Identification of Dermacentor Andersoni Saliva Proteins That

IDENTIFICATION OF DERMACENTOR ANDERSONI SALIVA PROTEINS THAT

MODULATE MAMMALIAN PHAGOCYTE FUNCTION

LWIINDI MUDENDA

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY College of Veterinary Medicine

MAY 2015

To the faculty of Washington State University:

The members of the committee appointed to examine the

dissertation of LWIINDI MUDENDA find it satisfactory and recommend that

it be accepted.

______Wendy Brown, MPH, PhD., Chair

______Kelly Brayton, PhD.

______Jeb Owen, PhD.

______Guy Palmer, DVM, PhD, Dr.med.vet (honoris causa)

______Glen Scoles, PhD.

ACKNOWLEDGEMENTS

I am grateful to my major faculty advisor Dr Wendy C. Brown and my PhD committee members, Dr Kelly A. Brayton, Dr. Jeb P. Owen, Dr Guy H Palmer, and Dr Glen A. Scoles for their guidance from proposal development and experimental design to execution of my PhD research.

Their invaluable comments and suggestions have greatly influenced my approach to resolving scientific questions and they have helped me to acquire the research skills that I will need to face future challenges.

I am thankful to past and present members of the Brown, Brayton, and Scoles labs for their contribution to my laboratory skills development and training. I wish to thank other members of the

Veterinary Microbiology and Pathology department for their willingness to answer technical questions and offer solutions wherever possible.

I am immensely grateful to the Fulbright Program and the Paul G. Allen School for Global

Animal for funding my PhD program.

iii

IDENTIFICATION OF DERMACENTOR ANDERSONI SALIVA PROTEINS THAT

MODULATE MAMMALIAN PHAGOCYTE FUNCTION

Abstract

by Lwiindi Mudenda, BVM., MSc., Ph.D. Washington State University May 2015

Chair: Wendy C. Brown

Ticks are obligate blood sucking parasites which transmit a wide range of pathogens worldwide including protozoa, bacteria and viruses. Additionally, tick feeding alone may result in anemia, dermatosis and toxin-induced paralysis. Dermacentor andersoni is a species of tick found in the western United States that transmits pathogens of public health importance including Rickettsia rickettsii, Francisella tularensis, and Colorado Tick Fever Virus, as well as Anaplasma marginale, a rickettsial pathogen that causes economic losses in both the dairy and beef industries worldwide. D. andersoni ticks are obligate blood sucking parasites that require a blood meal through all stages of their lifecycle. During feeding, ticks secrete factors that modulate both innate and acquired immune responses in the host which enables them to feed for several days without detection. The pathogens transmitted by ticks exploit these immunomodulatory properties to facilitate invasion of and replication in the host. Molecular characterization of these immunomodulatory proteins secreted in tick saliva offers an opportunity to develop novel anti-tick vaccines as well as anti-inflammatory drug targets. To this end we performed deep sequence analysis on unfed ticks and ticks fed for 2 or 5 days. The pooled data generated a database of 21,797 consensus sequences. Salivary gland gene expression levels of unfed ticks were compared to 2- and 5-day fed ticks to identify genes upregulated early during tick feeding. Next we performed mass spectrometry on saliva from 2- and

5-day fed ticks and used the database to identify 677 proteins. We cross referenced the protein data with the transcriptome data to identify 157 proteins of interest for immunomodulation and blood feeding. Both proteins of unknown function and known immunomodulators were identified. We expressed four of these proteins and tested them for inhibition of macrophage activation and/or cytokine expression in vitro. The results showed diverse effects of the various test proteins on the inflammatory response of mouse macrophage cell lines. The proteins upregulated some cytokines while downregulating others. However, all the proteins upregulated the regulatory cytokine IL-10.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

DEDICATION ...... x

CHAPTER ONE ...... 1

LITERATURE REVIEW ...... 1

REFERENCES ...... 6

CHAPTER TWO ...... 10

ABSTRACT ...... 10

INTRODUCTION...... 11

MATERIALS AND METHODS ...... 12

RESULTS ...... 17

DISCUSSION ...... 21

REFERENCES ...... 26

TABLES ...... 34

FIGURES ...... 38

CHAPTER THREE ...... 42

ABSTRACT ...... 42

INTRODUCTION...... 42

MATERIALS AND METHODS ...... 44

RESULTS ...... 47

DISCUSSION ...... 49

REFERENCES ...... 53

TABLES ...... 57

FIGURES ...... 58

vii

LIST OF TABLES

CHAPTER TWO

Table 1. Genes and primers for qPCR...... 34

Table 2. Pump methods for the 1st and 2nd dimension separations...... 35

Table 3. Differential gene transcription...... 36

Table 4. D. andersoni saliva proteins homologous to previously reported immunomodulators ...... 37

CHAPTER THREE

Table 1 Primers used for protein expression ...... 57

viii

LIST OF FIGURES

CHAPTER TWO

Figure 1: Venn diagram representing the number of transcripts observed at different feeding time

points...... 38

Figure 2: Cluster analysis of transcript data...... 39

Figure 3: Bootstrap median gene expression ratios...... 41

CHAPTER THREE

Figure 1: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and Iris...... 58

Figure 2: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and DaSG6931...... 59

Figure 3: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and Insect cell culture supernatant...... 60

Figure 4: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and Salivary gland extract...... 61

Figure 5: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and DaSG3273...... 62

Figure 6: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and DaSG9580...... 63

Figure 7: Cytokine expression profile of cells that were treated with LPS only compared with cells

that were treated with LPS and DaSG4532...... 64

DEDICATION

This dissertation is dedicated to my late mother Doris H. Mudenda, my father Philemon Mudenda,

and to my siblings; Dean, Milambo, Moono and Katiba for their love and support.

CHAPTER ONE

LITERATURE REVIEW

Ticks are obligate, hematophagous parasites that have a worldwide distribution. They can feed on their hosts for days and even weeks without being detected by the host immune system

(Deruaz et al., 2008; Kramer et al., 2008). Their immunoevasion is achieved through a wide range of proteins that are secreted in their saliva (Ribeiro and Francischetti, 2003; Schwalie and Schultz,

2009). Previous studies have shown that tick salivary proteins are able to inhibit or modulate both innate and adaptive immune responses. As a result, the infectious organisms carried by ticks exploit these immunoevasion mechanisms to proliferate within the host and cause disease. The organisms transmitted by ticks cause infection in both humans and animals. Tickborne diseases affecting humans include Rocky Mountain spotted fever, Colorado tick fever, Lyme disease, babesiosis, monocytic and granulocytic erlichiosis, tickborne encephalitis, etc. Tickborne diseases are also responsible for huge economic losses in the livestock industry. The economically important diseases of livestock include East Coast fever, heartwater, louping ill, cattle fever, babesiosis and anaplasmosis, which destroy vital sources of protein (meat and milk), draft power, transport and fibre

(Sonenshine, 1991). In 1997, global losses due to tick-borne diseases of livestock were estimated to be between $13.9 and $18.7 billion (de Castro, 1997).

Dermacenter andersoni ticks transmit diseases of public health importance including Rocky

Mountain spotted fever, tularemia, Colorado tick fever (Boppana et al., 2009) and cause tick paralysis (Parola and Raoult, 2001). Additionally, D. andersoni ticks transmit Anaplasma marginale, a rickettsial pathogen that causes economic losses in both the dairy and beef industries worldwide.

The losses caused by anaplasmosis are a result of low weight gain, reduction in milk production, abortion, the cost of treatment, and mortality (Morley and Hugh-Jones, 1989; Kocan et al., 2003).

D. andersoni ticks belong to the family Ixodidae, and are three-host ticks that require a blood meal through all stages of their life cycle. The ticks attach to their host using their hypostome which is armed with rows of recurved teeth (Sonenshine, 1991). In order to feed, the ticks cut the host’s epidermis and insert their mouth parts into the wound site (Kramer et al., 2008; Reck et al., 2009). It is expected that such injuries to the dermis would trigger wound healing responses which consist of inflammation, coagulation, and migration of immune and reparative cells including fibroblasts, resulting in tick rejection (Ribeiro and Fransischetti, 2003; Steen et al., 2006; Kramer et al., 2008;

Reck et al., 2009). However, ticks are able to persist on their host (Schwalie and Schultz, 2009).

During feeding, blood ingestion alternates with expulsion of saliva into the host (Gregson, 1960; as reviewed by Sonenshine, 1991). Tick saliva contains hundreds of different proteins and other pharmacologically active molecules (Chmelar et al., 2008). The molecules in tick saliva confer antihemostatic, anti-inflammatory, and immunomodulatory properties (Guo et al., 2009). They suppress both the innate and adaptive immune responses. Innate immune mechanisms are the earliest barriers against invading organisms and comprise cellular and non-cellular components (Murphy,

2008; Woolard and Frelinger, 2008). Cells of the innate immune system include neutrophils, monocytes, macrophages, dendritic cells, basophils, eosinophils, mast cells, gamma delta T cells, and natural killer cells. Phagocytic cells (macrophages, neutrophils and dendritic cells) are the first to respond to infectious agents. They contribute to an effective immune response by engulfing and degrading microbes, as well as presenting antigen to T cells, thereby generating pro-inflammatory and adaptive immune responses. In addition to fighting infection, the innate immune system also plays an important role during cutaneous injury by promoting wound repair. Macrophages play a central role in all stages of wound healing and modulate the function of the different cell types

2 involved in this process by producing several cytokines and growth factors (Rodero and

Khosrotehrani, 2010; Delavary et al., 2011). Macrophages produce pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α, growth factors including TGF-β and PDGF that stimulate the proliferation of fibroblasts and keratinocytes (Rodero and Khosrotehrani, 2010; Delavary et al.,

2011), and chemokines (IL-8, MIP-1α, etc.) that attract inflammatory cells to the site of the wound.

The saliva components may be further classified as described in the review by Steen and others (2006). Firstly, there are enzymes such as apyrases which hydrolyse ADP and prevent ADP- induced coagulation (Valenzuela et al., 2002, Ribeiro and Francischetti, 2003). Secondly, there are enzyme inhibitors such as Ixolaris, a serine protease inhibitor which inhibits the tissue factor (TF) - blood coagulation factor VIIa complex, thereby inhibiting blood coagulation (Francischetti et al.,

2002). Serine protease inhibitors have anticoagulant and / or immunosuppressive activities (Prevot et al., 2009). Thirdly, some salivary proteins are homologous to and seem to mimic host proteins. An example is the peptide produced by the salivary glands of the tick Amblyomma americanum known as the macrophage migration inhibitory factor (Bowen et al., 2010).. The fourth category includes the immunoglobulin-binding proteins which have been reported to be present in the saliva of

Rhipicephalus appendiculatus and are believed to protect the ticks from ingested host immunoglobulins (Wang and Nuttall, 1999). A fifth class, namely, amine-binding lipocalins, is thought to inhibit the inflammatory and vasoactive amines produced by the host (Mans et al., 2008).

The sixth group is the receptor agonist or antagonists such as platelet fibrinogen receptor antagonist that inhibits platelet aggregation (Kazimírová and Štibrániová, 2013). Other classes include the cement components, cardiotoxic and neurotoxic factors, and cytokine expression modulators (Steen et al., 2006). Cytokine expression modulators reportedly bind leukocyte membrane receptors and modulate cytokine expression at the transcriptional level. An example of a cytokine expression modulator is Salp 15, an Ixodes scapularis saliva protein that binds to DC-SIGN on dendritic cells,

3 subsequently activating a Raf-1/MEK-dependent signaling cascade that results in downregulation of pro-inflammatory cytokines induced by TLR-2 and TLR-4 ligands, as well as B. burgdoferri (Hovius et al., 2008).

Studies with Ixodes ricinus ticks have shown that tick saliva impairs the phagocytic activity of macrophages and their production of NO and cytokines (Kyckova and Kopecky, 2006).

Furthermore, Ixodes scapularis saliva has been reported to cause reduced expression of β2 integrins,

− impaired adherence and reduced production of 02 by neutrophils, resulting in reduced killing of

Borrelia burgdoferi (Guo et al., 2009). Although some of the molecules in some tick species have been identified, the majority of proteins in tick saliva, and their full impact on the host-tick interaction remain unknown (Alarcon-Chaidez et al., 2003; Reck et al., 2009). Additionally, some of the proteins that have properties described above have only been identified in tick salivary gland extracts, but have not been confirmed to be present in tick saliva. Furthermore, some proteins have not yet been characterized; hence, their sequences do not appear in the databases (Steen et al., 2006).

The pharmacological properties of tick saliva proteins are many, but they cannot be exploited for therapeutic use until the proteins are well characterized. For this reason, we performed proteomics informed by transcriptomics to identify novel proteins found in the saliva of Dermacentor andersoni

(Mudenda et al., 2014).

The problem of tickborne diseases is further compounded by the fact that the use of chemical acaricides for tick control has resulted in the selection of acaricide-resistant tick populations

(Mulenga et al. 2007, Lambson et. al, 2005). However, studies have shown that repeated tick infestation of the vertebrate host provokes a vigorous immune response directed against tick salivary proteins that thwarts tick feeding and impairs pathogen transmission (Narasimhan et al., 2007).

Proteins that are known to invoke such an immune response include cement protein (Labuda et al.,

2006) and subolesin (de la Fuente et al., 2006). Molecular characterization and identification of novel

D. andersoni saliva proteins with immunomodulatory properties will contribute towards the quest for new vaccine targets (Brake et al., 2010). Moreover, the pharmacological properties of tick saliva proteins have potential for therapeutic use in other clinical conditions which are not related to tickborne diseases, such as autoimmune and chronic inflammatory diseases (Steen et al., 2006). For example, studies have shown that a saliva protein (OMCI) found in the soft tick, Ornithodorous moubata can inhibit complement hemolytic activity in humans and rodents, as well as experimental autoimmune myasthenia gravis induced by passive transfer in rats (Hepburn et al., 2007).

Myasthenia gravis is an antibody-mediated autoimmune disease of the neuromuscular junction (Hock et al., 2001). This protein has potential for use as a therapeutic agent for the treatment of complement mediated inflammatory diseases. Tick saliva may therefore provide a solution to resolving the problem of tick-borne diseases, as well as other diseases of vascular origin (Ribeiro and

Fransischetti, 2003).

REFERENCES

Alarcon-Chaidez, F. J., U. U. Muller-Doblies, and S. Wikel. 2003. Characterization of a recombinant

immunomodulatory protein from the salivary glands of Dermacentor andersoni. Parasite

Immunol. 25:69-77.

Boppana, V. D., S. T. Francisco, J. Alarcon-Chaidez, A. J. Adler and S. K. Wikel. 2009. Blood

feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces

interleukin-4 expression by cognate antigen responding CD4+ T cells. Parasites &

Vectors 2:47 doi: 10.1186/1756-3305-2-47.

Bowen, C.J., Jaworski, D.C., Wasala, N.B., and L. B. Coons. 2010. Macrophage migration

inhibitory factor expression and protein localization in Amblyomma americanum (Ixodidae).

Experiment. Applied. Acarol. 50(4):343-352.

Chmelar, J., C. J. Oliveira, P. Rezacova, I. M. B. Francischetti, Z. Kovarova, G. Pejler, P.

Kopacek, J. M. C. Ribeiro, M. Mares, J. Kopecky and M. Kotsyfakis. 2011. A tick

salivary protein targets cathepsin G and chymase and inhibits host inflammation and

platelet aggregation. Blood 117(2):736-744. de Castro, J.J. 1997. Sustainable tick and tickborne disease control in livestock improvement in

developing countries. Veterinary Parasitology 71:77-97. de la Fuente, J., Almazán, C., Blas-Machado, U., Naranjo, V., Mangold, A.J., Blouin, E.F., Gortazar,

C., and K.M. Kocan. 2006. The tick protective antigen, 4D8, is a conserved protein involved

in modulation of tick blood ingestion and reproduction. Vaccine. 24(19):4082-95.

Delavary, B. M., W. M. van der Veer, M. van Egmond, F. B. Niessen and R. H. J. Beelen. 2011.

Macrophages in skin injury and repair. Immunobiol. 216:753-762.

Deruaz, M., A. Frauenschuh, A. L. Alessandri, J. M. Dias, F. M. Coelho, R. C. Russo, B. R. Ferreira,

G. J. Graham, J, P. Shaw, T. N. C. Wells, M. M. Texeira, C. A. Power, and A.E.I.

Proudfoot. 2008. Ticks produce highly selective chemokine binding proteins with anti-

inflammatory activity. J. Exp. Med. 205(9)2019-2031.

Francischetti, I. M. B., J. G. Valenzuela, J. F. Andersen, T. N. Mather, and J. M. C. Ribeiro. 2002.

Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland

of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the

inhibition of factor VIIa/tissue factor complex. Blood 99: 3602-3612.

Guo, X., C. J. Booth, M. A. Paley, X. Wang, K. DePonte, E. Fikrig, S. Narasimhan, and R. R.

Montgomery. 2009. Inhibition of neutrophil function by two tick salivary proteins. Infect.

Immun. 77(6):2320-2329.

Hoch, W., Mcconville, J., Helms, S., Newsom-Davis, J., Melms, A., and A. Vincent. 2001. Auto-

antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without

acetylcholine receptor antibodies. Nature Medicine 7, 365 – 368.

Hovius, J.W., de Jong, M.A, den Dunnen, J., Litjens, M., Fikrig E, van der Poll, T., Gringhuis, S.I.,

and T.B. Geijtenbeek. 2008. Salp15 binding to DC-SIGN inhibits cytokine expression by

impairing both nucleosome remodeling and mRNA stabilization. PLoS Pathog. 4(2):e31.

Kazimírová, M. and I. Štibrániová. 2013. Tick salivary compounds: their role in modulation of host

defences and pathogen transmission. Front. Cell. Infect. Microbiol. 3: 43.

Kocan, K. M., J. de la Fuente, A. A. Guglielmone, and R. D. Melendez. 2003. Antigens and

alternatives for control of Anaplasma marginale infection in cattle. Clinical microbiology

Reviews. 16(4)698-712.

Kramer, C., Z. Nahmias, D. D. Norman, T. A. Mulvihill, L. B. Coons, and J. A. Cole. 2008.

Dermacenter variabilis: regulation of fibroblast migration by tick salivary gland extract and

saliva. Exp. Parasitol. 119:391-397.

Kyckova, K., and J. Kopecky. 2006. Effect of tick saliva on mechanisms of innate immune response.

J. Med. Entomol. 43(6):1208-1214.

Labuda, M., Trimnell, A.R., Lickova, M., Kazimirova, M., Davies, G.M., Lissina, O., Hails, R.S.,

and P.A. Nuttall. 2006. An antivector vaccine protects against a lethal vector-borne

pathogen. PLoS Pathog. 2(4):e27.

Lambson, B., V. Nene., M. Obura, T. Shah, P. Pandit, O. Ole-MoiYoi, K. Delroux, S. Welburn, R.

Skilton, E. de Villiers, and R. Bishop. 2005. Identification of candidate sialome components

expressed in ixodid tick salivary glands using secretion signal complementation in

mammalian cells. Insect Mol. Biol. 14(4):404-414

Mans, B.J., Ribeiro, J.M.C., and J.F. Andersen. 2008. Structure, function, and evolution of biogenic

amine-binding proteins in soft ticks. J. Biol. Chem. 283(27): 18721–18733.

Morley, R. S., and M.E. Hugh-Jones. 1989. The cost of anaplasmosis in the Red River Plains and

south-east areas of Louisiana. Veterinary Research Communications, 13 (5), 349-358.

Mudenda, L., Pierlé, S. A., Turse, J.E., Scoles G.A., Purvine S.O., Nicora C.D., Clauss T.R.,

Ueti M.W., Brown W.C., and K.A Brayton. 2014. Proteomics informed by

transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva. Int. J

Parasitol. 44(13):1029-37.

Mulenga, A., M. Blandon, and R. Khumthong. 2007. The molecular basis of the Amblyomma

americanum tick attachment phase. Exp. Appl. Acarol. 41:267-287.

Murphy, K., Travers, P., Walport, M., 2008. Innate Immunity. In: Lawrence, E. (Ed.), Janeway's

Immuno biology, Garland Science, New York and London, pp. 39.

Narisimhan, S., K. Deponte, N. Marcantonio, X. Liang, T. E. Royce, K. F. Nelson, C. J. Booth, B.

Koski, J. F. Anderson, F. Kantor and E. Fikrig. 2007. Immunity against Ixodes scapularis

salivary proteins expressed within 24 hours of attachment thwarts tick feeding and impairs

Borrelia transmission. PLoS ONE 2(5):e451.

Parola, P., and D. Raoult. 2001. Ticks and tickborne bacterial diseases in humans: an emerging

infectious threat. Clin. Infect. Dis. 32(6):897-928.

Prevot, P., A. Beschin, L. Lins, J. Beaufays, A. Grosjean, L. Bays, B. Adam, M. Brossard, R.

Basseur, K. Z. Boudjeltia, L. Vanhamme, and E. Godfroid. 2009. Exosites mediate the anti-

inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus.

FEBS J. 276:3235- 3246.

Ribeiro, J. M. C., and I. M. B. Francischetti. 2003. Role of arthropod saliva in blood feeding: sialome

and post-sialome perspectives. Annu. Rev. Entomol. 48:73-88.

Reck Jr. J., M. Berger, F. S. Marks, R. B. Zingali, C. W. Canal, C. A. S. Ferreira, J. A. Guimaraes

and C. Termignonoi. 2009. Pharmacological action of tick saliva upon haemostasis and the

neutralization ability of sera from repeatedly infested hosts. Parasit. 136:1339-1349.

Rodero M. P. and K. Khosrotehrani. 2010. Skin wound healing modulation by macrophages. Int. J.

Exp. Pathol. 3(7)643-653.

Schwalie, P. C., and J. Schultz. Positive selection in tick saliva proteins of the salp 15 family. 2009.

J. Mol. Evol. 68:186-191.

Sonenshine, D.E., 1991. Pharmacodynamics of blood sucking in ticks, Biology of ticks Vol. 1,

Oxford University Press. Inc, New York, pp. 137.

Steen, N.A., S. C. Barker, and P. F. Alewood. 2006. Proteins in the saliva of the Ixodida (ticks):

pharmacological features and biological significance. Toxicon 47: 1-20.

Valenzuela J. G., I. M. B. Francischetti, V. M. Pham, M. K. Garfield, T. N. Mather, and J. M. C.

Ribeiro. 2002. Exploring the sialome of the tick Ixodes scapularis. J. Exp. Biol. 205:2843-

2864.

Wang, H. and P.A. Nuttall. 1999. Immunoglobulin binding proteins in ticks: new target for vaccine

development against a blood-feeding parasite. Cell. Mol. Life Sci. 56:286–295.

Woolard, M. D., and J. A. Frelinger. 2008. Outsmarting the host: bacteria modulating the immune

response. Immunol. Res. 41:188-202.

CHAPTER TWO

PROTEOMICS INFORMED BY TRANSCRIPTOMICS IDENTIFIES NOVEL SECRETED

PROTEINS IN DERMACENTOR ANDERSONI SALIVA

ABSTRACT

Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western

United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis.

Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defenses such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick.

Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop anti-tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify

D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomics investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared to two- and five-day fed ticks to identify genes upregulated early during tick feeding. We cross referenced the proteomics data with the transcriptome data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

INTRODUCTION

Ticks are obligate hematophagous parasites that transmit a wide range of pathogens worldwide including protozoa, bacteria and viruses. They can feed on their hosts for days and even weeks without being detected by the host immune system (Deruaz et al., 2008; Kramer et al., 2008).

Immune evasion is achieved through a wide range of proteins that are secreted in saliva (Ribeiro and

Francischetti, 2003; Francischetti et al., 2009; Schwalie and Schultz, 2009). Tick salivary proteins are able to inhibit or modulate both innate and adaptive immune responses (Juncadella et al., 2007;

Schuijt et al., 2008; Guo et al., 2009). Infectious organisms carried by ticks exploit these immunoevasion mechanisms to proliferate within the vertebrate host and cause disease

(Ramamoorthi et al., 2005). Dermacentor andersoni ticks are important vectors of human and animal pathogens including Rickettsia rickettsii (Rocky Mountain Spotted Fever) (Niebylski et al., 1999),

Colorado tick fever virus (Florio et al., 1950), Francisella tularensis (tularemia) (Reif et al., 2011) and Anaplasma marginale (bovine anaplasmosis) (Kocan et al., 1985). Control of tick infestation in livestock is typically by application of chemical acaricides, which has resulted in the selection of acaricide-resistant tick populations, contamination of meat and milk products, and is an important source of environmental pollution, that kills non-target organisms (Kunz and Kemp, 1994; Lambson et al., 2005; de la Fuente et al., 2007; Mulenga et al., 2007b).

Tick saliva contains hundreds of different proteins and other pharmacologically active molecules that confer antihemostatic, anti-inflammatory, and immunomodulatory properties (Guo et al., 2009; Chmelar et al., 2011). Molecular characterization and identification of novel D. andersoni saliva proteins could provide targets for drug development, in addition to new and improved anti-tick vaccine targets as an environmentally friendly alternative to the use of acaricides. Although a few tick saliva proteins have been previously identified, the majority of proteins in saliva and their full impact on the host-tick interaction remain unknown. Moreover, some of the proteins described have

11 only been identified in salivary gland extracts but have not been confirmed to be secreted into saliva

(Steen et al., 2006). The objective of this study was to identify novel, potentially immunosuppressive proteins that are upregulated early during feeding and secreted in the saliva of D. andersoni ticks. As the D. andersoni genome has not been sequenced, we established an expressed sequence tag (EST) library with over 21,000 consensus sequences from unfed, two-day fed and five-day fed ticks and used a method which relies on an all-frame translation of a transcriptome dataset to generate a database of predicted proteins (Evans et al., 2012). Evans and colleagues (Evans et al., 2012) demonstrated that de novo transcriptome assembly generates a data set that can be used to identify virtually all detectable proteins using a method known as proteomics informed by transcriptomics.

We used our EST library to identify 677 proteins expressed in D. andersoni tick saliva by mapping peptide sequences obtained from two-day and five-day fed tick saliva to the EST database following mass spectrometry. This comprehensive and novel catalog of D. andersoni saliva proteins serves as a foundation for research on the development of novel anti-tick vaccines, which could also diminish transmission of tick-borne pathogens.

MATERIALS AND METHODS

Tick Feeding

The D. andersoni ticks used in this study were from the Reynold’s Creek stock maintained at the USDA Agricultural Research Service tick unit in Moscow, Idaho. Adult female Dermacentor andersoni ticks were allowed to feed on Holstein calves to study differential protein expression during tick feeding. All experiments involving animals were approved by the University of Idaho,

Institutional Animal Care and Use and Biosafety Committees (Protocol Numbers, IACUC: 2013-66,

Biosafety: B-010-13) in accordance with institutional guidelines based on the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Tick Salivary Gland EST Database

Unfed ticks as well as ticks fed for two or five days were dissected under a microscope, using a pair of forceps and sharp, sterile razor blade as previously described by Mulenga (Mulenga et al.,

2007a). Salivary glands were teased out, washed in Hank’s balanced salt solution, and immersed in

5-10 volumes of RNAlater (Applied Biosystems, USA). Samples were left in RNAlater at 4°C overnight to allow thorough penetration of the tissue by RNAlater solution and stored at -80°C after removing the supernatant, as per manufacturer’s instructions.

Total RNA was extracted from 40 pairs of salivary glands for each cohort of ticks using TRI

Reagent solution (Applied Biosystems, USA). Then, mRNA was isolated from total RNA using the

Ambion Poly (A) Purist kit in accordance with the manufacturer’s instructions. A cDNA library was prepared in nine steps; fragmentation of RNA, double stranded cDNA synthesis, fragment end repair,

AMPure bead preparation, adaptor ligation, small fragment removal, library quantitation, cDNA library quality assessment and preparation of working aliquots, according to the Genome Sequencer

FLX manual. The cDNA library was then sequenced using the 454 pyrosequencer (Roche, Germany) with GS FLX Titanium series reagents. Sequences were assembled from all cohorts to generate a database using CLC Genomics workbench 6.5’s de novo assembly tool (Gnerre et al., 2011). The algorithm was applied using the following parameters: word size of 20 nt, bubble size of 50 nt and the minimum contig length was 200 bp. Annotation was aided by BLASTX. The cut-off criteria for protein prediction were set as E-value ≤e-20, identity ≥ 45 %, and ≥ 60 % coverage to the corresponding best hit (Zeng and Extavour, 2012). Sequences have been deposited in GenBank’s

Sequence Read Archive with accession number SRP041899.

Comparative Transcriptional Analysis and K-means Clustering

Reads from each sample were mapped against the EST database using CLC Genomics

Workbench 6.5. (CLC Bio, Denmark). Gene expression levels were determined by counting the number of reads that mapped back to each contig. Transcripts were quantified in reads per kilobase of exon model per million mapped reads (RPKM) as described by Mortazavi (Mortazavi et al., 2008) and assigned P-values using Kal’s test. Although there is no genome on which to map the sequences, the error rate of the assembly algorithm used in this study is estimated to be low, thus we consider that the success rate is high enough for appropriate abundance measurements using RPKM where

RPKM = total exon reads/mapped reads (millions) x exon length (kb) (Misner et al., 2013).

Transcription values were compared and analyzed following adjustment of their distribution by quantile normalization (Pierlé et al., 2013). Fold change was calculated by comparison of normalized transcript expression values (RPKM values) at the different feeding time points. We used

K-means clustering to assign the mean transcriptional value of each gene to the cluster whose center is nearest. Lloyd’s algorithm was used for these experiments (Lloyd, 1982). Euclidean distance was used as distance metric; five partitions were used to generate the clusters. For each gene, the mean gene expression value over all input samples was subtracted. Normalized expression values were used for clustering.

Quantitative PCR

In order to validate the RNAseq data we performed quantitative PCR (qPCR) for a set of genes. Total RNA was extracted from 40 pairs of pooled salivary glands from unfed, two-day and five-day fed ticks respectively using TRI Reagent solution (Applied Biosystems, USA), treated with the TURBO DNA-free kit (Dnase treatment), and subsequently used for first strand cDNA synthesis with Superscipt VILO cDNA Synthesis Kit (Invitrogen, USA). The primers used for qPCR are listed in Table 1. The qPCR conditions were 50°C for 2 min, 95 °C for 10 min, 44 cycles of 95 °C for

15 sec, 60 °C for 1 min and 72 °C for 7 min and a final hold at 4 °C. All reactions were done in

14 triplicate using 0.4 ul of 10 µM primers per well, and 0.5 ng cDNA per well. The qPCR data were analyzed using the Relative Expression Software Tool (REST) and BootstRatio to assess the statistical significance of the gene expression ratios across different techniques (Pfaffl et al., 2002;

Clèries et al., 2012). The data were normalized using DaSG8252 as a reference gene.

Tick Saliva Sample Preparation

Saliva was collected from two-day fed and five-day fed ticks after stimulating salivation by injecting the ticks with 5-10 µl of 10 mg/ml dopamine in 1.2 % NaCl solution, using a 50 µl syringe and 12.7 x 0.33 mm needle. Saliva was collected using a 10 µl capillary tube fitted over the ticks’ mouth parts, and kept on ice. All the saliva collected was pooled into a microfuge tube and stored at -

80°C. Saliva was collected from 200 five-day fed ticks and 400 two-day fed ticks.

The protein concentration of the saliva was determined using Pierce BCA protein assay

(Pierce, Rockford, IL). Urea (all chemicals purchased through Sigma Aldrich, St. Louis, MO, unless otherwise stated) was then added to the sample so that the final urea concentration in the sample was

8 M. Dithiothreitol (DTT) was then added to a final concentration of 10 mM. After incubating the sample in a thermo mixer at 37°C for 1 hour with constant shaking, iodoacetamide was added to reach a concentration of 40 mM. The sample was incubated again as described above. Following incubation, the sample was diluted with 50 mM ammonium bicarbonate, pH 8.0 to bring the concentration of urea in the sample to 1.5 M, and adjusted to 1 mM CaCl2. Sample digestion was performed by adding trypsin at a ratio of 1:50 (trypsin to protein) and incubating at 37°C for 3 hours.

The sample was desalted by passing the sample through a 1 ml C18 SPE column (Supelco,

Bellefonte, PA). Briefly, the columns were conditioned with methanol and rinsed with acidified water (0.1 % trifluoroacetic acid (TFA)). The sample solution was put through the column at a rate no faster than 1 ml / min. The column was washed with 95:5 H2O: Acetonitrile (ACN), 0.1 % TFA and allowed to dry. Samples were eluted in 1 ml 80:20 ACN: H2O, 0.1 % TFA. The sample was

15 concentrated to a volume of about 50-100 µl in a speed vac. Protein concentration was determined by performing the BCA protein assay, and the sample was stored at -80°C until needed for two dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analysis.

Two Dimensional LC-MS/MS Analysis

Tick saliva was separated into 15 fractions by 2D-LC. 2D-LC was performed by applying the acidified saliva peptide mixture to a strong cation exchange (SCX) column (first dimension separation) followed by a binary gradient to high salt to elute the peptides. Fractions of peptides were loaded onto a reverse phase HPLC column which removes salt while performing a second separation of peptides based on their hydrophobicity. The 2D-LC system was custom built using two Agilent

1200 nanoflow pumps and one 1200 capillary pump (Agilent Technologies, Santa Clara, CA), Valco valves (Valco Instruments Co., Houston, TX,USA), and a PAL auto sampler (Leap Technologies,

Carrboro, NC, USA). Custom software allowed for full automation of parallel event coordination and near 100% MS duty cycle by use of two trapping columns and two analytical columns. The columns were made by slurry packing media into fused silica (Polymicro Technologies Inc.,

Phoenix, AZ, USA) using a 0.5 cm sol-gel frit for media retention (Maiolica et al., 2005).

The first dimension SCX column consisted of 5 µm Polysulfoethyl A (PolyLC Inc.,

Columbia, MD, USA), 15 cm x 360 µm o.d. x 150 µm i.d. The trapping columns were 5 µm Jupiter

C18 (Phenomenex, Torrence, CA), 4 cm x 360 µm o.d. x 150 µm i.d. Second dimension reversed- phase columns comprised 3-µm Jupiter C18 (Phenomenex, Torrence, CA, USA), 35-cm x 360 µm o.d. x 75 µm i.d. Mobile phases consisted of 0.1 mM NaH2PO4 (A) and 0.3 M NaH2PO4 (B) for the first dimension and 0.1 % formic acid in water (A) and 0.1 % formic acid acetonitrile (B) for the second dimension (Table 2).

MS analysis was performed using a LTQ Orbitrap Velos mass spectrometer (Thermo

Scientific, San Jose, CA, USA) outfitted with a custom-built electrospray ionization (ESI) interface.

Electrospray emitters were custom made using 150 µm o.d. x 20 µm i.d. chemically etched fused silica as described by Kelly (Kelly et al., 2006). Heated capillary temperature of 325°C, and spray voltage of 2.4 kV were used. Data was acquired for 100 minutes, starting 50 minutes after sample injection and 10 minutes into the gradient. Orbitrap spectra (automatic gain control (AGC) 1x106) were collected from 400-2000 m/z at a resolution of 60k followed by data dependant ion trap collision induced dissociation (CID) MS/MS (collision energy 35 %, AGC 3x104) of the ten most abundant ions. A dynamic exclusion time of 60 sec was used to discriminate against previously analyzed ions.

Protein Identification

RNAseq data was used to inform protein identification. Proteins were identified by mapping peptide masses and/or sequences to our Dermacentor andersoni EST database following all-frame translation into amino acid sequences, and using SEQUEST (Thermo Finnigan, USA). SEQUEST filtering criteria included a mass tolerance of ± 1-3, ΔCn 0.1, Xcorr 1.5 for charge states of +1 and

+2, and an Xcorr of 2.5 for a charge state of +3. Peptide assignments to MS/MS spectra were validated by the generating function (MS-GF) cut off ≤ 1E−9 and peptide prophet score of ≥ 0.5.

MS-GF values are a peptide spectral probability that assigns a statistical significance to each individual spectral identification (Kim et al., 2008). An MS-GF cut off ≤ 1E−9 gives a false discovery rate (FDR) of ≤ 1% (Kim et al., 2008). Peptide prophet computes a probability of being correct for each peptide assignment to a spectrum and was used to validate SEQUEST search results

(Keller et al., 2002; Ma et al., 2012). Identified peptides were searched against a reversed sequence decoy database to determine FDR values at given cut offs.

RESULTS

Development of an EST Database

Messenger RNA was extracted from salivary glands from unfed, two-day fed and five-day fed ticks and sequenced using Roche’s 454 sequencing technology. The number of sequence reads obtained for each of the feeding time points (day 0, day 2 and day 5) were 156,620, 157,647 and

318,000 respectively. The data from all three time points was pooled and assembled resulting in

21,797 unique sequences. Twenty-eight sequences were not annotated as a gene or transcript, resulting in 21,769 sequences that were further analyzed. The distribution of 21,506 sequences at the three time points sampled is illustrated in Fig. 1. The remaining 263 sequences had very low representation such that following normalization of gene expression they had a value of zero at all three time points.

The total number of transcripts that were assigned a putative function was 2,810 with an additional 706 being annotated as conserved hypothetical proteins (Supplemental file 1). Genes upregulated early during feeding included those coding for metabolic proteins, genes involved in endocytosis or nutrient uptake, reproduction and cytoskeletal network proteins, transporter proteins, splicing and transcription factors, signaling molecules, and proteins required for successful feeding and immunomodulation including genes coding for immunosuppressant protein p36, serine/threonine protein kinases, cement protein, suppressor of cytokine signaling, lipocalins, cystatin, and serpin 8.

Differential Transcription

We were interested in identifying transcripts that were up-regulated early during feeding and still present at day five, but absent or low at day zero, as well as those transcripts upregulated early during feeding (at two days) that were low or absent at five days but whose proteins are present at both two and five days, assuming this set of transcripts would include immunomodulators. Table 3 summarizes the differential transcription of genes at the different feeding time points. Genes were classified as differentially expressed if their fold change was >2 with a p<0.05. The number of genes

18 upregulated at two days when compared with day zero was 7,132. Among these, the number of genes that were still upregulated at five days was 2357.

Cluster Analysis

We partitioned our comparative transcriptional analyses into 5 clusters (Fig. 2) in order to visualize the expression patterns of the different genes. Clusters 2 and 3 represent expression patterns that are not of interest to us in the current study: they contain genes that are high in unfed ticks and downregulated early during feeding, suggesting that they are not required for host immune suppression and successful feeding. Clusters 1 and 5, on the other hand, represent the ideal gene expression patterns for immunomodulation of host responses and successful feeding of the tick.

These genes are upregulated at day-two and their transcripts are still present at day-five. The transcription patterns observed in clusters 1 and 5 can also be seen in cluster 4, however genes in cluster 4 are regulated with a smaller magnitude. Supplemental file 1 shows the affiliation of different genes to the clusters shown in Fig. 2.

All the genes in cluster 1 are unknown. There is a conserved hypothetical protein

(DaSG10689) in cluster 2 while the rest of the genes are unknown. Two genes in cluster 3 have been assigned putative function; DaSG5281 (ribosomal protein L17) and DaSG10972 (cement protein

RIM36), the rest are unknown. Only six genes belong to cluster 5 and their functions are not known.

The rest of the 2810 genes with assigned putative function, 706 genes coding for conserved hypothetical proteins, and other genes with unknown function belong to cluster 4.

Quantitative PCR results

In order to validate the gene expression levels observed following deep sequencing we performed qPCR on nine genes listed in Table 1. The genes were selected to include various expression patterns across the three feeding time points. DaSG8252, a ribosomal protein S1 RNA binding domain-

19 containing protein was used as a reference gene to normalize the data. Based on our RNAseq data,

DaSG8252 is constitutively expressed at a stable rate across the different feeding time points. In support of the use of ribosomal genes as housekeeping genes, Koci (Koči et al., 2013) found that ribosomal proteins S4 and L13A showed a highly stable expression pattern over a 7-day feeding period in female Ixodes scapularis ticks. Ribosomal genes appear to be reliable reference genes in ticks as previously reported (Scharlaken et al., 2008; Nijhof et al., 2009; Mamidala et al., 2011).

Following qPCR, changes in transcription were analyzed using REST. We compared transcriptional activity at day zero versus day two, day zero versus day five and lastly, day two versus day five. At each time point, six out of the nine genes tested had a similar expression pattern to our RNA sequence data, and out of the six consistent genes, four had statistically significant ratios (p<0.05).

This result was then confirmed by BootstsRatio analyses, which showed that 70% of the genes had statistically significant transcriptional trends across the different techniques (Fig. 3). Sequencing depth is known to have an effect on the correlation of gene expression levels. Good congruency may be achieved with genes that have high numbers of reads mapped while genes that are less strongly transcribed may exhibit lower reproducibility (Mortazavi et al., 2008). Consequently, some transcripts may be assessed with good precision, while others may be hard to measure reliably through the different techniques (Łabaj et al., 2011) The set of genes that were selected for qPCR had a range of RPKM values, and at some of the time points the RPKM values were normalized to zero.

Interestingly, incongruent results were most often obtained when comparing time points where the

RPKM value was normalized to zero in only one of the time points. Thus considering that the set of genes selected for qPCR had low relative transcription levels at some time points, this result suggests that for less strongly transcribed genes, about 70% will be reliably measured through the different techniques.

Identified Saliva Proteins

Saliva was collected from two-day fed and five-day fed ticks for LCMS/MS and subsequent identification of proteins. The 30 fractions collected from LC generated a total of 19,460 MS/MS spectra that passed the MSGF filter. These spectra mapped to 2,104 unique peptide sequences. Of these peptides, 1,847 mapped to a total of 677 proteins translated from our EST database described above (Supplemental file 2). To our knowledge, this is the most comprehensive search yet for the individual proteins produced in D. andersoni tick saliva. The differential expression of the identified proteins showed that 140 proteins were detected only at two days, 165 at five days, and 372 proteins were detected at both time points. Cross referencing with the transcriptome data identified 148 proteins that are upregulated early and present at both 2 and 5 days of feeding and/or have sequence identity to previously reported immunomodulators (15[Table 4]) for a total of 157 genes/proteins of interest. Of the 677 identified proteins, 136 (20%) have homologs in the public databases, based on a

BLAST cut-off:

DISCUSSION

Proteomics informed by transcriptomics

A few studies have previously reported the use of transcriptomics to inform proteomics in other tick species. A recent study examined proteins from homogenized Dermacentor reticularis larvae and identified 287 proteins following 1D gel electrophoresis and LC-MS/MS (Villar et al.,

2014). A second study compared proteins in the saliva of partially and fully engorged Rhipicephalus

(Boophilus) microplus (Tirloni et al., 2014). This study employed 1D gel electrophoresis and LC-

MS/MS and identified 187 tick proteins and 68 host/bovine proteins. A third study used 1D gel electrophoresis and LC-MS/MS to analyze saliva from male and female Ornithodorus moubata, and identified a total of 193 proteins with only ten of these proteins being found in both sexes (Diaz-

Martin et al., 2014). A proteomic analysis of saliva from pilocarpine- or dopamine-induced salivation

21 in fed female Rhipicephalus sanguineus ticks identified 19 tick proteins and 56 host proteins

(Oliveira et al., 2013). While each of these studies had a different focus, it is interesting to note they report categories of proteins with functional annotations and biological processes that are consistently identified in all the studies and are also represented in our data (Supplemental file 2 and

3). These broad categories of proteins include genes/proteins that encode metabolic process, signaling, regulation, transporter activity structural molecules, electron carrier activity enzyme regulator activity, catalytic activity, reproduction, binding, etc., and lastly unknown proteins. Tirloni et al. (2014) also identified some of the immunomodulators discussed in this study including serpins, cystatin, thyropins, metalloproteases, serine carboxipeptidase and alpha-2-macroglobulin. They reported differences in the saliva of partially and fully engorged ticks fed for 17 and 20 days.

Similarly, we found that some proteins were only expressed at two days of feeding, and others at five days. All of these studies report a total number of identified proteins far below our total of 677. The

D. andersoni proteome may therefore, be the most comprehensive Ixodidae tick saliva proteome yet.

The differences in the number of identified proteins may be attributed to differences in fractionation methods (Villar et al., 2014) as well as the instrumentation used for mass spectrometry.

Identified proteins

The innate immune response occurs within four hours of tissue injury (Murphy et al., 2008) thus ticks must overcome the host defense mechanisms, which include hemostasis, inflammation and cell mediated immunity to acquire a blood meal (Sonenshine, 1991). Previous studies have shown that an array of pharmacologically active tick saliva proteins is secreted within the first 24 hours of attachment (Narisimhan et al., 2007). The main objective of this study was to identify proteins that are secreted in D. andersoni saliva during feeding; particularly those proteins that modulate the host immune response. Given that the D. andersoni genome has not been sequenced, we developed an

EST library to aid in identifying the saliva proteins following mass spectrometry. Comparative

RNAseq analyses were employed to identify genes that were upregulated early during tick feeding assuming they would include genes that were important for immunomodulation. As has been previously reported in other systems we found many genes to be differentially regulated over time, with the majority (more than 80% of the transcripts [18,253]) being of unknown function (Ribeiro et al., 2006; Mercado-Curiel et al., 2011; McNally et al., 2012). We cross referenced the transcriptome with protein data to identify proteins for testing as immunomodulators. Almost all the genes (650) corresponding to identified proteins were found in cluster 4, with 12 genes in cluster 1, and four in cluster 5 (Fig. 2). The number of genes corresponding to protein data in clusters 2 and 3 were one and nine. The 157 proteins of interest are those that were upregulated early during feeding (at day- two) and they were found in clusters 1, 4 and 5.

In order to identify proteins that are secreted in saliva and potentially required for successful feeding, including those involved in immunomodulation, we chose two feeding time points within the slow feeding phase (day two and day five) for collection of saliva from D. andersoni ticks. Day two was the earliest time point at which we could collect a sufficient amount of tick saliva for mass spectrometry due to technical challenges with this system (Chen et al., 2012). Day five was chosen because the ticks would have established their feeding site by that time, and thus they would be secreting proteins necessary for maintenance of the feeding site. After day five, ticks feed rapidly to repletion as can be inferred from the rate of increase in live weight of female D. andersoni during feeding (Bergman et al., 2000).

An additional criterion for identifying the proteins of interest was based on bioinformatics to include proteins with sequence identity to known immunomodulators, and to exclude proteins with sequence identity to non-immunosuppressors or other irrelevant proteins. Among the functionally annotated proteins that may be involved in suppression of host immune response to ticks were catalase, serpin 8 precursor, alpha-2-macroglobulin, tumor rejection antigen (gp96), serine carboxypeptidase and thioredoxin peroxidase, which were present in the two-day and five-day fed

23 saliva, and were upregulated at 2 days. The remaining proteins; tumor rejection antigen (gp96), metalloprotease, immunosuppressant protein p36, lospins 8 and 9, thyropin precursor, serpin-1 precursor, and intracellular cystatin were upregulated at day 5, and were present in tick saliva either at both two and five days of feeding or at a single feeding time point (day two or day five).

Protein p36 was first reported as an immunosuppressant protein from the salivary glands of

D. andersoni ticks (Bergman et al., 2000; Ribeiro et al., 2011) as it was shown to suppress concanavalin A-stimulated splenocyte proliferation (Alarcon-Chaidez et al., 2003). Homologues of immunosuppressant protein p36 have been reported for Amblyomma variegatum (Nene et al., 2002),

Haemaphysalis longicornis (Konnai et al., 2009), and Rhipicephalus appendiculatus (Nene et al.,

2004). Cystatins are cysteinyl protease inhibitors (Ribeiro et al., 2011). A salivary cystatin that targets cathepsin L, sialostatin L, was reported to have anti-inflammatory action and to reduce proliferation of cytotoxic leukocytes (Kotsyfakis et al., 2006). Serpins, on the other hand, are serine protease inhibitors that have been shown to inhibit platelet aggregation and inflammation (Chmelar et al., 2011; Mulenga et al., 2013). Lospins are lone star tick serpins that have been reported for

Amblyomma amercanum (Mulenga et al., 2007a). Thyropins, though structurally different from cystatins, are also cysteine protease inhibitors (Lenarčič and Bevec, 1998) classified as thyroglobulin type-1 domain inhibitors (Lenarčič and Bevec, 1998). Catalase and thioredoxin peroxidase quench reactive oxygen species. In addition, thioredoxin peroxidase converts macrophages to an alternatively activated phenotype that produces high levels of the regulatory cytokine interleukin 10 (IL-10) and low levels of the pro-inflammatory cytokine IL-12 (Donnelly et al., 2005). Although alpha-2- macroglobulin induces T cell proliferation, and activation and proliferation of macrophages in vertebrates, “tick saliva alpha-2-macroglobulin may be linked to interference in inflammation and immunomodulation” (Tirloni et al., 2014). Similarly, Tumor rejection antigen gp96 reportedly induces T cell proliferation and secretion of interferon-γ (IFN-γ) at low doses, and promotes a Th2- like immunoregulatory phenotype with an increased IL10:IFN-γ ratio at higher doses in rats (Mirza

24 et al., 2006). Several enzymes present in the saliva of blood-feeding arthropods including apyrases, esterases, glucosidases, phospholipase A2, metalloproteases, kininases, and serine proteases are said to assist blood feeding (Francischetti et al., 2009). Thus some of the predicted enzymes on our protein list such as salivary gland metalloprotease, serine threonine kinase and hydrolases may be involved in the modulation of host defense mechanisms during tick feeding. The majority (80%) of the identified proteins are of unknown function. Out of the 677 identified proteins, 136 (20%) have homologs in the public databases. Our list of saliva proteins will be useful in future studies to aid in the development of anti-inflammatory drug targets as well as new tick control measures.

In summary, using proteomics informed by transcriptomics, we have generated the most comprehensive set of proteins ever detected in D. andersoni saliva. To identify proteins involved in immunomodulation, we analyzed our data set to identify genes/proteins that are up-regulated early and secreted in saliva. These candidates include both known and unknown proteins and provide a list of genes/proteins for further study of immunosuppression. The identified saliva proteins may be exploited as targets for development of anti-inflammatory drugs and anti-tick vaccines.

REFERENCES

Alarcon-Chaidez, F.J., Muller-Doblies, U.U., Wikel, S., 2003. Characterization of a recombinant

immunomodulatory protein from the salivary glands of Dermacentor andersoni. Parasite

Immunol 25, 69-77.

Bergman, D.K., Palmer, M.J., Caimano, M.J., Radolf, J.D., Wikel, S.K., 2000. Isolation and

Molecular Cloning of a Secreted Immunosuppressant Protein from Dermacentor andersoni

Salivary Gland. J Parasitol 86, 516-525.

Chen, G., Severo, M.S., Sohail, M., Sakhon, O.S., Wikel, S.K., Kotsyfakis, M., Pedra, J.H.F., 2012.

Ixodes scapularis saliva mitigates inflammatory cytokine secretion during Anaplasma

phagocytophilum stimulation of immune cells. Parasit Vectors 5, 229.

Chmelar, J., Oliveira, C.J., Rezacova, P., Francischetti, I.M.B., Kovarova, Z., Pejler, G., Kopacek, P.,

Ribeiro, J.M.C., Mares, M., Kopecky, J., Kotsyfakis, M., 2011. A tick salivary protein targets

cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood 117,

736-744.

Clèries, R., Galvez, J., Espino, M., Ribes, J., Nunes, V., de Heredia, M.L., 2012. BootstRatio: A

web-based statistical analysis of fold-change in qPCR and RT-qPCR data using resampling

methods. Comput Biol Med 42, 438-445. de la Fuente, J., Almazan, C., Canales, M., de la Lastra, J.M.P., Kocan, K.M., Willadsen, P., 2007. A

ten-year review of commercial vaccine performance for control of tick infestations on cattle.

Anim Health Res Rev 8, 23–28.

Deruaz, M., Frauenschuh, A., Alessandri, A.L., Dias, J.M., Coelho, F.M., Russo, R.C., Ferreira, B.R.,

Graham, G.J., Shaw, J.P., Wells, T.N.C., Teixeira, M.M., Power, C.A., Proudfoot, A.E.I.,

2008. Ticks produce highly selective chemokine binding proteins with anti-inflammatory

activity. J Exp Med 205, 2019-2031.

Diaz-Martin, J., Diaz-Lopez, A., Moreno-Bueno, G., Castilla, M.A., Rosa-Rosa, J.M., Cano, A.,

Palacios, J., 2014. A core microRNA signature associated with inducers of the epithelial-to-

mesenchymal transition. J Pathol 232, 319-329.

Donnelly, S., O'Neill, S.M., Sekiya, M., Mulcahy, G., Dalton, J.P., 2005. Thioredoxin Peroxidase

Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages. Infect

Immun 71, 166-173.

Evans, V.C., Barker, G., Heesom, K.J., Fan, J., Bessant, C., Matthews, D.A., 2012. De novo

derivation of proteomes from transcriptomes for transcript and protein identification. Nat

Methods 9, 1207-1211.

Florio, L., Miller, M.S., Mugrage, E.R., 1950. Colorado tick fever: Isolation of the virus from

Dermacentor andersoni in nature and a laboratory study of the transmission of the virus in

the tick. J Immunol 64, 257-263.

Francischetti, I.M.B., Sá-Nunes, A., Mans, B.J., Santos, I.M., Ribeiro, J.M.C., 2009. The role of

saliva in tick feeding. Front Biosci 14, 2051-2088.

Gnerre, S., Maccallum, I., Przybylski, D., Ribeiro, F.J., Burton, J.N., Walker, B.J., Sharpe, T., Hall,

G., Shea, T.P., Sykes, S., Berlin, A.M., Aird, D., Costello, M., Daza, R., Williams, L., Nicol,

R., Gnirke, A., Nusbaum, C., Lander, E.S., Jaffe, D.B., 2011. High-quality draft assemblies

of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci U S A

108, 1513-1518.

Guo, X., Booth, C.J., Paley, M.A., Wang, X., DePonte, K., Fikrig, E., Narasimhan, S., Montgomery,

R.R., 2009. Inhibition of neutrophil function by two tick salivary proteins. Infect Immun 77,

2320-2329.

Juncadella, I.J., Garg, R., Ananthnarayanan, S.K., Yengo, C.M., Anguita, J., 2007. T-cell signaling

pathways inhibited by the tick saliva immunosuppressor, Salp15. FEMS Immunol Med

Microbiol 49, 433-438.

Keller, A., Nesvizhskii, A.I., Kolker, E., Aebersold, R., 2002. Empirical statistical model to estimate

the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74,

5383-5392.

Kelly, R.T., Page, J.S., Luo, Q., Moore, R.J., Orton, D.J., Tang, K., Smith, R.D., 2006. Chemically

Etched Open Tubular and Monolithic Emitters for Nanoelectrospray Ionization Mass

Spectrometry. Anal Chem 78, 7796-7801.

Kim, S., Gupta, N., Pevzner, P.A., 2008. Spectral Probabilities and Generating Functions of Tandem

Mass Spectra: A Strike against Decoy Databases. J Proteome Res 7, 3354-3363.

Kocan, K.M., Baron, S.J., Ewing, S.A., Hair, J.A., 1985. Transmission of Anaplasma marginale by

adult Dermacentor andersoni during feeding on calves. Am J Vet Res 46, 1565-1567.

Koči, J., Šimo, L., Park, Y., 2013. Validation of Internal Reference Genes for Real-Time

Quantitative Polymerase Chain Reaction Studies in the Tick, Ixodes scapularis (Acari:

Ixodidae). J Med Entomol 50, 70-84.

Konnai, S., Nakajima, C., Imamura, S., Yamada, S., Nishikado, H., Kodama, M., Onuma, M.,

Ohashi, K., 2009. Suppression of cell proliferation and cytokine expression by HL-p36, a tick

salivary gland-derived protein of Haemaphysalis longicornis. Immunology 126, 209-219.

Kotsyfakis, M., Sá-Nunes, A., Francischetti, I.M.B., Mather, T.N., Andersen, J.F., Ribeiro, J.M.C.,

2006. Antiinflammatory and immunosuppressive activity of Sialostatin L, a salivary cystatin

from the tick Ixodes scapularis. J Biol Chem 281, 26298-26307.

Kramer, C., Nahmias, Z., Norman, D.D., Mulvihill, T.A., Coons, L.B., Cole, J.A., 2008.

Dermacenter variabilis: regulation of fibroblast migration by tick salivary gland extract and

saliva. Exp Parasitol 119, 391-397.

Kunz, S.E., Kemp, D.H., 1994. Insecticides and acaricides: resistance and environmental impact. Rev

Sci Tech 13, 1249-1286.

Łabaj, P.P., Leparc, G.G., Linggi, B.E., Markillie, L.M., Wiley, S.H., Kreil, D.P., 2011.

Characterization and improvement of RNA-Seq precision in quantitative transcript

expression profiling. Bioinformatics 27, i383-i391.

Lambson, B., Nene, V., Obura, M., Shah, T., Pandit, P., Ole-MoiYoi, O., Delroux, K., Welburn, S.,

Skilton, R., de Villiers, E., Bishop, R., 2005. Identification of candidate sialome components

expressed in ixodid tick salivary glands using secretion signal complementation in

mammalian cells. Insect Mol Biol 14, 403-414.

Lenarčič, B., Bevec, T., 1998. Thyropins – New structurally related proteinase inhibitors. Biol Chem

379, 105-111.

Lloyd, S.P., 1982. Least Squares Quantization in PCM. IEEE Transactions on Information Theory

28, 129-137.

Ma, K., Vitek, O., Nesvizhskii, A.I., 2012. A statistical model-building perspective to identification

of MS/MS spectra with PeptideProphet. BMC Bioinformatics 13, S1.

Maiolica, A., Borsotti, D., Rappsilber, J., 2005. Self-made frits for nanoscale columns in proteomics.

Proteomics 5, 3847-3850.

Mamidala, P., Rajarapu, S.P., Jones, S.C., Mittapalli, O., 2011. Identification and validation of

reference genes for quantitative real-time polymerase chain reaction in Cimex lecturarius. J

Med Entomol 48, 947-951.

McNally, K.L., Mitzel, D.N., Anderson, J.M., Ribeiro, J.M.C., Valenzuela, J.G., Myers, T.G.,

Godinez, A., Wolfinbarger, J.B., Best, S.M., Bloom, M.E., 2012. Differential salivary gland

transcript expression profile in Ixodes scapularis nymphs upon feeding or flavivirus

infection. Ticks Tick Borne Dis 3, 18-26.

Mercado-Curiel, R.F., Palmer, G.H., Guerrero, F.D., Brayton, K.A., 2011. Temporal characterization

of the organ-specific Rhipicephalus microplus transcriptional response to Anaplasma

marginale. Int J Parasitol 41, 851-860.

Mirza, S., Muthana, M., Fairburn, B., Slack, L.K., Hopkinson, K., Pockley, A.G., 2006. The stress

protein gp96 is not an activator of resting rat bone marrow-derived dendritic cells, but is a

costimulator and activator of CD3+ T cells. Cell Stress Chaperones 11, 364-378.

Misner, I., Bicep, C., Lopez, P., Halary, S., Bapteste, E., Lane, C.E., 2013. Sequence comparative

analysis using networks: software for evaluating de novo transcript assembly from next-

generation sequencing. Mol Biol Evol 30, 1975-1986.

Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., Wold, B., 2008. Mapping and quantifying

mammalian transcriptomes by RNA-Seq. Nat Methods 5, 621-628.

Mulenga, A., Blandon, M., Khumthong, R., 2007a. The molecular basis of the Amblyomma

americunum tick attachment phase. Exp Appl Acarol 41, 267-287.

Mulenga, A., Khumthong, R., Blandon, M.A., 2007b. Molecular and expression analysis of a family

of the Amblyomma americanum tick Lospins. J Exp Biol 210, 3188-3198.

Mulenga, A., Kim, T., Ibelli, A.M.G., 2013. Amblyomma americanum tick saliva serine protease

inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that

delays plasma clotting and inhibits platelet aggregation. Insect Mol Biol 22, 306-319.

Murphy, K., Travers, P., Walport, M., 2008. Innate Immunity. In: Lawrence, E. (Ed.), Janeway's

Immuno biology, Garland Science, New York and London, pp. 39.

Narisimhan, S., Deponte, K., Marcantonio, N., Liang, X., Royce, T.E., Nelson, K.F., Booth, C.J.,

Koski, B., Anderson, J.F., Kantor, F., Fikrig, E., 2007. Immunity against Ixodes scapularis

salivary proteins expressed within 24 hours of attachment thwarts tick feeding and impairs

Borrelia transmission. PLoS One 2, e451.

Nene, V., Lee, D., Quackenbush, J., Skilton, R., Mwaura, S., Gardner, M.J., Bishop, R., 2002. AvGI,

an index of genes transcribed in the salivary glands of the ixodid tick Amblyomma

Variegatum. Int J Parasitol 32, 1447-1456.

Nene, V., Lee, D., Kang'a, S., Skilton, R., Shah, T., de Villiers, E., Mwaura, S., Taylor, D.,

Quackenbush, J., Bishop, R., 2004. Genes transcribed in the salivary glands of female

Rhipicephalus appendiculatus ticks infected with Theileria parva. Insect Biochem Mol Biol

34, 1117-1128.

Niebylski, M.L., Peacock, M.G., Schwan, T.G., 1999. Lethal effect of Rickettsia rickettsii on its tick

vector (Dermacentor andersoni). Appl Environ Microbiol 65, 773-778.

Nijhof, A.M., Balk, J.A., Postigo, M., Jongejan, F., 2009. Selection of reference genes for

quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus

appendiculatus ticks and determination of the expression profile of Bm86. BMC Mol Biol

10, 112.

Oliveira, C.J., Anatriello, E., de Miranda-Santos, I.K., Francischetti, I.M., Sa-Nunes, A., Ferreira,

B.R., Ribeiro, J.M., 2013. Proteome of Rhipicephalus sanguineus tick saliva induced by the

secretagogues pilocarpine and dopamine. Ticks Tick Borne Dis 4, 469-477.

Pfaffl, M.W., Horgan, G.W., Demfle, L., 2002. Relative expression software tool (REST) for group-

wise comparison and statistical analysis of relative expression results in real-time PCR.

Nucleic Acids Res 30, e36.

Pierlé, S.A., Hammac, G.K., Palmer, G.H., Brayton, K.A., 2013. Transcriptional pathways associated

with the slow growth phenotype of transformed Anaplasma marginale BMC Genomics 14,

272.

Ramamoorthi, N., Narasimhan, S., Pal, U., Bao, F., Yang, X.F., Fish, D., Anguita, J., Norgard, M.V.,

Kantor, F.S., Anderson, J.F., Koski, R.A., Fikrig, E., 2005. The Lyme disease agent exploits

a tick protein to infect the mammalian host. Nature 436, 573-577.

Reif, K.E., Palmer, G.H., Ueti, M.W., Scoles, G.A., Margolis, J.J., Monack, D.M., Noh, S.M., 2011.

Dermacentor andersoni transmission of Francisella tularensis subsp. novicida reflects

bacterial colonization, dissemination, and replication coordinated with tick feeding. Infect

Immun 79, 4941-4946.

Ribeiro, J.M.C., Francischetti, I.M.B., 2003. Role of arthropod saliva in blood feeding: sialome and

post-sialome perspectives. Annu Rev Entomol 48, 73-88.

Ribeiro, J.M.C., Alarcon-Chaidez, F., Francischetti, I.M.B., Mans, B.J., Mather, T.N., Valenzuela,

J.G., Wikel, S.K., 2006. An annotated catalog of salivary gland transcripts from Ixodes

scapularis ticks. Insect Biochem Mol Biol 36, 111-129.

Ribeiro, J.M.C., Anderson, J.M., Manoukis, N.C., Meng, Z., Francischetti, I.M.B., 2011. A further

insight into the sialome of the tropical bont tick, Amblyomma variegatum. BMC Genomics

12, 136.

Scharlaken, B., de Graaf, D.C., Goossens, K., Brunain, M., Peelman, L.J., Jacobs, F.J., 2008.

Reference gene selection for insect expression studies using quantitative real-time PCR: the

head of the honeybee, Apis mellifera, after a bacterial challenge. J Insect Science 8, 33.

Schuijt, T.J., Hovius, J.W.R., van Burgel, N.D., Ramamoorthi, N., Fikrig, E., van Dam, A.P., 2008.

The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi

sensu lato isolates. Infect Immun 76, 2888-2894.

Schwalie, P.C., Schultz, J., 2009. Positive selection in tick saliva proteins of the salp 15 family. J

Mol Evol 68, 186-191.

Sonenshine, D.E., 1991. Pharmacodynamics of blood sucking in ticks, Biology of ticks Vol. 1,

Oxford University Press. Inc, New York, pp. 137.

Steen, N.A., Barker, S.C., Alewood, P.F., 2006. Proteins in the saliva of the Ixodida (ticks):

pharmacological features and biological significance. Toxicon 47, 1-20.

Tirloni, L., Reck, J., Terra, R.M., Martins, J.R., Mulenga, A., Sherman, N.E., Fox, J.W., Yates, J.R.,

3rd, Termignoni, C., Pinto, A.F., Vaz Ida, S., Jr., 2014. Proteomic analysis of cattle tick

Rhipicephalus (Boophilus) microplus saliva: a comparison between partially and fully

engorged females. PLoS One 9, e94831.

Villar, M., Popara, M., Ayllon, N., Fernandez de Mera, I.G., Mateos-Hernandez, L., Galindo, R.C.,

Manrique, M., Tobes, R., de la Fuente, J., 2014. A systems biology approach to the

characterization of stress response in Dermacentor reticulatus tick unfed larvae. PLoS One 9,

e89564.

Zeng, V., Extavour, C.G., 2012. ASGARD: an open-access database of annotated transcriptomes for

emerging model arthropod species. Database (Oxford) 2012, bas048.

TABLES

Table 1. Genes and primers for qPCR.

Table 2. Pump methods for the 1st and 2nd dimension separations.

SCX Fractionation in 1st Reversed-phase Separation in 2nd Dimension Dimension

Fraction %Ba Start %Ba End Time (min) %Ba Event

1 0 0 -40 0 Trap/Wash 2 0.0 1.5 0 0 3 1.5 3.0 2 8 4 3.0 4.0 10 NA Start Acq. 5 4.0 5.0 20 12 6 5.0 6.0 75 35 7 6.0 7.0 97 60 8 7.0 8.0 100 85 9 8.0 9.0 103 0 10 9.0 10.0 110 NA End Acq. 11 10.0 11.0 12 11.0 14.0 13 14.0 17.0 14 17.0 20.0 15 17.0 70.0 Ba =Semi-continuous gradient

Table 3. Differential gene transcription.

* Includes genes with higher calculated fold changes that that were not significant

Table 4. D. andersoni saliva proteins homologous to previously reported immunomodulators

FIGURES

Figure 1: Venn diagram representing the number of transcripts observed at different feeding time points.

Figure 2: Cluster analysis of transcript data. K means clusters were generated using normalized transcript expression values (RPKM). The Y-axis represents normalized expression (RPKM) values

39 while x-axis represents the 3 different feeding time point designated unfed (day 0), early (2-day fed) and late (5-day fed). The cluster number is shown in the top right hand corner of each panel. Clusters

1, 4 and 5 contained transcripts with relative expression patterns of interest, while genes falling into clusters 2 and 3 contained transcripts that were downregulated relative to day 0 and were not of interest.

Figure 3: Bootstrap median gene expression ratios.

The whisker box plots show the comparison between RNAseq data and qPCR data for the zero to day two comparison. The Y-axis represents the median expression ratios while the X-axis represents each gene tested. The boxes and whiskers show the distribution of the permutated ratios. The ratios for 7 out of 10 of the genes are very similar as can be observed from the tight boxes and whiskers.

CHAPTER THREE

EFFECTS OF DERMACENTOR ANDERSONI SALIVA PROTEINS ON MACROPHAGE

IMMUNE FUNCTION IN VITRO

ABSTRACT

Tick saliva proteins have many pharmacological properties including anti-hemostasis and immunomodulation that enable ticks to feed without rejection by the host immune system.

Macrophages are resident in the skin and are among the immune cells that are inhibited by tick saliva proteins. We chose four Dermacentor andersoni saliva proteins that have sequence homology to previously reported immunomodulators and were upregulated during tick feeding for immunomodulation testing. The proteins were expressed using a baculovirus expression system and tested on lipopolysaccharide stimulated macrophage cell lines. A mouse cytokine antibody array was used for detecting expression of multiple cytokines from the conditioned cell culture media. We found that the recombinant D. andersoni saliva proteins upregulated some cytokines and downregulated others, with differential effects on cytokine expression. More interesting, all proteins upregulated the immunosuppressive cytokine IL-10.

INTRODUCTION

Phagocytes (neutrophils, macrophages and dendritic cells) contribute to an effective immune response by engulfing and degrading microbes, as well as presenting parasite-derived molecules

(antigen) to T cells, thus generating a pro-inflammatory and adaptive immune response.

Macrophages are resident in skin and play a central role in all (early and late) stages of wound healing. The inflammatory response can be initiated by macrophages in response to infection and/or

42 tissue damage (Murphy, 2008). Activated macrophages secrete a range of pro-inflammatory cytokines including IL-1β, TNF-α, IL-6, CXCL8, and IL-12. Cytokines recruit immune cells and mediate host immune responses to infection or tissue injury (Saukkonen et al., 1990). The role of the inflammatory response in wound healing is to phagocytize and remove infectious organisms and debris, and release factors responsible for the recruitment and division of cells involved in the proliferative phase and repair of injured tissues (Eming et al., 2007). Additionally, macrophages and

- neutrophils produce toxic products such as nitric oxide (NO) and the superoxide anion (O2 ) that kill phagocytized microorganisms (Murphy, 2008). Tick saliva has been reported to induce expression of the regulatory cytokine IL-10 and inhibit production of the proinflammatory cytokine TNF-α by macrophages (Prevot et al., 2009; Leboulle et al., 2002; Gakwisa et al., 2001). The pharmacological properties of tick saliva proteins are many, but they cannot be exploited for therapeutic use or targeted in anti-tick vaccines until the proteins are well characterized. We previously reported the identification of 677 proteins in Dermacentor andersoni tick saliva, and 157 of these proteins were of interest for immunomodulation testing (Mudenda et al., 2014). In this study, we expressed four proteins using an insect cell expression system and tested them on murine macrophage cell lines to determine which proteins have an effect on the production of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12, macrophage chemotactic protein-1

(MCP-1), MCP-5, etc., and the regulatory cytokine IL-10. The four proteins were selected for immunomodulation testing because they were upregulated during tick feeding and have sequence homology to immunomodulators previously reported in other systems (Ixodes scapularis, Hottentotta judaicus, Dermacentor variabilis, Haemaphysalis longicornis). These proteins include DaSG6931

(serine protease inhibitor), DaSG3273 (protein kinase C inhibitor), DaSG9580 (cystatin) and

DaSG4532 (protein disulfide isomerase-1).

MATERIALS AND METHODS

Protein expression

Total RNA was extracted from 40 pairs of pooled salivary glands from five-day fed ticks using TRI Reagent solution (Applied Biosystems, USA), treated with the TURBO DNA-free kit

(Dnase treatment), and subsequently used for first strand cDNA synthesis with Superscipt VILO cDNA Synthesis Kit (Invitrogen, USA). Primers (Table 1) were then designed to generate full length

PCR products using a thermostable proofreading DNA polymerase. Proteins were subsequently expressed using a baculovirus expression system from Life Technologies, USA, according to the manufacturer’s instructions. Briefly, genes of interest were amplified and their blunt-end PCR products were cloned into a donor C-terminal-His pfastBac /CT-TOPO vector bearing an Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedrin (PH) promoter for expression in insect cells, and then transformed into One Shot® Mach1™ T1R Chemically Competent E. coli for analysis. Following analysis of transformants for correct orientation and reading frame, the recombinant pFastBac™ TOPO® vector (1 ng (5 µl)) was used for transformation into 100 µl

MAX Efficiency DH10Bac™ competent E. coli cells that contain a baculovirus shuttle vector

(bacmid) and a helper plasmid, and allows generation of a recombinant bacmid following transposition of the pFastBac™ expression construct. The recombinant bacmid DNA was then transfected into SF9 insect cells (Invitrogen) to generate baculovirus for subsequent protein expression. For protein expression, SF9 cells were infected with the recombinant bacculovirus at a multiplicity of infection (MOI) of 10 and incubated at 27°C for 72 hours. Expressed histidine-tagged recombinant proteins were analyzed by SDS PAGE and western blotting using Penta-His-specific mouse monoclonal antibody (Life Technologies, USA) as the primary antibody for detection. The recombinant proteins were then affinity purified on nickel columns using the ProBond™ purification

44 system (Invitrogen, USA) and dialyzed against phosphate buffered saline (PBS). The dialyzed samples were concentrated using Vivaspin Turbo 15 centrifugal concentrators (Sartorius Stedim

Biotech, Germany).

Two other proteins; the Ixodes ricinus immunosuppressor protein (Iris) and DaSG6931 were expressed at GenScript USA Inc., similarly, using an insect cell expression system.

Cell Culture

All handling of cell lines was done under sterile conditions in a biological safety cabinet.

Insect Cell Culture

A single vial of SF9 insect cells was removed from liquid nitrogen and thawed in a 37°C water bath by agitating the cryovial until only a small ice crystal was remaining. The cryovial was decontaminated with 70% alcohol and placed in a biological safety cabinet, and then the cells were transferred to a sterile 15 ml conical tube. The cells were then resuspended by drop-wise addition of serum free culture medium (Sf-900™ III SFM from Life Technologies, USA) that was pre-warmed to room temperature while swirling the tube. Next, the cells were seeded into a T-75 flask at a seeding density of 2 × 104–5 × 104 viable cells/cm2. The volume of medium per flask was about 15 -

20 ml. The flask was then placed with loosened caps into a 26–28°C, non-humidified, non-CO2 incubator. The cells were passaged when they were in mid-log phase of growth and about 90% confluent. The seeding density was as above (2 × 104–5 × 104 viable cells/cm2).

Macrophage cell culture (J774.A1 cells)

Mouse macrophage cells (J774.A1) were grown in complete Dulbecco’s Modified Eagle

Medium (cDMEM) with high glucose (4,500mg/L), containing 10% heat inactivated fetal bovine serum and gentamycin. The cells were cultured in 15-20 ml cDMEM contained in T75 tissue culture

45 flasks at a seeding density of 2 x 106 , in a humid incubator with 5% carbon dioxide at 37°C. Seeding density was determined after counting cells with a hemocytometer (Damiani et al., 1980).

The treatment groups included cells cultured in cDMEM with LPS only, LPS with negative control protein (Sf9 insect cell culture supernatant), LPS with Iris (positive control), LPS with salivary gland extract, and LPS with each one of the test proteins DaSG6931, DaSG3273, DaSG9580 and DaSG4532.

Mouse cytokine antibody assay

Murine macrophage cell lines (J774A.1 cells) were pre-incubated at 37°C for 24 hours.

Each well contained 5 x 105 cells in 1 ml cDMEM. After 24 hours, the cells were washed with sterile

PBS and then supplied with 1 ml per well fresh cDMEM. The cells were then treated with one of the following treatments; 1 µg/ml LPS only, 1 µg/ml LPS plus Sf9 insect cell culture supernatant containing 20 µg/ml protein concentration (negative control), 1 µg/ml LPS plus 400 nM Ixodes ricinus immunosuppressant protein (positive control), LPS plus salivary gland extract containing 20

µg/ml protein concentration, and 1 µg/ml LPS plus 400 nM test protein (DaSG6931, DaSG3273,

DaSG9580) and 200 nM DaSG4532. Following treatment, the cells were incubated for another 24 hours at 37°C with 5% CO2. Cell supernatants were then collected from each well, centrifuged, and screened for cytokine expression levels. The cytokine activity in cell supernatants was determined using the RayBio® Mouse cytokine Antibody Array (Cat#AAM-CYT-1) kit. The RayBio Mouse cytokine Antibody Array is a membrane-based antibody array that can be used for screening and comparing expression levels of many cytokines in a single assay. The total number of cytokines screened using this kit was 22 including granulocyte-colony stimulating factor (G-CSF), granulocyte- macrophage colony stimulating factor (GM-CSF), IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-12p40p70,

IL-12p70, IL-13, IL-17, IFN-γ, monocyte chemoattractant protein 1 (MCP-1), MCP-5, Rantes, stem cell factor (SCF), sTNFRI, TNF-α, thrombopoietin (THPO) and vascular endothelial growth factor

(VEGF). Relative amounts of TNF-α, IL-6, IL-12, MCP-1, MCP-5, and IL-10 and other cytokines in the cytokine array were derived by determining the cytokine signal intensities in the cell supernatants using densitometry. Densitometry analysis was performed at RayBiotech, Inc., USA, using the excel based RayBiotech analysis tool. The positive control spots (on the membranes) have a controlled amount of biotinylated antibody printed onto the array and were used for normalizing data when comparing signal intensity between samples, as well as orienting the arrays. Each antibody was spotted on the array in duplicate.

Statistical analysis was performed using two-tailed t-test to compare the differences between

LPS treated samples with those that were treated with LPS plus test proteins, and to compare controls with treated groups. P < 0.05 was considered statistically significant.

RESULTS

Protein expression

Five proteins including 4 test proteins designated DaSG6931, DaSG3273, DaSG9580,

DaSG4532 and a positive control (Iris) were expressed using the baculovirus expression system. SF9 insect cells were used for generation of recombinant baculovirus following transfection with recombinant bacmid DNA, and for subsequent protein expression.

Immunomodulation assay

We compared the cytokine expression levels of cells that were treated with LPS only with those that were treated with LPS and the positive control protein Iris (Fig. 1). We found that Iris significantly (p<0.05) downregulated GM-CSF. While we expected that Iris would upregulate IL-10,

47 it was interesting to observe that a number of other cytokines were also significantly upregulated

(p<0.05) including IL-2, IL-3, IL-4, IL-5, IL-9, IL-12p40/p70, IL-13, IL-17, and IFN-γ. The IL-

12p40 molecule was more highly expressed than the IL-12p70 hetreodimer of IL-12p35/p40. Next, we compared the cytokine expression levels of cells that were treated with LPS only with those that were treated with LPS and DaSG6931 (Fig. 2). DaSG6931 significantly downregulated 1L-12p70, while the cytokines IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12p40, IL-13, and IL-17, were upregulated. The cytokine expression profile induced by DaSG6931 was very similar to that induced by Iris.. The insect cell culture supernatant was used as a negative control because the test proteins in this study were expressed using an insect cell expression system. When the cytokine expression levels of the cells that were treated with LPS and insect cell culture supernatant were compared with those that were treated with LPS only (Fig. 3), the results showed an increase in the number of downregulated cytokines in contrast with both Iris and DaSG6931. The cytokines that were significantly downregulated included IL-2, IL-3, IL-4, IL-9, IL-17, IL-12p70, SCF, and GM-CSF.

Two cytokines were significantly upregulated; sTNFRI and VEGF. Similarly, the cells that were treated with LPS and salivary gland extract downregulated even more cytokines when compared with cells that were treated with LPS only, including GM-CSF, IL-2, IL-12p40, IL-17, IFN-γ, MCP-1,

SCF, TNF-α, and VEGF (Fig. 4). The cytokine IL-6 was significantly upregulated. The cytokines IL-

12p40 (that is part of the IL-12p70 heterodimer as well as IL-23 heterodimer) and IL-10 were significantly upregulated in the cells that were treated with the proteins DaSG3273, DaSG9580 and

DaSG4532. In addition, DaSG3273 and DaSG4532 upregulated TNF-α (p<0.05). The cytokines IL-

2, IL-3, and SCF were significantly downgulated by DaSG3273 (Fig. 5) and DaSG9580 (Fig. 6). In addition, DaSG3273 downregulated IL-12p70, sTNFRI, and VEGF, while DaSG9580 downregulated

IFN-γ and MCP-1. The cytokine IFN-γ was also significantly downregulated by DaSG4532 (Fig. 7).

In order to determine if the effect of the insect cell culture supernatant (negative control) was different from the effect of the three proteins that were expressed in our lab, we compared the cytokine expression results of each of the three proteins with those of the insect cell culture supernatant. We observed significant differences. DaSG3273 had significantly different expression levels for IL-12p40, IL-10, thrombopoietin, TNF-α and VEGF that were significantly upregulated by the protein, and MCP-5, SCF, sTNFRI, TNF-α, and IL-12p70 that were significantly downregulated.

The differences between DaSG9580 and the negative control involved a different set of cytokines than the one listed for DaSG3273 above which included GM-CSF, IL-3, IL-10, IL-12p40 that were significantly upregulated, and IL-5, SCF, STNRI, thrombopoietin and VEGF that were significantly downregulated with protein treatment. The differences for DaSG4532 were also diverse and include

IL-6, IL-12p40, IL-12p70, and IL-13, that were significantly upregulated, and IL-5, MCP-5, sTNFRI,

TNF-α and VEGF that were significantly downregulated.

DISCUSSION

A number of tick saliva proteins with immunomodulatory properties have been previously identified including serine protease inhibitors (serpins), cystatins, chemokine binding proteins, protein kinase inhibitors, etc. (Prevot et al, 2009; Oliveira et al., 2010; Horka et al., 2012). Serpins are widely distributed in living organisms including humans, arthropods, nematodes, plants, viruses, etc. (Irwin et al., 2000; Gettins, 2002). They belong to a superfamily of proteins with a primary structure similarity within a region of approximately 350 residues that corresponds to a conserved tertiary structural domain (Gettins, 2002). Serpins are found intracellularly as well as extracellularly and have diverse functions that include but are not limited to proteinase inhibition (Silverman et al.,

2001). Differences in the patterns of sequence conservation of serpins within phylogenetically

49 distinct groups can be correlated with the divergence of structure and function (Irwin et al., 2000).

Ixodes ricinus immunosuppressor (Iris) is a serine protease inhibitor (Serpin) found in the saliva of

Ixodes ricinus ticks. Iris is reported to interfere with both the host immune response and hemostasis.

We chose Iris as our positive control because it was previously reported to bind to monocytes/macrophages and inhibit secretion of TNF-α (Prevot et al., 2009). In agreement with this trend, in our study Iris slightly reduced the expression levels of TNF-α and another pro-inflammatory cytokine, MCP-1 (Fig.1) even though the difference was not statistically significant. Additionally,

Iris upregulated a number of signaling molecules including the cytokines IL-4, IL-5, and IL-13 that favor the differentiation of T cells to a TH2 phenotype (Murphy et al., 2008). This observation is in agreement with literature as tick saliva has previously been reported to promote the development of

TH2 responses (Skallova et al., 2008). The effect of Iris on the LPS stimulated mouse cell lines was similar to that of DaSG6931 which is also a serine protease inhibitor. Both Iris and the serine protease inhibitor DaSG6931 significantly upregulated IL-17 while IL-6 was also upregulated, although not significantly. Il-17 induces the production of proinflammatory cytokines including IL-6,

G-CSF, and TNF-α (Onishi and Gaffen, 2010). Previous reports have shown that Salp 1, an Ixodes scapularis saliva protein that modulates CD4+ T cell activation by inhibiting IL-2 (Anguita et al.,

2002) is able to induce a TH17 response in the presence of IL-6 when IL-2 is repressed (Juncadella et al., 2014). However, in the presence of IL-2, IL-6 induces a TH2 response. In this study, the control protein Iris and DaSG6931 induced an increase in both IL-6 and IL-2, thereby potentially promoting a TH2 response in agreement with previous reports that tick saliva proteins induce a TH2 response

(Skallova et al., 2008). Consistent with a TH2 response, IL-4, IL-5 and IL-13 were also upregulated.

The rest of the proteins that were tested had appreciably different effects from what was observed for Iris and the serine protease 1 inhibitor protein DaSG6931. The effects of the proteins

DaSG3273, DaSG9580 and DaSG4532 were similar to salivary gland extract than the positive

50 control protein Iris. This difference may be attributed to the diverse signaling pathways used by saliva proteins to modulate the responses of immune cells (Lieskovska and Kopecky, 2012).

We selected and tested DaSG3273, a protein that is homologous to protein kinase C inhibitor because the protein kinase C family of proteins play important roles in signaling pathways that are important for cellular growth, differentiation, homeostasis, and immune responses (Beltman et al.,

1996; Weerd et al., 2014). We found that the effect of this protein on LPS-stimulated murine macrophage cell lines was similar to that of cystatin. Cystatins are yet another large family of proteins with diverse biological activities (Zhou et al.2006). They are cysteine protease inhibitors that inhibit proteases involved in antigen processing (Karim et al., 2005). An Ixodes scapularis tick cystatin, Sialostatin L that is effective in the prevention of experimental asthma inhibits IL-2, IL-9 and the TH2 cytokines IL-4, IL-5, and IL-13 (Horka et al., 2012). In agreement, we found that the protein DaSG9580 that has sequence homology to cystatin inhibits all of these cytokines. We also chose to test DaSG4532, which has homology to protease disulphide isomerase (PDI) because it was upregulated during tick feeding, a role for this protein in tick feeding has not yet been demonstrated.

Reports from several systems suggest that PDI is immunogenic and therefore promotes inflammation. However, one report suggests that PDI regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vascular smooth muscle and endothelial cells, and that this regulation is thiol redox-dependent (Laurindo et al., 2008). NADPH oxidase plays an important role in innate immunity through generation of reactive oxygen species that are microbicidal (Segal, 2008).

In this study, we did not test for inhibition of reactive oxygen species, but we did observe that the protein DaSG4532 that has sequence homology to PDI upregulated IL-10, as well as IL-12p40 whereas it significantly downregulated the proinflammatory cytokine IFN-γ. This shows that PDI may play an immunomodulatory role during tick feeding.

As expected, all proteins that were tested upregulated IL-10. The cytokine IL-10 suppresses immune responses through various mechanisms that act differently for different types of antigen

51 presenting cells (Mittal and Roche, 2015). All tested proteins also upregulated IL-12p40 while IL-

12p70 alone was either unchanged or downregulated. This is consistent with antibody binding to the

IL-12p40 molecule rather than the IL-12p70 heterodimer. Since IL-12p40 is also a constituent of IL-

23, this could also account for the increase in anti-IL-12p40 antibody binding (the cytokine array kit did not test for IL-23). Previous reports have shown that the IL-12p40 homodimer is antagonistic to

IL-12 activity when secreted by antigen presenting cells in the absence of p35 (Kalinski et al., 2001).

This significant increase of IL-12p40 induced by all tick proteins suggests that the tick saliva proteins upregulate the homodimer IL-12p40 to inhibit IL-12 activity and the subsequent TH1 responses. This theory should be investigated further in future experiments.

We found it surprising that the proteins DaSG3273, DaSG9580 and DaSG4532 increased the expression of TNF-α despite an increase in the regulatory cytokine IL-10. Similarly, while the immunoregulatory cytokine IL-10 and TH2 cytokines were upregulated, the pro-inflammatory cytokine IFN-γ was also significantly upregulated by Iris More work will be done in future to verify this finding.

REFERENCES

Anguita, J., Ramamoorthi, N., Hovius, J.W.R., Das, S., Thomas, V., Persinski, R., Conze, D.,

Askenase, P.W., Rincón, M., Kantor, F.S., and E. Fikrig. 2002. Salp15, an Ixodes scapularis

Salivary Protein, Inhibits CD4+ T Cell Activation. Immun. 16:849–859.

Beltman, J., McCormick, F., and Cook, S.J. 1996. The Selective Protein Kinase C Inhibitor, Ro-31-

8220, Inhibits Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression,

Induces c-Jun Expression, and Activates Jun N-terminal Kinase. J. Biol. Chem.

271(43):27018 –27024.

Damiani, G., C. Kiyotaki, W. Soeller, M. Sasada, J. Peisach and B. R. Bloom. 1980.

Macrophage variants in oxygen metabolism. J. Exp. Med. 152: 808-822. de Weerd, A., Kho, M., Kraaijeveld, R., Zuiderwijk, J., Weimar, W., and Baan, C. 2014. The protein

kinase C inhibitor sotrastaurin allows regulatory T cell function. Clin Exp Immunol.

175(2): 296–304.

Eming S. A., T. Krieg, and J. M. Davidson. 2007. Inflammation in Wound Repair: Molecular

and Cellular Mechanisms. J. Invest. Dermatol. 127:514–525.

Gettins, Peter G. W. 2002. Serpin Structure, Mechanism, and Function. Chem. Rev.

102: 4751-4803.

Horka, H., Staudt, V., Klein, M., Taube, C., Reuter, S., Dehzad, N., Andersen, J.F., Kopecky,

J., Schild, H., Kotsyfakis, M., Hoffmann, M., Gerlitzki, B., Stassen, M., Bopp, T., and E.

Schmitt. 2012. The Tick Salivary Protein Sialostatin L Inhibits the Th9-Derived Production

of the Asthma-Promoting Cytokine IL-9 and Is Effective in the Prevention of Experimental

Asthma. J. Immunol. 188(6): 2669-2676

Irving, J. A, Pike, R. N, Lesk, A. M., Whisstock, J. C. 2000. Phylogeny of the serpin

superfamily: implications of patterns of amino acid conservation for structure and

function. Genome Res. 10(12):1845-64.

Juncadella, I.J., Bates, T.C., Suleiman, R., Monteagudo-Mera, A., Olson Jr, C.M., Navasa, Olivera,

E.R., Osborne, B.A.,and J. Anguita. 2010. The tick saliva immunosuppressor, Salp15,

contributes to Th17- induced pathology during Experimental Autoimmune

Encephalomyelitis. Biochem. Biophys. Res. Commun. 402(1): 105–109.

Kalinski, P., Vieira, P.L., Schuitemaker, J.H.N., de Jong, E.C., and M.L. Kapsenberg. 2001.

Prostaglandin E2 is a selective inducer of interleukin-12 p40 (IL-12p40) production and an

inhibitor of bioactive IL-12p70 heterodimer. Blood 97: 3466-3469.

Karim, S., Miller, N.J., Valenzuela, J., Sauer, J.R., Mather, T.N., 2005. RNAi-mediated gene

silencing to assess the role of synaptobrevin and cystatin in tick blood feeding. Biochem.

Biophys. Res. Commun. 334:1336–1342.

Langhansová, H., Bopp, T., Schmitt, E., and J. Kopecky. 2015. Tick saliva increases production

of three chemokines including monocyte chemoattractant protein-1, a histamine-releasing

cytokine. Parasite Immunol. 37: 92–96.

Laurindo, F.R.M., Fernandes, D.C., Amanso, A.M., Lopes, L.R., and C.X. C. Santos. 2008. Novel

Role of Protein Disulfide Isomerase in the Regulation of NADPH Oxidase Activity:

Pathophysiological Implications in Vascular Diseases. Antioxidants & Redox Signaling. Vol.

10, Number 6.

Lieskovska, J., and J. Kopecky. 2012. Effect of tick saliva on signalling pathways activated by TLR-

2 ligand and Borrelia afzelii in dendritic cells. Parasite Immunol. 34: 421–429.

Mittal, S.K., and P.A. Roche. 2015. Suppression of antigen presentation by IL-10. Current Opinion in

Immunol. 34:22-27.

Mudenda, L., Pierlé, S. A., Turse, J.E., Scoles G.A., Purvine S.O., Nicora C.D., Clauss T.R.,

Ueti M.W., Brown W.C., and K.A Brayton. 2014. Proteomics informed by

transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva. Int. J

Parasitol. 44(13):1029-37.

Murphy, K., Travers, P., and M. Walport. 2008. Innate immunity. In: Lawrence, E. (Ed.),

Janeway’s Immuno biology. Garland Science, New York and London, pp. 39–108 and

555-592.

Oliveira, C.J., Carvalho, W.A., Garcia, G.R., Gutierrez, F.R., de Miranda, S.I.K., Silva, J.S., and B.R

Ferreira. 2010. Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like

receptor-2 expression as potential targets. Vet Parasitol. 167(2-4):288-97.

Onishi, R.M., and S.L., Gaffen. 2010. Interleukin-17 and its target genes: mechanisms of interleukin-

17 function in disease. Immunol. 129(3): 311–321.

Prevot, P., A. Beschin, L. Lins, J. Beaufays, A. Grosjean, L. Bays, B. Adam, M. Brossard, R.

Basseur, K. Z. Boudjeltia, L. Vanhamme, and E. Godfroid. 2009. Exosites mediate the

anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes

ricinus. FEBS J. 276:3235-3246.

Saukkonen, B., S. Sande, C. Cioffe, S. Wolpe, B. Sherry, A. Cerami and E. Tuomanen. 1990.

The role of cytokines in the generation of inflammation and tissue damage in

experimental gram-positive meningitis. J. Exp. Med.171:439-448.

Segal, A.W. 2008. The function of the NADPH oxidase of phagocytes and its relationship to other

NOXs in plants, invertebrates, and mammals. Int. J. Biochem. Cell. Biol. 40(4-3): 604–

618.

Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin, P.B., Gettins, P.G., Irving,

J.A., Lomas, D.A., Luke, C.J., Moyer, R.W., Pemberton, P.A., Remold-O’Donnell, E.,

Salvesen, G.S., Travis, J., Whisstock, J.C., 2001. The serpins are an expanding

superfamily of structurally similar but functionally diverse proteins: evolution,

mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem.

276: 33293–33296.

Skallová, A., Iezzi, G., Ampenberger, F., Kopf, M., and J. Kopecky. 2008. Tick Saliva Inhibits

Dendritic Cell Migration, Maturation, and Function while Promoting Development of Th2

Responses. J Immunol. 180:6186-6192.

Zhou, J., Ueda, M., Umemiya, R., Battsetseg, B., Boldbaatar, D., Xuan, X., and K. Fujisaki. 2006.

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression

patterns in relation to innate immunity. Insect Biochem. Mol. Biol. 36:527–535

TABLES

Table 1 Primers used for protein expression

Gene Forward primer Reverse Primer DaSG3273 (Protein Kinase C CGA TGG CTA CCG AGG TTC AGA GCC CGG AGG CCA GCC inhibitor) DaSG9580 (Cystatin) GAT GTG GAG GCG AAG CTG AAC CTG GAA ATG CTC GAG TGT GTC TTC C AAG DaSG4532 (disulfide ATG CCT TTG GCT GGT AGA GTT AAG TTC AAC ATG CTT CTT GAC CTG isomerase 1) CTT TGC GTC T

FIGURES

70,000,000.00 LPS only vs LPS + Iris

60,000,000.00

50,000,000.00

40,000,000.00

30,000,000.00 * * * Signal intensity Signal 20,000,000.00 * * * LPS only * * * * LPS + Iris 10,000,000.00 * 0.00

Cytokine-specific antibodies

Figure 1 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and Iris.

70,000,000.00 LPS only vs LPS + DaSG6931

60,000,000.00

50,000,000.00

40,000,000.00

30,000,000.00 * * LPS only

Signal Signal intensity 20,000,000.00 * * * LPS + DaSG6931 * * * 10,000,000.00

0.00

Pos

SCF

IL-5 IL-2 IL-3 IL-4 IL-6 IL-9

Neg

IL-10 IL-13 IL-17

GCSF

VEGF

TNF-a

MCP-1 MCP-5

RANTES

sTNFRI

GM-CSF

IL-12p70

IFN-gamma

IL-12p40p70 Thrombopoietin Cytokine-specific antibodies Figure 2 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and DaSG6931

70,000,000.00 LPS only vs LPS + insect cell supernatant

60,000,000.00

* 50,000,000.00

40,000,000.00

30,000,000.00 LPS only Signal Signal intensity LPS + Insect cell 20,000,000.00

10,000,000.00 * * * * * * * 0.00 *

Cytokine-specific antibodies Figure 3 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and Insect cell culture supernatant.

70,000,000.00 LPS only vs LPS + Salivary gland extract

60,000,000.00 * 50,000,000.00 *

40,000,000.00

30,000,000.00 Signal Signal intensity 20,000,000.00 LPS only LPS + SG extract 10,000,000.00 * * * * * * * *

0.00

Pos

SCF

IL-2 IL-3 IL-4 IL-5 IL-6 IL-9

Neg

IL-10 IL-13 IL-17

GCSF

VEGF

TNF-a

MCP-1 MCP-5

RANTES

sTNFRI

GM-CSF

IL-12p70

IFN-gamma

IL-12p40p70 Thrombopoietin Cytokine-specific antibodies

Figure 4 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and Salivary gland (SG) extract.

70,000,000.00 LPS only vs LPS + DaSG3273

60,000,000.00

50,000,000.00

40,000,000.00

30,000,000.00 * Signal Signal intensity LPS only 20,000,000.00 * * LPS + DaSG3273 10,000,000.00 * * * * *

0.00 *

Pos

SCF

IL-6 IL-2 IL-3 IL-4 IL-5 IL-9

Neg

IL-10 IL-13 IL-17

GCSF

VEGF

TNF-a

MCP-1 MCP-5

RANTES

sTNFRI

GM-CSF

IL-12p70

IFN-gamma

IL-12p40p70 Thrombopoietin Cytokine-specific antibodies

Figure 5 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and DaSG3273.

70,000,000.00 LPS only vs LPS + DaSG9580

60,000,000.00 *

50,000,000.00

40,000,000.00 *

30,000,000.00 Signal Signal intensity LPS only 20,000,000.00 * * LPS + DaSG9580 10,000,000.00 * * *

0.00 *

Pos

SCF

IL-5 IL-2 IL-3 IL-4 IL-6 IL-9

Neg

IL-10 IL-13 IL-17

GCSF

VEGF

TNF-a

MCP-1 MCP-5

RANTES

sTNFRI

GM-CSF

IL-12p70

IFN-gamma

IL-12p40p70 Thrombopoietin Cytokine-specific antibodies

Figure 6 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and DaSG9580.

70,000,000.00 LPS only vs LPS + DaSG4532

60,000,000.00

50,000,000.00

40,000,000.00

30,000,000.00 Signal Signal intensity LPS only 20,000,000.00 LPS + DaSG4532

10,000,000.00 * * *

0.00

Pos

SCF

IL-6 IL-2 IL-3 IL-4 IL-5 IL-9

Neg

IL-10 IL-13 IL-17

GCSF

VEGF

TNF-a

MCP-1 MCP-5

RANTES

sTNFRI

GM-CSF

IL-12p70

IFN-gamma

IL-12 p40p70 Thrombopoietin Cytokine-specific antibodies

Figure 7 Cytokine expression profile of cells that were treated with LPS only compared with cells that were treated with LPS and DaSG4532.