Interferon Regulatory Factor 7 (IRF7) in Systemic Lupus Erythematosus

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in the Department of Pharmacology & Systems Physiology

of the College of Medicine

Mark Verba

B.S. Widener University

2017

Committee:

Leah Kottyan, Ph.D. (Chair)

Terry Kirley, PhD

Jo El Schultz, PhD

Abstract

Systemic Lupus Erythematosus (SLE) is a complex, chronic autoimmune disease involving multiple organ systems, which can range in severity from mild to life threatening commensurate with organ system involvement. Research suggests that an increase in production of pro- inflammatory cytokines, particularly, Type I Interferon (IFN-I) is a key mediator in the pathogenesis of SLE. This process is critically regulated by Interferon Regulatory Factor 7 (IRF7), a transcription factor that finely regulates transcription of interferon stimulated genes, and therefore production of IFN-I. Faulty regulation of IFN-stimulated genes by IRF7 leads to aberrant production of IFN-I, and therefore plays an essential role in the pathogenesis of SLE. This thesis highlights the importance of rs1131665, a genetic variant at position 412 within the putative inhibitory domain of the IRF7 gene, changing a glutamine (412Q, risk) to an arginine (412R, protective). This variant is statistically associated with the risk of developing SLE. A Luciferase

Reporter Assay confirms increased interferon stimulated response element (ISRE) reporter activity in the risk IRF7 relative to the protective IRF7 in vitro. Enzyme-linked immunosorbent assay

(ELISA) assays show genotype dependent production of all IFN-α subtypes, with increased IFN-

α production demonstrated by the risk variant at 3 and 24 hour post-stimulation with resiquimod.

RNA-sequencing analysis shows increased IFN-α gene expression, and interferon-responsive gene expression in the IRF7 risk variant, confirming the role of the IRF7 (412Q) risk variant in the pathogenesis of SLE. Due to the lack of curative treatment for SLE patients, this thesis highlights the importance of IRF7, and serves as a plausible foundation for future targeted therapy against

IRF7, and IFN-I.

III

Preface and Acknowledgements

I would like to express a sincere thank you to Dr. Leah Kottyan, Dr. Terry Kirley, Dr. Jo

El Schultz, and Dr. Katie Hobbing for demonstrating consistent support and encouragement throughout the duration of the thesis, and being a critical part of the committee.

Particularly, I extend a whole-hearted thank you to Molecular and Developmental Biology graduate student Ellen Javier, whose leadership, sedulousness, and dedication to this project has gone well beyond boundaries, leaving absolutely no ambiguity that any of the data presented herein could be presented without her exceedingly hard work and leadership as she continues to advance this project.

Furthermore, I would like to show gratitude to Katelyn Dunn, Mehak Chawla, Carmy

Forney, Omer Donmez, Hope Rowden, Amber Sauder, and the rest of the Kottyan Lab. I would also like to thank Dr. Matt Weirauch and his lab for their hard work and consistent help not only in this project, but many of the concurrent projects in the lab. I would like to thank the

Department of Pharmacology and Systems Physiology at the University of Cincinnati as well as the Center for Autoimmune Genomics and Etiology (CAGE) Department at the Cincinnati

Children’s hospital.

Table of Contents Page

1. List of Figures and Tables V

2. Background 1

3. GWAS, Genetic Etiology, and Genetic Drivers of SLE 1

4. Interferons are Primary Drivers of SLE 3

5. Toll- Like Receptors and Signaling 6

6. Interferon Regulatory Factor 7 (IRF7) 9

7. Hypothesis 13

8. Methods 15

9. HEK-Blue Cell Line 15

10. Pharmacological Agent: Resiquimod 15

11. Dual Luciferase Reporter Assay and Plasmid Design 18

12. ELISA to measure IFN alpha 18

13. RNA-Sequencing Analysis 19

14. Results 20

15. Future studies 23

16. Conclusions 24

17. Abbreviations 26

18. Bibliography 27

List of Figures and Tables Figures: Page 1. Three mechanisms of IFN signaling 5

2. The SLE risk rs1131665 (Q412R) 11

3. Luciferase Reporter Assay from Fu et al. 12

4. HEK-blue cells and our experimental setup 14

5. Pharmacologic properties of resiquimod (R-848) 16

6. Activation of NF-kB by Resiquimod 17

7. Overexpression of IRF7 and genotype dependent production of IFN-I. 22

BACKGROUND

Systemic Lupus Erythematosus (SLE) is a complex, multisystem autoimmune disease harboring widespread clinical presentations, and can range in severity from mild to life- threatening. SLE is estimated to affect 51 per 100,000 people in the USA, impacts women nine times more frequently than men, and has an onset period between 16 and 55 years of age [2].

Symptoms of SLE are broad but can include rash, swelling in the joints, fever, and other morbidities including, but not limited to, cardiovascular disease, osteoporosis, and kidney disease

(Lupus Nephritis) [4,5]. Due to the widespread clinical presentation of SLE, properly diagnosing and treating patients with SLE remains a challenge and occurs on a patient-to-patient basis.

Currently, there is no cure for SLE, and treatment generally consist of immunosuppressive drugs, corticosteroids, and targeted therapy; treating the symptoms of the disease but failing to treat the underlying cause [1]. Therefore, elucidating the pathogenic mechanisms that underlie SLE is of utmost clinical relevance, and will increase the likelihood of a curative treatment for SLE patients.

GWAS, Genetic Etiology, and Genetic Drivers of SLE

Genome wide association studies (GWAS) comparing cases and controls have been pivotal in the understanding of SLE. Due to their large statistical power (p<5x10-8), these GWAS have allowed researchers to determine which genes play a crucial role in the development of SLE. Over the last decade, the power of these GWAS have provided some valuable insight into the pathogenesis of SLE, identifying 91 loci containing variants that can be attributed to the development of SLE. Many additional associated loci are yet to be clearly validated [6]. Taken together, there a three lines of evidence strongly supporting the role of genetics in the etiology of

SLE: 1) the evidence of established genetic loci 2) studies of monozygotic twins which show a

SLE disease concordance between 24 and 50 percent, and 3) studies demonstrating that siblings of patients who have SLE are 30 times more likely to have SLE relative to individuals which lack a sibling affected by SLE [2].

Consistent with the clinical presentation of SLE, the results of these GWAS have consistently identified loci involved with immunomodulatory, inflammatory, and autoimmune functions. Unlike many other diseases which are primarily monogenic, in which one gene is responsible for the diseased phenotype, SLE has been suggested to derive from a combination of modest effects from many genes. However, it is worth noting that roughly 1 percent of patients who present with SLE suffer from monogenic disease. Although a very small percentage of patients have clinical manifestations of SLE through a monogenetic mechanism, often times these single gene defects expose powerful pathogenic drivers of the diseased phenotype, which is the case in SLE patients. Two examples of these monogenic drivers of SLE include mutations associated with TREX1, and C1 complement protein (particularly C1q, C1r/s). TREX1 is a 3’ exonuclease that is primarily responsible for the degradation of ssDNA and dsDNA. Mutations in

TREX1 (inherited or de novo mutants) are commonly associated with Aicardi-Goutières syndrome, and resemble a lupus-like phenotype through immunologic dysregulation [6,8]. This lack of ability to degrade ssDNA and dsDNA in patients with TREX1 mutations allows for this nuclear

DNA to activate pattern recognition receptors (PRR’s), leading to downstream activation of innate immunity and autoimmunity via production of type I Interferon which is of particular importance for this thesis, as it is one of the primary drivers of SLE. Subsequently, the complement system is composed of membrane-bound soluble proteins that have been considered a critical component of innate immunity, opsonizing foreign pathogens to elicit an appropriate immune response, and leading to the formation of a membrane attack complex which destroys the pathogen. The

2 complement system is composed of three primary pathways (classical, alternative, and lectin) which all result in the cleavage of C3 to C3a through C3 convertase leading to the recruitment of other compliment proteins involved in lytic processes and phagocytosis [9]. Each of these complement pathways differ in their initiation mechanisms. Upon initiation of the classical pathway, C1q interacts with substrate, leading to the activation of C1r/s serine proteases. As stated previously, individuals with deficiencies of C1 subcomponents have a high risk of developing of

SLE, with >90% of individuals developing the disease with mutations in complement proteins.

Although the TREX1 and C1 mutations are two very rare examples of monogenic defects in SLE, the importance of single gene defects is important when analyzing the SLE phenotype. The focus of this discussion will primarily be on protein coding variants of Interferon Regulatory Factors

(IRF’s) which are involved with the production of type 1 interferon, a polypeptide cytokine involved in innate and adaptive immunity.

Interferons are Primary Drivers of SLE

Interferons (IFNs) are an important class of cytokines that were identified over 60 years ago and named on the basis of their ability to interfere with viral replication [12]. IFN’s can be categorized into three main families, type I IFN, type II IFN, and type III IFNs. Type I interferons are ubiquitously expressed and have been shown to be 1) mediating innate and adaptive immunity through mechanisms relating to B and T cell differentiation, 2) promoting the expression of MHC

Class I and MHC II, and 3) upregulating costimulatory molecules on dendritic cells and monocytes

[11]. The type I IFN family consist of IFN-α (which has 13 subtypes), IFN-β, IFN-ε, IFN-κ, IFN-ω in humans, and IFN-γ and IFN-t in pigs and cattle [13]. Type II IFN is unrelated to the other two families of interferons but has been shown to play a pivotal role as a cytokine when released from activated Natural Killer cells or T cells. Type III IFN (IFN-λ) is the least characterized of the three

IFN families, primarily due to its limited tissue distribution [14]. Type I IFNs are the most established class (particularly IFN-α and IFN-β), as they play a key role in numerous disease pathologies.

The focus of this thesis will be on Type I IFN regulation by interferon regulatory factors

(IRFs), as type I IFNs are widely accepted as a key facilitator in SLE patients. Specifically, IFN-

α has been shown to be elevated in over 50% of patients with SLE, with level of production correlating with disease severity, flare up and tissue involvement [11]. As this area of research continues to expand, many diseases are now being termed “Interferonopathies” or diseases with a type I IFN gene signature, highlighting the importance of these cytokines in the pathogenesis of many diseases, particularly autoimmune diseases such as rheumatoid arthritis and SLE. Although type I IFNs are expressed across many different cell types, plasmacytoid dendritic cells (pDCs) have demonstrated to be the primary producer of IFN- α [2]. This becomes particularly important when considering the complexities and regulation of type I IFN signaling (Figure 1).

Type I IFN signaling can occur downstream of numerous receptor-mediated pathways, with the two primary pathways being Toll-like receptor (TLR)-mediated, and Interferon receptor- mediated (IFNAR1 and IFNAR2).

Figure 1: Three mechanisms of IFN signaling. The receptor on the left is IFNAR1/IFNAR2, which is the signaling pathway for IFN-I. This pathway leads to production of IFN stimulated genes, a major component in SLE. Negishi H, Taniguchi T, Yanai H. The Interferon (IFN) Class of Cytokines and the IFN Regulatory Factor (IRF) Transcription Factor Family. Cold Spring

Harb Perspect Biol. 2018 Nov 1;10(11)

Toll- Like Receptors and Signaling

Toll-like receptors are expressed on many immune cells including dendritic cells, macrophages and B-cells. Under homeostatic conditions, the primary pathway for production of type I IFN is through TLR-mediated signaling. TLR’s are the largest family of the pattern recognition receptors (PRRs), and are effectively able to detect pathogen-associated-molecular patterns (PAMPs) and initiate a signaling cascade in response to receptor activation. There are 10

TLRs in human, and 13 TLRs in mouse. These PAMPs include evolutionary characteristics of the invading pathogen that can be distinguished from self. These characteristics include presence of lipopolysaccharides (LPS), flagellin, and other peptidoglycans [15]. TLRs can be further classified based upon their receptor localization, whether they are present on the cell surface, or present as endosomal receptors. TLRs expressed on the cell surface include TLR1, TLR2, TLR4, TLR5,

TLR6, and TLR10 which generally recognize these common PAMPs, while TLRs expressed on the endosomal membrane include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 which can recognize microbial-derived nucleic acids owing to a much broader pathogen recognition [16].

TLR4 is a unique receptor within the family as it can be expressed on the cell surface or can be expressed through an endosome. Although the ability to recognize microbial nucleic acids allows for a much broader range of pathogen recognition, it also increases the likelihood of these receptors to recognize self, and initiate a cascade of cytokine production leading to autoimmunity [17].

IRF7 signals downstream of two endosomal TLRs: TLR7 and TLR9. These TLRs are of importance as their signaling cascade is finely regulated by IRF7, culminating in the transcription of type I IFN. TLR7 has been shown to be activated by ssRNA from RNA viruses, as well as the imidazoquinoline derivative resiquimod which is used in our study to initiate the signaling cascade in vitro [15]. TLR9 has been shown to be activated by CpG-DNA, unmethylated cytosine-guanosine

DNA motifs in bacterial or viral DNA which is recognized as foreign by TLR9, and thus activating the receptor. TLR7 and TLR9 both signal through the myeloid differentiation adaptor protein

MYD88. Upon ligand binding, MYD88 adapter protein interacts with IRAK4, which phosphorylates IRAK1 and IRAK2. This leads to the recruitment of TRAF6 which leads to polyubiquitination of other proteins including TAB1 and TAB2, activating mitogen-activated protein kinases (MAPKs) resulting in the downstream activation of transcription factors including

AP-1, CREB, and NF-kB which ultimately lead to transcription of pro-inflammatory cytokines.

More importantly, following stimulation of TLR7 and TLR9, the MYD88 – IRAK complex can lead to the recruitment of TRAF3, leading to the activation of IKKa – IRAK complex, phosphorylating IRF7, which is then translocated into the nucleus where it induces the transcription of type I IFN [20]. Conclusively, the faulty regulation of these pathways can lead to overproduction of cytokines, and particularly type I IFN, leading to autoimmunity. Interestingly,

IRF7 has restricted tissue distribution, but is present largely in plasmacytoid dendritic cells

(pDC’s), the same cell type that is responsible for large quantities of IFN-α production.

Type I IFN can also signal through TLR-independent pathways, as demonstrated in the case in IFNAR1 and IFNAR2 heterodimeric receptor signaling. This receptor utilizes type I IFN as an endogenous ligand, and signals through members of the Janus activated kinase family, specifically TYK2 which is constitutively associated with IFNAR1, and JAK1 which is associated with IFNAR2. In this signaling cascade, ligand-induced dimerization of the receptor allows for trans-phosphorylation of key tyrosine resides within the tyrosine kinase domains regulatory loop, allowing for increased substrate accessibility [18]. IFNAR1 is critical in this ligand induced dimerization. IFNAR1 has been shown to have a large N-glycosylated ectodomain, as well as an important intracellular region of 100 residues; both of which are critical for ligand binding. The

7 importance of these domains was demonstrated in mouse studies devoid of IFNAR1, resulting in mice that were unable to respond to type I IFN [18]. Following autophosphorylation and activation of the receptor, the associate JAKs (JAK1 and TYK2) lead to the phosphorylation and activation of STATs, particularly STAT1 and STAT2, which can form either homodimers or heterodimers.

STAT1 has been shown to be phosphorylated at serine residue 727 (Ser727) for full transcriptional activation [13]. Following STAT dimerization, the phosphorylated STAT dimer translocates to the nucleus, where it forms a trimeric complex called ISGF3, which includes STAT1, STAT2, and

IRF9. In the nucleus, this complex interacts and binds to Interferon Stimulated Response Elements

(ISREs) in the promotor regions of certain IFN inducible genes. Furthermore, IRF2, has been shown to be a key attenuator of ISGF3 transcriptional activation. Studies have shown that the absence of IRF2 leads to increased type I IFN signaling [13, 14].

In summary, the combination of type I IFN signaling in a TLR-dependent, and TLR- independent manner, if not regulated properly by IRFs, can lead to aberrant production of type I

IFN, contributing to the hallmarks of SLE pathogenesis. As type I IFNs are produced through

TLR7 and TLR9 receptor signaling, a process critically regulated by IRF7, aberrant production of pro-inflammatory cytokines and type I IFN allows for the further activation of IFNAR1 and

IFNAR2, which leads to the transcription of hundreds of IFN stimulated genes. This culminates in a series of positive feedback loops between TLRs and IFNAR1/2, which can quickly lead to abnormal signaling and may cause an overproduction of type I IFN leading to SLE pathogenesis.

Being that these processes are in large part regulated by IRF, we aim to elucidate the role protein coding mutations in IRF7 have on transcriptional regulation and production of type I IFN.

Interferon Regulatory Factor-7 (IRF7)

IRF7 is activated through pathogen-associated molecular patterns receptors (PAMPRs) including intracellular Toll-like receptors (TLRs) -7 and -9. It is also activated through adaptors

MyD88/TRAF6/TRAF3 and cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors

(RLRs) through MAVS/TRAF6/TRAF3 [22]. Like other IRFs, inactive IRF7 locates to the cytoplasm until a signaling event leads to its transactivation (via phosphorylation), dimerization, and translocation into the nucleus [23,24]. Activated IRF7 can homodimerize or heterodimerize with other activated IRFs (such as IRF5 and IRF3, which are activated through overlapping receptors)

[22]. Once in the nucleus, IRF7 dimers bind regulatory regions of the DNA and forms a transcriptional complex with other co-activators [22,25,26]. When a large region of IRF7 containing amino acids 247–467 is deleted from IRF7, it is constitutively active as measured by dimerization, nuclear translocation, DNA binding, and gene expression [27]. A second more refined mutant study of IRF7 further suggests that the 412 position is critical for transactivation of IRF7 [28]. This key position at 412aa is the focus of this thesis project.

There are four isoforms of IRF7. Isoforms a and b have identical DNA binding domains and are the most highly expressed isoforms in immune cells. The scope of the thesis will focus on isoform a in B-cells and plasmacytoid dendritic cells (pDC’s). Unbiased genomic analyses have revealed an enrichment of activating chromatin marks and expressed gene products in B-cells at lupus risk loci, including the IRF7 locus [29,30]. B-cells are critical cells in the development and pathogenesis of lupus. Patients with lupus have many autoantibodies produced from B cells that have eluded immunological tolerance. These autoantibodies are often pathogenic in patients with lupus and can result in immune complex deposition and inappropriate, self-directed inflammatory responses [31]. IRF7, along with IRF3, drives Type I IFN production [22,27,32,33]. Type I IFNs are

9 critical mediators of disease pathoetiology in SLE, and specifically pDCs are the largest producer of this cytokine family [34,35] .

Our group recently refined previous studies identifying a systemic lupus erythematosus

(SLE or lupus) risk locus that contains a genetic variant that changes an amino acid (Q412R) in the inhibitory domain of IRF7 (see Figure 2). IRF7 is a transcription factor (TF) critical for production of type I interferon (IFN). The Q412R genetic variant at the IRF7 SLE risk locus has been found in multiple independent genetic studies of lupus risk at a robust, genome-wide significant level [35-41]. Our analysis also shows that the Q412R variant explains most of the genetic association at this locus (Figure 2).

Published studies establish that 412Q IRF7 has increased transcriptional activity as compared to 412R when each of these variants are overexpressed in the HEK cell line that contains a luciferase reporter under the control of a promoter containing a DNA sequence that IRFs are known to bind – an interferon-sensitive response element (ISRE) (Figure 3) [37].

Figure 2. The SLE risk rs1131665 (Q412R) is shown in the context of the conserved domains in the IRF7 protein. DBD: DNA binding domain; CAD: constitutive activation domain; VAD: virus-activated domain; ID: inhibitory domain; SRD: signal response domain.

Figure 3. Luciferase Reporter Assay from Fu et al., confirming the role of the risk variant

412Q relative to 412R. (Fu Q et al. Association of a functional IRF7 variant with systemic lupus erythematosus. Arthritis and rheumatism. 2011;63(3):749-54.

Hypothesis and Goal of this study

This thesis tested the overall hypothesis that genetic risk at IRF7 contributes to transcriptional dysregulation of disease relevant type I IFNs in clinically- relevant cells to increase SLE risk. The goal of this thesis was to demonstrate that a SLE risk variant in the inhibitory domain of IRF7 binds interferon stimulated response elements (ISREs) and regulates the expression of type I interferons in a genotype-dependent manner (Figure 4).

Figure 4: HEK-blue cells which overexpress TLR7/8 in their endosomal membrane were used for our experiments. Resiquimod was used to stimulate TLR-7 and produce IFN-I

METHODS

HEK-Blue Cell Line

For all of the experiments in this thesis, HEK-blue cells (Invivogen) were used. This cell line is derived from HEK293 Cell line, and was selected for its stable overexpression of TLR-‘s -

7 and -8. These cell lines allow for the activation of endosomal expressed TLR-7. This activation leads to downstream transcription of inflammatory genes. The signaling cascade in HEK-blue cells can be stimulated by numerous ligands, including gardiquimod, imiquimod, and resiquimod

(R848). Resiquimod (1ug/mL), chosen for its dual activation of TLR-‘s -7 and -8, was incubated with each passage of cells.

Pharmacological Agent: Resiquimod

Resiquimod is an imidazoquinoline derivative and is characterized based upon its anti-viral activity. Resiquimod is a Toll-Like receptor agonist, and stimulates the immune system through activation of TLR’s -7 -8. Upon activation, resiquimod induces the production of IFN-α, as well as other pro-inflammatory cytokines through activation of transcription factors. Resiquimod is not FDA approved, but is approved in EU for use in patients with the indications of cutaneous T-

Cell lymphoma. 1 ug/mL of resiquimod was used for our experiments based upon other studies suggesting that maximum NF-κB induction in human TLR -7 occurred at a resiquimod concentration of 1ug/mL (Figure 4, Figure 6). Pharmacologic properties of resiquimod are presented in Figure 5 [42].

Figure 5: Pharmacologic properties of resiquimod (R-848) National Library of Medicine

(US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for

CID 159603, Resiquimod.

Figure 6: Activation of NF-kB by Resiquimod downstream of TLRs 7, 8, and 9. Jurk, M.,

Heil, F., Vollmer, J. et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 3, 499 (2002).

Dual-Luciferase Reporter Assay and Plasmid Design

The Dual-Luciferase Reporter assay allowed us to determine the transcriptional differences between the two IRF7 variants (412Q and 412R). For our luciferase plasmid, we selected a vector expressing ISRE followed by our luciferase gene (pISRE-Luc). For our IRF7 plasmid, we selected a plasmid consisting of the wild-type IRF7, and created our IRF7 variants using site- directed mutagenesis (412Q and 412R). DNA-sequencing allowed us to confirm that our site- directed mutagenesis yielded the proper IRF7 variants.

A Dual-Luciferase assay was utilized for its inherent ability to normalize the data for variables such as starting cell number, ending cell number, and importantly transfection efficiency. The transfection of 1 ug of our IRF7 variant (Risk-412Q, or Non-Risk 412R) and 1 ug of our pISRE-

Luciferase reporter plasmid. To account for differences in cell number and transfection efficiency,

1 ug of a Renilla luciferase as an internal control plasmid was also co-transfected. After stimulating cells with resiquimod, the per well ratio of ISRE-Luc experimental activity to Renilla luciferase control activity accounted for well-to-well variability. Luciferase activity was measured after a 24- hour period using GloMax microplate reader. After measuring the FireFly luciferase activity,

NanoDLR Stop & Glo Reagent was added, and the Renilla control luciferase was measured.

Enzyme-linked immunosorbent assay (ELISA) to measure IFN alpha

ELISA is a biochemistry assay that can measure an analyte such as a cytokine. In experiments herein, we measure a subset of type I IFNs call IFN alpha. In our studies, an antibody that recognizes multiple IFN alphas was used to quantify the cytokine in the supernatant of stimulated cells. Specifically, we used ELISA to determine how the levels of IFN-α were varied

18 between 412Q, 412R, and HEK-blue cells (Control). An enzymatic reaction allows for the quantitative measurement of antibody-substrate binding using an absorbance measurement on a

GloMAX microplate reader. The IFN-alpha ELISA Kit was purchased from ThermoFisher, and

IFN-α detection antibodies were diluted according to the suggested manufacture specifications.

Being that HEK-blue cells do not express IRF7, one plasmid was transfected (412Q or 412R) into

5 different wells of HEK-blue cells for each genotype. The supernatant of the cells was isolated following stimulation with resiquimod, and IFN-α concentration was quantified based on a standard curve of known concentrations of IFN-alpha. To determine the difference between the groups, absorbance was measured for the different IRF7 genotypes at 0 hours, 3 hours, and 24 hours.

RNA-Sequencing Analysis

RNA was collected from cells 3 hours post-transfection, and was used to determine genotype- dependent expression of genes involved in the viral defense/IFN-I pathway. Experimentally, HEK- blue cells were stimulated with resiquimod after an IRF7 plasmid was transfected (either 412Q or

412R) into 5 different wells of the HEK-blue cells. The RNA of those cells was isolated, and paired end RNA-sequencing was performed. A bioinformatic analysis was conducted with the help of Dr.

Matt Weirauch laboratory using DESeq2.

RESULTS

We transfected constructs with IRF7 risk and non-risk expression plasmids into a cell line with constitutive TLR7 expression (HEK-blue cells). After stimulation with 1ug/mL of resiquimod, a compound with induces the synthesis of IFN-type I through the TLR 7/8 pathway, we extracted

RNA and cell supernatants at 3 and 24 hours post stimulation (Figure 7B). This concentration of resiquimod was chosen due to previous studies that show concentration dependence in HEK cells, with peak activation of NF-ĸB at 1ug/mL [21]. For our experiments, resiquimod was incubated with our cell culture of HEK-blue cells. We performed an ISRE luciferase reporter assay to confirm the previous results from another lab (Figure 3, Figure 7A). These data confirmed increased luciferase reporter activity downstream of ISRE signaling from the Q variant of IRF7 compared to the R variant. ELISA from the supernatant of cells demonstrated that cells transfected with 412Q IRF7 secreted 2.5-fold more interferon alpha than cells transfected with 412R IRF7 (Figure 7B). When looking at production of IFN-α over time in Figure 7B, we can see that at 3 and 24 hour time points, the 412Q IRF7 produced more IFN-α when compared to 412R IRF7. Furthermore, we can see that production of IFN-α remained elevated between the 3 hour and 24 hour time points in the

412Q IRF7 while the 412R IRF7 appears to return to baseline level at 24 hours. This data strongly supports genotype dependent production of IFN-α, but it is worth noting the potential impact of differential transactivation times between the two variants. If differences in dimerization or phosphorylation sites exist between the 412Q IRF7 and 412R IRF7, the rate at which IFN-α is produced could differ between the variants, and is worth considering. We then performed RNA- seq on the RNA from the cells and identified a strong genotype-dependent increase in the expression of Type-I interferons and interferon responsive genes at 3 hours (Figure 7C, red genes).

In Figure 7C, the genes shown in red (IFNB1, IFNA8, IFNA1, IFNA2, IFNL3, IFNA16, IFNA7,

IFNA6, IFNA21, IFNA4, IFNA14, IFNA17 and IFNA10) are involved with expression of Type I

IFN, while the remaining genes, also shown in red (OAS2, IFITM1, DDX58, IFIH1, CCL5,

IFITM3, GBP1, MX2, BST2, TRIM22) are IFN-responsive genes and show increased gene expression. The genes shown in grey show no significant differences in gene expression between the 412Q IRF7 and 412R IRF7. The RNA-seq analysis showed that there were no differences in the transcription of the mRNA encoding 412Q and 412R IRF7 in the overexpression studies, revealing a biological rather than a quantitative difference in gene expression. Altogether, these studies identified genotype-dependent production of type I IFN RNA and proteins at both time points (Figure 7).

Figure 7. Overexpression of IRF7 in in vitro cell culture has demonstrated genotype dependent production of IFN-I. We transiently transfected HEK-blue cells (293T cells with stable overexpression of TLR7) with either the risk or the non-risk form of IRF7 under a strong promoter. Cells were then stimulated with a viral mimic molecule, resiquimod (R848) for up to 24 hours. A) ISRE-luciferase reporter plasmid was co-transfected with IRF7. Luciferase read after 3 hours B) IFN-alpha ELISA measured all 14 IFN-alpha subtypes. C) RNA sequencing shows transcriptional differences between HEK-blue cells overexpressing risk and non-risk IRF7.

FUTURE STUDIES

The next steps in this study will be assessment of genotype-dependent IRF7 transcriptional regulatory activity in immune cells. Our study used a cell line that is commonly used in molecular biology due to its ability to grow well and be transfected with overexpression constructs. In the next set of experiments, our lab will need to show that IRF7 also increases type I IFN in plasmacytoid dendritic cells, B-cells, and macrophages.

Additionally, our lab will need to identify the sequence IRF7 binds to in the DNA of the human genome and test the hypothesis that the binding of IRF7 is genotype-dependent. If confirmed, then we may find that 412Q IRF7 binds places in the genome that are different than

412R IRF7 after the same extracellular signaling. For example, we hypothesize that 412Q IRF7 binds uniquely to more inflammatory genes than 412R IRF7. There are currently no antibodies that are specific to IRF7 and can be used with chromatin immunoprecipitation experimental protocols. To assess the mechanism of genotype-dependent binding to the genome, 412Q and 412R

IRF7 constructs will be made in which IRF7 is linked to a fluorescent epitope tag called green fluorescent protein (GFP), because antibodies against GFP have proven to be excellent for use in chromatin immunoprecipitation studies in other systems. Genotype-dependent binding could be due to several other mechanisms including genotype-dependent dimerization of IRF7 with other

IRF7 molecules or other IRF transcription factors. It could also be due to differences in the extent of nuclear localization of IRF7 after activation via phosphorylation. GFP-tagged IRF7 reagents will allow assessment of nuclear localization and dimerization.

CONCLUSIONS

In this master’s thesis, I sought to review the current literature on IRF and type-I interferon signaling and worked with lab members to test preliminary hypotheses about SLE-risk genotype- dependent IRF7 activity. Our results are consistent with our working hypothesis that SLE-risk variants containing a mutation of a specific amino acid in IRF7 leads to increased type-I interferon signaling. Previous literature has established that type-I interferon signaling increases lupus risk and mediates inflammation in patients with lupus.

Notably, it is important to specify the potential impact of this project, and how it advances the field of SLE research, particularly in regard to therapeutics and diagnostic procedures. IRF7 plays an extremely important role in the pathogenesis of SLE based upon all the collected data presented within this thesis. Being that there is no curative treatment, a targeted therapy against

IRF7 or IFN-I appears to be reasonable. More so, determining some of the key factors in nuclear translocation of IRF7, such as phosphorylation sites and dimerization interactions, may alleviate some of the complications of targeted therapy. Also worth considering, are targeted therapies against IFNAR1 and IFNAR2. As stated previously, a positive feedback loop exists between TLR- dependent, and TLR-independent signaling if not critically regulated by IRF7. With that being said, the production of other pro-inflammatory cytokines via IFNAR signaling may play a key role in the pathogenesis of SLE. By designing a targeted therapy against the large ectodomain where the IFN-I binds in IFNAR1/2, the production of these cytokines would be halted, alleviating some of the systemic inflammation present in SLE patients. Overall, this thesis undoubtedly highlights the importance of numerous mechanistic pathways of SLE, and serves as a foundation for more in depth research to be conducted.

We found genotype-dependent responses to resiquimod in our in vitro studies. As resiquimod is used in the context of some cancers and viral infections in other countries, our data would support a hypothesis that resiquimod would have increased activity in subjects with 412Q

IRF7 compared to 412R. In SLE, the drug hydroxychloroquine is used as a therapeutic because it can reduce intracellular TLR signaling. If SLE patients with 412Q IRF7 have more robust intracellular TLR signaling, then it is possible that 1) hydroxychloroquine would be more efficacious in these patients and 2) SLE patients with 412Q IRF7 may benefit from a higher dose of hydroxychloroquine if TLR expression is also increased in the disease population with the risk

IRF7. Any of these patient-specific hypotheses would require validation in vitro using patient- derived cells and in animal models before they could be considered for clinical studies in humans.

Altogether, the results in this master’s thesis provide supportive evidence for the role of

412Q IRF7 in increasing inflammatory signaling and the production of type I IFNs. In conjunction with additional studies which are forthcoming from the Kottyan laboratory, these data lay a foundation for a new mechanistic understanding of the etiology of SLE in subjects with the SLE risk genotype at rs1134665.

Glossary

1. Enzyme-linked Immunosorbent assay – ELISA

2. Interferon Regulatory Factor 7 – IRF7

3. Interferon Stimulated Response Elements – ISRE

4. Interferons – IFN’s

5. Plasmacytoid Dendritic Cells – pDC’s

6. Resiquimod – R-848

7. Systemic Lupus Erythematosus – SLE

8. Toll-Like Receptors - TLR’s

BIBLIOGRAPHY

1. U.S. Food & Drug Administration. “Lupus therapies Continue to Evolve.” 2017 2. Bertsias, George, Ricard Cervera, and Dimitrios T. Boumpas. "Systemic Lupus Erythematosus: pathogenesis and clinical features." EULAR textbook on rheumatic diseases 5 (2012): 476-505. 3. Centers for Disease Control and Prevention; Division of Population Health. (Reviewed 2018) 4. González LA, Alarcón GS. The evolving concept of SLE comorbidities. Expert Rev Clin Immunol. 2017 Aug;13(8):753-768. doi: 10.1080/1744666X.2017.1327353. Epub 2017 May 22. PMID: 28471690. 5. National Institute of Diabetes and Digestive and Kidney Diseases Health Information center. “Lupus and Kidney Disease. 2017 6. Vaughn SE, Kottyan LC, Munroe ME, Harley JB. Genetic susceptibility to lupus: the biological basis of genetic risk found in B cell signaling pathways. J Leukoc Biol. 2012 Sep;92(3):577-91. doi: 10.1189/jlb.0212095. Epub 2012 Jun 29. PMID: 22753952; PMCID: PMC3748338. 7. Li W, Titov AA, Morel L. An update on lupus animal models. Curr Opin Rheumatol. 2017 Sep;29(5):434-441. doi: 10.1097/BOR.0000000000000412. PMID: 28537986; PMCID: PMC5815391. 8. Fye JM, Orebaugh CD, Coffin SR, Hollis T, Perrino FW. Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome. J Biol Chem. 2011 Sep 16;286(37):32373-82. doi: 10.1074/jbc.M111.276287. Epub 2011 Aug 1. PMID: 21808053; PMCID: PMC3173215. 9. Birmingham DJ, Hebert LA. The Complement System in Lupus Nephritis. Semin Nephrol. 2015 Sep;35(5):444-54. doi: 10.1016/j.semnephrol.2015.08.006. PMID: 26573547. 10. Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev Immunol. 2014 Jan;14(1):36-49. doi: 10.1038/nri3581. PMID: 24362405; PMCID: PMC4084561. 11. Jefferies CA. Regulating IRFs in IFN Driven Disease. Front Immunol. 2019 Mar 29;10:325. doi: 10.3389/fimmu.2019.00325. PMID: 30984161; PMCID: PMC6449421. 12. Chasset, François, and Laurent Arnaud. "Targeting interferons and their pathways in systemic lupus erythematosus." Autoimmunity reviews 17.1 (2018): 44-52. 13. Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. 2005 May;5(5):375-86. doi: 10.1038/nri1604. PMID: 15864272. 14. Negishi, Hideo, Tadatsugu Taniguchi, and Hideyuki Yanai. "The interferon (IFN) class of cytokines and the IFN regulatory factor (IRF) transcription factor family." Cold Spring Harbor Perspectives in Biology 10.11 (2018): a028423. 15. O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol. 2013 Jun;13(6):453-60. doi: 10.1038/nri3446. Epub 2013 May 17. PMID: 23681101.

16. Urban-Wojciuk Z, Khan MM, Oyler BL, Fåhraeus R, Marek-Trzonkowska N, Nita-Lazar A, Hupp TR, Goodlett DR. The Role of TLRs in Anti-cancer Immunity and Tumor Rejection. Front Immunol. 2019 Oct 22;10:2388. doi: 10.3389/fimmu.2019.02388. PMID: 31695691; PMCID: PMC6817561. 17. Wu J, Chen ZJ. Innate immune sensing and signaling of cytosolic nucleic acids. Annu Rev Immunol. 2014;32:461-88. doi: 10.1146/annurev-immunol-032713-120156. PMID: 24655297. 18. Ragimbeau J, Dondi E, Alcover A, Eid P, Uzé G, Pellegrini S. The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression. EMBO J. 2003 Feb 3;22(3):537-47. doi: 10.1093/emboj/cdg038. PMID: 12554654; PMCID: PMC140723. 19. Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014 Sep 25;5:461. doi: 10.3389/fimmu.2014.00461. PMID: 25309543; PMCID: PMC4174766. 20. Dhillon B, Aleithan F, Abdul-Sater Z, Abdul-Sater AA. The Evolving Role of TRAFs in Mediating Inflammatory Responses. Front Immunol. 2019 Feb 4;10:104. doi: 10.3389/fimmu.2019.00104. PMID: 30778351; PMCID: PMC6369152. 21. J Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, Lipford G, Bauer S. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol. 2002 Jun;3(6):499. doi: 10.1038/ni0602-499. PMID: 12032557. 22. Ning S, Pagano JS, Barber GN. IRF7: activation, regulation, modification and function. Genes and immunity. 2011;12(6):399-414. doi: 10.1038/gene.2011.21. PubMed PMID: 21490621; PMCID: 4437765. 23. Yang H, Lin CH, Ma G, Baffi MO, Wathelet MG. Interferon regulatory factor-7 synergizes with other transcription factors through multiple interactions with p300/CBP coactivators. The Journal of biological chemistry. 2003;278(18):15495-504. doi: 10.1074/jbc.M212940200. PubMed PMID: 12604599. 24. Wathelet MG, Lin CH, Parekh BS, Ronco LV, Howley PM, Maniatis T. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol Cell. 1998;1(4):507-18. PubMed PMID: 9660935. 25. Qin BY, Liu C, Lam SS, Srinath H, Delston R, Correia JJ, Derynck R, Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation. Nat Struct Biol. 2003;10(11):913-21. doi: 10.1038/nsb1002. PubMed PMID: 14555996. 26. Qin BY, Liu C, Srinath H, Lam SS, Correia JJ, Derynck R, Lin K. Crystal structure of IRF-3 in complex with CBP. Structure. 2005;13(9):1269-77. doi: 10.1016/j.str.2005.06.011. PubMed PMID: 16154084. 27. Lin R, Mamane Y, Hiscott J. Multiple regulatory domains control IRF-7 activity in response to virus infection. The Journal of biological chemistry. 2000;275(44):34320-7. doi: 10.1074/jbc.M002814200. PubMed PMID: 10893229. 28. Sathish N, Zhu FX, Golub EE, Liang Q, Yuan Y. Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein

ORF45. The Journal of biological chemistry. 2011;286(1):746-56. doi: 10.1074/jbc.M110.150920. PubMed PMID: 20980251; PMCID: 3013033. 29. Hu X, Kim H, Stahl E, Plenge R, Daly M, Raychaudhuri S. Integrating autoimmune risk loci with gene-expression data identifies specific pathogenic immune cell subsets. American journal of human genetics. 2011;89(4):496-506. doi: 10.1016/j.ajhg.2011.09.002. PubMed PMID: 21963258; PMCID: 3188838 30. Trynka G, Sandor C, Han B, Xu H, Stranger BE, Liu XS, Raychaudhuri S. Chromatin marks identify critical cell types for fine mapping complex trait variants. Nat Genet. 2013;45(2):124- 30. doi: 10.1038/ng.2504. PubMed PMID: 23263488; PMCID: 3826950 31. Waldman M, Madaio MP. Pathogenic autoantibodies in lupus nephritis. Lupus. 2005;14(1):19- 24. PubMed PMID: 15732283.

32. Ikushima H, Negishi H, Taniguchi T. The IRF family transcription factors at the interface of innate and adaptive immune responses. Cold Spring Harbor symposia on quantitative biology. 2013;78:105-16. doi: 10.1101/sqb.2013.78.020321. PubMed PMID: 24092468.

33. Miyagawa F, Tagaya Y, Ozato K, Asada H. Essential Requirement for IFN Regulatory Factor 7 in Autoantibody Production but Not Development of Nephritis in Murine Lupus. J Immunol. 2016;197(6):2167-76. doi: 10.4049/jimmunol.1502445. PubMed PMID: 27527596.

34. Salloum R, Niewold TB. Interferon regulatory factors in human lupus pathogenesis. Translational research : the journal of laboratory and clinical medicine. 2011;157(6):326-31. doi: 10.1016/j.trsl.2011.01.006. PubMed PMID: 21575916; PMCID: 3096827.

35. Salloum R, Franek BS, Kariuki SN, Rhee L, Mikolaitis RA, Jolly M, Utset TO, Niewold TB. Genetic variation at the IRF7/PHRF1 locus is associated with autoantibody profile and serum interferon-alpha activity in lupus patients. Arthritis Rheum. 2010;62(2):553-61. doi: 10.1002/art.27182. PubMed PMID: 20112359; PMCID: PMC2832192.

36. Akahoshi M, Nakashima H, Sadanaga A, Miyake K, Obara K, Tamari M, Hirota T, Matsuda A, Shirakawa T. Promoter polymorphisms in the IRF3 gene confer protection against systemic lupus erythematosus. Lupus. 2008;17(6):568-74. doi: 10.1177/0961203308089340. PubMed PMID: 18539711.

37. Fu Q, Zhao J, Qian X, Wong JL, Kaufman KM, Yu CY, Hwee Siew H, Tan Tock Seng Hospital Lupus Study G, Mok MY, Harley JB, Guthridge JM, Song YW, Cho SK, Bae SC, Grossman JM, Hahn BH, Arnett FC, Shen N, Tsao BP. Association of a functional IRF7 variant with systemic lupus erythematosus. Arthritis and rheumatism. 2011;63(3):749-54. doi: 10.1002/art.30193. PubMed PMID: 21360504; PMCID: PMC3063317.

38. Lin LH, Ling P, Liu MF. The potential role of interferon-regulatory factor 7 among Taiwanese patients with systemic lupus erythematosus. The Journal of rheumatology. 2011;38(9):1914- 9. doi: 10.3899/jrheum.101004. PubMed PMID: 21632682.

39. International Consortium for Systemic Lupus Erythematosus G, Harley JB, Alarcon-Riquelme ME, Criswell LA, Jacob CO, Kimberly RP, Moser KL, Tsao BP, Vyse TJ, Langefeld CD, Nath SK, Guthridge JM, Cobb BL, Mirel DB, Marion MC, Williams AH, Divers J, Wang W, Frank SG, Namjou B, Gabriel SB, Lee AT, Gregersen PK, Behrens TW, Taylor KE, Fernando M, Zidovetzki R, Gaffney PM, Edberg JC, Rioux JD, Ojwang JO, James JA, Merrill JT, Gilkeson GS, Seldin MF, Yin H, Baechler EC, Li QZ, Wakeland EK, Bruner GR, Kaufman KM, Kelly JA. Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci. Nat Genet. 2008;40(2):204-10. doi: 10.1038/ng.81. PubMed PMID: 18204446; PMCID: PMC3712260.

40. Niewold TB, Kelly JA, Kariuki SN, Franek BS, Kumar AA, Kaufman KM, Thomas K, Walker D, Kamp S, Frost JM, Wong AK, Merrill JT, Alarcon-Riquelme ME, Tikly M, Ramsey- Goldman R, Reveille JD, Petri MA, Edberg JC, Kimberly RP, Alarcon GS, Kamen DL, Gilkeson GS, Vyse TJ, James JA, Gaffney PM, Moser KL, Crow MK, Harley JB. IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus. Annals of the rheumatic diseases. 2012;71(3):463-8. doi: 10.1136/annrheumdis-2011-200463. PubMed PMID: 22088620; PMCID: PMC3307526.

41. Li P, Cao C, Luan H, Li C, Hu C, Zhang S, Zeng X, Zhang F, Zeng C, Li Y. Association of genetic variations in the STAT4 and IRF7/KIAA1542 regions with systemic lupus erythematosus in a Northern Han Chinese population. Hum Immunol. 2011;72(3):249-55. doi: 10.1016/j.humimm.2010.12.011. PubMed PMID: 21167895.

42. PubChem. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 159603, Resiquimod; [cited 2020 Aug. 17].