with GzmB positive mast cell location in lesional atopic dermatitis human skin samples, which found GzmB positive mast cells to be located throughout the dermis and specifically increased at sites where immune infiltration was found.22 Macrophages and mast cells contribute to GzmB production and secretion in human RD skin tissues and the GzmB producing macrophages and mast cells are located throughout the dermis. Mast cell degranulation can recruit neutrophils to the irradiated site through release of IL-8.48 Since mast cells can influence neutrophil recruitment and activation, we wanted to observe the neutrophil levels in our human RD skin samples. We did not see many neutrophils within the human RD skin and there was no difference in neutrophil levels in the human RD skin when compared to the healthy human skin. However, since the samples were observed to be chronic RD by a dermatologist-scientist with pathology training, the inflammation is expected to be in a late stage of inflammation. Thus the lack of neutrophils is expected as neutrophil infiltration tends to wind down after acute inflammatory response.117 The human RD skin samples are only a snap shot of one point in the progression of RD and thus we would need to observe human skin effected by radiation at an earlier time point in wound healing to observe the neutrophil mast cell interaction in human RD. 66 A major function of the skin is to provide a barrier to the external environment. E-cadherin assists in cell-cell adhesion and thus contributes to barrier function. E-cadherin can be cleaved by GzmB. GzmB mediated loss of E-cadherin is observed in many barrier dysfunction diseases, including atopic dermatitis and blood-retinal barrier dysfunction.22,93 Thus, we explored the levels of E-cadherin in human RD skin and found a significant decrease in E-cadherin staining in the human RD skin when compared to healthy human skin. In acute RD, the loss of E-cadherin was observed in a murine RD model.20 The study also suggests that although keratinocyte apoptosis may contribute to the loss of E-cadherin, most keratinocytes remained viable post-radiation exposure. This suggests the loss of E-cadherin from radiation exposure is not due to keratinocyte apoptosis but from factors which target E-cadherin degradation.20 The study also observed reduced E-cadherin staining in human RD samples, which supports our findings. The study obtained human skin samples from head and neck cancer patients after going through 10 or 23 fractions of 2 Gy dose of radiation. They looked at levels of E-cadherin in the RD tissues using immunofluorescence. After 10 fractions of 2 Gy dose (total dose is 20 Gy, over 2 weeks of treatment) there was no observed loss of E-cadherin. However, after 23 fractions of 2 Gy radiation (total dose is 46 Gy, over 4 weeks of radiation) E-cadherin loss is observed, however, not quantified. 20 In this thesis, I analyzed and quantified the loss of E-cadherin to observe the magnitude which E-cadherin is lost due to radiation exposure. By doing this I was able to detect a 76% loss in E-cadherin staining in the human RD skin samples compared to the healthy skin samples. Filaggrin is an important protein within the stratum corneum that plays a role in epithelial barrier function which prevents trans-epidermal water loss. Trans-epidermal water loss has been detected in RD patients and is an indication of barrier dysfunction.18,63 Since GzmB cleaves 67 filaggrin and is present in barrier dysfunction disease, like human atopic dermatitis, we looked at the level of filaggrin in human RD skin tissues.22 There was a significant decrease of filaggrin staining in the human RD skin when compared to healthy human skin. The loss of E-cadherin and filaggrin in RD is similar to the loss of E-cadherin and filaggrin in lesional atopic dermatitis where GzmB was also elevated.22 The loss of E-cadherin and filaggrin appeared to be located where GzmB is elevated in RD. This indicates the role of GzmB in epithelial barrier disruption through the proteolytic cleavage of E-cadherin and filaggrin in the skin. Fibrosis is a common chronic effect of RD and can be characterized by dermal thickening, scarring, atrophy, and hair loss.51 Decorin is an extracellular matrix protein which is anti-fibrotic through its ability to inhibit the bioavailability of TGF-β.33 The loss of decorin has not been well studied in RD. However, in an in vitro RD model, a down-regulation of decorin at the mRNA and protein level was observed in γ-irradiated human breast stromal fibroblasts.68 Thus, the levels of decorin was explored in human RD skin. There was a significant decrease of decorin in the human RD skin when compared to healthy human control skin. The loss of decorin in human RD skin samples, which have an increase in GzmB, was expected since lesional atopic dermatitis human skin samples showed a loss of decorin and an elevation of GzmB.22 In human pressure skin injury, where GzmB was elevated, almost a complete loss of decorin was observed in the dermis. This supports our findings in our RD human skin samples. Collagen disorganization was also observed in pressure injuries. An increase in collagen disorganization was observed in pressure injury skin compared to control and further confirms the loss of decorin, through GzmB cleavage, can lead to poor wound healing and greater severity if injury.116 Collagen organization may be of interest for future directions within RD human skin tissues to further explore delayed wound healing and fibrosis. 68 WT and GzmB-/- mice were induced with a model of RD to evaluate the RD severity, immune cell infiltration, and decorin expression. Since we wanted to explore both acute and chronic RD in a mouse model, a more severe phenotype was desired and thus we followed models from other RD studies.111, 113, 114 A large single 40 Gy dose can produce a severe RD phenotype in an established mouse RD model. One model irradiated mice limbs with different doses of radiation to indicate which dose was required for 50% of the mice to develop moist desquamation and severe fibrosis. 35.8 Gy was required for 50% of the mice to display moist desquamation, while 39.8 Gy was required for 50% of the mice to develop severe fibrosis.111 In another mouse RD model, hind limbs of mice were irradiated with either fractionated doses or a single doses of radiation. This was done to compare which radiation exposure produced a greater RD phenotype. The single 40 Gy dose of radiation caused the mice ankles to be more swollen from 2-4 weeks post-radiation exposure compared to a single 20 Gy radiation exposure or fractionated radiation exposures.112 Another RD mouse model irradiated mouse dorsal skin with a single dose of 2, 10, 20, 40, or 80 Gy to establish the best dose to test their proteomic experiments. The model showed that a single 40 Gy dose was ideal since it induced phenotypical outcomes that are close to the clinical observations in human RD. The 40 Gy dose showed moist desquamation over the entire irradiated area with a few zones of necrosis by three weeks post-radiation exposure. Wounds were completely healed by day 28 and a noticeable scar could be observed.113 Another mouse RD model came to a similar conclusion to continue with a single exposure of 45 Gy radiation dose on mouse dorsal skin for their RD models. This study compared a single exposure of either 8.5, 17, 25, or 45 Gy. They found 45 Gy developed the most severe RD starting at day five. The study claims that a single 45 Gy was used for their future studies since it showed clinical effects in the skin, such as desquamation and erythema.40 69 Thus, we chose to irradiate the mice with a single 40 Gy dose to the dorsal skin of the mice. To find a good time point for ending the study, we wanted to wait long enough to see severe RD with clinical observation but not lose the inflammation response for histological applications. We looked at other RD mouse models where a large RD severity was observed and inflammatory response was still present. In a mouse RD model where mouse dorsal skin was irradiated with a single 40 Gy dose, higher severity scores were observed in irradiated skin from day 7-14.113 In another mouse RD model where the mouse dorsal skin was irradiated with a single 40 Gy dose of radiation, RD severity was prominent at day 14, trans-epidermal water loss was increased at day 14, and inflammation was still present .114 Thus day 14 was chosen as the endpoint for acute RD mouse model looking at both WT and GzmB-/- mice. Day 4 was chosen to see an earlier time point within acute RD to capture early GzmB expression from early infiltrated cells. To determine an end point for the preliminary chronic RD mouse model we looked at chronic RD mouse models in literature. As mentioned, a previous study showed 39.8 Gy was required for 50% of the mice to develop severe fibrosis so a 40 Gy dose was used in our study.111 In a mouse model where the right hind leg is irradiated with a single 30 Gy exposure, TGF-β and fibrosis can be seen in the skin 90 days post-radiation exposure.41 Another model showed that 40 Gy was an ideal dose since it induced clinical outcomes that are close to the clinical observations. In this model wounds were completely healed by day 28 and a noticeable scar can be observed.113 However in another mouse model of RD where a single 45 Gy was delivered to the dorsal skin of the mouse, cytokines and inflammatory cells were still largely increased at day 28 indicating a prolonged healing phase may occur and complete chronic RD may not be seen until a later timepoint.40 To balance the probability for a prolonged acute RD response we brought our mice model out to day 46 (6.5 weeks). 70 We explored acute RD using a mouse model brought out to day 14 and included day 4. The severity of the mice was scored every other day up to day 14 in both WT and GzmB-/- mice using the clinical scoring system adapted from Douglas and Fowler (1976)115 , and described elsewhere.40 The severity of RD was reduced in the GzmB-/- mice between days 4 and 12 when compared to the WT mice group. Severe RD is observed from scoring if the mice display dry or wet desquamation (1.5 severity score and up) which can be caused by a disruption in the epithelial barrier. GzmB can cleave barrier proteins, both E-cadherin and filaggrin, which can contribute to epithelial barrier disruption. Thus, a genetic deletion of GzmB was hypothesized to lower severity scores so the results of this part of the study is as expected.22 The first thing to explore in the mice tissues at both day 4 and 14 post irradiation was the level of GzmB as this can indicate which stage GzmB is present. WT mice 14 days post-radiation exposure showed elevated GzmB levels when compared to the WT non-irradiated mice. Most of the GzmB staining is found in the dermis. Originally we hypothesized GzmB would be present at day 4 since another mouse model found some inflammatory cytokines, IL-1α, MCP-1, TNF-α, and IL-6 to be upregulated as early as 2 days post-radiation exposure.41 The inflammatory cytokines can recruit inflammatory cells which were expected to produce GzmB. However there was no significant increase in GzmB in the WT mice 4 days post-radiation exposure when compared to the non-irradiated mice skin. The time points are only a snap shot of the immune response in RD, so it is still unknown when GzmB expression first occurs in irradiated skin. However GzmB expression can be predicted to occur between day 4 and 14 post a single 40 Gy radiation exposure to mice skin. Radiation induces mast cell degranulation which corresponds to tissue changes.52 Since mast cells co-localized to GzmB in human RD skin tissues, we wanted to determine whether 71 mast cells were a source of GzmB in irradiated murine skin. Since GzmB was only elevated at day 14, we only observe GzmB positive mast cells at day 14. Most of the GzmB positive mast cells are found in the dermis which is similar to where mast cells are located in the skin of both a dermatitis murine model and UV irradiated mice model.22,34 In a pressure injury mouse model, mast cells in the dermis were a major source of GzmB and showed both intact and degranulated phenotypes in which GzmB appeared to be released from the cell.116 This is similar with the RD mouse model and indicates that mast cells can be a source of GzmB in mice skin exposed to radiation. There was no significant difference in amount of mast cells in WT or GzmB-/- mice at day 4 and 14 post-radiation exposure when compared to non-irradiated control WT and GzmB-/- mice. Most of the mast cells were found in the dermis. With the lack of an increase in mast cell numbers, the initial increase in GzmB secretion could be contributed to the activation of the skin resident mast cells rather than the immune cell recruitment of mast cells. The difference in mast cell levels in the human RD skin tissues versus the mice RD model may be due to a difference in human versus mice RD phenotype or may indicate a difference in acute RD (mice model) versus chronic RD (human model). However, mast cells are one of the skin resident immune cell populations, and since ionizing radiation exposure causes degranulation of mast cells, the presence of mast cells in the human and mice RD skin and the co-localization to GzmB indicates that mast cells may be a contributor to GzmB expression in both human and mice RD.52,53 Future RD studies should explore the mast cell differences in acute versus chronic RD mouse models and include various time points, including day 8-10 post-radiation exposure and later then day 14. This may give us insight to when the mast cells are activated and if the number of mast cells ever increase in this RD mouse model. 72 There was a trend showing a potential decrease in the level of mast cells in the GzmB-/- mice at day 14 when compared to the WT mice 14 days post-radiation exposure. This trend was not observed in other disease models which have looked at GzmB-/- mice. In a study looking at dermatitis, there was no difference in the number of mast cell recruitment observed between WT and GzmB-/- mice.22 In a UV irradiation mouse model, the levels of mast cells also did not change in UV irradiated GzmB-/- mice dorsal skin compared to UV irradiated WT skin.34 The difference between the results of the studies could be due to a difference in disease progressions in UV irradiation and dermatitis versus RD. The difference could also be due to the time point chosen. The difference in mast cells in WT versus GzmB-/- mice was also only a trend and not significant. This could be due to the low number of mice in each group or to the time point chosen. Future RD mice model studies should investigate levels of mast cells between WT and GzmB-/- mice to confirm a significant loss of mast cells is found in GzmB-/- mice subject to radiation. Subsets of CD3+ T-cells are known to secrete GzmB and are known as an infiltrated immune cell in wound healing.75 Since there was no increase in mast cell levels, and to explore if there are other immune cell infiltrations to the skin, CD3+ T-cells were analyzed. There was a significant loss of CD3+ staining in the WT and GzmB-/- mice at day 4 and 14 post-radiation exposure when compared to the WT and GzmB-/- non-irradiated control mice. Although this was not as I hypothesized, since an increase of CD3+ T-cells may contribute to the increase of GzmB levels, the loss of CD3+ T-cells in the early stage of RD may be explained. Previous studies have demonstrated that T-cells, and B-cells are highly sensitive to radiation compared to other cell types including monocytes and macrophages.56 Further, the number of T-cells declined after the first day of radiation exposure and remained low. The monocytes did not decline as extensively 73 as the T-cells.56 A study looking at CD3+ T-cell levels in the blood of patients after radiation exposure to the Chernobyl disaster showed less CD3+ T-cells in the blood compared to the controls.55 Also in a mice study where mice were exposed to a 17.9 Gy radiation dose to the skin, macrophage inflammation was prevalent with fewer lymphocytes at 14 days post-radiation exposure.54 However, the time points are only a snap shot of the immune response of RD so CD3+ T-cells may still increase or at least resume to non-irradiated levels eventually, however it can be predicted this probably occurs after 14 days post a single 40 Gy radiation exposure to mice skin. Since CD3 is a pan-T cell marker, CD8+ and CD4+ populations should be included in future studies to observe the role of T-cells in RD. Neutrophils, a infiltrated immune cell in RD, have been shown to be recruited by IL-8 which is secreted by mast cells through degranulation.48 Since mast cells co-stain with GzmB in RD mice tissues, neutrophil infiltration is an interest. There was a significant increase of neutrophil staining in the WT and GzmB-/- mice at 14 days post-radiation exposure when compared to the WT and GzmB-/- non-irradiated control mice. There was also a significant increase in neutrophil staining in the WT mice at 4 days post-radiation exposure when compared to the WT non-irradiated control mice. The increase in neutrophil staining indicates there is an increase in neutrophil infiltration which is expected after radiation exposure.40,49 The increase in neutrophil infiltration at day 14, which is the same time point when mast cells are co-staining with GzmB and where GzmB expression is increased, indicates that neutrophils and mast cell degranulation may be connected and the neutrophil infiltration could have partly occurred due to the activation of mast cells. Mast cells promote neutrophil recruitment to the site of injury through degranulation. When mast cells degranulate, potentially through radiation exposure, IL-8 can be released into the environment which is a strong chemoattractant for neutrophils.48,53 74 Recruited neutrophils produce more inflammatory cytokines, including IL-1α and TNF-α which are inflammatory cytokines that can increase more inflammatory signals resulting in more inflammation.48 There was also a trend of less neutrophils in the GzmB-/- mice at day 14 post-radiation exposure when compared to the WT mice at day 14 post-radiation exposure. This trend was only observed in a mouse model of autoimmune blistering where both WT and GzmB-/- mice were subject to an epidermolysis bullosa acquisita disease model. Although the total infiltrated cell counts were comparable in both GzmB-/- mice with epidermolysis bullosa acquisita and WT mice with epidermolysis bullosa acquisita, neutrophil infiltration was lower by 40% in GzmB-/- mice when compared to WT mice.89 The mouse homolog for IL-8, which is a chemoattractant for neutrophils, was significantly lower in the GzmB-/- mice with epidermolysis bullosa acquisita when compared to the WT mice with epidermolysis bullosa acquisita. When primary mouse epidermal cells were incubated with extracellular GzmB, an increase in the mouse homolog for IL-8 was found, indicating GzmB may play a role in regulating neutrophil recruitment.89 This may explain the difference in neutrophil levels found in our RD GzmB-/- mice. However, the difference in neutrophils between WT and GzmB-/- mice groups was also not significant and was only a trend. This could be due to the low number of mice in each group or the time point chosen. Future RD mice model studies should investigate levels of neutrophils between WT and GzmB-/- mice to confirm a significant loss of neutrophils cells is found in GzmB-/- mice subject to RD as well as explore IL-8 levels within the model. To assess scarring in a RD mice model, a preliminary WT and GzmB-/- mouse model of RD was conducted out to day 46. The GzmB-/- mice 46 days post-radiation exposure showed less scar area when compared to the WT mice 46 days post-radiation exposure. The decrease in scar 75 area due to inhibition of GzmB in a mouse model has been seen before in a burn mouse model. The mice that received the GzmB inhibitor treatment showed less scar area versus the control group.35 This supports our findings that when GzmB is deleted in a chronic injury model, the area of the scar can be mitigated. GzmB can cleave an anti-fibrotic protein, decorin, which can result in a pro-fibrotic factor, TGF-β, to become bioavailable and can contribute to scar formation.33 Since decorin loss was found in human RD, decorin levels were analyzed in the murine model at 46 days post-radiation exposure. There was a significant increase of decorin staining in the GzmB-/- mice at 46 days post-radiation exposure when compared to the WT mice at 46 days post-radiation exposure. The RD murine results are similar to the burn mouse model described above, the scar area and decorin loss is reduced when GzmB activity is removed through an inhibitor (as in the burn model) or through genetic deletion (as in our RD model).35 The loss of decorin has been seen in many other animal models that use GzmB-/- mice subject to various diseases. In a mouse model of dermatitis, GzmB-/- mice showed less decorin loss when compared to the WT mice.22 In a pressure injury mouse model, aged WT mice showed more decorin loss when compared to aged GzmB-/- mice when subject to a burn.116 A similar result was also found in a UV irradiation mouse model where WT and GzmB-/- mice were exposed to UV radiation for up to 20 weeks. The irradiated GzmB-/- mice maintained their decorin while the irradiated WT mice showed a loss of decorin.34 The study showed that the loss of GzmB expression can result in a protective effect against decorin cleavage thus resulting in less scar tissue in chronic RD. Future studies should explore other fibrotic factors including TGF-β and fibronectin in chronic murine RD models. 76 Since the neutrophils were a cell type that was elevated in both WT and GzmB-/- mice at day 14 post-radiation exposure, we explored if the neutrophil infiltration continued to day 46 post-radiation exposure. The neutrophil infiltration did not continued to day 46 post-radiation exposure in either the WT or GzmB-/- mice. This was expected and helps confirm our chronic RD model, since by day 46 the mice were displaying scarring which is a sign of chronic RD. Thus the lack of neutrophils helps confirm a chronic phenotype with little inflammation or at least late stage inflammation.117 A limitation of this study includes the time points selected for the mice model. Originally the time points, 14 and 4 days post-radiation exposure, were selected due to previous mice models indicating severe RD with clinical observations at day 14 and an inflammation response at day 4. At the day 14 time point in our mice model, the mice skin appeared, histologically, to have some form of epidermal ulceration which is an indication of desquamation. One of the GzmB-/- mice retained most of the epidermis with a small section of epidermal ulceration. Another GzmB-/- mice tissue showed it still had about one to two cell length of epidermis covering the dermis. The rest of the mice for both WT and GzmB-/- mice samples had epidermal ulceration covering almost the whole harvest tissue. Since the two samples, which contained more epidermis, were found in the GzmB-/- mice, this could indicate less epidermal destruction, however the number of mice in this study was low and these two mice did not make a significant difference. Additionally, a study which looked at wound healing time points in the mouse foot pad after a single dose of 40 Gy radiation, found that there is a two-phase response in acute RD. The first phase is a destructive phase during which cell-cell disruption occurs from day 0 to day 9 post-radiation exposure. The second phase is a regenerative phase during which keratinocytes over proliferate and try to rebuild the barrier which can start from day 9 to day 18 post-radiation 77 exposure.20 Since re-epithelization can start as early as day 9, the keratinocytes found on the edges of the ulceration area in our mice skin tissues could have begun to produce more E-cadherin to replace the E-cadherin which had been lost in the damage stage. With the ulceration found in the day 14 mice skin tissues, and with the keratinocytes found at the edges of the ulceration potentially beginning the healing phase, the cleavage of E-cadherin and filaggrin could not be observed at day 14 and should be observed at an earlier time point like day 8 post-radiation exposure. Although the time point for acute RD may have been too late to observe the epidermal barrier dysfunction, the severity scores and immune cell infiltration can be observed and thus provide valuable information in mice models of RD and contribute to the evidence which GzmB can play an important role in RD severity. More time points should be further explored to pin point specific GzmB contributions to RD pathophysiology. Future studies will be focused on aged mice. Since age can be a contributor to severe RD development, introducing an aging murine model to the RD murine model could indicate if the age of the mice effects the severity of RD. Using APOE knockout mice with GzmB/APOE double knockout mice in our RD murine model would allow for investigation on whether aging can influence RD severity. The mice used were also only female mice since they can be group housed with greater success and minimize the chance for further injuries from fighting. Although there has been limited studies showing males and female develop RD differently, there could be differences in females versus males.8 Thus, further studies should include male mice to observe if there are any sex differences in RD severity and wound healing. The limitations from using immunohistochemistry include only observing known proteins, limited to proteins which antibodies are available and reliable, and limitations on understanding the mechanism of action. Although the mechanism of action cannot be concluded exclusively from 78 immunohistochemistry, the stage of RD development can be very detailed on cell location, loss of proteins, and the presence of GzmB. Further studies could utilize flow cytometry, enzyme-linked immunosorbent assays, gene expression assays, and extracellular GzmB inhibitors to characterize the role of GzmB in RD. Another limitation of the current study is the lack of information of the human RD samples. Only the sex, age, and location of the RD patients was the information provided for each sample. There were also only six RD human skin samples since RD is not a commonly biopsied disease. Since we had limited information, we had a dermatologist-scientist with pathology training assess the samples and confirm if the samples were chronic or acute. All of the samples were chronic with one sample potentially at a late acute/early chronic stage (Table 2). We also only had a small sample size for the healthy control human skin, however normal healthy human skin is not normally biopsied, thus with the small sample size and little information, rigorous unpaired nonparametric statistical tests were run on all samples (Wilcoxon test). Human RD skin samples are hard to acquire and thus any information from human RD skin tissues can add valuable information towards the RD mechanism within human RD skin. By applying rigorous unpaired nonparametric testing, we can still see significant evidence on the location, level, source, and role of GzmB expression in human RD. 79 Chapter 5: Conclusion and Future Directions The results of my studies suggest that GzmB is present in human RD skin tissue. The loss of both E-cadherin and filaggrin in the human RD skin tissue suggests that GzmB could contribute to barrier dysfunction in RD. The loss of decorin in the human RD and the murine RD model indicates that GzmB may also contribute to scarring. The increase in both macrophages and mast cells in the human RD skin tissues further suggest that both cell types contribute to RD pathophysiology. The co-staining of GzmB to macrophages and mast cells indicates that macrophages and mast cells are a source of GzmB in human RD. In the RD mouse model, the increase of GzmB in the mice skin at 14 day post-radiation exposure demonstrates that GzmB is present in this model and that it is a good model to use to look at the functional role of GzmB with GzmB-/- mice. The co-staining of GzmB and TBO staining indicates that mast cells produce GzmB in the RD mouse model. The increase in neutrophils in the irradiated mice show an influx in inflammation. All of the findings contribute to furthering the understanding of the RD mechanism and can shed a light on the role of GzmB in RD severity. The work presented provides the foundation for future RD studies looking at more extensive RD mechanism and the role of GzmB in the RD phenotype. Future studies should continue to look at the effects of GzmB on different stages of RD. Looking at more time points and more fibrosis markers within chronic RD, such as TGF-β levels and fibronectin, will help further explore the contribution of GzmB to fibrosis and scarring in RD. An earlier time point in the mice model of RD, like day 8-10, could show a difference in barrier protein levels, such as E-cadherin and filaggrin, since there may be less epidermal loss. Additionally, the earlier time point in the mice model will help pin point exactly when GzmB is introduced into the skin post-radiation exposure. Future studies should also observe the level and 80 effect of GzmB after multiple radiation exposures in a mouse model and compare the results to the single radiation exposure mouse model. Using a GzmB inhibitor could increase our knowledge on the ability of extracellular GzmB to be a novel therapeutic target for RD. The findings in this thesis identify a novel role for GzmB in RD through cleavage of barrier proteins and an anti-fibrotic factor, decorin. The findings also suggest what cell types are producing GzmB in irradiated skin and that by removing GzmB, the severity of RD can be mitigated. Future work should consider characterizing more GzmB substrates in various RD models and time points. A summary of key thesis findings are highlighted in the figure below (Figure 19). Figure 19. Summary of findings. Skin is exposed to radiation. Inflammatory cell (neutrophils, mast cells, and macrophages) is observed. Mast cells and macrophages secrete GzmB into the extracellular space. Extracellular GzmB may cleave E-cadherin and filaggrin in the epidermis and contribute to barrier disruption. An increase in barrier disruption can result in dryness, itchiness, and inflammation in the skin which can further increase RD severity. Extracellular GzmB can also cleave decorin which can result in disorganization of collagen leading to fibrosis and scarring. 81 References 1. Miller, K. D. et al. Cancer treatment and survivorship statistics, 2019. CA. Cancer J. Clin. 69, 363–385 (2019). 2. Maddocks-Jennings, W., M Wilkinson, J. & Shillington, D. 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Thesis/Dissertation
eng
Pathology and Laboratory Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Granville, David James
Granzyme B : a novel therapeutic target for radiation dermatitis
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