CORNEAL WOUND HEALING: CELLS and MOLECULES By

CORNEAL WOUND HEALING: CELLS AND MOLECULES By

Sri Magadi

DISSERTATION

In partial fulfilment of requirements for the degree of DOCTOR OF PHILOSOPHY In PHYSIOLOGICAL OPTICS AND VISION SCIENCE

Presented to the Graduate Faculty of the College of Optometry University of Houston

August, 2016

Approved: ______Alan R. Burns, PhD (Chair)

______Rachael Redfern, OD., PhD

______Samuel D Hanlon, OD., PhD

______William Miller, OD., PhD

______Rolando Rumbaut, MD., PhD

Committee in charge

Dedication

I dedicate this dissertation to my loving parents, Prema and Ragu Chetlur, my amazing husband and best friend Sudarshan Magadi, my two beautiful daughters Siri Magadi and

Sneha Magadi, my teachers, friends, and entire family who have supported me throughout my life and academic endeavors. I couldn’t have progressed and succeeded in achieving my dreams without you, and I am thankful for your love and support.

Acknowledgments

I would like to thank Dr. Alan Burns, who has been an amazing mentor to me, given me the freedom to think, and guided me through the difficult terrains of the Ph. D program.

He was patient and understanding as he helped me navigate through my experiments and offered me beneficial advice that helped me improve as a student and a scientist. Dr.

Burns, I am truly grateful to you for your friendship and mentorship.

I thank Dr. Sam Hanlon; whose friendship I cherish. Dr. Hanlon let me express and discuss my ideas, encouraged me to accomplish and surpass my goals, and supported me throughout my academic journey in the Ph.D. program.

I am grateful to my committee members: Dr. Rachel Redfern, who over the years has been a tremendous support and a great friend to me; Dr. Rolando Rumbaut, who has guided me and helped me with my experiments; and Dr. William Miller, who has helped me understand the clinical aspect of vision science and broadened my perspective of the field.

I thank our lab mom Evelyn Brown for her kindness, understanding, and assistance. I thank my friends and lab members at UHCO, who are like family to me, for their unwavering support. I am very fortunate to have met you all and grateful for your friendship.

iii

I thank Dr. Laura Frishman for her dedication to the Ph. D program and giving me a chance to become part of it.

I thank Dr. Steven Nielsen at the University of Texas at Dallas for giving me an opportunity to perform research and broadening my thinking while encouraging me to begin my Ph.D. I also thank my friends and faculty members at the University of Texas at

Dallas for their encouragement and support.

Lastly, I give special thanks to my family for standing by my side and helping me pursue my academic goals. My husband and my best friend Sudarshan Magadi has always encouraged me to conquer my obstacles and peruse my goals in life. His selfless and unconditional love, the support from my loving parents and amazing children has been a pillar of strength and ultimately has helped me accomplish my goals. My faculty, friends, family, and mentors are an integral part of my success and I am ever grateful for your friendship and support.

CORNEAL WOUND HEALING: CELLS AND MOLECULES By

Sri Magadi

DISSERTATION

In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In PHYSIOLOGICAL OPTICS AND VISION SCIENCE

Presented to the Graduate Faculty of the College of Optometry University of Houston

August, 2016

Abstract

Purpose: Corneal abrasion elicits an acute inflammatory response involving neutrophil

(PMN) and platelet extravasation from the limbus. While these cells deliver important growth factors that promote wound healing, resolution of the inflammatory response (i.e.,

PMN clearance) is also needed to restore corneal homeostasis. This dissertation evaluated mechanisms regulating platelet recruitment after corneal injury, PMN migration in the abraded mouse corneal stroma and PMN clearance during the resolution of inflammation.

Methods: Adult C57BL/6J mice were anesthetized and a 2mm central corneal abrasion was made using a golf-spud/Alger brush. As a means of dysregulating platelet extravasation, approaches were used that build on the established observation that platelet recruitment is linked to PMN recruitment. Some mice received an intraperitoneal injection of anti-Ly6G antibody to deplete circulating PMNs while others received topical applications of recombinant interleukin-20 (rIL-20) to blunt inflammation. To determine if platelet extravasation was dependent on CD18 expression levels, two groups of mutant mice were studied, one with intermediate-high CD18 expression (CD18hypo

(I-H)) and one with low-intermediate CD18 expression (CD18hypo (L-I)). To evaluate inflammation, excised corneas were immunolabelled and PMNs, platelets, mast cells, and degranulated mast cells were counted and limbal vessel diameters were measured. To evaluate PMN surface contacts with other cells and the extracellular matrix, excised corneas were imaged using serial block-face scanning electron microscopy. To study

PMN migration in vivo, time-lapse sequences were collected using the Heidelberg Retinal

Tomographer III with Rostock Cornea module (HRT-RCM).

Results: Following a central corneal abrasion, IL-20-treated mice and anti-Ly6G treated mice showed reduced PMN and platelet extravasation. PMN extravasation in CD18hypo

(I-H) mice was similar to WT mice while platelet extravasation was diminished (~80%) only in CD18hypo (L-I) mice. Passive transfer of WT PMNs into CD18hypo (L-I) mice did not restore platelet extravasation to WT levels. In all cases, mice with diminished platelet recruitment showed diminished limbal vessel dilatation and reduced mast cell degranulation (p≤ 0.05).

8h post-injury, majority (70%) of paralimbal PMNs close to the limbus moved circumferentially with an average speed of 3.1±0.4µm/min, significantly less than the

6.1±0.3µm/min speed of PMNs at the paralimbus/closer to the center of the cornea where majority (60%) of these PMNs showed oriented migration toward the wound (p≤0.05).

Serial block-face data provided evidence for a circumferential orientation of corneal cells and collagen at the paralimbus. Here, PMN surfaces predominantly contacted collagen

(~60%), and keratocytes (~21%). At the center of the cornea, PMN numbers were maximal at 24h and markedly reduced by 72h post-injury. 24h post-injury, 64% of central PMNs were migrating away from the center which was significantly higher than that observed at 48h post-injury (p≤0.05). 52% of parawound PMNs were observed migrating away from the center of the cornea and by 72h this percentage dropped to 25%.

30h-72h post-injury macrophage numbers increased and macrophage phagocytosis of

PMNs was clearly evident at the limbus.

Conclusion: While PMNs and platelets share a strong co-dependence in their recruitment after corneal abrasion, it appears that vessel dilatation is only required for platelet extravasation. Vessel dilatation appears to be mediated by mast cell degranulation which seems to require PMN CD18. The observation that many extravascular PMNs at the limbus are not oriented toward to the wound but are instead oriented circumferentially is novel. Given that the mouse cornea possesses an annular ring of collagen at the paralimbus, and collagen accounts for the majority of PMN surface contact, this collagen ring may account for the observed circumferential migration pattern of limbal PMNs during corneal inflammation. The clearance of PMNs during the resolution of inflammation appears to involve migration away from the center of the cornea and removal via macrophage phagocytosis.

Table of Contents

Abstract ...... 1 Table of Contents ...... 4 List of Figures and Legends ...... 7 List of Tables ...... 10 Abbreviations ...... 11

Chapter 1: Introduction ...... 13 1.1: Importance of research in corneal wound healing ...... 13 1.2: Overview of the cornea ...... 14 1.2.1: Corneal Development ...... 14 1.2.2: Differences between the mouse and human cornea ...... 14 1.2.3: Corneal Anatomy...... 15 1.3: Overview of inflammation ...... 17 1.3.1: Acute and chronic inflammation ...... 17 1.3.2: Adhesion molecules and immune cells during inflammation ...... 19 1.3.3: Neutrophils ...... 20 1.3.4: Platelets...... 21 1.4: Corneal wound in mice ...... 21 1.5: Cytokines...... 24 1.5.1: The IL-10 super family of cytokines ...... 25 1.5.2: IL-20 sub-family of cytokines ...... 27 1.5.3: Role of IL-20 subfamily in mouse corneal wound healing ...... 28

Chapter 2: Aim 1- Evaluate mechanisms regulating platelet recruitment after corneal injury ...... 31 2.1: Introduction ...... 31 2.2: Methods ...... 34 2.2.1: Choice of animal model...... 34 2.2.2: Corneal abrasion model ...... 35 2.2.3: Immunohistochemistry and antibodies used ...... 36

2.2.4: Imaging and counting platelets and mast cells ...... 37 2.2.5: Counting PMNs in the injured cornea ...... 39 2.2.6: Counting mast cells at the limbal region ...... 40 2.2.7: Imaging the limbus and diameter measurements ...... 41 2.2.8: IL-20 administration ...... 43 2.2.9: Administration of anti-Ly6G antibody ...... 43 2.2.10: Isolation of PMNs...... 43 2.2.11: Statistical Analysis ...... 44 2.3: Results ...... 45 2.3.1: Evaluate the role of anti-Ly6G in mice after corneal injury ...... 45 2.3.1.1: Evaluate PMN and platelet co-dependent recruitment after corneal abrasion in mice treated with anti-Ly6G...... 45 2.3.1.2: Evaluate venule and arteriole diameters in wounded mice treated with anti-Ly6G antibody ...... 49 2.3.1.3: Evaluate Mast cells in wounded mice treated with anti-Ly6G antibody .. 51 2.3.4: Evaluate the role of IL-20 in mice after corneal injury ...... 53 2.3.4.1: Role of IL-20 on platelet recruitment after corneal abrasion in mice ...... 53 2.3.4.2: Evaluate venule and arteriole diameters in wounded mice treated with IL- 20...... 57 2.3.4.3: Evaluate Mast cells in wounded mice treated with IL-20 ...... 59 2.3.5: Evaluate the role of CD18 on platelet recruitment during corneal inflammation in mice...... 60 2.3.5.1: Initial Pilot study to evaluate limbal vessel diameters in CD18 hypo mice ...... 60 2.3.5.2: Evaluate the role of CD18 in platelet extravasation ...... 63 2.3.5.3: Evaluate the role of CD18 on limbal venule and arteriole diameters ...... 65 2.3.5.4: Evaluate the role of CD18 on limbal mast cells ...... 70 2.3.6: Evaluate if platelet extravasation can be restored in PMN reconstituted mice 73 2.4: Discussion ...... 79

Chapter 3: Aim2: Neutrophil migration and resolution of inflammation ...... 84 3.1: Introduction ...... 84 3.2: Methods ...... 87

3.2.1: Choice of animal model...... 88 3.2.2: Corneal abrasion model ...... 88 3.2.3: Imaging with the Scanning Electron Microscope (SEM)...... 89 3.2.4: In vivo imaging the cornea with the Heidelberg Retinal Tomographer III with Rostock Cornea module...... 95 3.2.5: Statistical Analysis ...... 99 3.3: Results ...... 100 3.3.1: PMN migration and resolution of inflammation after corneal abrasion ...... 100 3.3.1.1: PMN migration during the early phase of inflammation after corneal injury ...... 102 3.3.1.2: PMN migration during the late phase of inflammation after corneal injury ...... 105 3.3.1.3: Resolution of inflammation after corneal injury...... 116 3.4: Discussion ...... 130

Chapter 4: Conclusions ...... 134 Chapter 5: Future directions ...... 138 References ...... 140

List of Figures and Legends

Figure I-1: Inflammatory cascade after corneal injury in the mouse...... 23

Figure I-2: IL-10 family of cytokines have receptor overlaps...... 26

Figure I-3: IL-20 sub-family of cytokines...... 27

Figure M-2.1: Central corneal wound in C57BL/6J mice...... 35

Figure M-2.2: Intact basement membrane after corneal wound...... 36

Figure M-2.3: Corneal flat mount of an adult C57BL/6J mouse...... 37

Figure M-2.4: Stitched panels of the limbal vessels used for platelet counts...... 38

Figure M-2.5: Intravascular and extravascular platelets...... 39

Figure M-2.6: Corneal regions for counting PMNs...... 40

Figure M-2.7: Limbal region selected for counting mast cells...... 40

Figure M-2.8: Counting mast cells and degranulated mast cells...... 41

Figure M-2.9: Measurement of venule and arteriole diameters ...... 42

Figure C-2.1: Anti-Ly6G antibody reduced circulating PMNs...... 46

Figure C-2.2: Reduced PMN counts across the injured cornea in anti-Ly6G mice. 46

Figure C-2.3: Platelet counts were reduced in anti-Ly6G treated mice...... 47

Figure C-2.4: Platelet extravasation in anti-Ly6G treated mice...... 48

Figure C-2.5: Limbal vessel dilatation in anti-Ly6G treated ...... 50

Figure C-2.6: Mast cells in mice treated with anti-Ly6G antibody ...... 52

Figure C-2.7: Topical IL-20 reduced platelet recruitment after corneal abrasion. .. 54

Figure C-2.8: PMN and Platelet extravasation is inhibited in IL-20 treated mice. .. 55

Figure C-2.9: Limbal vessel dilatation in IL-20 treated mice after corneal injury. . 58

Figure C-2.10: Mast cells in mice treated with IL-20 after corneal injury...... 59

Figure C-2.11: Pilot study: Limbal vessel dilation is reduced in CD18hypo mice .... 62

Figure C-2.12: Extravasated platelets in CD18hypo mice with intermediate-high (I-

H) and low-intermediate (L-I) levels of CD18 ...... 64

Figure C-2.13: Limbal venule and arteriole diameters in CD18hypo mice with intermediate-high (I-H) and low-intermediate (L-I) levels of CD18...... 69

Figure C-2.14: Limbal mast cell and degranulated limbal mast cells in CD18hypo mice with intermediate-high (I-H) and low-intermediate (L-I) levels of CD18 ...... 72

Figure C-2.15: Extravasated platelets in CD18hypo mice reconstituted either with wild-type or Cd18hypo PMNs 24h after corneal injury...... 74

Figure C-2.16: Limbal vessel diameters in CD18hypo mice reconstituted either with wild-type or Cd18hypo PMNs ...... 76

Figure C-2.17: Limbal mast cell and degranulated limbal mast cell counts in

CD18hypo mice reconstituted either with wild-type or CD18hypo PMNs ...... 78

Figure C-2.18: Possible explanation of Aim1 results...... 82

Figure M-3.1: Serial block-face for imaging with the SEM...... 90

Figure M-3.2: Scanning Electron Microscope (SEM)...... 94

Figure M-3.3: In vivo imaging using Heidelberg Retinal Tomographer III with

Rostock Cornea module (HRT-RCM)...... 96

Figure M-3.4: Determination of PMN migration angle...... 98

Figure C-3.1: In vivo HRT-RCM imaging of PMN migration patterns within the paralimbus of the injured mouse cornea...... 101

Figure C-3.2: Tracking PMN speed using in vivo HRT-RCM 8h post-injury...... 103

Figure C-3.3: PMN speed 24h after corneal injury...... 107

Figure C-3.4: PMN speed 30h after corneal injury...... 108

Figure C-3.5: PMN speed 36h after corneal injury ...... 109

Figure C-3.6: PMN speed 48h after corneal injury...... 110

Figure C-3.7: PMN speed 72h after corneal injury...... 112

Figure C-3.8: Comparing excised and fixed 24h post-wound fluorescently labelled

PMN images to in vivo confocal images acquired 24h after injury...... 115

Figure C-3.9: 48h post-injury in vivo HRT-RCM images ...... 116

Figure C-3.10: Comparing fluorescently labelled and confocal images 72h post- injury...... 117

Figure C-3.11: Macrophage phagocytosis 72h post-injury...... 119

Figure C-3.12: PMN migration during early (8h) and late phase of inflammation

(24h-72h)...... 123

Figure C-3.13: Identification of structures in a SEM serial block-face section...... 124

Figure C-3.14: PMN surface contact measurements...... 126

Figure C-3.15: Extravasated PMNs at 18h post-injury...... 128

Figure C-3.16: Circumferential orientation of cells and structures at the limbus.. 129

List of Tables

Table 1: PMN movement 8h post-injury...... 104

Table 2: Average PMN speed (in µm/min) at different regions of the cornea 24h -

72h after central corneal abrasion...... 113

Table 3: Percentage of PMN movement at various corneal regions 24h, 30h, 36h,

48h, and 72h post-injury...... 114

Table 4: PMN and macrophage counts at different corneal regions...... 120

Table 5: PMN surface contacts with other cells and structures 8h post-injury ...... 127

Abbreviations

ALL ………………………… Anterior limiting lamina ANOVA ………………………… Analysis of Variance APC ………………………… Allophycocyanin ………………………… The Association for Research in ARVO Vision and Ophthalmology BSA ………………………… Bovine serum albumin CD ………………………… Cluster of differentiation DAPI ………………………… 4',6-diamidino-2-phenylindole ECM ………………………… Extracellular matrix FITC ………………………… Fluorescein isothiocyanate g ………………………… Gram GP1bα ………………………… Glycoprotein I b alpha h ………………………… Hour(s) ………………………… Heidelberg retinal tomographer III HRT-RCM with rostock cornea module I.P ………………………… Intraperitoneal IBD ………………………… Inflammatory bowel disease ICAM-1 ………………………… Intercellular adhesion molecule-1 Ig ………………………… Immunoglobulin G IL ………………………… Interleukin IL-20R ………………………… IL-20 receptor JAK ………………………… Janus Kinase JAM ………………………… Junction adhesion molecule KDa ………………………… Kilo Dalton L ………………………… Limbus LASIK ………………………… Laser-assisted in situ keratomileusis LFA-1 ………………………… Lymphocyte function-associated antigen M ………………………… Molar

MAC-1 ………………………… Macrophage-1 antigen mg ………………………… Milligram ml ………………………… Milliliter mM ………………………… Millmolar MMP ………………………… Matrix metalloprotinase ng ………………………… Nanogram PAF ………………………… Platelet activating factor PBS ………………………… Phosphate buffered saline PE ………………………… Phycoerythrin ………………………… Platelet endothelial cell adhesion PECAM-1 molecule-1 PL ………………………… Paralimbus PLL ………………………… Posterior limiting lamina PMN ………………………… Polymorph nuclear PRK ………………………… Photorefractive keratectomy PW ………………………… Parawound rIL-20 ………………………… Recombinant IL-20 SE ………………………… Standard Error of mean SEM ………………………… Scanning electron microscope ………………………… Signal transducer and activator of STAT transcription TNF-α ………………………… Tumor necrosis factor-alpha VEGF ………………………… Vascular endothelial growth factor WC ………………………… Wound center WT ………………………… Wild-type µl ………………………… Microliter μm ………………………… Micrometer

Chapter 1: Introduction

1.1: Importance of research in corneal wound healing

The cornea is our window to the world. It is the clear surface that covers the eye and protects it from the external environment. It receives visual input from the outside world and aids in focusing the light rays into the eye. It is a highly organized tissue accounting for almost two-thirds of the eye’s refractive power. Clearly, the cornea plays a major role in vision.

While the cornea’s placement in front of the eye is ideal for receiving visual stimuli, its interaction with the environment makes it vulnerable to injury. Physical damage, environmental stress, corneal dystrophies, infection, and refractive surgeries injure the cornea. Also, contact lens wear can physically damage the corneal epithelium

(Li et al., 2002; Poggio et al., 1989; Yamamoto et al., 2002). The unsanitary and improper handling of contact lenses can further lead to infections (Morgan et al., 2005;

Robertson and Cavanagh, 2008). Prolonged exposure to sunlight, also known as phototoxicity, damages the corneal epithelium (Kronschlager et al., 2015; Mallet and

Rochette, 2013; Srivastav et al., 2016). Moreover, corneal cross-linking treatments and surgeries such as LASIK and PRK can damage the cornea (Mastropasqua et al., 2014;

Mohamed-Noriega et al., 2014; Reinstein et al., 2012; Ti and Tan, 2001; Trost et al.,

2013; Wang et al., 2015; Zhang, 2012). Additionally, genetic disorders such as Fuchs’ dystrophy damage the corneal layers (Acar et al., 2016; Akhtar et al., 2015; Ljubimov et al., 2002; Maeng et al., 2015; Pfister, 2004), while Dry Eye Disease brought about by environmental stress, infections, or corneal surgeries causes a loss in tear film production,

13 damages corneal nerves; and if severe, it can negatively affect vision (Chao et al., 2015;

Narayanan et al., 2013; Redfern et al., 2013; Sutu et al., 2016; Wegener et al., 2015).

Therefore, studying the cornea’s response to injury and its ability to heal provides the basis for developing novel therapeutic strategies. Understanding molecular and cellular events during corneal wound healing will provide insights into corneal repair and restoration of vision.

1.2: Overview of the cornea

1.2.1: Corneal Development

The human cornea is composed of cells derived from the ectoderm. The corneal epithelium originates from the surface ectoderm, while the neural ectoderm makes the endothelium and stromal keratocytes (Graw, 2010; Hay, 1980; Sevel and Isaacs, 1988).

Corneal nerves in the stroma and epithelium come from the neural crest and ectodermal placode-derived trigeminal ganglion (Lwigale, 2015). The superficial mesenchymal cells form the anterior limiting lamina (ALL), also known as Bowman’s membrane. The corneal stroma is derived from mesenchymal tissue, and the posterior limiting lamina

(PLL) or Descemet’s membrane is derived from the deep mesenchymal cells within the stroma (Hagedoorn, 1928; O'Rahilly, 1975; Wulle and Lerche, 1969).

1.2.2: Differences between the mouse and human cornea

The mouse is a commonly used animal in varied fields of research. Although there are similarities such as a stratified epithelium, presence of a corneal stroma including keratocytes, and endothelium to list a few, structural differences exist between the mouse

14 and human cornea. Apart from the obvious differences in eye size, the mouse cornea lacks the collagenous primary stroma during fetal development. Furthermore, in the stroma, sub-epithelial fibrils are not organized into bundles in the adult mouse (Haustein,

1983). The human corneal epithelium is also thicker than that of the mouse. The general consensus in the literature suggests the mouse lacks an anterior limiting lamina (or

Bowman’s layer) (Henriksson et al., 2009).

1.2.3: Corneal Anatomy

The human cornea consists of five layers: the epithelium, anterior limiting lamina (ALL), stroma, posterior limiting lamina (PLL), and endothelium. These corneal layers differ in thickness and function.

The corneal epithelium is the outermost surface of the cornea in constant contact with the external environment. It is made up of a stratified epithelium which consists of

2-3 layers of superficial cells, 2-3 layers of wing cells, and a monolayer of basal cells. It is ~ 60µm thick (DelMonte and Kim, 2011; Reinstein et al., 2008). The epithelium functions as a barrier and protects the eye from foreign materials such as dust, water and bacteria. It further prevents infection and aids in wound healing by releasing cytokines and other molecules (Li et al., 2011b; McDermott et al., 2003; Redfern et al., 2011), thus restoring corneal homeostasis (Yang et al., 2000). Basal cells in the corneal epithelium secrete and adhere to the basement membrane. A nerve plexus (so called “sub-basal” plexus) is located just above this basement membrane and gives rise to vertical nerves which branch out into the stratified epithelium.

The anterior limiting lamina, also known as the Bowman’s layer, lies just below the epithelium. It is ~ 17µm thick in the center and ~ 20µm thick in the periphery (Tao et al., 2011). It was thought to act as a barrier between the epithelium and the corneal stroma, preventing epithelial cells from contacting stromal cells. But more recent thoughts suggests it provides structural support for the cornea while helping to maintain corneal transparency (Lagali et al., 2009).

The stroma is the thickest corneal layer, located below the anterior limiting lamina. Approximately 90% of the cornea’s thickness can be attributed to the stroma. It is

~ 460µm thick (Reinstein et al., 2009). Keratocytes are cells within the stroma. The stroma can be divided into anterior and posterior layers, which differ from each other in arrangement, width, and thickness of collagen lamellae. The stroma is made up of regularly arranged collagen fibrils that contribute to the maintenance of corneal transparency (Bron, 2001). Dermatan sulfate proteoglycans such as decorin are involved in interfibrillar spacing and lamellar adhesion properties of collagen in the cornea.

Keratan sulfate proteoglycans such as lumican, mimecan, and keratocan are essential in the regulation of the diameter of the collagen fibrils (Michelacci, 2003). Thousands of proteins reside in the stroma and help in maintaining corneal transparency and homeostasis (Xuan et al., 2016).

The posterior limiting lamina, or Descemet’s membrane, is located inferior to the stroma. It is ~ 12µm thick (Johnson et al., 1982). It is primarily made up of type VIII

16 collagen and is suggested to provide structural support to the cornea (Tamura et al.,

1991).

The endothelium is the innermost corneal layer composed of a single layer of hexagonal squamous cells. It maintains corneal transparency by pumping out excess fluid that leaks into the stroma, thereby preventing corneal edema (Hamann, 2002; Verkman,

2006). In the human cornea, endothelial cells do not regenerate. Rather, the loss of these cells is managed by the enlargement and migration of surrounding cells, resulting in endothelial polymorphism and polymegethism.

1.3: Overview of inflammation

Inflammation is the body’s complex response to injury or infection. Inflammation is derived from the word “inflammare” with means “setting on fire.” The well-known cardinal signs of inflammation are heat (calor), redness (rubor), swelling (tumor), pain

(dolor), and a loss of function (functio laesa) (Rocha e Silva, 1994; Scott et al., 2004). It is widely accepted that the initial four cardinal signs were added by Celsus and the fifth one by Galen (Rather, 1971).

1.3.1: Acute and chronic inflammation

The body’s initial response to injury or insult is known as acute or innate inflammation which occurs for a shorter duration and involves cells and molecules such as PMNs, platelets, monocytes, chemokines and cytokines (Wilson et al., 2001). The recruitment of leukocytes in response to injury and an increase in blood flow followed by vasodilatation, are some of the hallmark features of the acute phase. The relaxation of smooth muscles

17 facilitates the increase in blood flow to the venules. Histamine release primarily from mast cells, the presence of other vasodilators like bradykinins and leukotrienes, release of pro-inflammatory cytokines like IL-1 and TNF result in the blood vessels to become leaky thereby increasing vascular permeability (Yoshikai, 2001). Acute inflammation can also be mediated by the activation of classic, alternate and lectin pathways of the complement system leading to PMN activation, release of leukotrienes, histamine and increased vascular permeability. PMNs and platelets extravasate from the blood vessels via post-capillary venules (Burns et al., 2005; Dimasi et al., 2013) and deliver beneficial growth factors to aid wound healing and tissue repair (Borregaard et al., 2007; Li et al.,

2006b). Thus acute inflammation can be beneficial to wound healing. Acute inflammation can result in complete resolution and regaining tissue homeostasis, or progress to chronic inflammation. Persistent or chronic injuries usually progress to chronic inflammation as evidenced by a longer duration and involvement of T and B- cells, macrophages, plasma cells and antigen specific antibody production. Chronic inflammation is characterized by tissue damage, angiogenesis, and tissue repair via scar formation (fibrosis) (Jackson et al., 1997). In this dissertation, I studied acute inflammation involving the recruitment of beneficial leukocytes after a central corneal abrasion.

During injury or pathogen invasion, the body’s complex response involves a cascade of molecular events initiated by the release of cytokines and other proteins.

Vascular changes and the activation of the innate immune response lead to the extravasation of PMNs and platelets out of blood vessels into the site of injury.

Leukocyte extravasation occurs in a step-wise process. In response to injury, many adhesion molecules, including the selectin family, are activated on the vascular endothelium. Activated PMN capture to the vascular endothelium is initiated by leukocyte L-Selectin. Once PMNs are captured, their rolling on the endothelium is facilitated by P-Selectin. The binding of leukocyte CD18 to endothelial intercellular adhesion molecule-1 (ICAM-1) helps in firm adhesion of PMNs to the vascular endothelium. Transmigration of the PMNs out of blood vessels into the extravascular space is facilitated by other adhesion molecules such as vascular Platelet Endothelial

Adhesion Molecule-1 (PECAM-1) , Junctional Adhesion Molecule (JAM), and CD99

(Choi et al., 2009; Smith and Anderson, 1991; Zarbock and Ley, 2008).

1.3.2: Adhesion molecules and immune cells during inflammation

CD18 is a β2 integrin present on the surface of PMNs. CD11a/CD18 (lymphocyte function-associated antigen (LFA-1)), CD11b/CD18 (Macrophage-1 antigen (MAC-1)),

CD11c/CD18, and CD11d/CD18 are the four members of the CD18 family (Gahmberg et al., 2009; Tan, 2012). CD11a/CD18 (LFA-1), CD11b/CD18 (MAC-1) are present on all myeloid cells, but CD11c/CD18 and CD11d/CD18 are present on antigen presenting cells

(Asada et al., 1991; Diamond et al., 1995; Gahmberg and Fagerholm, 2002; Gahmberg et al., 1997; Kurzinger et al., 1981). CD18 plays a crucial role in PMN firm adhesion and transmigration (Arnaout, 1990; Gahmberg, 1997; Green et al., 2006; Sarantos et al.,

2008; Shamri et al., 2005; Smith et al., 1988; Willeke et al., 2001; Zen et al., 2011).

MAC-1, a member of the CD18 family, plays a key role in PMN apoptosis (Diamond et al., 1993; Rubel et al., 2003; Walzog et al., 1997; Yan et al., 2004). Following PMN

19 activation, the normally low surface expression of CD18 increases (Albelda et al., 1994;

Paugam et al., 1997). Both LFA-1 and MAC-1 can bind endothelial Intercellular

Adhesion Molecule-1 (ICAM-1) and other adhesion molecules such as Junctional

Adhesion Molecule (JAM) (Figueroa et al., 2015; Malik, 1993). MAC-1 can bind GP1bα present on platelets. Recent studies show that CD18 binds to ICAM-1 on keratocytes in the cornea and facilitates their direct surface interactions (Burns et al., 2005; Gagen et al.,

2010; Petrescu et al., 2007).

1.3.3: Neutrophils

PMNs are hematopoietic cells from the myeloid lineage. (PMNs) have a multi-lobed nucleus hence the name polymorph nuclear cells. The primary, secondary and tertiary granules in PMN cytoplasm contain growth factors such as Vascular Endothelial Growth

Factor-A (VEGF-A), antimicrobial defensins, and proteases (Bainton et al., 1987;

Borregaard et al., 2007). Upon activation, PMNs degranulate, releasing their contents into the surrounding space. Quiescent PMNs are ~7µm in diameter. There are about 4.5 X109

PMNs in an adult human and they have an average half-life of ~5 days. PMNs play a very important role in wound healing and tissue repair, as they contain essential growth factors in their granules (Borregaard and Cowland, 1997; Cohen et al., 2015). In the cornea, when PMNs encounter pathogens, one of the many ways they kill the pathogens is by phagocytosing them and releasing anti-bacterial peptides from their granules

(McDermott, 2009). These anti-bacterial peptides can act upon phagocytosed, as well as bystander pathogens in the surrounding extracellular matrix. Another mechanism to clear the cornea of pathogens is the release of PMN extracellular traps (NETs) to engulf the

20 disease-causing microorganism (Kolaczkowska and Kubes, 2013). Apart from releasing

VEGF-A, PMNs in the cornea release proteases and matrix metalloprotinase (MMP) 9

(Chakrabarti and Patel, 2005) important for corneal repair.

1.3.4: Platelets

Platelets are ~1-2µm in diameter. They originate from megakaryocytes (Hartwig and

Italiano, 2003; Italiano and Shivdasani, 2003; Machlus et al., 2014; Sadoul, 2015) and contain α, dense and λ granules (Harrison and Cramer, 1993; Tamagawa-Mineoka, 2015).

There are about 150000-400000/µl of blood in humans with a half-life of 8-10 days.

Although platelets are well-known for their role in thrombosis formation, they also play a role in tissue repair as they carry growth factors in their granules. Platelet activation occurs by their binding to the extracellular matrix, after which they degranulate and release their contents into the extracellular space. In the cornea, platelets are important for keratocyte regeneration (Lam et al., 2015), and corneal wound healing is delayed in the absence of platelets. The release of growth factors and VEGF-A from activated platelet granules promote corneal epithelial cell and nerve recovery. In response to injury,

PMNs and platelets extravasate from blood vessels. It is suggested that platelets bind to

PMNs, and this interaction primes the PMNs to enhance PMN functions in response to microorganism invasion, and boost PMN extravasation (Page and Pitchford, 2013).

1.4: Corneal wound in mice

A healthy and transparent cornea is vital for viewing the world. The cornea comes in contact with the external environment, which invariably exposes it to pathogens on a regular basis. Therefore, an intact cornea is essential to form a physical barrier, and

21 protect our eyes from infection. The intricate arrangement of the layers in the cornea and the collagen fibrils, as well as the cellular and molecular composition of the cornea, account for maintaining corneal transparency. Corneal abrasions due to accidents, infections, corneal dystrophies, contact lens wear, and refractive surgeries can injure the cornea, as evidenced by loss of corneal epithelial cells, nerves, and stromal keratocytes at the site of injury (Li et al., 2011a; Petrescu et al., 2007), and elicit an inflammatory response. Rapid re-epithelialization is essential to ward off infections from invading pathogens. Keratocyte regeneration is vital for stromal repair and the maintenance of corneal transparency (Burns et al., 2005; Gagen, 2012).

In response to injury, leukocyte recruitment is initiated by the release of cytokines, chemokines, and upregulation of specific cell adhesion molecules by vascular endothelial cells (Choi et al., 2009; Smith and Anderson, 1991; Zarbock and Ley, 2008).

Leukocytes preferentially extravasate at post-capillary venules where the shear forces favor leukocyte adhesion (Bienvenu and Granger, 1993; Kim and Sarelius, 2004).

In a mouse model of central corneal abrasion, the removal of the central epithelium elicits an acute inflammatory response involving PMN and platelet extravasation from the limbal blood vessels surrounding the avascular cornea (Hayashi et al., 2010; Li et al., 2011a; Petrescu et al., 2007). This recruitment of PMNs and platelets is essential, as it helps in wound healing, tissue regeneration, and nerve regeneration (Li et al., 2006b; Marrazzo et al., 2011). The magnitude of the response is regulated by a coordinated cascade of inflammatory cells and mediators (Li et al., 2011a; Li et al.,

2011b). However, excessive or chronic inflammation results in tissue damage. Pro and anti-inflammatory mediators and cytokines act in tandem to achieve the delicate balance of initiating the innate response and facilitating wound healing. The increase in anti- inflammatory mediators, while inhibiting pro-inflammatory mediators and cytokines helps to prevent tissue damage (Jaeschke and Hasegawa, 2006).

Figure I-1: Inflammatory cascade after corneal injury in the mouse.

Representative illustration showing some of the cells and molecules

involved in the initiation and propagation of innate inflammation and

leukocyte extravasation. (Figure courtesy of CW Smith)

As shown in Figure 1.1, a central corneal epithelial abrasion initiates the migration of γδ T cells from the limbus to the corneal epithelium (Byeseda et al., 2009;

Li et al., 2011b), and the release of Interleukin-22 (IL-22) (Li et al., 2011b), a cytokine that can induce motility and division in epithelial cells (Laurence et al., 2008). The released IL-22, in response to the injury, stimulates epithelial secretion of the chemokine

CXCL1 (Liang et al., 2010; Stacey et al., 2014). Since CXCL1 is a potent chemoattractant of PMNs, PMN recruitment is enhanced. Since PMN and platelet recruitment is co-dependent, platelet extravasation is also increased. Growth and other essential factors are released, thus benefitting corneal wound healing (Li et al., 2011b).

Overall, the inflammatory cascade is orchestrated by the release of pro-inflammatory cytokines.

1.5: Cytokines

Cytokines are small secreted proteins about 8-40KDa (Dinarello, 2000) released by cells.

They act upon the same, other nearby, or distant cells to affect inflammation. Cytokines modulate the inflammatory response by regulating host response to injury, insult, or infection. Depending on the end-result, cytokines can be classified as pro-inflammatory or anti-inflammatory. Pro-inflammatory cytokines increase inflammatory response to injury by upregulating genes or by activating other small proteins such as chemokines or chemoattractants. Pro-inflammatory cytokines such as Interleukin-1 (IL-1) or Tumor

Necrosis Factor (TNF) increase innate inflammation by enhancing vascular permeability to facilitate the extravasation of PMNs from blood vessels (Zhang and An, 2007). Anti- inflammatory cytokines on the other hand decrease inflammation either by suppressing

24 gene upregulation of pro-inflammatory molecules and cytokines (Dinarello, 2002) or by inhibiting the release of chemokines and chemoattractants.

An imbalance in pro and anti- inflammatory cytokines enhances the inflammatory response in eye diseases (Pflugfelder et al., 2013). The cytokine IL-33 is suggested to play a role in initiating allergy induce conjunctivitis (Asada et al., 2015). Imbalance between pro and inflammatory cytokines alters disease progression in autoimmune uveitis, an inflammatory disease of the eye (Horai and Caspi, 2011). As observed in the above studies, cytokines play a very crucial role in modulating the inflammatory response.

1.5.1: The IL-10 super family of cytokines

The cytokine IL-22 belongs to the IL-10 super family. Other members of this family include IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29 (Blumberg et al., 2001; Ouyang et al., 2011). The IL-10 family of cytokines are part of the class II family of cytokines as they interact with class II receptors via interactions with the non- receptor tyrosine kinase Janus Kinase (JAK) family (Ferrao et al., 2016; Kotenko and

Pestka, 2000). This family of cytokines plays a wide role during injury or inflammation starting with initiating host defense and launching an immune response to aid with wound healing and tissue remodeling.

The genetic similarity between the members of the IL-10 family is the basis for grouping them together. This family of cytokines function via similar signaling pathways and also share their receptors (Commins et al., 2008; Fickenscher et al., 2002).

Figure I-2: IL-10 family of cytokines have receptor overlaps. IL-20

binds with either IL-20 R1/IL-20R2 or IL-22R1/IL-20R2. IL-10, IL-22,

IL-28, and IL-29 all share the receptor IL10R2. This biological similarity

among these cytokines forms the basis for grouping them as the IL-10

super family. Figure adopted from Logston et, al. 2012.

IL-10 is very important for resolution of inflammation as it suppresses inflammation and helps to prevent tissue damage during bacterial and viral infections

(Mege et al., 2006; Ouyang et al., 2011). Similar to its role in inflammatory bowel disease (IBD), the IL-10 family (IL-10, IL-19, IL-20, IL-22, and IL-24) is protective during liver inflammation by suppressing production of pro-inflammatory cytokines and

26 increasing cell proliferation (Ouyang et al., 2011; Zenewicz et al., 2007). Thus the IL-10 family of cytokines play a very important role in modulating inflammation and aiding tissue healing.

1.5.2: IL-20 sub-family of cytokines

The IL-20 subfamily consisting of IL-19, IL-20 IL-22, IL-24 and IL-26 resides within the larger IL-10 superfamily (Blumberg et al., 2001; Eidenschenk et al., 2014; Rutz et al.,

2014). Similar to the IL-10 super family, the IL-20 subfamily receptors are heterodimers and members share common receptor subunits. Members of the IL-20 family enable interactions between innate leukocytes and epithelial cells (Rutz et al., 2014; Zdanov,

2010).

Figure I-3: IL-20 sub-family of cytokines. Receptors are heterodimeric

and members share receptor sub-units. Figure adopted from Logston et,

al. 2012.

This dissertation focused on IL-20, a member of the IL-20 subfamily. IL-20 plays a role in various diseased tissues and binding to its receptor activates a STAT3- dependent pathway and promotes cell proliferation in human keratinocyte and glioblastoma cell line (Blumberg et al., 2001; Chen and Chang, 2009). Based on the increase in IL-20 levels, it is suggested that IL-20 is pro-inflammatory in diseases such as psoriasis (Lowes et al., 2007; Otkjaer et al., 2005; Romer et al., 2003) and rheumatoid arthritis (Kragstrup et al., 2015; Kragstrup et al., 2008). IL-20 and the IL-20 subfamily induce genes regulating cell proliferation, participate in tissue repair and wound healing

(Sa et al., 2007) as well as promote host protection by releasing antimicrobial peptides

(Wolk et al., 2008) in keratinocytes. Further, a dysregulation in IL-20 signaling could lead to primary open angle glaucoma (Wirtz and Keller, 2016). The above studies suggest that the effects of IL-20 appear to be tissue specific.

1.5.3: Role of IL-20 subfamily in mouse corneal wound healing

In the mouse cornea, similar to any other tissue in the body, injury elicits an inflammatory response. The innate defense response involves the upregulation of pro- inflammatory cytokines such as IL-22, and the release of PMN chemoattractant CXCL1.

PMNs and platelets emigrate out of limbal venules and PMNs migrate into the corneal stroma (Li et al., 2006b; Petrescu et al., 2007). The migrating leukocytes not only defend the cornea against possible pathogen invasion, but also benefit wound healing as they deliver growth factors like VEGF-A (Li et al., 2006a) essential in promoting regeneration of epithelial cells, nerves, and keratocytes (Lam et al., 2015).

Interleukin-22 (IL-22) is released from infiltrating γδ T cells in response to injury

(Sabat et al., 2014) . Mouse experiments in which a neutralizing anti-IL-22 antibody was applied topically after corneal abrasion, not only significantly reduced PMN recruitment, but also delayed epithelial wound closure (Li et al., 2011b). γδ T cells are the major source of IL-22 in the mouse cornea. Experiments involving topical application of recombinant IL-22 after corneal injury in γδ T cell knockout mice restored corneal wound healing (Li et al., 2011b). IL-22 plays an important role in corneal wound healing.

IL-22 RA is a receptor subunit which binds IL-22 and the mouse corneal epithelium shows positive staining for the IL-22RA subunit (Li et al., 2011b). As shown in the IL-20 subfamily figure in the introduction section (Figure I-3), IL-22RA subunit not only binds IL-22 but can also couple with IL-20RB and bind IL-20 (Rutz et al.,

2014). Corneal basal epithelial cells stain positively for the IL-20 receptor subunit IL-

20RA. Two heterodimeric receptors IL-20RA/IL-20RB and IL-22RA/IL-20RB can bind

IL-20, and a single heterodimer receptor IL-22RA1/IL-10RB can bind IL-22 (Rutz et al.,

2014). Thus the receptor subunit IL-22RA is shared by both IL-20 and IL-22 and the corneal epithelium also stains positively for IL-22RA. After corneal abrasion, epithelial cells and keratocytes show positive staining for IL-20 (Wanyu Zhang and Sri Magadi, manuscript submitted).

Experimental interventions that limit PMN and platelet recruitment typically result in delayed wound healing (Li et al., 2006b; Nagaoka et al., 2000). However, we

29 now have unpublished data showing topical application of IL-20 to the injured cornea sustains normal epithelial healing and nerve regeneration while inhibiting the potent chemoattractant CXCL1 levels thereby suppressing PMN recruitment by about 70%

(Wanyu Zhang and Sri Magadi, manuscript submitted) suggesting IL-20 has therapeutic potential in the treatment of corneal injuries. Before we finalize that conclusion, it is important to determine whether platelet recruitment is normal in the presence of recombinant IL-20 topically applied to the wounded cornea (evaluated in Aim 1).

The early response to corneal insult is mediated by neutrophil (PMN) and platelet extravasation form the limbal vasculature and the extensive infiltration of PMNs into the wounded cornea. These PMNs and platelets are essential for wound healing and tissue repair. While the initiation of innate inflammation with PMN migration to the site of injury is essential, it is just as important to have mechanisms for their removal to avoid the onset of chronic inflammation since the very defense mechanism that seeks to protect the tissue can also destroy tissues (evaluated in Aim 2).

The purpose of this dissertation was to evaluate mechanisms regulating platelet recruitment after corneal injury, evaluate PMN migration in the abraded mouse cornea and determine the mechanism by which central PMNs “disappear” from cornea during resolution of inflammation.

Chapter 2: Aim 1- Evaluate mechanisms regulating platelet recruitment after corneal injury

2.1: Introduction

PMN-platelet co-dependent recruitment originated with experiments in which an anti-PMN antibody, GR1, was given to the mouse prior to corneal abrasion to reduce

PMNs from the circulation (Li et al., 2006a). Injecting anti-PMN antibody, GR1, into the mouse prior to corneal abrasion diminishes PMNs from circulation and markedly blunts

PMN and platelet recruitment into the injured cornea. It is a well-used method for performing experiments using neutropenic mice (Li et al., 2011a; Queen and Satchell,

2013; Singh et al., 2012).It is now known that the GR1 antibody is not specific for PMNs and can affect certain mononuclear cells as well. Anti-Ly6G antibody is very specific to

PMNs (Daley et al., 2008). The first part of this aim was to evaluate PMN and platelet recruitment after corneal abrasion in mice treated with anti-Ly6G.

The early response to corneal insult is mediated by PMN and platelet extravasation form the limbal vasculature and the extensive infiltration of PMNs into the wounded cornea (Gan et al., 1999; Hanlon et al., 2014). These leukocytes are essential for wound healing and tissue repair (Li et al., 2006b) as they deliver several growth factors into the cornea. The magnitude of the response is regulated by a coordinated cascade of inflammatory cells and mediators (Kolaczkowska and Kubes, 2013; Li et al.,

2011a; Li et al., 2011b). One such mediator is the cytokine IL-20 which belongs to the

IL-20 sub-family of the IL-10 super-family. Previous studies show topical application of

IL-20 inhibits PMN recruitment without delaying wound closure (unpublished Wanyu

Zhang). The second part of this aim was to evaluate the effect of IL-20 on platelet recruitment after corneal injury in mice.

CD18 is a β2 integrin heterodimer and two members of this family, lymphocyte function-associated antigen (LFA-1) a.k.a. CD11a/CD18 and Macrophage-1 antigen

(Mac-1) a.k.a. CD11b/CD18 are present on PMNs (Dimasi et al., 2013; Tonnesen, 1989).

LFA-1-dependent adhesion to the inflamed endothelium is necessary for efficient emigration of PMNs at sites of inflammation, and Mac-1-dependent adhesion enhances phagocytosis (Li et al., 2006). After a central corneal injury, CD18 “hypo” mice express low levels of CD18, show normal PMN recruitment but delayed wound healing with little or no platelet recruitment (Lam et al., 2015). The third part of this aim was to further evaluate the role of CD18 on platelet recruitment during corneal inflammation in mice.

Corneal epithelial abrasion elicits an acute inflammatory response involving emigration of PMNs and platelets from limbal vasculature. The release of growth factors from extravasated PMNs and platelets is beneficial to wound healing. Blood vessel dilatation is a hallmark of inflammation (Gutterman et al., 2016; Hersh and Bodey,

1970). In a separate, but related study in our lab, we found that sustained limbal vessel dilation was blunted in mast cell deficient mice (kitw-sh/w-sh) (~75%), there was a marked delay in PMN extravasation, and little or no platelet extravasation (Hargrave, unpublished).

Arterioles have more smooth muscle than venules and can actively change their diameter, whereas venules are passive (Granger DN, 2010). Vascular resistance to blood flow follows Poiseuille's Law (Resistance = 8휂퐿/휋푅4 where η = viscosity of blood, L= length of the blood vessel, R=radius of the blood vessel) (Pfitzner, 1976; S P Sutera,

1993). Clearly, the resistance to blood flow is inversely proportional to the 4th power of the radius of the vessel, a very small change in arteriole diameter can result in a large change in blood flow which in turn will affect venule diameters. Hence changes in both arteriole and venule diameters were evaluated in this study following corneal injury.

Mast cells are in close proximity to the limbal blood vessels. Apart from their well-known role during allergic reactions, in response to injury, activated mast cells release vasoactive molecules (Krystel-Whittemore et al., 2015; Kunder et al., 2011) such as histamine which binds the vascular endothelial cells and arterioles (Payne et al., 2004) causing the blood vessels to dilate thereby increasing vascular permeability (Krystel-

Whittemore et al., 2015; Metcalfe et al., 1981). Hence mast cells as well as degranulated mast cells were evaluated in this study.

This aim tested the hypothesis that PMN-platelet extravasation is important for corneal wound healing and evaluated mechanisms regulating platelet recruitment after corneal injury. Specifically this aim further investigated the co-dependency of platelet-

PMN recruitment in mice treated with anti-Ly6G and IL-20 and examined whether mast cells play a role in modulating inflammation, and if PMN CD18 plays a role in platelet extravasation in our mouse model of corneal injury.

2.2: Methods

Eight-twelve week old adult C57BL/6J mice were used for all experiments. Experiments were conducted at the Children’s Nutrition Research Center (CNRC) at the Baylor

College of Medicine and the Michael E. DeBakey VA Medical Center, Houston, TX. All mice were treated following both institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

2.2.1: Choice of animal model

The inflammatory response that follows a corneal abrasion is complex and impossible to replicate in an in vitro setting and the animal model of choice is the mouse. Although there are differences between mouse and human eyes, there are many similarities and the mouse is routinely used by many investigators to study the cornea. Our lab has published numerous studies using the mouse model of corneal injury and these studies provide the necessary background data and rationale for pursuing the experiments outlined in the current study.

Previous studies have shown that corneas of C57BL/6J mice under the age of 8 weeks are still developing (Hanlon et al., 2011). Older mice (e.g., 6 months) show significant age-related reductions in epithelial nerve density (Wang et al., 2012).

Nerve density is relatively stable from 8-12 weeks of age and mice over 8 weeks of age are considered to have fully matured with respect to corneal thickness (Hanlon et al.,

2011). Hence 8-12 week old mice were used for all studies because the cornea is considered mature.

2.2.2: Corneal abrasion model

A central corneal wound was made according to a previously established protocol (Li et al., 2006b). Briefly, adult C57BL/6J wild-type mice (n=4/group) were purchased from

Jackson Laboratory (Bar Harbor, ME). Mice were anesthetized by intraperitoneally injecting pentobarbital sodium solution (50mg/kg) and a 2mm diameter central corneal epithelial region was marked by a trephine and mechanically debrided with a golf club spud or alger brush under a dissecting microscope (Figure M-2.1) while taking care not to penetrate the basement membrane (Figure M-2.2). The white dotted line in Figure M-2.1 marks the edge of the wound. Black arrow in Figure M-2.2 is pointing at the wound edge.

Corneal epithelial cells can be seen to the right of the black arrow while white arrows are pointing at the intact uninterrupted basement membrane. The inflammatory response differs depending on the size, shape or depth of the wound. Therefore care was taken to maintain consistency in wounding the mice.

Figure M-2.1: Central corneal wound in C57BL/6J mice. Cornea is

stained with fluorescein (green) to mark the wound edge (white dash line)

Figure M-2.2: Intact basement membrane after corneal wound. Plastic

cross-section of the cornea stained with Toluidine Blue. Wound edge

(black arrow) with intact basement membrane (white arrows). Bar =

15µm.

2.2.3: Immunohistochemistry and antibodies used

Twenty-four hours post corneal injury, mice were euthanized and whole eyes were fixed in phosphate buffered saline (PBS) containing 2% paraformaldehyde (PFA) for fifteen minutes. Corneas along with the limbal blood vessels were excised and further fixed in

PBS-buffered 2% PFA for forty-five minutes and then washed three times, five minutes per wash in PBS. Corneas were permeabilized with 0.1% Triton-X for thirty minutes and blocked for thirty minutes with PBS containing 1% bovine serum albumin (BSA). Fc receptors were blocked to avoid non-specific binding of antibody. Four incisions were made in the corneas to make radial cuts and the corneas were incubated overnight with fluorescently labelled anti-mouse antibodies (Figure M-2.3) PE or APC conjugated anti-

CD31 which labels vascular endothelial PECAM-1 (BD Bioscience, San Jose, CA) for 36 detection of blood vessels, DAPI to label all cell nuclei, and TRITC conjugated anti-

CD41 which labels GPIIb for detection of platelets (BD Bioscience, San Jose, CA). FTIC conjugated Avidin (ebiosciences, San Diego, CA) and APC conjugated CD80 (BD

Bioscience, San Jose, CA) were used to label mast cells. Purified anti-Ly6G (Rat IgG2a, clone 1A8), (BD Bioscience, San Jose, CA) was used for antibody depletion of circulating PMNs.

Figure M-2.3: Corneal flat mount of an adult C57BL/6J mouse.

Limbal vessels (*) are stained with anti-CD31 antibody to label all blood

vessels. Leukocytes were counted and vessel diameters were measured

using images from this region. Bar = 90 µm.

2.2.4: Imaging and counting platelets and mast cells

The entire corneal limbus was divided into four quadrants. Z-stacks of panels of each immunostained quadrant were imaged using a 20X lens mounted on a DeltaVison Core microscope system (Applied Precision, Issequah, WA). The captured panels were

37 deconvolved and stitched together using SoftWorx software. The stitched image of each quadrant labeled with fluorescent antibodies against blood vessels and platelets (Figures

M-2.4 and M-2.5), and FITC-avidin to stain mast cell granules, was used for counting intra- and extra-vascular platelets and limbal mast cells (Figures M-2.7 and Figure M-

2.8).

Figure M-2.4: Stitched panels of the limbal vessels used for platelet

counts. Several panels of images in the limbal region were deconvolved

and stitched to get a single image of an entire quadrant shown in the image

above. Limbus is fluorescently labelled for blood vessels (anti-CD31, red)

and platelets (anti-CD41, green). Bar = 60µm.

Figure M-2.5: Intravascular and extravascular platelets. Limbal blood

vessels (red) and platelets (green) are labelled in the cornea 24h after a

central corneal abrasion (A&B). Panel B shows platelets only without

blood vessels present in panel A. Arrows point to platelets inside blood

vessels and arrowheads show platelets outside limbal vasculature.

2.2.5: Counting PMNs in the injured cornea

In response to a central corneal injury, PMNs emigrate out of the limbal blood vessels (* in Figure M-2.6), migrate through the stroma and reach the corneal center. Hence PMNs were counted at various corneal regions (Figure M-2.6).

Figure M-2.6: Corneal regions for counting PMNs. PMN counts made

at L=limbus, PL=Paralimbus, PW=Parawound, and WC=Wound center.

2.2.6: Counting mast cells at the limbal region

Figure M-2.7: Limbal region selected for counting mast cells 24h post-

injury. Corneal limbal vessels were fluorescently labelled (anti-CD31,

red). Mast cells very close to the limbus were counted in the limbal region

marked by the white dashed line. Bar = 60µm.

Figure M-2.8: Counting mast cells and degranulated mast cells 24h

post-injury. Corneal limbal mast cells were fluorescently labelled (anti-

avidin, green). The entire z-stack of the limbal mast cell images were

analyzed and a limbal mast cell with more than 10 extracellular granules

(white arrows) within one mast cell diameter was recorded as a

degranulated mast cell. Bar = 15 µm.

2.2.7: Imaging the limbus and diameter measurements

The entire corneal limbus was divided into four quadrants. Z-stacks of panels of each immunostained quadrant was imaged using a 20X lens mounted on a DeltaVison Core

Spectris microscope system (Applied Precision, Issequah, WA). The captured panels were deconvolved and stitched together using SoftWorx software. The stitched image of all the quadrants labelled for blood vessels was used for venule and arteriole diameter measurements. Limbal venule and arteriole diameters were measured at 50µm intervals along the entire vessel length (Figure M-2.9) and all values were graphed using a cumulative frequency plot.

Figure M-2.9: Measurement of venule and arteriole diameters 24h

post-injury. Several panels of images in the limbal region are deconvolved

and stitched to get a single image of an entire quadrant. The limbus is

fluorescently labelled for blood vessels (anti-CD31, red). Bar = 60µm (A).

Figure (B), the highlighted part of the image with dashed lines in figure

(A) is enlarged and rotated 90°. Venule (arrow) and arteriole (star)

diameters were measured at 50µm intervals (B) through the entire length

of the corneal limbus. Bar = 40µm.

2.2.8: IL-20 administration

After corneal abrasion, over a 24h period, once every 4h, wounded mice were given topical applications (200ng/ml) of recombinant mouse IL-20 (rIL-20) (R & D Systems,

Minneapolis, MN) dissolved in phosphate buffered saline (PBS) (200ng/mL) and control mice received PBS.

2.2.9: Administration of anti-Ly6G antibody

24h prior to injury, mice received a single intraperitoneal (I.P) injection of 5mg of anti-

Ly6G antibody (BD Bioscience, San Jose, CA) prepared in 1ml of PBS.

2.2.10: Isolation of PMNs

CD18 hypo mice with low levels of CD18 expression, as well as wild-type mice corneas, were wounded as explained in the general methods section above. Isolated wild-type

PMNs with normal levels of CD18 and PMNs from mice expressing low levels of CD18 were infused into CD18 hypo mice. A density gradient centrifuge protocol established by

Swamydas et.al., (Swamydas and Lionakis, 2013) was used to isolate the PMNs. Briefly, mouse bone marrow cells were collected from the femur and the tibia and then centrifuged at 1,400 rpm at 4°C for 7 minutes. The cells were then re-suspended in sterile

PBS, centrifuged for 30minutes at 2,000 rpm at room temperature for separating PMNs.

PMNs were counted using flow cytometry. 200,000 PMNs were injected into each mouse via the tail vein.

2.2.11: Statistical Analysis

Data were analyzed using a student’s t-test or a one-way or two-way analysis of variance

(ANOVA) with a Tukey post-test for pair-wise multiple comparisons. Data are expressed as mean±SE and P values ≤0.05 were considered statistically significant.

2.3: Results

2.3.1: Evaluate the role of anti-Ly6G in mice after corneal injury

The issue of PMN-platelet co-dependent recruitment originated with experiments in which an anti-PMN antibody, GR1, reduced PMN and platelet recruitment into the injured cornea. GR1 antibody is not specific for PMNs, and anti-Ly6G antibody specifically reduces the animal of PMNs (Daley et al., 2008). Hence, to investigate the effect of specifically inhibiting PMN recruitment into the injured cornea, PMNs, platelets, and limbal mast cells were counted and limbal vessel diameters were measured following treatment with anti-Ly6G antibody.

2.3.1.1: Evaluate PMN and platelet co-dependent recruitment after corneal abrasion in mice treated with anti-Ly6G.

The purpose of this study was to determine the effect of anti-Ly6G treatment on PMN and platelet co-dependent recruitment in our mouse model of corneal injury. The data suggest that anti-Ly6G antibody administration reduced circulating PMNs (Figure C-2.1).

24h after corneal injury in anti-Ly6G treated mice, PMN counts at all the regions of the cornea were significantly reduced when compared to controls (Figure C-2.2). Previous studies in the lab showed a codependency in platelet and PMN recruitment. Antibody depletion of platelets reduced PMN recruitment and vice versa in our mouse model of corneal injury (Li et al., 2006b). Since anti-Ly6G treated mice had significantly reduced

PMN extravasation, platelets were evaluated in these mice as detailed in the methods section (Figure M-2.5). Results showed a significant reduction in platelet extravasation

(~80%) (Figures C-2.3 and C-2.4) in mice treated with anti-Ly6G antibody.

Figure C-2.1: Anti-Ly6G antibody reduced circulating PMNs.

Unpublished data courtesy of Wanyu Zhang. Data compiled and analyzed by Sri Magadi. (n =4, p≤ 0.01).

Figure C-2.2: Reduced PMN counts across the injured cornea in anti-

Ly6G mice. Compared to wild-type mice, anti-Ly6G treated mice had significantly less PMNs at different corneal regions. (n=4, p≤0.05).

A B

Figure C-2.3: Platelet counts were reduced in anti-Ly6G treated mice.

Mice treated with anti-Ly6G antibody (A) compared to wild-type controls

(B). The image shows limbal blood vessels (red) platelets (green) 24h after

a central corneal abrasion. Bar = 40µm.

Figure C-2.4: Platelet extravasation in anti-Ly6G treated mice. 24h after corneal injury, total platelets which accounts for platelets inside and outside the limbal vessels (A) and extravascular (outside) platelets (B).

(n=4, p≤0.05).

2.3.1.2: Evaluate venule and arteriole diameters in wounded mice treated with anti- Ly6G antibody

Vasodilation is a hallmark feature of acute inflammation. Therefore limbal vessel diameters were measured as detailed in the methods section (Figure M-2.9). A significant reduction in both limbal venule and arteriole diameters was observed in mice treated with anti-Ly6G antibody (Figure C-2.5).

Figure C-2.5: Limbal vessel dilatation in anti-Ly6G treated mice after corneal injury. Figures A and C are graphs of the cumulative distribution functions. 24h

post-injury, venule (A & B) and arteriole (C &D) diameters were

measured. (n=4, p≤0.0001).

2.3.1.3: Evaluate Mast cells in wounded mice treated with anti-Ly6G antibody

In wildtype mice, mast cells are in close proximity to limbal vessels and in response to injury, they release histamine. Binding of histamine to the blood vessels increases vascular permeability. Hence non-degranulated mast cells and degranulated mast cells at the limbus were evaluated as detailed in the methods section (Figure M-2.7 & Figure M-

2.8). Although mast cell numbers in anti-Ly6G treated mice were not different than wild- types, these mice had significantly reduced degranulating mast cells (Figure C-2.6).

Figure C-2.6: Mast cells in mice treated with anti-Ly6G antibody after corneal injury. Wild-type mice were treated with anti-Ly6G antibody 24h after corneal abrasion. Limbal mast cell (A) and degranulated mast cells

(B) were counted at the corneal limbus. (n=4, p≤0.001).

2.3.4: Evaluate the role of IL-20 in mice after corneal injury

Interleukin-20 (IL-20) is a member of the IL-20 subfamily (Figure I-3) and in epidermal tissues, it plays a significant role in wound healing (Blumberg et al., 2001). To investigate the role of IL-20, mice were wounded (Figure M-2.1 and Figure M-2.2) and

IL-20 was administered as described in the methods section (2.2.8). PMNs, platelets and limbal mast cells were counted and limbal vessel diameters were measured (Figure M-2.5

– Figure M-2.9).

2.3.4.1: Role of IL-20 on platelet recruitment after corneal abrasion in mice

After corneal abrasion, epithelial cells and keratocytes show positive staining for IL-20, remarkably, topical application of IL-20 inhibits CXCL1 levels thereby inhibiting PMN recruitment while sustaining normal epithelial healing and nerve regeneration (Wanyu

Zhang and Sri Magadi, manuscript submitted).Whether platelet recruitment is normal under these conditions and accounts for the normal rate of wound closure is unclear.

Since previous experiments as well as the anti-Ly6G treated mice showed a co- dependency of PMN-platelet recruitment, and since PMN recruitment is reduced in IL-20 treated mice, platelets were evaluated in mice treated with IL-20.

The anti-inflammatory effects of IL-20 in our mouse model of corneal injury was supported by data which showed a significant reduction (~70%) in extravasated platelets in IL-20 treat mice 24h after corneal abrasion (Figures C-2.7 and C-2.8).

Figure C-2.7: Topical IL-20 reduced platelet recruitment after corneal abrasion.

Limbal blood vessels (red) platelets (green) of an adult C57BL/6J wild- type mouse cornea 24h after a 2mm central corneal abrasion treated with

PBS (A&B) or recombinant mouse IL-20(C&D). Panels B and D show platelets only without blood vessels present in figures A&C, respectively.

Long arrows point to platelets inside blood vessels and short arrows show platelets outside the limbal vasculature. Bar = 40µm.

Figure C-2.8: PMN and Platelet extravasation is inhibited in IL-20 treated mice. 24h after corneal abrasion, total platelets (I=platelets inside blood vessels and 0=extravasated platelets) were evaluated (n=4, p≤0.05).

(PMN data provided by Wanyu Zhang) (A). Extravasated platelet (outside platelets) counts are shown in (B) (n==4, p≤0.05).

Since IL-20 treated mice showed diminished PMN and platelet recruitment after corneal injury, limbal vessel diameters were measured in these mice. The data showed a significant decrease in venule and arteriole diameters in the presence of IL-20 in mice after corneal abrasion (Figure C-2.9). Since vessel dilatation is associated with mast cell degranulation, limbal mast cells and degranulated mast cells were evaluated in IL-20 treated mice. Although mast cell numbers in IL-20 treated mice were not different than wild-types, these mice had significantly fewer degranulating mast cells (Figure C-2.10).

2.3.4.2: Evaluate venule and arteriole diameters in wounded mice treated with IL-20

Figure C-2.9: Limbal vessel dilatation in IL-20 treated mice after corneal injury. Figures A and C are graphs of the cumulative distribution functions. 24h post-injury, venule (A & B) and arteriole (C &D) diameters were measured. (n=4, p≤0.0001).

2.3.4.3: Evaluate Mast cells in wounded mice treated with IL-20

Figure C-2.10: Mast cells in mice treated with IL-20 after corneal

injury. Wild-type mice were treated with IL-20 24h after corneal

abrasion. Limbal mast cell (A) and degranulated mast cells (B) were

counted at the corneal limbus. (n=4, p≤0.001).

2.3.5: Evaluate the role of CD18 on platelet recruitment during corneal inflammation in mice.

Data from the IL-20 and anti- Ly6G groups of mice showed that reduced limbal vessel dilation was associated with low PMN and platelet recruitment in our mouse model of corneal injury. Since previous studies showed that CD18hypo mice have normal PMN extravasation but reduced platelet extravasation (Lam et al., 2015), this mouse model was used to tease out the role of CD18 on platelet extravasation.

2.3.5.1: Initial Pilot study to evaluate limbal vessel diameters in CD18 hypo mice with low levels of CD18 and known to have reduced platelet recruitment

The initial pilot study in CD18hypo mice with normal PMN but blunted platelet extravasation evaluated limbal vessel diameters to determine whether platelet extravasation was associated with limbal vessel dilation. Limbal venule and arteriole diameters were measured as detailed in the methods section (Figure M-2.9). The data showed a significant decrease in venule and arteriole diameters in these CD18hypo mice after corneal abrasion (Figure C-2.11).

Figure C-2.11: Pilot study: Limbal vessel dilation is reduced in

CD18hypo mice with known reduction in platelet extravasation after corneal injury. Figures A and C are graphs of the cumulative distribution functions. 24h post-injury, venule (A & B) and arteriole (C &D) diameters were measured. (n=3, p≤0.0001). Venule and arteriole diameters in both the uninjured wild-type and CD18hypo mice were not significantly different.

2.3.5.2: Evaluate the role of CD18 in platelet extravasation

Since the pilot study showed an association between limbal vessel dilation and platelet extravasation, the role of CD18 on platelets in injured CD18hypo mice corneas was further investigated. Generally CD18 levels in CD18hypo mice are reduced by ~80%

(Wilson et al., 1993). The study conducted in 2015 evaluated CD18hypo mice after corneal injury. Platelet counts and limbal vessel diameter measurements were higher in these mice and I could not replicate the pilot study. Upon further investigation, flow cytometry and genotyping data leaned toward genetic contamination in these mice.

However these mice had intermediate to high (I-H) levels of CD18 compromising the interpretation of these data. For this reason, a new group of CD18hypo mice were purchased in 2016 and they were confirmed to have low-intermediate (L-I) levels of

CD18. These mice were monitored by genotyping for the neomycin cassette to ensure there was no change in genotype. In both the groups, 24h post-injury, corneas were excised and platelet extravasation, limbal venule and arteriole diameters as well as mast cells counts were recorded as detailed in the methods section (Figure M-2.5 – Figure M-

2.9).

24h after corneal injury there was no significant difference in platelet extravasation, in mice with intermediate-high levels of CD18 (Figure C-2.12) whereas platelet extravasation was significantly reduced in CD18hypo mice with low-intermediate levels of CD18 (P≤0.001). One must use caution while interpreting results. Three mice with intermediate-high levels of CD18 were used for the experiment and we need at least

14 mice to detect a significant difference in platelet extravasation in these mice. Anti-

Ly6G and IL-20 treated mice showed an association with leukocyte recruitment and changes in limbal vessel dilatations, limbal vessel diameters were measured in both the

CD18 (I-H) and CD18 (L-I) groups of mice (Figure C-2.13).

Figure C-2.12: Extravasated platelets in CD18hypo mice with

intermediate-high (I-H) and low-intermediate (L-I) levels of CD18 24h

after corneal injury. 24h after corneal abrasion, platelet extravasation in

mice with intermediate-high levels of CD18 (A, n=3) and in CD18hypo

mice with low-intermediate levels of CD18 (B) was evaluated (n=4,

P≤0.001).

2.3.5.3: Evaluate the role of CD18 on limbal venule and arteriole diameters

The increase in average limbal venule diameter in wild-type mice was not statistically different from the increase observed in CD18hypo mice with intermediate to high (I-H)

CD18 levels (Figure C-2.13 panels A & B). At 24h post-injury, the small increase in venule (Figure C-2.13 E&F) and arteriole (Figure C-2.13 G&H) diameters in CD18hypo mice with low-intermediate (L-H) levels of CD18 was not statistically significant.

Although average arteriole diameters in CD18hypo mice with intermediate to high (I-H) showed statistical significance (Figure C-2.13 C& D), the increase in average arteriole diameter in CD18hypo mice with intermediate to high (I-H) CD18 levels was greater than that observed in CD18hypo mice with low-intermediate (L-H) levels of CD18

(Figure C-2.13 C&D, G&H).

Figure C-2.13: Limbal venule and arteriole diameters in CD18hypo mice with intermediate-high (I-H) and low-intermediate (L-I) levels of

CD18. Figures A, C, E and G are graphs of the cumulative distribution functions. 24h post-injury, venule (A&B, E&F, n=3) and arteriole (C&D,

G&H) diameters were measured. (n=4) (p≤0.0001).

2.3.5.4: Evaluate the role of CD18 on limbal mast cells

Limbal mast cell numbers were reduced in mice with intermediate-high (I-H)

CD18 levels but mast cell degranulation was not significantly different than the wild-type controls 24h post-injury. In contrast, CD18hypo mice with low-intermediate levels (L-I) of CD18 had significantly fewer limbal mast cells as well as significantly less number of degranulating mast cells 24h after corneal injury (Figure C-2.14).

Figure C-2.14: Limbal mast cell and degranulated limbal mast cells in

CD18hypo mice with intermediate-high (I-H) and low-intermediate

(L-I) levels of CD18. 24h after corneal injury, limbal mast cells (A &C) and degranulated mast cells (B &D) were counted at the corneal limbus.

(CD18hypo (I-H), n=3 & CD18hypo (L-I), n=4, p≤0.001).

2.3.6: Evaluate if platelet extravasation can be restored in PMN reconstituted mice

Further, reconstitution experiments to see if vessel dilation and platelet recruitment can be restored in CD18 hypo mice with low levels of CD18 were conducted by injecting isolated PMNs either from wild-type or CD18hypo mice (L-I). The method for isolation of PMNs is detailed in the general methods section (2.2.10). Extravasated platelets, limbal vessel diameters, limbal mast cells and degranulated mast cells in the limbal region were evaluated.

Extravasated platelet counts was not significantly different in CD18hypo mice treated with wild-type or with CD18hypo PMNS, but both the groups had significantly less platelets outside the limbal vasculature compared to wild-type controls (Figure C-

2.15, P≤0.01). In wild-type mice, the average limbal venule diameter increased by ~70%

(p≤0.0001) and arteriole diameter by ~114%. The increase in venule (Figure C-2.16, A-

B) and arteriole diameter (Figure C-2.16, C-D) was not statistically different from CD18 hypo mice that received an infusion of CD18hypo PMNs. Since vessel dilatation appears to be a constant feature associated with platelet extravasation, and mast cell degranulation is associated with vessel dilatations, limbal mast cells were counted. Mast cell numbers were significantly reduced in the CD18hypo group treated with WT PMNs and CD18

PMNs (Figure C-2.17 A, p≤0.05). Degranulated mast cells in both the reconstituted

CD18 hypo mice were significantly less (Figure C-2.17 B, p≤ 0.01) than wild-type mice.

Figure C-2.15: Extravasated platelets in CD18hypo mice reconstituted either with wild-type or Cd18hypo PMNs 24h after corneal injury.

Extravasated platelet counts was not significantly different in CD18hypo reconstituted mice. (CD18hypo+WTPMN n=3, CD18hypo+CD18PMN n=4, P≤0.01).

Figure C-2.16: Limbal vessel diameters in CD18hypo mice reconstituted either with wild-type or Cd18hypo PMNs 24h after corneal injury. Figures A & C are graphs of the cumulative distribution functions. 24h post-injury, venule (A&B) and arteriole (C&D) diameters were measured. (CD18hypo+WTPMN n=3, CD18hypo+CD18PMN n=4, p≤0.0001).

B D

Figure C-2.17: Limbal mast cell and degranulated limbal mast cell counts in CD18hypo mice reconstituted either with wild-type or

CD18hypo PMNs 24h after corneal injury. 24h after corneal injury, limbal mast cells (A) and degranulated mast cells (B) were counted at the corneal limbus. (CD18hypo+WTPMN n=3, CD18hypo+CD18PMN n=4, p≤0.001).

2.4: Discussion

The aim of this study was to investigate mechanisms regulating platelet recruitment after corneal injury which was achieved by evaluating three mouse models of corneal abrasion. The first study evaluated the role of anti-Ly6G treatment in mice corneal inflammation after corneal injury. Mice treated with anti-Ly6G antibody which specifically reduces circulating PMNs were evaluated to determine what effect this has on PMN and platelet co-dependent recruitment into the injured cornea. Circulating

PMNs were significantly reduced in anti-Ly6G treated mice. Twenty-four hours after corneal injury, compared to wild-type mice, anti-Ly6G treated mice had ~80% reduction in extravasated platelets and these mice had significantly less PMNs at different regions in the cornea. Limbal vessel dilation was reduced and there were fewer degranulated mast cells which correlates with reduced vessel dilatation suggesting degranulating mast cells are likely important for vasodilation. In our mouse model of corneal injury, the data support the co-dependency of PMN and platelet recruitment during corneal injury and a reduction in platelet recruitment is associated with reduced limbal vessel diameters and mast cell degranulation.

The second part of this study investigated the role of IL-20 on platelet recruitment in our mouse model of corneal injury. Topically applied IL-20 resulted in a ~70% reduction in both PMN and platelet recruitment as compared to PBS controls. The co- dependency of PMN and platelet recruitment during corneal injury is reiterated here.

Hence, in the cornea, after injury, IL-20 is anti-inflammatory as it can limit the inflammatory response and yet sustain normal rates of epithelial and nerve recovery

(Wanyu Zhang and Sri Magadi, manuscript submitted), a remarkable finding since reduced platelet or PMN recruitment invariably resulted in delayed wound healing and diminished nerve regeneration as the leukocytes deliver essential growth factors. This study also sought to determine whether platelet recruitment is normal in IL-20 treated mice and accounts for the normal rate of wound closure or if IL-20 independently aids in wound healing. The data are consistent with a direct healing effect for IL-20 since a significant reduction in platelet and PMN recruitment did not result in diminished wound healing and nerve regeneration after corneal injury suggesting IL-20 has therapeutic potential in the treatment of corneal injuries.

The aim of the final part of the study was to evaluate the role of CD18 on platelet recruitment during corneal inflammation in our mouse model of corneal injury. The initial pilot study established that vessel dilatation was coincident with platelet but not

PMN extravasation since CD18hypo mice have normal PMN extravasation. To further evaluate the role of CD18 on platelets, injured CD18hypo mouse corneas were analyzed in the study conducted in 2015. The pilot study data could not be replicated in these mice. Flow cytometry and genotyping data lean toward genetic contamination in these mice. However these mice were not exactly wild-types and were used to evaluate the role of CD18. Data with intermediate to high (I-H) levels of CD18 belong to these mice.

Therefore, a new group of CD18hypo mice was purchased in 2016 which had low- intermediate (L-I) levels of CD18. 24h after corneal injury, mice with intermediate-high levels of CD18 had no significant difference in platelet extravasation, venule diameters, and limbal mast cell degranulation whereas platelet extravasation, vessel diameters and

80 mast cell numbers as well as degranulated mast cells were significantly reduced in

CD18hypo mice with low-intermediate levels of CD18.

To see if vessel dilation and platelet recruitment can be restored, CD18 hypo mice with low levels of CD18 were injected with isolated PMNs either from wild-type or

CD18hypo mice. Both the groups had significantly less platelets outside the limbal vasculature and degranulated mast cells. No significant increase in venule diameter and a slight significant increase in arteriole diameter was observed in the reconstituted mice.

The failure of wild-type PMNs to rescue platelet extravasation during the reconstitution experiments can be due to the fact that bone marrow derived PMNs are immature. The reconstituted wild-type PMNs could have decreased function if their maturation in the eye was hampered. Also, PMNs can get activated during the isolation procedure and removed from circulation. Further, PMN extravasation is normal in CD18hypo mice.

Hence the new reconstituted PMNs could have encountered a competitive environment or resistance to extravasate from the limbal vasculature and be removed from the blood stream.

Analyzing the results from Aim 1 (Figure C-2.18), it follows that platelet extravasation and limbal vessel diameters are reduced in IL-20 and anti-Ly6G treated mice. These mice had same number of mast cells as wild-types, but had significantly reduced degranulating mast cells. Previous studies reveal that CD18 binding activates

PMNs (Liles et al., 1995; Schymeinsky et al., 2007). Activated PMNs release molecules like defensins and platelet activating factor (PAF) (Gallo et al., 1997; Jonsson et al.,

2011; Mocsai, 2013) which can activate mast cells as well as cause vasodilation (Nilsson et al., 2000; Wang et al., 2016) which dilates the venules and they become leaky. IL-20 and anti-Ly6G treated mice showed diminished PMN extravasation, therefore less release in defensins/PAF could have resulted in less degranulated mast cells. Hence the expectation is that vessel dilation and extravasated platelets would be reduced in these mice.

Figure C-2.18: Possible explanation of Aim1 results.

Platelets, but not PMN extravasation, appear to require venule dilation

which is dependent on mast cell degranulation. CD18 on PMNs is

necessary for mast cell degranulation.

In our mouse model of corneal injury, PMN extravasation was normal in

CD18hypo mice. Hence defensins/PAF release would be expected to be comparable to wild-type controls. However, CD18 is necessary for mast cell differentiation and homing

(Rosenkranz et al., 1998). In this study, CD18hypo mice with higher levels of CD18 had no statistical difference in PMN recruitment, platelet extravasation, vessel diameters or mast cell degranulation compared to wild-type controls. CD18hypo mice known to have reduced levels of CD18 have normal PMN recruitment, diminished platelet recruitment, reduced vessel dilation, fewer mast cells and more importantly show a reduction in degranulating mast cells. Hence platelets, but not PMN extravasation, requires venule dilation which may be dependent on mast cell degranulation. CD18 on PMNs appears to be necessary for mast cell degranulation. Therefore, the data suggest CD18 on PMNs is necessary for platelet extravasation.

Chapter 3: Aim2: Neutrophil migration and resolution of inflammation

Evaluate neutrophil (PMN) migration in the abraded mouse cornea and determine the mechanisms by which central neutrophils (PMNs) disappear from the cornea during resolution of inflammation

3.1: Introduction

The early response to corneal insult is mediated by the extensive infiltration of PMNs into the wounded cornea (Gan et al., 1999; Hanlon et al., 2014; Jaeschke and Hasegawa,

2006; Kolaczkowska and Kubes, 2013). PMNs contribute to the first line of defense in response to injury or infection. It is well documented that these infiltrating PMNs are essential for wound healing and tissue repair (Kolaczkowska and Kubes, 2013; Li et al.,

2006b; Zhang et al., 2016) as they deliver growth factors like VEGF-A into the cornea.

The magnitude of the response is regulated by a coordinated cascade of inflammatory cells and mediators (Kolaczkowska and Kubes, 2013; Li et al., 2011a; Li et al., 2011b).

The extracellular matrix (ECM) of the corneal stroma is largely made up of type I collagen tightly-packed into regularly arranged lamella (Assouline et al., 1992; Nakayasu et al., 1986). The stromal cells known as keratocytes lie within the interlamellar spaces separating the lamella and constitute an extensive cellular network with cell-cell junctions for adhesion(desmosomes) and communication (gap junctions) (Assouline et al., 1992; Watsky, 1995). While moving through the interlamellar space of the stromal

ECM, 50% of the surface of the migrating PMN is in contact with collagen and 40% with keratocytes (Gagen, 2012; Gagen et al., 2010; Petrescu et al., 2007).The extent of these

84 surface contacts, and their contribution to PMN migration are modulated via the integrin family, β1, β2, and β3 integrins (Hanlon et al., 2014; Petrescu et al., 2007).

Two hours after a central corneal abrasion in mice, PMNs can be seen extravasating from the limbal vasculature, migrating to the wound edge by about 6h, and accumulating in center of the wound with infiltration peaking between 12h to18h (Li et al., 2006a). PMN numbers reduce in the central corneal stroma by 48h and are almost absent by 72h when inflammation is largely resolved. This is a host-regulated mechanism modulated by cytokines and other molecules (Filep and El Kebir, 2009; Gronert, 2010;

Liclican and Gronert, 2010). While the initiation of innate inflammation with PMN recruitment to the site of injury is essential, it is just as important to have mechanisms for their removal to avoid the onset of chronic inflammation since the very defense mechanism that seeks to protect the tissue can also destroy tissues.

In our mouse model of central corneal abrasion, the mechanism behind the disappearance of PMNs from the wound center is unknown but might be explained by macrophage phagocytosis. However, macrophage numbers within the central corneal stroma are relatively low prior to injury and show only a modest increase (30%) after 48h after corneal injury (courtesy of Samuel Hanlon and unpublished pilot study observations). Hence, it seems unlikely that macrophage phagocytosis by itself could account for the disappearance of thousands of PMNs. Another possibility is that PMNs could leave the central cornea, through a process of reverse migration and return to the limbus. The limbal vasculature is invested with a large number (much greater than that

85 found in the central cornea: (Zhang, 2014) of perivascular macrophages which could decrease PMN numbers through phagocytosis. Alternatively, PMNs could re-enter the vasculature and leave via the circulation. Several publications document reverse migration of PMNs in other tissues and species and re-entry into the cremaster vasculature in mice following ischemia-reperfusion injury has been attributed to loss of cell polarization. (Woodfin et al., 2011, Colom B et al., 2015).

The Heidelberg Retinal Tomography (HRT) is a diagnostic tool for in vivo evaluation of the ocular structures. Although initially developed to view structures like the retina and the optic nerve at the back of the eye, the HRT with the Rostock the

Cornea Module (HRT-RCM) allows in vivo imaging of the cornea. Observations using the HRT in our lab suggest PMNs at the early stage of inflammation in the corneal stroma migrate with a speed of ~7µm/minute (Hanlon et al., 2014) . As mentioned above, peak

PMN migration is around 12h-18h which is a very difficult time-point to track PMNs.

Since, at 8h post-injury, PMNs migrate but not in very large numbers and therefore was the chosen time-point during which PMN migration is in progress, but there is no crowding of cells and hence the goal of tracking individual PMN can be achieved.

Corneal injury initiates the migration of PMNs out of the limbal vasculature toward the wound where these PMNs travel within the interlamellar space where a large percentage of their surface is in contact with collagen (50%) and keratocytes (40%)

(Gagen et al., 2010; Petrescu et al., 2007). The surface contacts of PMNs at the paralimbal or the region very close to the limbal region is unknown. Hence 8h and 18h

86 post injury corneas were fixed as per the protocol mentioned in the methods section and serial block-face images were collected using the Tescan-Gatan3View SEM (Figure M-

3.1 and Figure M-3.2).

The inflammatory response in the normal cornea contributes to wound healing and nerve regeneration by the release of growth factors from PMNs and the disappearance of PMNs during resolution of inflammation is thought to be a regulated process (Gronert, 2010; Schwab et al., 2007). Dysregulation in the clearance of PMNs leads to delayed wound healing and prolonged inflammation leads to destructive chronic inflammation (Filep and El Kebir, 2009; Gronert, 2010; Martin et al., 2015). Hence a complete understanding of corneal wound healing can only come about if we have a better understanding of both the early and late phases of corneal inflammation. This aim evaluated the hypothesis that resolution of inflammation involves the clearance or

“disappearance” of PMNs by determining how PMNs change as they move toward the central lesion, their migration patterns and the surface contacts they make with the corneal stromal cells.

3.2: Methods

Eight-twelve12 week old adult C57BL/6J male mice were used for all experiments.

Experiments were conducted at the University of Houston, College of Optometry,

Houston, TX. Mice were treated following the University of Houston guidelines and the

ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

3.2.1: Choice of animal model

The inflammatory response that follows a corneal abrasion is complex and impossible to replicate in an in vitro setting and the animal model of choice is the mouse. Although there are differences between mouse and human eyes, there are many similarities such as a stratified epithelium, presence of a corneal stroma including keratocytes, and endothelium to list a few and the mouse is routinely used by many investigators to study the cornea. Our lab has published numerous studies using the mouse model of corneal injury and these studies provide the necessary background data and rationale for pursuing the experiments outlined in the current study.

Previous studies have shown that corneas of C57Bl/6J mice under the age of 8 weeks are still developing. Older mice (e.g., 6 months) show significant age-related reductions in epithelial nerve density (Wang et al., 2012). Nerve density is relatively stable from 8-12 weeks of age and mice over 8 weeks of age are considered to have fully matured with respect to corneal thickness (Hanlon et al., 2011). Hence 8-12 week old mice were used in all studies because the cornea is considered mature.

3.2.2: Corneal abrasion model

A central corneal wound was made according to a previously established protocol (Li et al., 2006b). Briefly, adult C57BL/6J wild type mice were purchased from Jackson

Laboratory (Bar Harbor, ME). Mice were anesthetized by intraperitoneally injecting ketamine (75mg/Kg body weight) and xylazine (7.5mg/Kg body weight). A 2mm diameter central corneal epithelial region was marked by a trephine and mechanically

88 debrided with an alger brush with a 0.5mm burr (Alger Equipment Co., Inc., Lago Vista,

TX) under a dissecting microscope while taking care not to penetrate the basement membrane (Figure M-2.1 and Figure M-2.2) . Eye lashes were trimmed to prevent hindrances while imaging using the Heidelberg Retinal Tomographer III with Rostock

Cornea module (HRT-RCM).

3.2.3: Imaging with the Scanning Electron Microscope (SEM)

Corneas were excised and fixed at 8h and 18h post-abrasion. The limbal vessels are anterior to Schlemm’s canal and they extend laterally toward the cornea, but only up to the point that Descemet’s membrane (posterior limiting lamina; PLL) begins. In other words, Schlemm’s canal and the PLL are landmarks that define the limits of the limbal vessel zone. Blocks of adult C57BL/6J mouse corneal tissue were processed using a modified protocol to enhance specimen contrast. Briefly corneas were fixed in 0.1M sodium cacodylate buffer containing 2.5% glutaraldehyde and four radial cuts were used to divide the cornea into four individual quadrants.

A B

Figure M-3.1: Serial block-face for imaging with the SEM. (A) Corneal

flat mount of an adult C57BL/6J mouse, limbus (red). (B) Individual

quadrant of the cornea mounted in the epoxy resin block (light grey) and

oriented for serial block face imaging. Arrow is pointing to the cutting

edge of the block face.

The tissues were infiltrated with heavy metals (e.g., osmium tetroxide, uranyl acetate, and lead aspartate) and washed extensively with sodium cacodylate buffer and distilled water. The tissues were dehydrated in serial dilutions of acetone from 30% -

100% and then incubated in Embed 812 resin for several days to allow the resin to completely penetrate the tissue. The detailed protocol followed was as follows:

1) Mouse whole eyes were placed in primary fixative (0.1M Sodium cacodylate,

2.5% glutaraldehyde, 20M calcium chloride) for 15 minutes. Corneas were

dissected and then returned to the fixative for an additional 1hour and 45minutes.

Prepare primary fixative: 10 mL of primary fixative pH 7.4

5 mL 0.2M Sodium cacodylate buffer

1mL 25% glutaraldehyde

20 µL of 1M calcium chloride

4 mL distilled water

2) Corneas were washed 5 times, 3 minutes/wash in 10 mL 0.1M sodium cacodylate

buffer containing 20 µL of 1M calcium chloride.

3) A solution containing 3% potassium ferrocyanide in 0.3M cacadylate buffer with

4mM calcium chloride combined with equal volume of 4% aqueous osmium

tetroxide for 1hour on ice was prepared as follows

0.06 g potassium ferrocyanide

2mL 0.2M sodium cacodylate

8 µL 1M calcium chloride

To 2 mL of ferrocyanide fix, 2 mL 4% osmium tetroxide in distilled water was

added.

4) After corneas were washed in distilled water, they were placed in freshly prepared

osmium solution for 1hour on ice.

5) While incubating the tissues with osmium, fresh thiocarbohydrazide (TCH)

solution was prepared by dissolving 0.2 gm thiocarbohydrazide (Ted Pella) to 10

mL ddH20 and place in a 60˚ C oven for 1 hour, (agitate by swirling gently every

10 minutes to facilitate dissolving). The TCH solution is filtered through a 0.22

um Millipore syringe filter right before use.

6) Corneas were washed in ddH20 5 times, 3 minutes/wash and then placed in the

filtered TCH solution for 20 minutes at room temperature.

7) After rinsing the corneas in in ddH20 5 times, 3 minutes/wash, they were placed in

2% osmium tetroxide for 30 minutes.

8) Corneas were washed in ddH20 5 times, 3 minutes/wash and then placed in 1%

uranyl acetate overnight at ~4°C.

9) Next day, after rinsing in ddH20 5 times, 3 minutes/wash, the corneas were

incubated in Walton’s lead aspartate solution and placed in the oven for

30minutes. Walton’s lead aspartate staining was made as follows: 0.066 gm of

lead nitrate is dissolved in 10 mL 0.03M aspartic acid solution (0.04g aspartic

acid in 10 mL distilled water). pH, was adjusted to 5.5 by adding 1N KOH

dropwise while monitoring the pH. The clear lead aspartate solution was placed in

a 60˚ oven for 30 minutes.

10) Corneas were washed in ddH20 5 times, 3 minutes/wash, placed in the lead

aspartate solution and then placed in the oven for 30 minutes.

11) Corneas were washed in ddH20 5 times, 3 minutes/wash and dehydrated using

freshly prepared acetone series (30%, 50%, 70%, 90%, and 100%) for 10 minutes/

dilution. This dehydration step removes water from the tissues, a necessary step

since epoxy resins are not miscible with water.

12) Corneas were then placed in a series of Embed 812 epoxy resin and acetone

dilutions, Embed812: Acetone; (1:3 (for 4h), 1:1 (overnight), 3:1 (8h), 100% resin

(overnight), and 100% fresh resin next day (6h).

13) Corneas were embedded in Embed812 resin and placed in oven for 2 days.

14) Corneal tissue blocks were trimmed and mounted for imaging with the SEM

15) The tissues were carefully oriented with the limbus against the cutting edge of the

mold (Figure M-3.1) filled with resin and cured overnight in the oven at 65° C.

The limbus was serially sectioned using the Gatan 3View system mounted inside

the SEM. Serial block-face ultrastructural images (transverse sections, Figure M-

3.2) were collected at 100nm intervals and viewed as registered Z-stacks. From

each quadrant, 50 serial images of the limbus were selected, each separated by a

distance of 500nm (total = 25 µm Z stack). Surface contacts for 10 PMNs from

each image stack were evaluated. Electron micrographs were viewed in ImageJ.

Using the dimensions of the serial images from the SEM, the scale was set in

ImageJ and PMN surface contact with other cells was measured using the ImageJ

measurement tool.

Figure M-3.2: Scanning Electron Microscope (SEM). The Gatan 3View

system (blue arrow) consists of a microtome mounted inside the scanning

electron microscope (SEM) (visible here on the left monitor, orange

arrow) cuts serial sections while the SEM images the block face. The red

arrow is pointing to the SEM inner chamber where the tissue block is

placed for cutting. White arrow is pointing to the acquired

electronmicrograph.

To study early inflammation in vivo, 8h after central corneal abrasion, injured mice were anesthetized by intra-peritoneal (IP) injection of ketamine (100mg/Kg body weight) and xylazine (10mg/Kg body weight). This dose of ketamine and xylazine is

94 required to maintain anesthesia during image collection with the HRT-RCM. The first aim of this study was to evaluate early PMN migration patterns and evaluate the PMN surface contacts at the paralimbus, very close to the limbal region. Hence PMNs were imaged at 8h post-injury. The second aim was to evaluate the “disappearance” mechanisms of PMNs during the resolution of inflammation. Hence mouse corneas were imaged at 24h, 30h, 36h, 48h, and 72h post injury.

3.2.4: In vivo imaging the cornea with the Heidelberg Retinal Tomographer III with

Rostock Cornea module

PMN extravasation peaks around 12h-18h at which time thousands of PMNs infiltrate into the cornea and is very difficult to track PMNs. Hence the 8h time-point was chosen to reflect a time when PMN migration is in progress, but there is no crowding of cells and hence tracking individual PMNs can be achieved. After focusing on the cornea with the

HRT-RCM, the cornea was applanated with very little force so as to stabilize the images

(Figure M-3.3). Time-lapse image sequences were collected for 10 minutes by collecting six 100-second scan sequences at 1 frame per second, in a 400µm ×400µm image frame of the HRT-RCM. Eight hour post-injury, time-lapse sequences were collected at the paralimbal very close to the limbal region. At 24h, 48h, and 72h time-lapse sequences were collected at the center, parawound and the paralimbus very close to the limbus. All sequences in the paralimbal regions, were collected in the superior quadrant due to the ease of accessibility. Scan depth was carefully chosen to remain within the anterior stroma where the majority of infiltrating PMNs are found (Petrescu et al., 2007). Volume scans encompassing the entire depth of the cornea were collected at 9 different regions

95 which included the center to the periphery of the cornea and were used to obtain PMN and macrophage counts.

Figure M-3.3: In vivo imaging using Heidelberg Retinal Tomographer

III with Rostock Cornea module (HRT-RCM). The mouse’s head was

positioned comfortably so as to allow it to protrude and be accessible for

imaging with the HRT-RCM while the remaining triangular piece of the

centrifuge tube served to support the head. The red insulating foam helped

maintain body temperature.

The six separate time-lapse sequences were concatenated and then registered using the ImageJ Linear Stack Alignment with SIFT plugin. A custom MatLab program was used to semi-automatically track ten randomly selected PMNs to obtain speed, velocity and migration angle relative to the wound center. In the post-stabilized time- lapse sequences, the location of each PMN was manually selected and then the center of

96 the same PMN was marked through the entire sequence to track PMN movement. As per convention, non-motile cells with speed less than 1µm/min were not tracked (Hanlon et al., 2014; Smith, 2000; Werr et al., 1998) . Further cells that were close to the frame edge or cells that were not visible through the entire sequence were not tracked. The technique for analyzing these parameters is established in the Burns’ lab (Hanlon et al., 2014).

One of the typical ways to describe cell motility is the cell speed (CS) which is calculated by dividing the distance travelled by the cell (path length) by the time taken to travel that distance. The straight-line displacement of the PMN from its initial to final location divided by time taken for the displacement is termed as cell velocity (CV)

(Beltman et al., 2009; Hanlon et al., 2014).

∑푚=∞ √(푥 −푥 )2+(푦 −푦 )2 Cell Speed (CS) = 푚=1 푚+1 푚 푚+1 푚 푇

√(푥 −푥 )2+(푦 −푦 )2 Cell Velocity (CV) = 푛 푚 푛 푚 푇

(Adapted from Hanlon et al., 2014)

Where m= initial location and n= final location of the PMN, T= time taken to travel the distance

At each time-point the average cell speed and velocity were calculated.

To determine the direction of movement, the migration angle (MA) was determined for each PMN. A grid as shown below (Figure M-3.4) was drawn. The angle between points A & B (∟AOB) and C & D (∟COD) is ±30°. The angle between points

A&C (∟AOC) and B&D (∟BOD) is ±150°.

Figure M-3.4: Determination of PMN migration angle. The grid was

used to determine the direction of movement of PMNs at different corneal

regions after corneal injury.

The custom MatLab program tracks the path of each PMN. Analyzing both the cell path from the MatLab program and the HRT sequences collected, the direction of

PMN motion was obtained. If the PMN path was between ∟AOB, the angle of displacement was considered to be -15° and was +15° if the path was to the right of the mid-point O between ∟COD. Similarly, if the PMN path was below mid-point O (i.e. between ∟AOC), the displacement angle was considered to be +150° and the PMN was considered to be moving toward the wound. If the PMN path was at the top of the mid-

98 point O (i.e. between ∟BOD), the angle was considered to be -150° and the PMN was labelled as moving away from the wound.

Using the grid, the mid-point O was placed at the starting location of individual

PMNs and the resultant direction of movement was recorded for each PMN at every time-point.

3.2.5: Statistical Analysis

Data were analyzed using a student’s t-test or a one-way or two-way analysis of variance

(ANOVA) with a Tukey post-test for pair-wise multiple comparisons. Data are expressed as mean±SE and P values ≤0.05 were considered statistically significant.

3.3: Results

3.3.1: PMN migration and resolution of inflammation after corneal abrasion

In our model of central corneal abrasion, in response to injury, PMNs extravasate the limbal vasculature, reach the wound edge at 6h and peak at the center by 18h. Previous studies evaluated PMN migration in the corneal stroma adjacent to the wound at 8h post- injury. PMN migration behavior at the peripheral cornea has not been examined. Hence

PMN migration patterns at the paralimbus and regions closer to the limbus were evaluated in vivo using HRT-RCM. Initial observations showed two different PMN migrating patterns at the paralimbus (Figure C-3.1).

To investigate further, PMNs at the two paralimbal regions were extensively evaluated: PMNs at the paralimbus, very close to the limbal vessels (orange arrows) and paralimbal PMNs (blue arrows) moving toward the center in injured corneas.

100

Figure C-3.1: In vivo HRT-RCM imaging of PMN migration patterns within the paralimbus of the injured mouse cornea. (A) Image of

PMNs at the paralimbus, very close to the limbal vessels (orange arrows) and more centrally oriented paralimbal PMNs (blue arrows) moving toward the center in injured corneas. The white dashed line denotes the paralimbal region very close to the limbus (left of the dashed line) and the more central paralimbal region (right of the white dashed line). Bar =

50µm. (B) Corneal wholemount shows the average direction of PMN movement circumferential (orange arrows) and radial toward the center

(blue arrows). Bar = 90µm.

101

3.3.1.1: PMN migration during the early phase of inflammation after corneal injury

To study early PMN migration from the limbus toward the wound, migration speed and pattern was evaluated 8h after corneal injury (Figure C-3.2 and Table 1).

Paralimbus/close to limbus

Cell Speed = 3.1µm/min

102

Paralimbus closer to the center Cell Speed = 6.1µm/min

Figure C-3.2: Tracking PMN speed using in vivo HRT-RCM 8h post- injury. Image sequences at the paralimbus close to the limbus (A) and at a paralimbal region nearer to the center (B). The black arrows indicate the resultant direction of PMN movement. L is the limbal region and C is toward the center.

103

Toward Away Circumferential center center Ambiguous Paralimbal close to limbal region 69±4.3 * 13±8.3 8±4.5 10±4.6 Paralimbal close to center 28±14.9 61±14.6 ** 7±5.1 3±2.1

Table 1: PMN movement 8h post-injury.

PMN migration patterns at paralimbal, very close to limbal regions and

those at the paralimbal, closer to center were evaluated. Data are mean

percentages of PMNs ±SE (n=3). * = p≤0.05 compared to

circumferential movement at paralimbus close to center. ** = p≤0.05

compared to movement toward center

The average PMN speed at the paralimbal region, very close to the limbal region was significantly reduced (3.1±0.4µm/min, p≤0.001) than that at the more centrally positioned paralimbal region which was 6.1±0.3µm/min (Figure C-3.2 & table 1). A significantly higher percentage (p≤0.05) of the PMNs close to the limbal region moved circumferentially (~70%) compared to the majority of the more centrally positioned paralimbal PMNs (~61%, p≤0.05)) which moved toward the wound (Table 1 & Figure C-

3.12).

104

3.3.1.2: PMN migration during the late phase of inflammation after corneal injury

48h post-abrasion, in our mouse model of corneal injury, PMNs were greatly reduced at the center. In an attempt to evaluate this “disappearance” of PMNs during the late phase of inflammation, abraded corneas were evaluated at different time-points and at different corneal regions between 24h – 72h post-wounding (Figures C-3.3-C-3.7).

105

A 24 hours

Center Cell Speed = 3.9 µm/min

24 hours

Parawound

Cell Speed = 4.6µm/min

106

24 hours

Paralimbus

Cell Speed = 4.1µm/min

Figure C-3.3: PMN speed 24h after corneal injury. PMNs were tracked using in vivo HRT-RCM sequences at center (A), parawound (B) and at the paralimbal region (C). The black arrows indicate the resultant direction of movement. “C” indicates the direction of the center of the cornea and

“L” the limbus (n=3).

107

Figure C-3.4: PMN speed 30h after corneal injury. In vivo tracking of

PMNs using HRT-RCM sequences at paralimbal close to limbal region.

The black arrow indicates the resultant direction of movement. “C” indicates the direction of the center of the cornea and “L” the limbus.

108

Figure C-3.5: PMN speed 36h after corneal injury. In vivo tracking of

PMNs using HRT-RCM sequences at the paralimbal close to limbal region. The black arrow indicates the resultant direction of movement. “C” indicates the direction of the center of the cornea and “L” the limbus.

109

48 hours

Center Cell Speed = 3.1µm/min

B 48 hours Parawound

Cell Speed = 3.0µm/min

Figure C-3.6: PMN speed 48h after corneal injury. In vivo tracking of

PMNs using HRT-RCM sequences at center (A) and parawound (B). The black arrow indicates the resultant direction of movement. “C” indicates the direction of the center of the cornea and “L” the limbus.

110

72 hours

Parawound

Cell Speed = 2.9µm/min

111

72 hours

Paralimbal

Cell Speed = 2.4µm/min

Figure C-3.7: PMN speed 72h after corneal injury. PMNs were tracked using in vivo HRT-RCM sequences at parawound (A) and paralimbal regions (B). “C” indicates the direction of the center of the cornea and “L” the limbus.

112

Paralimbus, Center Parawound close to limbus 24h 4.9±1.02 4.5±0 * 4.7±0.2 ** 30h N/A N/A 4.5±0.3 36h N/A N/A 3.5±0.3 48h 3.2±0.1 3.3±0.3 N/A 72h 0 2.6±0.3 2.9±0.5

Table 2: Average PMN speed (in µm/min) at different regions of the

cornea 24h -72h after central corneal abrasion.

24h-72h PMNs tracked at the paralimbus were closer to the limbus (n=2-

5). * and ** = p≤0.05 compared to PMN speed 72h.

PMN average speed at the parawound and paralimbus, very close to the limbus regions 24h after injury was significantly higher than that observed at 72h post-injury at both the regions (Table2, p≤0.05). PMN speed at all the other regions as well as time- points did not show statistical significance. 72h after injury, the average speed of the

PMNs at the center was less than 1µm/min and hence these PMNs could not be tracked.

24h after corneal abrasion, 64% of the central PMNs were migrating away from the center which was significantly higher than that observed at 48h post-injury (p≤0.05).

52% of the parawound PMNs were observed migrating away from the center of the cornea and by 72h this percentage had dropped to 25% while macrophage numbers increased (Table 3 and Table 4). At each time-point during the late phase of inflammation

(24h-72h) at different corneal regions, a high percentage of PMNs were seen moving away from the center (Table 3).

113

Center Toward Away Circumferential center center Ambiguous 24h 3.8±2.7 12.2±0.4 64.6±0.8 * 19.5±3.9 48h 0 11.5±5.2 1.6±1.1 87±6.3 72h 0 0 0 0

Parawound Toward Away Circumferential center center Ambiguous 24h 6.2±0.2 11±3.6 52.6±3.9 30.3±7.7 48h 5.6±3.9 3.3±2.4 34.1±10.4 57±16.7 72h 0 1.2±0.1 25±17.7 67.9±22.7

Paralimbus Toward Away Circumferential center center Ambiguous 24h 38±5.7 5±4.6 37±1.2 20±4.7 30h 24.8±1.8 8.2±3.1 57.4±1.1 9.6±2.06 36h 26.9±3.7 6.4±1.2 37.3±6.2 29.3±4.3 72h 0 1.4±0.1 25±17.7 63.9±25.5

Table 3: Percentage of PMN movement at various corneal regions

24h, 30h, 36h, 48h, and 72h post-injury

24h-72h PMNs tracked at the paralimbus were closer to the limbus

(n=2-3). * = p≤0.05 compared to movement away from center 48h post-

injury

To further confirm PMN orientation and migration pattern, fluorescently labelled as well as HRT images were evaluated. PMNs near and just below the limbus appear to be elongated and horizontally (circumferentially) oriented where as PMNs further away from the limbal region appear to be radially oriented (Figures C-3.8-C-3.10).

114

A B L L L L

C D L L L

Figure C-3.8: Comparing excised and fixed 24h post-wound

fluorescently labelled PMN images to in vivo confocal images acquired

24h after injury. Fluorescence images (A, C) of PMNs labelled with

PMN antibody anti-Ly6G-FITC (green) and in vivo HRT-RCM images (B,

D). L=limbal region and C=center of the cornea (panel B). Fluorescently

labelled images bar = 92µm and HRT images bar = 50µm.

115

3.3.1.3: Resolution of inflammation after corneal injury

Macrophages also called “big eaters” are known for their role in phagocytosis of dead cells and debris. To evaluate if macrophage phagocytosis could be a plausible explanation for the decrease in PMN numbers at the center, PMNs and macrophages were counted at various corneal regions. Macrophage numbers were significantly higher at 72h compared to earlier time-points (p≤0.01) (Table 4). PMN infiltration into the central cornea increased by 24h and the numbers reduce by about 72h whereas macrophage numbers increased between 30h – 72h after corneal injury (Figure C-3.9 - Figure C-3.11).

At the center of the cornea, PMN numbers were maximal at 24h and markedly reduced by 72h post-injury (p≤0.01). Macrophage numbers at the center and parawound regions increased between 30h-72h post-injury and were significantly higher at 72h compared to

48h (p≤0.01). Macrophage phagocytosis of PMNs was clearly evident at the limbus

(Figure C-3.11).

A B L B

Figure C-3.9: 48h post-injury in vivo HRT-RCM images at center (A)

and paralimbus/ very close to limbal region (B). PMN (white arrows),

macrophages (black arrows) and the limbus (L). Bar = 50µm. 116

A BB L L

C DD

Figure C-3.10: Comparing fluorescently labelled and confocal images

72h post-injury. Cornea labelled with PMN antibody anti-Ly6G-FITC

(green, A&C) and in vivo HRT-RCM image (B&D, grey). L=limbal

region. In all images, black arrows indicate macrophages and white

arrows are pointing at PMNs. The banded appearance in panel C is

probably folds in the cornea and does not distract from the presence of

117

PMNs. Fluorescently labelled images bar = 92µm and HRT images bar =

50µm.

A D C

B E C

C F C

118

G H

Figure C-3.11: Macrophage phagocytosis 72h post-injury. Corneas were stained with macrophage marker anti-F4/80 (red) and with anti-

Ly6G to stain PMNs (green). Panels A, B, C, G show PMNs in the center and the parawound PMNs are shown in panels D, E, F, H. White arrows are pointing at PMNs and black arrows reveal macrophages. The SEM image (I) shows a PMN (red arrow) engulfed inside a macrophage (black arrow) and two other PMNs (white arrows) in contact with the macrophage. Bar = 14µm

119

PMNs in PMNs in Macrophages in Macrophages in center/mm2 paralimbus /mm2 center/mm2 parawound/mm2 8 h 293±20.8 4850±666 ** 0 31±3 24 h 5817±500 * 1150±106 6±1 53±3 30 h 1745±142 1125±171 15±5 59±3 36 h 1133±149 668±72 22±4 58±8 48 h 1100±98 727±51 56±4 78±7 72 h 150±29 950±49 165±14 *** 143±3 ***

Table 4: PMN and macrophage counts at different corneal regions. Using in vivo HRT-RCM volume scan images, PMN and macrophage

numbers were counted at the center, parawound and paralimbal areas.

(p≤0.01 (n= 2-3). * = p≤0.01 compared to 8h post-injury, ** p≤0.01

compared to 24h post-injury, *** p≤0.01 compared to 24h- 48h post-

injury

Summarizing the data thus far

During early phase of inflammation (8h time-point), PMNs predominantly move toward the center and move away from center during the late phase of inflammation (24h

– 72h) (Figure C-3.12). PMN numbers reduce in the central corneal stroma by 48h and are almost absent by 72h when inflammation is resolving. The mechanism behind the disappearance of PMNs from the wound center is unknown but might be explained by macrophage phagocytosis.

120

121

122

Figure C-3.12: PMN migration during early (8h) and late phase of

inflammation (24h-72h). Percentage of PMNs migrating at different

corneal regions and their direction of migration is indicated by colored

arrows and lines (n=2-3).

Previous studies in the lab showed paralimbal PMNs move through the interlamellar space of the stromal extra cellular matrix (ECM) where 50% of the surface of the migrating PMN is in contact with collagen and 40% with keratocytes (Gagen,

2012; Gagen et al., 2010; Petrescu et al., 2007). Surface contacts PMNs make at the limbus are unknown. Since PMN speed close to the limbus was slower than that at the more centrally positioned paralimbal region (Figure C-3.2 & Table 1), it raised a possibility that PMNs closer to the limbus make different surface contacts compared to the PMNs migrating toward the wound in the corneal stroma. Therefore, PMN surface contacts were evaluated using the SEM serial block-face imaging (Figures C-3.13 & C-

123

3.14). Table 5 shows ~60% of the PMN surface near the limbus contacted collagen which was greater than that of the PMNs migrating toward the wound in the corneal stroma (50%) observed during previous experiments (Gagen et al., 2010; Petrescu et al.,

2007).

Figure C-3.13: Identification of structures in a SEM serial block-face

section. Blood vessel (red arrow) extravascular PMNs (black arrows) and

the collagen lamellae (orange arrow) in a SEM serial block-face image.

Bar= 10µm.

124

125

Figure C-3.14: PMN surface contact measurements. Five PMNs are

identified from the serial block-face images from the SEM for surface

contact measurements (A). PMN surface contacts (B) with collagen (1),

another PMN (2), macrophage (3), blood vessel (4), and keratocyte (5).

(n=3). Bar= 10µm.

126

PMN surface contacts were measured in three different mouse corneas and results were expressed as mean±SE. The percent of PMN surface contact with other cells or structures is also shown in the table below.

PMN PMN profile percent contact length (µm) contact Collagen 24.9±6.8 60.1 Keratocytes 8.8±2.2 21.3 Macrophages 4.4±1.9 10.5 Blood vessels 1.2±0.8 2.8 other PMNs 1.24±0.9 3 RBCs 0.2±0.1 0.5 Platelets 0.04±0.04 0.1 Nerves 0 0 ECM 0.7±0.7 1.7 Mast cells 0.1 4.9

Table 5: PMN surface contacts with other cells and structures 8h post-injury

(n=3).

In vivo analysis of mouse corneas using HRT-RCM showed that PMNs moved circumferentially at the paralimbus very close to the limbus region (Figure C-3.12). SEM serial block-face images were evaluated to confirm the circumferential migration pattern of the PMNs close to the limbus. SEM images show circumferentially oriented PMNs.

Collagen, and keratocytes appear to be circumferentially arranged (Figure C-3.15).

To further confirm the orientation of the PMNs very close to the limbus, lengths and widths from 15 PMNs were measured 18h post-injury. After analyzing serial sections

127 from the SEM, the maximum length of the PMN in the x-plane was recorded as the PMN length and the maximum measurement of the PMN in the z-plane was recorded as its width. Data suggest that the average PMN length was ~7±1 µm width was ~ 4±1 µm

(Table 6). Since PMNs were more elongated in the x-plane than the z-plane, these PMNs were considered to be oriented circumferentially.

Figure C-3.15: Extravasated PMNs at 18h post-injury. In the

electronmicrograph, red arrow is pointing at a blood vessel with a red

blood cell inside it and black arrow is pointing at extravasated PMNs.

128

Figure C-3.16: Circumferential orientation of cells and structures at the limbus. High magnification of the image above shows Blood vessel (red arrow) at close proximity to PMNs which are very close to the limbal region (black arrows) and the collagen and keratocytes (orange arrow). Bar= 10µm.

129

3.4: Discussion

The aim of this study was to investigate PMN migration in our mouse model of corneal injury and determine the mechanism by which central PMNs disappear from cornea during resolution of inflammation.

Registered HRT-RCM image sequences of early PMN migration showed that the

PMN speed 8h post-injury at the more central portion of the paralimbus was significantly higher (6.1±0.3µm/min) compared to the PMN speed at the paralimbus very close to limbal region (3.1±0.4µm/min, (p≤0.001)) . Predominantly PMNs at the paralimbal very close to the limbal region moved circumferentially (~70%) and paralimbal PMNs close to the center were predominantly moving toward the wound (~61%).

The mouse limbus/paralimbus is known to have an annulus of circumferentially oriented collagen which has been suggested to play a role in maintaining corneal shape and strength (Hayes et al., 2007; Meek and Knupp, 2015; Sheppard et al., 2010). This band of collagen is about 300-400µm wide in the mouse (Hayes et al., 2007). We hypothesized that this annular ring of collagen may impose an orientation on the keratocytes and PMNs. In vivo imaging of live mice corneas suggest that the PMNs at the paralimbus near the limbal region moved at a lesser speed in a circumferential pattern compared to the PMNs in the more central portions of the paralimbus. The circumferential orientation of PMNs and collagen was further suggested by our serial block-face imaging. PMN surfaces predominantly had contacts with collagen, and keratocytes and also with other cells and structures like macrophages, blood vessels,

130 platelets, nerves, and the extracellular matrix (ECM). The major surface contacts for

PMNs were collagen and keratocytes. Given that collagen is circumferentially oriented at the limbus/paralimbus, the annular ring of collagen may also impose a circumferential orientation on the keratocytes. Taken together, circumferentially arranged collagen and keratocytes at the limbus/paralimbus may provide a possible explanation for the observed circumferential migration pattern PMNs during the early phase of inflammation.

Two hours after corneal abrasion, PMNs begin to extravasate form the limbal vasculature, begin to migrate to the wound edge by about 6h, and the infiltration peaks between 12h-18h (Li et al., 2006a). PMN numbers reduce in the central corneal stroma by 48h and are almost absent by 72h when inflammation is resolving. The mechanism behind the disappearance of PMNs from the wound center is unknown but might be explained by macrophage phagocytosis. Clearance of dead cells and pathogens by macrophages is well-known. However, macrophage numbers within the central corneal stroma are relatively low prior to injury and show only a modest increase (20%) after injury. Hence, it seems unlikely that macrophage phagocytosis could account for the disappearance of thousands of PMNs. Another possibility is that PMNs could leave the central cornea, return to the limbus, re-enter the vasculature and leave via the circulation.

Several publications document reverse migration of PMNs in other tissues and species where the disruption of polarization of PMNs causes them to reverse migrate in to the cremaster blood vessels in mice following ischemia-reperfusion injury (Woodfin et al.,

2011, Colom B et al., 2015).

131

24h after corneal injury, there was no significant difference in PMN migration speed in the corneal regions. However, at 72h post-injury, PMN speed was significantly reduced at the parawound and paralimbal/ region very close to the limbal region when compared to the speed at 24h (4.5±0 vs. 2.6±0.3 & 4.7±0.2 vs. 2.9±0.5 respectively).

Since there were very few non-motile PMNs at the center at 72h after injury and their average speed was less than 1µm/min, they could not be tracked. PMN speed could not be obtained at the center. Interestingly, 24h post-injury, the PMN migration angle suggested most of the PMNs (~64%) were moving away from the central wound area. At all the time-points (24h-72h), at the parawound and at the paralimbal areas, PMNs were either moving away from the center or their movement was random or ambiguous. This random movement was observed more frequently at the center and parawound at the 48h time-point and 72h time-point at the parawound and paralimbal areas.

This phenomenon was not only observed in vivo, but also suggested by fluorescence imaging where differences were observed in PMN orientation at the paralimbus and parawound areas. Further presence of macrophages was observed at the center, parawound and paralimbal regions at all time-points, and more at 72h post-injury as seen in both in vivo HRT-RCM imaging and in vitro by antibody labelled immunohistochemistry. Data from the SEM provided evidence for PMN phagocytosis by macrophages at regions close to the limbus.

Summarizing the data (Figure C-3.12), during the early phase of inflammation, a higher percentage of PMNs were moving circumferentially at a lower speed at the

132 paralimbal very close to the limbal region and radially at significantly higher speed toward the wound in the paralimbal region. Many studies have evaluated PMN speed in various tissues and species. PMN migration speed in injured zebra fish embryos was ~20-

30 µm/min(Lam and Huttenlocher, 2013) where as it was ~7 µm/min in mice with endotoxin-induced uveitis (Planck et al., 2008). Further, PMN speed in mouse cremaster muscle was ~15 µm/min (Lerchenberger et al., 2013). In different species and tissues,

PMN speed ranged between 7 -30µm/min and PMN migration speed (~4-6 µm/min) in the cornea appears to be at the lower limit of the range. Since PMNs were still migrating to the wound area, PMN numbers were higher at the paralimbal region when compared to the center of the cornea. During the late phase of inflammation, at 24h, majority of the

PMNs were at the center, hence there was a rise in PMNs numbers at the center compared to the paralimbal region. Further, at 24h majority of the PMNs were moving away from the center toward the limbal region and there were few macrophages in the central and parawound regions of the cornea. Hence at 24h post-injury, to a larger extent, reverse migration can be considered for PMN disappearance at the center. Macrophages were seen at the center and parawound regions at all the time-points from 30h-72h.

During the late phase of inflammation, PMN numbers reduced in the center as macrophage numbers increased, and ~ 50% -25% of the PMNs were moving away from the center. PMNs at the Paralimbal/ close to limbal area were moving toward the limbus or away from the center and a smaller percentage were moving circumferentially. Hence

PMN disappearance between 30h-72h time-points can be a contribution of both PMN reverse migration and PMN phagocytosis by macrophages at the center or parawound regions.

133

Chapter 4: Conclusions

While the initiation of innate inflammation with PMN recruitment to the site of injury is essential, it is just as important to have mechanisms for their removal to avoid the onset of chronic inflammation since the very defense mechanism that seeks to protect the tissue can also destroy tissues. The purpose of this dissertation was to evaluate mechanisms regulating platelet recruitment and evaluate neutrophil (PMN) migration in the abraded mouse cornea and determine the mechanism by which central neutrophils

(PMNs) disappear from cornea during resolution of inflammation.

In our mouse model of corneal abrasion, platelet extravasation and limbal vessel diameters were reduced in IL-20 or anti-Ly6G treated mice. These mice had same number of mast cells as wild-types, but had significantly reduced degranulating mast cells. Previously published reports reveal that CD18 binding activates PMNs (Liles et al.,

1995; Schymeinsky et al., 2007). Activated PMNs release molecules like defensins/platelet activating factor (PAF) which can activate mast cells as well as cause vasodilation thereby, venules could dilate and become leaky. IL-20 and anti-Ly6G treated mice had low PMN extravasation, and therefore likely reduced release of defensins/PAF could have resulted in less degranulated mast cells, Indeed, vessel dilation and extravasated platelets were diminished. PMN extravasation was normal in CD18hypo mice. Hence defensins/PAF release might be expected to be comparable to wild-type

134 controls. However, CD18 is necessary for mast cell differentiation and homing

(Rosenkranz et al., 1998). In this study, CD18hypo mice with higher levels of CD18 had no statistical difference in PMN recruitment, platelet extravasation, vessel diameters or mast cell degranulation compared to wild-type controls. CD18hypo mice known to have reduced levels of CD18 had normal PMN recruitment, diminished platelet recruitment, reduced vessel dilation, fewer mast cells and more importantly show a reduction in degranulating mast cells. Hence platelets, but not PMN extravasation, requires venule dilation which may be dependent on mast cell degranulation. CD18 on PMNs appears to be necessary for mast cell degranulation and may explain why CD18 is necessary for platelet extravasation.

In our mouse model of central corneal abrasion, during the early phase of inflammation, a higher percentage PMNs were moving circumferentially at a lower speed (~4 µm/min) at the paralimbal very close to the limbal region and radially at a higher speed (~6 µm/min) toward the wound in the paralimbal region. PMN migration speed in injured zebra fish embryos was ~20-30 µm/min(Lam and Huttenlocher, 2013) where as it was ~7 µm/min in mice with endotoxin-induced uveitis (Planck et al., 2008).

Further, PMN speed in mouse cremaster muscle was ~15 µm/min (Lerchenberger et al.,

2013). In different species and tissues, PMN speed ranged between 7 -30µm/min.

Corneal injury results in an edematous stroma. Hence PMN speed was higher (~6

µm/min) at the paralimbus, close to the center. The loosely spaced collagen bundles close to the limbus probably should favor fast PMN movement, but the slower speeds likely reflect the increased surface contact with annular collagen (60%) compared to less

135 collagen contact (50%) outside the limbus (Gagen et al., 2010; Petrescu et al., 2007) . As inflammation resolved, during the late phases of inflammation (24h-72h) PMN speed was slow again, probably due to the reduction in stromal edema and the obstruction caused by the tightly organized collagen lamellae.

Since PMNs were still migrating to the wound area, PMN numbers were higher at the paralimbal region when compared to the center of the cornea. During the late phase of inflammation, at 24h, majority of the PMNs were at the center, hence there was a rise in

PMNs numbers at the center compared to the paralimbal region. Further, at 24h majority of the PMNs were moving away from the center toward the limbal region and there were no macrophages in the cornea. Hence at 24h post-injury, reverse migration is considerable for PMN disappearance at the center. 30h and 36h after corneal abrasion, majority of the PMNs at the Paralimbal/ close to limbal area were moving toward the limbus or away from the center and a smaller percentage were moving circumferentially at both the time points. Few macrophages are seen at the center at the 30h, and 36h time- point. At the 48h time-point, PMN numbers reduced in the center, macrophage numbers increased, and although a higher percentage of PMN movement was ambiguous, about

30% moved away from the center. At 72h post-injury, PMN numbers reduced at the center while macrophage numbers increased significantly at all the corneal regions.

However, about 25% of the PMNs moved away from the center. Hence PMN disappearance at 30h, 36h, 48h and 72h time-point appears to be the result of reverse migration and phagocytosis by macrophages at the center or parawound regions.

136

In the clinic, the usual treatment option after LASKI surgery or infection is steroids or mast cell stabilizers like visine to reduce redness in the eye due to allergies.

Applying the results of this dissertation, it could be suggested that the use of steroids or mast cell stabilizers block beneficial innate inflammation thereby hampering tissue repair and regeneration. Therefore, if available, other treatment options should be considered and anti-inflammatory drugs should be used cautiously. Accumulation of inflammatory cells also known as peripheral infiltrates are observed in patients in the eye (Dr. William

Miller, personal communication). They appear to be slightly elongated near the limbus and circular near the center. Contact guidance is necessary for PMN movement. Novel results from aim2 show PMNs moving circumferentially at a slower speed near the limbus and at a higher speed, radially near the center. Perhaps PMN speed as well as orientation and migration patterns contribute to the difference in shape observed in these peripheral infiltrates.

This dissertation has brought forth novel findings such as extravascular PMNs at the limbus are oriented circumferentially, resolution of inflammation involves reverse

PMN migration and macrophage phagocytosis, PMN-platelet co-dependent recruitment is

CD18 dependent and vessel dilatation is necessary for platelet extravasation. Given the significant role of PMNs in corneal wound healing and nerve recovery, studying PMN extravasation will provide new information on PMN migration that will enhance our understanding of the basic inflammatory process. Establishing a link between CD18, mast cells, vessel dilation and platelet recruitment will increase our understanding of how inflammation is regulated, a process we have shown is critical for efficient corneal wound healing.

137

Chapter 5: Future directions

The first aim of this dissertation was to evaluate mechanisms regulating platelet recruitment in the abraded mouse cornea. In our mouse model of corneal abrasion, the study suggested that platelet but not PMN extravasation requires venule dilation. CD18, a

β2 integrin present on PMNs. CD18hypo mice known to have reduced levels of CD18 have normal PMN recruitment, diminished platelet recruitment, reduced vessel dilation, fewer mast cells and more importantly show a reduction in degranulating mast cells.

CD18 is necessary for mast cell degranulation. Mast cell degranulation is necessary for vessel dilation. Our data suggest that CD18 on PMNs is necessary for platelet extravasation. Future studies can evaluate the role of CD18 in CD18 knock-out mice.

CD11a/CD18 (LFA-1) and CD11b/CD18 (MAC-1) are two members of the CD18 family. Isolated PMNs from either LFA-1 or MAC-1 knockout mice can be incubated with isolated primary mast cells. Histamine release can be measured using flow cytometric assays to evaluate a LFA-1 or MAC-1 dependent mechanism for histamine release by mast cells. The current study also shows that reconstitution with bone marrow- derived wildtype PMNs did not restore platelet extravasation CD18hypo mice. The study could be repeated with mature PMNs isolated from peripheral blood. As well, the timing of their injection into the recipient mouse could be adjusted and even given 3-4 hours after wounding. This would ensure that the injected PMNs would encounter inflamed limbal endothelium capable of supporting PMN emigration.

This dissertation evaluated PMN migration during the early and late phases of inflammation in our mouse model of central corneal abrasion. Evaluating the late phases

138 of inflammation when inflammation is resolving, the study revealed that PMN disappearance in the corneal regions may involve reverse migration of PMNs back to the limbal region where they could be phagocytosed by paralimbal macrophages. Although not investigated in this dissertation, limbal extravascular PMN disappearance may include PMN re-entry into the blood stream. The disappearance of PMNs could also occur through phagocytosis by macrophages at the center or parawound regions. While the study found evidence for PMN phagocytosis by macrophages at regions close to the limbus, keratocytes are also capable of PMN phagocytosis (Burns, personal communication) and their contribution needs to be considered. Further studies need to evaluate if PMNs return back to the blood stream and are thus removed from the cornea during the resolution of inflammation. Further, PMN migration during the early phase of inflammation involves β1, β2, and β3 integrins. Studies can evaluate if β1 or β3 integrins are also involved in PMN reverse migration which can be achieved by antibody blockade

(Hanlon et al., 2014).

Over all, these studies will help develop effective means of modulating the inflammatory response with a goal of improving corneal wound recovery.

139

References

Acar, B.T., Bozkurt, K.T., Duman, E., Acar, S., 2016. Bilateral cloudy cornea: is the usual suspect congenital hereditary endothelial dystrophy or stromal dystrophy? BMJ

Case Reports 2016 doi:10.1136/bcr-2015-214094.

Akhtar, S., Alkatan, H.M., Kirat, O., Khan, A.A., Almubrad, T., 2015. Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission

Elec Tomo Microsc Microanal 21, 666-679.

Albelda, S.M., Smith, C.W., Ward, P.A., 1994. Adhesion molecules and inflammatory injury. FASEB J 8, 504-512.

Arnaout, M.A., 1990. Structure and function of the leukocyte adhesion molecules

CD11/CD18. Blood 75, 1037-1050.

Asada, M., Furukawa, K., Kantor, C., Gahmberg, C.G., Kobata, A., 1991. Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Biochemistry 30, 1561-1571.

Asada, Y., Nakae, S., Ishida, W., Hori, K., Sugita, J., Sudo, K., Fukuda, K., Fukushima,

A., Suto, H., Murakami, A., Saito, H., Ebihara, N., Matsuda, A., 2015. Roles of Epithelial

Cell-Derived Type 2-Initiating Cytokines in Experimental Allergic Conjunctivitis. Invest

Ophthalmol Vis Sci 56, 5194-5202.

140

Assouline, M., Chew, S.J., Thompson, H.W., Beuerman, R., 1992. Effect of growth factors on collagen lattice contraction by human keratocytes. Invest Ophthalmol Vis Sci

33, 1742-1755.

Bainton, D.F., Miller, L.J., Kishimoto, T.K., Springer, T.A., 1987. Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J Exp Med

166, 1641-1653.

Beltman, J.B., Maree, A.F., de Boer, R.J., 2009. Analysing immune cell migration. Nat

Rev Immunol 9, 789-798.

Bienvenu, K., Granger, D.N., 1993. Molecular determinants of shear rate-dependent leukocyte adhesion in postcapillary venules. Am J Physiol 264, H1504-1508.

Blumberg, H., Conklin, D., Xu, W.F., Grossmann, A., Brender, T., Carollo, S., Eagan,

M., Foster, D., Haldeman, B.A., Hammond, A., Haugen, H., Jelinek, L., Kelly, J.D.,

Madden, K., Maurer, M.F., Parrish-Novak, J., Prunkard, D., Sexson, S., Sprecher, C.,

Waggie, K., West, J., Whitmore, T.E., Yao, L., Kuechle, M.K., Dale, B.A.,

Chandrasekher, Y.A., 2001. Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell 104, 9-19.

Borregaard, N., Cowland, J.B., 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503-3521.

141

Borregaard, N., Sorensen, O.E., Theilgaard-Monch, K., 2007. Neutrophil granules: a library of innate immunity proteins. Trends Immunol 28, 340-345.

Bron, A.J., 2001. The architecture of the corneal stroma. The British journal of ophthalmology 85, 379-381.

Burns, A.R., Li, Z., Smith, C.W., 2005. Neutrophil migration in the wounded cornea: the role of the keratocyte. Ocul Surf 3, S173-176.

Byeseda, S.E., Burns, A.R., Dieffenbaugher, S., Rumbaut, R.E., Smith, C.W., Li, Z.,

2009. ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing. Am J Pathol 175, 571-579.

Chakrabarti, S., Patel, K.D., 2005. Regulation of matrix metalloproteinase-9 release from

IL-8-stimulated human neutrophils. J Leukoc Biol 78, 279-288.

Chao, C., Stapleton, F., Zhou, X., Chen, S., Zhou, S., Golebiowski, B., 2015. Structural and functional changes in corneal innervation after laser in situ keratomileusis and their relationship with dry eye. Graefes Arch Clin Exp Ophthalmol 253, 2029-2039.

Chen, W.Y., Chang, M.S., 2009. IL-20 is regulated by hypoxia-inducible factor and up- regulated after experimental ischemic stroke. J Immunol 182, 5003-5012.

142

Choi, E.Y., Santoso, S., Chavakis, T., 2009. Mechanisms of neutrophil transendothelial migration. Front Biosci 14, 1596-1605.

Cohen, H.C., Frost, D.C., Lieberthal, T.J., Li, L., Kao, W.J., 2015. Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-gamma in polymorphonuclear leukocyte primary and tertiary granule release. Biomaterials 50, 47-

55.

Commins, S., Steinke, J.W., Borish, L., 2008. The extended IL-10 superfamily: IL-10,

IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J Allergy Clin Immunol 121, 1108-

1111.

Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S., Albina, J.E., 2008. Use of

Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leuko Biol 83, 64-

70.

DelMonte, D.W., Kim, T., 2011. Anatomy and physiology of the cornea. J Cataract

Refract Surg 37, 588-598.

Diamond, M.S., Alon, R., Parkos, C.A., Quinn, M.T., Springer, T.A., 1995. Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1). J Cell Biol 130, 1473-

1482.

143

Diamond, M.S., Garcia-Aguilar, J., Bickford, J.K., Corbi, A.L., Springer, T.A., 1993. The

I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J Cell Biol 120, 1031-1043.

Dimasi, D., Sun, W.Y., Bonder, C.S., 2013. Neutrophil interactions with the vascular endothelium. Internat Immunopharm 17, 1167-1175.

Dinarello, C.A., 2000. Proinflammatory cytokines. Chest 118, 503-508.

Dinarello, C.A., 2002. The IL-1 family and inflammatory diseases. Clin Exp Rheumatol

20, S1-13.

Eidenschenk, C., Rutz, S., Liesenfeld, O., Ouyang, W., 2014. Role of IL-22 in microbial host defense. Curr Top Microbiol Immunol 380, 213-236.

Ferrao, R., Wallweber, H.J., Ho, H., Tam, C., Franke, Y., Quinn, J., Lupardus, P.J., 2016.

The Structural Basis for Class II Cytokine Receptor Recognition by JAK1. Structure 24,

897-905.

Fickenscher, H., Hor, S., Kupers, H., Knappe, A., Wittmann, S., Sticht, H., 2002. The interleukin-10 family of cytokines. Trends Immunol 23, 89-96.

Figueroa, C.D., Matus, C.E., Pavicic, F., Sarmiento, J., Hidalgo, M.A., Burgos, R.A.,

Gonzalez, C.B., Bhoola, K.D., Ehrenfeld, P., 2015. Kinin B1 receptor regulates

144 interactions between neutrophils and endothelial cells by modulating the levels of Mac-1,

LFA-1 and intercellular adhesion molecule-1. Innate Immun 21, 289-304.

Filep, J.G., El Kebir, D., 2009. Neutrophil apoptosis: a target for enhancing the resolution of inflammation. J Cell Biochem 108, 1039-1046.

Gagen, 2012. Cellular and Molecular Mechanisms of Corneal Inflammation and Wound

Healing, College of Optometry. University of Houston, Houston, TX.

Gagen, D., Laubinger, S., Li, Z., Petrescu, M.S., Brown, E.S., Smith, C.W., Burns, A.R.,

2010. ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse. Exp Eye Res 91, 676-684.

Gahmberg, C.G., 1997. Leukocyte adhesion: CD11/CD18 integrins and intercellular adhesion molecules. Curr Opin Cell Biol 9, 643-650.

Gahmberg, C.G., Fagerholm, S., 2002. Activation of leukocyte beta2-integrins. Vox Sang

83 Suppl 1, 355-358.

Gahmberg, C.G., Fagerholm, S.C., Nurmi, S.M., Chavakis, T., Marchesan, S., Gronholm,

M., 2009. Regulation of integrin activity and signalling. Biochim et Biophys Acta 1790,

431-444.

145

Gahmberg, C.G., Tolvanen, M., Kotovuori, P., 1997. Leukocyte adhesion--structure and function of human leukocyte beta2-integrins and their cellular ligands. FEBS 245, 215-

232.

Gallo, R.L., Kim, K.J., Bernfield, M., Kozak, C.A., Zanetti, M., Merluzzi, L., Gennaro,

R., 1997. Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse. J Biol Chem 272, 13088-13093.

Gan, L., Fagerholm, P., Kim, H.J., 1999. Effect of leukocytes on corneal cellular proliferation and wound healing. Invest Ophthalmol Vis Sci 40, 575-581.

Granger DN, S.E., 2010. Inflammation and the Microcirculation. Morgan & Claypool

Life Sciences, San Rafael, CA Chapter 3 .

Graw, J., 2010. Eye development. Curr Topics Develop Biol 90, 343-386.

Green, C.E., Schaff, U.Y., Sarantos, M.R., Lum, A.F., Staunton, D.E., Simon, S.I., 2006.

Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium. Blood 107, 2101-2111.

Gronert, K., 2010. Resolution, the grail for healthy ocular inflammation. Exp Eye Res 91,

478-485.

146

Gutterman, D.D., Chabowski, D.S., Kadlec, A.O., Durand, M.J., Freed, J.K., Ait-Aissa,

K., Beyer, A.M., 2016. The Human Microcirculation: Regulation of Flow and Beyond.

Circ Res 118, 157-172.

Hagedoorn, A., 1928. The Early Development Of The Endothelium Of Descemet's

Membrane, The Cornea And The Anterior Chamber Of The Eye. Br J Ophthalmol 12,

479-495.

Hamann, S., 2002. Molecular mechanisms of water transport in the eye. Int Rev Cytol

215, 395-431.

Hanlon, S.D., Patel, N.B., Burns, A.R., 2011. Assessment of postnatal corneal development in the C57BL/6 mouse using spectral domain optical coherence tomography and microwave-assisted histology. Exp Eye Res 93, 363-370.

Hanlon, S.D., Smith, C.W., Sauter, M.N., Burns, A.R., 2014. Integrin-dependent neutrophil migration in the injured mouse cornea. Exp Eye Res 120, 61-70.

Harrison, P., Cramer, E.M., 1993. Platelet alpha-granules. Blood Rev 7, 52-62.

Hartwig, J., Italiano, J., Jr., 2003. The birth of the platelet. J Thromb Haemost 1, 1580-

1586.

147

Haustein, J., 1983. On the ultrastructure of the developing and adult mouse corneal stroma. Anat Embryol 168, 291-305.

Hay, E.D., 1980. Development of the vertebrate cornea. Int Rev Cytol 63, 263-322.

Hayashi, Y., Call, M.K., Chikama, T.-i., Liu, H., Carlson, E.C., Sun, Y., Pearlman, E.,

Funderburgh, J.L., Babcock, G., Liu, C.-Y., Ohashi, Y., Kao, W.W.-Y., 2010. Lumican is required for neutrophil extravasation following corneal injury and wound healing. J Cell

Sci 123, 2987-2995.

Hayes, S., Boote, C., Lewis, J., Sheppard, J., Abahussin, M., Quantock, A.J., Purslow, C.,

Votruba, M., Meek, K.M., 2007. Comparative study of fibrillar collagen arrangement in the corneas of primates and other mammals. Anat Rec 290, 1542-1550.

Henriksson, J.T., McDermott, A.M., Bergmanson, J.P., 2009. Dimensions and morphology of the cornea in three strains of mice. Invest Ophthalmol Vis Sci 50, 3648-

3654.

Hersh, E.M., Bodey, G.P., 1970. Leukocytic mechanisms in inflammation. Ann Rev Med

21, 105-132.

Horai, R., Caspi, R.R., 2011. Cytokines in autoimmune uveitis. J Interferon Cytokine Res

31, 733-744.

148

Italiano, J.E., Jr., Shivdasani, R.A., 2003. Megakaryocytes and beyond: the birth of platelets. J Thromb Haemost 1, 1174-1182.

Jackson, J.R., Seed, M.P., Kircher, C.H., Willoughby, D.A., Winkler, J.D., 1997. The codependence of angiogenesis and chronic inflammation. FASEB J 11, 457-465.

Jaeschke, H., Hasegawa, T., 2006. Role of neutrophils in acute inflammatory liver injury.

Liver Int 26, 912-919.

Johnson, D.H., Bourne, W.M., Campbell, R.J., 1982. The ultrastructure of Descemet's membrane. I. Changes with age in normal corneas. Arch Ophthalmol 100, 1942-1947.

Jonsson, F., Mancardi, D.A., Kita, Y., Karasuyama, H., Iannascoli, B., Van Rooijen, N.,

Shimizu, T., Daeron, M., Bruhns, P., 2011. Mouse and human neutrophils induce anaphylaxis. J Clin Invest 121, 1484-1496.

Kim, M.B., Sarelius, I.H., 2004. Role of shear forces and adhesion molecule distribution on P-selectin-mediated leukocyte rolling in postcapillary venules. Am J Physiol 287,

H2705-2711.

Kolaczkowska, E., Kubes, P., 2013. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13, 159-175.

149

Kotenko, S.V., Pestka, S., 2000. Jak-Stat signal transduction pathway through the eyes of cytokine class II receptor complexes. Oncogene 19, 2557-2565.

Kragstrup, T.W., Greisen, S.R., Nielsen, M.A., Rhodes, C., Stengaard-Pedersen, K.,

Hetland, M.L., Hørslev-Petersen, K., Junker, P., Østergaard, M., Hvid, M., Vorup-Jensen,

T., Robinson, W.H., Sokolove, J., Deleuran, B., 2015. The interleukin-20 receptor axis in early rheumatoid arthritis: novel links between disease-associated autoantibodies and radiographic progression. Arthritis Res Ther 18, 61.

Kragstrup, T.W., Otkjaer, K., Holm, C., Jørgensen, A., Hokland, M., Iversen, L.,

Deleuran, B., 2008. The expression of IL-20 and IL-24 and their shared receptors are increased in rheumatoid arthritis and spondyloarthropathy. Cytokine 41, 16-23.

Kronschlager, M., Talebizadeh, N., Yu, Z., Meyer, L.M., Lofgren, S., 2015. Apoptosis in

Rat Cornea After In Vivo Exposure to Ultraviolet Radiation at 300 nm. Cornea 34, 945-

949.

Krystel-Whittemore, M., Dileepan, K.N., Wood, J.G., 2015. Mast Cell: A Multi-

Functional Master Cell. Front Immunol 6, 620.

Kunder, C.A., St John, A.L., Abraham, S.N., 2011. Mast cell modulation of the vascular and lymphatic endothelium. Blood 118, 5383-5393.

150

Kurzinger, K., Reynolds, T., Germain, R.N., Davignon, D., Martz, E., Springer, T.A.,

1981. A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure. J Immunol 127, 596-602.

Lagali, N., Germundsson, J., Fagerholm, P., 2009. The role of Bowman's layer in corneal regeneration after phototherapeutic keratectomy: a prospective study using in vivo confocal microscopy. Invest Ophthalmol Vis Sci 50, 4192-4198.

Lam, F.W., Phillips, J., Landry, P., Magadi, S., Smith, C.W., Rumbaut, R.E., Burns,

A.R., 2015. Platelet recruitment promotes keratocyte repopulation following corneal epithelial abrasion in the mouse. PLoS One 10, e0118950.

Lam, P.Y., Huttenlocher, A., 2013. Interstitial leukocyte migration in vivo. Curr Opin

Cell Biol 25, 650-658.

Laurence, A., O'Shea, J.J., Watford, W.T., 2008. Interleukin-22: a sheep in wolf's clothing. Nat Med 14, 247-249.

Lerchenberger, M., Uhl, B., Stark, K., Zuchtriegel, G., Eckart, A., Miller, M., Puhr-

Westerheide, D., Praetner, M., Rehberg, M., Khandoga, A.G., Lauber, K., Massberg, S.,

Krombach, F., Reichel, C.A., 2013. Matrix metalloproteinases modulate ameboid-like migration of neutrophils through inflamed interstitial tissue. Blood 122, 770-780.

151

Li, L., Ren, D.H., Ladage, P.M., Yamamoto, K., Petroll, W.M., Jester, J.V., Cavanagh,

H.D., 2002. Annexin V binding to rabbit corneal epithelial cells following overnight contact lens wear or eyelid closure. CLAO J 28, 48-54.

Li, Z., Burns, A.R., Han, L., Rumbaut, R.E., Smith, C.W., 2011a. IL-17 and VEGF are necessary for efficient corneal nerve regeneration. Am J Pathol 178, 1106-1116.

Li, Z., Burns, A.R., Miller, S.B., Smith, C.W., 2011b. CCL20, γδ T cells, and IL-22 in corneal epithelial healing. FASEB J 25, 2659-2668.

Li, Z., Burns, A.R., Smith, C.W., 2006a. Two waves of neutrophil emigration in response to corneal epithelial abrasion: distinct adhesion molecule requirements. Invest

Ophthalmol Vis Sci 47, 1947-1955.

Li, Z., Rumbaut, R.E., Burns, A.R., Smith, C.W., 2006b. Platelet response to corneal abrasion is necessary for acute inflammation and efficient re-epithelialization. Invest

Ophthalmol Vis Sci 47, 4794-4802.

Liang, S.C., Nickerson-Nutter, C., Pittman, D.D., Carrier, Y., Goodwin, D.G., Shields,

K.M., Lambert, A.J., Schelling, S.H., Medley, Q.G., Ma, H.L., Collins, M., Dunussi-

Joannopoulos, K., Fouser, L.A., 2010. IL-22 induces an acute-phase response. J Immunol

185, 5531-5538.

152

Liclican, E.L., Gronert, K., 2010. Molecular circuits of resolution in the eye. Sci World J

10, 1029-1047.

Liles, W.C., Ledbetter, J.A., Waltersdorph, A.W., Klebanoff, S.J., 1995. Cross-linking of

CD18 primes human neutrophils for activation of the respiratory burst in response to specific stimuli: implications for adhesion-dependent physiological responses in neutrophils. J Leukoc Biol 58, 690-697.

Ljubimov, A.V., Atilano, S.R., Garner, M.H., Maguen, E., Nesburn, A.B., Kenney, M.C.,

2002. Extracellular matrix and Na+,K+-ATPase in human corneas following cataract surgery: comparison with bullous keratopathy and Fuchs' dystrophy corneas. Cornea 21,

74-80.

Lowes, M.A., Bowcock, A.M., Krueger, J.G., 2007. Pathogenesis and therapy of psoriasis. Nature 445, 866-873.

Lwigale, P.Y., 2015. Corneal Development: Different Cells from a Common Progenitor.

Prog Mol Biol Trans Sci 134, 43-59.

Machlus, K.R., Thon, J.N., Italiano, J.E., Jr., 2014. Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular processes mediating platelet formation. Br J Haem 165, 227-236.

153

Maeng, Y.S., Lee, G.H., Choi, S.I., Kim, K.S., Kim, E.K., 2015. Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts. BMC Med Genomics 8, 74.

Malik, A.B., 1993. Endothelial cell interactions and integrins. New Horiz 1, 37-51.

Mallet, J.D., Rochette, P.J., 2013. Wavelength-dependent ultraviolet induction of cyclobutane pyrimidine dimers in the human cornea. Photochem Photobiol Sci 12, 1310-

1318.

Marrazzo, G., Bellner, L., Halilovic, A., Li Volti, G., Drago, F., Dunn, M.W.,

Schwartzman, M.L., 2011. The role of neutrophils in corneal wound healing in HO-2 null mice. PLoS One 6, e21180.

Martin, K.R., Ohayon, D., Witko-Sarsat, V., 2015. Promoting apoptosis of neutrophils and phagocytosis by macrophages: novel strategies in the resolution of inflammation.

Swiss Med Wkly 145, w14056.

Mastropasqua, L., Lanzini, M., Curcio, C., Calienno, R., Mastropasqua, R., Colasante,

M., Mastropasqua, A., Nubile, M., 2014. Structural modifications and tissue response after standard epi-off and iontophoretic corneal crosslinking with different irradiation procedures. Invest Ophthalmol Vis Sci 55, 2526-2533.

154

McDermott, A.M., 2009. The role of antimicrobial peptides at the ocular surface. Ophthal

Res 41, 60-75.

McDermott, A.M., Redfern, R.L., Zhang, B., Pei, Y., Huang, L., Proske, R.J., 2003.

Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells. Invest Ophthalmol

Vis Sci 44, 1859-1865.

Meek, K.M., Knupp, C., 2015. Corneal structure and transparency. Prog Ret Eye Res 49,

1-16.

Mege, J.L., Meghari, S., Honstettre, A., Capo, C., Raoult, D., 2006. The two faces of interleukin 10 in human infectious diseases. Lancet 6, 557-569.

Metcalfe, D.D., Kaliner, M., Donlon, M.A., 1981. The mast cell. Crit Rev Immunol 3,

23-74.

Michelacci, Y.M., 2003. Collagens and proteoglycans of the corneal extracellular matrix.

Braz J Med Biolo Res 36, 1037-1046.

Mocsai, A., 2013. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med 210, 1283-1299.

155

Mohamed-Noriega, K., Riau, A.K., Lwin, N.C., Chaurasia, S.S., Tan, D.T., Mehta, J.S.,

2014. Early corneal nerve damage and recovery following small incision lenticule extraction (SMILE) and laser in situ keratomileusis (LASIK). Invest Ophthalmol Vis Sci

55, 1823-1834.

Morgan, P.B., Efron, N., Brennan, N.A., Hill, E.A., Raynor, M.K., Tullo, A.B., 2005.

Risk factors for the development of corneal infiltrative events associated with contact lens wear. Invest Ophthalmol Vis Sci 46, 3136-3143.

Nagaoka, T., Kaburagi, Y., Hamaguchi, Y., Hasegawa, M., Takehara, K., Steeber, D.A.,

Tedder, T.F., Sato, S., 2000. Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression. Am J Pathol 157, 237-247.

Nakayasu, K., Tanaka, M., Konomi, H., Hayashi, T., 1986. Distribution of types I, II, III,

IV and V collagen in normal and keratoconus corneas. Ophthal Res 18, 1-10.

Narayanan, S., Redfern, R.L., Miller, W.L., Nichols, K.K., McDermott, A.M., 2013. Dry eye disease and microbial keratitis: is there a connection? Ocul Surf 11, 75-92.

Nilsson, G., Metcalfe, D.D., Taub, D.D., 2000. Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response.

Immunology 99, 314-319.

156

O'Rahilly, R., 1975. The prenatal development of the human eye. Exp Eye Res 21, 93-

112.

Otkjaer, K., Kragballe, K., Funding, A.T., Clausen, J.T., Noerby, P.L., Steiniche, T.,

Iversen, L., 2005. The dynamics of gene expression of interleukin-19 and interleukin-20 and their receptors in psoriasis. Br J Dermatol 153, 911-918.

Ouyang, W., Rutz, S., Crellin, N.K., Valdez, P.A., Hymowitz, S.G., 2011. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Ann Rev

Immunol 29, 71-109.

Page, C., Pitchford, S., 2013. Neutrophil and platelet complexes and their relevance to neutrophil recruitment and activation. Int Immunopharm 17, 1176-1184.

Paugam, C., Chollet-Martin, S., Dehoux, M., Chatel, D., Brient, N., Desmonts, J.M.,

Philip, I., 1997. Neutrophil expression of CD11b/CD18 and IL-8 secretion during normothermic cardiopulmonary bypass. J Cardiothor Vasc Anesth 11, 575-579.

Payne, G.W., Madri, J.A., Sessa, W.C., Segal, S.S., 2004. Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle. FASEB J 18, 280-286.

Petrescu, M.S., Larry, C.L., Bowden, R.A., Williams, G.W., Gagen, D., Li, Z., Smith,

C.W., Burns, A.R., 2007. Neutrophil interactions with keratocytes during corneal

157 epithelial wound healing: a role for CD18 integrins. Invest Ophthalmol Vis Sci 48, 5023-

5029.

Pfister, R.R., 2004. Permanent corneal edema resulting from the treatment of PTK corneal haze with mitomycin: a case report. Cornea 23, 744-747.

Pfitzner, J., 1976. Poiseuille and his law. Anaesthesia 31, 273-275.

Pflugfelder, S.C., Corrales, R.M., de Paiva, C.S., 2013. T helper cytokines in dry eye disease. Exp Eye Res 117, 118-125.

Planck, S.R., Becker, M.D., Crespo, S., Choi, D., Galster, K., Garman, K.L., Nobiling,

R., Rosenbaum, J.T., 2008. Characterizing extravascular neutrophil migration in vivo in the iris. Inflammation 31, 105-111.

Poggio, E.C., Glynn, R.J., Schein, O.D., Seddon, J.M., Shannon, M.J., Scardino, V.A.,

Kenyon, K.R., 1989. The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. New Engl J Med 321, 779-783.

Queen, J., Satchell, K.J., 2013. Promotion of colonization and virulence by cholera toxin is dependent on neutrophils. Infect Immun 81, 3338-3345.

158

Rather, L.J., 1971. Disturbance of function (functio laesa): the legendary fifth cardinal sign of inflammation, added by Galen to the four cardinal signs of Celsus. Bull N Y Acad

Med 47, 303-322.

Redfern, R.L., Patel, N., Hanlon, S., Farley, W., Gondo, M., Pflugfelder, S.C.,

McDermott, A.M., 2013. Toll-like receptor expression and activation in mice with experimental dry eye. Invest Ophthalmol Vis Sci 54, 1554-1563.

Redfern, R.L., Reins, R.Y., McDermott, A.M., 2011. Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells. Exp Eye Res 92, 209-

220.

Reinstein, D.Z., Archer, T.J., Gobbe, M., 2012. The history of LASIK. J Refract Surg

28, 291-298.

Reinstein, D.Z., Archer, T.J., Gobbe, M., Silverman, R.H., Coleman, D.J., 2008.

Epithelial thickness in the normal cornea: three-dimensional display with Artemis very high-frequency digital ultrasound. J Refract Surg (Thorofare, N.J. : 1995) 24, 571-581.

Reinstein, D.Z., Archer, T.J., Gobbe, M., Silverman, R.H., Coleman, D.J., 2009. Stromal thickness in the normal cornea: three-dimensional display with artemis very high- frequency digital ultrasound. J Refract Surg (Thorofare, N.J. : 1995) 25, 776-786.

159

Robertson, D.M., Cavanagh, H.D., 2008. The Clinical and Cellular Basis of Contact

Lens-related Corneal Infections: A Review. Clin Ophthal 2, 907-917.

Rocha e Silva, M., 1994. A brief survey of the history of inflammation. 1978. Agents

Actions 43, 86-90.

Romer, J., Hasselager, E., Norby, P.L., Steiniche, T., Thorn Clausen, J., Kragballe, K.,

2003. Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine a or calcipotriol. J Invest

Dermatol 121, 1306-1311.

Rosenkranz, A.R., Coxon, A., Maurer, M., Gurish, M.F., Austen, K.F., Friend, D.S.,

Galli, S.J., Mayadas, T.N., 1998. Impaired mast cell development and innate immunity in

Mac-1 (CD11b/CD18, CR3)-deficient mice. J Immunol 161, 6463-6467.

Rubel, C., Gomez, S., Fernandez, G.C., Isturiz, M.A., Caamano, J., Palermo, M.S., 2003.

Fibrinogen-CD11b/CD18 interaction activates the NF-kappa B pathway and delays apoptosis in human neutrophils. Europ J Immunol 33, 1429-1438.

Rutz, S., Wang, X., Ouyang, W., 2014. The IL-20 subfamily of cytokines--from host defence to tissue homeostasis. Nat Rev Immunol 14, 783-795.

S P Sutera, a.R.S., 1993. The History Of Poiseuille Law. Ann Rev Fluid Mech 25, 1-19.

160

Sa, S.M., Valdez, P.A., Wu, J., Jung, K., Zhong, F., Hall, L., Kasman, I., Winer, J.,

Modrusan, Z., Danilenko, D.M., Ouyang, W., 2007. The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis. J Immuno 178, 2229-2240.

Sabat, R., Ouyang, W., Wolk, K., 2014. Therapeutic opportunities of the IL-22-IL-22R1 system. Nat Rev Drug Discov 13, 21-38.

Sadoul, K., 2015. New explanations for old observations: marginal band coiling during platelet activation. J Thromb Haemost 13, 333-346.

Sarantos, M.R., Zhang, H., Schaff, U.Y., Dixit, N., Hayenga, H.N., Lowell, C.A., Simon,

S.I., 2008. Transmigration of neutrophils across inflamed endothelium is signaled through LFA-1 and Src family kinase. J Immunol 181, 8660-8669.

Schwab, J.M., Chiang, N., Arita, M., Serhan, C.N., 2007. Resolvin E1 and protectin D1 activate inflammation-resolution programmes. Nature 447, 869-874.

Schymeinsky, J., Mocsai, A., Walzog, B., 2007. Neutrophil activation via beta2 integrins

(CD11/CD18): molecular mechanisms and clinical implications. Thromb Haemost 98,

262-273.

Scott, A., Khan, K.M., Cook, J.L., Duronio, V., 2004. What is "inflammation"? Are we ready to move beyond Celsus? Br J Sports Med 38, 248-249.

161

Sevel, D., Isaacs, R., 1988. A re-evaluation of corneal development. Trans Amer

Ophthalmol Soc 86, 178-207.

Shamri, R., Grabovsky, V., Gauguet, J.M., Feigelson, S., Manevich, E., Kolanus, W.,

Robinson, M.K., Staunton, D.E., von Andrian, U.H., Alon, R., 2005. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol 6, 497-506.

Sheppard, J., Hayes, S., Boote, C., Votruba, M., Meek, K.M., 2010. Changes in corneal collagen architecture during mouse postnatal development. Invest Ophthalmol Vis Sci 51,

2936-2942.

Singh, P., Hu, P., Hoggatt, J., Moh, A., Pelus, L.M., 2012. Expansion of bone marrow neutrophils following G-CSF administration in mice results in osteolineage cell apoptosis and mobilization of hematopoietic stem and progenitor cells. Leukemia 26, 2375-2383.

Smith, C.W., 2000. Possible steps involved in the transition to stationary adhesion of rolling neutrophils: a brief review. Microcirc 7, 385-394.

Smith, C.W., Anderson, D.C., 1991. PMN adhesion and extravasation as a paradigm for tumor cell dissemination. Cancer Metastasis Rev 10, 61-78.

Smith, C.W., Rothlein, R., Hughes, B.J., Mariscalco, M.M., Rudloff, H.E., Schmalstieg,

F.C., Anderson, D.C., 1988. Recognition of an endothelial determinant for CD 18-

162 dependent human neutrophil adherence and transendothelial migration. J Clin Invest 82,

1746-1756.

Srivastav, A.K., Mujtaba, S.F., Dwivedi, A., Amar, S.K., Goyal, S., Verma, A.,

Kushwaha, H.N., Chaturvedi, R.K., Ray, R.S., 2016. Photosensitized rose Bengal- induced phototoxicity on human melanoma cell line under natural sunlight exposure. J

Photochem Photobiol 156, 87-99.

Stacey, M.A., Marsden, M., Pham, N.T., Clare, S., Dolton, G., Stack, G., Jones, E.,

Klenerman, P., Gallimore, A.M., Taylor, P.R., Snelgrove, R.J., Lawley, T.D., Dougan,

G., Benedict, C.A., Jones, S.A., Wilkinson, G.W., Humphreys, I.R., 2014. Neutrophils recruited by IL-22 in peripheral tissues function as TRAIL-dependent antiviral effectors against MCMV. Cell Host Microbe 15, 471-483.

Sutu, C., Fukuoka, H., Afshari, N.A., 2016. Mechanisms and management of dry eye in cataract surgery patients. Curr Opin Ophthalmol 27, 24-30.

Swamydas, M., Lionakis, M.S., 2013. Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. Journal of visualized experiments : JoVE, e50586.

Tamagawa-Mineoka, R., 2015. Important roles of platelets as immune cells in the skin. J

Dermatol Sci 77, 93-101.

163

Tamura, Y., Konomi, H., Sawada, H., Takashima, S., Nakajima, A., 1991. Tissue distribution of type VIII collagen in human adult and fetal eyes. Invest Ophthalmol Vis

Sci 32, 2636-2644.

Tan, S.M., 2012. The leucocyte beta2 (CD18) integrins: the structure, functional regulation and signalling properties. Biosci Rep 32, 241-269.

Tao, A., Wang, J., Chen, Q., Shen, M., Lu, F., Dubovy, S.R., Shousha, M.A., 2011.

Topographic thickness of Bowman's layer determined by ultra-high resolution spectral domain-optical coherence tomography. Invest Ophthalmol Vis Sci 52, 3901-3907.

Ti, S.E., Tan, D.T., 2001. Recurrent corneal erosion after laser in situ keratomileusis.

Cornea 20, 156-158.

Tonnesen, M.G., 1989. Neutrophil-endothelial cell interactions: mechanisms of neutrophil adherence to vascular endothelium. J Invest Dermatol 93, 53s-58s.

Trost, A., Schrodl, F., Strohmaier, C., Bogner, B., Runge, C., Kaser-Eichberger, A.,

Krefft, K., Vogel, A., Linz, N., Freidank, S., Hilpert, A., Zimmermann, I., Grabner, G.,

Reitsamer, H.A., 2013. A new nanosecond UV laser at 355 nm: early results of corneal flap cutting in a rabbit model. Invest Ophthalmol Vis Sci 54, 7854-7864.

Verkman, A.S., 2006. Aquaporins in endothelia. Kidney Int 69, 1120-1123.

164

Walzog, B., Jeblonski, F., Zakrzewicz, A., Gaehtgens, P., 1997. Beta2 integrins

(CD11/CD18) promote apoptosis of human neutrophils. FASEB J 11, 1177-1186.

Wang, J., Chung, J.L., Schuele, G., Vankov, A., Dalal, R., Wiltberger, M., Palanker, D.,

2015. Safety of cornea and iris in ocular surgery with 355-nm lasers. J Biomed Optics 20,

095005.

Wang, M., Shibamoto, T., Kuda, Y., Tanida, M., Zhang, T., Song, J., Kurata, Y., 2016.

The responses of pulmonary and systemic circulation and airway to anaphylactic mediators in anesthetized BALB/c mice. Life Sci 147, 77-84.

Watsky, M.A., 1995. Keratocyte gap junctional communication in normal and wounded rabbit corneas and human corneas. Invest Ophthalmol Vis Sci 36, 2568-2576.

Wegener, A.R., Meyer, L.M., Schonfeld, C.L., 2015. Effect of Viscous Agents on

Corneal Density in Dry Eye Disease. J Ocul Pharma Therap 31, 504-508.

Werr, J., Xie, X., Hedqvist, P., Ruoslahti, E., Lindbom, L., 1998. beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo. J Exp Med

187, 2091-2096.

Willeke, T., Scharffetter-Kochanek, K., Gaehtgens, P., Walzog, B., 2001. A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils. Biorheol 38, 89-100.

165

Wilson, R.W., Ballantyne, C.M., Smith, C.W., Montgomery, C., Bradley, A., O'Brien,

W.E., Beaudet, A.L., 1993. Gene targeting yields a CD18-mutant mouse for study of inflammation. J Immunol 151, 1571-1578.

Wilson, S.E., Mohan, R.R., Mohan, R.R., Ambrosio, R., Jr., Hong, J., Lee, J., 2001. The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog Retin Eye Res 20, 625-637.

Wirtz, M.K., Keller, K.E., 2016. The Role of the IL-20 Subfamily in Glaucoma.

Mediators Inflamm 2016, 4083735.

Wolk, K., Witte, K., Witte, E., Proesch, S., Schulze-Tanzil, G., Nasilowska, K., Thilo, J.,

Asadullah, K., Sterry, W., Volk, H.-D., Sabat, R., 2008. Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes. J Leukoc Biol 83, 1181-1193.

Wulle, K.G., Lerche, W., 1969. Electron microscopic observations of the early development of the human corneal endothelium and Descemet's membrane.

Ophthalmologica 157, 451-461.

Xuan, M., Wang, S., Liu, X., He, Y., Li, Y., Zhang, Y., 2016. Proteins of the corneal stroma: importance in visual function. Cell Tissue Res 364, 9-16.

166

Yamamoto, K., Ladage, P.M., Ren, D.H., Li, L., Petroll, W.M., Jester, J.V., Cavanagh,

H.D., 2002. Effect of eyelid closure and overnight contact lens wear on viability of surface epithelial cells in rabbit cornea. Cornea 21, 85-90.

Yan, S.R., Sapru, K., Issekutz, A.C., 2004. The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration. Immunol Cell Biol 82, 435-446.

Yang, H., Reinach, P.S., Koniarek, J.P., Wang, Z., Iserovich, P., Fischbarg, J., 2000.

Fluid transport by cultured corneal epithelial cell layers. Br J Ophthalmol 84, 199-204.

Yoshikai, Y., 2001. Roles of prostaglandins and leukotrienes in acute inflammation caused by bacterial infection. Curr Opin Infect Dis 14, 257-263.

Zarbock, A., Ley, K., 2008. Mechanisms and consequences of neutrophil interaction with the endothelium. Am J Pathol 172, 1-7.

Zdanov, A., 2010. Structural analysis of cytokines comprising the IL-10 family. Cytokine

Growth Factor Rev 21, 325-330.

Zen, K., Guo, Y.L., Li, L.M., Bian, Z., Zhang, C.Y., Liu, Y., 2011. Cleavage of the

CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis. Blood 117, 4885-4894.

167

Zenewicz, L.A., Yancopoulos, G.D., Valenzuela, D.M., Murphy, A.J., Karow, M.,

Flavell, R.A., 2007. Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation. Immunity 27, 647-659.

Zhang, J.-M., An, J., 2007. Cytokines, inflammation, and pain. Int Anesthesiol Clin 45,

27-37.

Zhang, W., 2014. MACROPHAGES IN CORNEAL EPITHELIAL WOUND

HEALING, COllege of Optometry. University of Houston, Houston, TX, p. 153.

Zhang, Y., Li, L., Liu, Y., Liu, Z.R., 2016. PKM2 released by neutrophils at wound site facilitates early wound healing by promoting angiogenesis. Wound Repair Regen 24,

328-336.

Zhang, Z.Y., 2012. Corneal endothelial damage in the relatively thin cornea after collagen cross-linking treatment. Cornea 31, 967; author reply 967-968.

168