ABSTRACT TOKARZ, DEBRA ANN. Tripartite Motif Containing 9 As A

ABSTRACT

TOKARZ, DEBRA ANN. Tripartite Motif Containing 9 as a Novel Innate Immune Response Gene and Mediator of Macrophage Migration. (Under the direction of Dr. Jeffrey Yoder).

Innate immunity provides the first line of defense against invading pathogens.

Pathogens are sensed by pattern recognition receptors (PRRs) present on cells throughout the

body, which recognize diverse pathogen associated molecular patterns (PAMPs). Activation

of PRRs by PAMPs turns on the expression of numerous genes that regulate innate immune

responses. A critical component of this response is the recruitment and activation of

macrophages and neutrophils to sites of infection and inflammation. These phagocytic cells

are highly adept at engulfing and killing pathogens, as well as recruiting additional

inflammatory cells. However, excessive or prolonged infiltration of tissues by neutrophils

and macrophages is implicated in the pathogenesis of a variety of inflammatory disorders.

Therefore, understanding how migration of these leukocytes is regulated will facilitate the design of novel therapeutic strategies for modulating inflammation. Recently, we identified tripartite motif containing 9 (trim9) as a gene with increased transcript levels in larval zebrafish following exposure to different PAMPs. TRIM9 is a member of the tripartite motif family of RING E3 ubiquitin ligases. TRIM9 is highly expressed in neurons and, until recently, was considered to be brain specific. We find that TRIM9 is expressed in larval zebrafish neutrophils and macrophages and in human myeloid cell lines, and responds transcriptionally to PAMP stimulation in these cells. In neurons, TRIM9 serves as an intracellular mediator of axon migration in response to the chemoattractant Netrin, a role conserved in invertebrates and mammals. We hypothesized that TRIM9 may act as a mediator of migration in neutrophils and macrophages during the immune response. Using mosaic transgenic zebrafish larvae we show that disrupting Trim9 ubiquitin ligase activity in macrophages alters cell morphology and significantly reduces in vivo macrophage migration.

Our findings support a role for TRIM9 in mediating migration in cell types other than neurons, and suggest that ubiquitination of TRIM9 substrates may be critical for this function in macrophages. Identifying the TRIM9 protein interaction network in macrophages will further elucidate the mechanisms of TRIM9 function within these cells and reveal new potential targets for modulating leukocyte migration and inflammation.

by Debra Ann Tokarz

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Comparative Biomedical Sciences

Raleigh, North Carolina

2016

APPROVED BY:

______Dr. Jeffrey A. Yoder Dr. Samuel Jones Committee Chair

______Dr. Matthew Koci Dr. Barbara Sherry

DEDICATION

To my parents, for their faith in me

and

To my husband, for his patience

BIOGRAPHY

Debra Tokarz was raised in southeast Michigan by her parents, Ray and Sandi, along

with her younger brother J.R. and her older half-siblings Rebecca, Bryant, Andrew, Matt,

Kristopher, and James. Having been preceded in the world by her hell-raising eldest brothers

who brought notoriety to the Tokarz name in her home town of St. Clair Shores, Debra was

forced to become an exceptionally well-behaved child. This she accomplished by spending most of her time with her nose in books, a habit that lead to extremely thick glasses and early aspirations of becoming a writer. However, she fell in love with biology in junior high, and hasn’t looked back since.

Debra attended college at the University of Michigan in Ann Arbor, MI, where she earned a B.S. in Cell and Molecular Biology. While there she worked as a research assistant in the laboratory of Dr. John Fink, where she helped analyze genes for mutations associated with inherited neurologic disorders. So began her interest in biomedical research, which continues to this day.

Forced to come to grips with choosing an actual career path as college came to an end, she fell back on her love of animals and chose to attend veterinary school at Michigan

State University. Through her courses at vet school and through summer research projects,

Debra was introduced to veterinary pathology and chose to specialize in this area. She completed a residency in anatomic veterinary pathology at the Veterinary Medical Teaching

Hospital at the University of California-Davis and was boarded in September 2011.

iii

During her pathology residency, Debra learned about host-pathogen interactions and the immune response. She decided she wanted to learn more and chose to do her PhD training at North Carolina State University in the lab of Dr. Jeff Yoder. In the Yoder lab,

Debra has enjoyed doing great research with great people and a great model organism, the zebrafish. Upon completing her PhD training, she is very excited to be staying on at NC State as a comparative veterinary pathologist.

ACKNOWLEDGMENTS

I am incredibly grateful for the outstanding mentorship and guidance provided by my

advisor, Dr. Jeff Yoder. I am also grateful for the help and insight provided by my committee

members: Dr. Sam Jones, Dr. Barbara Sherry, and Dr. Matthew Koci. I am also thankful for funding support provided by the NCSU Comparative Medicine and Translational Research

Training Program (NIH T32 OD011130) and the American Association of Immunologists

Careers in Immunology Fellowship.

To all the members of the Yoder lab – Ivan, Hayley, Jess, Dustin, Amanda, Amyn,

Ashley – you made it a joy to come to lab each day! Many others shared their technical expertise with me throughout this work and I am grateful for their help, especially Shannon

Chiera, Dr. Katie Sheats, Dr. Stephanie Gupton, and Dr. Eva Johannes.

I would never be where I am today without the love and support of my family,

especially my parents, Ray and Sandi Tokarz, who have stood by me 100% in all of my

endeavors. Finally, I cannot express enough how thankful I am for my husband, Kip. His

love, his patience, and his humor have all been a source of strength for me through all of my

training. He truly deserves a degree for each one he has seen me through!

TABLE OF CONTENTS

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

INTRODUCTION...... 1

CHAPTER 1: LITERATURE REVIEW ...... 4

The Innate Immune Response ...... 4

Pattern Recognition Receptor Signalling ...... 4

Toll-like Receptors (TLRs) ...... 5

Retinoic Acid-Inducible Gene I-Like Receptors (RLRs) ...... 8

Nucleotide Binding Domain and Leucine Rich Repeat Containing

Receptors (NLRs) ...... 10

The inflammatory response ...... 14

Macrophage and Neutrophil Migration...... 16

Chemotaxis: a Primer ...... 17

Extravasation...... 18

Intracellular Signaling and Cytoskeletal Dynamics of Chemotaxis ...... 23

The Zebrafish as a Model of Innate Immunity ...... 26

Tripartite Motif (TRIM) Protein Family ...... 28

Ubiquitination ...... 29

TRIMs and Innate Immunity ...... 31

Tripartite Motif Containing 9 (TRIM9) ...... 33

References ...... 43

CHAPTER 2: DISRUPTION OF TRIM9 FUNCTION ABROGATES MACROPHAGE

MOTILITY IN VIVO ...... 71

Abstract ...... 73

Introduction ...... 74

Materials and Methods ...... 77

Results ...... 81

Array results...... 81

Trim9 transcript levels increase after immune stimulation ...... 83

Disruption of Trim9 function results in defective macrophage chemotaxis ...... 84

Disruption of Trim9 alters macrophage morphology and motility ...... 85

Discussion...... 86

References ...... 98

Supporting Information ...... 104

SI Materials and Methods ...... 104

SI References ...... 116

CHAPTER 3: CONCLUSIONS AND FUTURE DIRECTIONS ...... 117

References ...... 122

APPENDIX ...... 124

TRIM9 Expression in Neutrophils ...... 125

Introduction...... 125

Materials and Methods ...... 127

vii

Results ...... 130

Neutrophils specifically express the short isoform of TRIM9 ...... 130

Immune agonists induce changes in Trim9 transcript levels in neutrophils ...... 131

Discussion ...... 132

References ...... 134

viii

LIST OF TABLES

CHAPTER 1

Table 1. Human pattern recognition receptors and their ligands...... 38

APPENDIX

Table 1. Taqman assays used for quantitative RT-PCR...... 137

Table 2. RACE primers and conditions...... 137

LIST OF FIGURES

CHAPTER 1

Fig. 1. PRR stimulation initiates intracellular signaling pathways that lead to

transcription factor activation...... 41

CHAPTER 2

Fig. 1. Immune agonist exposure and summary of microarray results...... 90

Fig. 2. Immune agonist exposure induces increased trim9 transcript levels in

macrophages...... 92

Fig. 3. Macrophage-specific disruption of Trim9 function results in reduced cellular

chemotaxis in vivo...... 93

Fig. 4. In vivo disruption of Trim9 function in macrophages significantly alters cell

shape...... 95

Fig. 5. In vivo disruption of Trim9 function in macrophages significantly disrupts

macrophage velocity...... 96

Fig. S1. Heat map of 1110 genes with significantly different transcript levels (p < 0.05)

after exposure to either Pam3CSK4 or PolyIC at any time point...... 107

Fig. S2. Distribution of genes with significantly altered transcript levels during

Pam3CSK4 exposure...... 109

Fig. S3. Distribution of genes with significantly altered transcript levels during

PolyIC exposure...... 110

Fig. S4. Heat map of gene expression data for subgroups of genes responsive

primarily to PolyIC or Pam3CSK4...... 111

Fig. S5. Activation of CXCR4 pathway...... 112

Fig. S6. Activation of the Inflammatory Network...... 113

Fig. S7. Transcript level changes for genes of the Inflammatory Network...... 114

Fig. S8. In vivo disruption of Trim9 function in macrophages significantly alters cell

shape...... 115

APPENDIX

Fig. 1. Neutrophils express a single detectable isoform of TRIM9...... 138

Fig. 2. Immune agonist exposure does not alter the TRIM9 isoform profile in

DMSO-differentiated HL60 cells...... 139

Fig. 3. Neutrophil TRIM9 transcripts encode short isoforms of TRIM9...... 140

Fig. 4. Immune agonist exposure induces changes in trim9 transcript levels in larval

neutrophils...... 142

Fig. 5. Immune agonist exposure induces changes in TRIM9 transcript levels in

human neutrophil-like HL60 cells...... 143

INTRODUCTION

Vertebrate immune systems are classically considered as comprised of two arms:

innate immune responses and adaptive immune responses. Innate immune responses are

characterized by constitutive and rapidly-inducible (within minutes to hours) defenses that

have limited pathogen specificity. Adaptive immune responses can take several days to be

fully effective for a primary response, but provide highly pathogen specific immunity.

Furthermore, genetic alterations in lymphocytes, the effector cells of the adaptive immune response, generate long-lived, antigen-specific cell subsets, thereby providing immunological

“memory”. Both arms contribute in providing a fully effective immune response, but innate

immune responses provide the first line of defense against invading pathogens and signal for

induction of adaptive immunity.

Innate immune defenses are provided by barrier surfaces, such as skin and mucous

membranes, along with a contigent of host immune cells made up of granulocytes

(neutrophils, eosinophils, basophils), mast cells, monocytes, macrophages, dendritic cells,

and natural killer cells. These cells specialize in detection and elimination of pathogens and

virally-infected and cancerous cells. Among these cells types, neutrophils and

monocytes/macrophages are the major innate immune effector cells. They are phagocytic,

allowing them to engulf pathogens and other cells. Neutrophils make up the largest

percentage of circulating white blood cells in humans (Frazier and Drzymkowski 2016), and

are the first cells recruited from the blood to sites of infection in tissues (Soehnlein, Lindbom

and Weber 2009). Tissue resident macrophages are often the first immune cells to encounter

invading pathogens, and are major producers of pro-inflammatory molecules (Duque 2014).

Monocytes circulating in the blood are also recruited into tissues where they differentiate into macrophages and bolster the inflammatory response (van Furth and Cohn 1968, Ginhoux and

Jung 2014). Macrophages also act as a bridge linking activation of the innate and adaptive immune responses through antigen presentation to lymphocytes and cytokine production

(Rosenthal and Shevach 1973, Medzhitov and Janeway 1997).

Innate immune responses are controlled by a complex network of intracellular and intercellular signals. Molecular signals derived from pathogens or damaged host tissues are sensed by receptors on leukocytes and other cells. In response, a host of immune-related genes are expressed and effect the recruitment and activity of leukocytes in the tissues. While migration of neutrophils and macrophages into tissues is critical for host defense, excessive infiltration can damage tissues or perpetuate inflammation and is implicated in an array of acute and chronic inflammatory disorders (Chávez-Sánchez, et al. 2014, Steinberg, et al.

1994, Kinne, et al. 2000). Modulating neutrophil and macrophage migration is among the strategies for preventing and treating such disorders. Neutrophils and macrophages are guided to sites of inflammation by extracellular chemical signals called chemoattractants.

Drug therapies that block chemoattractant receptors have yielded mixed results due to the array of chemoattractants that may be active during inflammation (Russo, Garcia and Teixera

2010, Horuk 2009). Identifying novel intracellular mediators of leukocyte migration may yield new targets for modulating inflammation.

We recently identified a gene, tripartite motif containing 9 (trim9), that is upregulated in larval zebrafish during the innate immune response (Heffelfinger 2010). TRIM9 is a

member of the tripartite motif protein family, which is characterized by having amino terminal really interesting new gene (RING), Bbox, and Coiled-coil domains. The RING domain imparts E3 ubiquitin ligase activity (Joazeiro and Weissman 2000). The presence of various carboxy terminal domains in TRIM proteins can impart additional functions (Short and Cox 2006). TRIM9 has recently been identified as a modulator of the intracellular signals that lead to transcriptional activation during the innate immune response (Qin, et al.

2016, Shi, et al. 2014). However, in neurons, TRIM9 mediates intracellular signals that guide axon migration (Alexander, et al. 2010, Hao, et al. 2010, Song, et al. 2011, Winkle, et al.

2014). We hypothesized that TRIM9 may also act as an intracellular mediator of leukocyte migration. We utilized mosaic transgenic zebrafish and a novel in vivo migration assay to test this hypothesis in zebrafish macrophages.

CHAPTER 1: LITERATURE REVIEW

The Innate Immune Response

Innate immune responses provide the first lines of defense against invading pathogens.

Immediate defenses are provided through physical barriers, such as skin and mucous membranes, as well as constitutively produced cellular products, such as antimicrobial peptides present in barrier surface secretions. Additional molecular and cellular innate

immune responses can be rapidly induced within minutes to hours if pathogens breach these

initial barriers. The sensing of pathogens by cellular receptors signals the production of pro-

inflammatory cytokines and chemokines that in turn lead to recruitment and activation of

innate immune effector cells, of which macrophages and neutrophils are the predominant cell

types. The phagocytic capability of macrophages and neutrophils enables them to engulf, kill,

and process pathogens, making them critical to pathogen containment as well as further

inflammatory cell recruitment. Equally critical is that the activities of these cells are closely regulated to prevent excessive inflammation which can damage host tissues.

Pattern Recognition Receptor Signaling

Pattern recognition receptors (PRRs) are germline-encoded sensors, present on cell surfaces and within the cytoplasm, that provide a means of recognizing pathogens (Janeway

1989). PRRs sense pathogen associated molecular patterns (PAMPs) that are present in

bacteria, viruses, fungi and protozoa, but are absent or compartmentalized in host cells

(Mehdzitov 2000, Mogensen 2009). There are several families of PRRs in vertebrates, of which the best characterized are the toll-like receptors (TLRs), retinoic acid inducible gene I- like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD) and leucine rich repeat containing receptors (NLRs). TLRs are expressed on the cell surface or the lumenal surface of endosomes (Nishiya and DeFranco 2004), while NLRs and RLRs are cytoplasmic receptors (Inohara, Ogura and Nunez 2002, Yoneyama, Kikuchi and Matsumoto, et al. 2005). The binding of TLRs and RLRs by their respective ligands turns on intracellular signaling cascades that lead to activation of the NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) and IRF (interferon regulatory factor) transcription factors. NF-kB and IRFs stimulate expression of pro-inflammatory genes and interferons, respectively, that are necessary for an effective immune response (Pahl 1999, Honda and

Taniguchi 2006). Activation of NLRs is also implicated in modulating NF-κB and IRF activity, as well as initiating activation of a protein complex called the inflammasome that mediates maturation of pro-inflammatory cytokines.

Toll-like Receptors (TLRs)

Humans have 10 TLRs, each recognizing distinct ligands (Table 1) (Chuang and Ulevitch

2001, Chuang and Ulevitch 2000, Rock, et al. 1998, Takeuchi, Kawai and Sanjo, et al. 1999).

TLRs show broad tissue expression, although the highest levels of TLR expression tend to occur in leukocytes (Zarember and Godowski 2002). TLR1, TLR2, TLR4, TLR5, and TLR6

are present at the cell surface, enabling them to sense extracellular pathogens (Nishiya and

DeFranco 2004). TLR4 has been widely studied for its role in binding lipopolysaccharide

(LPS), a component of gram-negative bacterial cell walls (Chow, et al. 1999). A variety of

microbe-derived lipoproteins and glycoproteins are recognized by TLR2, alone or in

combination with TLR1 or TLR6 (Aliprantis, et al. 1999, Schwandner, et al. 1999, Hajjar, et

al. 2001, Takeuchi, Sato, et al. 2002, Ozinsky, et al. 2000, Takeuchi, Kawai and Mühlradt, et

al. 2001). TLR5 binds the bacterial protein flagellin (Hayashi, et al. 2001). Nucleic acids are

sensed by the endosomally located TLR3, TLR7, TLR8, and TLR9. TLR3 and TLR7/8 bind

viral-derived double-stranded and single-stranded RNA, respectively (Alexopolou, et al.

2001, Heil, et al. 2004). TLR9 recognizes unmethylated CpG DNA, which is abundant in

microbial genomes, but infrequent in mammalian DNA (Hemmi, et al. 2000). The ligand for

TLR10 has not been identified.

TLRs bind ligands via leucine rich repeats and dimerize to initiate a series of downstream signaling cascades (Botos, Segal and Davies 2011). Toll IL-1 Receptor (TIR) domains in the cytoplasmic tail of TLRs interact with TIR-domain containing adaptor molecules to activate two possible pathways: a MyD88 (myeloid differentiation primary-response protein 88)- dependent pathway utilized by all TLRs except TLR3, and a MyD88-indepent pathway utilized by TLR3 and TLR4 (Fig. 1) (Akira, Hoshino and Kaisho 2000, Oshiumi, et al. 2003,

Yamamoto, Sato, et al. 2003, Kawai, Takeuchi, et al. 2001).

In the MyD88-dependent pathway, activated MyD88 recruits IRAKs (IL-1 receptor- associated kinases) that in turn activate TRAF6 (TNF-receptor associated factor 6) (Gohda,

Matsumura and Inoue 2004, Wang, et al. 2001, Wensche, et al. 1997, Suzuki, et al. 2002).

TRAF6 activates 2 key downstream signaling proteins via ubiquitination: TAK1

(transforming growth factor activated protein kinase 1; also known as MAP3K7) and NEMO

(NF-κB essential modifier; also known as IKKγ or IKBKG) (Sun, et al. 2004, Wang, et al.

2001). TAK1 in turn activates the inhibitor of NF-κB (IκB)-kinase (IKK) complex, which consists of NEMO along with IKKα and IKKβ (Wang, et al. 2001). The NF-κB family consists of five members that function as homo- and heterodimers. NF-κB dimers are kept in an inactive state in the cytoplasm via binding to members of the IκB family. Activated IKK complex phosphorylates IκB proteins, which are then degraded, freeing NF-κB to translocate to the nucleus (Brown, et al. 1993). In parallel, TAK1 can also phosphorylate members of the

MAPKK (mitogen-activated protein kinase kinase) family, which drive a separate signaling cascade leading to activation of additional pro-inflammatory transcription factors

(Moriguchi, et al. 1996, Wang, et al. 1997, Chang and Karin 2001).

In the MyD88-independent pathway used by TLR3 and TLR4, the adaptor protein TRIF

(TIR-domain containing adaptor inducing IFN-β) activates two distinct signaling cascades

(Fig. 1). TRIF interaction with TRAF6 leads to NF-κB activation as described above (Jiang,

Mak and Li 2004). TRIF also interacts with TRAF3 (Oganesyan, et al. 2006, Häcker, et al.

2006). A complex of TRAF3, TANK (TRAF family member associated NF-κB activator), and NEMO facilitates activation of the IKK-related kinases: TBK1 (TANK-binding kinase 1) and IKKε (Oganesyan, et al. 2006, Häcker, et al. 2006, Guo and Cheng 2007, Chariot, et al.

2002, Clark, et al. 2011, Wang, Li and Dorf 2012). TBK1 and IKKε phosphorylate IRF3 and

IRF7, which form homo- and heterodimers, translocate to the nucleus, and activate transcription of the type I interferons (IFNs), which include the IFN-α gene family and the

IFN-β gene (Sharma, et al. 2003, Lin, et al. 1998, Yoneyama, Suhara, et al. 1998, Weaver,

Kumar and Reich 1998, Sato, et al. 2000).

Retinoic Acid-Inducible Gene I-Like Receptors (RLRs)

There are three identified members in the RLR family: RIG-I (retinoic acid-inducible

gene I; also known as DDX58), MDA5 (melanoma differentiation associated factor 5; also

known as IFIH1), and LGP2 (laboratory of genetics and physiology 2; also known as

DHX58) (Yoneyama, Kikuchi and Natsukawa, et al. 2004). All three bind double-stranded

RNA (dsRNA) (Table 1) (Murali, et al. 2008, Yoneyama, Kikuchi and Natsukawa, et al.

2004, Kato, Takeuchi and Mikamo-Satoh, et al. 2008, Takahasi, et al. 2009). DsRNA is not

normally present in the cytoplasm of host cells, but makes up the genome of certain viruses

and can be generated as a viral replication intermediate. RIG-I preferentially recognizes short

dsRNA fragments while MDA5 preferentially recognizes long dsRNA fragments (Kato,

Takeuchi and Mikamo-Satoh, et al. 2008, Yoneyama, Kikuchi and Natsukawa, et al. 2004).

In addition RIG-I and LGP2 bind single-stranded RNA with uncapped 5’ triphosphate ends that may be present in viral RNA intermediates, but are mostly absent in host cytosolic RNA

(Hornung, et al. 2006, Takahasi, et al. 2009, Pichlmair, et al. 2006). However, the presence

of short double-stranded segments induced by complementation within the single-stranded

RNA may be necessary for this interaction (Schlee, et al. 2009). Differential binding of RNA by RIG-I and MDA5 allows for detection of different viruses in vivo (Kato, Takeuchi and

Sato, et al. 2006).

RIG-I and MDA5 have two amino terminal caspase activation and recruitment domains

(CARDs), a central DExD/H RNA helicase domain, and a carboxy terminal regulatory

domain (RD). LGP2 shares a similar structure except that it lacks the CARDs. Studies

suggest LGP2 may act as a negative regulator of RIG-I, while promoting activity of MDA5

(Pippig, et al. 2009, Yoneyama, Kikuchi and Matsumoto, et al. 2005, Venkataraman, et al.

2007, Rothenfusser, et al. 2005). The helicase and RD regions contribute to RNA binding,

while the CARDs mediate downstream signaling (Yoneyama, Kikuchi and Natsukawa, et al.

2004, Pippig, et al. 2009, Takahasi, et al. 2009, Saito, et al. 2007). In the absence of bound

RNA, the RD region in RIG-I acts to sequester the CARDs and prevent signaling (Saito, et

al. 2007). Upon RNA binding, RIG-I adopts an “open” conformation, multimerizes, and

interacts via its CARDs with the CARD of MAVS (mitochondrial antiviral signaling protein;

also known as IPS-1/CARDIF/VISA) (Saito, et al. 2007, Meylan, et al. 2005, L. Xu, et al.

Mol Cell, Kawai, Takahashi, et al. 2005). MAVS is an adaptor protein present on

peroxisomes, the outer mitochondrial membrane, and mitochondria associated membranes

(Dixit, et al. 2010, Horner, et al. 2011, Seth, et al. 2005). The ligand-bound RIG-I or MDA5

are recruited to MAVS, which acts as a platform for assembly of multiple proteins in a

signaling complex that leads to activation of NF-κB and IRFs (Fig. 1) (Michallet, et al. 2008,

Ohman, et al. 2009, Horner, et al. 2011). Key intermediates in the NF-κB and IRF activation pathways, as described for TLRs, are also recruited to the MAVS signaling complex, including TRAF6 (L. Xu, et al. Mol Cell), TRAF3 (Horner, et al. 2011, Saha, et al. 2006),

TANK (Guo and Cheng 2007), NEMO (Zhou, et al. 2014, T. Zhao, et al. 2007), and IKKε

(Meylan, et al. 2005, Ohman, et al. 2009).

Nucleotide binding domain and leucine rich repeat containing receptors (NLRs)

The NLR family in humans has 22 members (Table 1) (Ting, et al. 2008). They have a

common protein domain organization characterized by an amino terminal protein-protein

interaction domain, a central NOD domain (also known as the NACHT domain), and

carboxy terminal leucine rich repeats (LRRs) (Koonin and Aravind 2000). The N-terminal

protein-protein interaction domains vary and are used to classify NLRs into 5 subfamilies:

NLRA (CIITA is the only member), NLRB (NAIP is the only member), NLRC (NOD1/2,

NLRC3/4/5; members have an amino terminal CARD domain), NLRP (NLRP1-14; members have an amino terminal pyrin domain), and NLRX (NLRX1 is the only member) (Ting, et al.

2008). The NOD domain mediates homotypic oligomerization of NLRs (Inohara, Koseki, et

al. 2000, Faustin, et al. 2007). The LRRs mediate ligand interactions and play an

autoregulatory role (Inohara, Koseki, et al. 2000, Girardin, Tournebize, et al. 2001, Khare, et

al. 2012, Cui, Zhu, et al. 2010, Martinon, Burns and Tschopp 2002).

Although a number of NLR members are implicated in inflammatory signaling in

response to PAMPs, direct interaction with PAMPs has only been demonstrated for a few

NLRs (Table 1). NOD1 directly binds diaminopimelic acid-containing peptides mainly

present in peptidoglycans from Gram-negative bacteria (Girardin, Boneca and Carneiro, et al.

2003, Laroui, et al. 2011). NOD2 binds the muramyl dipeptide (MDP) motif common to

peptidoglycans of all bacteria (Girardin, Boneca and Viala, et al. 2003, Grimes, et al. 2012).

NAIP binds to a subunit of the type III secretion system of Chromobacterium violaceum (Y.

Zhao, et al. 2011).

NOD1 and NOD2 promote NF-κB activation. Ligand-binding by NOD1 or NOD2 induces

homo-oligomerization and CARD-CARD interaction with RIPK2 (receptor interacting serine/threonine kinase 2; also known as RIP2/RICK) (Laroui, et al. 2011, Girardin,

Tournebize, et al. 2001, Inohara, Koseki, et al. 2000, Park, et al. 2007). RIPK2 in turn interacts with and activates NEMO, leading to NF-κB activation via the IKK complex

(Girardin, Tournebize, et al. 2001, Inohara, Koseki, et al. 2000) (Fig. 1).

A greater number of NLRs have emerged as negative regulators of PRR signaling pathways. NLRC3 attenuates LPS-induced NF-κB activation in macrophages by reducing the active (K63-polyubiquitinated) form of TRAF6 (Schneider, et al. 2012). NLRC5 can negatively regulate NF-κB activation by sequestering IKKα and IKKβ to prevent formation of the IKK complex (Cui, Zhu, et al. 2010, Meng, et al. 2015). LPS-induced activation of

TRAF6 (via TLR4) results in ubiquitination of NLRC5, preventing its inhibitory interaction with the IKK subunits (Meng, et al. 2015). NLRC5 has also been shown to interact with the

CARD region of RIG-I and prevent its interaction with MAVS and subsequent interferon activation (Cui, Zhu, et al. 2010). In complement, NLRX1 binds the CARD region of MAVS and prevents its interaction with RIG-I or MDA5 (Moore, et al. 2008). In addition, NLRX1 negatively regulates TLR-induced NF-κB activation by interacting with the IKK complex and blocking its kinase activity (Xia, et al. 2011). NLRP4 interacts with IKKα and inhibits its activation, preventing NF-κB activity (Fiorentino, et al. 2002). More recently, NLRP4 was found to prevent type I interferon induction via TBK1 by promoting TBK1 ubiquitination and degradation (Cui, Li, et al. 2012).

Inflammasomes are complexes of oligomerized proteins consisting of a sensor protein, an

adaptor protein, and pro-caspase-1 (Martinon, Burns and Tschopp 2002, Lu and Wu 2014).

NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 have been reported as sensor

molecules for initiating inflammasome assembly. The canonical adaptor protein is ASC

(apoptosis-associated speck-like protein containing a CARD; also known as PYCARD), which undergoes pyrin-pyrin domain interactions with NLRP molecules and CARD-CARD interactions with pro-caspase-1 (Martinon, Burns and Tschopp 2002, Lu and Wu 2014,

Wang, Manji, et al. 2002). NRLC4, which contains an amino terminal CARD rather than a pyrin domain, and NLRP1, which has a carboxy terminal CARD, are able to directly interact with pro-caspase-1 and assemble ASC-independent inflammasomes (Faustin, et al. 2007,

Poyet, et al. 2001). The consequence of NLR inflammasome assembly is cleavage of pro- caspase-1 to mature caspase-1, which in turn is able to cleave pro-interleukin-1β (pro-IL-1β) and pro-IL-18 to their active forms (Martinon, Burns and Tschopp 2002, Kuida, et al. 1995).

IL-1β and IL-18 are then released from the cell. Binding of IL-1β and IL-18 to their cognate receptors can activate the MyD88-dependent signaling pathway (Burns, et al. 1998, Adachi, et al. 1998). An important effect of IL-18 is promoting expression of IFNγ (Okamura, et al.

1995), which acts as an activator of macrophages to enhance macrophage microbicidal activity (Nathan, et al. 1983).

The NLR inflammasomes can be triggered by a variety of PAMPs dependent upon the

NLR involved (Table 1). The NLRP1 inflammasome is triggered by the bacterial peptidoglycan component MDP in an ATP-dependent manner in a minimal in vitro system

(Faustin, et al. 2007), suggesting NLRP1 may directly bind MDP. In vivo experiments in

mice indicate that NOD2, whose ligand is also MDP (Girardin, Boneca and Viala, et al.

2003), may act as a coactivator in NLRP1 inflammasomes (Hsu, et al. 2008). The NLRP3 inflammasome is one of the most studied and can be activated by a wide variety of pathogens and PAMPs (Mariathasan, et al. 2006, Lamkanfi, Malireddi and Kanneganti 2009, Gross, et al. 2009, Kanneganti, et al. 2006), as well as by host-derived molecules indicative of tissue injury, such as hyaluronan and cholesterol crystals (Yamasaki, et al. 2009, Duewell, et al.

2010). The exact mechanism of NLRP3 activation is unclear. The structural diversity among the many stimulants of NLRP3 makes it unlikely that they act as direct ligands (Schroder and

Tschopp 2010). Potassium efflux has been proposed as the common triggering event for

NLRP3 activity linking these divergent agonists (Muñoz-Planillo, et al. 2013). Bacterial acylated lipopeptides trigger inflammasome activation by NLRP7, although direct interaction between NLRP7 and acylated lipopeptides could not be demonstrated, suggesting NLRP7 is activated indirectly (Khare, et al. 2012). The NLRC4 (also known as IPAF) inflammasome is induced by subunits of bacterial type III secretion systems and by flagellin (Miao, et al. 2010,

Y. Zhao, et al. 2011). Interestingly, direct sensing of these ligands likely occurs via another

NLR member, NAIP (Y. Zhao, et al. 2011). In vitro and in vivo studies support a role for

NLRP6 (also known as PYPAF5) and NLRP12 (also known as PYPAF7/Monarch-1) in inflammasome activation: each colocalized with ASC and induced pro-caspase-1 activity

(Grenier, et al. 2002, Wang, Manji, et al. 2002, Chen, et al. 2011, Vladimer, et al. 2012).

However, other studies have shown that both NLRP6 and NLRP12 act as inhibitors of NF-

κB activation (Anand, et al. 2012, Williams, et al. 2005). Whether the activities of NLRP6 or

NLRP12 are stimulus-dependent remains unclear.

The inflammatory response

Together the PRR families provide the cell with a means of sensing a diverse array of pathogens at the extracellular and intracellular level and generating signals to initiate an immune response. For the majority of PRRs, these signals converge to activate transcription

factors, notably NF-κB and IRF, that in turn upregulate transcription of immune response

genes.

IRF3 and IRF7 activate transcription of the type I interferons (IFNs), which include the

IFN-α gene family and the IFN-β gene (Sharma, et al. 2003, Lin, et al. 1998, Yoneyama,

Suhara, et al. 1998, Weaver, Kumar and Reich 1998, Sato, et al. 2000). Type I IFNs are

secreted from the cell and activate the type I IFN receptor, leading to transcriptional

upregulation of interferon stimulated genes (ISGs) whose functions mediate antiviral cellular

activity (Der, et al. 1998, Platanias 2005, Yoneyama, Suhara, et al. 1996, Muller, et al. 1993,

Colamonici, et al. 1994). While IRF3 is constitutively produced in cells, IRF7 expression is

induced by type I IFN signals, providing a positive feedback loop that is important for

maintaining IFN production (Sato, et al. 2000, Marie, Durbin and Levy 1998).

NF-κB acts to upregulate the expression of over 150 genes, many of them immune-

related (Pahl 1999). Among the genes upregulated by NF-κB are key cytokines (intercellular

signaling molecules) that mediate innate immune responses: TNF-α (tumor necrosis factor

α), IL-6, and IL-1β (Hiscott, et al. 1993, Libermann and Baltimore 1990, Shimizu, et al.

1990, Collart, Baeuerle and Vassalli 1990). These cytokines have widespread effects on

vasculature and leukocytes that collectively promote leukocyte movement into tissues, as

discussed in further detail in the next section.

Activation of transcription factors and altered gene expression occurs rapidly, within

minutes to hours of PRR stimulation. Viral infection or double-stranded RNA exposure can

induce IRF3 activation within 3 hours with subsequent IFNα/β production 6-12 hours post exposure (Sharma, et al. 2003, Weaver, Kumar and Reich 1998, Sato, et al. 2000,

Yamamoto, Sato, et al. 2003). NF-κB activity is induced less than 30 minutes post exposure of cells to PAMPs or pathogens, and increases in cytokine expression, including TNF-α, IL-6 and IL-1β, are evident within 1 hour (Han, et al. 2002, Yamamoto, Sato, et al. 2003). A biphasic nature of NF-κB activity has been noted in several studies, with an initial period of

NF-κB activation and gene upregulation occurring 0.5-3 hours post stimulation and a second period 8-12 hours post stimulation (Han, et al. 2002). This biphasic activity may relate to the activity of genes induced by NF-κB (Han, et al. 2002). Indeed, TNF-α, IL-6 and IL-1β are upregulated by NF-κB and can in turn induce NF-κB activation upon binding to their cognate receptors, allowing for positive feedback loops (Adachi, et al. 1998, Burns, et al. 1998,

Shimizu, et al. 1990). While this can allow for quick amplification of immune signals, it also highlights the need for close regulation of these signaling pathways to prevent intractable inflammation. The complexity of the intracellular signaling pathways utilized by PRRs allows for numerous potential checkpoints. Negative regulation by some of the NLRs discussed above gives just a few examples of how cells have evolved ways to modulate these signaling pathways. Uncovering novel modulators of these pathways will help fine tune our understanding of how inflammation is controlled.

Macrophage and Neutrophil Migration

In order to fend off pathogens, it is critical that neutrophils and macrophages be able to get to the site of infection or tissue damage. Neutrophils and monocytes circulating in the blood must cross the vascular wall into the tissues (extravasation). In tissues, monocytes differentiate into macrophages (van Furth and Cohn 1968, Ginhoux and Jung 2014).

Neutrophils and macrophages must then navigate the extracellular matrix toward the inflammatory focus. This process requires a complex interplay of extracellular signals relayed through cell surface receptors to induce changes in adhesion molecules and the cytoskeleton.

The importance of neutrophil and monocyte/macrophage migration to the immune response is evidenced by the severe and recurrent infections suffered by individuals with disorders in leukocyte adhesion and motility (Badalato 2013). On the other end of the spectrum, exuberant tissue infiltration by neutrophils and macrophages is implicated in the pathogenesis of a variety of inflammatory disorders that negatively impact human health, from acute respiratory distress syndrome to atherosclerosis (Chávez-Sánchez, et al. 2014,

Steinberg, et al. 1994). As such, immunodulatory therapies that target neutrophil and macrophage chemotaxis are an active area of investigation for the treatment of such inflammatory conditions. Yet certain strategies for inhibiting leukocyte chemotaxis, such as antagonizing chemoattractant receptors, have met with little clinical success (Horuk 2009,

Russo, Garcia and Teixera 2010). Identifying novel strategies for modulating inflammation

will require understanding the signaling mechanisms that mediate neutrophil and macrophage

migration.

Chemotaxis: a primer.

Chemotaxis is the directed migration of cells along an extracellular chemical gradient, as

opposed to chemokinesis, which is the random (undirected) migration of cells in response to

an extracellular chemical signal (Keller, et al. 1977). Leukocytes use chemotaxis to get to

sites of inflammation or infection by moving up gradients of chemoattractants, made up of a

variety of host- and pathogen-derived molecules. Some well-characterized chemoattractants

that drive influx of neutrophils and macrophages during the innate immune response are

bacterially-derived formyl peptides (Schiffman 1975) and the complement component C5A

(Snyderman, et al. 1975).

A large subfamily of cytokines also act as chemoattractants, and are referred to as chemokines. During an innate immune response, macrophages, monocytes, and endothelial cells are important sources of chemokine production in response to TNFα, IL-1β, or PAMP stimulation (Sica, et al. 1990, Wen, Rowland and Derynck 1989, Wuyts, et al. 2003,

Yoshimura, Yuhki, et al. 1989), thus providing high concentrations at the source of inflammation. Chemokines have been demonstrated to bind to glycosaminoglycans, a component of proteoglycans found on cell surfaces and extracellular matrix components throughout the body. This binding is thought to facilitate the establishment of chemokine

gradients in tissues and in the blood stream, and has been shown to be critical to chemokine

function in vivo (Proudfoot, et al. 2003).

Different chemokines act on different leukocytes. IL-8 (also known as CXCL8) is a major neutrophil chemoattractant (Matsushima, et al. 1998, Yoshimura, Matsushima, et al.

1987). MCP-1 (monocyte chemoattractant protein-1; also known as CCL2), MIP-1α

(macrophage inflammatory protein-1α; also known as CCL3), and CCL5 (C-C chemokine ligand 5; also known as RANTES) are potent chemoattractants for monocytes and macrophages (Rollins, Walz and Baggiolini 1991, Uguccioni, et al. 1995). These chemokines play important roles in guiding neutrophils and monocytes out of the vasculature and through the tissues.

Extravasation

During the initial inflammatory response, neutrophils and monocytes exit the vasculature

mainly at postcapillary venules where they must traverse a layer of endothelium and then the

basement membrane and pericytes that make up the vascular wall (Majno, Palade and

Schoefl 1961). The process of extravasation occurs in a series of steps termed the leukocyte

adhesion cascade (Ley, et al. 2007). In the initial step, neutrophils and monocytes are

captured from the blood stream, tether to the endothelium, and begin to roll along it. This

process is mediated by interactions between P-selectin and E-selectin molecules expressed on

activated endothelium and their ligand, PSGL-1 (P-selectin glycoprotein-1), expressed on

leukocytes (Moore, et al. 1995, Sperandio, et al. 2003, Hidalgo, et al. 2007, Moore, et al.

1992, McEver, et al. 1989). Selectin molecules are absent from the endothelial surface unless

stimulated. P-selectin (also known as GMP-140) is constitutively expressed and stored in cytoplasmic granules that allow for rapid surface expression in response to inflammatory mediators such as histamine (McEver, et al. 1989). P-selectin expression is also upregulated by TNF-α (Gotsch, et al. 1994). Expression of E-selectin (also known as ELAM-1) is induced in response to TNF-α and IL-1β (Bevilacqua, et al. 1989, Huang and Eniola-Adefeso

2012). Another selectin, L-selectin, is expressed on leukocytes (Lewinsohn, Bargatze and

Butcher 1987). L-selectin binding to PSGL-1 mediates leukocyte-leukocyte interactions, facilitating secondary tethering to leukocytes already adhered to endothelium (Bargatze, et al.

1994). Interactions between E-selectin on the endothelium and ESL-1 (E-selectin ligand-1) and CD44 on leukocytes also contribute to rolling (Hidalgo, et al. 2007).

In the next phase of the leukocyte adhesion cascade, leukocytes slowly roll and ultimately arrest and form firm adhesions to the endothelium. The aptly named integrins play an integral role in this process. Integrins are transmembrane adhesion receptors that link extracellular ligands on cells and in the extracellular matrix to the actin cytoskeleton, providing points of traction for cell movement and stabilization (Burridge and Chrzanowska-Wodnicka 1996).

Integrins are composed of α and β chain heterodimers that undergo signal mediated conformational changes that affect their affinity for extracellular ligands (Harburger and

Calderwood 2009). Engagement of PSGL-1 by E-selectin or P-selectin leads to partial activation of the β2 integrins LFA-1 (lymphocyte function-associated antigen-1; also known

as αLβ2, CD11a/CD18) and Mac-1 (macrophage-1; also known as αMβ2, CD11b/CD18) on

leukocytes (Zarbock, Lowell and Lay 2007, Kuwano, et al. 2010, Ma, et al. 2004). The

partially activated LFA-1 and Mac-1 engage ICAM-1 (intercellular adhesion molecule-1) on endothelial cells with intermediate affinity to help slow rolling (Zarbock, Lowell and Lay

2007, Kuwano, et al. 2010, Ma, et al. 2004).

Detection of chemokines and chemoattractants provides an additional signal for the leukocyte to firmly attach to the endothelium by further enhancing integrin binding affinity.

For example, CXCL1 (C-X-C motif chemokine ligand 1; also known as GROα) expressed by activated endothelial cells engages its cognate receptor, CXC receptor 2 (CXCR2), on neutrophils to induce the high-affinity conformation of LFA-1, which then strongly binds

ICAM-1 to arrest the neutrophil (Zarbock, Lowell and Lay 2007). Similarly, MCP-1 binding to its receptor, CCR2, on monocytes induces the high-affinity conformation of LFA-1 and adhesion (Maus, et al. 2002). Interactions between VLA-4 (very late antigen-4; also known as α4β1 integrin) and its ligand VCAM-1 (vascular adhesion molecule-1) are also important for monocyte-endothelial adhesion (Huo, Hafezi-Moghadam and Ley 2000). CXCL12 and formyl peptide induce the high-affinity conformation of VLA-4 on monocytes to induce arrest on VCAM-1 (Chan, Hyduk and Cybulsky 2001). The intracellular signals mediating integrin activation are complex, and likely ligand dependent (Ley, et al. 2007). A variety of intracellular signaling mechanisms and cytoskeletal adaptors are implicated in the inside-out

signals that activate integrins (Hyduk, et al. 2007, Sampath, Gallagher and Pavalko 1998,

Jones, Wang, et al. 1998, Jones, Knaus, et al. 1998, Kuwano, et al. 2010). Over 40 proteins are postulated to be involved directly, with a resulting network of over 900 proteins potentially involved (Ley, et al. 2007).

Following firm adhesion, neutrophils and monocytes spread out and crawl along the

endothelium to the endothelial cell junction (Phillipson, et al. 2006, Schenkel, Mamdouh and

Muller 2004). In vivo studies indicate Mac-1/ICAM1 interactions predominantly mediate crawling on activated endothelium (Phillipson, et al. 2006, Sumagin, Prizant, et al. 2010).

The crawling leukocytes form numerous ICAM-1 enriched attachments to the underlying endothelium (Shulman, et al. 2009) that allow resistance to the shear forces applied by blood flow, enabling cells to move independent of the direction of blood flow (Phillipson, et al.

2006). The exact criteria by which a leukocyte identifies an optimal site for transendothelial migration are not entirely clear, although morphology of the endothelial cell junction and focal enrichment of adhesion molecules may contribute (Sumagin and Sarelius 2010).

Transendothelial migration occurs in two ways: through endothelial cell junctions

(paracellular) or directly through the endothelial cell (transcellular). The paracellular route is most commonly used (Woodfin, Voisin, et al. 2011). Several factors optimize transendothelial migration via the paracellular route. One is contraction of activated endothelial cells to widen the interendothelial gap (Goeckler and Wysolmerski 1995, Saito, et al. 1998). Opening of this gap is also facilitated by redistribution of VE-cadherin (vascular endothelial-cadherin) from the interendothelial junction (Del Maschio, et al. 1996, Allport,

Muller and Luscinskas 2000, Shaw, et al. 2001). VE-cadherin is a transmembrane protein, specifically expressed by endothelial cells, that participates in homotypic interactions via its extracellular domain to create adherens junctions between endothelial cells (Lampugnani, et al. 1992, Breviario, et al. 1995). Reversible removal of VE-cadherin from focal regions of the endothelial cell junction through endocytosis enables leukocyte transmigration (Allport,

Muller and Luscinskas 2000, Del Maschio, et al. 1996, Wessel, et al. 2014). Finally,

leukocytes are able to interact with other adhesion molecules that normally contribute to

maintaining interendothelial contacts and effectively use them as anchor points to move

through the junction. These molecules include PECAM1 (platelet/endothelial adhesion

molecule-1; also known as CD31), CD99, and JAM (junctional adhesion molecule) family

members (Muller, et al. 1993, Schenkel, Mamdouh and Chen, et al. 2002, Woodfin, Voisin,

et al. 2011, Woodfin, Reichel, et al. 2007). The mechanisms by which leukocytes cross the

endothelium via the transcellular route are not well understood (Feng, et al. 1998). Studies in

lymphocytes suggest that leukocyte contacts with ICAM-1-enriched foci on the endothelial cell recruit vesiculovacuolar organelles that form channels to create a transcellular pore

(Carman, et al. 2007, Millán, et al. 2006).

Having traversed the endothelial cell layer, the leukocyte must finally cross the layer of basement membrane and pericytes that make up the vessel wall. Recent studies have shown that the basement membrane contains regions of reduced extracellular matrix components that are aligned with pericyte gaps and are preferential sites, at least for neutrophils, for crossing the vessel wall (Wang, Voisin, et al. 2006, Voisin, Pröbstl and Nourshargh 2010).

The leukocyte-endothelial interactions at the endothelial cell junction prepare the leukocyte

to move through the basement membrane. PECAM-1 engagement mobilizes α6β1 to the

leukocyte cell surface, where it acts as a receptor for laminin, a major matrix protein in the

basement membrane (Dangerfield, et al. 2002). Pericytes also express adhesion molecules

similar to those expressed by the endothelium, such as ICAM-1 and VCAM-1, which

facilitate leukocyte movement through the vessel wall (Proebstl, et al. 2012, Verbeek, et al.

1995). Finally, cell surface proteases expressed by leukocytes may also aid in movement

through the basement membrane by cleaving matrix proteins (Wang, Dangerfield, et al.

2005).

Once leukocytes have exited the vasculature into the interstitium, in vivo and in vitro

studies indicate that movement through the three dimensional extracellular matrix is largely

independent of integrin mediated adhesions (Friedl, Zänker and Bröcker 1998, Lammerman, et al. 2008, Van Goethem, et al. 2010). Rather leukocytes rely primarily on the protrusion

and retraction of the actin cytoskeleton to propel and push the cell through pores in the

extracellular matrix along chemoattractant gradients (Lammerman, et al. 2008).

Intracellular signaling and cytoskeletal dynamics of chemotaxis

Upon sensing a chemoattractant gradient, leukocytes organize their actin cytoskeleton in

such a way that the cell becomes polarized with a leading edge and a trailing edge oriented

along the chemoattractant gradient. At the leading edge, the cell extends pseudopods that

consist of a core of actin filaments that physically push the cell membrane forward. This

process requires complex and coordinated intracellular signals connecting the activation of

the chemoattractant receptor to the actin cytoskeleton.

The majority of leukocyte chemoattractants signal through seven-transmembrane domain

G-protein-coupled receptors (Boulay, et al. 1997). As the name implies, these receptors are coupled on their cytosolic face to heterotrimeric G-proteins that, upon activation of the receptor, dissociate into Gα and Gβγ subunits (Lattin, et al. 2007). The free Gβγ subunit

activates phosphatidylinositol (PI) 3՛ kinase (PI3K) (Brock, et al. 2003). PI3K

phosphorylates PI-(3,4)-bisphosphate (PIP2) to PI-(3,4,5)-triphosphate (PIP3) at the cytosolic

leaflet of the plasma membrane. PIP3 rapidly distributes in a polarized manner to the leading

edge of chemotaxing cells (Servant, et al. 2000, Nishio, et al. 2007). Polarization of PIP3 is

the result of both positive feedback signaling in the front compartment of the cell (Wang, et

al. 2002) and dephosphorylation of PIP3 in the sides and rear of the cell by SHIP1 (Src

homology 2 domain-containing inositol-5-phosphatase 1) and PTEN (phosphatase and tensin

homolog) (Nishio, et al. 2007, Heit, et al. 2008). Studies in Ship1-/- mice indicate polarized

localization of PIP3 to the leading edge of the cell is necessary for proper actin filament

organization (Nishio, et al. 2007).

Localization of PIP3 is important because it acts as a docking site for proteins containing

pleckstrin homology (PH) domains (G. Shaw 1996), providing a means for localizing the

activity of downstream mediators. Along the leading edge of the cell, PH-domain containing guanine nucleotide exchange factors (GEFs) activate the GTP-binding proteins RAC1/2 (ras- related C3 botulinum toxin substrate 1/2) and CDC42 (cell division cycle 42) (Kunisaki, et al. 2006, Lawson, et al. 2011, Li, et al. 2003), which in turn mediate polymerization of actin filaments (F-actin) in an organized core that pushes the plasma membrane forward in a protruding pseudopod (Kunisaki, et al. 2006, Allen, et al. 1998, Srinivasan, et al. 2003).

Actin molecules are maintained as monomers (G-actin) in the cytoplasm in conjunction with the protein profilin (Pollard and Cooper 1984). A protein complex called ARP2/3 (actin- related protein 2/3) mediates addition of the monomers to the barbed end of preexisting actin filaments to form a branching actin network within the pseudopod (Mullins, Heuser and

Pollard 1998, Weiner, et al. 1999). Two protein complexes containing WASP (Wiskott-

Aldrich syndrome protein) family members link RAC and CDC42 to actin polymerization.

CDC42 activates the WASP-containing complex (Cammer, et al. 2009), enabling WASP to

interact with G-actin and the ARP2/3 complex (Higgs and Pollard 2000, Machesky and Insall

1998). Activated RAC interacts with a larger protein complex containing WAVE (WASP-

family verprolin homologous protein) (Suetsugu, et al. 2006). WAVE simultaneously

interacts with PIP3 at the leading edge and mediates ARP2/3 activation (Suetsugu, et al.

2006).

WASP and, more recently, WAVE complex members have also been found to interact with members of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family of proteins, which consists of VASP, MENA (mammalian Ena; also known as ENAH), and

EVL (Enah/Vasp-like) (Chen, et al. 2014, Havrylenko, et al. 2015, Castellano, et al. 2001).

Ena/VASP proteins mediate actin polymerization, albeit in straight rather than branched filaments (Hansen and Mullins 2010, Gertler, et al. 1996), and localize to the leading edge of

chemotaxing cells, including neutrophils and macrophages (Neel, et al. 2009, Chen, et al.

2014). VASP interaction with the activated WASP or WAVE complex synergistically

enhances actin assembly and cell motility with ARP2/3 (Chen, et al. 2014, Havrylenko, et al.

2015, Castellano, et al. 2001).

While signals mediating actin polymerization in the front part of the cell act to protrude

the cell membrane forward, signals in the rear compartment of the cell (the uropod) mediate

contraction of the cytoskeleton to withdraw the cell’s trailing end and push the nucleus

forward (Lammerman, et al. 2008). This signaling is mediated by another GTP-binding

protein, RhoA, whose activity is confined to the uropod in polarized cells (J. Xu, et al. 2003).

RhoA activity leads to activation of myosin (J. Xu, et al. 2003), an actin binding protein that mediates contraction of the actin cytoskeleton (Vicente-Manzanares, et al. 2009).

Finally, it must be noted that the signaling cascades described above are not the sole means by which chemoattractant signals manifest the cytoskeletal rearrangements that move the cell, and different chemoattractants may utilize different mediators. Neutrophils utilize a

MAP kinase mediated pathway to prioritize signals from different chemoattractants (Heit, et al. 2008). The activity of different phospholipases contributes to chemotaxis in neutrophils and monocytes downstream of IL-8 and MCP-1, respectively (Wu, et al. 2012, Cathcart

2009). VASP is required for IL-8 induced neutrophil chemotaxis, but dispensable for chemotaxis induced by MIP1α (Neel, et al. 2009). Clearly, we are just beginning to uncover the complexity underlying regulation of chemotaxis.

The Zebrafish as a Model of Innate Immunity

Over the past two decades, zebrafish (Danio rerio) have emerged as powerful vertebrate animal models for studying innate immune function. Despite over 400 million years of evolution (Kumar and Hedges 1998), many key features of the innate immune response are shared between zebrafish and humans. Zebrafish express TLRs, NLRs, and RLRs (Jault,

Pichon and Chluba 2004, Laing, et al. 2008, Nie, et al. 2005). Further, signaling molecules downstream of PRRs are present and functionally conserved between zebrafish and humans

(van der Sar, Stockhammer, et al. 2006, Stein, et al. 2007, Nie, et al. 2005, Phelan, Mellon

and Kim 2005). Conserved ligand recognition for some zebrafish TLRs and RIG-I has also been demonstrated (Nie, et al. 2005, Ribeiro, et al. 2010, Phelan, Mellon and Kim 2005).

In addition to their small size, high fecundity, and genetic tractability, zebrafish are a unique model for studying innate immunity because their ex utero development provides easy access to the organism during a time when innate immune responses are fully functional and capable of defending the organism against pathogens in the absence of a functional adaptive immune response (Lam, et al. 2004). By 72 hours post fertilization (hpf), zebrafish possess fully functional neutrophils and macrophages capable of directed migration to a site of infection or inflammation, phagocytosis and bacterial killing (Herbomel, Thisse and

Thisse 1999, Ellet, et al. 2011, Lieschke, et al. 2001). Transgenic zebrafish lines with fluorescently labeled neutrophils and macrophages combined with the optical transparency of larval zebrafish facilitate unprecedented in vivo studies of these effector cells (Ellet, et al.

2011, Mathias, et al. 2006, Renshaw, et al. 2006).

Because of their small size, larval zebrafish provide a means of assessing transcriptional changes at the whole organism level. Stimulation of PRRs leads to altered transcription of genes which play a role in mediating innate immune responses. Therefore, analyzing the suite of genes whose transcription levels are altered in response to PRR stimulation may reveal novel mediators of innate immunity. We recently tested this hypothesis using larval zebrafish (Heffelfinger 2010). An RNA microarray strategy was used to define the whole organism transcriptome in 72 hpf zebrafish larvae exposed to either of two complementary

PAMPs: triacylated lipopeptide (Pam3CSK4), a TLR1/2 agonist inducing NF-κB (Ribeiro, et al. 2010), and polyinosinic-polycytidylic acid (PolyIC), a synthetic dsRNA analog known to

activate TLR3 and RLRs and induce interferon along with NF-κB (Phelan, Mellon and Kim

2005, Kato, Takeuchi and Mikamo-Satoh, et al. 2008). Transcriptome analysis identified a

set of common response genes with human homologs that included both known and novel

innate immune response genes (Heffelfinger 2010). One gene identified by this list was tripartite motif containing 9 (trim9). Trim9 transcripts increased in the larval zebrafish in response to either PAMP at 8 hours post exposure, similar to the timing of late NF-κB gene induction (Han, et al. 2002). At the time of this study, Trim9 had no known immune function, and was considered to be brain specific (Li, et al. 2001, Tanji, et al. 2010, Berti, et al. 2002). However, Trim9 is a member of the tripartite motif protein family, a large family of multifunctional proteins, in which several members are known innate immune mediators, highlighting the potential of Trim9 as a novel innate immune mediator.

Tripartite Motif (TRIM) Protein Family

The tripartite motif (TRIM) family is a large family of E3 ubiquitin ligases found across metazoans (Sardiello, et al. 2008). Over 70 members are present in humans (Sardiello, et al.

2008), while over 200 have been identified in zebrafish (Boudinot, et al. 2011). With few exceptions, the family is defined by the presence of three conserved protein motifs in the amino terminus: a really interesting new gene (RING) domain followed by one to two Bbox domains and a Coiled-coil region, in that order. The RING domain is responsible for imparting E3 ubiquitin ligase activity (Joazeiro and Weissman 2000). Bboxes are zinc finger structures unique to the TRIM family, but their function remains unclear. The Coiled-coil

region mediates hetero- and homotypic interactions (Reymond, et al. 2001). TRIMs possess a

variety of carboxy terminal domains which have been used to subclassify them into families,

although phylogenetic subclassifications have also been proposed (Short and Cox 2006,

Ozato, et al. 2008, Marin 2012). TRIM family members serve many diverse cellular

functions, and a number have been implicated in innate immunity.

Ubiquitination

Ubiquitination is a posttranslation protein modification in which covalent linkages are

formed between lysine residues in ubiquitin and a target protein substrate. The process of

ubiquitination of a protein substrate involves three enzymes: E1, E2, and E3 (Hershko,

Heller, et al. 1983). E1 is an ubiquitin-activating enzyme that catalyzes formation of a thiol-

ester bond between the carboxy terminal glycine of ubiquitin and the E1 enzyme itself in an

ATP-dependent manner (Haas and Rose 1982). The human genome encodes 2 ubiquitin-

activating E1 enzymes, which in turn interact with particular sets of the E2 conjugases (Jin,

et al. 2007). When E1 and E2 enzymes bind, the ubiquitin is transferred from E1 to a cysteine

residue in the E2 conjugase (Pickart 2001). There are at least 38 E2 conjugase genes in the human genome (Ye and Rape 2009), and they act as a bridge between the E1 and the E3 enzymes. The E2 conjugase binds to an E3 ubiquitin ligase and the ubiquitin is transferred to the target, typically the ε-amino group of a lysine within either the target protein or ubiquitin itself (for polyubiquitination) (Pickart 2001).

Protein substrate specificity for ubiquitination is largely provided by the E3 ligase enzymes, which directly interact with the substrate protein. There are over 600 E3 ubiquitin ligases encoded in the human genome, and they largely group into two families: the HECT

(Homologous to E6-AP Carboxy Terminus) domain-containing proteins and the RING domain-containing proteins, which include the TRIM proteins. The RING E3 ligases catalyze the direct transfer of ubiquitin from the E2 conjugase to the substrate protein, and therefore must interact simultaneously with the ubiquitin loaded E2 conjugase and the protein substrate. In TRIMs, the RING domain is necessary and sufficient for interaction with the E2 conjugase, and individual TRIM proteins have been shown to interact with particular subsets of the E2 conjugases (Napolitano, Jaffray, et al. 2011). As with other RING proteins, the additional motifs present in TRIM proteins mediate binding of the substrate protein, and potentially other proteins within a complex (Reymond, et al. 2001, Napolitano, Jaffray, et al.

2011, Joazeiro and Weissman 2000).

Ubiquitination was initially discovered for its role in targeting tagged proteins for proteasomal degradation (Hershko, Ciechanover, et al. 1980, Wilkinson, Urban and Haas

1980). More recently, ubiquitination has become recognized as an important cell signaling and trafficking mechanism. The ubiquitin molecule is a 76 amino acid long protein containing seven lysine residues at positions 6, 11, 27, 29, 33, 48, and 63. There is evidence that the position of the lysine residue participating in the ubiquitin linkage, as well as the length of the ubiquitin chain determine the fate of the tagged protein. The canonical signal for protein degradation by the 26S proteasome is a K48-linked polyubiquitin chain of at least

4 ubiquitin molecules (Chau, et al. 1989, Thrower, et al. 2000). Polyubiquitin tags with K63

or K27 linkages activate intermediates in the NF-κB and IRF signaling pathways (Sun, et al.

2004, Zhou, et al. 2004, Huye, et al. 2007, Arimoto, et al. 2010). Monoubiquitination can act

as a signal for receptor internalization, endosomal trafficking, and DNA polymerase

recruitment (Mukhopadhyay and Riezman 2007, Bienko, et al. 2005).

In addition to their role as E3 ligases for ubiquitin, some TRIM proteins have been found

to act as E3 ligases for other ubiquitin-like tags such as SUMO (small ubiquitin-like modifier), Nedd8 (neural precursor cell-expressed developmentaly downregulated gene 8),

and ISG15 (interferon-induced 15 kDa protein). Auto-SUMOylation of TRIM19 (also called

PML) is necessary for its nuclear localization and function (Zhong, et al. 2000). TRIM40

inhibits the activity of NEMO, a protein whose activity is required for activation of NF-κB,

via neddylation of NEMO (Noguchi, et al. 2011). TRIM25 mediates ligation of ISG15 to 14-

3-3σ protein in vivo, although the functional consequences of this ISGylation are not clear

(Zou and Zhang 2005).

TRIMs and Innate Immunity

TRIM family members serve diverse cellular functions, and many have been implicated

in innate immune responses, particularly antiviral responses. Studies have shown that a

number of TRIM proteins are capable of restricting viral entry, replication and release (Uchil,

et al. 2008, Turelli, et al. 2001, Eldin, et al. 2009, Gao, et al. 2009, Allouch, et al. 2011). For

a few of the TRIM members, restriction occurs through viral interaction. One of the most

studied is TRIM5α of African green monkeys and macaques. TRIM5α from these species is

able to restrict HIV-1 infection via recognition of the viral capsid by specific residues in its

carboxy terminal domain, with subsequent activation of TAK1 kinase complex leading to

NF-κB activation (Stremlau, et al. 2004, Nakayama, et al. 2005, Pertel, et al. 2011). TRIM28

is able to restrict retroviral replication by mediating silencing of the viral promoter (Wolf and

Goff 2007). TRIM21 has been shown to act as a cytoplasmic IgG receptor able to bind and neutralize antibody-opsonized adenovirus (Mallery, et al. 2010, Keeble, et al. 2008).

Other TRIM members affect the activation of NF-κB and interferon signaling pathways.

In a study by Versteeg and colleagues (Versteeg, et al. 2013), over 50% of the tested TRIM

proteins were able to induce activation of one or both pathways in an in vitro cell-based

reporter assay. For some of these TRIM proteins, the mode of action for this activity has been

elucidated. Several TRIM proteins regulate RIG-I, the cytoplasmic viral RNA sensor, and its

downstream signaling complex. TRIM4 and TRIM25 mediate K63-linked polyubiquitination

of RIG-I to facilitate its binding with the downstream signal mediator MAVS, which subsequently turns on downstream NF-κB and IRF3 signaling (Gack, et al. 2007, Yan, et al.

2014). TRIM14 recruits NEMO to the MAVS signaling complex on mitochondria downstream of RIG-I activation (Zhou, et al. 2014). TRIM44 also interacts with MAVS and was shown to prevent ubiquitination and degradation of MAVS (Yang, et al. 2013). TRIM32 and TRIM56 have both been implicated in mediating polyubiquitination to activate STING, another member of the MAVS signaling complex (Tsuchida, et al. 2012, Zhang, et al. 2012).

Following interferon activation, TRIM6 activates IKKε via formation of unanchored K48- linked polyubiquitin chains (Rajsbaum, Versteeg, et al. 2014).

A growing number of TRIM proteins have also been demonstrated to modulate the activity of intermediates in the NF-κB activation pathway. TRIM8, TRIM20, and TRIM23 act as positive regulators of NF-κB. TRIM8 mediates K63-linked polyubiquitination of

TAK1 to activate it (Li, et al. 2011). Following cleavage by caspase-1 downstream of inflammasome activation, the N-terminal fragment of TRIM20 (also called MEFV/Pyrin) interacts with IκB-α and the p65 subunit of NF-κB to promote their degradation and nuclear translocation, respectively (Chae, et al. 2008). TRIM23 mediates K27-linked polyubiquitination of NEMO, which in turn activates the IKK complex (Arimoto, et al.

2010). Other TRIM proteins negatively regulate NF-κB activity. TRIM19 (also called PML) sequesters NF-κB in the nucleus in PML bodies, suppressing NF-κB transcriptional activity

(Wu, et al. 2003). TRIM21, TRIM27, and TRIM40 act to suppress IKK complex activity

(Niida, Tanaka and Kamitani 2010, Zha, et al. 2006, Noguchi, et al. 2011).

Tripartite Motif Containing 9 (TRIM9)

TRIM9 (also known as Spring/MADD2/RNF91) is a Class I TRIM, as defined by Short and Cox (Short and Cox 2006). It has the canonical tripartite motif with a RING, two Bbox domains, and a Coiled-coil region at the amino terminus, while the carboxy terminus contains a COS box, a fibronectin type 3 (FN3) domain, and a SPRY/B30.2 domain. The

COS box mediates microtubule binding (Short and Cox 2006). FN3 and SPRY domains are involved in protein-protein interactions. At least two protein isoforms of TRIM9 are expressed in mice, rats, and humans (Li, et al. 2001, Tanji, et al. 2010, Winkle, et al. 2014,

Qin, et al. 2016). The long form contains all protein domains, while the short form lacks the

SPRY domain (Qin, et al. 2016, Winkle, et al. 2014). Although initially considered to be

brain specific (Berti, et al. 2002, Tanji, et al. 2010, Li, et al. 2001), TRIM9 transcript and

protein expression has been detected in non-neural cells, including leukocytes (Qin, et al.

2016, Versteeg, et al. 2013, Rajsbaum, Stoye and O'Garra 2008, Carthagena, et al. 2009).

TRIM9 was initially identified as a binding partner of SNAP25 (synaptosome-associated

protein of 25 kDa) (Li, et al. 2001). SNAP25 is part of a SNARE complex that facilitates

vesicle fusion and release at neuronal synapses (Sutton, et al. 1998). TRIM9 binds SNAP25

via its Coiled-coil region, preventing SNAP25 interaction with the other SNARE

components, thereby acting as a negative regulator of vesicle exocytosis (Li, et al. 2001).

This study found that TRIM9 was present in the soluble cytosolic portion of neurons, as well

as in association with cytoskeletal components (Li, et al. 2001).

Subsequently, three separate groups identified a role for the TRIM9 homologs in

Caenorhabditis elegans and Drosophila in mediating axon growth guidance (Hao, et al.

2010, Alexander, et al. 2010, Song, et al. 2011). TRIM9 was required for axon branching, and also muscle arm extension, toward the chemoattractant UNC-6 (also known as Netrin-1)

(Alexander, et al. 2010, Hao, et al. 2010, Song, et al. 2011). TRIM9 was found to bind via its

SPRY domain to the UNC-6 receptor UNC-40 (also known as DCC) (Hao, et al. 2010,

Alexander, et al. 2010), and chemotaxis deficits were specific for this chemoattractant/receptor combination in TRIM9 mutants (Hao, et al. 2010, Song, et al.

2011). Although the RING domain was not involved in this interaction, expression of RING- deleted TRIM9 (ΔRING-TRIM9) or of mutant TRIM9 lacking ligase function in an

otherwise wild-type C. elegans recapitulated guidance defects, indicating ΔRING-TRIM9

acts in a dominant negative manner (Alexander, et al. 2010). C. elegans TRIM9 was also

found to interact with UNC-73 (Alexander, et al. 2010), a downstream mediator of UNC-40

whose vertebrate homolog is TRIO, a GEF implicated in RAC1 activation (van Rjissel, et al.

2012). TRIM9 mutants also failed to localize MIG-10, another downstream mediator of

UNC-40 signals that regulates actin polymerization (Hao, et al. 2010, Song, et al. 2011).

Winkle and colleagues extended these findings to mammals, demonstrating that TRIM9 regulates Netrin-1-induced axon branching in a manner that is dependent upon SNAP25-

mediated vesicle exocytosis (Winkle, et al. 2014). The interaction between TRIM9 and DCC

is also conserved in humans (Winkle, et al. 2014). Interestingly, while ΔRING-TRIM9 in

mouse neurons acted in a dominant negative manner to block Netrin-1-induced axon branching as occurred in C. elegans, this did not phenocopy complete loss of TRIM9. In contrast, TRIM9-deficient neurons showed increased constitutive axon branching (Winkle, et

al. 2014).

In neurons, TRIM9 localizes to tips of filopodia, which are F-actin enriched protrusions at the leading edge of the migrating axon (Winkle, et al. 2014), placing TRIM9 in a critical region for regulating cytoskeletal dynamics. A subsequent study identified VASP as a target of TRIM9 ubiquitination (Menon, et al. 2015). As described earlier, VASP mediates processive actin polymerization and localizes to the leading edge of migrating cells (Hansen and Mullins 2010, Chen, et al. 2014). TRIM9, via its Coiled-coil domain, binds VASP and ubiquitinates it, affecting VASP localization within filopodia. Netrin-1 stimulation promotes

de-ubiquitination of VASP, which can then move to the filopodial tips and polymerize F- actin (Menon, et al. 2015).

Just within the past year, an entirely new role for TRIM9 as a modulator of innate immune signaling has been reported. Through an interaction with β-TRCP (β transducin repeat containing protein), TRIM9 negatively regulates NF-κB activation (Shi, et al. 2014).

β-TRCP is a member of a protein complex that ubiquitinates phosphorylated IκBα, marking it for degradation and allowing NF-κB to translocate to the nucleus (Liang, Zhang and Sun

2006). Via a binding site in its amino terminus, TRIM9 interacts with β-TRCP, preventing it from associating with the protein complex and carrying out ubiquitin ligation (Shi, et al.

2014). In contrast to its negative regulation of NF-κB, TRIM9 acts as a positive regulator of interferon activation (Versteeg, et al. 2013, Qin, et al. 2016). Upon viral infection, TRIM9 undergoes K63-linked autoubiquitination and oligomerization, and subsequently acts with

GSK3-β (glycogen synthase kinase 3 β) to enhance activation of TBK1, which in turn activates IRF3/7 (Qin, et al. 2016). Interestingly, this function is specifically carried out by the short isoform of TRIM9.

While these studies demonstrate a role for TRIM9 in the innate immune response, the majority were performed in neural, fibroblast, or lung-derived cell lines. In vivo studies on

TRIM9 immune function, and specifically within neutrophils and macrophages, are lacking.

Furthermore, TRIM proteins, with their various protein domains and isoforms, are known to be multifunctional (Napolitano and Meroni 2012). TRIM9 associates with proteins, such as

VASP, that regulate chemotaxis and cytoskeletal dynamics not just in neurons but in leukocytes, making it a strong candidate as a regulator of neutrophil and macrophage

chemotaxis. Zebrafish provide an optimal model for studying this potential function of

TRIM9 and further elucidating its role in the innate immune response.

Table 1. Human pattern recognition receptors and their ligands

Gene symbol Gene name Ligand Reference(s) TLRs TLR1 Toll-like receptor 1 Triacylated lipopeptides (Takeuchi, Sato, et al. 2002) (Pam3CSK4)* TLR2 Toll-like receptor 2 Triacylated lipopeptide (Aliprantis, et al. 1999), (Hajjar, et al. (Pam3CSK4)*, diacylated 2001), (Ozinsky, et al. 2000), lipopeptide**, phenol (Schwandner, et al. 1999), (Takeuchi, soluble modulin**, Sato, et al. 2002), (Takeuchi, Kawai and zymosan**, peptidoglycan, Mühlradt, et al. 2001) lipotechoic acid, TLR3 Toll-like receptor 3 dsRNA, PolyIC (Alexopolou, et al. 2001) TLR4 Toll-like receptor 4 Lipopolysaccharide (Chow, et al. 1999) TLR5 Toll-like receptor 5 Flagellin (Hayashi, et al. 2001) TLR6 Toll-like receptor 6 diacylated lipopeptide**, (Hajjar, et al. 2001), (Ozinsky, et al. phenol soluble modulin**, 2000), (Takeuchi, Kawai and Mühlradt, zymosan** et al. 2001) TLR7 Toll-like receptor 7 ssRNA (Heil, et al. 2004) TLR8 Toll-like receptor 8 ssRNA (Heil, et al. 2004) TLR9 Toll-like receptor 9 Unmethylated CpG DNA (Hemmi, et al. 2000) TLR10 Toll-like receptor 10 Unknown RLRs RIG-I Retinoic acid-inducible gene I dsRNA, 5’PPP ssRNA, (Kato, Takeuchi and Mikamo-Satoh, et PolyIC (LMW) al. 2008), (Yoneyama, Kikuchi and Natsukawa, et al. 2004), (Hornung, et al. 2006), (Pichlmair, et al. 2006),

Table 1 continued.

RLRs continued MDA5 Melanoma differentiation dsRNA, polyI:C (HMW) (Kato, Takeuchi and Mikamo-Satoh, associated 5 et al. 2008), (Takahasi, et al. 2009) LGP2 Laboratory of genetics and dsRNA, 5’PPP ssRNA (Takahasi, et al. 2009), (Murali, et al. physiology 2 2008) NLRs CIITA Class II major histocompatibility Unknown complex transactivator NAIP NLR family apoptosis inhibitory Cprl protein of (Y. Zhao, et al. 2011) protein Chromobacterium violaceum NOD1 Nucleotide binding oligomerization Diaminopimelic acid- (Girardin, Boneca and Carneiro, et al. domain containing, 1 containing peptides 2003), (Laroui, et al. 2011) NOD2 Nucleotide binding oligomerization Muramyl dipeptide (Girardin, Boneca and Viala, et al. domain containing, 2 2003), (Grimes, et al. 2012) NLRC3 NLR family, CARD domain Unknown containing 3 NLRC4 NLR family, CARD domain NAIP (Y. Zhao, et al. 2011) containing 4 NLRC5 NLR family, CARD domain Unknown containing 5 NLRP1 NLR family, pyrin domain Muramyl dipeptide (Faustin, et al. 2007) containing 1 NLRP2 NLR family, pyrin domain Unknown containing 2 NLRP3 NLR family, pyrin domain Direct ligand undetermined containing 3

Table 1 continued.

NLRs continued NLRP4 NLR family, pyrin domain Unknown containing 4 NLRP5 NLR family, pyrin domain Unknown containing 5 NLRP6 NLR family, pyrin domain Unknown containing 6 NLRP7 NLR family, pyrin domain Bacterial acylated (Khare, et al. 2012) containing 7 lipoproteins (indirectly) NLRP8 NLR family, pyrin domain Unknown containing 8 NLRP9 NLR family, pyrin domain Unknown containing 9 NLRP10 NLR family, pyrin domain Unknown containing 10 NLRP11 NLR family, pyrin domain Unknown containing 11 NLRP12 NLR family, pyrin domain Unknown containing 12 NLRP13 NLR family, pyrin domain Unknown containing 13 NLRP14 NLR family, pyrin domain Unknown containing 14 NLRX1 NLR family member X1 Unknown * Recognized by heterodimer of TLR1/2 ** Recognized by heterodimer of TLR 2/6 LMW: low molecular weight, HMW: high molecular weight

Figure 1. PRR stimulation initiates intracellular signaling pathways that lead to transcription factor activation. Ligation of TLRs initiates MyD88-dependent and MyD88- independent pathways. TLR4 activates both pathways and is shown here as a representative TLR. The MyD88-dependent pathway leads to NF-κB activation and MAPKK activation. The MyD88-independent pathway, initiated by TRIF, leads to activation of NF-κB and IRF3. RIG-I, like MDA-5, binds dsRNA and activates NF-κB and IRF3 via MAVS. Among the NLRs, NOD1 and NOD2 activate NF-κB via RIPK2. Other NLRs, however, block NF-κB and IRF3 activation. This figure is adapted from Mogensen (2009).

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CHAPTER 2: DISRUPTION OF TRIM9 FUNCTION ABROGATES MACROPHAGE MOTILITY IN VIVO

Debra A. Tokarza, Amy K. Heffelfingera, Dereje D. Jimaa,b, Jamie Gerlacha, Radhika N.

Shaha, Ivan Rodrigueza, Ashley A. Fletchera, Shila K. Nordonea,c, J. Mac Lawc,d, Steffen

Heberc,e and Jeffrey A. Yodera,b,c

aDepartment of Molecular Biomedical Sciences, bCenter for Human Health and the

Environment, cComparative Medicine Institute, dDepartment of Population Health and

Pathobiology, eDepartment of Computer Science, North Carolina State University, Raleigh,

NC USA

Experiments to generate the microarray data presented in this chapter were performed by

Amy K. Heffelfinger, with additional support in design and analysis provided by Dereje D.

Jima, Radhika N. Shah, Ashley A. Fletcher, Ivan Rodriguez, Shila K. Nordone, J. Mac Law, and Steffen Heber. I performed the transcriptional studies on Trim9, designed the Trim9 transgenes, created the mosaic transgenic zebrafish and in vivo migration assays used, and performed the imaging and analysis. Under my supervision, Jamie Gerlach performed macrophage migration assays in response to Pam3CSK4.

Abstract

The vertebrate immune response is comprised of multiple molecular and cellular

components that interface to provide an adequate defense against pathogens. Although much

is known on how individual molecules or cells respond to infection, an understanding of the

whole-organism response to pathogen exposure remains unresolved, due to the dynamic

complexity of the immune system and its interdependent innate and adaptive functionality.

Zebrafish larvae provide a unique model for overcoming this obstacle as larvae can

successfully defends themselves from pathogens while lacking a functional adaptive immune

system during the first few weeks of life, making it possible to examine exclusively the

innate immune response in a whole-organism context. It was hypothesized that the transcriptional response of zebrafish larvae to immune agonists would identify known immune-response genes as well as reveal genes that mediate innate immunity in novel ways.

In order to test this hypothesis, zebrafish larvae were exposed to the chemically diverse

immune agonists, polyinosine-polycytidylic acid (PolyIC) and synthetic triacylated

lipoprotein (Pam3CSK4) and transcriptome analyses completed using microarray analyses.

This strategy successfully identified known immune response genes, as well as genes that

had not been implicated in immune function, including the E3 ubiquitin ligase, tripartite

motif 9 (trim9). Although Trim9 expression has been described as “brain specific”, here we

demonstrate elevated levels of trim9 transcripts in macrophages after immune stimulation. As

Trim9 has been implicated in axonal migration, we investigated and demonstrate that

disruption of Trim9 function impairs macrophage chemotaxis and cellular architecture in

vivo. These results demonstrate that Trim9 mediates cellular movement and migration in

macrophages as well as neurons.

Introduction

The innate immune system is capable of mounting a rapid and potent inflammatory

response that is critical as a first line of defense against invading pathogens. Innate immune

responses are induced when pathogens are sensed by a variety of cellular pattern recognition

receptors (PRRs). Several classes of PRRs are present in vertebrates, among which the toll-

like receptors (TLRs) are the best characterized. TLRs bind diverse pathogen associated

molecular patterns (PAMPs) present in bacteria, viruses, and other microbes (Mogensen

2009). Binding triggers intracellular signaling cascades that activate NF-κB (nuclear factor of

kappa light polypeptide gene enhancer in B-cells) and IRFs (interferon regulatory factors), which in turn alter expression of a wide array of genes that mediate intra- and intercellular responses to enable the recruitment and activation of innate immune effector leukocytes to sites of infection or inflammation. Among these leukocytes, macrophages and neutrophils play a key role in innate immune responses because of their ability to phagocytose and kill microorganisms. In addition, macrophages act as important coordinators of the immune response through production of pro-inflammatory cytokines and antigen presentation to lymphocytes (Duque 2014, Rosenthal and Shevach 1973).

While innate immune responses are critical for host defense, excessive leukocyte activation and recruitment can lead to tissue damage and compromise organ function.

Dysregulated innate immune responses are implicated in the pathogenesis of various acute and chronic inflammatory conditions that reduce patient survival and health-related quality of life (Casellas, et al. 1999, Rubenfeld, et al. 2005, Uchino, et al. 2005, Cheung, et al. 2006).

Identifying novel innate immune response genes and defining the regulatory networks that control macrophage and neutrophil activity will inform new therapeutic strategies for modulating inflammation. Because an effective innate immune response requires the coordination of multiple tissues and cells types, it is ideal to study this response in the context of the whole organism.

Zebrafish (Danio rerio) are an increasingly utilized model for studying vertebrate immune responses due to their small size, high fecundity, short generation time, and reference genome. Importantly, the zebrafish immune system shares many conserved features with mammalian immune systems (Lieschke, et al. 2001, van der Haart, Spaink and Meijer

2012, Zapata, et al. 2006, Wittamer, et al. 2011). By 72 hours post fertilization (hpf),

zebrafish express critical innate immune components such as TLRs (van der Sar,

Stockhammer, et al. 2006) and fully functional neutrophils and macrophages (Ellett, et al.

2011, Herbomel, Thisse and Thisse 1999). The ex utero development of zebrafish larvae

allows easy access to the whole organism during this window of time, which is weeks before

development of a functional adaptive immune response (Lam, et al. 2004). This facilitates

focused investigation of innate immune responses on the whole organism level.

Although several groups have recently utilized transcriptome analysis to investigate

the immune response in larval zebrafish, these studies have focused on the response to

specific bacterial or viral pathogens and/or known immune-related genes (van der Sar,

Spaink, et al. 2009, Ordas, et al. 2011, Briolat, et al. 2014, Yang, et al. 2015, Stockhammer, et al. 2009). To identify novel innate immune response genes, we defined the whole organism transcriptional response of larval zebrafish exposed to two different PAMPs: polyinosine-polycytidylic acid (PolyIC), agonist of the viral PAMP sensor TLR3

(Alexopolou, et al. 2001, Phelan, Mellon and Kim 2005, Matsuo, et al. 2008), and a synthetic triacylated lipoprotein (Pam3CSK4), agonist of the bacterial PAMP sensor TLR2 (Aliprantis

1999, Ribeiro, et al. 2010). Through analysis of this transcriptome we identified a list of common larval innate immune response genes with orthologous genes in human. One candidate novel innate immune response gene, tripartite motif 9 (trim9), was chosen for further analysis.

Trim9 is a member of the large tripartite motif protein family defined by the presence of 3 amino-terminal protein motifs: a really interesting new gene (RING) domain, Bbox domains, and a coiled coil region. The RING domain imparts E3 ubiquitin ligase activity

(Joazeiro and Weissman 2000). Trim9 is abundantly expressed in neurons of the cerebral cortex and was previously considered to be “brain specific” (Li, et al. 2001, Berti, et al. 2002,

Tanji, et al. 2010). Here we demonstrate that trim9 transcripts can be detected in zebrafish and human macrophages, and that trim9 transcript levels increase within these cells after immune stimulation. Further, we demonstrate a potential role for Trim9 in regulating macrophage motility and cellular architecture in vivo in zebrafish. These results highlight the zebrafish as a comparative immunology tool and identify Trim9 as a novel intracellular mediator in macrophages via its ubiquitin ligase activity.

Materials and Methods

Immune agonist exposure and RNA isolation

At 72 hpf, zebrafish larvae were exposed to 10 μg/ml PolyIC (Invivogen), 5 μg/ml

Pam3CSK4 (Invivogen) or no agonist (negative control) by immersion for 4, 8, 12, 24 or 36

hrs. Exposures were executed in 24-well plates with 10 larvae per well in a volume of 2.0 ml.

RNA was isolated from euthanized larvae using TRIzol reagent (Invitrogen) and further

purified by RNeasy MinElute Cleanup (Qiagen). RNA quantity and quality was verified with

a NanoDrop ND1000 spectrophotometer and an Agilent 2100 Bioanalyzer. RNA (2 μg) was reverse transcribed using Superscript III (Invitrogen). Each treatment group and time point for microarray analyses included RNA pooled from 30 larvae (3 wells) and four biological replicates using different clutches of embryos.

Microarrays

Microarray slides were custom-designed 4 x 44 k zebrafish arrays (Agilent Technologies) as described (Stockhammer, et al. 2009) and included 43,371 probes (GEO Accession

GPL7735). RNA was labeled (Cy3) and hybridized to arrays using standard methods

(Cogenics, Research Triangle Park, NC). Image analyses and the calculations of spot intensities were performed using Agilent’s Feature Extraction software version 8.5 (Agilent

Technologies). Details of array analyses are provided in Supporting Information.

Isolation of zebrafish macrophages

Zebrafish macrophages were isolated from Tg(mpeg1:EGFP) transgenic zebrafish in

which the mpeg1 promoter drives macrophage-specific expression of enhanced green fluorescent protein (EGFP) (Ellet, et al. 2011). At 120 hpf, groups of 75-100

Tg(mpeg1:EGFP) transgenic larvae were exposed by immersion to 10 μg/ml PolyIC, 5 μg/ml

Pam3CSK4 or no agonist for 8 or 12 hours. Following exposure, larvae pooled by treatment group were disaggregated into single cell suspensions as previously described (Manoli and

Driever 2012) and sorted for EGFP-positive cells using a Dako Cytomation MoFlo cytometer. RNA was isolated from sorted cells using the Qiagen RNeasy Micro kit.

Cell Culture

Human promonocytic U-937 cells (ATCC CRL-1593.2) were cultured in RPMI-1640 media (Corning cellgro) supplemented with 10% fetal bovine serum (Atlanta Biologicals).

Differentiation was induced by 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) stimulation for 24 hours. Differentiated cells were rested for 6 days then stimulated with either 0.1 ug/ml Pam3CSK4, 10 ug/ml PolyIC, or 0.1 ug/ml ultrapure lipopolysaccharide

(LPS) from E. coli 0111:B4 (Invivogen). At 4, 8, and 12 hours post exposure cells were lysed and RNA isolated using TRIzol reagent according to manufacturer’s protocol.

Construction of zebrafish transgenes

The mpeg1 promoter was amplified from an mpeg1:mCherry plasmid provided by

Graham Lieschke. Full length and RING-deleted trim9 were amplified from a zebrafish trim9 cDNA clone (ATCC 10330554; IMAGE clone ID 6968453). Teschovirus 2A peptide and

EGFP were amplified from a p3E-2A-EGFPpA plasmid provided by Kristen Kwan. The pDestTol2pA2 vector and pCS2FA-transposase were obtained in the Tol2kit (Kwan, et al.

2007) from Koichi Kawakami. pDestTol2pA2 was restriction digested with XhoI and ClaI

(Thermo Scientific). Amplicons were cloned into the linearized pDestTol2pA2 vector using

Gibson Assembly Master Mix (New England BioLabs) according to manufacturer instructions. Tol2 transposase mRNA was reverse transcribed from the pCS2FA-transposase plasmid (Urasaki, Morvan and Kawakami 2006, Kawakami, et al. 2004) using mMessage mMachine SP6 kit (Ambion).

In vivo chemotaxis assay

Double transgenic zebrafish were generated on the Tg(mpeg1:mCherry) background in which the mpeg1 promoter drives macrophage-specific expression of mCherry (Ellet, et al.

2011). Tg(mpeg1:mCherry) embryos were injected with 50 pg of the Tg(mpeg1:EGFP),

Tg(mpeg1:trim9,EGFP) or Tg(mpeg1:ΔRINGtrim9,EGFP) transgene plasmid (see Fig. 4) and 50 pg of Tol2 transposase mRNA at the single cell stage to generate mosaic EGFP transgenic larvae on the stable mCherry background. At 72 hpf, larvae were anesthetized in

0.016% Tricaine in egg water and microinjected in the left otic vesicle with a 1 nl volume of

phosphate buffered saline (PBS) containing either 100 pg PolyIC or 50 pg Pam3CSK4.

Injected larvae were recovered in fresh egg water at 28 °C. At 2 hours post-injection, larvae

were reanesthetized and mounted in 1% low melting point agarose. Stacked fluorescence

images of the left otic vesicle and a 250-µm segment of the tail just caudal to the urogenital

opening were acquired with a laser scanning confocal microscope (AZ-C2+, Nikon

Instruments, Melville, NY). Within the otic vesicle and tail, macrophages expressing

mCherry or mCherry with EGFP were counted using NIS-Elements AR 4.11 software

(Nikon). The percentage of macrophages co-expressing EGFP was determined in the otic vesicle and tail, and then compared to obtain a migration index:

% = % 𝑜𝑜𝑜𝑜 𝐸𝐸𝐸𝐸𝐸𝐸𝑃𝑃 − 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑖𝑖𝑖𝑖 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 Outlines and circularity indices for individual𝑜𝑜𝑜𝑜 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 − macrophages𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑚𝑚in𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 vivo inℎ𝑎𝑎 unstimulated𝑔𝑔𝑔𝑔𝑔𝑔 𝑖𝑖𝑖𝑖 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 mosaic

transgenic zebrafish larvae were obtained using the NIS-Elements AR 4.11 software.

Time lapse microscopy

For time lapse images, 72 hpf mosaic transgenic larvae were anesthetized and injected in

the otic vesicle as above, then mounted in 1% low melting point agarose. Fluorescent images

were taken every 30 seconds for 2 hours on a Zeiss Lightsheet Z.1 (Jena, Germany) with s-

CMOS PCO.edge cameras (PCO AG, Kelheim, Germany) and processed using ZEN black

Imaging software (Zeiss). Cell tracking was performed using ImageJ and the MTrackJ plug-

in (Meijering, Dzyubachyk and Smal 2012). Migration tracks for cells visible for the full 2

hours of imaging were plotted using the Ibidi Chemotaxis and Migration Tool plug-in for

Image J (ibidi.com/software/chemotaxis_and_migration_tool/).

Statistical analyses

For real time PCR analyses, Student’s T tests were performed to compare control and treatment fold changes. Data from in vivo larval zebrafish macrophage assays were compared using a one-way ANOVA and Tukey’s HSD or Steel-Dwass post hoc test. For all statistical tests, results were considered significant if p < 0.05.

Results

Array results

In order to identify genes that are transcriptionally responsive to immune stimuli in vivo, zebrafish larvae were independently exposed to two chemically distinct immune agonists,

Pam3CSK4 and PolyIC. At 4, 8, 12, 24 or 36 hr post exposure (hpe) RNA was isolated from pooled larvae and subjected to microarray analyses. This strategy identified 574 and 1035 genes that were differentially expressed (as compared to control) at one or more time points after exposure to 5 μg/mL Pam3CSK4 or 10 μg/mL PolyIC, respectively (Fig. 1A-C and

Figs. S1-S3). Hierarchal clustering of these genes reveals that more than 50% of these genes

displayed altered transcript levels at 8 and 12 hpe. Smaller groups of genes were observed to

have increased transcript levels either at early (4 hpe) or later (24 or 36 hpe) timepoints. One

group of genes displayed increased transcript levels at early (4 hpe) and late (24 and/or 36

hpe) time points (Fig. S1). Interestingly, 27 genes were observed to have increased transcript

levels at multiple time points after exposure to PolyIC (as compared to Pam3CSK4 exposure)

(Fig. S4). This group includes NF-κB repressor factor (nkrf) and CCCTC-binding factor

(ctcf), which is a host cell restriction factor for Kaposi’s sarcoma-associated virus (Li, et al.

2014). Four genes were identified with increased transcript levels at 12 hpe for Pam3CSK4

(as compared to PolyIC) including mitochondrial pyruvate carrier 1 (mpc1) which plays a

role in glycolysis (Bricker, et al. 2012) (Fig. S4). An interrogation of this complete gene list

revealed that eight members of the canonical CXCR4 chemokine receptor pathway were

increased significantly representing an activation of this pathway (Fig. S5). The top gene

network identified from this list of genes is the Inflammatory (NF-κB) Network confirming

that exposure of zebrafish larvae to the selected concentrations of Pam3CSK4 and PolyIC

activates immune signaling pathways (Fig. S6-S7).

In order to identify zebrafish genes that could be considered common immune response

genes, we restricted the gene list to sequences that displayed significant changes in transcript

levels for both immune agonists and represented defined zebrafish genes. Of the 230

zebrafish sequences that responded to both agonists, 121 represented defined genes including

known immune response genes (e.g. mmp9, mmp13a, c3a.1 and il17d) as well as genes with no described immune function such as trim9 (Fig. 1D).

Trim9 transcript levels increase after immune stimulation

Trim9 has been shown to play a significant role in axonal migration (Alexander, et al.

2010, Hao, et al. 2010, Song, et al. 2011, Winkle, et al. 2014), therefore, we hypothesized that Trim9 also may play a role in the motility of immune cells of the zebrafish larvae.

Results from quantitative RT-PCR (qPCR) confirmed the increase in trim9 transcript levels

in larval zebrafish following 8 hour exposures to Pam3CSK4 or PolyIC (Fig. 2A). Because

macrophages are one of the major innate immune effector cells active in the larval zebrafish

(Herbomel, Thisse and Thisse 1999, Lieschke, et al. 2001), we sought to determine whether

trim9 transcripts were expressed in and responded to immune stimulation within these cells.

EGFP-expressing macrophages were isolated from Tg(mpeg1:EGFP) (Ellett, et al. 2011) transgenic larvae, following 8 or 12 hours of exposure of the whole larvae to Pam3CSK4 or

PolyIC. In macrophages, trim9 transcript levels increased following exposure to either agonist, particularly at 12 hpe (Fig. 2B).

TRIM9 is highly conserved across vertebrate species. To determine whether a similar transcriptional response occurred in human macrophages, we exposed macrophage-like cells derived from the human U937 monocytic cell line (Baek, et al. 2009, Liu and Wu 1992,

Sundstrom 1976) to both agonists used in the zebrafish, as well as lipopolysaccharide (LPS) a TLR4 agonist (Chow, et al. 1999). We detected significant increases in TRIM9 transcript levels in response to Pam3CSK4 and LPS to a similar extent and in a similar time frame as seen in the larval zebrafish macrophages (Fig. 2C). In contrast to the zebrafish, no transcriptional response was detected for PolyIC in the U937 macrophage-like cells. These

results show that immune stimulation increases TRIM9 transcripts in macrophages across

species, suggesting the potential for a conserved TRIM9 function in activated macrophages.

Disruption of Trim9 function results in defective macrophage chemotaxis

In neurons, TRIM9 is necessary for axon branching and attraction in response to the

chemoattractant Netrin-1/UNC-6 (Alexander, et al. 2010, Hao, et al. 2010, Song, et al. 2011,

Winkle, et al. 2014). During an inflammatory response leukocytes similarly migrate along

chemoattractant gradients to reach sites of inflammation or infection, therefore we

hypothesized that Trim9 may function to mediate chemotaxis in macrophages. To test this

hypothesis we designed a transgenic approach to disrupt Trim9 function in zebrafish

macrophages in vivo by using the macrophage-specific mpeg1 gene promoter (Ellett, et al.

2011) to drive expression of truncated Trim9 lacking the RING domain necessary for ubiquitin ligase activity (∆RINGTrim9) (Fig. 3). A teschovirus 2A peptide sequence (Kim, et al. 2011) was incorporated into the transgenes to allow for coexpression of EGFP.

Transgenes were introduced into Tg(mpeg1:mCherry) embryos to yield mosaic transgenic fish in which all macrophages express mCherry and a subset coexpress EGFP, indicating expression of the experimental transgene (Fig. 3A). To assess macrophage chemotaxis in vivo, mosaic transgenic zebrafish larvae were injected at 72 hpf in the otic vesicle with either

Pam3CSK4 or PolyIC and macrophage migration into the otic vesicle was quantified using fluorescence microscopy. In Caenorhabditis elegans and in cultured cortical neurons of

mouse, expression of RING-deleted TRIM9 acts in a dominant negative manner to abolish

the response to Netrin (Alexander, et al. 2010, Winkle, et al. 2014). Interestingly, we found

that macrophages expressing ∆RINGTrim9 had a significant reduction in migration to the

injected otic vesicle relative to macrophages expressing full length Trim9, or EGFP alone,

regardless of the inciting agonist (Fig. 3B-C). These results indicate that loss of Trim9 function negatively impacts macrophage chemotaxis.

Disruption of Trim9 alters macrophage morphology and motility

Imaging of individual transgenic macrophages in vivo revealed a stark contrast in morphology induced by ∆RINGTrim9 expression. Migrating macrophages extend cellular protrusions called pseudopods formed by polymerization of actin filaments that push the cell membrane forward (Hartwig and Stossel 1985). Whereas macrophages expressing the control

(EGFP) or full length Trim9 transgenes were typically elongated to stellate due to formation of multiple pseudopod extensions, macrophages expressing ∆RINGTrim9 were significantly more circular with smooth cell contours (Fig. 4; Fig. S8).

Time lapse microscopy was employed to visualize the migration of transgenic macrophages in vivo. In accordance with our migration assay data, macrophages expressing

∆RINGTrim9 show significant reductions in velocity and travel limited distances over the 2 hour imaging period compared to macrophages expressing the control or full length Trim9 transgenes (Fig. 5). Further, while macrophages expressing the control or full length Trim9 transgenes exhibit highly dynamic pseudopod extension and retraction over the course of imaging, macrophages expressing ∆RINGTrim9 generally lack pseudopod extensions, or

occasionally exhibit short, thin cytoplasmic extensions (movies not shown). Taken together,

these data suggest that Trim9 ubiquitin ligase function may be necessary for proper

macrophage pseudopod formation and motility.

Discussion

Gene expression studies combining zebrafish embryo infection models with

transcriptome analyses have been successfully employed to identify genes that are

transcriptionally responsive to immune stimuli (Lü, et al. 2015, van der Vaart, Spaink and

Meijer 2012, Ordas, et al. 2011, Wu, et al. 2010, Hegedus, et al. 2009, van der Sar, Spaink, et

al. 2009, Stockhammer, et al. 2009) and have demonstrated the utility of the zebrafish model

for identifying known immune response genes that are conserved with human (van der Vaart,

Spaink and Meijer 2012). By assessing transcriptional changes in zebrafish larvae after

exposure to chemically distinct immune agonists we have also identified known immune

response genes including those involved in the CXCR4 signaling pathway and in the

Inflammatory (NF-κB) network.

An important goal of our study was to identify genes that had not been implicated in immune function and are well conserved in humans. To that end we chose to investigate

Trim9, which is highly conserved between zebrafish and humans but at the time of our transcriptional analysis had no known immune function. Recently, dual roles for Trim9 in regulating NF-κB and IRF3 activation have been identified. NF-κB and IRF3 activate transcription of pro-inflammatory genes and interferons, respectively, downstream of PRRs

and other immune receptors. TRIM9 negatively regulates NF-κB activation (Shi, et al. 2014), but positively regulates IRF3 activation (Qin, et al. 2016). This provides additional validation of Trim9 as an innate immune mediator and of our zebrafish screen as a tool for identifying novel immune response genes.

TRIM9 transcripts increase in macrophages following stimulation with agonists for TLR2

(Pam3CSK4), TLR3 (PolyIC), and TLR4 (LPS). These results demonstrate for the first time a TRIM9 transcriptional response to PRR stimulation in vivo and in macrophages. The transcriptional response to Pam3CSK4 was similar across species. LPS was not tested in the zebrafish as they are highly tolerant to LPS (Novoa, et al. 2009). PolyIC failed to induce a

TRIM9 transcriptional response in human cells. This could represent a species difference in

TRIM9 regulation, or reflect differences in whole organism versus cell-direct stimulation. A common transcription factor activated by each of these receptors is NF-κB (Aliprantis, et al.

1999, Alexopolou, Holt, et al. 2001, Chow, et al. 1999). The timing of the TRIM9

transcriptional response at 8-12 hours post stimulation is consistent with late phase NF-κB-

activated genes (Han, et al. 2002). This late phase is likely due to a second wave of NF-κB activation induced by early immune response genes (Han, et al. 2002). This could account for the trim9 transcriptional response in macrophages even when agonists were not applied directly, as in the larval zebrafish. Given that TRIM9 negatively regulates NF-κB activation

(Shi, et al. 2014), this could serve as a negative feedback mechanism for regulating inflammatory signals.

Our data demonstrate that in addition to regulating innate immune signaling pathways,

Trim9 also mediates cellular architectural dynamics and motility in macrophages. In

invertebrate models and in mice, TRIM9 mediates axon migration toward the

chemoattractant Netrin-1/UNC-6 (Alexander, et al. 2010, Hao, et al. 2010, Song, et al. 2011,

Winkle, et al. 2014). Neurons expressing ΔRINGTRIM9 fail to extend axon branches along a

Netrin-1 gradient, but are otherwise able to respond normally to other chemoattractant

signals (Winkle, et al. 2014, Hao, et al. 2010). In contrast to neurons, macrophages

expressing ΔRINGTrim9 show abnormal cell morphology with a lack of cell protrusions

with or without immune stimulation. Further, they showed significant reductions in migration

velocity resulting in failure to migrate to an inflammatory site regardless of the inciting

agonist. These findings suggest that Trim9 plays a broader role in cell motility in

macrophages, rather than mediating chemotaxis toward a specific chemoattractant, as in

neurons. TRIM9 in neurons directly interacts with the DCC netrin-1 receptor to mediate

Netrin-1 signaling (Winkle, et al. 2014). In human peripheral blood leukocytes, DCC is not detectable by immunohistochemistry or qPCR (Ly, et al. 2005), suggesting TRIM9 could have alternative means of regulation and, therefore, function in leukocytes.

Defining the mechanism by which TRIM9 effects macrophage motility will require further experiments, however published reports suggest TRIM9 may modulate cytoskeletal dynamics. TRIM9 contains a C-terminal subgroup one signature (COS) box that mediates microtubule binding (Short and Cox 2006). To date the role of microtubule binding in

TRIM9 function remains unknown. In neurons, TRIM9 mediates axon migration through interactions with vasodilator-stimulated phosphoprotein (VASP) (Menon, et al. 2015). VASP functions to lengthen actin polymers (Hansen and Mullins 2010) and localizes to the tips of actin-rich protrusions called filopodia at the leading edge of migrating axons (Winkle, et al.

2014). Ubiquitination of VASP by TRIM9 limits VASP mobility to filopodia tips (Menon, et al. 2015). Interestingly, ΔRINGTRIM9 showed prominent colocalization with VASP in neurons, an interaction which was otherwise transient (Menon, et al. 2015), suggesting that

ΔRINGTRIM9 binds and holds VASP, potentially preventing VASP function. VASP is expressed in macrophages and mediates cytoskeletal changes required for macrophage phagocytosis (Coppolino, et al. 2001), making it a strong candidate for mediating TRIM9 function in macrophage motility. VASP has been shown to mediate chemoattractant-specific chemotaxis in neutrophils (Eckert and Jones 2007, Neel, et al. 2009). Whether TRIM9 is required for neutrophil migration will be an important question to address in future experiments.

Acknowledgements

We thank Annemarie Meijer and Herman Spaink (University of Leiden) for their custom array design; Graham Lieschke (Monash University) and David Traver (University of

California at San Diego) for transgenic lines; and Kristen Kwan (University of Utah) and

Koichi Kawakami (National Institute of Genetics, Japan) for Tol2 plasmids. This research was supported by funding from the National Institutes of Health [R21 AI076829 (J.A.Y.),

T32 OD011130 (D.A.T.), T32 GM008776 (A.K.H), and P30 ES025128], the NC State

University College of Veterinary Medicine (J.A.Y.), and by an American Association of

Immunologists Careers in Immunology Fellowship (D.A.T. and J.A.Y.).

Fig 1. Immune agonist exposure and summary of microarray results. (A) Zebrafish larvae (72 hpf) were exposed to 5 μg/mL Pam3CSK4, 10 μg/mL PolyIC or no agonist for 4, 8, 12, 24 or 36 hr. RNA was isolated from larvae at each time point and employed for microarray analyses. (B-C) Exposure to Pam3CSK4 or PolyIC led to the identification of 574 and 1035 genes, respectively, that had significantly increased or decreased transcript levels at one or more time point (as compared to control larvae) (Figs. S1-S3). Venn diagrams indicate the numbers of genes with altered transcript levels at each time point. (D) Heat map of genes for which transcript levels were altered by exposure to both Pam3CSK4 and PolyIC.

Fig 2. Immune agonist exposure induces increased TRIM9 transcript levels in macrophages. (A) Relative trim9 transcript levels in zebrafish larvae after 8 hr of exposure to 5 μg/mL Pam3CSK4 or 10 μg/mL PolyIC. Larval exposure was initiated at 72 hpf. (B) Relative trim9 transcript levels in larval zebrafish macrophages after 8 and 12 hr exposures to 5 μg/mL Pam3CSK4 or 10 μg/mL PolyIC. Zebrafish larvae (120 hpf) of the Tg(mpeg1:EGFP) transgenic line were exposed to immune agonists and EGFP+ macrophages isolated by cell sorting. (C) The human promonocytic cell line U937 was differentiated to a macrophage-like phenotype and exposed to 10 ug/ml PolyIC, 0.1 ug/ml Pam3CSK4 or 0.1 ug/ml LPS for 4, 8 or 12 hr. Relative transcript levels were determined by qPCR. A,C: mean ± SEM, N=3, *p<0.05.

Fig 3. Macrophage-specific disruption of Trim9 function results in reduced cellular chemotaxis in vivo. (A) Three transgenes were constructed that employed the macrophage- specific promoter of the mpeg1 gene (Ellett, et al. 2011). The Tg(mpeg1:trim9,EGFP) transgene expresses full-length zebrafish Trim9 and EGFP from the same transcript but produces both proteins via a viral 2A peptide cleavage site (Kim, et al. 2011). The Tg(mpeg1:∆RINGTrim9,EGFP) transgene expresses both zebrafish Trim9 that lacks the RING domain and EGFP. The Tg(mpeg1:EGFP) transgene expresses EGFP. When these transgenes are injected into 1-cell zebrafish embryos of the stable transgenic line Tg(mpeg1:mCherry) the resultant larvae are mosaic with all macrophages expressing mCherry (Ellett, et al. 2011) but only a subpopulation of macrophages expressing Tg(mpeg1:trim9,EGFP), Tg(mpeg1:∆RINGTrim9,EGFP) or Tg(mpeg1:EGFP). Trim9 protein domains include RING, B-box (BB), coiled coil (CC), COS, FN3 and SPRY (Short and Cox, 2006). (B-C) The cellular migration index for chemotaxis towards PolyIC or Pam3CSK4 is shown for zebrafish macrophages expressing Tg(mpeg1:trim9,EGFP), Tg(mpeg1:∆RINGTrim9,EGFP) or Tg(mpeg1:EGFP) on the Tg(mpeg1:mCherry) genetic background as compared to macrophages within the same individual expressing only mCherry. N=20-39. *p<0.05.

Fig 4. In vivo disruption of Trim9 function in macrophages significantly alters cell shape. (A) Zebrafish macrophages expressing Tg(mpeg1:EGFP), Tg(mpeg1:trim9,EGFP) or Tg(mpeg1:∆RINGtrim9,EGFP) on the Tg(mpeg1:mCherry) background (see Fig. 3) were photographed to assess cell shape. Images shown are representative of ten cells photographed per transgene (Fig. S8). (B) Circularity scores are shown for each individual cell. Scale: 0-1, where 1 indicates a perfect circle. N=17. *p<0.05.

Fig 5. In vivo disruption of Trim9 function in macrophages significantly disrupts macrophage velocity. Time-lapse video recordings were collected documenting the in vivo movement of zebrafish macrophages expressing Tg(mpeg1:EGFP), Tg(mpeg1:trim9,EGFP) or Tg(mpeg1:∆RINGtrim9,EGFP) (see Fig. 3). (A-B) The mean and maximum velocities of individual macrophages are displayed. N=9-16. *p>0.05 (C) Plots of 2-hour migration tracks for individual macrophages. N=6-8.

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Supporting Information

SI Materials and Methods

Zebrafish. Adult zebrafish were maintained in a recirculating aquarium facility (Aquatic

Habitats, Apopka, FL) at 28 °C with a 10 hr light/14 hr dark cycle and fed a commercial grade zebrafish diet. Wildtype zebrafish were obtained from EkkWill Waterlife Resources

(Ruskin, FL). Transgenic zebrafish lines Tg(mpeg1:mCherry) and Tg(mpeg1:EGFP) (Ellett, et al. 2011) were kind gifts from Graham Lieschke (Monash University) and David Traver

(University of California at San Diego). Zebrafish embryos were obtained by natural spawning and maintained at 28 °C in egg water (0.5 mg/L methylene blue and 60 mg/L aquarium salt mixture). Larvae were euthanized in 0.02% Tricaine methanesulfonate

(Finquel MS-222; Argent Chemical). Zebrafish husbandry and experiments involving live animals were approved by the North Carolina State University Institutional Animal Care and

Use Committee.

Determination of agonist levels. Prior to microarray analyses, zebrafish larvae (72 hpf) were exposed to 1-5 μg/mL Pam3CSK4 or 5-50 μg/mL PolyIC for 24 and 36 hr and their phenotypes observed and the transcriptional response of the interleukin 1, beta (il1b) and the myxovirus (influenza) resistance A (mxa) genes assessed by qPCR. The concentrations of agonists selected for microarray analyses induced increased transcript levels of the il1b gene

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(5 μg/ml Pam3CSK4) or the mxa gene (10 μg/ml PolyIC) while yielding no overt phenotype

or detectable microscopic damage (as assessed by a Board Certified Veterinary Pathologist).

Microarray data preprocessing and analyses. An initial quality assessment of the raw microarray data was conducted and control spots, outliers, and spots with low average intensity were removed. To reduce the effect of inhomogeneous background hybridization across arrays the background correction method normexp was applied (Ritchie, et al. 2007).

This model-based approach uses Agilent’s background estimates to compute positive, corrected hybridization intensities. Normexp was implemented in the Bioconductor

(http://www.bioconductor.org) package limma (Smyth 2005). Quantile normalization between arrays was implemented (in limma) in order to reduce technical variation. Finally, hybridization intensities were log2-transformed and averaged for all replicate spots on the array.

Statistical modeling and extraction of differential gene expression. The effect of immune agonist exposure on larval transcript levels was investigated by analyzing array data using a linear model with two factors: 1) agonist treatment (Pam3CSK4, PolyIC, and control) and 2) time after exposure (4, 8, 12, 24 and 36 hpe). To accommodate for the dissimilarity between biological replicates, treatment and control samples of each replicate were paired. Using the preprocessed hybridization intensities, changes in transcript levels between each treatment and control group for each time point were computed via an empirical Bayes moderated paired t-test (Smyth 2004) using the Bioconductor package limma. Candidate genes for

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differential expression with a p value of less than 0.05 were selected. To account for multiple

testing, the false discovery rate of the extracted candidate gene list was computed from the p-

value distribution of the corresponding genes using the Bioconductor package qvalue (Storey

2002).

To visualize expression patterns using heat-maps, the log-transformed average expression of the agonist treated samples were subtracted from the untreated control samples at each time point. Hierarchical cluster analyses of gene expression was used to group genes with similar expression pattern using Gene Cluster 3.0 program (de Hoon, et al. 2004) and heat-

maps were generated using Java Treeview program (Saldanha 2004). Gene pathway and

network analyses were conducted using Ingenuity Pathway Analysis (Qiagen).

Quantification of trim9 transcript levels. cDNA was synthesized using Invitrogen

Superscript III First Strand cDNA Synthesis kit. qPCR was performed using Applied

Biosystems Taqman Universal PCR Master Mix II and Taqman Gene Expression assays for zebrafish trim9 (Dr03081570_m1) and ef1α (Dr03432748_m1) and human TRIM9

(Hs00364838_m1) and ACTB (Hs01060665_g1). Fold changes in transcript levels were calculated using the 2-∆∆Ct method (Livak and Schmittgen 2001).

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Fig. S1. Heat map of 1110 genes with significantly different transcript levels (p < 0.05) after exposure to either Pam3CSK4 or PolyIC at any time point. Gene clusters with similar expression patterns are annotated on the right.

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Fig. S2. Distribution of genes with significantly altered transcript levels during Pam3CSK4 exposure. Total number of genes with significantly different transcript levels for each time point after Pam3CSK4 exposure is indicated in the histogram on the left. The size of the subsets of genes with significantly different transcript levels in one (red), two (blue), three (purple) or five (green) time points after agonist exposure is indicated in the histogram above.

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Fig. S3. Distribution of genes with significantly altered transcript levels during PolyIC exposure. Total number of genes with significantly different transcript levels for each time point after PolyIC exposure is indicated in the histogram on the left. The size of the subsets of genes with significantly different transcript levels in one (red), two (blue), three (purple), four (orange) or five (green) time points after agonist exposure is indicated in the histogram above.

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Fig. S4. Heat map of gene expression data for subgroups of genes responsive primarily to (A) PolyIC or (B) Pam3CSK4.

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Fig. S5. Activation of CXCR4 pathway. (A) Components of the human CXCR4 pathway are indicated. Genes identified with transcriptional changes in zebrafish larvae after exposure to Pam3CSK4 or PolyIC are outlined in pink. (B) Heat map of transcript levels of the zebrafish genes in the CXCR4 signaling network.

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Fig. S6. Activation of the Inflammatory Network. Components of the human NF-κB network are indicated. Genes identified with transcriptional changes in zebrafish larvae after exposure to Pam3CSK4 or PolyIC are outlined in pink.

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Fig. S7. Transcript level changes for genes of the Inflammatory Network. Genes from the Inflammatory Network that displayed altered transcript levels in zebrafish larvae after exposure to immune agonists. Histograms reflect changes (log ratio) in transcript levels at different time points after exposure to Pam3CSK4 or PolyIC (red = increase, green = decrease).

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Fig. S8. In vivo disruption of Trim9 function in macrophages significantly alters cell shape. Zebrafish macrophages expressing Tg(mpeg1:EGFP), Tg(mpeg1:trim9,EGFP) or Tg(mpeg1:∆RINGtrim9,EGFP) on the Tg(mpeg1:mCherry) background (see Fig. 3) were photographed to assess cell shape.

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SI References de Hoon, MJ, S Imoto, J Nolan, and S Miyano. "Open source clustering software." Bioinformatics 20 (2004): 1453–1454.

Ellett, F, L Pase, JW Hayman, A Andrianopoulos, and GJ Lieschke. "mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish." Blood 117(4) 2011: e49-56.

Livak, KJ, and TD Schmittgen. "Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2-ddCT Method." Methods 25 (2001):402-408.

Renshaw, SA, CA Loynes, DM Trushell, S Elworthy, PW Ingham, and MK Whyte. "A transgenic zebrafish model of neutrophilic inflammation." Blood 108(13) (2006): 3976-3978.

Ritchie, ME, J Silver, A Oshlack, M Holmes, D Diyagama, A Holloway, and GK Smyth. "A comparison of background correction methods for two-colour microarrays." Bioinformatics 23(20) (2006): 2700-2707.

Saldanha, AJ. "Java Treeview--extensible visualization of microarray data." Bioinformatics 20 (2004): 3246–3248.

Smyth, GK. "Linear models and empirical bayes methods for assessing differential expression in microarray experiments." Stat Appl Genet Mol Biol 3 (2004): Article 3.

Storey, JD. "A direct approach to false discovery rates." Journal of the Royal Statistical Society Series B (2002): 479-498.

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CHAPTER 3: CONCLUSIONS AND FUTURE DIRECTIONS

The purpose of this work was to establish a role for Trim9 in the vertebrate innate

immune response. Trim9 was initially identified by our lab as a gene with increased

transcript levels in larval zebrafish exposed to immune agonists. Based on the published role

of TRIM9 in mediating axon migration, we hypothesized that it may also mediate migration in immune cells. To test this hypothesis, we quantified TRIM9 expression in immune cells of zebrafish and humans following immune stimulation and tested Trim9 function in macrophage migration in vivo in the zebrafish model.

This work demonstrates for the first time that TRIM9 transcripts are increased

downstream of PRR activation in macrophages, both in vivo in the zebrafish and in human

cell culture. The TRIM9 transcriptional response occurs at 8-12 hours post exposure to the immune agonist. Our data and others show that macrophages arrive at sites of tissue inflammation or infection within 2 hours (Ellett, et al. 2011, Herbomel, Thisse and Thisse

1999), indicating that upregulation of Trim9 occurs after macrophages have arrived at sites of inflammation. Increased expression of Trim9 may act as a feedback mechanism for NF-κB

and IRF3 transcription factors. Both of these transcription factors can be activated in

response to PRR stimulation. TRIM9 negatively regulates NF-κB by sequestering β-TrCP and thereby preventing degradation of IκBα (Shi, et al. 2014). TRIM9 positively regulates

IRF3 activation by TBK1 and GSK3β following viral infection (Qin, et al. 2016).

Upregulation of Trim9 would therefore favor suppression of NF-κB-mediated gene activation and promotion of interferon activity. However, it must be noted that only the short isoform of

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TRIM9 can mediate IRF3 activation, while both long and short isoforms of Trim9 can inhibit

NF-κB (Qin, et al. 2016). The quantitative RT-PCR assays used in these studies do not differentiate between the known transcript variants of TRIM9. Preferential production of the splice variants encoding the long isoform of TRIM9 over the short isoform could favor negative regulation of NF-κB over positive regulation of interferon.

This work introduces Trim9 as a new potential regulator of macrophage motility.

Motility is critical for macrophage function because macrophages must be able to move through tissues to reach sites of infection or inflammation. On the other hand, preventing macrophage infiltration of tissues could be an effective way of treating inflammatory disorders. A better understanding of how macrophage motility is controlled will help inform novel strategies for modulating inflammation. To that end, defining the mechanisms by which Trim9 functions in macrophages may reveal novel protein networks or regulatory mechanisms that control macrophage motility.

Migrating macrophages polarize to form a trailing uropod and a leading edge with pseudopods formed by a meshwork of polymerized actin filaments (Hartwig and Stossel

1985). Macrophages expressing ΔRINGTrim9 lack pseudopod extensions and exhibit markedly reduced motility, suggesting defects in actin remodeling and/or cell polarization. In neurons, Trim9 mediates Netrin-guided axon migration by linking activation of the Netrin receptor to downstream mediators of cytoskeletal changes at the leading edge of the migrating axon (Alexander, et al. 2010, Menon, et al. 2015, Song, et al. 2011). Taken together, I propose that Trim9 participates in a protein network that coordinates cytoskeletal

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dynamics at the leading edge of migrating macrophages, and that its ubiquitin ligase activity

is critical for this function.

Several future lines of experimentation could help test this hypothesis.

Characterization of the cytoskeleton in macrophages with and without functional Trim9

would help determine whether disruption of cytoskeletal remodeling contributes to the

phenotype of the macrophages in the Tg(mpeg1:ΔRINGTrim9,EGFP) transgenic zebrafish.

Lifeact is an F-actin specific probe (Riedl, et al. 2008, Lemieux, et al. 2014) that has recently

been used to label F-actin in zebrafish in vivo in a cell-specific manner (Lam, et al. 2015),

and could be used to address this question in our transgenic zebrafish model. In addition, an in vitro cell culture model in which TRIM9 could be knocked down, or ΔRINGTRIM9 could be expressed, would allow more detailed imaging of actin and microtubule cytoskeletal remodeling during migration. Ideally, this would be done in a human myeloid cell line, such as the U937, to determine if the role of TRIM9 in immune cell migration is conserved across species. Attempts to perform these experiments in U937 cells in our lab were hindered by low transfection efficiency. Alternative cell lines and transfection methods could be explored.

Characterizing the subcellular localization of TRIM9 in resting and migrating macrophages would also help determine if it is spatially related to the leading edge. The transgenes used in our studies were designed to have coexpression of fluorescent protein rather than fusing it to Trim9 in order to prevent the fluorescent protein from interfering with protein function. Having established the expected phenotype in cells expressing full length or

ΔRINGTrim9, transgenes with fluorescent proteins fused to Trim9 or ΔRINGTrim9 could be

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used to assess subcellular localization of Trim9 in migrating macrophages in vivo. In parallel, immunofluorescence studies in human primary macrophages or the U937 cell line could be done to assess subcellular localization of endogenous TRIM9. In HeLa cells transfected with tagged TRIM9, TRIM9 subcellular localization changed following viral infection (Versteeg, et al. 2013). Studies in the human cells would establish whether similarities exist across species, and allow for more controlled examination of TRIM9 localization in resting versus stimulated macrophages.

Finally, identifying protein interacting partners and ubiquitination targets for TRIM9 in macrophages may help elucidate its mechanism of action and reveal other potential mediators controlling macrophage motility. Attempts to co-immunoprecipitate endogenous

TRIM9 from U937 cell lysates were unsuccessful do to issues with antibody specificity. An alternative strategy would be to pull down interacting proteins from lysates using affinity- tagged TRIM9. This has the advantage of avoiding use of an antibody. The disadvantage is that it does not allow for protein interactions in their native environment in the cell, and may not pull down proteins that weakly or transiently interact. Importantly, transient interaction between E3 ubiquitin ligases and their substrates is common and provides a challenge in identifying these interactions (Harper and Tan 2012). As such, TRIM9 may be an ideal candidate for proximity-dependent biotin identification. In this method a promiscuous prokaryotic biotin ligase is fused to the protein of interest, in this case TRIM9, and expressed in mammalian cells (Roux, et al. 2012). Upon addition of biotin to the culture media, the biotin ligase will biotinylate proteins that interact with or are in very close proximity to the fused protein. The biotinylated proteins can then be isolated and identified by mass

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spectrometry. This method could be used to screen for potential interacting partners of

TRIM9.

In summary, this work identifies TRIM9 as a novel mediator in macrophage function and broadens the potential role of TRIM9 in mediating cell migration to cells outside of the nervous system. Although the work presented here focuses on the role of TRIM9 in macrophages, it supports recent findings that TRIM9 is more ubiquitously expressed than previously considered (Qin, et al. 2016). Moving forward, knowledge gleaned about TRIM9 function in macrophage motility could be applicable to other cells and vice versa.

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Harper, JW, and MM Tan. "Understanding Cullin-RING E3 biology through proteomics- based substrate identification." Mol Cell Prot 11 (2012): 1541-1550.

Herbomel, P, B Thisse, and C Thisse. "Ontogeny and behaviour of early macrophages in the zebrafish embryo." Development 126 (1999): 3735-3745.

Lam, P-y, S Mangos, JM Green, J Reiser, and A Huttenlocher. "In vivo imaging and characterization of actin microridges." PLoS ONE 10(1) (2015): e0115639.

Lemieux, MG, D Janzen, R Hwang, J Roldan, I Jarchum, and DA Knecht. "Visualization of the actin cytoskeleton: different F-actin-binding probes tell different stories." Cytoskeleton (Hoboken) 71(3) (2014): 157-169.

Menon, S, et al. "The E3 ubiquitin ligase TRIM9 is a filopodia off switch required for netrin- dependent axon guidance." Dev Cell (2015): 698-712.

Qin, Y, Q Liu, S Tian, W Xie, J Cui, and R-F Wang. "TRIM9 short isoform preferentially promotes DNA and RNA virus-induced production of type I interferon by recruiting GSK3B to TBK1." Cell Res, Feb (2016): [Epub ahead of print] doi:10.1038/cr.2016.27.

Riedl, J, et al. "Lifeact: a versatile marker to visualize F-actin." Nat Methods 5(7) (2008): 605.

Roux, KJ, DI Kim, M Raida, and B Burke. "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells." J Cell Biol 196(6) (2012): 801-810.

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Shi, M, et al. "Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9." Nat Communications 5 (2014): 1-14.

Song, S, et al. "TRIM-9 functions in the UNC-6/UNC-40 pathway to regulate ventral guidance." J Genet Genom 38 (2011): 1-11.

Versteeg, GA, et al. "The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors." Immunity 38 (2013): 384- 398.

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APPENDIX

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TRIM9 Expression in Neutrophils

Introduction

Neutrophils are the first leukocytes recruited from circulation into tissues in response to infection or inflammation, and their activity may promote subsequent movement of monocytes into the tissues (Soehnlein, Lindbom and Weber 2009). Failure to resolve neutrophilic inflammation can, however, lead to tissue injury and is implicated in a number of chronic inflammatory diseases (Kruger, et al. 2015). Identifying novel regulators of neutrophil function may inform new strategies for modulating neutrophil activity in inflammatory diseases.

The tripartite motif (TRIM) family is a large protein family characterized by the presence of three amino-terminal protein domains: a really interesting new gene (RING) domain, Bbox domains, and a Coiled-coil region. The RING domain imparts E3 ubiquitin ligase activity which provides substrate specificity for ubiquitin conjugase enzymes (Joazeiro and Weissman 2000). TRIMs possess a variety of carboxy terminal domains that further diversify their function. A number of TRIMs have been implicated in innate immunity.

In a screen of larval zebrafish for innate immune response genes, we identified tripartite motif 9 (trim9) as a gene with increased transcript levels in response to the immune agonists

Pam3CSK4 and polyinosine-polycytidylic acid (PolyIC). Pam3CSK4 is a synthetic triacylated lipopeptide that mimics bacterial cell wall components and is a ligand for the toll- like receptor (TLR) 2/1 heterodimer (Aliprantis, et al. 1999). PolyIC is a mimic of viral

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double-stranded RNA and is a ligand for TLR3 and the retinoic acid inducible gene I-like receptors (RLRs) (Kato, et al. 2008, Yoneyama, et al. 2004, Alexopolou, et al. 2001).

Stimulation of these receptors turns on intracellular signaling cascades that result in activation of transcription factors, notably nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and interferon regulatory factor 3 (IRF3) (Aliprantis, et al. 1999,

Michallet, et al. 2008, Alexopolou, et al. 2001). Recently, TRIM9 was found to mediate these signaling pathways, acting as a negative regulator of NF-κB activation and a positive regulator of IRF3 activation (Shi, et al. 2014, Qin, et al. 2016). In neutrophils, TLR activation induces production of pro-inflammatory cytokines and chemokines and promotes antimicrobial activity of neutrophils by increasing phagocytosis and priming reactive oxygen species production (Hayashi, Means and Luster 2003). Changes in TRIM9 expression within neutrophils could affect intracellular signaling and neutrophil function induced by TLR stimulation.

To our knowledge, TRIM9 expression has not been characterized in neutrophils. We examined TRIM9 expression in mammalian neutrophils and quantified the transcriptional response of TRIM9 in neutrophils following in vivo immune agonist exposure in the zebrafish and direct, in vitro exposure in human neutrophil-like HL60 cells. We find that TRIM9 is expressed in mammalian neutrophils in an isoform specific manner and that neutrophil

TRIM9 transcript levels are differentially altered by immune agonist exposure.

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Materials and Methods

Zebrafish. Adult zebrafish were maintained in a recirculating aquarium facility (Aquatic

Habitats, Apopka, FL) at 28 °C with a 10 hr light/14 hr dark cycle and fed a commercial grade zebrafish diet. The transgenic Tg(mpo:EGFP) zebrafish line (Renshaw, et al. 2006) was obtained from Steven Renshaw (University of Sheffield). Zebrafish embryos were obtained by natural spawning and maintained at 28 °C in egg water (0.5 mg/L methylene blue and 60 mg/L aquarium salt mixture). Larvae were euthanized in 0.02% Tricaine methanesulfonate (Finquel MS-222; Argent Chemical). Zebrafish husbandry and experiments involving live animals were approved by the North Carolina State University

Institutional Animal Care and Use Committee.

RNA and protein isolation from primary neutrophils. Canine peripheral blood samples were obtained from the Clinical Studies Core at the North Carolina State University College of Veterinary Medicine. Canine neutrophils were isolated from canine peripheral blood as previously described (Li, et al. 2011). Human primary neutrophils were provided by the lab of Sam Jones (North Carolina State University). Mouse neutrophils were isolated from bone marrow samples provided by Stephanie Gupton. Bone marrow cell pellets were treated with hypotonic saline to lyse red blood cells then separated on a Histopaque 1119/1077 gradient.

Cells from the gradient interface were washed in phosphate buffered saline/0.5% heat inactivated fetal bovine serum (Atlanta Biologicals). RNA was isolated using TRIzol reagent

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according to manufacturer’s protocol. For protein, cells were lysed in RIPA buffer for 30

minutes on ice followed by brief sonication.

Cell culture. Human promyeloblast HL-60 cells were cultured in RPMI-1640 media

(Corning cellgro) supplemented with 15% fetal bovine serum (Atlanta Biologicals).

Differentiation was induced by addition of 3% DMSO for 6 days (Collins, et al. 1978).

Differentiated cells were stimulated with either 0.1 ug/ml Pam3CSK4, 50 ug/ml PolyIC, or

0.1 ug/ml ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4 (Invivogen) in 12 well plates with ~1 x 106 cells/well. At 4, 8, and 12 hours post exposure (hpe) RNA was isolated

using TRIzol reagent according to manufacturer’s protocol. For protein, cells were lysed in

RIPA buffer for 30 minutes on ice followed by brief sonication.

Larval zebrafish PAMP exposure and isolation of neutrophils. At 120 hr post fertilization

(hpf), groups of 75-100 Tg(mpo:EGFP) zebrafish larvae (Renshaw, et al. 2006) were exposed by immersion to 10 μg/ml PolyIC, 5 μg/ml Pam3CSK4 or no agonist for 8 or 12 hours. Following exposure, larvae pooled by treatment group were disaggregated into single cells suspensions as previously described (Manoli and Driever 2012) and suspensions sorted for EGFP-positive cells using a Dako Cytomation MoFlo cytometer. RNA was isolated from sorted cells using the Qiagen RNeasy Micro kit.

Western blot. Cell lysates were run on 10% SDS-polyacrylamide gels and transferred to

PVDF membranes. Blots were blocked for 1 hour in TBST buffer (50 mM Tris-HCl, 150

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mM NaCl, 0.1% Tween-20) with 5% nonfat milk then incubated overnight at 4°C with

polyclonal rabbit anti-TRIM9 antibody diluted 1:1000 in blocking buffer, followed by

incubation with horseradish peroxidase conjugated goat anti-rabbit IgG antibody (Cell

Signal) diluted 1:5000 for 1 hour at room temperature. Antibody was detected using an

enhanced chemiluminescent detection system (Bio-rad). Primary antibodies used were a commercial rabbit polyclonal antibody raised against recombinant human TRIM9 amino acids 1-350 (10786-1-AP; Proteintech) and a rabbit polyclonal antibody raised against recombinant murine Trim9 amino acids 158-271 (Winkle, et al. 2014) that was provided by

Stephanie Gupton.

Rapid amplification of cDNA ends (RACE). TRIM9 transcripts were amplified by 3ʹ and 5ʹ

RACE using the GeneRacer kit (Invitrogen) with total RNA isolated from human primary neutrophils. Gene specific primers and cycling conditions used are listed in Table 2.

Quantitative RT- PCR. cDNA was synthesized using Invitrogen Superscript III First Strand cDNA Synthesis kit. Quantitative real-time PCR (qPCR) was performed using Taqman

Universal PCR Master Mix II (Applied Biosystems) and Taqman Gene Expression assays

(Applied Biosystems; Table 1). Gene expression fold changes were calculated using the

2-∆∆Ct method (Livak and Schmittgen 2001).

Statistical analyses. For real time PCR analyses, Student’s T tests were performed to compare control and treatment fold changes. Results were considered significant if p < 0.05.

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Results

Neutrophils specifically express the short isoform of TRIM9

Formerly, TRIM9 was considered to be specifically expressed in brain tissue (Berti,

et al. 2002, Li, et al. 2001, Shi, et al. 2014, Tanji, et al. 2010). Protein expression of TRIM9

has since been confirmed in multiple tissues and cell types, including human peripheral blood

mononuclear cells and the human monocytic cell line THP-1 (Qin, et al. 2016). Protein

expression of TRIM9 in neutrophils, however, has not been examined. To evaluate TRIM9

protein expression in neutrophils, western blotting for TRIM9 was performed on lysates from

human, canine, and mouse primary neutrophils, as well as from human promyeloblast HL60

cells treated with DMSO to differentiate them to a neutrophil-like phenotype.

Alternative RNA splice variants encode at least two protein isoforms of TRIM9 in human, mouse, and rat brain (Li, et al. 2001, Tanji, et al. 2010, Winkle, et al. 2014). One isoform is an approximately 80 kDa protein containing the full complement of TRIM9 protein domains, while the second, shorter isoform of approximately 60 kDa lacks the final carboxy terminal SPRY domain (Winkle, et al. 2014, Qin, et al. 2016). Although two isoforms were detected in rat brain, only a single isoform between 55-70 kDa was detected in mammalian neutrophils (Fig. 1). This did not change with immune agonist exposure (Fig. 2).

To determine if TRIM9 transcripts preferentially encoded a single isoform, rapid

amplification of cDNA ends (RACE) was used to amplify full length TRIM9 transcripts from

human primary neutrophil total RNA. Three different TRIM9 transcripts were amplified from

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human neutrophils. Two of the transcripts differ in the 3ʹ untranslated region, but encode the same protein isoform (neutrophil isoform A). This isoform matched the known short human

TRIM9 isoform 2 (NCBI refseq NP_443210.1) and lacked the C-terminal SPRY domain

(Fig. 3). The third transcript encodes a protein (neutrophil isoform B) that differs from either of the known human TRIM9 isoforms and lacks the carboxy terminal half of the SPRY domain (Fig. 3). These findings along with the Western blot data suggest that neutrophils do not express full length TRIM9.

Immune agonists induce changes in Trim9 transcript levels in neutrophils

In whole zebrafish larvae, trim9 transcript levels increase in response to both

Pam3CSK4 and PolyIC at 8 hpe (Heffelfinger 2010). To determine whether this transcriptional response occurs in neutrophils, we isolated EGFP-positive neutrophils from

Tg(mpo:EGFP) larvae following 8 or 12 hours of exposure of the whole larvae to

Pam3CSK4 or PolyIC. In response to PolyIC stimulation, trim9 transcript levels did increase in neutrophils, but at a later time point than in whole the larvae (Fig. 4). Unexpectedly, trim9 transcripts decreased in neutrophils in response to Pam3CSK4 at 8 hpe, and to a lesser extent at 12 hpe.

To determine if a similar response occurs in human neutrophils, DMSO-differentiated

HL60 neutrophil-like cells were exposed to Pam3CSK4, PolyIC, or lipopolysaccharide (LPS) for 4, 8, or 12 hours and TRIM9 transcripts levels quantified by qPCR. TRIM9 transcript levels also showed agonist-specific responses in the human cells, but in a pattern almost

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opposite of that in the zebrafish. TRIM9 transcript levels were significantly reduced in response to PolyIC at 8 hpe, but increased in response to LPS (Fig. 5). Taken together, these findings suggest that in neutrophils TRIM9 may be subject to immune agonist-specific modes of regulation.

Discussion

This work is the first to demonstrate TRIM9 expression in neutrophils. There are two known protein isoforms of TRIM9, a full length isoform and a short isoform lacking the carboxy terminal SPRY domain. The TRIM9 variants expressed in neutrophils encode proteins lacking all or part of the SPRY domain. Interestingly, the recently identified role of

TRIM9 as a positive regulator of type I interferon signaling is mediated specifically by the short isoform of TRIM9 (Qin, et al. 2016), while negative regulation of NF-κB can be

mediated by either isoform (Qin, et al. 2016, Shi, et al. 2014). Thus, both functions could be

carried out by the TRIM9 isoforms we identified in neutrophils. The only reported function

of the SPRY domain of TRIM9 is mediating interaction with DCC Netrin-1 receptor (DCC).

DCC is a receptor for the chemoattractant Netrin-1/UNC-6 and TRIM9 mediates axon migration toward Netrin-1/UNC-6 during neuronal development (Alexander, et al. 2010,

Hao, et al. 2010, Winkle, et al. 2014). DCC is not detectable in human granulocytes by immunohistochemistry or real time PCR (Ly, et al. 2005). If identified, other functions of

TRIM9 that require the SPRY domain could be perturbed in neutrophils.

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TRIM9 is expressed at relatively lower protein levels in neutrophils than in brain.

TRIM9 is easily detectable in 5 μg of rat brain lysate, while 40 μg of human neutrophil lysate

was required to detect a TRIM9 signal. It is possible that full length TRIM9 could be

expressed at lower levels that we could not detect. It is also possible that not all TRIM9

transcripts were amplified by RACE. A northern blot of human neutrophil RNA could help

ascertain if additional transcript species are present.

In contrast to macrophages in which immune agonists generally induced increases in

TRIM9 transcript levels, the TRIM9 transcriptional response in neutrophils differed between immune agonists and across species. Differences seen across the species could reflect differences in the route of immune agonist exposure (directly on the cells in culture versus whole organism exposure in the zebrafish), or could reflect evolutionary divergence in signaling between zebrafish and human neutrophils. Cellular expression of TLR3 could

influence the response to PolyIC in neutrophils. The high molecular weight PolyIC used in these experiments is a ligand for TLR3 and MDA-5 (Kato, et al. 2008, Alexopolou, et al.

2001). HL60 cells do not express TLR3 protein, although they do express MDA-5 (Berger, et al. 2012). Primary human neutrophils also do not express TLR3 (Hayashi, Means and

Luster 2003). In zebrafish, TLR3 mRNA expression is fairly ubiquitous and is detectable in blood (Jault, Pichon and Chluba 2004), but whether expression occurs in zebrafish neutrophils has not, to our knowledge, been determined. If the role of Trim9 in regulating type I interferon (IFN) activation is conserved in zebrafish, increased transcript levels in response to PolyIC could act as a positive feedback loop to promote antiviral activity.

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Aside from its recently identified role in modulating NF-κB and IRF3 activation,

TRIM9 has been primarily studied in neurons where it mediates Netrin-guided axon migration and regulates calcium-dependent, SNARE-mediated synaptic vesicle exocytosis

(Alexander, et al. 2010, Hao, et al. 2010, Li, et al. 2001, Winkle, et al. 2014, Song, et al.

2011). Both such functions could be transferable to neutrophils. Neutrophils must migrate in response to chemoattractant signals to reach sites of tissue infection and inflammation.

Neutrophils contain a variety of cytoplasmic granules whose contents are released during an inflammatory response in a regulated manner. Release of at least some of these granules is mediated by SNARE complexes (Mollinedo, et al. 2006). Since TRIM9 is expressed in neutrophils and immune stimulation alters transcript levels, the function of TRIM9 in neutrophils warrants further investigation.

Acknowledgements

We thank Dr. Stephanie Gupton (University of North Carolina – Chapel Hill) for providing mouse bone marrow and an anti-Trim9 antibody and Jessica Powell (University of

Surrey) for her help performing qPCR for the HL60 cells.

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Table 1. Taqman assays used for quantitative RT-PCR.

Target Taqman Gene Expression Assay # Danio rerio trim9 Dr03081570_m1 Danio rerio ef1α Dr03432748_m1 Homo sapiens TRIM9 Hs00364838_m1 Homo sapiens ACTB Hs01060665_g1

Table 2. RACE primers and conditions.

Annealing temp, Primer Sets Sequence (5ʹ-3ʹ) Enzyme # of cycles HsTrim9_3ʹRACE_F TGCCCTCATCGATGCCCTCAACAGAAGAA Titan 72 °C, 5 cycles GeneRacer 3ʹ Primer GCTGTCAACGATACGCTACGTAACG 70 °C, 5 cycles 68 °C, 5 cycles 65 °C, 10 cycles Trim9_nested_3ʹRACE_F CAAGCTGAAGGTGGTTCGAGATCAGAT Titan Same as above GeneRacer 3ʹ Nested Primer CGCTACGTAACGGCATGACAGTG Trim9_5ʹRACE_R3 ATCTGATCTCGAACCACCTTCAGCTTG Kapa 72 °C, 5 cycles GeneRacer 5ʹ Primer CGACTGGAGCACGAGGACACTGA 70 °C, 5 cycles 68 °C, 15 cycles 65 °C, 15 cycles Trim9_5ʹRACE_R4 TTCTTCTGTTGAGGGCATCGATGAGGGCA Kapa Same as above GeneRacer 5ʹ Nested Primer GGACACTGACATGGACTGAAGGAGTA Titan: Titanium Taq DNA polymerase (Clontech) Kapa: Kapa2G Robust HotStart DNA polymerase with GC buffer (Kapa Biosystems)

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Fig. 1. Neutrophils express a single detectable isoform of TRIM9. Rat brain lysate (5 μg) and lysates (40 μg) from HL60 cells differentiated with 3% DMSO to a neutrophil phenotype, human peripheral blood neutrophils (PBN), canine PBN, and mouse bone marrow neutrophils (BMN) were immunoblotted with anti-Trim9 antibody that recognizes short and long isoforms of TRIM9. HL60 cells and human and canine PBN were immunoblotted with a commercial rabbit polyclonal antibody raised against recombinant human TRIM9 amino- terminus. Mouse BMN was immunoblotted with rabbit polyclonal antibody raised against recombinant murine TRIM9 amino acids 158-271 (Winkle, et al. 2014).

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Fig. 2. Immune agonist exposure does not alter the TRIM9 isoform profile in DMSO- differentiated HL60 cells. HL60 cells were treated with 3% DMSO to induce a neutrophil- like phenotype then exposed to 0.1 μg/ml Pam3CSK4, 50 μg/ml PolyIC, or 0.1 μg/ml LPS for 8 hours. Lysates (30 μg) were immunoblotted with a commercial rabbit polyclonal antibody raised against recombinant human TRIM9 amino-terminus that recognizes both isoforms of TRIM9.

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Fig. 3. Neutrophil TRIM9 transcripts encode short isoforms of TRIM9. TRIM9 transcripts were amplified from human primary neutrophil RNA by 3ʹ and 5ʹ RACE. Three different transcripts were identified encoding two different protein isoforms. Two transcripts encode isoform A, which is identical to the known human TRIM9 isoform 2 (NP_443210.1). The second neutrophil isoform (B) does not match either of the known human TRIM9 isoforms. Splice variation in this isoform results in a frame shift and early stop codon that truncates the SPRY domain. Isoform 1 (NP_055978.4) represents the full length TRIM9.

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Neutrophil_isoA MEEMEEELKCPVCGSFYREPIILPCSHNLCQACARNILVQTPESESPQSHRAAGSGVSDY TRIM9_iso2_NP_443210.1 MEEMEEELKCPVCGSFYREPIILPCSHNLCQACARNILVQTPESESPQSHRAAGSGVSDY Neutrophil_isoB MEEMEEELKCPVCGSFYREPIILPCSHNLCQACARNILVQTPESESPQSHRAAGSGVSDY TRIM9_iso1_NP_055978.4 MEEMEEELKCPVCGSFYREPIILPCSHNLCQACARNILVQTPESESPQSHRAAGSGVSDY ************************************************************ RING domain Neutrophil_isoA DYLDLDKMSLYSEADSGYGSYGGFASAPTTPCQKSPNGVRVFPPAMPPPATHLSPALAPV TRIM9_iso2_NP_443210.1 DYLDLDKMSLYSEADSGYGSYGGFASAPTTPCQKSPNGVRVFPPAMPPPATHLSPALAPV Neutrophil_isoB DYLDLDKMSLYSEADSGYGSYGGFASAPTTPCQKSPNGVRVFPPAMPPPATHLSPALAPV TRIM9_iso1_NP_055978.4 DYLDLDKMSLYSEADSGYGSYGGFASAPTTPCQKSPNGVRVFPPAMPPPATHLSPALAPV ************************************************************

Neutrophil_isoA PRNSCITCPQCHRSLILDDRGLRGFPKNRVLEGVIDRYQQSKAAALKCQLCEKAPKEATV TRIM9_iso2_NP_443210.1 PRNSCITCPQCHRSLILDDRGLRGFPKNRVLEGVIDRYQQSKAAALKCQLCEKAPKEATV Neutrophil_isoB PRNSCITCPQCHRSLILDDRGLRGFPKNRVLEGVIDRYQQSKAAALKCQLCEKAPKEATV TRIM9_iso1_NP_055978.4 PRNSCITCPQCHRSLILDDRGLRGFPKNRVLEGVIDRYQQSKAAALKCQLCEKAPKEATV ************************************************************ Bbox 1 Neutrophil_isoA MCEQCDVFYCDPCRLRCHPPRGPLAKHRLVPPAQGRVSRRLSPRKVSTCTDHELENHSMY TRIM9_iso2_NP_443210.1 MCEQCDVFYCDPCRLRCHPPRGPLAKHRLVPPAQGRVSRRLSPRKVSTCTDHELENHSMY Neutrophil_isoB MCEQCDVFYCDPCRLRCHPPRGPLAKHRLVPPAQGRVSRRLSPRKVSTCTDHELENHSMY TRIM9_iso1_NP_055978.4 MCEQCDVFYCDPCRLRCHPPRGPLAKHRLVPPAQGRVSRRLSPRKVSTCTDHELENHSMY ************************************************************ Bbox 2 Neutrophil_isoA CVQCKMPVCYQCLEEGKHSSHEVKALGAMWKLHKSQLSQALNGLSDRAKEAKEFLVQLRN TRIM9_iso2_NP_443210.1 CVQCKMPVCYQCLEEGKHSSHEVKALGAMWKLHKSQLSQALNGLSDRAKEAKEFLVQLRN Neutrophil_isoB CVQCKMPVCYQCLEEGKHSSHEVKALGAMWKLHKSQLSQALNGLSDRAKEAKEFLVQLRN TRIM9_iso1_NP_055978.4 CVQCKMPVCYQCLEEGKHSSHEVKALGAMWKLHKSQLSQALNGLSDRAKEAKEFLVQLRN ************************************************************ Coiled coil region Neutrophil_isoA MVQQIQENSVEFEACLVAQCDALIDALNRRKAQLLARVNKEHEHKLKVVRDQISHCTVKL TRIM9_iso2_NP_443210.1 MVQQIQENSVEFEACLVAQCDALIDALNRRKAQLLARVNKEHEHKLKVVRDQISHCTVKL Neutrophil_isoB MVQQIQENSVEFEACLVAQCDALIDALNRRKAQLLARVNKEHEHKLKVVRDQISHCTVKL TRIM9_iso1_NP_055978.4 MVQQIQENSVEFEACLVAQCDALIDALNRRKAQLLARVNKEHEHKLKVVRDQISHCTVKL ************************************************************ COS box Neutrophil_isoA RQTTGLMEYCLEVIKENDPSGFLQISDALIRRVHLTEDQWGKGTLTPRMTTDFDLSLDNS TRIM9_iso2_NP_443210.1 RQTTGLMEYCLEVIKENDPSGFLQISDALIRRVHLTEDQWGKGTLTPRMTTDFDLSLDNS Neutrophil_isoB RQTTGLMEYCLEVIKENDPSGFLQISDALIRRVHLTEDQWGKGTLTPRMTTDFDLSLDNS TRIM9_iso1_NP_055978.4 RQTTGLMEYCLEVIKENDPSGFLQISDALIRRVHLTEDQWGKGTLTPRMTTDFDLSLDNS ************************************************************ FN3 domain Neutrophil_isoA PLLQSIHQLDFVQVKASSPVPATPILQLEECCTHNNSATLSWKQPPLSTVPADGYILELD TRIM9_iso2_NP_443210.1 PLLQSIHQLDFVQVKASSPVPATPILQLEECCTHNNSATLSWKQPPLSTVPADGYILELD Neutrophil_isoB PLLQSIHQLDFVQVKASSPVPATPILQLEECCTHNNSATLSWKQPPLSTVPADGYILELD TRIM9_iso1_NP_055978.4 PLLQSIHQLDFVQVKASSPVPATPILQLEECCTHNNSATLSWKQPPLSTVPADGYILELD ************************************************************

Neutrophil_isoA DGNGGQFREVYVGKETMCTVDGLHFNSTYNARVKAFNKTGVSPYSKILVLQTSEGKALQQ TRIM9_iso2_NP_443210.1 DGNGGQFREVYVGKETMCTVDGLHFNSTYNARVKAFNKTGVSPYSKTLVLQTSEGKALQQ Neutrophil_isoB DGNGGQFREVYVGKETMCTVDGLHFNSTYNARVKAFNKTGVSPYSKTLVLQTSEVAWFAF TRIM9_iso1_NP_055978.4 DGNGGQFREVYVGKETMCTVDGLHFNSTYNARVKAFNKTGVSPYSKTLVLQTSEVAWFAF ********************************************** ******* : SPRY domain Neutrophil_isoA YPSERELRGI------TRIM9_iso2_NP_443210.1 YPSERELRGI------Neutrophil_isoB DPGSAHSDIILSNDNLTVTCSSYDDRVVLGKTGFSKGIHYWELTVDRYDNHPDPAFGVAR TRIM9_iso1_NP_055978.4 DPGSAHSDIILSNDNLTVTCSSYDDRVVLGKTGFSKGIHYWELTVDRYDNHPDPAFGVAR *.. . *

Neutrophil_isoA ------TRIM9_iso2_NP_443210.1 ------Neutrophil_isoB MDVMKDVMLGKDDRAWAMYVDNNRSWFMHNNSHTNRYPAAPKPSLLHVGF------TRIM9_iso1_NP_055978.4 MDVMKDVMLGKDDKAWAMYVDNNRSWFMHNNSHTNRTEGGITKGATIGVLLDLNRKNLTF

Neutrophil_isoA ------TRIM9_iso2_NP_443210.1 ------Neutrophil_isoB ------TRIM9_iso1_NP_055978.4 FINDEQQGPIAFDNVEGLFFPAVSLNRNVQVTLHTGLPVPDFYSSRASIA

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Fig. 4. Immune agonist exposure induces changes in trim9 transcript levels in larval zebrafish neutrophils. Relative trim9 transcript levels in larval zebrafish neutrophils after 8 and 12 hr exposures of whole larvae to 5 μg/ml Pam3CSK4 or 10 μg/ml PolyIC. Representative of two biological replicates.

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Fig. 5. Immune agonist exposure induces changes in TRIM9 transcript levels in human neutrophil-like HL60 cells. Relative TRIM9 transcript levels in HL60 cells treated with 3% DMSO to induce neutrophil-like phenotype then exposed to 0.1 μg/ml Pam3CSK4, 50 μg/ml PolyIC, or 0.1 μg/ml LPS for 4, 8 , or 12 hours. Mean ± SEM, N = 3. *p<0.05

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