Inflammatory Cytokines in Jet Propulsion Fuel-8 Induced Irritant Contact Dermatitis in Male Fischer Rats

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Thomas Joseph Kannanayakal Wright State University

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This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. INFLAMMATORY CYTOKINES IN JET PROPULSION FUEL-8 INDUCED IRRITANT CONTACT DERMATITIS IN MALE FISCHER RATS

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

THOMAS J. KANNANAYAKAL M.S., University of Madras, 2003

2009 Wright State University

WRIGHT STATE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

April 24, 2009

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION

BY Thomas J. Kannanayakal ENTITLED Inflammatory Cytokines in Jet Propulsion Fuel-8

Induced Irritant Contact Dermatitis in Male Fischer Rats BE ACCEPTED IN PARTIAL

FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science.

------Barbara E. Hull, Ph.D. Thesis Director

------David Goldstein, Ph.D. Department Chair Committee on Final Examination

------Barbara E. Hull, Ph.D.

------James N. McDougal, Ph.D.

------Cheryl Conley, Ph.D.

------Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies

Abstract

Kannanayakal, Thomas J. M.S., Department of Biological Sciences, Wright State University, 2009. Inflammatory cytokines in Jet Propulsion fuel-8 induced Irritant Contact Dermatitis in Male Fischer rats.

Contact dermatitis is one of the most common occupational diseases.

Absenteeism of the affected workers and associated health care costs makes this disease

an economically important issue. Irritant contact dermatitis (ICD) is a common contact

dermatitis observed among workers. As each category of irritants triggers the

inflammatory process by different method, studying the mechanism of ICD is difficult.

Information on the mechanism of action of an irritant is indispensable for the

development of effective prophylactic and therapeutic measures. In this study, we have

investigated the early changes in rat skin induced by jet fuel-8 (JP-8). Although previous

studies have described the cytokines involved in the latter phase of the inflammatory process induced by JP-8, there is no information on the molecular mechanisms that leads

to the downstream events. Gallucci et al., (2004) and Chatterjee et al., (2006) has shown

that JP-8 induces elevated levels of interleukin-1α (IL-1α) in skin and cultured

keratinocytes, respectively. The preformed and stored proinflammatory cytokine, IL-1α

that has autocrine and paracrine functions, appear to trigger the cascade of events that leads to the JP-8-induced contact dermatitis. We hypothesize that JP-8 induces increased levels of IL-1α at early post-exposure time points. To test our hypothesis, we applied JP-

8 in an occlusive patch for 1 h on the rat skin and measured IL-1 at 0, 1, 2, 3, 4, and 8 h post exposure. Neutrophils infiltrate the skin during the late phase of the inflammatory process; therefore we studied the neutrophil infiltration during the selected time points

iii

and measured the levels of CXCL2, the cytokine associated with the infiltration of

neutrophils. Preformed and stored IL-1 might play an important role in the JP-8 induced

gene expression in skin. We hypothesized that that blocking of IL-1 receptor will inhibit

the JP-8 induced gene expression pattern. To test our hypothesis, we delivered IL-1 receptor antagonist into the skin before JP-8 treatment. We found that At 3 h post exposure, IL-1α was reduced in JP-8 exposed skin and the selected time points were too early in the inflammatory cascade to reveal infiltration of neutrophils, although there was a slight increase in the level of CXCL2 at 2 h post exposure. Because IL-1β has similar biological effects, we measured this cytokine from the same samples used for IL-1α quantification. IL-1β did not significantly change throughout the study. The decrease in the level of IL-1α implies that JP-8 induces the release of the preformed IL-1α. IL-1 receptor antagonist did not cause significant changes in the gene expression pattern. We conclude that the release of the preformed IL-1α by JP-8 is one of the important events at the early time points of the JP-8-induced cascade of events.

Table of Contents

Page

Introduction

Contact Dermatitis 1

Jet Fuel-8 2

Skin Structure 2

Cytokines in Irritant Contact Dermatitis 3

Cytokine expression in response to JP-8 exposure 6

Potential mediators in JP-8 induced inflammation 7

Significance and Hypotheses 8

Materials and Methods

Animals 12

Exposure study 12

Dissection of skin 13

Total Protein isolation and estimation 13

ELISA 14

Histology 15

Receptor antagonist 17

Tempol 17

Intradermal injection 17

Immunohistochemistry 17

Image analysis 18

Gene expression studies 18

Results

Interleukin-1 expression in response to JP-8 exposure 21

Neutrophil infiltration study

Expression of CXCL2 22

Histology 24

Intradermal injection induced changes in skin

Histological changes 27

Morphological changes 29

Detection of injected interleukin-1 receptor antagonist in the epidermis 30

Effect of blocking IL-1α pathway in JP-8 exposed skin 31

Related studies

Reactive oxygen species 33

Autocrine effect of IL-1α in JP-8 induced inflammatory process 38

Discussion 41

Conclusion 46

Related studies 46

Future directions

Changes in ions in response to JP-8 exposure 48

Study of efficacy of IL-1RA 48

Appendix

ELISA optimization table

For interleukin 1 alpha 49

For interleukin 1 beta 50

For CXCL2 51

Summary of the quality control reports from GeneChips 52

List of transcripts changed in gene expression study 52

References 61

vii

List of Figures

Page

1. Structure of skin 2

2. Hypothetical sequence of events in ICD 4

3. Exposure study setup 12

4. Exposure study time point 13

5. Image Analysis 18

6. Injection, exposure and skin collection time points 19

7. Interleukin 1 alpha expression 21

8. CXCL2 expression 22-23

9. Control and JP-8 exposed skin 24-26

10. Bleb formed by Intradermal injection 27

11. Histology of skin after intradermal injection 28

12. Fluorescent Microscopy of IL-1 receptor antagonist and 30

Keratin-14

13. Fluorescent Microscopy of IL-1 receptor antagonist and IL-1 31

receptor

14. IL-1 alpha expression in response to JP-8 exposure after 33-35

tempol treatment in each replication

15. IL-1 alpha expression in response to JP-8 exposure after 35

tempol treatment combined data

16. IL-1 beta expression in response to JP-8 exposure after tempol 36-37

treatment in each replication

viii

17. IL-1 beta expression in response to JP-8 exposure after tempol 38

treatment combined data

18. Injection, exposure and collection of skin 39

19. IL-1 alpha expression in response to JP-8 exposure after 40

interleukin 1 receptor antagonist treatment

List of Tables

Page

1. Sandwich ELISA standard protein, capture and detection 14

antibodies

2. Dehydration and embedding steps 15

3. Staining steps 16

4. Length of epidermis in the injected skin and control skin 29

5. Interleukin-1 alpha expression in pg/mg of protein 39

6. ELISA optimization table for IL-1 alpha 49

7. ELISA optimization table for IL-1 beta 50

8. ELISA optimization table for CXCL2 51

9. Summary of the quality control reports form Affymetrix 52

Rat230_2.0 GeneChips

10. List of transcripts changed in gene expression study 52

List of Abbreviations

ACD Allergic Contact Dermatitis

CXCL2 Macrophage inflammatory protein 2α

ELISA Enzyme Linked Immunosorbent Assay

GM-CSF Granulocyte-macrophage colony-stimulating factor

ICD Irritant Contact Dermatitis

IL Interleukine

IL-1 RA IL-1 receptor antagonist

JP-8 Jet Propulsion fuel – 8

NATO North Atlantic Treaty Organization

NF-κB Nuclear factor-κB

ROS Reactive Oxygen Species

TNF Tumor Necrosis Factor

Acknowledgements

I would like to thank Dr. Barbara E. Hull for her guidance in the undertaking and completion of this project. I am grateful for the knowledge and scientific wisdom she has shared with me.

I would also like to thank the members of my committee, Dr. James N.

McDougal, for providing materials, lab space and guidance on cytokine and gene expression studies, and Dr. Cheryl Conley for her valuable feedback in this project.

I deeply appreciate the support of the Department of Pharmacology and

Toxicology, and the Department of Biological Sciences. I thank Drs Michael Leffak,

Courtney Sulentic, Randal Gallucci (The University of Oklahoma), Wayne Carmichael,

David Cool, Andrea Hoffman and Don Cipollini for their time and guidance.

I would like to express my thanks to my friends and family who gave invaluable assistance and who kept me sane through it all. My special thanks to Carol Garrett,

Theresa Kannanayakal, Richard Salisbury, Eric Romer and Shannon Romer.

xii

Introduction

Contact Dermatitis

An inflammatory response in the skin resulting from contact with external factors

is termed contact dermatitis (Fyhrquist-Vanni et al., 2007). Contact dermatitis forms

30% of all occupational diseases (Diepgen and Coenraads, 1999). Worldwide about 4 million working days are lost each year due to absenteeism of the affected workers

(English, 2001; English, 2004). The economic damage from health care costs and loss in

labor hours due to contact dermatitis makes it an important issue. Contact dermatitis

triggered by antigen is termed as Allergic Contact Dermatitis (ACD) (Beltrani and

Beltrani, 1997). A non-immunologic, local, reversible, inflammatory reaction due to

repeated or single contact to the skin is referred to as Irritant Contact Dermatitis (ICD)

(Corsini and Galli, 2000). ICD is the most common form of contact dermatitis in the work environment (Elsner, 1994). Although ICD is a major health problem, the molecular mechanism underlying this process is yet to be clearly understood (Chew and

Maibach, 2006). Jet propulsion fuel-8 (JP-8) causes contact dermatitis (Gallucci et al.,

2001; McDougal et al., 2007). A Single exposure of skin to JP-8 triggered gene expression of inflammatory proteins (McDougal et al., 2007). Contact dermatitis resulting from an allergic reaction requires multiple exposures. Therefore, the changes documented in the single-exposure gene expression studies imply that JP-8 may cause

ICD. In this study, we have investigated the change in the levels of inflammatory signaling proteins in an attempt to document the mechanism by which JP-8 induces ICD.

Jet fuel-8

JP-8 is used in aircraft, ground vehicles, cooking stoves, and personnel heaters by the militaries of the United States and the North Atlantic Treaty Organization (NATO).

About 5 billion gallons of JP-8 is used every year, and there is a widespread exposure of the military personal. JP-8, a kerosene based fuel, is a complex mixture of hydrocarbons containing about 200 aromatic and aliphatic hydrocarbons. Because JP-8 is a performance based fuel, the composition of the components may differ from region to region (Subcommittee on Jet-Propulsion Fuel, 2003). Therefore, the US Air Force provided pooled samples, collected from different regions, for toxicological studies.

Skin Structure:

Figure 1: Cartoon of normal human skin showing epidermis (E), dermis (D), subcutaneous fat layer (SCFL), and skin appendages including hair follicle (HF), sebaceous gland (SG), eccrine sweat gland (ESG), Meissner’s corpuscle (MC), pacinian corpuscle (PC), blood vessels (BV), sweat glandn pore (P), nerve fiber (NF), free nerve ending (FNe). Picture and legend adapted from (Tobin, 2006).

Skin is the largest organ of the body. As the interface between the external environment and our body, the skin is the first organ to encounter environmental stressors

(Tobin, 2006). Skin acts as a protective barrier to many physical, chemical, and biological stressors. The epidermis and the dermis form the skin (Feinkel and Woodley,

2001). The outer epidermal layer is formed of stratified, keratinized epithelium. The keratin producing cells, keratinocytes, are the major cell type found in the epidermis.

The antigen presenting Langerhans cells, the pigment producing Melanocytes, and the sensory Merkel cells are the other type of cells present in the epidermis (Tobin, 2006).

The dermis, a connective tissue that supports the epidermis, is composed of fibrous and amorphous extracellular matrix. The dermis may be divided into two main regions: the upper papillary dermis, which consists of high density of fibroblastic cells capable of more rapid proliferation, and the lower reticular dermis composed of large- diameter collagen and elastin fibrils. The dermis consists of epidermal derived appendages, neurovascular networks, sensory receptors, and dermal cells that include the bone marrow-derived mast cells and macrophages. The mast cells and macrophages play an active role in allergic reactions. Monocytes from blood migrate to the dermis and differentiate into macrophages. The phagocytic macrophages may act as antigen presenting cells (Seeley et al., 1995; Tobin, 2006).

Cytokines in irritant contact dermatitis

The mechanism of chemically induced ICD is yet to be documented (Corsini and

Galli, 1998). The hypothetical sequence of events is shown in figure 2. ICD is identified only by excluding other causes, such as ACD, by skin patch testing (Elsner, 1994).

Substances that can dissolve keratin, dehydrate the skin and cause physical injury in the skin cells induces ICD (Beltrani and Beltrani, 1997). Substances such as detergents,

solvents, and oils cause inflammation through different mechanisms, making the study of

biochemical mechanisms involved in skin irritation extremely complex (Corsini and

Galli, 1998). ICD is a complex process that involves epidermal and dermal cells as well

as migrating leukocytes. During inflammation, the epidermis and the dermis communicate with each other by secretion of cytokines. Cytokines are signaling molecules ranging from 6 kDa to 60 kDa. Chemicals that trigger ICD, in addition to damaging the tissue, can also alter or induce the release of cytokines (Corsini and Galli,

1998). The chemicals can also induce epidermal keratinocytes to release stored

inflammatory cytokines IL-1 and TNFα. (McKenzie and Sauder, 1990). The following

figure shows the hypothetical sequence of events occurring in ICD.

Figure 2: Hypothetical sequence of events in ICD, adapted from (Corsini and Galli, 1998).

The important cytokines in ICD are the interleukin-1 family and Tumor necrosis

factor-α (TNF-α) (Chew and Maibach, 2006). The IL-1 family consists of IL-1α , IL-1β

and an IL-1 receptor antagonist (IL-1ra). In the skin, IL-1 is produced by dendritic cells, keratinocytes, Langerhans cells, melanocytes, fibroblasts and endothelial cells (Dinarello,

1994). IL-1 is released from skin in response to irritants such as benzalkonium chloride

and sensitizers such as oxazolone (Wilmer et al., 1994). The biological effects of IL-1α

and IL-1β are very similar although they share only 30% of amino acid sequence

(Dinarello, 1996). IL-1α and IL-1β are synthesized as 33 kDa precursor molecules.

Both the pro-IL-1α and mature IL-1α are biologically active. IL-1α lacks the

hydrophobic leader sequence that is required for secretion, which may be the reason that

preformed IL-1α is stored in the skin cells until the membrane integrity of the cell is

compromised (Dinarello, 1997). The IL-1β precursor is not biologically active until it is

processed by IL-1β converting enzyme (Dinarello, 1996). Mature IL-1β is secreted from

the cell. IL-1α and IL-1β are initiators of the inflammatory cascade as they can rapidly

trigger the transcription of a number of genes such as activator protein-1, NF-κB, and

nuclear factor IL-6 (Bankers-Fulbright et al., 1996). IL-1α and IL-1β also up-regulate

the expression of cellular adhesion molecules on vascular endothelial cells (Detmar et al.,

1992). There are two types of IL-1 receptors, type I and type II. IL-1 triggers signal

transduction by binding to IL-1 receptor type I. The type II receptor does not produce a

known biological effect and acts as a decoy receptor in the negative feedback loop. IL-

1ra binds to the type II receptor without triggering signal transduction. When IL-1ra

binds to the receptor it prevents the interaction of IL-1α and IL-1β with the receptors.

Thus, IL-1ra provides negative feedback for IL-1 signaling (Feinkel and Woodley,

2001).

TNF-α, a multifunctional cytokine, is produced in skin by dendritic cells,

keratinocytes, fibroblasts and mast cells (Kock et al., 1990; McKenzie and Sauder, 1990).

Protein and mRNA levels of TNF-α were increased when normal human keratinocytes,

were exposed to ultra violet-B light and lipopolysacchride in vitro (Kock et al., 1990).

TNF-α up-regulates cellular adhesion molecules on the surface of vascular endothelial

cells, thereby contributing to the accumulation of leukocytes at the sites of inflammation

(Detmar et al., 1992). TNF-α also stimulates the secretion of other inflammatory

cytokines such as IL-1 and IL-6 resulting in the amplification of the biological effects

(Luheshi and Rothwell, 1996). The binding of TNF-α to its receptor increases the

activity of specific target genes such as nuclear factor kappa B (NF-κB). NF-κB

regulates the transcription of other inflammatory molecules such as IL-1 (Bazzoni and

Beutler, 1996; Corsini et al., 1997; Hohmann et al., 1990; Karin and Ben-Neriah, 2000).

Although TNFα is an important cytokine in the inflammatory process, irritants like

benzalkonium chloride do not appear to induce significant TNFα production in the skin

(Corsini and Galli, 1998).

Cytokine expression in response to JP-8 exposure

JP-8 is one of the fuel oils primarily used by the military. JP-8 causes contact dermatitis (Gallucci et al., 2004; Kabbur et al., 2001; McDougal et al., 2007; Rogers et al., 2001; Rogers et al., 2004). Brief exposure to JP-8 causes inflammation in skin that

may be due to change in homeostasis of osmotic, oxidative, and membrane stability

(McDougal et al., 2007). Cultured human neonatal epidermal keratinocytes up-regulated

IL-8 when treated with 0.1% JP-8 for 24 h (Witzmann et al., 2005). JP-8 induced increased expression of TNFα in hairless mice, and in cultured EpiDerm human skin

model (Chatterjee, A. et al., 2006). Three hundred µL of JP-8 applied on the back of rats

in the neck region for seven days, induced up-regulation of cytokines such as growth

related oncogene-α and –β, monocyte chemotactic protein-1, macrophage inflammatory

protein -1α and -1β, eotaxin, IL-1β, and IL-6 (Gallucci, R.M. et al., 2004). After 1 h

exposure of JP-8 to rat skin, mRNA expression of amphiregulin, CCL2, CCL3, CCL4,

CXCL1, CXCL2, IL-10, and IL-6 increased (McDougal, et al., 2007). mRNA levels of

S100 calcium binding protein A9 and CCL20 increased after 3 h post exposure

(McDougal, et al., 2007).

Potential mediators in JP-8 induced inflammation

Another possible trigger in JP-8 induced ICD is reactive oxygen species (ROS).

Skin, being at the interface between the environment and the body, is often exposed to

pro-oxidant agents resulting in the generation of reactive oxygen species (Svobodova et

al., 2006). ROS are also generated in the skin as a part of self defense mechanism against

invading pathogens. Endogenously, ROS are produced in keratinocytes by aerobic

respiration, enzymatic oxidation, xenobiotics and by infiltrating leukocytes (Darr and

Fridovich, 1994; Fuchs and Milbradt, 1994). However, excessive production of ROS is

harmful for the tissue. The antioxidant network in skin prevents the production of ROS

and reduces damage to the tissue. Superoxide dismutase converts reactive radicals to

hydrogen peroxide and molecular oxygen. Catalase, an enzyme of the antioxidant

7 network, breaks down the hydrogen peroxide into water and oxygen. Another antioxidant enzyme is glutathione peroxidase (coupled with glutathione), which removes harmful hydrogen peroxides from the tissue. Non-enzymatic systems such as alpha- tocopherol (vitamin E), ascorbate (vitamin C) and urate regulate the activity of ROS. In the cells (Winyard et al., 2000). ROS produces injurious effect when it overwhelms the antioxidant capacity of the skin (Briganti and Picardo, 2003) and plays a significant role in the inflammatory process.

In the inflammatory cascade, ROS and cytokines are linked by Nuclear Factor-κB

(NF-κB) complex. The activation of NF-κB by ROS is one of the early events in the inflammatory reaction (Portugal et al., 2007). Removal of the inhibitory protein IκB allows the translocation of NF-κB resulting in the activation of target genes (Allen and

Tresini, 2000). The genes for TNFα, IL-1, IL-6, IL-8 and inducible nitrous oxide synthase (iNOS) are transcriptional targets for NF-κB. TNFα and IL-1 act as activators of NF-κB resulting in the positive feedback cytokine loop (Fuchs et al., 2001).

Significance and Hypotheses

IL-1α is an important inflammatory signaling protein which is preformed and stored in keratinocytes (Dinarello, 1997). Gallucci et al., (2004) has shown that chronic exposure of JP-8 induced elevated level of IL-1β in the skin. Twenty four hour after exposure to JP-8, cultured human keratinocytes upregulated IL-1α; however, the cytokine was below detectable level in the skin of the rats (Chatterjee et al., 2006).

McDougal et al., (2007) has shown that 1h exposure of skin to JP-8 induces the mRNA expression of signaling proteins associated with IL-1α pathway. The role of IL-1α in

vivo, at the early time points after JP-8 exposure is yet to be documented. In this study,

we have investigated the changes in the level of IL-1α at the early time points after 1 h post exposure of JP-8. The gene expression studies (McDougal et al., 2007) and

keratinocytes cell culture studies (Chatterjee et al., 2006) suggest that the level of IL-1α

may increase after brief exposure to JP-8. We hypothesized that the IL-1 level

increases in rat skin after 1 h exposure of JP-8. Our Specific aim 1 was to measure

the IL-1α and IL-1β in control and JP-8 exposed skin at 0, 1, 2, 3, 4, and 8 post exposure time point.

JP-8 induced inflammation is associated with neutrophil infiltration (Gallucci et

al., 2004). CXCL2 induces neutrophil infiltration (Watanabe et al., 1991). After 1, 4 and

8h exposure to JP-8, levels of CXCL2 mRNA increased 54.9, 26.2, and 16.8 fold

respectively (McDougal et al., 2007). The expression of CXCL2 and neutrophil

infiltration indicates the advanced stage of inflammatory process. In UV induced ICD,

neutrophil infiltration occurred before 8 h in the dermis; within 24 h neutrophils

infiltrated the epidermis (Fisher et al., 2001). To identify the early indications of the

inflammatory process we monitored the time points at which CXCL2 was expressed and

neutrophil infiltration occurred. Although there was significant up-regulation of the

CXCL2 gene immediately after JP-8 exposure (McDougal et al., 2007), the protein expression of the signaling molecule and its impact on neutrophil infiltration at the early time points after JP-8 exposure were yet to be documented. CXCL2 protein expression may be increased after the 1 h exposure of JP-8, followed by neutrophil infiltration. We hypothesized that protein levels of CXCL2 will increase followed by neutrophil

infiltration after 1 h exposure of JP-8 and tested it in the following aims 2 and 3.

Specific aim 2 was to measure CXCL2 in control and JP-8 exposed skin at 0, 1, 2, 3,

4, and 8 post exposure time points, while Specific aim 3 was to identify the time

point at which neutrophils infiltrate into the skin.

One-hour exposure of JP-8 to rat skin was shown to result in changing of the transcripts of proteins involved in ERK/MAPK, IL-6, PDGF, P38, MAPK, IGF-1, EGF, IL-2,

JAK/stat, and Neurotrophin/Trk pathways (McDougal et al., 2007). A one-minute JP-8 exposure has been shown to induce up-regulation of genes involved in basal transcription and translations, and down-regulation of genes related to DNA repair, metabolism, and keratin in cultured human epidermal keratinocytes (Chou et al., 2006). The changed transcripts are monocyte chemotactic protein-1 (ccl2), cyclin-dependent kinase inhibitor

1A (CDKN1A), DNA-damage-inducible transcript 3 (DDIT3), Dual specificity

phosphatase 1 (DUSP1), DUSP6, v-fos, FBJ murine osteosarcoma viral oncogene homolog (FOS), heat shock 27kDa protein 1 (HSPB1), jun oncogene (JUN), v-myc myelocytomatosis viral oncogene homolog (MYC), Phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2), Suppressorof Cytokine Signaling 3 (SOCS3), tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein - gamma polypeptide

(YWHAG) (McDougal et al., 2007). If the gene expression related to inflammatory process, is triggered by IL-1α, that has been preformed and stored in epidermis, then blocking the receptor of IL-1α should change the gene expression pattern. We

hypothesized that blocking of IL-1 receptor will inhibit the JP-8 induced gene expression pattern. The changes in the levels of the cytokines and the gene expression pattern induced by the cytokine will help us to understand the molecular mechanism of

JP-8 induced skin inflammation. Understanding the molecular mechanism of the inflammatory process is an important step to develop therapeutic and preventive measures of JP-8 induced skin inflammation.

Materials and Methods

Animals: Male Fischer rats (CDF/CrlBR, Charles River Laboratories, 250-350 g)

was housed one per cage with 12 h day and night cycle. The rats were acclimatized for 3

days before the study. Food and water was provided ad libitum. The number of animals

was selected based on the recommendation from Statistical Consulting Center at Wright

State University.

Exposure study: A Hill Top ChamberTM (Lomir Biomedical, Inc., Quebec

Canada) containing five hundred microliters of JP-8 or an empty chamber (sham) was placed on the skin. The chamber was held by duct tape as shown in Figure 3.

Figure 3: Exposure setup. The length of the ruler in the picture is 15 cm.

After the exposure period, the animals were humanely killed using CO2 at time points

shown in Figure 4. The exposed region was marked with a permanent marker.

0 h 1 h 2 h 3 h 4 h 8 h 12 h 18 h 24 h

JP-8 Exposure (striped region)

Figure 4: Timeline for exposure study

Dissection of skin: After removing the Hill Top Chamber, we removed the

residual JP-8 using cotton gauze. For the control, we wiped the skin under the chamber

using cotton gauze. We marked the area under the chamber using a permanent marker.

The exposed skin was surgically removed after humanely killing the animals using CO2.

The underlying fascia and fat of the skin were removed using a single-edged razor blade

(Industrial Razor Blades – single edged No. 9, VWR Scientific, Media, PA). An eight- millimeter biopsy punch (using Uni-Punch, Plymouth Meeting, PA) was taken from the injected region of the skin. The biopsy punches were flash frozen in liquid nitrogen.

Total protein isolation and estimation: One hundred mg of frozen tissue was pulverized in Bessman tissue pulverizer (Fisher Scientific, Pittsburgh, PA) pre-cooled with liquid nitrogen. The tissue was homogenized using Tissue Tearor (Model 985-370,

Biospec Products) in a round-bottom 5 mL tube containing 1 mL of PBS with 10 uL protease inhibitors (Sigma #P8340, St. Louis, MO). During homogenization, the sample the tube was kept in a small beaker full of ice to reduce heat build up. The homogenate was transferred to 1.5 ml microcentrifuge tubes and spun at 13,000 g for 15 minutes at 4

ºC. The supernatant was used for total protein estimation and ELISA. Protein estimation 13

was done by Bradford method using Protein Assay Kit from Bio-Rad Laboratories,

Hercules, CA. We used SpectraMax Plus 384 (Molecular Devises, Sunnyvale, CA) to

read the colorimetric changes at 595 µm in the assay.

ELISA: Sandwich ELISA was used to quantitate IL-1α, IL-1β, and CXCL2

protein levels. The antibodies and standards were purchased from R & D systems,

Minneapolis, MN. Table 2 provides the information on the proteins used for the assay.

Table 1: Primary and detection antibodies Cytokine Capture Detection Standard antibody antibody 1 IL-1α MAB 500 BAF 500 500 RL 2 IL-1β AF501NA BAF 501 501 RL 3 CXCL2 MAB 525 BAF 525 525 RL

The lowest dilution of antibody pair from the optimization table (Table 6 to 8

given in the appendix) was selected and tested for consistency of the standard curve. The

process was repeated for each lot of the antibody. The optimized amount of capturing antibody, diluted in 1x PBS, 100 μL per well, was incubated at room temperature

overnight. Each well was washed with wash buffer (1x PBS with 0.5% Tween 20) three

times using a plate washer (ELx50, Bio-teck, Winooski, VT). Antibody coated wells were blocked using 1% BSA. Blocking was carried out at room temperature for an hour.

We washed the wells three times with wash buffer after blocking. Standards or sample in

an appropriate dilution were added to each well (100 µL) and incubated for 2 hours at

room temperature. After washing the wells three times in wash buffer, detection

antibody, in the recommended dilution, was added to each well (100 µL) and incubated

for 2 hours in room temperature. The wells were then washed three times with wash buffer and the substrate (DY998, R&D systems, Minneapolis, MN) was added to each

well (100 μL). After 30 minutes incubation with the substrate, 50 µL of stop solution

(DY994, R&D systems, Minneapolis) was added to each well and the plates were gently

tapped to ensure through mixing. The chromogenic reaction in the wells was read using a

SpectraMax Plus 384 (Molecular Devises Sunnyvale, CA) at 450 nm.

Histology: The tissue was fixed in 4% formaldehyde for 4 hours. The fixed tissue

was dehydrated in ethanol and embedded in paraffin by series of steps given in the Table

Table 2: Dehydration and embedding steps Steps Tissue transferred to: Time 1 50% Ethanol 1 hour 2 70% Ethanol 1 hour 3 95% Ethanol 1 hour 4 100% Ethanol 45 minutes 5 100% Ethanol 45 minutes 6 100% Ethanol 45 minutes 7 Xylene 1 45 minutes 8 Xylene 2 45 minutes 9 Xylene and paraffin 45 minutes 10 Paraffin 1 45 minutes 11 Paraffin 2 45 minutes

Dehydrated tissues were embedded in paraffin blocks and cooled overnight at room temperature. After cooling, the blocks were kept in the refrigerator overnight, trimmed, and sectioned 7 µm thick. The sections were transferred to a warm water bath containing gelatin to remove the wrinkles. Gelatin aids the attachment of sections to the slides. The mounted sections were kept in oven, set at 40 °C overnight, to remove excess paraffin. The sections were then stained with hematoxylin and eosin and/ or Masson

Trichrome using the steps given in Table 3 a, b and c.

Table 3a: Staining steps common to hematoxylin and eosin, and Masson Trichrome (Humason, 1979) Steps Sections transferred to: Time 1 Xylene 2 minutes 2 Xylene 2 minutes 3 100% Ethanol 2 minutes 4 100% Ethanol 2 minutes 5 95% Ethanol 2 minutes 6 70% Ethanol 2 minutes 7 Rinse in tap water 2 minutes

Table 3b: Staining steps for hematoxylin and eosin Steps Sections transferred to: Time 8 Hematoxylin 30 seconds 9 Distilled water 3 minutes 10 50% Ethanol 2 minutes 11 70% Ethanol 2 minutes 12 Eosin 7 seconds 13 95% Ethanol 2 minutes 14 100% Ethanol 2 minutes 15 100% Ethanol 2 minutes 16 Xylene 2 minutes 17 Xylene 2 minutes 18 Mount in permount 2 minutes

Table 3c: Staining steps for Masson Trichrome Steps Sections transferred to: Time 8 Iron alum 2 minutes 9 Running water 5 minutes 10 Distilled water 2 minutes 11 Hematoxylin 30 seconds 12 Running tap water 2 minutes 13 Acid Fuschin 15 seconds 14 Distilled water 2 minutes 15 Ponceau de Xylidene 4 minutes 16 Tap water 1 minute 17 Phosphomolybdic acid 5minutes 18 Fast green 30 seconds 19 Acid water 1 minute 20 70% Ethanol 1 minute 21 95% Ethanol 1 minute 22 100% Ethanol 2 minutes 23 100% Ethanol 2 minutes 24 Xylene 2 minutes 25 Xylene 2 minutes 26 Mount in permount 2 minutes

Receptor antagonist: We used Kineret (Anakinra), from Amgen, Thousand

Oaks, CA, as IL-1α receptor antagonist.

Tempol: We used tempol (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl) from

Sigma (catalog #56516), St. Louis, MO, antioxidant in our related study.

Intradermal injection: The rats were anesthetized using Isoflurane (1-chloro

2,2,2-trifluoroethyl difluoromethyl ether) before intradermal injection. After carefully clipping the fur on the back of the rat, a needle (0.3 mm thick and 19 mm long) was inserted subcutaneously, about 1.5 centimeters away from the site of injection with the tip of the needle brought up towards the epidermis. We injected 100 µl of receptor antagonist or phosphate buffered saline. By this method of intradermal injection, we prevented puncturing the barrier layer (stratum corneum) and reduced the possibility of the JP-8 entering through a disrupted barrier.

Immunohistochemistry: One hour after the injection, we removed the skin and flash froze it in liquid nitrogen. The frozen tissues were sectioned at 20 µm and fixed in

4% paraformaldehyde. The paraformaldehyde fixed sections were permeabilized using

0.1% triton X-100 for 2 minutes. The tissue was blocked with 5% donkey serum for 1h, and 1% non-fat dry milk for half an hour. We incubated the tissue with the primary antibodies at room temperature overnight. The primary and secondary antibodies were optimized and appropriate working concentrations were selected. For optimization the antibodies we used semi-log dilution of the antibodies including the recommended concentration as the median value in the series. Primary antibodies were Spring

Bioscience #E2624 ((rabbit anti-human, , 0.67 µg/ml) for Keratin-14, which is specific for basal keratinocytes; Gene Tex #1.384.92.17.19 (mouse anti-human, 10 µg/ml), for IL-

1ra; and R&D Systems #AF771 (goat anti-mouse, 1 µg/ml) for IL-1R1. Antibodies raised

in donkey (Molecular Probes #A21207, #A31570, #A11055) labeled with Alexa Fluor

594 (red), 555 (orange) or 488 (green) for Keratin-14, IL-2ra, and IL-1R1 , respectively

were used as secondary antibodies. Vectashield mounting media with DAPI (H-1200)

was used and the slides were viewed with a Leica fluorescent microscope.

Image analysis: We used Image-proplus software (version 3.0 for windows,

Media cybernetics, Silver spring, MD) to measure the length of the epidermis. The

histological-photomicrographs of the skin were loaded to the software and using a tracing

tool the contour of the outermost nucleated layer of the epidermis was traced. The length

of the traced line was calculated using the software.

Tracing of epidermsis

1 cm

Figure 5. Image Analysis: Epidermis of 1 cm skin was measured using a tracing tool from the Image proplus software. The dark curved line in the image is the tracing line.

Gene expression study: We injected 100 µl of IL-1α receptor antagonist or the phosphate buffered saline in the rat skin. After one hour, we placed 500 µl of JP-8 on the

TM. injected area using Hill top chamber We removed the chambers after 1 h and humanely killed the animals using CO2, dissected the skin, removed the underlying

muscle fascia and flash froze the skin in liquid nitrogen (figure 5). Total RNA isolation was carried out used TRIreagent (Molecular Research Center, Cincinnati, OH). The isolated RNA was cleaned up using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The concentration and the quality of total

RNA was determined using the Experion RNA Standard Sensitivity chips in the Experion

Automated Electrophoresis System (Bio-Rad, Hercules, CA).

Removal of JP-8 Collection of skin IL-1α ra injection 1 h post exposure treatment

Striped region: exposure period (1h)

Figure 6: Injection, exposure and skin collection time points

To generate double-stranded cDNA we used a Superscript II cDNA synthesis kit

(Invitrogen, Carlsbad, CA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter binding site (Invitrogen). The double-stranded cDNA was used to prepare labeled cRNA. The labeling of cRNA was done in vitro transcription using a T7 polymerase (MEGAscript T7 kit; Ambion) in the presence of biotin-11-CTP and biotin-

16-UTP (Enzo Diagnostics, Farmingdale, NY) and purified using RNeasy columns

(Qiagen). The labeled (biotinylated) cRNA was fragmented and hybridized to rat 230 2.0

GeneChip arrays (Affymetrix, Santa Clara, CA). The gene chips contain probe sets for

about 31,000 genes. As per the recommended protocol we carried out chip hybridization,

washing, and staining. The chips were scanned and the digitalized image data was

processed using GCOS software from Affymetrix. The data were analyzed using

GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA).

Results

Interleukin-1 expression in response to JP-8 exposure

To test whether JP-8 induces the increase of IL-1 in skin, we measured IL-1α and

JP-8 induced interleukin 1 alpha expression after 3h of exposure 70.00

60.00

50.00

40.00

protein 30.00

20.00

10.00 pg of cytokine /ug of soluble soluble of /ug cytokine of pg 0.00 Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Control JP-8

Figure 7a: Experiment to determine the effect of JP-8 on IL-1α: IL-1α was reduced at 3h post exposure in JP-8 exposed skin.

JP-8 induced interleukin 1 alpha expression after 3h of exposure 20.00 18.00 16.00 14.00 12.00 10.00 protein 8.00 6.00 4.00 2.00 pg of cytokine /ug of soluble soluble of /ug cytokine of pg 0.00 Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Control JP-8

Figure 7b: Repetition of the experiment to determine the effect of JP-8 on IL-1α at 3h post exposure.

IL-1β at 0, 1, 2, 3, 4, and 8 h post exposure to JP-8. Six rats were used in each experiment. Each rat received control and JP-8 exposure. We expected to see increases in the level of IL-1 in rat skin after 1 h of exposure to JP-8. A consistent trend in the reduction of IL-1α was observed at 3h post exposure in JP-8 exposed skin (Figure 7a and

7b). IL-1α did not show a consistent change at other time points (graph not shown). IL-

1β did not change throughout the study (graph not shown).

Neutrophil infiltration study:

To test whether JP-8 induces the increase of, CXCL2 in skin we measured the

cytokine at 0, 1, 2, 3, 4, and 8 h post exposure period of JP-8.

JP-8 induced CXCL2 expression after 2h of JP-8 exposure 2.50

2.00

1.50

protein 1.00

0.50 pg of cytokine /ug of soluble soluble of /ug cytokine of pg 0.00 Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Control JP-8

Figure 8a: Experiment to determine the effect of JP-8 on CXCL2: CXCL2 was increased at 2h post exposure in JP-8 exposed skin.

JP-8 induced CXCL2 expression after 2h of exposure 0.45 0.40 0.35 0.30 0.25

protein 0.20 0.15 0.10 0.05 pg of cytokine /ug of soluble soluble of /ug cytokine of pg 0.00 Rat 1 Rat 2 Rat 3 Rat 4 Rat 5 Rat 6 Control JP-8

Figure 8b: Repetition of the experiment to determine the effect of JP-8 on CXCL2 at 2h post exposure.

Six rats were used in each experiment. Each rat received control and JP-8 exposure. A consistent trend in the increase of CXCL2 was observed at 2h post exposure in JP-8

exposed skin (Figure 8a and 8b). CXCL2 did not show a consistent change at other time

points (graph not shown).

Epidermis Epidermis

Dermis

Collagen fibers

Gaps

Figure 9a: 8h post exposure control Figure 9b: 8h post exposure JP-8 treated No increase in cell density is observed indicating lack of inflammatory infiltration of neutrophils. The gaps in dermis and different shades of the collagen fibers are inconsistently seen in the control and JP-8 exposed skin. Bar =40 µm

Epidermis Epidermis

Dermis Dermis

Figure 9c: 12h post exposure control Figure 9b: 12h post exposure JP-8 treated No increase in cell density is observed indicating lack of inflammatory infiltration of neutrophils. The gaps in dermis and different shades of the collagen fibers are inconsistently seen in the control and JP-8 exposed skin. Bar =40 µm

Epidermis Epidermis

Dermis

Figure 9e: 24h post exposure control Figure 9f: 24h post exposure JP-8 treated No increase in cell density is observed indicating lack of inflammatory infiltration of neutrophils. The gaps in dermis and different shades of the collagen fibers are inconsistently seen in the control and JP-8 exposed skin. Bar =40 µm

In the hematoxylin and eosin stained sections, the cell density between the control and

the JP-8 exposed skin was not significantly different when observed under light

microscope. Figure 9 shows representative photomicrographs from 8, 12, and 24 h post

exposure. After the exposure period, we observed mild erythema under the JP-8 patch.

In most of the cases, the erythema faded away within 1h. In 8, 12, and 24h post exposure time points we did not find inflammatory neutrophil infiltration.

Intradermal Injection

Intradermal injection induced changes in skin

Histological Changes

We studied the possibility of using intradermal injection to deliver biomolecules for pharmacological manipulation of skin. As the epidermis is thin, injection of biomolecules was made in the dermis. The intradermal injection resulted in the

appearance of a bleb similar to the artificially created blister (Figure 10).

Figure 10: Bleb produced by modified intradermal injection on the back of rat. Fur was clipped before the injection. The ruler is graduated in centimeters. A bleb resulting from 100 µL was approximately 1 cm in diameter (the distance between the arrows)

In the artificially created blisters, the epidermal and dermal layers are separated.

(Kennedy et al., 1999). The signaling sequence would be hindered if there is a separation of the epidermis from the dermis. To understand whether intradermal injection would result in the separation of epidermis and dermis, we studied the histological changes after intradermal injection.

1 1 2 2

Figure 11a Figure 11b

Figure 11a: Light Microscopy of control skin stained with Mason’s Trichrome. 1 - Epidermis, 2 – dermis Figure 11a: Light Microscopy of IL-1 RA injected skin stained with Mason’s Trichrome. 1 - Epidermis, 2 - Gaps in dermis resulted from (100 µL) injection Bar = 10 µm

An intradermal injection looks like a blister (Figure 11a). However, the intradermal injection is different from a blister (Figure 11b), because there is no separation of epidermis from dermis. The injected fluid created small pocket in the dermis by stretching the collagen rich layer. The pockets of the injected fluid are formed near the site of injection which appears as a bleb from outside. The contour of the epidermis in the injected skin (Figure 11b) is less wavy compared to control skin (Figure

11a). Dermis in the injected tissue has gaps surrounded by collagen fibers which have lost the network-like pattern observed in the control skin but the epidermis appears intact.

Morphological changes: Image analysis study of epidermis

To understand the morphological changes in the skin after intradermal injection we measured the length of the epidermis in control skin and injected skin.

Table 4: Length of epidermis in the injected skin and control skin Rat Anatomical Replication Length of epidermis in Length of epidermis number region injected skin (mm) in control skin (mm) 1 1.351 1.822 1 Upper back 2 1.738 1.975

1 1.558 1.685 2 Upper back 2 1.507 1.737

1 1.495 1.786 3 Upper back 2 1.565 1.661

1 1.317 1.724 4 Upper back 2 1.289 1.764

1 1.503 1.679 2 Lower back 2 1.525 1.788

1 1.304 1.592 3 Lower back 2 1.326 1.638

1 1.337 1.743 4 Lower back 2 1.452 1.658

Average 1.477 1.732

Variance 0.017 0.009

One millimeter section of the skin was observed under the microscope. Using the image analyzer software, we traced the topmost nucleated cell layer of the epidermis. The

traced line was measured and the length of the line was recorded using the software.

Measuring the length of the epidermis, in one unit length of the skin, in the injected skin

and control skin helps us to understand whether intradermal injection has induced

stretching of the epidermis. The injections were made in the upper back and lower back

of the animals. We observed that intradermal injection made at various regions of the

back of the rat induced stretching of the epidermis and did not induce separation of

epidermis and dermis.

Detection of injected Interleukin-1 receptor antagonist in skin

To test whether the injected antagonist could reach the epidermis we probed the

antagonist in the epidermis, after injection, using flurophore tagged antibody. Control

tissue (Figure 12 a) and IL-1ra injected tissue (Figure 12 b) were both treated with

primary and secondary antibody of IL-1ra and Keratin-14.

1 1

Figure 12a Figure 12b

Figure 12: Fluorescent Microscopy of control skin stained for Keratin-14 (red) in basal membrane and IL-1ra (orange). 1 - Basal membrane (a), and IL-1ra injected skin stained for Keratin-14 (red) and IL-1ra (orange). 1 - Basal membrane, 2 - IL-1ra in epidermis (b). Bar = 5 µm

Figure 13a Figure 13b Figure 13c

Figure 13: Fluorescent Microscopy of IL-1ra injected skin immunostained for IL-1ra (orange) (a), of IL-1ra injected skin immunostained for IL-1R1 (green) (b) and of IL-1ra injected skin showing co-localization of IL-1R1 (green) and IL-1ra(orange) (c) . Bar = 5 µm

Compared to the control tissue, the injected tissue shows immunostaining for IL-1ra

(orange) in the epidermis and dermis. Keratin-14, in the basal membrane, appeared in both the control and injected tissue. Figure 13 a shows the presence of IL-1ra (orange) in the injected skin. In Figure 13 b, we can see the IL-1R1 (green). Figure 13 c is the overlay of Figure 13A and Figure 13 B showing the co-localization of IL-1R1 and the injected IL-1ra.

Effect of blocking IL-1α pathway in JP-8 exposed skin

If preformed IL-1α plays a role in the initial stage then blocking the activity of the

cytokine should modify the early sequence of events. To investigate the effect of IL-1α

RA (Kineret) on gene expression to JP-8 exposure, we injected 100 µl of IL-1α RA or phosphate buffered saline in to the rat skin. We did not see changes in the ERK/MAPK,

IL-6, PDGG, P38, MAPK, IGF-1, EGF, IL-2, JAK/stat, and Neurotrophin/Trk pathways induced by IL-1ra after 1 h exposure of JP-8. A complete list of changed gene is provided in the appendix (Table 11). Out of 31,000 genes only 170 genes show at least

31 two fold difference. About 5 % of the genes may change by chance. Any change that is less than 5 % may not be considered as reliable change in the gene expression. We found that only 0.55 % genes have changed in our study. Injection of the IL-1α RA did not change significant number of genes in 1h exposure of JP-8 to rat skin.

Related studies:

Reactive oxygen species:

To understand the role of ROS in JP-8 induced inflammation, we delivered an antioxidant, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl) into the skin

before JP-8 exposure. For our study, we intradermally injected one hundred micro liters

of phosphate buffered saline (sham) or tempol, into the skin. Five hundred microliters JP-

8, in a hill top chamberTM or an empty chamber was placed on the injected region for 1 h.

After 2 h post exposure period the rats were humanly killed, using CO2, and the skin was

dissected. Interleukin 1 was measured as described earlier.

The results obtained 1) for IL-1 alpha for each rat is shown in figures 14 (A-F)

and the combined data for 6 rats, is shown in figure 15, and 2) for IL-1 beta, the raw data is shown for each rat in figures 16 (A-F) and the combined data for 6 rats, is shown in figure 17. Figures 14 and 16 shows the trend in the cytokine expression. The figures are as follows:

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 1

35 30 25 20 15 protein 10 5 pg of cytokine/ug of soluble of cytokine/ug of pg 0

1 Control 1 Tempol 5 mM 1 Tempol 25 mM 1 Tempol 50 mM

Figure 14 A. Replication 1: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 2

15 10 5

0 pg of cytokine/ug of soluble protein of cytokine/ug of pg 2 Control 2 Tempol 5 mM 2 Tempol 25 mM 2 Tempol 50 mM Figure 14 B. Replication 2: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 3

25 20 15 protein 10 5 0 pg of cytokine/ug of soluble of cytokine/ug of pg 3 Control 3 Tempol 5 mM 3 Tempol 25 mM 3 Tempol 50 mM Figure 14 C. Replication 3: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 4

25 20 15

0 pg ofpg cytokine/ug of soluble protein 4 Control 4 Tempol 5 mM 4 Tempol 25 mM 4 Tempol 50 mM Figure 14 D. Replication 4: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 5

30 25 20 15

protein 10 5 0 pg of cytokine/ug of soluble of soluble ofpg cytokine/ug 5 Control 5 Tempol 5 mM 5 Tempol 25 mM 5 Tempol 50 mM

Figure 14 E. Replication 5: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1 alpha expression in Tempol treatment after JP-8 exposure Rat 6

protein 10

0 soluble of cytokine/ug of pg 6 Control 6 Tempol 5 mM 6 Tempol 25 mM 6 Tempol 50 mM

Figure 14 F. Replication 6: Effect of tempol in JP-8 induced IL-1α changes in skin

IL-1alpha expression in Tempol treatment after JP-8 exposure

25 20

15 protein 10

soluble of cytokine/ug of pg 0 Control Tempol 5 mM Tempol 25 mM Tempol 50 mM

Figure 15: Graphical representation of combined data. Error bar represents SD of n=6

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 1

60.00

50.00 40.00

30.00 protein 20.00

10.00

soluble of cytokine/ug of pg 0.00 1 Control 1 Tempol 5 mM 1 Tempol 25 mM 1 Tempol 50 mM

Figure 16 A. Replication 1: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 2

70.00 60.00 50.00

40.00 30.00

20.00

10.00

0.00 pg of cytokine/ug of soluble protein of cytokine/ug of pg 2 Control 2 Tempol 5 mM 2 Tempol 25 mM 2 Tempol 50 mM Figure 16B. Replication 2: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 3

70.00 60.00 50.00 40.00 30.00 protein 20.00 10.00 0.00 soluble of cytokine/ug of pg 3 Control 3 Tempol 5 mM 3 Tempol 25 mM 3 Tempol 50 mM Figure 16C. Replication 3: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 4

60.00

50.00

40.00 30.00 protein 20.00 10.00

pg of cytokine/ug of soluble of cytokine/ug of pg 0.00

4 Control 4 Tempol 5 mM 4 Tempol 25 mM 4 Tempol 50 mM Figure 16 D. Replication 4: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 5

60.00 50.00 40.00

30.00 protein 20.00

10.00

soluble of cytokine/ug of pg 0.00 5 Control 5 Tempol 5 mM 5 Tempol 25 mM 5 Tempol 50 mM Figure 16 E. Replication 5: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure Rat 6

45.00 40.00 35.00 30.00 25.00 20.00 protein 15.00 10.00 5.00

pg of cytokine/ug of soluble of cytokine/ug of pg 0.00

6 Control 6 Tempol 5 mM 6 Tempol 25 mM 6 Tempol 50 mM Figure 16 F. Replication 6: Effect of tempol in JP-8 induced IL-1β changes in skin

IL-1 beta expression in Tempol treatment after JP-8 exposure

70 60 50 40 30 protein 20 10 pg of cytokine/ug of soluble of cytokine/ug of pg 0

Control Tempol 5 mM Tempol 25 mM Tempol 50 mM Figure 17: Graphical representation of combined data. There is no significant difference between the groups. Error bar represents ±SD of n= 6

We observed a consistent trend of higher level of IL-1α in 5 mM and 50 mM compared to control and tempol 25 mM treated skin. However there was no significant change of expression of IL-1α and IL-β in JP-8 exposed skin in the tempol treatment. Interestingly tempol, a well known ROS scavenger, did not produce a dose response. For future study, efficacy of tempol in our model has to be evaluated. We suggest the use of

Lipopolysaccharide (LPS) triggered ROS system to evaluate the effectiveness of tempol.

LPS is an inducer of ROS. Studying the inflammatory response of LPS in tempol pre- treated skin will aid us to understand the tempol’s ROS scavenging efficacy in our model.

In future, the study should be repeated with LPS as a positive control to better understand the current data.

Autocrine effect of IL-1α in JP-8 induced inflammatory process

To understand the role of preformed IL-1α we injected IL-1α receptor antagonist (IL-1α

RA) into the skin before JP-8 exposure. IL-1α RA was injected into the skin in an

attempt to block the activity of IL-1 Since we found decrease in IL-1α at 3 h post

exposure to JP-8, we selected the time point for this study.

We injected IL-1α RA and after 1h we did the exposure. The exposure period was 1 h. Three hours post exposure we humanely killed the animals, dissected the skin and estimated the cytokine levels as discussed earlier (Figure 18).

Removal of JP-8 Collection of skin IL-1α RA injection 3 h post exposure treatment

Striped region: exposure period (1h)

Figure 18: Injection, exposure and collection of skin

Table 5: IL-1α expression in pg/mg of protein Replication RA Placebo JP-8 Control Rat 1 3.497 4.130 4.520 5.538 Rat 2 3.883 4.428 6.998 5.222 Rat 3 4.837 5.013 6.419 8.388 Rat 4 3.675 5.872 4.619 6.783 Rat 5 3.782 4.991 4.034 4.688 Rat 6 3.162 4.566 4.957 3.619 Average 3.806 4.833 5.258 5.706 SD 0.565 0.611 1.176 1.674 CV 14.844 12.638 22.370 29.332

IL-1 alpha expression

8.000 7.000 6.000 5.000 4.000

protein 3.000 2.000 1.000 pg of cytokine / mg of cytokine of pg 0.000 Treatments

IL-1 RA and JP-8 Placebo and JP-8 JP-8 Empty patch

Figure 19: IL-1 alpha expression in response to JP-8 exposure after interleukin 1 receptor antagonist treatment. Error bar represents ±SD of n= 6

The preliminary result shown in figure 19 and table 6 shows that the receptor

blocking did not significantly alter the IL-1α in control and JP-8 exposed skin. The efficacy of the receptor blocker has to be studied before we can make a conclusion in this study.

Discussion

In our study, we have shown that the level of IL-1α decreases in rat skin after 1h occlusive exposure of JP (Figure 7). Chatterjee et al., (2006) have suggested that the level of IL-1α may increase after brief exposure of JP-8 to keratinocytes in vitro.

Epidermal layer, where IL-1α is preformed and stored is primarily composed of keratinocytes. We hypothesized that the IL-1 level increases in rat skin after 1 h exposure of JP-8. At the 3h post exposure IL-1α decreased (Figure 7a and 7b). IL-1α did not show a consistent change in other time points. IL-1β levels did not show a significant change at any time point. In normal murine skin, IL-1α was present in the dermis and nucleated epidermal layers in a diffused pattern (Wood et al., 1996). Kabbur et al., (2001) have reported increases in the level of IL-1α after 1h exposure of skin to JP-

8. Interestingly, our results indicate that preformed, stored IL-1α is released from the skin at 3 h in response to JP-8 exposure. IL-1α when bound to IL-1 type I receptors in keratinocytes, activates interleukin-1 receptor associated kinase-1 pathway inducing downstream events of the inflammatory cascade (Sanmiguel et al., 2009; Stylianou and

Saklatvala, 1998). IL-1 regulates gene expression and cellular processes through activation of the transcription factors NF-κB and activator protein 1 complex (Verstrepen et al., 2008). In response to mechanical insult to the epidermal layer, IL-1α level increased within 10 minutes and remained elevated at 2 and 4h time points. As IL-1α release induces further synthesis of the cytokine in keratinocytes (Lee et al., 1991), we expected to see increasing levels of IL-1 after JP-8 exposure. However, throughout the post-exposure study, IL-1α levels did not increase in the JP-8 exposed skin. To prevent

JP-8 contamination during protein isolation we removed the JP-8 residues on the skin by

wiping the skin with cotton gauze thoroughly. However JP-8 enters into the skin (Kim et

al., 2006)and skin remains in the tissue until removed by the system. Therefore, one of

the probable reasons for the reduced amount of IL-1α at the early post-exposure time points could be the presence of JP-8 in sample denaturing the cytokine or interfering with the estimation process of the cytokine. To check whether the residual quantity of JP-8 interferes with the assay, we may have to identify the quantity of JP-8 or its components

left at each time points and spike the assay with the estimated quantity of JP-8 or its

components. We found in literature that the detection of elevated levels of cytokines in

cultured human keratinocytes and in vivo study was not affected after JP-8 exposure

study. Cultured human keratinocytes after JP-8 exposure up-regulated IL-1α protein

level at 24 h post exposure period (Chatterjee et al., 2006). IL-1β protein level was

elevated in the 7-day chronic exposure study. The cytokine estimation was carried out

after 2 h following exposure to JP-8 (Gallucci et al., 2004). Based on the above two

studies it appears that measurement of IL-1 in the media or in the tissue is not impaired

by the presence of residual amount of JP-8.

We did not see changes in the levels of IL-1β in skin after JP-8 exposure.

Chronic exposure of JP-8 induced increased levels of IL-1β in skin (Gallucci et al.,

2004). Sanmiguel et al., (2009) have suggested that IL-1α may play a role in leukocyte

recruitment in skin following the release of the cytokine, and IL-1β could play a role in

the maintenance of inflammatory process. The exact difference in the role of IL-1α and

IL-1β in skin inflammation is yet to be established, although the suggestion of Sanmiguel

et al., (2009) may explain the increased expression of IL-1β in the chronic exposure study

(Gallucci et al., 2004) and lack of change in the level of IL-1β in our acute exposure

study.

Inflammation is characterized by the infiltration of neutrophils. CXCL2 is a

cytokine induced by the proinflammatory cytokines. CXCL2, also known as cytokine-

induced neutrophil chemoattractant-3, is the major neutrophil chemotactic factors in rats

(Murakami et al., 1997). CXCR2, the receptor of CXCL2, is expressed in normal

epidermis and the level of the cytokine increases during the inflammation (Zaja-

Milatovic and Richmond, 2008). In skin, the mRNA level of CXCL2 increased after 1h exposure of JP-8 (McDougal et al., 2007). In our study, we found an increase of CXCL2

at 2h post exposure time point (Figure 8). Compared to the quantity of IL-1 in rat skin,

the level of CXCL2 is very low. Although there is slight increase in CXCL2 at the 2h

time point we did not see significant neutrophil infiltration in JP-8 exposed skin.

Gallucci et al., (2004) have observed that repeated exposure of JP-8 for 7 days

induced neutrophil infiltration in rat skin. We did not find neutrophil infiltration in the

JP-8 exposed skin in 8, 12, 24 h post exposure. UV rays, an irritant, induce neutrophil

infiltration within 8h of skin exposure. The lack of neutrophil infiltration in the skin

suggests that single exposure of JP-8 may not induce infiltration of leukocytes or the

inflammatory infiltration of cells does not occur before 24 h after the exposure.

The reduction of IL-1α in JP-8 exposed skin indicates the release of preformed

and stored cytokine. If the released IL-1α is responsible for the trigger of the JP-8

induced inflammatory cascade, then blocking of the pathway should induce changes in

the downstream events. To block the IL-1 activity of IL-1α we delivered IL-1 receptor antagonist into the skin. The intradermal injection produced a bleb similar in appearance

to a blister created by suction method (Kennedy et al., 1999). In the artificially created

blister, the epidermis and the dermis are separated by a fluid filled space. To study the

events in the inflammatory process we have to maintain the integrity of the epidermis and

dermis. The cell signaling between the two skin layers is indispensable for the

inflammatory pathway. Therefore, if the intradermal injection results in the separation of

the epidermis and dermis then we may not be able to use the model to study JP-8 induced

inflammation. We studied the tissue sections of the skin under microscope after intradermal injection to understand the changes induced by intradermal injection.

Compared to control sections large gaps were found in the dermis (Figure 11) between

the collagen fibers in the injected skin. The collagen fibers in the papillary dermis region

appear to be stretched. The epidermis in the injected skin was stretched as a result of

bleb formation. We compared the length of the epidermis in the control skin and the

injected skin (Table 5). We measured the length of the epidermis in 1 mm section of

skin. In the control skin, the contour of epidermis is convoluted and measured longer

than the epidermis of the skin from bleb. The intradermal injection did not separate the

epidermis and dermis and found to be compatible for our study. However, the

intradermal injection can be considered successful only if the delivered molecules reache

the epidermis. In skin, IL-1 receptors are predominantly concentrated in the epidermis

(Figure 13b). Since the epidermis is thin, the injection has to be made in the dermis close

to epidermis. The tight junctions (Furuse et al., 2002) in the epidermis may hinder the

movement of the antagonist into the upper layers.

To study whether the injected antagonist could pass the basal layer of the

epidermis and bind to the receptor, we probed the antagonist after injection. We detected

the injected antagonist above the basal membrane of the epidermis (Figure 12b). We

tagged the basal membrane with keratine 14 antibody (Figure 12a). Keratin-14 is found

in the basal layer of epidermis. Therefore, detection of the antagonist in the layers above

the keratin-14 tagged region indicates that the antagonist could pass through the basal membrane of the epidermis. We probed the injected skin with the IL-1 receptor and IL-1

receptor antagonist antibody (IL-1ra) in the same sections. The antibodies for both the receptors and the receptor antagonist were in close proximity indicating higher probability of the binding of the receptor and the antagonist (Figure 12c).

To understand the role of IL-1 pathway in JP-8 induced gene expression pattern, we delivered IL-1ra into the skin before JP-8 exposure. If preformed IL-1α plays a role in the initial stage then blocking the activity of the cytokine should modify the early sequence of events. We did not see changes in genes expression pattern induced by 1h exposure of JP-8 (Table 11). The inflammatory genes that are up-regulated in our study are Wnt, Lef1, and orosmucoid1 In the skin, Wnt and Lef1 are essential for development of hair follicles (Reddy et al., 2001). Wnt 5a is up-regulated in the lesion of psoriatic

skin (Reischl et al., 2007). Lef1 is a DNA-binding protein which is regulated by beta-

catenin (cytoskeletal component and transcription factor), the downstream effector of

canonical Wnt signaling (Nusse, 1999). Beta-catenin is a gene expressed during the

inflammatory process. However, a change in beta-catenin gene was not observed in this

study. The detailed process of interaction of beta-catenin and Wnt signaling is yet to be

understood. IL-1 beta alters beta-catenin to increase vascular permeability during

inflammation (Barbieri and Weksler, 2007). Orosomucoid1 or Alpha-1-acid glycoprotein

(AGP) is a natural anti-inflammatory and immunomodulatory agent (Williams et al.,

1997). The antagonist used in the study is a drug developed for treatment of arthritis in humans. The efficacy of the antagonist in a rat model is yet to be clearly understood.

Conclusion

One-hour occlusive JP-8 exposure does not induce neutrophil infiltration by 24 h post exposure. The change in the level of CXCL2, although small compared to IL-1α levels, indicates that the cytokine could play a role at the early stage of the inflammatory cascade. We conclude that the one of the early events after the JP-8 exposure might be the release of the preformed and stored IL-1 α.

Related Studies

IL-1 is released from skin in response to irritants such as benzalkonium chloride and sensitizers such as oxazolone (Wilmer et al., 1994). The released cytokine could further induce is production of IL-1 through the autocrine loop. To understand the autocrine role of preformed IL-1α released in response to JP-8 exposure, we delivered the IL-1ra into the skin before JP-8 exposure. Phosphate buffered saline was injected as sham treatment. JP-8 was applied in a patch over the injected regions. An additional empty patch or patch containing JP-8 was placed on the non-injected region of the skin to study and compare the basal level of the cytokine without injection. As there is a decrease in the amount of cytokine at 3h post exposure time point we measured the level of IL-1α at 3h post exposure time point. There is no statistical significant difference between the four groups. Until the efficacy of the human IL-1ra is tested on rat system, we have at least two possible reasons for the outcome. One possibility is that the

delivered receptor antagonist may not have efficiently bound to the receptor and failed to

bring its effect. The second probability is that the change in the cytokine level might be

occurring at a different time point. Evaluating the efficacy of the antagonist and the

repeating the study is necessary to interpret the current data.

We also used tempol, an antioxidant to understand if any dose response reduction

effect on the expression of IL-1α. Tempol, can be delivered intradermally into the skin to

scavenge ROS. It has been has shown that in cultured fibroblasts, tempol reduced UV

induced ROS generation (Yan et al., 2005). We delivered tempol intradermally before

JP-8 exposure. The IL-1α and IL-1β levels (figure 15 and 17) in sham treatment and tempol treated skin is not significantly different after JP-8 exposure. However, there is a trend in the expression (figure 14 and 16) of the cytokines. Interestingly 5 mM and 50 mM of tempol injection induced the increased expression of the IL-1α and IL-1β compared to sham and 25 mM tempol injection. Lipopolysaccharide (LPS), a bacterial lipoglycan induces ROS synthesis. To confirm the results we will have to repeat the experiment with a positive control such as lipopolysaccharide, a bacterial lipoglycan that induces ROS synthesis.

Future direction

Changes in ions in response to JP-8 exposure

Preformed IL-1α, a known inflammation inducer in skin, was believed to play an

important role in ICD. In our study, we have shown that the level of IL-1α decreases on

exposure to JP-8. IL-1α has no leader peptide to aid secretion, and the mechanism of

release of IL-1α is not yet documented. JP-8 on entering the dermal layer may change the ionic balance as a result membrane disruption (Monteiro-Riviere et al., 2004). The change in the ionic balance associated with membrane disruption may in turn can induce the release of IL-1α. Studying the changes in ionic balance in skin may help us to understand the molecular changes that happen after JP-8 exposure(Denda et al., 2000).

Changes in pH, calcium, potassium, sodium and magnesium ions may be studied by blotting skin sections on to a gel containing chemical indicators. For future study, understanding the role of ionic change in the release of IL-1α will provide understanding of the early events in JP-8 induced inflammatory process.

Study of efficacy of IL-1 RA

Documenting the efficacy of the receptor antagonist may be an area of interest for future studies. The blocking effect of the selected antagonist has to be studied. Injection of IL-1α will trigger inflammation (Willet et al., 2002). Studying the inflammatory response induced by IL-1α in the IL-1 receptor antagonist treated tissue will be useful in understanding the blocking effect of the receptor antagonist.

Appendix

ELISA Optimization tables (7-9):

Table 6. ELISA Optimization table for IL-1 alpha: Monoclonal Capture (MAB 500)/ Polyclonal Detection (BAF 500)

100 ng/ml detection ab 150 ng/ml detection ab

1 2 3 4 5 6 7 8 9 10 11 12

0.50 0.50 1.0 1.0 1.50 1.50 0.50 0.50 1.0 1.0 1.50 1.50 μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero A standard standard standard standard standard standard standard standard standard standard standard standard 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL B standard standard standard standard standard standard standard standard standard standard standard standard 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL C standard standard standard standard standard standard standard standard standard standard standard standard 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL D standard standard standard standard standard standard standard standard standard standard standard standard Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero E standard standard standard standard standard standard standard standard standard standard standard standard 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL F standard standard standard standard standard standard standard standard standard standard standard standard 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL G standard standard standard standard standard standard standard standard standard standard standard standard 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL 1ng/mL H standard standard standard standard standard standard standard standard standard standard standard standard

200 ng/ml detection ab 250 ng/ml detection ab

Table 7. ELISA Optimization table for IL-1 beta: Polyclonal Capture (AF-501-NA)/ Polyclonal Detection (BAF 501)

50 ng/ml detection ab 100 ng/ml detection ab

1 2 3 4 5 6 7 8 9 10 11 12

0.75 0.75 1.0 1.0 1.25 1.25 0.75 0.75 1.0 1.0 1.25 1.25 μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero A standard standard standard standard standard standard standard standard standard standard standard standard 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL B standard standard standard standard standard standard standard standard standard standard standard standard 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL C standard standard standard standard standard standard standard standard standard standard standard standard 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL D standard standard standard standard standard standard standard standard standard standard standard standard Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero E standard standard standard standard standard standard standard standard standard standard standard standard 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL F standard standard standard standard standard standard standard standard standard standard standard standard 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL G standard standard standard standard standard standard standard standard standard standard standard standard 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL 8ng/mL H standard standard standard standard standard standard standard standard standard standard standard standard

200 ng/ml detection ab 400 ng/ml detection ab

Table 8. ELISA Optimization table for CXCL2: Monoclonal Capture (MAB 525)/ Polyclonal Detection (BAF 525)

100 ng/ml detection ab 200 ng/ml detection ab

1 2 3 4 5 6 7 8 9 10 11 12

2μg/mL 2μg/mL 4μg/mL 4μg/mL 6μg/mL 6μg/mL 2μg/mL 2μg/mL 4μg/mL 4μg/mL 6μg/mL 6μg/mL Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Capture Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero A standard standard standard standard standard standard standard standard standard standard standard standard 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL B standard standard standard standard standard standard standard standard standard standard standard standard 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL C standard standard standard standard standard standard standard standard standard standard standard standard 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL D standard standard standard standard standard standard standard standard standard standard standard standard

Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero Zero E standard standard standard standard standard standard standard standard standard standard standard standard 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL 1 ng/mL F standard standard standard standard standard standard standard standard standard standard standard standard 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL 2 ng/mL G standard standard standard standard standard standard standard standard standard standard standard standard 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL 4 ng/mL H standard standard standard standard standard standard standard standard standard standard standard standard

300 ng/ml detection ab 400 ng/ml detection ab

Table 9: Summary of the quality control reports form Affymetrix Rat230_2.0 GeneChips Metric Sample N Mean SD Minimum Maximum Background PBS 3 65.73 0.90 63.07 67.70 Kineret 3 67.93 0.70 65.30 69.50 Noise (Raw Q) PBS 3 2.15 0.08 2.06 2.20 Kineret 3 2.17 0.05 2.11 2.22 % Present PBS 3 65.56 0.70 64.70 66.20 Kineret 3 65.83 0.50 65.30 66.40 % Absent PBS 3 31.77 0.83 31.10 32.70 Kineret 3 31.50 0.45 31.00 31.90 % marginal PBS 3 2.67 0.05 2.60 2.70 Kineret 3 2.63 0.15 2.50 2.80 3'/5' Ratio PBS 3 1.48 0.42 1.06 1.90 GAPDH Kineret 3 1.14 0.02 1.12 1.17

Table 10: List of transcripts changed in gene expression study: Gene name Fold change Description 1 LOC502543 3.675 similar to SPRR1b 2 Lef1 3.671 lymphoid enhancer binding factor 1 transmembrane 4 superfamily member 3 Tm4sf4 3.658 4 4 Tdh_predicted 3.6 L-threonine dehydrogenase (predicted) v-myc myelocytomatosis viral related oncogene, neuroblastoma derived 5 Mycn 3.406 (avian) similar to retinoic acid-responsive 6 RGD1307551_predicted 3.094 protein; STRA6 (predicted) similar to retinoic acid-responsive 7 RGD1307551_predicted 3.033 protein; STRA6 (predicted) 8 2.977 Transcribed locus

Gene name Fold change Description 9 LOC499291 2.918 LOC499291 10 2.896 Transcribed locus DnaJ (Hsp40) homolog, subfamily C, 11 Dnajc6_predicted 2.815 member 6 (predicted) 12 2.798 Transcribed locus 13 Lef1 2.725 Lymphoid enhancer binding factor 1 Wingless-type MMTV integration site 14 Wnt5a 2.724 5A similar to Myb proto-oncogene protein 15 LOC498982 2.66 (C-myb) similar to hypothetical protein 16 RGD1308116_predicted 2.657 MGC42105 (predicted) 17 2.493 Transcribed locus 18 Selenbp1 2.429 selenium binding protein 2 19 LOC499663 2.391 LOC499663 20 Dlx1_predicted 2.39 distal-less homeobox 1 (predicted) 21 2.378 22 2.282 similar to retinoic acid-responsive 23 RGD1307551_predicted 2.232 protein; STRA6 (predicted) 24 Tagln3 2.222 transgelin 3 25 2.213 26 2.212 Desmocollin 2 (predicted) lecithin-retinol acyltransferase ; hypothetical gene supported by 27 Lrat ; LOC497758 2.204 NM_022280 Similar to KIAA0802 protein 28 2.08 (predicted) 29 Cdh22 2.055 cadherin 22

Gene name Fold change Description Msh homeo box homolog 2 30 Msx2 2.051 (Drosophila) msh homeo box homolog 2 31 Msx2 2.049 (Drosophila) 32 Orm1 2.044 orosomucoid 1 similar to 4921507I02Rik protein 33 RGD1304723_predicted 2.036 (predicted) 34 Ptch 2.019 Patched homolog 1 (Drosophila) 35 LOC498911 2.005 LOC498911 36 Mg29 0.498 Mitsugumin 29 (predicted) 37 Fhl1 0.495 four and a half LIM domains 1 similar to ankyrin repeat and SOCs 38 LOC361187 0.495 box-containing protein 5 39 Hmox1 0.495 heme oxygenase (decycling) 1 40 0.484 Nebulin (predicted) 41 Ca3 0.482 carbonic anhydrase 3 42 LOC499702 0.479 similar to Synaptopodin-2 (Myopodin) Bol, boule-like (Drosophila) 43 0.479 (predicted) 44 Pfkm 0.474 phosphofructokinase, muscle 45 RGD:620885 0.471 fast myosin alkali light chain 46 Pdlim7 0.466 PDZ and LIM domain 7 Similar to 106 kDa O-GlcNAc transferase-interacting protein 47 0.465 (predicted) heat shock 27kD protein family, 48 Hspb3 0.465 member 3 potassium inwardly rectifying channel, 49 RGD:69247 0.462 subfamily J, member 11

Gene name Fold change Description solute carrier family 2 (facilitated 50 Slc2a4 0.46 glucose transporter), member 4 51 0.45 52 Pfkm 0.448 phosphofructokinase, muscle Similar to myocytic induction/differentiation originator 53 0.447 (predicted) 54 0.446 Transcribed locus SET and MYND domain containing 1 55 Smyd1_predicted 0.442 (predicted) 56 0.429 Transcribed locus 57 Myl2 0.426 myosin, light polypeptide 2 dihydrolipoamide branched chain 58 Dbt 0.425 transacylase E2 similar to ankyrin repeat domain- 59 LOC503446 0.424 containing SOCS box protein 12 60 0.421 61 Mybpc1 0.419 myosin binding protein C, slow type obscurin, cytoskeletal calmodulin and 62 Obscn 0.414 titin-interacting RhoGEF 63 Tpm1 0.408 Tropomyosin 1, alpha similar to RIKEN cDNA 1110001E17 64 RGD1307901_predicted 0.406 (predicted) 65 Tnni1 0.402 troponin I, skeletal, slow 1 66 Art1_predicted 0.395 ADP-ribosyltransferase 1 (predicted) nebulin-related anchoring protein 67 Nrap_predicted 0.395 (predicted) 68 0.39 Transcribed locus 69 RGD1307979_predicted 0.388 similar to muscle-derived protein

Gene name Fold change Description MDP77 variant 1 (predicted) 70 Ckm 0.387 creatine kinase, muscle 71 LOC498066 0.386 similar to putative SH3BGR protein 72 Tpm1 0.385 tropomyosin 1, alpha 73 RGD:1303245 0.384 reticulon 2 (Z-band associated protein) 74 0.376 Transcribed locus 75 Tnnc2_predicted 0.373 troponin C2, fast (predicted) 76 Pygm 0.373 muscle glycogen phosphorylase 77 Myl3 0.372 myosin, light polypeptide 3 78 0.368 Transcribed locus similar to ankyrin repeat and SOCs 79 LOC361187 0.368 box-containing protein 5 80 Myh4 0.365 myosin, heavy polypeptide 4 similar to muscle-derived protein 81 RGD1307979_predicted 0.364 MDP77 variant 1 (predicted) 82 RGD1305774_predicted 0.363 hypothetical LOC287353 (predicted) heat shock protein, alpha-crystallin- 83 Hspb6 0.363 related, B6 Cardiomyopathy associated 1 84 0.361 (predicted) 85 0.356 Transcribed locus myxovirus (influenza virus) resistance 86 Mx1 0.354 1 87 0.354 Similar to mKIAA0613 protein 88 Tnni1 0.349 troponin I, skeletal, slow 1 DNA-damage-inducible transcript 4- 89 Ddit4l 0.349 like 90 Aqp4 0.348 Aquaporin 4 91 RGD1311701_predicted 0.346 similar to RIKEN cDNA 1110030K22

Gene name Fold change Description (predicted) 92 Capn3 0.339 calpain 3 93 Trdn 0.338 triadin 94 Tmod4_predicted 0.335 tropomodulin 4 (predicted) 95 Tnni2 0.334 troponin 1, type 2 myosin, heavy polypeptide 7, cardiac 96 Myh7 0.33 muscle, beta 97 0.325 Sarcalumenin (predicted) 98 0.324 Transcribed locus 99 Actn3 0.319 actinin alpha 3 100 Csrp3 0.318 cysteine-rich protein 3 101 Hrc 0.316 histidine rich calcium binding protein 102 0.315 ATPase, Ca++ transporting, cardiac 103 Atp2a1 0.314 muscle, fast twitch 1 104 0.313 Similar to OTTMUSP00000000621 105 0.308 Transcribed locus Aspartate-beta-hydroxylase 106 0.308 (predicted) protein phosphatase 1, regulatory 107 Ppp1r1a 0.307 (inhibitor) subunit 1A phosphodiesterase 4D interacting 108 Pde4dip 0.306 protein (myomegalin) bol, boule-like (Drosophila) 109 Boll_predicted 0.306 (predicted) striated muscle activator of Rho- 110 Stars 0.305 dependent signaling 111 Tnnt3 0.304 troponin T3, skeletal, fast 112 RGD1306989_predicted 0.304 similar to DKFZP566O084 protein

Gene name Fold change Description (predicted) 113 Myh1 0.301 type 2X myosin heavy chain 114 Lao1_predicted 0.301 L-amino acid oxidase 1 (predicted) Transcribed locus, strongly similar to NP_009035.3 ribosomal protein L10a; 60S ribosomal protein L10a [Homo 115 0.3 sapiens] 116 Myom2 0.3 Myomesin 2 117 LOC498440 0.294 similar to myozenin 1 118 0.293 Transcribed locus 119 Eno3 0.293 enolase 3, beta similar to Leiomodin 1 (Leiomodin, muscle form) (64 kDa autoantigen D1) (64 kDa autoantigen 1D) (64 kDa autoantigen 1D3) (Thyroid-associated ophthalmopathy autoantigen) (Smooth 120 LOC296935 0.292 muscle leiomodin) (SM-Lmod) sine oculis homeobox homolog 1 121 Six1 0.291 (Drosophila) 122 0.286 123 RGD1306911_predicted 0.285 similar to CG10671-like (predicted) 124 Trim63 0.284 tripartite motif-containing 63 125 Pgam2 0.283 phosphoglycerate mutase 2 Transcribed locus, strongly similar to NP_077248.1 popeye domain 126 0.283 containing 3 [Mus musculus] 127 Pygm 0.277 muscle glycogen phosphorylase similar to RIKEN cDNA 1110011D13 128 RGD1311911_predicted 0.276 (predicted)

Gene name Fold change Description 129 Ttn 0.275 titin 130 Myom1 0.275 myomesin 1 (skelemin) 185kDa 131 0.273 Transcribed locus 132 Pygm 0.27 muscle glycogen phosphorylase 133 Enh 0.269 Enigma homolog 134 0.268 Transcribed locus 135 0.265 Transcribed locus adenosine monophosphate deaminase 136 Ampd1 0.263 1 (isoform M) Troponin C, cardiac/slow skeletal 137 0.261 (predicted) titin immunoglobulin domain protein 138 Ttid_predicted 0.258 (myotilin) (predicted) 139 Pvalb 0.258 parvalbumin similar to tropomyosin 1, embryonic 140 MGC109519 0.254 fibroblast - rat 141 0.245 Myopalladin (predicted) myosin light chain kinase 2, skeletal 142 Mylk2 0.244 muscle calcium channel, voltage-dependent, 143 Cacng1 0.241 gamma subunit 1 myosin binding protein C, fast-type 144 Mybpc2_predicted 0.241 (predicted) Creatine kinase, mitochondrial 2, 145 Ckmt2 0.241 sarcomeric 146 Mb 0.241 myoglobin 147 Rpl3l_predicted 0.24 ribosomal protein L3-like (predicted) 148 Smpx 0.239 small muscle protein, X-linked 149 Rgmc_predicted 0.238 RGM domain family, member C

Gene name Fold change Description (predicted) 150 0.237 myosin, heavy polypeptide 4, skeletal 151 Myh4_predicted 0.234 muscle (predicted) myosin heavy chain, polypeptide 6 ; myosin, heavy polypeptide 7, cardiac 152 Myh6 ; Myh7 0.233 muscle, beta 153 Ryr1 0.228 ryanodine receptor 1, skeletal muscle 154 Myf6 0.225 myogenic factor 6 kelch repeat and BTB (POZ) domain 155 Kbtbd10 0.225 containing 10 156 Rnf30_predicted 0.223 ring finger protein 30 (predicted) apolipoprotein B editing complex 2 157 Apobec2_predicted 0.218 (predicted) 158 0.217 Bol, boule-like (Drosophila) 159 0.215 (predicted) 160 Trdn 0.208 Triadin 161 Actn2_predicted 0.206 actinin alpha 2 (predicted) cytochrome c oxidase, subunit VIa, 162 Cox6a2 0.203 polypeptide 2 myosin regulatory light chain 2, 163 Mlc2 0.199 ventricular/cardiac muscle isoform Cytochrom c oxidase subunit VIII-H 164 Cox8h 0.186 (heart/muscle) 165 Trdn 0.185 triadin 166 Ttn 0.176 titin 167 Myoz2_predicted 0.176 myozenin 2 (predicted) 168 Tnnt1 0.168 troponin T1, skeletal, slow

Gene name Fold change Description 169 0.158 Transcribed locus myosin, heavy polypeptide 7, cardiac 170 Myh7 muscle, beta

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