A Dual Role for REV-ERB Alpha in Th17 Cell Mediated Immune Response

Home , ARNTL, GATA3, PER1, PER3, Period (gene), STAT4

UNIVERSITY OF CALIFORNIA, SAN DIEGO

A Dual Role for REV-ERB alpha in Th17 Cell Mediated Immune Response

A dissertation submitted in partial satisfaction of the

requirements for the degree

Doctor of Philosophy

Biology

Fang-Chen Chang

Committee in charge:

Professor Ye Zheng, Chair Professor Jack Bui Professor Christopher K. Glass Professor Lifan Lu Professor Clodagh O’Shea Professor Elina Zuniga

2016

Fang-Chen Chang, 2016

The dissertation of Fang-Chen Chang is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

Chair

University of California, San Diego

2016

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DEDICATION

To my parents, for the opportunities they afforded me, and their unwavering support. And to Edmond, who spent many beautiful weekend afternoons waiting in the Salk parking lot.

EPIGRAPH

First, there is a mountain, then there is no mountain, then there is.

—Traditional Buddhist saying, via Donovan (1967)

TABLE OF CONTENTS

SIGNATURE PAGE ...... iii DEDICATION ...... iv EPIGRAPH ...... v TABLE OF CONTENTS ...... vi LIST OF FIGURES ...... viii LIST OF TABLES ...... x ACKNOWLEDGEMENTS ...... xi VITA ...... xii ABSTRACT OF THE DISSERTATION ...... xiii Chapter One: Introduction ...... 1 1.1 Th17 cells ...... 1 1.1.1 CD4+ T helper cells ...... 1 1.1.2 Th17 effector function and cytokine production ...... 3 1.1.3 Transcriptional regulation of Th17 differentiation ...... 4 1.1.4 The role of Th17 cells in multiple sclerosis ...... 6 1.2 REV-ERBS ...... 8 1.2.1 Nuclear hormone receptors ...... 8 1.2.2 REV-ERBs ...... 8 1.2.3 Endogenous and synthetic REV-ERB ligands ...... 10 1.2.4 Regulation of the circadian rhythm ...... 11 1.2.5 Regulation of metabolism ...... 12 1.2.6 Regulation of immune functions ...... 14 1.3 Figures ...... 16 Chapter 2: Material and Methods ...... 18 2.1 Mice ...... 18 2.2 In vitro CD4 T cell differentiation ...... 18 2.3 Flow cytometry ...... 19 2.4 Reverse transcription and quantitative PCR ...... 20 2.5 Western blot ...... 20 2.6 Retroviral transduction ...... 21 2.7 Dual luciferase reporter assay ...... 21

2.8 DNA binding assay ...... 21 2.9 Chromatin Immunoprecipitation ...... 22 2.10 ChIP-sequencing ...... 22 2.11 RNA-sequencing and data analysis ...... 23 2.12 EAE models ...... 23 2.13 Histology analysis ...... 24 2.14 SR9009 treatment ...... 25 Chapter 3: Identification of REV-ERBa as a novel regulator in Th17 differentiation ...... 27 3.1 Results ...... 27 3.2 Tables and Figures ...... 32 Chapter 4: The functional role of REV-ERBa in Th17 cell mediated autoimmune response ...... 44 4.1 Results ...... 44 4.2 Tables and Figures ...... 48 Chapter 5: In vitro and in vivo characterizations of REV-ERBa deficiency ...... 56 5.1 Results ...... 56 5.2 Tables and Figures ...... 59 Chapter 6: Discussion ...... 65 6.1 Summary of findings ...... 65 6.3 Bridging the gain and loss of function phenotypes ...... 68 6.4 Conclusion ...... 72 6.5 Figures ...... 73 References ...... 76

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LIST OF FIGURES

Figure 1.1 Effector CD4+ T cell lineages ...... 16 Figure 1.2 REV-ERBs and RORs play essential and opposing roles in the regulation of the molecular clock ...... 17 Figure 3.1 REV-ERBa is up-regulated in Th17 cells ...... 32 Figure 3.2 REV-ERBa is up-regulated in human Th17 cells ...... 33 Figure 3.3 Ectopic REV-ERBa expression inhibits Th17 differentiation ...... 34 Figure 3.4 REV-ERBs inhibit RORγt-dependent IL-17A expression ...... 35 Figure 3.5 Analysis of different REV-ERBα domains that are involved in repression of IL-17A expression ...... 36 Figure 3.6 REV-ERBa over expression inhibits the expression of Th17 signature genes ...... 37 Figure 3.7 REV-ERBa directly inhibits IL17a expression ...... 38 Figure 3.8 Figure 2.8 REV-ERBa binds to a RORE within the il17a locus ...... 39 Figure 3.9 Figure 2.9 REV-ERBa binds to the endogenous Il17a locus ...... 40 Figure 3.10 REV-ERBa binds to the endogenous Il17a locus ...... 41 Figure 3.11 REV-ERBa and RORgt compete for binding to the Il17a locus ...... 42 Figure 4.1 Doxycycline induces transgenic REV-ERBα expression in vivo, but does not affect EAE disease progression ...... 48 Figure 4.2 In vivo induction of REV-ERBa expression in Th17 cells suppresses EAE disease progression ...... 49 Figure 4.3 Doxycycline treatment does not affect homing of transferred CD4+ T cells ...... 50 Figure 4.4 REV-ERB agonist SR9009 inhibits in vitro Th17 differentiation ...... 51 Figure 4.5 REV-ERB agonist SR9009 inhibits in vitro human Th17 differentiation52 Figure 4.6 REV-ERB agonist SR9009 enhances NCoR recruitment to the Il17a locus ...... 53 Figure 4.7 Treatment with REV-ERB agonist SR9009 suppresses EAE progression ...... 54 Figure 4.8 Treatment with REV-ERB agonist SR9009 prevents relapse of EAE . 55

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Figure 5.1 Knock down of REV-ERBα suppresses in vitro Th17 cell differentiation59 Figure 5.2 CD4Cre REV-ERBa/b flfl mice have a deletion of the DBD in REV- ERBa transcript ...... 60 Figure 5.3 REV-ERBα deficiency suppresses Th17 cell differentiation ...... 61 Figure 5.4 REV-ERBα deficiency ameliorates EAE disease progression ...... 62 Figure 5.5 REV-ERBα deficiency ameliorates EAE disease progression ...... 63 Figure 5.6 REV-ERBα represses Nfil3 expression ...... 64 Figure 6.1 SR9009 treatment does not cause aberrant weight loss in mice ...... 73 Figure 6.2 Working model 1 ...... 74 Figure 6.3 Working model 2 ...... 75

LIST OF TABLES

Table 2.1 KEGG pathway analysis ...... 42

ACKNOWLEDGEMENTS

I would like to thank Ye for giving me a chance at the very beginning and for his support and mentorship over the years. I couldn’t have asked for a better mentor or environment to learn in and grow as a scientist. My gratitude also goes out to each member of the Zheng lab, past and present. To Lauren and Sagar, who provided friendship and comraderie as we weathered grad school together; to Yuqiong, for all her help with experiments and setting an example of efficiency and hard work; to Xudong, for great conversations and insights; to Alert, for keeping the lab in perfect working condition and the interesting and educational conversations about politics, hiking and more. And to Yang, who slaved through long EAE sacs with me.

I’d also like to thank my committee for their insights, encouragements and guidance. The ideas and suggestions that came out of these conversations helped shape the direction of my projects.

Chapters 2-5, in full (with minor exceptions to conform to this dissertation), were submitted to Nature Medicine by Chang C, Zhao X, Solt L, Liang Y, Bapat

S, Cho H, Kamenacka T, Leblanc M, Atkins A, Yu R, Downes M, Burris T, Evans

R, Zheng. ‘The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease’. The dissertation author was the primary investigator and author of this paper.

VITA

2010 B.S. in Biology, Duke University

2012-2014 Graduate Teaching Assistant, University of California, San Diego

2016 Ph.D. in Biology, University of California, San Diego

PUBLICATIONS

Forte E, Salinas RE, Chang C, et al. The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells. J Virol. 2012 Jun;86(12):6889-98

Hernandez J, Chang C, et al. The CREB/CRTC2 Pathway Modulates Autoimmune Disease by Promoting Th17 Differentiation. Nature Communications. 2015 doi:10.1038/ncomms8216

In preparation

Chang C, Zhao X, Solt L, et al. The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease. (Under revision for resubmission to Nature Medicine)

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ABSTRACT OF THE DISSERTATION

A Dual Role for REV-ERB alpha in Th17 Cell Mediated Immune Response

Fang-Chen Chang

Doctor of Philosophy in Biology

University of California, San Diego, 2016

Professor Ye Zheng, Chair

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis(Korn,

Bettelli, Oukka, & Kuchroo, 2009; Weaver, Hatton, Mangan, & Harrington, 2007). The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORgt(Ivanov et al., 2006). Here we identify REV-ERBa

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(encoded by Nr1d1), a member of the nuclear hormone receptor (NHR) family, as a transcriptional repressor that antagonizes RORgt function in Th17 cells.

REV-ERBa binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORgt-dependent genes including Il17a and Il17f.

Furthermore, elevated REV-ERBa expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE), a Th17 cell- mediated autoimmune disease. These results suggest that modulating REV-

ERBa activity may hold therapeutic potential for treatment of Th17 cell-mediated autoimmune diseases.

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Chapter One: Introduction

1.1 Th17 cells

1.1.1 CD4+ T helper cells

CD4 T cells are critical components of the adaptive immune system.

Naïve T helper (Th) cells undergo clonal expansion and differentiation into effector cells upon activation by antigen-presenting cells (APCs). Each subset is characterized by their distinct effector functions and cytokines secreted. The differentiation process is directed by the cytokines present in the environment.

Effector Th cells were initially classified into two subsets, Th1 and Th2 (Lazarevic et al., 2011)(Dong and Flavell 2000; Glimcher and Murphy 2000). Th1 cells produce high levels of interferon-gamma (IFN-g) and initiates cell mediated inflammatory responses against intracellular bacteria (Scanga et al., 2002; Seki et al., 2002). Th1 differentiation and IFN-g production are induced by IL-12, and

IFN-g in turn leads to the activation of the transcription factor T-bet, which sustains Th1 development (Agnello et al., 2003). Th2 cells produce interleukin-4

(IL-4), IL-5, and IL-13 and are crucial for humoral and anti-parasite immunity

(Anthony, Rutitzky, Urban, Stadecker, & Gause, 2007). Th2 differentiation is driven by IL-4, in a STAT6-dependent manner, resulting in the activation of transcription factor GATA3.

1 2

In 1998, CD4+ T cells secreting IL-17 were first described (Jovanovic et al., 1998). The observation was followed by studies that officially established

Th17 cells as a third subset distinct from Th1 and Th2 cells (Park H. 2005;

Harrington L.E. 2005; Bettelli, Korn, and Kuchroo 2007; Chang and Dong 2007;

Weaver et al. 2006). The discovery of Th17 cells has shed light on many immune responses previously unexplained by the Th1/Th2 paradigm, such as certain fungal and extracellular bacterial infections like Candida albicans (W. Huang, Na,

Fidel, & Schwarzenberger, 2004) and Pseudomonas aeruginosa (Liu et al.,

2011). It has also enlightened our understanding of how autoimmune diseases such as multiple sclerosis, psoriasis and rheumatoid arthritis develop and progress. IL-17, a signature cytokine produced by Th17 cells, is expressed highly in the CNS lesions of patients with multiple sclerosis (Brucklacher-Waldert,

Stuerner, Kolster, Wolthausen, & Tolosa, 2009; Durelli et al., 2009; Tzartos et al.,

2008), in the skin of patients with psoriasis (Wilson et al., 2007), and in joint tissues from rheumatoid arthritis patients (Kotake et al., 1999). In accordance with human studies, the pathogenic role of Th17 cells has been established in animal models such as experimental autoimmune encephalomyelitis (EAE) (Cua et al., 2003; Langrish et al., 2005), collagen-induced arthritis (Lubberts et al.,

2004) and trinitrobenzene sulfuric acid- (TNBS) induced colitis (Z. Zhang, Zheng,

Bindas, Schwarzenberger, & Kolls, 2006).

Regulatory T cell (Treg) is another CD4 subset that was recently characterized. Tregs play an important role in maintaining immune tolerance and homeostasis by suppressing activation of excessive immune responses. The

3 development and maintenance of Tregs depend on constituent expression of the transcription factor Foxp3 (Rudensky, 2011; Sakaguchi, Yamaguchi, Nomura, &

Ono, 2008). In both human and mice, Foxp3 deficiency leads to the development of severe autoimmune inflammation. In vitro, naive T cells can be differentiated into induced-Tregs in the presence of IL-2 and TGFb, although the genetic profile of iTreg is not identical to natural Tregs. Treg’s crucial role in immune self- tolerance and homeostasis suggest it may possess the potential to treat allergy, autoimmune disease, tissue rejections and cancer.

1.1.2 Th17 effector function and cytokine production

The Th17 subset was initially characterized by its ability to secrete IL-17A, which is part of the IL-17 cyotokine family that includes IL-17B, IL-17C, IL-17D,

IL-17E (also known as IL-25) and IL-17F (Jin & Dong, 2013). IL-17A and IL-17F are the only two cytokines in the family that are produced by Th17 cells. They share 55% homology and display many similar functions (Hymowitz et al., 2001).

IL-17A and IL-17F can function individually or as heterodimers, suggesting there may be both distinct and synergistic functions (Chang & Dong, 2007; Wright et al., 2007). Both cytokines signal through the IL-17 receptor, which consists of IL-

17RA and IL-17RC (Toy et al., 2006). IL-17R is expressed on a broad range of cell types including epithelial cells. IL-17A and IL-17F exert their proinflammatory properties by inducing the expression of cytokines (IL-6, IL-8, GM-

CSF, G-CSF) (Fossiez et al., 1996; Jones & Chan, 2002), chemokines (CXCL1,

CXCL2, CXCL5, and CXCL8) (Laan et al., 1999) and metalloproteinases in these

4 cells. The recruitment and activating migration of neutrophils is particularly important for protection against Gram-negative bacteria and fungal infections

(Kolls & Lindén, 2004). And induction of antimicrobial-peptide production is crucial for maintaining the integrity of epithelial-barrier in mucosal host defense.

Th17 cells can also produce various other cytokines such as IL-21, IL-22,

GM-CSF, IL-9, IL-10, and IFN-g, some only in certain contexts. IL-21 is involved in the induction and expansion of Th17 cells (Nurieva et al., 2007). IL-22 was shown to be important for mucosal barrier immunity, and GM-CSF plays a crucial role in the pathogenicity of Th17 cells in autoimmune disorders (Codarri et al.,

2011; El-Behi et al., 2011), most likely by helping with the recruitment of macrophages, monocytes, and expansion of T cells (Croxford et al., 2015).

1.1.3 Transcriptional regulation of Th17 differentiation

Th17 differentiation requires transforming growth factor-beta (TGFb) and

IL-6 in mouse (Mangan et al., 2006; 2006) and is further sustained by IL-23. At low concentrations of TGFb, TGFb synergizes with IL-6 to promote IL-23 receptor and Th17 differentiation. However, more recent studies have shown that

Th17 differentiation can occur in the absence of TGFb (Ghoreschi et al., 2010).

Although IL-6 nor IL-23 alone can induce Th17 differentiation, the combination of the two, together with IL-1b, results in effective Th17 differentiation from naïve

CD4 T cells.

Th17 differentiation is initiated by the combination of activated TCR and cytokine receptors. The downstream signaling events lead to the activation of

5 transcription factors that induce expression of Th17 signature genes, including

Il17a and Il17f. One of the crucial signaling events involves the phosphorylation of STAT3. Similar to the roles of STATs in Th1 and Th2 differentiation, STAT3 selectively mediates Th17 differentiation (Wei, Laurence, Elias, & O'Shea, 2007;

Liang Zhou et al., 2007). STAT3 is required for IL-6 induction of IL-21 and regulates the expression of retinoic acid receptor-related orphan receptor gamma t (RORgt), which is the master regulator of Th17 differentiation. RORgt is part of the ROR subfamily of the nuclear receptor superfamily. The ROR family consists of ROR alpha, ROR beta and ROR gamma (Jetten, 2009). RORg has two isoforms, RORg and RORgt. They are both encoded by the Rorc gene and differ only at the N terminus (He, Deftos, Ojala, & Bevan, 1998). While RORg is expressed in a wide range of tissues, RORgt is only expressed in lymphoid cells.

RORgt is critical for lymph node formation during development (Eberl et al.,

2004; Kurebayashi et al., 2000; Sun et al., 2000), as well as Th17 differentiation and IL-17 production (Ivanov et al., 2006; Yang et al., 2008). Under neutral condition, ectopic expression of RORgt is sufficient to induce expression of IL-

17A, IL-17F, IL-23R and IL-22. And under Th17 polarizing conditions, IL-17 expression is diminished in CD4+ T cells from Rorg-deficient mice (Ivanov et al.,

2006). RORgt modulates Th17 differentiation by recognizing and binding to ROR response elements (ROREs) in the promoter and cis-regulatory elements upstream of the Il17a locus, and other Th17 signature genes. Both the 2kb Il17a promoter region and the conserved non-coding region 5 (CNS5, also called

CNS2) are required for optimal transcriptional activation of IL-17A. Both regions

6 contain ROREs and mutations in these sequences result in a decrease in Il17a activation (Yang et al., 2008; F. Zhang, Meng, & Strober, 2008).

In addition to RORgt, RORa is also expressed in Th17 cells, although its role in Th17 differentiation is less critical than that of RORgt. RORa-deficiency results in moderate decrease in IL-17A expression but coexpression of RORa with RORgt induces greater Th17 differentiation than ectopic Rorgt expression alone, indicating the two nuclear receptors work synergistically in the regulation of Th17 differentiation. More recently, IRF4, BATF and RUNX1-deficient mice were shown to exhibit impaired Th17 development and reduced susceptibility to autoimmunity, suggesting that they also play important roles in Th17 differentiation (Biswas et al., 2010; Schraml et al., 2009; F. Zhang et al., 2008).

1.1.4 The role of Th17 cells in multiple sclerosis

Th17 cells play a critical role in maintaining persistent inflammation in multiple sclerosis (MS). They can enter the encephalic compartment and penetrate the blood brain barrier to recruit inflammatory cells. C-C chemokine receptor 6 (CCR6) expression on the cell surface of Th17 cells binds to C-C chemokine ligand 20 (CCL20), which is expressed constitutively by vascular endothelium of the blood-cerebrospinal barrier, enabling Th17 cells to enter the brain (Reboldi et al., 2009). In the brain, Th17 cells secrete proinflammatory cytokines such as IL-17A, which aid in the recruitment and migration of other immune cells into the central nervous system (CNS) (Kebir et al., 2007). IL-17A may also interfere with the remyelinating process by promoting cell death in

7 myelin-forming cells (M. K. Paintlia, Paintlia, Singh, & Singh, 2011). However, as

EAE progression in mice is only lightly perturbed in IL-17A knockout mice and wild-type mice administered with IL-17A neutralizing antibodies (Hofstetter et al.,

2005), other cytokines secreted by Th17 cells may play crucial roles in EAE induction in concert with IL-17A. GM-CSF, IL-21 and IL-22, have all been shown to contribute to Th17 pathogenicity and EAE progression.

Recently, therapies targeting Th17-associated cytokines, such as IL-17A,

IL-23 and GM-CSF have been developed and clinical trials are being conducted for some of these strategies (Constantinescu et al., 2015; Garnock-Jones, 2015;

McInnes et al., 2015). But most of the efforts in targeting single cytokines have not shown promising results in MS, Crohn’s disease or rheumatoid arthritis.

Some efforts have instead focused on targeting the Th17 differentiation program as a whole. As the master regulator of Th17 differentiation, RORgt garners the most amount of interest. Many small molecule screens have ben conducted to find suitable drugs to target RORgt directly. Small molecules like Digoxin and

SR1001 were shown to bind to RORgt and inhibit transcription of its target genes, resulting in the suppression of Th17 differentiation and EAE progression in mice (Huh et al., 2011; Solt et al., 2011). These preclinical findings indicate that targeting the Th17 differentiation program through its key regulators holds potential for treatment of autoimmune diseases.

1.2 REV-ERBs

1.2.1 Nuclear hormone receptors

Nuclear hormone receptors form a class of ligand-activated proteins that act as on or off switches for gene transcription upon binding to specific sequences. These sequences are called hormone response elements (HRE) and typically contain two consensus hexameric half-sites. Nuclear hormone receptor proteins usually consist of a variable N-terminal region, a conserved DNA binding domain (DBD), a variable hinge region, a conserved ligand binding domain (LBD) and a variable C-terminal region (Kosztin, Izrailev, & Schulten, 1999). The binding of ligands to the LBD causes conformational changes to the protein, which initiates a cascade of down stream events, such as the recruitment of co- regulatory proteins to activate or repress transcription. Even though the endogenous ligands are known for only half of the nuclear receptors, many natural and synthetic ligands have been identified and developed to use as chemical tools to study the functions of these receptors and to probe their therapeutic potential.

1.2.2 REV-ERBs

REV-ERB alpha and beta (encoded by Nr1d1 and Nr1d2) are part of the family of nuclear receptors. REV-ERBa was first discovered. Because it is encoded on the opposite strand of the ERBA (also known as THRA) oncogene,

9 which encodes the thyroid hormone receptor-alpha, it was named REV-ERB alpha, which derived from ‘reverse strand of ERBA’ (Lazar, Hodin, Darling, &

Chin, 1989; Miyajima et al., 1989; 1988). REV-ERBa and REV-ERBb have similar temporal and special expression, consistent with their overlapping functions. They are both expressed in a wide array of tissues throughout the body and exhibit circadian expression pattern and functions (Guillaumond,

Dardente, Giguère, & Cermakian, 2005; Torra et al., 2000). Unlike most nuclear receptors, REV-ERBs lack the C-terminal AF2 region of the LBD (Bonnelye et al.,

1994; Dumas et al., 1994). Since the AF2 domain is required for most nuclear receptors to recognize co-activators, REV-ERB was generally thought to be a transcriptional repressor. Indeed, REV-ERBa was first identified as a negative regulator of the circadian clock gene Bmal1 (Preitner et al., 2002). Subsequently,

Ueda et al. (Ueda et al., 2002) described the oscillatory pattern of REV-ERB expression and characterized the Rev-ErbA/ROR response elements (AGGTCA) located in the promoter region of its target genes. REV-ERBs generally functions as monomers, but have been reported to form homodimers as well (Harding &

Lazar, 1995). Upon activation by the binding to its ligand, heme (Raghuram et al.,

2007; Rogers, Ying, & Burris, 2008), REV-ERBs recruit co-repressor, NCoR to repress the transcription of its target genes by way of histone deacetylation and condensation of chromatin (Harding & Lazar, 1995; Yin & Lazar, 2005). Although it’s be long assumed that REV-ERB’s repressive capability is dependent on direct interaction with DNA, a recent study proposed an alternative genomic mechanism by which REV-ERBs regulates its target genes (Y. Zhang et al.,

2015). Genome-wide cistrome analysis revealed that REV-ERBs can modulate gene expression by recruiting the HDAC3 co-repressor to sites where REV-ERB is tethered by cell type-specific transcription factors instead of direct binding to a specific DNA sequence (Y. Zhang et al., 2015).

1.2.3 Endogenous and synthetic REV-ERB ligands

REV-ERBs were initially categorized as orphan nuclear receptors. Then studies of E75, the Drosophila melanogaster orthologue of REV-ERB, uncovered a role for E75 as a Haem sensor (Reinking et al., 2005). This discovery led to efforts that confirmed the human receptor also binds to heme (Raghuram et al.,

2007; Yin et al., 2007). Mutation studies of REV-ERB identified a key amino acid residue in the protein that is necessary for heme binding and REV-ERB’s repressor function. Furthermore, reduced intracellular heme levels impaired the recruitment of the NCOR-HDAC3 co-repressor complex to REV-ERB target gene promoters and resulted in decreased repression of REV-ERB target gene BMAL1

(Rogers et al., 2008). Since heme levels are regulated in a circadian manner, heme availability may be one of the regulating factors for REV-ERB activity

(Rogers et al., 2008).

Crystal structures of REV-ERB bound to heme aided in our understanding of how the ligand interacts with the receptor (Gupta & Ragsdale, 2011; Pardee et al., 2009), which led to successful efforts in targeting REV-ERB with synthetic ligands. GSK4112 was the first published synthetic REV-ERB agonist. It increases the recruitment of NCoR to BMAL1 promoter in a dose dependent

11 manner (Kumar et al., 2010), however GSK4112 is limited by poor performance in vivo. SR9009 and SR9011 were subsequently identified and characterized as potent REV-ERB agonists with in vivo activity. Both compounds were shown to substantially affect circadian parameters though the modulation of REV-ERB

(Solt et al., 2012)(Solt et al. 2012). Since then, additional REV-ERB agonists have been described (Trump et al., 2013).

1.2.4 Regulation of the circadian rhythm

Circadian rhythms play an important role in all major physiological processes, such as sleep, feeding, blood pressure and immune functions

(Mohawk, Green, & Takahashi, 2012). At the molecular level, circadian rhythm is generated by transcriptional-translational feedback loops of a set of core clock genes. BMAL1 (also known as ARNTL) and CLOCK form heterodimers and induce the expression of the cryptochrome genes (CRY1 and CRY2) and the period genes (PER1, PER2 and PER3). As CRY and PER protein levels build up, they form heterodimers and begin to repress the expression of BMAL1 and

CLOCK. These feedback loops result in the self-sustaining oscillation of the core clock gene expressions, and in extension, downstream genes and cellular processes. REV-ERB plays a critical role in the regulation and maintenance of the molecular clock (Guillaumond et al., 2005). Previously it was thought to play a uREV-ERB represses the transcription of BMAL1 through direct interaction with

ROREs in the BMAL1 promoter. It is worth noting that REV-ERB’s own

12 expression is regulated by BMAL1-CLOCK heterodimers. REV-ERBa knockout mice have aberrant expression of Bmal1 and changes in both phase and period of their locomotor behavior (Preitner et al., 2002). REV-ERBb knockout mice have less prominent circadian phenotypes. But REV-ERBa/b double knockout mice are completely arrhythmic, similar to Bmal1-, Cry-, and Per-deficient mice

(Cho et al., 2012). Previously, REV-ERB was considered just a component of an accessory loop that modulates the pattern of the core clock genes, but these recent results suggest that it plays a more essential role than was previously appreciated, and should be considered a core clock gene.

In contrast to the REV-ERBs, RORs up-regulate BMAL1 expression (Sato et al., 2004) by binding to the same ROREs that REV-ERBs recognize. RORa deficiency in mice results in alterations in the circadian oscillator. In addition,

RORb and RORg have also been implicated in regulating the circadian rhythm

(André et al., 1998; Masana, Sumaya, Becker-Andre, & Dubocovich, 2007;

Takeda, Jothi, Birault, & Jetten, 2012). REV-ERBa and RORa are expressed anti-phase to each other, leading to alternating activation and repression of core clock genes (Preitner et al., 2002; Sato et al., 2004).

1.2.5 Regulation of metabolism

Since circadian rhythms are closely linked to metabolic pathways, alterations in core clock gene expressions lead to metabolic disorders like obesity and insulin resistance (Bass & Takahashi, 2010; Gamble & Young,

2013). As an integral component of the molecular clock, REV-ERBs play an

13 important role in regulating metabolic pathways. REV-ERBa was found to regulate lipid metabolism, glucose metabolism, adipogenesis, and oxidative capacity in skeletal muscle. APOA1, a component of high-density lipoprotein

(HDL) is regulated by REV-ERBa (Vu-Dac et al., 1998) and REV-ERBa deficient mice have dyslipiaemia (Raspè et al., 2002). On the other hand, REV-ERBb is involved in the regulation of fatty acid and lipid absorption, energy expenditure and lipogenesis in muscle (Ramakrishnan, Lau, Burke, & Muscat, 2005).

In hepatic glucose metabolism, REV-ERBα directly regulates the expression of the genes encoding the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PCK) and glucose-6-phosphatase

(G6PC)(Yin et al., 2007). REV-ERBa is also involved in regulating the oxidative capacity of skeletal muscle and mitochondrial biogenesis (Woldt et al., 2013). It is up-regulated in oxidative skeletal muscle and its deficiency leads to reduced mitochondrial content and oxidative function, which results in impaired exercise capacity (Woldt et al., 2013).

Lastly, REV-ERBs are also involved in adipogenesis. REV-ERBα expression is highly induced during adipogenesis(Chawla & Lazar, 1993), and ectopic expression of REV-ERBα in 3T3-L1 preadipocytes results in increased expression of markers of adipogenesis, and potentiated adipocyte differentiation(Fontaine et al., 2003). Interestingly, although REV-ERBα expression is required for adipogenesis in vitro, REV-ERBα deficiency in vivo is associated with increased adiposity and increased weight gain when fed on a high-fat diet(Delezie et al., 2012). This apparent discrepancy may be due to a

14 dual role for REV-ERBα in adipogenesis, where REV-ERBα protein levels are increased early in adipogenesis but substantially reduce in the late stages of the process to allow for efficient differentiation of the fat cells(J. Wang & Lazar,

2008).

1.2.6 Regulation of immune functions

Our understanding of the role of REV-ERB in immune cells has been lacking until recent years, with efforts focusing mainly on macrophages. REV-

ERBa has been shown to regulate the production and release of the proinflammatory cytokine interleukin-6 (IL-6) in macrophages (Gibbs et al., 2012).

And a recent genome-wide analysis of REV-ERB binding profiles in macrophages demonstrated that REV-ERBs regulate target gene expression through the repression of distal enhancers associated with macrophage-lineage- determining factors (Lam et al., 2013). These data suggest that REV-ERB plays an important role in macrophage cell function.

In a nuclear receptor screen in different Th cells, we noticed that REV-

ERBa expression was significantly higher in Th17 cells. Given REV-ERB and

ROR’s opposing roles in regulating circadian rhythm, in which they are known to bind the same RORE, we speculated that REV-ERBs could also play a role in

Th17 differentiation and function. Indeed, a recent study found perturbations in

Th17 differentiation in CD4+ T cells from REV-ERBa deficient mice (Yu et al.,

2013). Our initial experiments indicated a suppressive role of REV-ERBa in Th17

15 cells, where overexpression of REV-ERBa in Th17 conditions resulted in a decrease in IL17A producing cells (Figure 3.3).

As our understanding of the role Th17 cells play in inflammation and autoimmunity grows, targeted therapies that antagonize IL-17A and Th17 cells have garnered a lot of attention and effort. Initial studies have shown promise for several experimental autoimmune diseases (Curtis & Way, 2009; Uyttenhove &

Van Snick, 2006). Our studies identify REV-ERBa as a novel regulator of the

Th17 subset that play a key role in Th17 cell differentiation and function. The objective of this thesis research is to understand how REV-ERBs regulate and modulate Th17 differentiation, in the hopes of gathering more insights into how

Th17 cells are induced and maintained, as well as create a new avenue for potential treatments.

In the following chapters I will describe the findings that support the notion of REV-ERB being a novel and effective regulator of Th17 differentiation. In chapter 2, I will describe the material and methods by which all the studies in this thesis work were done; in chapter 3, I will discuss the findings we have on understanding the molecular mechanism by which REV-ERBs regulate Th17 differentiation; in chapter 4, I will examine REV-ERBa’s role in autoimmune disease models using gain of function approaches; in chapter 5, I will describe the REV-ERBa deficient phenotype in both cell-based assays and autoimmune disease models; finally, in chapter 6 I will summarize our data, discuss significance of our findings and explore future directions.

1.3 Figures

Th1 IFNg Cellular immunity

IL-12 T-bet

IL-4 Humoral immunity Th2 IL-5 IL-13 Allergies Naive IL-4 Gata3 CD4 IL-17A IL-6 Th17 IL-17F Tissue inﬂammation TGFb IL-21 Autoimmunity IL-22 RORgt IL-2 TGFb iTreg IL-10 TGFb Immune tolerance Foxp3

Figure 1.1 Effector CD4+ T cell lineages. T helper subsets are characterized by the cytokines they produce and their effector immune functions, as indicated on the right. Cytokines in the environment help drive differentiation by initiating signaling cascades that result in the activation of master regulators. Critical transcription factors such as RORgt for Th17 cells then drive expression of lineage specific cytokines and effector functions.

ROR REV-ERB

Clock, Bmal1

RORE

Per, Cry Rev-Eerb CLOCK BMAL1 CLOCK BMAL1 REV-ERB PER2 PER1 E-box RevDR2 CRY2 CRY1

Figure 1.2 REV-ERBs and RORs play essential and opposing roles in the regulation of the molecular clock. REV-ERBs act as repressors and RORs as activators for the regulation of core clock genes.

Chapter 2: Material and Methods

2.1 Mice

Rosa-M2rtTA, TRE-REV-ERBa, and 2D2 transgenic mice were purchased from Jackson Laboratory. The three transgenic mice were crossed to generate

Rosa-M2rtTAxTRE-RVBx2D2 mice. C57BL/6, SJL/J, and Ly5.1+ congenic mice were purchased from the Jackson Laboratory. All mice were maintained in the

Salk Institute SPF animal facility in accordance with institutional regulations.

2.2 In vitro CD4 T cell differentiation

For mouse CD4 T cell differentiation, total CD4 T cells were isolated from the spleen and lymph nodes using the Dynabeads CD4 Positive Isolation Kit

(Invitrogen). When indicated, enriched CD4 T cells were further sorted for naive cells (CD25- CD62L low CD44high). Cells were resuspended in Click’s medium

(Irvine Scientific) at 1 million cells per ml, and then plated in 24 well plates coated with Goat-Hamster IgG antibody (200ng/ml; MP Biomedicals) with the addition of soluble anti-CD3 (1mg/ml; 145-2C11) and anti-CD28 (1mg/ml; 37.51) from Bio X

Cell. Polarizing conditions for different T helper subsets are as following: Th1: mIL-2 (100U/ml; Tonbo), mIL-12 (20ng/ml; Peprotech) and anti-IL-4 (5mg/ml; Bio

X Cell). Th2: mIL-2 (100U/ml; Tonbo), mIL-4 (20ng/ml; Biolegend), anti-IFN-g and anti-IL-12 (5mg/ml; Bio X Cell). Th17: mIL-6 (20ng/ml; Biolegend), mIL-23

(20ng/ml; R&D), mIL-1b (20ng/ml; Peprotech), hTGF-b (2ng/ml; Peprotech), anti-

18 19

IFN-g and anti-IL-12 (5mg/ml; Bio X Cell). iTreg: mIL-2 (100U/ml; Tonbo) and hTGF-b (2ng/ml; Peprotech).

For human CD4 T cell differentiation, total CD4 T cells were isolated from

PBMC using the EasySep Human CD4+ T Cell Enrichment Kit (StemCell). Cells were plated at 1 million per ml in 24 well plates coated with 5ug/ml anti-human

CD3 and anti-human CD28 (Tonbo). For human Th1 differentiation, hIL-2

(100U/ml; Tonbo), hIL-12 (20ng/ml; Tonbo), anti-human IL-4 (10ug/ml;

Biolegend) were used. For human Th17 differentiation, hIL-6 (20ng/ml;Tonbo), hIL-23 (20ng/ml;Tonbo), hIL-1b (20ng/ml;Tonbo), hTGF-b (5ng/ml;Peprotech), anti-human IL-4 and anti-human IFN-g(10 mg/ml; Biolegend) were used. Cells were cultured for 6-8 days and then analyzed by FACS and RT-qPCR.

2.3 Flow cytometry

Cytokine expression was assessed by intracellular staining. The following antibodies were used for cell surface staining: anti-CD4-PerCPCy5.5 (RM4-5) and anti-CD62L-APC(MEL-14) from Tonbo Biosciences; anti-CD44PE(IM7) and anti-CD25-FITC(7D4) from Ebiosciences; for intracellular staining: anti-IL-17A-

PE(eBio17B7), anti-IFN-γ-APC (XMG1.2) and anti-Foxp3-FITC(FJK-16s) from

Ebiosciences. Cells were stimulated with PMA and ionomycin in the presence of brefeldin A (GolgiPlug; BD) for 5 hours at 37°C. Cells were then fixed with fixation and permeabilization reagents from BD or Ebioscience (for Foxp3 staining) and labeled with appropriate antibodies before being analyzed on a BD

FACSAria Cell Sorter. Results were analyzed with Flowjo (Tree Star). 20

2.4 Reverse transcription and quantitative PCR

Total RNA was isolated from CD4 T cells using TRIzol reagent (Life technologies). cDNA was synthesized with iScript Reverse Transcription

Supermix for RT-qPCR (Bio-rad) according to the manufacturer’s protocol, followed by qPCR using SYBR Green PCR Master Mix (Applied Biosystems).

Quantitative PCR was performed on an Applied Biosystems ViiA™ 7 Real-Time

PCR System with gene specific primers listed in Supplementary Table 1. The comparative threshold cycle method and an internal control (β-actin) were used to normalize the expression of genes of interest.

2.5 Western blot

Proteins from cell lysates were electrophoresed on a 10% SDS PAGE gel and transferred to PVDF membrane. Blots were probed with anti-T-bet (1:1,000;

BioLegend), anti-RORgt (1:500; Santa Cruz), anti- REV-ERBa (1:1000; described previously(Cho et al., 2012)), or anti-REV-ERBb (1:100; described previously(Cho et al., 2012)) antibodies, at 4 °C overnight followed by incubation for 1 h at room temperature with the appropriate secondary antibodies conjugated to horseradish peroxidase. Expression of β-Actin was used as loading control. Detection was performed using the SuperSignal West Femto

Maximum sensitivity Substrate kit (Thermo scientific) following the manufacturer’s protocol.

2.6 Retroviral transduction

HEK 293T cells were seeded at 0.5 million per well of a six well plate the day before transfection. 2mg total plasmid DNA was transfected via Fugene6 reagent (Promega) according to manufacturer’s protocol, which contained 0.8mg of pCL-Eco retroviral packaging plasmid and 1.2mg of expression plasmid. pCL-

Eco was a gift from Inder Verma(Naviaux, Costanzi, Haas, & Verma, 1996). Viral supernatant was harvested 48 and 72 hours post transfection. CD4+ T cells were cultured in Th17 polarizing condition and retroviral transduction was performed

24 and 48 hours post activation by incubating cells with viral supernatant in the presence of polybrene (4mg/ml; Millipore) and centrifuged at 2500 rpm for 90 minutes at 32°C.

2.7 Dual luciferase reporter assay

HEK 293 T cells were seeded at 20,000 cells per well in 96 well plates.

Luciferase assays were performed at 48 hours post transfection using the Dual-

Glo Lucifer's Assay System (Promega) according to the manufacturer’s protocol.

The luciferase reporter pGL4 mIL-17 2kb promoter+CNS-5 was purchased from

Addgene (plasmid #20128)(F. Zhang et al., 2008; 2008; 2008).

2.8 DNA binding assay

HEK293 T cells were transfected with 2 mg of pCDNA3 control vector,

Flag-tagged REV-ERBa, or HA-tagged RORgt using Fugene 6 (Promega) 22 according to manufacturer’s protocol. 48 hours post transfection, cells were harvested for nuclear protein extraction. The nuclear proteins were incubated with streptavidin agarose (Millipore) and biotinylated probes for 2 hours at 4°C, followed by washes. Proteins bound to agarose beads were assayed by Western blot with anti-Flag antibody (M2; Sigma) and anti-HA antibody (HA-7; Sigma).

The sequence of biotinylated Il17a CNS5 RORE oligo probe is 5’-

TCAGTTGCTGACCTTGATTCTA-3’.

2.9 Chromatin Immunoprecipitation

Naive CD4+ T cells were activated and polarized in Th17 condition for

3 days for ChIP experiments as described previously(Cho et al., 2012). All antibodies were used at 4mg/reaction. Mouse IgG control antibody was purchased from Santa Cruz Biotechnology. RORgt ChIP was performed with a combination of antibodies from Biolegend and Santa Cruz Biotechnologies.

REV-ERBa antibody was generated as previously described(Cho et al., 2012).

Primers spanning the regulatory regions of Il17a, Cry1, and Gmpr are described in Supplementary Table 2. Ct value for each sample was normalized to the corresponding input value.

2.10 ChIP-sequencing

ChIP-sequencing libraries were constructed and sequenced as described previously(Cho et al., 2012). Short DNA reads were demultiplexed using Illumina 23

CASAVA v1.8.2. Reads were aligned against the mouse mm9 reference genome using the Bowtie2 aligner with standard parameters that allow up to 2 mismatches in the read. Peak calling, motif analyses, and other data analysis were performed using HOMER, a software suite for ChIP-seq analysis as described previously(Cho et al., 2012). Visualization of ChIP-Seq results was achieved by uploading custom tracks onto the UCSC genome browser.

2.11 RNA-sequencing and data analysis

mRNA was extracted from Th17 cells on day 3 of in vitro differentiation.

RNA-sequencing libraries were prepared from 100ng total RNA (TrueSeq v2,

Illumina) and singled-ended sequencing performed on the Illumina HiSeq 2500, using bar-coded multiplexing and a 50 bp read length, yielding a median of

34.1M reads per sample. Read alignment and junction finding was accomplished using STAR(Dobin et al., 2013) and differential gene expression with Cuffdiff

2(Trapnell et al., 2013), utilizing UCSC mm9 as the reference sequence.

Student’s t test was performed to generate a list of differentially expressed genes

(p<0.05), which was then run through KEGG pathway analysis on DAVID(D. W.

Huang, Sherman, & Lempicki, 2009a; 2009b) to examine enriched functional groups. Heatmaps were generated on Matrix2png(Pavlidis & Noble, 2003).

2.12 EAE models

For active EAE, mice were immunized subcutaneously with 200ng of

MOG35-55 peptide in CFA, and received 200ng of Pertussis toxin intraperitoneally on days 0 and 2. Mice were monitored daily for disease progression using the following scoring criteria: 0, normal; 1, limp tail; 2, weakened hind limbs; 3, complete paralysis of one limb; 4- complete paralysis of both limbs with weakening of the front limbs. At the end point, half of the brain and spinal cord were preserved for histology. Lymphocytes were enriched from the other half of the tissues by Percoll gradient separation and analyzed for cytokine production by flow cytometry after 5 hours of PMA stimulation in the presence of Ionomycin and Golgiplug.

For passive EAE, CD4 T cells from Rosa-M2rtTAxTRE-RVBx2D2 mice were activated with plate-bound Goat-hamster IgG and soluble anti-CD3 and anti-CD28 under Th17 condition. After 3 days of culturing, the cells were re- stimulated overnight in the presence of IL-18 (20ng/ml; Fisher Scientific). In the following day, 2-3 million cells were adoptively transferred into wild type recipient mice. The recipient mice were given normal water or Doxycycline water to induce

REV-ERBa expression in transferred T cells.

2.13 Histology analysis

Mice were euthanized by CO2 asphyxia and perfused with PBS. The brains and spines were dissected and post-fixed in 10% buffered formalin for at 25 least 72 hours before processing. Vertebrall columns with spinal cords were decalcified before sectioning in situ. Multiple transverse sections of the cervical, thoracic, and lumbar spinal cord were prepared routinely and stained with H&E and Luxol Fast Blue. Histological sections were evaluated and scored for inflammation and neuronal degeneration. H&E-stained slides were assessed for inflammation using the following scoring system: (0) = no evidence of inflammation; (1) rare, scattered small foci of cellular inflammation; (2) multiple, isolated foci of cellular infiltration; (3) multiple, confluent foci of inflammation; and

(4) foci of necrosis and/or neutrophilic infiltration. Luxol Fast Blue-stained slides were assessed for neuronal degeneration or axonal swelling using the following scoring system: (0) normal; (1) minimal or few, scattered degenerative neurons;

(2) moderate, multifocal groups of degenerative neurons; (2) marked or large, multifocal degenerative neurons; and (4) severe or coalescing groups of degenerative neurons.

2.14 SR9009 treatment

For cell cultures, SR9009 was dissolved in DMSO and added at final concentrations of 5mM or 10mM. For EAE experiments, SR9009 was dissolved in 15% Cremaphor and 85% water at 10mg/ml. Mice were administered at 100 mg/kg body weight intraperitoneally daily.

Chapters 2, in full (with minor exceptions to conform to this dissertation), was submitted to Nature Medicine by Chang C, Zhao X, Solt L, Liang Y, Bapat S,

Cho H, Kamenacka T, Leblanc M, Atkins A, Yu R, Downes M, Burris T, Evans R, 26

Zheng. ‘The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease’. The dissertation author was the primary investigator and author of this paper.

Chapter 3: Identification of REV-ERBa as a novel regulator in Th17 differentiation

3.1 Results

Beyond their critical roles in Th17 cell differentiation, members of the ROR family are known to be key players in the circadian regulatory machinery(Jetten,

2009; Sato et al., 2004), where their transcriptional activator function is countered by a pair of repressors, REV-ERBa and REV-ERBb(Cho et al., 2012; Ueda et al.,

2002). In an effort to identify novel players in the nuclear hormone receptor superfamily that are involved in T cell function, we conducted expression profiling of NHRs in different T helper cell subsets. We noticed that, similar to RORgt,

REV-ERBa expression was uniquely up-regulated in Th17 cells at both mRNA and protein levels (Figure 3.1). Furthermore, REV-ERBa was more highly expressed in human Th17 cells relative to Th1 cells (Figure 3.2). The unique expression pattern of REV-ERBa suggested that it may play a role in the regulation of Th17 cells. Previous studies on circadian regulation demonstrated that by binding to the same RORE motifs RORs activate transcription of their target genes, whereas REV-ERBs act as repressors of the same targets(Cho et al., 2012; Ueda et al., 2002). We hypothesized that REV-ERBs may serve as a negative regulator of Th17 cell differentiation and function by antagonizing

RORgt.

27 28

To assess the role of REV-ERBs in Th17 cells, we examined the effects of ectopic expression of REV-ERBs on Th1 and Th17 cell differentiation. Retroviral expression of REV-ERBa during Th17 differentiation significantly suppressed IL-

17A production in T cells (Figure 3.3). The inhibitory effect of REV-ERBa is specific to Th17 cells, as it did not suppress IFN-g expression in Th1 cells.

Ectopic expression of REV-ERBb showed a modest negative impact on Th17 cells (Figure 3.3). Th17 differentiation can also be driven by ectopic expression of

RORgt in T cells cultured without Th17 polarizing cytokines(X. Wang et al., 2012;

Yang et al., 2008; F. Zhang et al., 2008). We found that co-expression of REV-

ERBa along with RORgt also led to significant decrease of IL-17A expression

(Figure 3.4), suggesting that REV-ERBa can suppress RORgt -dependent IL-17A expression. [Add RVB truncation experiment] Furthermore, transduction of various truncated forms of REV-ERBa demonstrated that the DNA-binding domain (DBD) is critical for REV-ERB to suppress Th17 differentiation (Figure

3.5). To evaluate the genome-wide effects of REV-ERBs’ ectopic expression in

Th17 cells, we performed RNA-sequencing analysis of Th17 cells retrovirally transduced with REV-ERBa, REV-ERBb or MigR1 control vector. KEGG pathway analysis of the differentially expressed genes indicated that REV-ERBa regulates genes involved in T cell receptor signaling, cytokine/chemokine signaling, as well as circadian rhythm regulation (Table 3.1). REV-ERBa transduced cells differentially expressed a number of Th17 cell signature genes compared with

MigR1 transduced cells, which include Il17a, Il17f, Il23r, Csf2, and Tgfb3.

Interestingly, most of these genes were significantly down-regulated by REV- 29

ERBa (Figure 3.6). REV-ERBb expression also suppressed most Th17 signature genes, but its impact was modest compared to REV-ERBa (Figure 3.6).

Therefore, we decided to focus on the role of REV-ERBa in suppressing Th17 cell differentiation and the expression of Th17 signature genes.

Since REV-ERBs and RORs both recognize ROREs, we hypothesized that REV-ERBa could directly interact with the Il17a locus and repress its transcription. RORE motifs located in CNS5 (also named CNS2), an enhancer

5kb upstream of the Il17a locus, are critical for optimal expression of Il17a(X.

Wang et al., 2012; Yang et al., 2008; F. Zhang et al., 2008). Using a reporter driven by the Il17a promoter and CNS5(F. Zhang et al., 2008), we measured luciferase activity after transfecting RORgt with or without REV-ERBa. Co-transfection of

REV-ERBa inhibited RORgt-dependent Il17a reporter activity in a dose dependent manner (Figure 3.7). To investigate whether REV-ERBa can directly bind to ROREs located at the Il17a locus, we performed an in vitro DNA binding assay. Biotinylated oligonucleotides containing the RORE motif derived from the

Il17a CNS5 enhancer were incubated with nuclear extracts from 293T cells transfected with either REV-ERBa or RORgt expressing plasmids. The

DNA:protein complexes were then precipitated with streptavidin beads, and

Western-blots were performed to detect precipitated REV-ERBa and RORgt. As shown in Figure 3.8, both REV-ERBa and RORgt bind to the RORE motif. To determine if the REV-ERBa:CNS5 interaction occurs in vivo, we performed chromatin immunoprecipitation (ChIP) experiments in Th17 cells with anti-REV-

ERBa and anti- RORgt antibodies. Indeed, both REV-ERBa and RORgt bound 30 to the CNS5 region in Th17 cells (Figure 3.9). These findings suggest that REV-

ERBa can directly repress Il17a expression by binding to the Il17a CNS5 enhancer.

To identify genome-wide REV-ERBa target genes in Th17 cells, we performed REV-ERBa ChIP-seq assays. As expected, the de novo REV-ERBa binding motif is highly similar to the established RORgt binding motif (Figure

3.10a). When compared to previously published RORgt ChIP-seq data, about

30% of the 8884 REV-ERBa binding sites in Th17 cells are also RORgt binding sites (Figure 3.10b)(Ciofani et al., 2012). KEGG pathway analysis of genes bound by both REV-ERBa and RORgt revealed that they are enriched with genes involved in T cell signal and cytokine/chemokine pathways (Figure 3.10c). In addition to Il17a, several other Th17 cell signature genes, including Il17f, Il23r, and Tgfb3, were identified as direct targets of REV-ERBa (Figure 3.10d). The finding that REV-ERBa binds to a large number of RORgt target genes suggests that REV-ERBa inhibits Th17 cell differentiation through direct suppression of the expression of key Th17 cell signature genes. To further test this hypothesis, we examined whether ectopic REV-ERBa expression decreases the binding of

RORgt to Il17a. Indeed, by ChIP-qPCR we saw significant decrease in RORgt enrichment at the Il17a locus in cells that were retrovirally transduced with REV-

ERBa (Figure 3.11a). Conversely, ectopic RORgt expression decreased REV-

ERBa binding at the Il17a locus (Figure 3.11b). Combined, these results show that that both RORgt and REV-ERBa bind to at least one RORE in the endogenous Il17a locus. Relative protein abundance of the two nuclear receptors 31 determine which transcription factor occupies the locus, and as a result, the expression level of IL17A. Thus increased REV-ERBa expression inhibits Th17 cell differentiation through out competing RORgt for Il17a and potentially other

Th17 signature genes.

Chapters 3, in full (with minor exceptions to conform to this dissertation), was submitted to Nature Medicine by Chang C, Zhao X, Solt L, Liang Y, Bapat S,

Cho H, Kamenacka T, Leblanc M, Atkins A, Yu R, Downes M, Burris T, Evans R,

Zheng. ‘The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease’. The dissertation author was the primary investigator and author of this paper.

3.2 Tables and Figures

a Tbet Foxp3 Gata3 b REV-ERBα 1.0 5 0.8 4 Th1 0.8 Th17 4 0.6 3 iTreg 3 0.6 0.4 2

2 0.4 n

o 0.2 1 i 1 0.2 ss e 0 0.0 0 Relative expression r 0.0 Day(s) 7 g g p 0 1 2 0 1 2 7 0 1 7 g re re 2 x Th Th Th Th re 0 1 2 3 4 Th Th1 iT Th Th1 iT Th Th Th iT e Naive Naive Naive Th1

e RORγt v i t RORγt REV-ERBα REV-ERBβ 0.8 a l

e 2.5 1.5 0.6

R 0.6 2.0 1.0 0.4 1.5 0.4 1.0 0.5 0.2 0.2 0.5 0.0 0.0 0.0 0.0 Day(s) 7 g 0 1 2 7 g 0 1 2 7 g 0 1 2 0 1 2 3 4 re re Th Th re Th Th Th T Th Th Th T Th Th1 iT Naive Th1 i Naive Th1 i Naive c Th1 Th17 Days 1 2 3 4 1 2 3 4 REV-ERBα

REV-ERBβ RORγ

Tbet

β-Actin

Figure 3.1 REV-ERBα is up-regulated in Th17 cells. (a) mRNA expression of REV-ERBα, REV-ERBβ, as well as T cell lineage specifying transcription factors T-bet, Gata3, RORγt, and Foxp3, in Th1, Th2, Th17 and iTreg cells differentiated for 3 days in vitro. (b) REV-ERBα and RORγt mRNA expression in Th1, Th17 and iTreg cells over 4 days of in vitro differentiation. (c) Protein expression of REV-ERBα, REV-ERBβ, RORγ and T-bet during in vitro differentiation of Th1 and Th17 cells over 4 days. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

hRORγt hTbet hREV-ERBα 15 60 3 *** P=0.06 10 2 40 Donor 5 20 **** 1 #1

0 0 0 20 60 8 * 15 *** 6 40 ** 10 4 20 Donor Relative expression 5 2 #2 0 0 0 Th1Th17 Th1Th17 Th1Th17

Figure 3.2 REV-ERBα is up-regulated in human Th17 cells. (a) mRNA expression of RORγt, T-bet, and REV-ERBα in human CD4+ T cells activated under Th1 and Th17 polarizing conditions for 6 days. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

IL-17A MIGR1 REV-ERBα REV-ERBβ 100 80 + γ 0 0 5.05e-3 60 N

Th1 F 40 I 20 99.8 99.7 99.8 % 0

*** 41.3 10.5 26.7 50 **** 40 A+ Th17 30 20 L-17

I 10

0.26 % 1.71 0.213 0 IFNγ α β B B R R Control-E -E EV EV R R

Figure 3.3 Ectopic REV-ERBα expression inhibits Th17 differentiation. FACS analysis of IL-17A and IFN-g expression in mouse CD4+ T cells activated under Th1 and Th17 polarizing conditions and transduced with MigR1, REV- ERBα or REV-ERBβ retroviral vectors. Data are representative of three independent experiments with triplicate wells for each condition. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

MigR1 RORγ 0.098 15.7

0.929 0.364 IL-17A

RORγ+REV-ERBα RORγ+REV-ERBβ 4.96 10.1

0.792 0.285

IFNγ

20 *

15 A+

10 L-17

% I 5

1 t β Rγ VB MigR O R R t+ Rγ O RORγt+RVBαR

Figure 3.4 REV-ERBs inhibit RORγt-dependent IL-17A expression. CD4+ T cells were cultured in the presence of IL-2, α-IL-12 and α-IFN-γ, and transduced with a control vector, RORγt, or RORγt with either REV-ERBα or REV-ERBβ at a 1:1 ratio. Cytokine production was examined on day 4 by flow cytometry. FACS plots shown are representative of 3 independent experiments. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05). 36

a 132 198 451 614 RVBα-FL 103 225 N terminus RVBα-DBD DBD 220 451 614 Hinge domain RVBα- N220 LBD 132 198 288 RVBα- C288 103 198 451 614 RVBα- N103

b MigR1 Revα-FL RVBα-DBD 27.6 13.1 8.36

0.475 3.92 1.77 A 7

1 Th17 - RVBα- N103 RVBα- C288 RVBα- N220 L I 1.07 9.75 21.2

16.4 0.693 0.604 IFNγ

Th17 30

% 20

L17 10 I

0 1 L D 3 8 0 -F α -DB -N10 -C28-N22 MigR α α α α RVB RVBRVB RVBRVB

Figure 3.5 Analysis of different REV-ERBα domains that are involved in repression of IL-17A expression. (a) Schematics of truncated versions of REV- ERBα. DBD: DNA binding domain. LBD: ligand binding domain. (b) CD4+ T cells were activated under Th17 cell polarizing condition and transduced with a control vector, full length REV-ERBα or truncated versions of REV-ERBα. Frequency of IL-17A and IFN-γ were examined by flow cytometry. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 37

1 2

1 1

# #

α α

B B

R R

E E

- -

V V

E E

MigR1 Control #1 Control MigR1

R R

MigR1 Control #2 Control MigR1

MigR1 Control #1 Control MigR1

MigR1 Control #1 Control MigR1 MigR1 Control #2 Control MigR1 MigR1 Control #2 Control MigR1

Il-17a Pim1 Vav3 Il-17f Spry1 Ccl3 Il-23r Stat2 Ncf1 Tgfb3 Stat1 Ccr5 Csf2 Il12rb Chemokine signaling Cc14 Ccl20 Il7r Cysltr1 Stat5a Cd40 Ltb4r1 Stat3 Inhba Furin Irf9 Il9 Fam124b Jak2 Fas Vax2 Akt2 Il1r2

Th17 differentiation and function Pik3ca Kit

Cry1 Jak-Stat pathway Akt3 Tnfsf11 Arnt1 Myc Il15ra Cd27 Npas2 Il2ra Tnfsf8 Cytokine-cytokine receptor interaction rhythm Bhlhe40 Socs2 Circadian Il2rb Il12rb2 Pik3cg Stat4 log fold change 2

Figure 3.6 REV-ERBα over expression inhibits the expression of Th17 signature genes. (a) Heat map of functional groups of differentially expressed genes in Th17 cells transduced with MigR1, REV-ERBα, or REV-ERBβ retroviral vectors. Relative fold change was normalized to the average of each row in the matrix. (b) KEGG pathway analysis of genes differentially expressed in REV- ERBα and MigR1 retrovirally transduced Th17 cells.

ORE ORE IL17A R R

CNS-5(CNS-2) 2kb promoter

b Il17a promoter+CNS5 3 ***

2 U F R

0 l t ) ) ) ) o γ 2 1 1 :1 tr : : : 0 n R (1 (1 (5 2 O ( Co R α α α α B VB VB VB V R R R R t+ t+ t+ t+ γ γ γ γ R R R R O O O O R R R R

Figure 3.7 REV-ERBα directly inhibits IL17a expression. (a) Schematic of RORE sites at the Il17a locus. (b) Luciferase assay of 293T cells co-transfected with an Il17a luciferase reporter, and combinations of RORγt and REV-ERBα at various ratios, with the amount of RORγt transfected remaining constant. Renilla luciferase activity was used as internal control. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Il17a RORE 10% input

-Flag t-HA -Flag t-HA α γ α γ

RVB ROR Control RVB ROR Control

Anti-HA

Anti-Flag

Figure 3.8 REV-ERBα binds to a RORE within the il17a locus. Binding of HA- tagged RORγt and Flag-tagged REV-ERBα to biotinylated DNA oligos containing RORE sequence in the Il17a CNS5 enhancer detected by Western-blot in CD4 T cells.

Il17a Cry1 Gmpr ** * ***

t **

u 1.0 1.0 1.0 p in

f 0.5 0.5 0.5 o

0.0 0.0 0.0 α t α α t G B γ G B γt G B γ Ig R R Ig R R Ig R R -E O -E O -E O V R V R V R E E E R R R

Figure 3.9 REV-ERBα binds to the endogenous Il17a locus. ChIP-qPCR to detect the binding of Il17a CNS5 enhancer, Cry1 (positive control) and Gmpr (negative control) by REV-ERBα and RORγt in Th17 cells. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 3.10 REV-ERBα binds to the endogenous Il17a locus. Peak analysis of Th17 cell REV-ERBα ChIP-seq data analyzed along with published RORγt ChIP-seq data. (a) Alignment of de novo generated REV-ERBα binding sequence to annotated RORγt binding sequence. (b) Trace analysis of ChIP-seq data visualized on the UCSC genome browser showing overlapping binding sites of REV-ERBα and RORγt at Il17a, Il17f, Il23r, Tgfb3, and Cry1 loci. (c) Venn diagram depicting the numbers of unique and shared genes bound by REV- ERBα and RORγt. (d) KEGG pathway analysis of REV-ERBα bounds genes. (g) (h) and (i) ChIP-qPCR to detect changes in REV-ERBα and RORγt binding to the Il17a locus in response to ectopic (h) REV-ERBα or (i) RORγt expression. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

a REV-ERBα RORγt 1.5 0.08 Control

t REV-ERBα 0.06 1.0

inpu 0.04 f o 0.5

% 0.02

0.0 0.00

Il17a Gmpr Il17a Gmpr b REV-ERBα RORγt 0.5 1.5

t 0.4 Control 1.0 RORγt 0.3 inpu f o

0.2 0.5 % 0.1

0.0 0.0

Il17a Gmpr Il17a Gmpr

Figure 3.11 REV-ERBα and RORγt compete for binding to the Il17a locus. (a) and (b) ChIP-qPCR to detect changes in REV-ERBα and RORγt binding to the Il17a locus in response to ectopic (a) REV-ERBα or (b) RORγt expression. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Table 3.1 KEGG pathway analysis. KEGG pathway analysis of genes differentially expressed in REV-ERBα and MigR1 retrovirally transduced Th17 cells.

KEGG Pathway Term Count P value T cell receptor signaling pathway 27 7.38E-07 Jak-STAT signaling pathway 28 3.29E-05

Chemokine signaling pathway 26 0.00334 Circadian rhythm 5 0.01452 Cytokine-cytokine receptor 27 0.05854 interaction

Chapter 4: The functional role of REV-ERBa in Th17 cell mediated autoimmune response

4.1 Results

Since high levels of REV-ERBa expression are inhibitory to Th17 cell differentiation in vitro, we explored whether constitutive REV-ERBa expression in

T cells could ameliorate Th17 cell mediated autoimmune diseases in vivo. We tested this hypothesis in EAE, a mouse model for multiple sclerosis, as Th17 cells play a critical role in EAE disease development. A tetracycline-inducible

REV-ERBa transgenic mouse (TRE- REV-ERBa/Rosa-M2rtTA)(Kornmann,

Schaad, Bujard, Takahashi, & Schibler, 2007) was crossed with a 2D2 TCR transgenic mouse, which carries a TCR that recognizes the MOG peptide (myelin oligodendrocyte glycoprotein)(Bettelli et al., 2003), to generate a triple transgenic mouse strain (TTg). T cells from the TTg mice express REV-ERBa constitutively under doxycycline treatment in vivo (Figure 4.1). CD4+ T cells from the TTg mice were activated in vitro under Th17 conditions for 4 days before being adoptively transferred into WT recipient mice to induce EAE. Mice were given doxycycline water to induce REV-ERBa expression in transferred 2D2 T cells, or normal water. The doxycycline treated group showed delayed EAE disease onset as well as slower disease progression, compared to mice that were given normal water

(Figure 4.2a). Elevated REV-ERBa expression did not affect homing or survival of the transferred CD4+ T cells (Figure 4.3). However, consistent with milder

44 45 disease progression observed in mice treated with doxycycline, the frequency of

IL-17A producing 2D2 T cells were significantly reduced in the spinal cord of these mice compared to controls (Figure 4.2b). Histopathology analysis showed that doxycycline treatment significantly reduced inflammation levels with a decreasing trend for demyelination in the spinal cord of these mice (Figure 4.2c).

These differences were dependent on REV-ERBa induction because the same doxycycline treatment of mice transferred with WT 2D2 T cells did not delay or ameliorate EAE disease progression (Figure 4.1b). Thus, increased expression of REV-ERBa in T cells can attenuate Th17 cell mediated EAE.

Structural studies have shown that REV-ERBs contain a ligand-binding domain, and their activity can be modulated by ligands(Woo et al., 2007). Heme, the prosthetic group in hemoglobin, was identified as an endogenous agonist that binds to REV-ERBs and potentiates their activity(Raghuram et al., 2007; Yin et al.,

2007). Efforts have also been made to generate synthetic REV-ERB agonists with higher specificity and fewer side effects. Two chemical compounds, SR9009 and

SR9011, bind specifically to REV-ERBs and modulate their activity, and exhibited favorable pharmacokinetic properties when tested in mice(Solt et al., 2012; Woldt et al., 2013). Since increased REV-ERBa expression suppresses Th17 cell differentiation and function, we tested if potentiating REV-ERB activity via agonist treatment could have a similar effect on Th17 cells. First, we cultured naïve mouse CD4 T cells under Th1, Th17, or iTreg differentiation conditions with or without SR9009. SR9009 treatment significantly inhibited Th17 cell differentiation in a dose dependent manner, but did not affect Th1 or iTreg 46 differentiation (Figure 4.4). Similarly, Th17 differentiation of human CD4+ T cells was significantly inhibited by SR9009 treatment (Figure 4.5). By ChIP-qPCR we saw that NCoR recruitment to the Il17a locus was increased in the presence of

SR9009, demonstrating that SR9009 can target the IL17A pathway directly via

REV-ERBs (Figure 4.6).

To test if the inhibitory effect of SR9009 on Th17 differentiation translates to in vivo mouse models, we immunized C57/BL6 mice with MOG peptide to induce EAE, followed by daily injection of SR9009 or vehicle control for 20 days.

Mice treated with SR9009 showed significantly delayed onset and slower progression of EAE compared to vehicle treated control group (Figure 4.7a), which corresponded to less IL17A production in the CNS (Figure 4.7b). To explore the potential of REV-ERB agonist in a more disease relevant setting, we explored the efficacy of REV-ERB agonist treatment in mice that have already developed EAE. We induced EAE in SJL mice, which upon PLP (myelin proteolipid protein) peptide immunization, exhibit disease progression in remitting and relapsing patterns mimicking the development of multiple sclerosis in humans. SR9009 treatment of SJL mice during the primary phase of EAE showed inhibitory effects similar to its effects in C57/BL6 mice (Figure 4.8). When

SR9009 was administered after the primary phase of EAE, SR9009-treated mice maintained their remitting state, whereas vehicle-treated control mice developed additional episodes of EAE symptoms (Figure 4.9a). Although IL17A production in the CNS at the experimental endpoint was not significantly lower in the

SR9009-treated mice, which may be due to smaller differences in disease scores 47 during subsequent relapses following the initial disease development, compared to those observed in experiments where treatment begins in the initial phase of

EAE. Nevertheless, these results demonstrate that modulating in vivo REV-ERB activity by its agonist SR9009 effectively suppresses development and progression of Th17 cell-mediated EAE. A previous study demonstrated that

REV-ERBa could suppress macrophage expression of IL-6, a key cytokine for

Th17 cell differentiation(Gibbs et al., 2012). Therefore, it is likely that SR9009 protects mice from EAE by targeting macrophages in addition to directly targeting

Th17 cells.

Chapters 4, in full (with minor exceptions to conform to this dissertation), was submitted to Nature Medicine by Chang C, Zhao X, Solt L, Liang Y, Bapat S,

Cho H, Kamenacka T, Leblanc M, Atkins A, Yu R, Downes M, Burris T, Evans R,

Zheng. ‘The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease’. The dissertation author was the primary investigator and author of this paper.

4.2 Tables and Figures

a b 4 Control Dox e

n 3 r

o 5 o i * sc ss

4 l e a r 2 c p 3 i n Ex

2 Cli e

v 1 i t 1 a l e 0 R Control Dox 0 0 5 12 15 20 24

Day(s)

Figure 4.1 Doxycycline induces transgenic REV-ERBα expression in vivo, but does not affect EAE disease progression. (a) Relative expression of doxycycline inducible transgenic REV- ERBα of CD4+ T cells sorted from the brain and spinal cord of mice treated with or without doxycycline. (b) EAE disease clinical scores of mice that received adoptive transfer of in vitro differentiated wild-type 2D2 Th17 cells, then treated with or without doxycycline (n=8 per group). Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05).

4 ** Control a 3.4 0.95 b 3 4 A+ Control 2 L-17 Dox % I 1 3 6.22 0 89.4 e r o 10 * sc

Dox l 2 0.294 a 8 c 1.02 i n γ+ ****

6 IL-17A N Cli 1 4 % IF

2 3.78 0 0 94.9 0 5 10 15 20 Control Dox IFNγ Day(s)

c H&E Luxol Fast Blue d

Inflammation Demyelination

4 * 4

Control 3 3 2 2

1 1 0 0

Control Dox Control Dox Dox

Bar = 200 um

Figure 4.2 In vivo induction of REV-ERBα expression in Th17 cells suppresses EAE disease progression. EAE was induced in C57/BL6 mice by adoptive transfer of in vitro differentiated Rosa-M2rtTAxTRE-REV-ERBax2D2 transgenic Th17 cells. Recipient mice were treated with or without Doxycycline water (n=7 per group) starting 2 days before Th17 cell adoptive transfer, and were monitored for EAE disease progression. Mice were analyzed on day 24 post transfer. (a) Clinical scores of mice induced with EAE. (b) FACS analysis of IL-17A and IFN-γ production of transferred 2D2 CD4+ T cells infiltrating in the CNS tissues. (c) Representative H&E and Luxol Fast Blue staining of the spinal cords to show the sites of immune cell infiltration (closed arrow) and demyelination (open arrow) (scale bar, 200 µm). (d) Pathological scores for the severity of inflammation and demyelination of the spinal cords. Data represents mean ± s.e.m. Statistical analyses were performed using two-way analysis of variance (ANOVA) for EAE clinical score analysis and two-tailed unpaired Student’s t-test for other analysis, comparing the indicated groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

a Spleen CNS 25 20 Control 20 Dox 15 15 % 4 10 10 CD 5 5

0 0 Host CD4 Transferred CD4 Host CD4 Transferred CD4 b

15k 20k

r 15k

be 10k m 10k nu

e 5k

C 5k

0 0 Host CD4 Transferred CD4 Host CD4 Transferred CD4

Figure 4.3. Doxycycline treatment does not affect homing of transferred CD4+ T cells. CD4+ T cell (a) percentage and (b) cell number of host and transferred 2D2 cells in the spleen and CNS at disease endpoint. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05).

a DMSO SR9009 5uM SR9009 10uM

0 0 0 Th1 98.7 97.9 95.9

39.2 26.1 14.7

IL-17A

Th17

0.0137 0 2.8e-3 IFNγ

IL-17A

iTreg 88.6 88.7 86.2

Foxp3

b Th1 Th17 iTreg 150 50 **** 100 ** + 40 + 80 A + 100 p3 g

30 x 60 N o L1-7 20 50 40 % F % I % IF 10 20

0 0 0 M M M M M M 5u 10u 5u 5u DMSO 10u 10u DMSO DMSO R9009R9009 R9009 S S R9009 R9009R9009 S S S S

Figure 4.4. REV-ERB agonist SR9009 inhibits in vitro Th17 differentiation. (a) Mouse CD4+ T cells were activated under Th1, Th17 and iTreg polarizing conditions and treated with DMSO or SR9009. IFN-γ, IL-17A and Foxp3 expression in Th1, Th17 and iTreg cells, respectively were analyzed by flow cytometry. (representative data, n=3) and quantified (b). Data represents mean ± s.e.m. Statistical analyses were performed using two-tailed unpaired Student’s t- test, comparing the indicated groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 52

hTh1 hTh17

20 20 **

15 + * 15 A + γ

N 10 10 L-17 % IF 5 % I 5

0 0

M M 5uM 5uM DMSO 10u DMSO 10u

Figure 4.5. REV-ERB agonist SR9009 inhibits in vitro human Th17 differentiation. IFN-γ and IL-17A production in human Th1 and Th17 polarized cells treated with either DMSO or SR9009. Data represents mean ± s.e.m. Statistical analyses were performed using two-tailed unpaired Student’s t-test, comparing the indicated groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

NCoR REV-ERBα 0.025 1.5 DMSO 0.020 t 1.0 SR9009 0.015 inpu f o 0.010 % 0.5 0.005

0.000 0.0

Il17a Gmpr Il17a Gmpr

Figure 4.6. REV-ERB agonist SR9009 enhances NCoR recruitment to the Il17a locus. ChIP-qPCR assay to detect enrichment of NCoR at the Il17a locus in response to SR9009. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

a b CNS

2.5 Vehicle 25 SR9009 20 2.0 7A+ 15 ** e L1 10 r o %I 1.5 **** 5 sc

l 0 a c i n 1.0 50 Cli

+ 40 g

0.5 N 30 F 20 %I 10 0.0 0 0 5 10 15 e 9 Day(s) Vehicl SR900

Figure 4.7. Treatment with REV-ERB agonist SR9009 suppresses EAE progression. (a) EAE disease progression of C57/BL6 mice that were immunized with MOG/CFA and treated with vehicle control or SR9009 (n=5 per group) via daily intra-peritoneal injection for 21 days. (b) IL-17A and IFN-γ production in the CNS at disease endpoint. Data represents mean ± s.e.m. Statistical analyses were performed using two-way analysis of variance (ANOVA) for EAE clinical score analysis and two-tailed unpaired Student’s t-test for other analysis, comparing the indicated groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

a b CNS 20 4 Vehicle 15

SR9009 7A+ 10 3 L1 e %I 5 r o

sc 0

l a

c 2

i 20 n Cli

+ 15 **** g

1 N 10 F

%I 5 0 0 0 5 0 5 0 5 0 5 0 e 1 1 2 2 3 3 4 cl ehi V Day(s) SR9009

Figure 4.8. Treatment with REV-ERB agonist SR9009 prevents relapse of EAE. (a) EAE disease progression of SJL mice immunized with PLP/CFA and treated with vehicle control (n=7) or SR9009 (n=8) daily starting after the primary phase of EAE as indicated by the arrow. (b) IL-17A and IFN-g production in the CNS at disease endpoint. Data represents mean ± s.e.m. Statistical analyses were performed using two-way analysis of variance (ANOVA) for EAE clinical score analysis and two-tailed unpaired Student’s t-test for other analysis, comparing the indicated groups (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Chapter 5: In vitro and in vivo characterizations of REV-ERBa deficiency

5.1 Results

Next we turned to REV-ERBa deficient models to further elucidate the role of REV-ERBa in Th17 cells. Having observed the suppressive role of REV-ERBa on Th17 cells in our gain of function studies, we expected to see enhanced Th17 differentiation in conditions of REV-ERBa deficiency. We began by assessing the effects of shRNA knockdown of REV-ERBa in Th17 cell differentiation. Two shRNAs with efficient REV-ERBa knockdown activity were used in these experiments (Figure 5.1). To our surprise, the frequency of IL-17A producing cells showed a moderate reduction in T cells transduced with a retroviral vector carrying REV-ERBa shRNA compared with T cells that were transduced with a control retroviral vector (Figure 5.1). Puzzled by this result, we utilized two REV-

ERBa deficient transgenic lines to further characterize and validate the loss of function phenotype. The first is a CD4Cre REV-ERBa/b flfl mouse, which is a

REV-ERBa DBD deficient knock-in, REV-ERBb knock out transgenic mouse

(Figure 5.2). Since we previous confirmed that the DBD is critical for REV-ERBa to inhibit Th17 differentiation (Figure 3.5), we considered these functional REV-

ERB knock-out mice. In in vitro Th17 differentiation assays we observed a moderate but consistent decrease in IL-17A production in CD4+ cells from the floxed mice (Figure 5.3). When we induced EAE in these mice along with CD4

56 57

Cre control mice, the floxed mice exhibited milder disease progression corresponding with lower levels of IL-17A production in the CNS (Figure 5.4). To eliminate the potential role that non-DBD domains of REV-ERBa may play in the observed phenotype, we generated BM chimera from REV-ERBa whole protein knock out mice, isolated CD4+ T cells from these mice and transferred them into

Rag KO mice. We then induced EAE in the recipient mice and again saw that the

REV-ERBa KO cohort had milder disease progression and less IL-17A production (Figure 5.5). Together these findings clearly show that REV-ERBa deficiency has a negative impact on Th17 differentiation and function, suggesting that a basal level of REV-ERB is required for an optimal Th17 response.

Although the results contradicted our initial prediction, they were consistent with recent findings by Hooper’s group. Hooper and colleagues showed that REV-ERBa deficient T cells were also defective in Th17 differentiation(Yu et al., 2013). It was proposed that in the absence of REV-ERBa, expression of NFIL3 increases, which in turn suppresses Th17 cell development by directly binding to the RORgt promoter and repressing its expression. In our studies, we found potential REV-ERBa binding sites in trace analysis of our REV-

ERBa ChIP-seq in Th17 cells. We also saw increased Nfil3 mRNA expression in

Th17 cells retrovirally transfected with REV-ERBa shRNAs, although the difference is modest and not always consistent across repeated experiments

(Figure 5.6).

These results suggest that REV-ERBa expression needs to be tightly controlled for robust Th17 cell differentiation. Insufficient REV-ERBa activity 58 leads to decreased Th17 cell differentiation potentially due to increased levels of

NFIL3 which suppresses RORgt expression; whereas at high levels REV-ERBa out-competes RORgt for regulatory binding sites in Th17 signature genes such as Il17a and Il17f, also resulting in the suppression of Th17 cell differentiation. It is worth noting that over-expression of REV-ERBa exerts a much stronger inhibitory effect on Th17 cells than decreased expression of REV-ERBa (Figure

3.3; Figure 5.3).

Chapters 5, in full (with minor exceptions to conform to this dissertation), was submitted to Nature Medicine by Chang C, Zhao X, Solt L, Liang Y, Bapat S,

Cho H, Kamenacka T, Leblanc M, Atkins A, Yu R, Downes M, Burris T, Evans R,

Zheng. ‘The nuclear receptor REV-ERBa modulates Th17 cell-mediated autoimmune disease’. The dissertation author was the primary investigator and author of this paper.

5.2 Tables and Figures

Control shRNA #1 shRNA #2 REV-ERBα

β-Actin

b REV-ERBα REV-ERBα c Control shRNA #1 shRNA #2 ** 30 * 25.8 19 20.7 IL-17A

1.96 1.93 2.35 %IL-17A+ 0 2 rol # IFNγ t A Con shRNAshRN #1

Figure 5.1 Knock down of REV-ERBα suppresses in vitro Th17 cell differentiation. (a) Confirmation of REV-ERBα shRNA knock down efficiency in Th17 cells by Western-blot. (b) CD4+ T cells were activated under Th17 polarizing condition and transduced with retroviruses carrying control shRNA, or one of the two REV-ERBα shRNAs. After 4 days of culturing, IL-17A and IFN- γ production was analyzed by flow cytometry. FACS plots shown represent 4 independent experiments. (c) Quantification. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01).

a b

DBD LBD WT

RVBa-full length ~70 RVBa fl/fl ~60 RVBa ﬂ/ﬂ Th17

Figure 5.2 CD4Cre REV-ERBa/b flfl mice have a deletion of the DBD in REV- ERBa transcript. (a) Schematic of full length and truncated REV-ERBas. (b) Detection of truncated REV-ERBa in Th17 cells from CD4Cre REV-ERBa/b flfl mice by western blot.

anti-CD3 concentration (ug/ml) 0.1ug

CD4 Cre 30 0.3ug e CD4 Cre RVB a/b flfl r

C 10.8 21.1

* IL-17A A

20 CD

L17 0.122 I * 0.0912 %

l f

10 l e f 6.83 13.7 r b / C

0 VB CD 3 1 3 1 R 1 0. 0. .0 .0 0.195 0.122 0 0 anti-CD3 concentration (ug/ml) IFNγ

Figure 5.3 REV-ERBα deficiency suppresses Th17 cell differentiation. CD4+ T cells from CD4Cre and CD4Cre REV-ERBa/b flfl mice were activated under Th17 polarizing condition with titrated concentration of anti-CD3. After 4 days of culturing, IL-17A and IFN-γ production was analyzed by flow cytometry. FACS plots shown (right panel) represent 3 independent experiments. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01).

a CD4 Cre Control b 4 CD4 Cre RVB a/b flfl 40 40 e r

o 30

3 γ A * N sc

e 20

L17 20 s

2 % IF % I ea 10 s 10 Di 1 0 0 l lfl lf f f /b /b a a 0 CD4 Cre CD4 Cre 0 2 4 6 8 9 1 2 3 4 5 6 7 8 9 0 1 2 1 1 1 1 1 1 1 1 1 2 2 2 RVB RVB Day(s) Cre Cre CD4 CD4

Figure 5.4 REV-ERBα deficiency ameliorates EAE disease progression. (a) EAE disease progression of CD4Cre control and CD4Cre REV-ERBa/b flfl mice that were immunized with MOG/CFA. (b) IL-17A and IFN-γ production in the CNS at disease endpoint. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01).

a b WT BM Chimera

4 RVBa KO BM Chimera 20 P = 0.11 60 e

r 3

15 γ A o 40 sc

IFN

L17 10 e I % s 2 20 %

ea 5 s

Di 1 0 0 a a a a

0 Chimer Chimer 0 5 7 9 1 3 5 7 9 1 3 5 BM 1 1 1 1 1 2 2 2 BM WT Day(s) WT RVBa KO BM Chimer RVBa KO BM Chimer

Figure 5.5 REV-ERBα deficiency ameliorates EAE disease progression. (a) EAE disease progression of WT BM chimera and REV-ERBα KO BM chimera mice that were immunized with MOG/CFA. (b) IL-17A and IFN-g production in the CNS at disease endpoint. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01).

a REV-ERBα

10kb

N l3

Nfil3

0.0020 **** ** 0.0015 U

F 0.0010 R

0.0005

0.0000 l ) ) tro Con

shRNA #1(new shRNA #2(sigma

Figure 5.6 REV-ERBα represses Nfil3 expression. (a) Trace analysis of ChIP- seq data visualized on the UCSC genome browser showing potential binding sites of REV-ERBα at the Nfil3 locus. (b) CD4+ T cells were activated under Th17 polarizing condition and transduced with retroviruses carrying control shRNA, or one of the two REV-ERBα shRNAs. After 4 days of culturing, transduced cells were sorted and lysed. mRNA expression of Nfil3 was examined by qPCR. Data represents mean ± s.e.m. Statistical analyses were performed using unpaired two-tailed Student's t test (*p<0.05, **p<0.01).

Chapter 6: Discussion

6.1 Summary of findings

REV-ERB and ROR’s antagonistic relationship is well documented in the context of circadian rhythm regulation and metabolic pathways. Both nuclear receptors recognize the same ROREs and regulate many of the same target genes. Our work revealed that the same relationship is present in Th17 cells as well. It is worth noting that although REV-ERBa and REV-ERBb share similar expression patterns and overlapping functions in other tissues, their roles seem to diverge in CD4+ T cells. REV-ERBa is exclusively expressed in Th17 cells whereas REV-ERBb is expressed in most Th subsets. Retroviral transduction of

REV-ERBa substantially reduced Th17 differentiation but Th17 cells expressing

REV-ERBb saw a more modest reduction. These data suggest non-overlapping roles and functions of REV-ERBa and REV-ERBb in CD4+ T cells.

Ectopic REV-ERBa expression substantially reduced IL-17A production and suppressed Th17 pathogenic signature genes. Our findings provide molecular mechanisms and epistasis evidence to support a role for REV-ERBa in the regulation of Th17 differentiation. We showed that REV-ERBa inhibits Th17 cells by binding to RORE motifs located in enhancer elements of Th17-related genes. Both REV-ERBa and RORgt can bind to the endogenous Il17a locus and elevating expression level of either nuclear receptor allows for it to out-compete the other for occupation of the CNS2 enhancer binding site.

65 66

In vivo studies support a role for REV-ERBa-mediated repression of Rorgt function in different models of EAE. In addition, we demonstrated the therapeutic potential of targeting REV-ERBa in Th17-mediated autoimmune diseases using a synthetic agonist, which was replicated in human Th17 cells. On the other hand, we characterized the REV-ERBa deficient phenotype in Th17 cell using several

REV-ERBa knockout models. We found that loss of REV-ERBa resulted in suboptimal Th17 response, suggesting a dual role for REV-ERBa in the regulation of Th17 differentiation and function. Overall, these studies define a new molecular pathway involved in the regulation of Th17 responses.

However, there are two aspects of the study that need to be addressed in more detail. The first is the specificity of the REV-ERBa agonist SR9009. And the second is the discrepancy between gain and loss of function phenotypes at first glance.

6.2 SR9009 specificity and toxicity

REV-ERBa is expressed in a broad range of tissues and cell types, such as the brain, muscle, liver, and heart. Other immune cells like macrophages also express high levels of REV-ERBa. Since the REV-ERB agonist SR9009 is delivered intraperitoneally, many REV-ERBa expressing cells could be affected.

It is important to examine whether the reduction in EAE severity we observed in drug treated mice was the result of perturbations of the Th17 pathways through

REV-ERBa, or due to drug toxicity and off-target effects. 67

SR9009 was used in two recent studies at the dose we used in our experiments (Solt et al., 2012; Woldt et al., 2013). In all cases there were no reports of toxicity at this dose. Furthermore we did not observe abnormal physiological or pathological symptoms in the mice that received the drug treatment. Weight tracking spanning the duration of our EAE experiments showed no aberrant weight loss in the mice treated with SR9009 compared to mice that received vehicle injection (Figure 6.1).

To address the question of specificity of SR9009, we transferred total

CD4+ T cells from REV-ERBa KO and REV-ERBa WT bone marrow chimeras into Rag KO mice, followed by MOG immunization to induce EAE. We then compared the effects of SR9009 treatment versus vehicle injection on these mice. We saw mild reduction of EAE in mice that received the agonist in the KO cohort. However, the results may have been confounded by REV-ERBa- expressing-cells in other tissues that were exposed to SR9009. For example, it’s known that REV-ERBa regulates IL-6 production in macrophages and IL-6 is a critical Th17 polarizing cytokine. Alternatively we could induce passive EAE in

REV-ERBa null mice by transferring Th17 cells derived from REV-ERBa WT and

KO mice, which would eliminate effects of SR9009 on all non-Th17 cells if the agonist were free of off-target effects. But REV-ERBa null mice often suffer from developmental defects that may also confound the outcome of the experiment.

In the end, we cannot to rule out the possibility that SR9009 exerts low grade toxicity or off-target effects that contributes to the amelioration of EAE disease state in mice. However, we were able to verify that SR9009 does indeed 68 modulate REV-ERBa activity in Th17 cells. ChIP-qPCR experiments in Th17 cells showed enrichment of NCoR recruitment to the Il17a locus in the presence of SR9009 (Figure 4.6). Together with the fact that the effects of SR9009 treatment on EAE mice were consistent with that of doxycycline induction of

REV-ERBa in mice, it seems reasonable to argue that SR9009 can indeed work through REV-ERBa to modulate Th17 mediated immune responses in some capacity.

6.3 Bridging the gain and loss of function phenotypes

Our gain of function studies indicated a repressive role for REV-ERBa in

Th17 cells. Therefore initially we expected REV-ERBa deficiency to have increased Th17 differentiation. Instead, we observed the opposite. We investigated the REV-ERBa loss of function phenotype thoroughly using two

REV-ERBa knock out mice: REV-ERBa KO bone marrow chimera and CD4 Cre

REV-ERBa/b fl/fl mice. In the former model, REV-ERBa is deleted completely in all blood cell lineages; in the latter, the DNA binding domain is deleted from REV-

ERBa. We conducted in vitro Th differentiations to observe differences in Th17 differentiation, as well as animal studies to look at the effect of REV-ERBa deficiency in the disease context. The loss of REV-ERBa modestly dampened

Th17 differentiation in vitro and significantly decreased EAE disease severity.

These results were consistent with findings from Yu et al. (Yu et al., 2013). In their study, Th17 differentiation was reduced in CD4+ T cells from REV-ERBa null mice, and IL-17A production in the small intestine of the same mice was 69 decreased. Yu and colleagues proposed that the transcription factor NFIL3, which is a known REV-ERBa target, suppresses Th17 cell development by directly binding and repressing the Rorgt promoter. Therefore REV-ERBa deficiency leads to elevated levels of NFIL3 and repression of Th17 cell differentiation. The mechanism proposed by Yu et al. presents REV-ERBa with dual roles and could explain why both REV-ERBa deficiency and REV-ERBa overexpression resulted in suppressed Th17 differentiation. In the loss of function scenario, NFIL3 is up-regulated, resulting in the suppression of RORgt expression and downstream IL-17A production; in the gain of function scenario, high levels of REV-ERBa compete directly with RORgt at the Il17a locus, overstepping the Nfil3 pathway and drastically inhibiting IL17A expression

(Figure 6.2). In our efforts to test this working model, we saw a moderate increase in NFIL3 expression in Th17 cells expressing shRNA against REV-

ERBa (Figure 5.6), although the differences were small and sometimes inconsistent between individual experiments. Our REV-ERBa ChIP-seq data indicated REV-ERBa enrichment at the Nfil3 locus, but more data is needed to establish functional relevance. In addition, we did not see clear evidence of perturbed RORgt expression in REV-ERBa deficient Th17 cells. Therefore further experimentations are needed to test the validity of this model.

Both in vitro and EAE experiments demonstrated impaired Th17 response in REV-ERBa deficient contexts. But the effects in vitro were quite modest compared to the phenotype observed in REV-ERBa knockout mice (Figure 5.3;

Figure 5.4; Figure 5.5). The fact that we saw only small reduction in cytokine 70 expression in cell-based assays but significant differences in disease outcome suggest that REV-ERBa may be regulating other cellular processes that affect

Th17 cell function, such as metabolic pathways that regulate survival, proliferation or migration (Figure 6.3). It is worth noting that direct repression of

IL-17A by REV-ERBa overexpression seems to bypass any other pathway that

REV-ERBa maybe be involved in, as the gain of function effect is much more dramatic than REV-ERBa deficient phenotypes.

Initial genome-wide expression profiling of Th17 cells from CD4 Cre REV-

ERBa/b fl/fl mice and their wild-type counterparts by RNA-seq did not yield obvious candidates. However, REV-ERBa may regulate a host of genes via an alternative mechanism that is independent of its DBD, which would not have been captured in our analysis using a REV-ERBa DBD-deficient knock-in mouse.

This mechanism was proposed in a recent study by Zhang and colleagues (Y.

Zhang et al., 2015), where they demonstrated that REV-ERBa regulates the molecular clock and metabolic pathways through two different mechanisms. The former is through interacting with DNA, and the latter through other tissue- specific transcription factors that regulate metabolic gene expression (Y. Zhang et al., 2015). In our gain of function studies, REV-ERBa’s inhibitory impact on

Th17 differentiation is largely dependent on its DNA-binding-domain (DBD)

(Figure 3.5). Results from luciferase assays, DNA-binding assays and ChIP experiments all confirmed that REV-ERBa binds directly to the endogenous Il17a locus and directly represses its expression. However, it is possible that in Th17 cells REV-ERBa also utilizes two distinct mechanisms to regulate two separate 71 sets of cellular pathways. Comparing ChIP-seq analysis of Th17 cells from REV-

ERBa DBD-deficient knock-in mice and REV-ERBa null mice could give us more insights into the transcriptional regulation of REV-ERBa in Th17 cells, and elucidate the mechanism by which REV-ERBa plays a positive role in Th17 differentiation.

Alternatively, but not mutually exclusive, timing could be a key factor in

REV-ERBa’s regulation of Th17 differentiation. During adipogeneis, Rev-erba gene expression declines initially followed by a subsequent increase. REV-ERBa protein levels are almost the opposite, increasing early in adipogenesis and decreasing drastically in adipocytes. The REV-ERBa protein is essential for the initial mitotic events that are necessary for adipogenesis. REV-ERBa protein is subsequently reduced due to increased degradation by the 26S proteasome. The reduction is necessary for adipocyte differentiation because REV-ERBa represses the expression of PPARg2, the master regulator of adipogenesis.

Therefore, contrary to what might be expected from REV-ERBa gene expression,

REV-ERBa protein levels must rise and then fall for adipocyte differentiation to take place (J. Wang & Lazar, 2008). Perhaps similar processes occur in Th17 cells. Early REV-ERBa expression may be important for priming the cell, potentially through chromatin remodeling to allow promoter access to Th17 signature genes. Subsequently, REV-ERBa expression decreases so it would not compete with RORgt for binding to genes like Il17a and Il17f. Indeed, REV-ERBa expression dynamics in Th17 cells are consistent with this hypothesis (Figure

3.1b and c). And further examination of the special and temporal expression of 72

REV-ERBa will present a more comprehensive picture of the role REV-ERba plays in Th17 biology.

6.4 Conclusion

In this study, we demonstrated a new role for REV-ERBa in the regulation of Th17 cell differentiation and function in addition to its established roles in circadian rhythm and metabolism. REV-ERBa is induced during Th17 cell differentiation and acts as both a facilitator and a suppressor of Th17 cells. On one hand, results from Hooper’s group suggest that normal RORgt expression is dependent on repression of Nfil3 by REV-ERBa. On the other hand, REV-ERBa directly competes with RORgt by binding to the RORE sites to repress the expression of key Th17 cell signature genes such as Il17a and Il17f. The regulatory function of REV-ERBa is heavily biased towards Th17 inhibition when

REV-ERBa is expressed at high levels or when its activity is enhanced by ligands

(sFig. 4). Elevated REV-ERBa expression in T cells or SR9009 treatment also suppresses development of EAE in vivo. Recently, concerted efforts have been made to identify RORa/g antagonists for treatment of Th17-related autoimmune diseases(Huh et al., 2011; Solt et al., 2011; Xiao et al., 2014). Our results suggest that a parallel strategy can be employed to develop REV-ERB agonists as drug candidates for Th17 cell-mediated autoimmune diseases. However, a lot remains unknown about the global transcriptional regulation of REV-ERBa in

Th17 cells. Additional studies utilizing ChIP-seq and bioinformatics may help us gain a deeper understanding of this novel player in Th17 mediated autoimmunity. 73

6.5 Figures

Vehicle 30 SR9009 100mg/kg

28 )

g 26 (m t 24 eigh W 22

20 0 6 8 1 3 5 7 1 1 1 1 Day(s)

Figure 6.1 SR9009 treatment does not cause aberrant weight loss in mice. Mice were weighed every few days spanning the duration of the EAE experiment.

Figure 6.2 Working model 1. REV-ERBa deficiency results in suppression of RORgt and IL-17A expression through the up-regulation of NFIL3. REV-ERBa overexpression oversteps NFIL3 by directly competing with RORgt for interaction with the Il17a locus.

Figure 6.3 Working model two. A basal level or early expression of REV-ERBa is required for optimal Th17 differentiation, potentially though the up-regulation of pro-Th17 pathways or chromatin remodeling to allow transcriptional access to Il17a and other Th17 genes. However, constitutive high expression of REV-ERBa later competes with RORgt for binding to Il17a and represses Th17 mediated immune response.

References

Agnello, Davide, Carla S R Lankford, Jay Bream, Akio Morinobu, Massimo Gadina, John J O'Shea, and David M Frucht. 2003. “Cytokines and Transcription Factors That Regulate T Helper Cell Differentiation: New Players and New Insights..” Journal of Clinical Immunology 23 (3) (May): 147–161.

André, E, F Conquet, M Steinmayr, S C Stratton, V Porciatti, and M Becker- André. 1998. “Disruption of Retinoid-Related Orphan Receptor Beta Changes Circadian Behavior, Causes Retinal Degeneration and Leads to Vacillans Phenotype in Mice..” The EMBO Journal 17 (14) (July 15): 3867–3877. doi:10.1093/emboj/17.14.3867.

Anthony, Robert M, Laura I Rutitzky, Joseph F Urban, Miguel J Stadecker, and William C Gause. 2007. “Protective Immune Mechanisms in Helminth Infection..” Nature Reviews. Immunology 7 (12) (December): 975–987. doi:10.1038/nri2199.

Bass, Joseph, and Joseph S Takahashi. 2010. “Circadian Integration of Metabolism and Energetics..” Science (New York, N.Y.) 330 (6009) (December 3): 1349–1354. doi:10.1126/science.1195027.

Bettelli, Estelle, Maria Pagany, Howard L Weiner, Christopher Linington, Raymond A Sobel, and Vijay K Kuchroo. 2003. “Myelin Oligodendrocyte Glycoprotein-Specific T Cell Receptor Transgenic Mice Develop Spontaneous Autoimmune Optic Neuritis..” The Journal of Experimental Medicine 197 (9) (May 5): 1073–1081. doi:10.1084/jem.20021603.

Biswas, Partha S, Sanjay Gupta, Emily Chang, Li Song, Roslynn A Stirzaker, James K Liao, Govind Bhagat, and Alessandra B Pernis. 2010. “Phosphorylation of IRF4 by ROCK2 Regulates IL-17 and IL-21 Production and the Development of Autoimmunity in Mice..” The Journal of Clinical Investigation 120 (9) (September): 3280–3295. doi:10.1172/JCI42856.

Bonnelye, E, J M Vanacker, X Desbiens, A Begue, D Stehelin, and V Laudet. 1994. “Rev-Erb Beta, a New Member of the Nuclear Receptor Superfamily, Is Expressed in the Nervous System During Chicken Development..” Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research 5 (12) (December): 1357–1365.

Brucklacher-Waldert, Verena, Klarissa Stuerner, Manuela Kolster, Julia Wolthausen, and Eva Tolosa. 2009. “Phenotypical and Functional Characterization of T Helper 17 Cells in Multiple Sclerosis..” Brain : a Journal of Neurology 132 (Pt 12) (December): 3329–3341. doi:10.1093/brain/awp289.

76 77

Chang, Seon Hee, and Chen Dong. 2007. “A Novel Heterodimeric Cytokine Consisting of IL-17 and IL-17F Regulates Inflammatory Responses..” Cell Research 17 (5) (May): 435–440. doi:10.1038/cr.2007.35.

Chawla, A, and M A Lazar. 1993. “Induction of Rev-ErbA Alpha, an Orphan Receptor Encoded on the Opposite Strand of the Alpha-Thyroid Hormone Receptor Gene, During Adipocyte Differentiation..” The Journal of Biological Chemistry 268 (22) (August 5): 16265–16269.

Cho, Han, Xuan Zhao, Megumi Hatori, Ruth T Yu, Grant D Barish, Michael T Lam, Ling-Wa Chong, et al. 2012. “Regulation of Circadian Behaviour and Metabolism by REV-ERB-Α and REV-ERB-Β..” Nature 485 (7396) (May 3): 123–127. doi:10.1038/nature11048.

Ciofani, Maria, Aviv Madar, Carolina Galan, Maclean Sellars, Kieran Mace, Florencia Pauli, Ashish Agarwal, et al. 2012. “A Validated Regulatory Network for Th17 Cell Specification..” Cell 151 (2) (October 12): 289–303. doi:10.1016/j.cell.2012.09.016.

Codarri, Laura, Gabor Gyülvészi, Vinko Tosevski, Lysann Hesske, Adriano Fontana, Laurent Magnenat, Tobias Suter, and Burkhard Becher. 2011. “RORγt Drives Production of the Cytokine GM-CSF in Helper T Cells, Which Is Essential for the Effector Phase of Autoimmune Neuroinflammation..” Nature Immunology 12 (6) (June): 560–567. doi:10.1038/ni.2027.

Constantinescu, Cris S, Aliya Asher, Waldemar Fryze, Wojciech Kozubski, Frank Wagner, Jehan Aram, Radu Tanasescu, et al. 2015. “Randomized Phase 1b Trial of MOR103, a Human Antibody to GM-CSF, in Multiple Sclerosis..” Neurology® Neuroimmunology & Neuroinflammation 2 (4) (August): e117. doi:10.1212/NXI.0000000000000117.

Croxford, Andrew L, Margit Lanzinger, Felix J Hartmann, Bettina Schreiner, Florian Mair, Pawel Pelczar, Björn E Clausen, Steffen Jung, Melanie Greter, and Burkhard Becher. 2015. “The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity..” Immunity 43 (3) (September 15): 502–514. doi:10.1016/j.immuni.2015.08.010.

Cua, Daniel J, Jonathan Sherlock, Yi Chen, Craig A Murphy, Barbara Joyce, Brian Seymour, Linda Lucian, et al. 2003. “Interleukin-23 Rather Than Interleukin-12 Is the Critical Cytokine for Autoimmune Inflammation of the Brain..” Nature 421 (6924) (February 13): 744–748. doi:10.1038/nature01355.

Curtis, Meredith M, and Sing Sing Way. 2009. “Interleukin-17 in Host Defence Against Bacterial, Mycobacterial and Fungal Pathogens..” Immunology 126 (2) (February): 177–185. doi:10.1111/j.1365-2567.2008.03017.x. 78

Delezie, Julien, Stéphanie Dumont, Hugues Dardente, Hugues Oudart, Aline Gréchez-Cassiau, Paul Klosen, Michèle Teboul, Franck Delaunay, Paul Pévet, and Etienne Challet. 2012. “The Nuclear Receptor REV-ERBα Is Required for the Daily Balance of Carbohydrate and Lipid Metabolism..” FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology 26 (8) (August): 3321–3335. doi:10.1096/fj.12- 208751.

Dobin, Alexander, Carrie A Davis, Felix Schlesinger, Jorg Drenkow, Chris Zaleski, Sonali Jha, Philippe Batut, Mark Chaisson, and Thomas R Gingeras. 2013. “STAR: Ultrafast Universal RNA-Seq Aligner..” Bioinformatics (Oxford, England) 29 (1) (January 1): 15–21. doi:10.1093/bioinformatics/bts635.

Dumas, B, H P Harding, H S Choi, K A Lehmann, M Chung, M A Lazar, and D D Moore. 1994. “A New Orphan Member of the Nuclear Hormone Receptor Superfamily Closely Related to Rev-Erb..” Molecular Endocrinology (Baltimore, Md.) 8 (8) (August): 996–1005. doi:10.1210/mend.8.8.7997240.

Durelli, Luca, Laura Conti, Marinella Clerico, Daniela Boselli, Giulia Contessa, Paolo Ripellino, Bruno Ferrero, Pierre Eid, and Francesco Novelli. 2009. “T- Helper 17 Cells Expand in Multiple Sclerosis and Are Inhibited by Interferon- Beta..” Annals of Neurology 65 (5) (May): 499–509. doi:10.1002/ana.21652.

Eberl, Gérard, Shana Marmon, Mary-Jean Sunshine, Paul D Rennert, Yongwon Choi, and Dan R Littman. 2004. “An Essential Function for the Nuclear Receptor RORgamma(T) in the Generation of Fetal Lymphoid Tissue Inducer Cells..” Nature Immunology 5 (1) (January): 64–73. doi:10.1038/ni1022. El-Behi, Mohamed, Bogoljub Ciric, Hong Dai, Yaping Yan, Melissa Cullimore, Farinaz Safavi, Guang-Xian Zhang, Bonnie N Dittel, and Abdolmohamad Rostami. 2011. “The Encephalitogenicity of T(H)17 Cells Is Dependent on IL- 1- and IL-23-Induced Production of the Cytokine GM-CSF..” Nature Immunology 12 (6) (June): 568–575. doi:10.1038/ni.2031.

Fontaine, Coralie, Guillaume Dubois, Yannick Duguay, Torben Helledie, Ngoc Vu- Dac, Philippe Gervois, Fabrice Soncin, et al. 2003. “The Orphan Nuclear Receptor Rev-Erbalpha Is a Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Target Gene and Promotes PPARgamma-Induced Adipocyte Differentiation..” The Journal of Biological Chemistry 278 (39) (September 26): 37672–37680. doi:10.1074/jbc.M304664200.

Fossiez, F, O Djossou, P Chomarat, L Flores-Romo, S Ait-Yahia, C Maat, J J Pin, et al. 1996. “T Cell Interleukin-17 Induces Stromal Cells to Produce Proinflammatory and Hematopoietic Cytokines..” The Journal of Experimental Medicine 183 (6) (June 1): 2593–2603.

Gamble, Karen L, and Martin E Young. 2013. “Metabolism as an Integral Cog in the Mammalian Circadian Clockwork..” Critical Reviews in Biochemistry and Molecular Biology 48 (4) (June): 317–331. doi:10.3109/10409238.2013.786672.

Garnock-Jones, Karly P. 2015. “Secukinumab: a Review in Moderate to Severe Plaque Psoriasis..” American Journal of Clinical Dermatology 16 (4) (August): 323–330. doi:10.1007/s40257-015-0143-7.

Ghoreschi, Kamran, Arian Laurence, Xiang-Ping Yang, Cristina M Tato, Mandy J McGeachy, Joanne E Konkel, Haydeé L Ramos, et al. 2010. “Generation of Pathogenic T(H)17 Cells in the Absence of TGF-Β Signalling..” Nature 467 (7318) (October 21): 967–971. doi:10.1038/nature09447.

Gibbs, Julie E, John Blaikley, Stephen Beesley, Laura Matthews, Karen D Simpson, Susan H Boyce, Stuart N Farrow, et al. 2012. “The Nuclear Receptor REV-ERBα Mediates Circadian Regulation of Innate Immunity Through Selective Regulation of Inflammatory Cytokines..” Proceedings of the National Academy of Sciences of the United States of America 109 (2) (January 10): 582–587. doi:10.1073/pnas.1106750109.

Guillaumond, Fabienne, Hugues Dardente, Vincent Giguère, and Nicolas Cermakian. 2005. “Differential Control of Bmal1 Circadian Transcription by REV-ERB and ROR Nuclear Receptors..” Journal of Biological Rhythms 20 (5) (October): 391–403. doi:10.1177/0748730405277232.

Gupta, Nirupama, and Stephen W Ragsdale. 2011. “Thiol-Disulfide Redox Dependence of Heme Binding and Heme Ligand Switching in Nuclear Hormone Receptor Rev-Erb{Beta}..” The Journal of Biological Chemistry 286 (6) (February 11): 4392–4403. doi:10.1074/jbc.M110.193466.

Harding, H P, and M A Lazar. 1995. “The Monomer-Binding Orphan Receptor Rev-Erb Represses Transcription as a Dimer on a Novel Direct Repeat..” Molecular and Cellular Biology 15 (9) (September): 4791–4802.

He, Y W, M L Deftos, E W Ojala, and M J Bevan. 1998. “RORgamma T, a Novel Isoform of an Orphan Receptor, Negatively Regulates Fas Ligand Expression and IL-2 Production in T Cells..” Immunity 9 (6) (December): 797–806.

Hofstetter, Harald H, Saleh M Ibrahim, Dirk Koczan, Niels Kruse, Andreas Weishaupt, Klaus V Toyka, and Ralf Gold. 2005. “Therapeutic Efficacy of IL- 17 Neutralization in Murine Experimental Autoimmune Encephalomyelitis..” Cellular Immunology 237 (2) (October): 123–130. doi:10.1016/j.cellimm.2005.11.002.

Huang, Da Wei, Brad T Sherman, and Richard A Lempicki. 2009a. “Systematic and Integrative Analysis of Large Gene Lists Using DAVID Bioinformatics Resources..” Nature Protocols 4 (1): 44–57. doi:10.1038/nprot.2008.211.

Huang, Da Wei, Brad T Sherman, and Richard A Lempicki. 2009b. “Bioinformatics Enrichment Tools: Paths Toward the Comprehensive Functional Analysis of Large Gene Lists..” Nucleic Acids Research 37 (1) (January): 1–13. doi:10.1093/nar/gkn923.

Huang, Weitao, Li Na, Paul L Fidel, and Paul Schwarzenberger. 2004. “Requirement of Interleukin-17A for Systemic Anti-Candida Albicans Host Defense in Mice..” The Journal of Infectious Diseases 190 (3) (August 1): 624–631. doi:10.1086/422329.

Huh, Jun R, Monica W L Leung, Pengxiang Huang, Daniel A Ryan, Michael R Krout, Raghu R V Malapaka, Jonathan Chow, et al. 2011. “Digoxin and Its Derivatives Suppress TH17 Cell Differentiation by Antagonizing RORγt Activity..” Nature 472 (7344) (April 28): 486–490. doi:10.1038/nature09978.

Hymowitz, S G, E H Filvaroff, J P Yin, J Lee, L Cai, P Risser, M Maruoka, et al. 2001. “IL-17s Adopt a Cystine Knot Fold: Structure and Activity of a Novel Cytokine, IL-17F, and Implications for Receptor Binding..” The EMBO Journal 20 (19) (October 1): 5332–5341. doi:10.1093/emboj/20.19.5332.

Ivanov, Ivaylo I, Brent S McKenzie, Liang Zhou, Carlos E Tadokoro, Alice Lepelley, Juan J Lafaille, Daniel J Cua, and Dan R Littman. 2006. “The Orphan Nuclear Receptor RORgammat Directs the Differentiation Program of Proinflammatory IL-17+ T Helper Cells..” Cell 126 (6) (September 22): 1121– 1133. doi:10.1016/j.cell.2006.07.035.

Jetten, Anton M. 2009. “Retinoid-Related Orphan Receptors (RORs): Critical Roles in Development, Immunity, Circadian Rhythm, and Cellular Metabolism..” Nuclear Receptor Signaling 7: e003. doi:10.1621/nrs.07003.

Jin, Wei, and Chen Dong. 2013. “IL-17 Cytokines in Immunity and Inflammation..” Emerging Microbes & Infections 2 (9) (September): e60. doi:10.1038/emi.2013.58.

Jones, Carol E, and Katie Chan. 2002. “Interleukin-17 Stimulates the Expression of Interleukin-8, Growth-Related Oncogene-Alpha, and Granulocyte-Colony- Stimulating Factor by Human Airway Epithelial Cells..” American Journal of Respiratory Cell and Molecular Biology 26 (6) (June): 748–753.

Jovanovic, D V, J A Di Battista, J Martel-Pelletier, F C Jolicoeur, Y He, M Zhang, F Mineau, and J P Pelletier. 1998. “IL-17 Stimulates the Production and 81

Expression of Proinflammatory Cytokines, IL-Beta and TNF-Alpha, by Human Macrophages..” Journal of Immunology (Baltimore, Md. : 1950) 160 (7) (April 1): 3513–3521.

Kebir, Hania, Katharina Kreymborg, Igal Ifergan, Aurore Dodelet-Devillers, Romain Cayrol, Monique Bernard, Fabrizio Giuliani, Nathalie Arbour, Burkhard Becher, and Alexandre Prat. 2007. “Human TH17 Lymphocytes Promote Blood-Brain Barrier Disruption and Central Nervous System Inflammation..” Nature Medicine 13 (10) (October): 1173–1175. doi:10.1038/nm1651.

Kolls, Jay K, and Anders Lindén. 2004. “Interleukin-17 Family Members and Inflammation..” Immunity 21 (4) (October): 467–476. doi:10.1016/j.immuni.2004.08.018.

Korn, Thomas, Estelle Bettelli, Mohamed Oukka, and Vijay K Kuchroo. 2009. “IL- 17 and Th17 Cells..” Annual Review of Immunology 27: 485–517. doi:10.1146/annurev.immunol.021908.132710.

Kornmann, Benoît, Olivier Schaad, Hermann Bujard, Joseph S Takahashi, and Ueli Schibler. 2007. “System-Driven and Oscillator-Dependent Circadian Transcription in Mice with a Conditionally Active Liver Clock..” PLoS Biology 5 (2) (February): e34. doi:10.1371/journal.pbio.0050034.

Kosztin, D, S Izrailev, and K Schulten. 1999. “Unbinding of Retinoic Acid From Its Receptor Studied by Steered Molecular Dynamics..” Biophysical Journal 76 (1 Pt 1) (January): 188–197. doi:10.1016/S0006-3495(99)77188-2.

Kotake, S, N Udagawa, N Takahashi, K Matsuzaki, K Itoh, S Ishiyama, S Saito, et al. 1999. “IL-17 in Synovial Fluids From Patients with Rheumatoid Arthritis Is a Potent Stimulator of Osteoclastogenesis..” The Journal of Clinical Investigation 103 (9) (May): 1345–1352. doi:10.1172/JCI5703.

Kumar, Naresh, Laura A Solt, Yongjun Wang, Pamela M Rogers, Gargi Bhattacharyya, Theodore M Kamenecka, Keith R Stayrook, et al. 2010. “Regulation of Adipogenesis by Natural and Synthetic REV-ERB Ligands..” Endocrinology 151 (7) (July): 3015–3025. doi:10.1210/en.2009-0800.

Kurebayashi, S, E Ueda, M Sakaue, D D Patel, A Medvedev, F Zhang, and A M Jetten. 2000. “Retinoid-Related Orphan Receptor Gamma (RORgamma) Is Essential for Lymphoid Organogenesis and Controls Apoptosis During Thymopoiesis..” Proceedings of the National Academy of Sciences of the United States of America 97 (18) (August 29): 10132–10137.

Laan, M, Z H Cui, H Hoshino, J Lötvall, M Sjöstrand, D C Gruenert, B E Skoogh, and A Lindén. 1999. “Neutrophil Recruitment by Human IL-17 via C-X-C 82

Chemokine Release in the Airways..” Journal of Immunology (Baltimore, Md. : 1950) 162 (4) (February 15): 2347–2352.

Lam, Michael T Y, Han Cho, Hanna P Lesch, David Gosselin, Sven Heinz, Yumiko Tanaka-Oishi, Christopher Benner, et al. 2013. “Rev-Erbs Repress Macrophage Gene Expression by Inhibiting Enhancer-Directed Transcription..” Nature 498 (7455) (June 27): 511–515. doi:10.1038/nature12209.

Langrish, Claire L, Yi Chen, Wendy M Blumenschein, Jeanine Mattson, Beth Basham, Jonathan D Sedgwick, Terrill McClanahan, Robert A Kastelein, and Daniel J Cua. 2005. “IL-23 Drives a Pathogenic T Cell Population That Induces Autoimmune Inflammation..” The Journal of Experimental Medicine 201 (2) (January 17): 233–240. doi:10.1084/jem.20041257.

Lazar, M A, R A Hodin, D S Darling, and W W Chin. 1989. “A Novel Member of the Thyroid/Steroid Hormone Receptor Family Is Encoded by the Opposite Strand of the Rat C-erbA Alpha Transcriptional Unit..” Molecular and Cellular Biology 9 (3) (March): 1128–1136.

Lazarevic, Vanja, Xi Chen, Jae-Hyuck Shim, Eun-Sook Hwang, Eunjung Jang, Alexandra N Bolm, Mohamed Oukka, Vijay K Kuchroo, and Laurie H Glimcher. 2011. “T-Bet Represses T(H)17 Differentiation by Preventing Runx1-Mediated Activation of the Gene Encoding RORγt..” Nature Immunology 12 (1) (January): 96–104. doi:10.1038/ni.1969.

Liu, Jialin, Yun Feng, Kun Yang, Qingyun Li, Liang Ye, Lizhong Han, and Huanying Wan. 2011. “Early Production of IL-17 Protects Against Acute Pulmonary Pseudomonas Aeruginosa Infection in Mice..” FEMS Immunology and Medical Microbiology 61 (2) (March): 179–188. doi:10.1111/j.1574- 695X.2010.00764.x.

Lubberts, Erik, Marije I Koenders, Birgitte Oppers-Walgreen, Liduine van den Bersselaar, Christina J J Coenen-de Roo, Leo A B Joosten, and Wim B van den Berg. 2004. “Treatment with a Neutralizing Anti-Murine Interleukin-17 Antibody After the Onset of Collagen-Induced Arthritis Reduces Joint Inflammation, Cartilage Destruction, and Bone Erosion..” Arthritis and Rheumatism 50 (2) (February): 650–659. doi:10.1002/art.20001.

Mangan, Paul R, Laurie E Harrington, Darrell B O'Quinn, Whitney S Helms, Daniel C Bullard, Charles O Elson, Robin D Hatton, Sharon M Wahl, Trenton R Schoeb, and Casey T Weaver. 2006. “Transforming Growth Factor-Beta Induces Development of the T(H)17 Lineage..” Nature 441 (7090) (May 11): 231–234. doi:10.1038/nature04754.

Masana, Monica I, Isabel C Sumaya, Michael Becker-Andre, and Margarita L 83

Dubocovich. 2007. “Behavioral Characterization and Modulation of Circadian Rhythms by Light and Melatonin in C3H/HeN Mice Homozygous for the RORbeta Knockout..” American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 292 (6) (June): R2357–67. doi:10.1152/ajpregu.00687.2006.

McInnes, Iain B, Philip J Mease, Bruce Kirkham, Arthur Kavanaugh, Christopher T Ritchlin, Proton Rahman, Désirée van der Heijde, et al. 2015. “Secukinumab, a Human Anti-Interleukin-17A Monoclonal Antibody, in Patients with Psoriatic Arthritis (FUTURE 2): a Randomised, Double-Blind, Placebo-Controlled, Phase 3 Trial..” Lancet (London, England) 386 (9999) (September 19): 1137– 1146. doi:10.1016/S0140-6736(15)61134-5.

Miyajima, N, R Horiuchi, Y Shibuya, S Fukushige, K Matsubara, K Toyoshima, and T Yamamoto. 1989. “Two erbA Homologs Encoding Proteins with Different T3 Binding Capacities Are Transcribed From Opposite DNA Strands of the Same Genetic Locus..” Cell 57 (1) (April 7): 31–39.

Miyajima, N, Y Kadowaki, S Fukushige, S Shimizu, K Semba, Y Yamanashi, K Matsubara, K Toyoshima, and T Yamamoto. 1988. “Identification of Two Novel Members of erbA Superfamily by Molecular Cloning: the Gene Products of the Two Are Highly Related to Each Other..” Nucleic Acids Research 16 (23) (December 9): 11057–11074.

Mohawk, Jennifer A, Carla B Green, and Joseph S Takahashi. 2012. “Central and Peripheral Circadian Clocks in Mammals..” Annual Review of Neuroscience 35: 445–462. doi:10.1146/annurev-neuro-060909-153128.

Naviaux, R K, E Costanzi, M Haas, and I M Verma. 1996. “The pCL Vector System: Rapid Production of Helper-Free, High-Titer, Recombinant Retroviruses..” Journal of Virology 70 (8) (August): 5701–5705.

Nurieva, Roza, Xuexian O Yang, Gustavo Martinez, Yongliang Zhang, Athanasia D Panopoulos, Li Ma, Kimberly Schluns, et al. 2007. “Essential Autocrine Regulation by IL-21 in the Generation of Inflammatory T Cells..” Nature 448 (7152) (July 26): 480–483. doi:10.1038/nature05969.

Paintlia, Manjeet K, Ajaib S Paintlia, Avtar K Singh, and Inderjit Singh. 2011. “Synergistic Activity of Interleukin-17 and Tumor Necrosis Factor-Α Enhances Oxidative Stress-Mediated Oligodendrocyte Apoptosis..” Journal of Neurochemistry 116 (4) (February): 508–521. doi:10.1111/j.1471- 4159.2010.07136.x.

Pardee, Keith I, Xiaohui Xu, Jeff Reinking, Anja Schuetz, Aiping Dong, Suya Liu, Rongguang Zhang, et al. 2009. “The Structural Basis of Gas-Responsive 84

Transcription by the Human Nuclear Hormone Receptor REV-ERBbeta..” PLoS Biology 7 (2) (February 24): e43. doi:10.1371/journal.pbio.1000043.

Pavlidis, Paul, and William Stafford Noble. 2003. “Matrix2png: a Utility for Visualizing Matrix Data..” Bioinformatics (Oxford, England) 19 (2) (January 22): 295–296.

Preitner, Nicolas, Francesca Damiola, Luis Lopez-Molina, Joszef Zakany, Denis Duboule, Urs Albrecht, and Ueli Schibler. 2002. “The Orphan Nuclear Receptor REV-ERBalpha Controls Circadian Transcription Within the Positive Limb of the Mammalian Circadian Oscillator..” Cell 110 (2) (July 26): 251–260.

Raghuram, Srilatha, Keith R Stayrook, Pengxiang Huang, Pamela M Rogers, Amanda K Nosie, Don B McClure, Lorri L Burris, Sepideh Khorasanizadeh, Thomas P Burris, and Fraydoon Rastinejad. 2007. “Identification of Heme as the Ligand for the Orphan Nuclear Receptors REV-ERBalpha and REV- ERBbeta..” Nature Structural & Molecular Biology 14 (12) (December): 1207– 1213. doi:10.1038/nsmb1344.

Ramakrishnan, Sathiya N, Patrick Lau, Les J Burke, and George E O Muscat. 2005. “Rev-Erbbeta Regulates the Expression of Genes Involved in Lipid Absorption in Skeletal Muscle Cells: Evidence for Cross-Talk Between Orphan Nuclear Receptors and Myokines..” The Journal of Biological Chemistry 280 (10) (March 11): 8651–8659. doi:10.1074/jbc.M413949200.

Raspè, Eric, Hélène Duez, Anethe Mansén, Coralie Fontaine, Catherine Fiévet, Jean-Charles Fruchart, Bjorn Vennström, and Bart Staels. 2002. “Identification of Rev-Erbalpha as a Physiological Repressor of apoC-III Gene Transcription..” Journal of Lipid Research 43 (12) (December): 2172–2179.

Reboldi, Andrea, Caroline Coisne, Dirk Baumjohann, Federica Benvenuto, Denise Bottinelli, Sergio Lira, Antonio Uccelli, Antonio Lanzavecchia, Britta Engelhardt, and Federica Sallusto. 2009. “C-C Chemokine Receptor 6- Regulated Entry of TH-17 Cells Into the CNS Through the Choroid Plexus Is Required for the Initiation of EAE..” Nature Immunology 10 (5) (May): 514– 523. doi:10.1038/ni.1716.

Reinking, Jeff, Mandy M S Lam, Keith Pardee, Heidi M Sampson, Suya Liu, Ping Yang, Shawn Williams, et al. 2005. “The Drosophila Nuclear Receptor E75 Contains Heme and Is Gas Responsive..” Cell 122 (2) (July 29): 195–207. doi:10.1016/j.cell.2005.07.005.

Rogers, Pamela M, Ling Ying, and Thomas P Burris. 2008. “Relationship Between Circadian Oscillations of Rev-Erbalpha Expression and Intracellular Levels of Its Ligand, Heme..” Biochemical and Biophysical Research Communications 85

368 (4) (April 18): 955–958. doi:10.1016/j.bbrc.2008.02.031.

Rudensky, Alexander Y. 2011. “Regulatory T Cells and Foxp3..” Immunological Reviews 241 (1) (May): 260–268. doi:10.1111/j.1600-065X.2011.01018.x.

Sakaguchi, Shimon, Tomoyuki Yamaguchi, Takashi Nomura, and Masahiro Ono. 2008. “Regulatory T Cells and Immune Tolerance..” Cell 133 (5) (May 30): 775–787. doi:10.1016/j.cell.2008.05.009.

Sato, Trey K, Satchidananda Panda, Loren J Miraglia, Teresa M Reyes, Radu D Rudic, Peter McNamara, Kinnery A Naik, Garret A FitzGerald, Steve A Kay, and John B Hogenesch. 2004. “A Functional Genomics Strategy Reveals Rora as a Component of the Mammalian Circadian Clock..” Neuron 43 (4) (August 19): 527–537. doi:10.1016/j.neuron.2004.07.018.

Scanga, Charles A, Julio Aliberti, Dragana Jankovic, Florence Tilloy, Soumaya Bennouna, Eric Y Denkers, Ruslan Medzhitov, and Alan Sher. 2002. “Cutting Edge: MyD88 Is Required for Resistance to Toxoplasma Gondii Infection and Regulates Parasite-Induced IL-12 Production by Dendritic Cells..” Journal of Immunology (Baltimore, Md. : 1950) 168 (12) (June 15): 5997–6001.

Schraml, Barbara U, Kai Hildner, Wataru Ise, Wan-Ling Lee, Whitney A-E Smith, Ben Solomon, Gurmukh Sahota, et al. 2009. “The AP-1 Transcription Factor Batf Controls T(H)17 Differentiation..” Nature 460 (7253) (July 16): 405–409. doi:10.1038/nature08114.

Seki, Ekihiro, Hiroko Tsutsui, Noriko M Tsuji, Nobuki Hayashi, Keishi Adachi, Hiroki Nakano, Shizue Futatsugi-Yumikura, et al. 2002. “Critical Roles of Myeloid Differentiation Factor 88-Dependent Proinflammatory Cytokine Release in Early Phase Clearance of Listeria Monocytogenes in Mice..” Journal of Immunology (Baltimore, Md. : 1950) 169 (7) (October 1): 3863– 3868.

Solt, Laura A, Naresh Kumar, Philippe Nuhant, Yongjun Wang, Janelle L Lauer, Jin Liu, Monica A Istrate, et al. 2011. “Suppression of TH17 Differentiation and Autoimmunity by a Synthetic ROR Ligand..” Nature 472 (7344) (April 28): 491–494. doi:10.1038/nature10075.

Solt, Laura A, Yongjun Wang, Subhashis Banerjee, Travis Hughes, Douglas J Kojetin, Thomas Lundasen, Youseung Shin, et al. 2012. “Regulation of Circadian Behaviour and Metabolism by Synthetic REV-ERB Agonists..” Nature 485 (7396) (May 3): 62–68. doi:10.1038/nature11030.

Sun, Z, D Unutmaz, Y R Zou, M J Sunshine, A Pierani, S Brenner-Morton, R E Mebius, and D R Littman. 2000. “Requirement for RORgamma in Thymocyte 86

Survival and Lymphoid Organ Development..” Science (New York, N.Y.) 288 (5475) (June 30): 2369–2373.

Takeda, Yukimasa, Raja Jothi, Veronique Birault, and Anton M Jetten. 2012. “RORγ Directly Regulates the Circadian Expression of Clock Genes and Downstream Targets in Vivo..” Nucleic Acids Research 40 (17) (September 1): 8519–8535. doi:10.1093/nar/gks630.

Torra, I P, V Tsibulsky, F Delaunay, R Saladin, V Laudet, J C Fruchart, V Kosykh, and B Staels. 2000. “Circadian and Glucocorticoid Regulation of Rev-Erbalpha Expression in Liver..” Endocrinology 141 (10) (October): 3799–3806. doi:10.1210/endo.141.10.7708.

Toy, Dean, David Kugler, Martin Wolfson, Tim Vanden Bos, Jesse Gurgel, Jonathan Derry, Joel Tocker, and Jacques Peschon. 2006. “Cutting Edge: Interleukin 17 Signals Through a Heteromeric Receptor Complex..” Journal of Immunology (Baltimore, Md. : 1950) 177 (1) (July 1): 36–39.

Trapnell, Cole, David G Hendrickson, Martin Sauvageau, Loyal Goff, John L Rinn, and Lior Pachter. 2013. “Differential Analysis of Gene Regulation at Transcript Resolution with RNA-Seq..” Nature Biotechnology 31 (1) (January): 46–53. doi:10.1038/nbt.2450.

Trump, Ryan P, Stefano Bresciani, Anthony W J Cooper, James P Tellam, Justyna Wojno, John Blaikley, Lisa A Orband-Miller, et al. 2013. “Optimized Chemical Probes for REV-ERBα..” Journal of Medicinal Chemistry 56 (11) (June 13): 4729–4737. doi:10.1021/jm400458q.

Tzartos, John S, Manuel A Friese, Matthew J Craner, Jackie Palace, Jia Newcombe, Margaret M Esiri, and Lars Fugger. 2008. “Interleukin-17 Production in Central Nervous System-Infiltrating T Cells and Glial Cells Is Associated with Active Disease in Multiple Sclerosis..” The American Journal of Pathology 172 (1) (January): 146–155. doi:10.2353/ajpath.2008.070690.

Ueda, Hiroki R, Wenbin Chen, Akihito Adachi, Hisanori Wakamatsu, Satoko Hayashi, Tomohiro Takasugi, Mamoru Nagano, et al. 2002. “A Transcription Factor Response Element for Gene Expression During Circadian Night..” Nature 418 (6897) (August 1): 534–539. doi:10.1038/nature00906.

Uyttenhove, Catherine, and Jacques Van Snick. 2006. “Development of an Anti- IL-17A Auto-Vaccine That Prevents Experimental Auto-Immune Encephalomyelitis..” European Journal of Immunology 36 (11) (November): 2868–2874. doi:10.1002/eji.200636662.

Vu-Dac, N, S Chopin-Delannoy, P Gervois, E Bonnelye, G Martin, J C Fruchart, V 87

Laudet, and B Staels. 1998. “The Nuclear Receptors Peroxisome Proliferator- Activated Receptor Alpha and Rev-Erbalpha Mediate the Species-Specific Regulation of Apolipoprotein a-I Expression by Fibrates..” The Journal of Biological Chemistry 273 (40) (October 2): 25713–25720.

Wang, Jing, and Mitchell A Lazar. 2008. “Bifunctional Role of Rev-Erbalpha in Adipocyte Differentiation..” Molecular and Cellular Biology 28 (7) (April): 2213– 2220. doi:10.1128/MCB.01608-07.

Wang, Xiaohu, Yibing Zhang, Xuexian O Yang, Roza I Nurieva, Seon Hee Chang, Sandra S Ojeda, Hong S Kang, et al. 2012. “Transcription of Il17 and Il17f Is Controlled by Conserved Noncoding Sequence 2..” Immunity 36 (1) (January 27): 23–31. doi:10.1016/j.immuni.2011.10.019.

Weaver, Casey T, Robin D Hatton, Paul R Mangan, and Laurie E Harrington. 2007. “IL-17 Family Cytokines and the Expanding Diversity of Effector T Cell Lineages..” Annual Review of Immunology 25: 821–852. doi:10.1146/annurev.immunol.25.022106.141557.

Wei, Lai, Arian Laurence, Kevin M Elias, and John J O'Shea. 2007. “IL-21 Is Produced by Th17 Cells and Drives IL-17 Production in a STAT3-Dependent Manner..” The Journal of Biological Chemistry 282 (48) (November 30): 34605–34610. doi:10.1074/jbc.M705100200.

Wilson, Nicholas J, Katia Boniface, Jason R Chan, Brent S McKenzie, Wendy M Blumenschein, Jeanine D Mattson, Beth Basham, et al. 2007. “Development, Cytokine Profile and Function of Human Interleukin 17-Producing Helper T Cells..” Nature Immunology 8 (9) (September): 950–957. doi:10.1038/ni1497.

Woldt, Estelle, Yasmine Sebti, Laura A Solt, Christian Duhem, Steve Lancel, Jérôme Eeckhoute, Matthijs K C Hesselink, et al. 2013. “Rev-Erb-Α Modulates Skeletal Muscle Oxidative Capacity by Regulating Mitochondrial Biogenesis and Autophagy..” Nature Medicine 19 (8) (August): 1039–1046. doi:10.1038/nm.3213.

Woo, Eui-Jeon, Dae Gwin Jeong, Mi-Youn Lim, Seung Jun Kim, Kyung-Jin Kim, Sei-Mee Yoon, Byoung-Chul Park, and Seong Eon Ryu. 2007. “Structural Insight Into the Constitutive Repression Function of the Nuclear Receptor Rev-Erbbeta..” Journal of Molecular Biology 373 (3) (October 26): 735–744. doi:10.1016/j.jmb.2007.08.037.

Wright, Jill F, Yongjing Guo, Amira Quazi, Deborah P Luxenberg, Frann Bennett, John F Ross, Yongchang Qiu, et al. 2007. “Identification of an Interleukin 17F/17A Heterodimer in Activated Human CD4+ T Cells..” The Journal of Biological Chemistry 282 (18) (May 4): 13447–13455. 88

doi:10.1074/jbc.M700499200.

Xiao, Sheng, Nir Yosef, Jianfei Yang, Yonghui Wang, Ling Zhou, Chen Zhu, Chuan Wu, et al. 2014. “Small-Molecule RORγt Antagonists Inhibit T Helper 17 Cell Transcriptional Network by Divergent Mechanisms..” Immunity 40 (4) (April 17): 477–489. doi:10.1016/j.immuni.2014.04.004.

Yang, Xuexian O, Bhanu P Pappu, Roza Nurieva, Askar Akimzhanov, Hong Soon Kang, Yeonseok Chung, Li Ma, et al. 2008. “T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors ROR Alpha and ROR Gamma..” Immunity 28 (1) (January): 29–39. doi:10.1016/j.immuni.2007.11.016.

Yin, Lei, and Mitchell A Lazar. 2005. “The Orphan Nuclear Receptor Rev- Erbalpha Recruits the N-CoR/Histone Deacetylase 3 Corepressor to Regulate the Circadian Bmal1 Gene..” Molecular Endocrinology (Baltimore, Md.) 19 (6) (June): 1452–1459. doi:10.1210/me.2005-0057.

Yin, Lei, Nan Wu, Joshua C Curtin, Mohammed Qatanani, Nava R Szwergold, Robert A Reid, Gregory M Waitt, et al. 2007. “Rev-Erbalpha, a Heme Sensor That Coordinates Metabolic and Circadian Pathways..” Science (New York, N.Y.) 318 (5857) (December 14): 1786–1789. doi:10.1126/science.1150179.

Yu, Xiaofei, Darcy Rollins, Kelly A Ruhn, Jeremy J Stubblefield, Carla B Green, Masaki Kashiwada, Paul B Rothman, Joseph S Takahashi, and Lora V Hooper. 2013. “TH17 Cell Differentiation Is Regulated by the Circadian Clock..” Science (New York, N.Y.) 342 (6159) (November 8): 727–730. doi:10.1126/science.1243884.

Zhang, Fuping, Guangxun Meng, and Warren Strober. 2008. “Interactions Among the Transcription Factors Runx1, RORgammat and Foxp3 Regulate the Differentiation of Interleukin 17-Producing T Cells..” Nature Immunology 9 (11) (November): 1297–1306. doi:10.1038/ni.1663.

Zhang, Yuxiang, Bin Fang, Matthew J Emmett, Manashree Damle, Zheng Sun, Dan Feng, Sean M Armour, et al. 2015. “Discrete Functions of Nuclear Receptor Rev-Erbα Couple Metabolism to the Clock..” Science (New York, N.Y.) (June 4). doi:10.1126/science.aab3021.

Zhang, Zili, Mingquan Zheng, Julie Bindas, Paul Schwarzenberger, and Jay K Kolls. 2006. “Critical Role of IL-17 Receptor Signaling in Acute TNBS-Induced Colitis..” Inflammatory Bowel Diseases 12 (5) (May): 382–388. doi:10.1097/01.MIB.0000218764.06959.91.

Zhou, Liang, Ivaylo I Ivanov, Rosanne Spolski, Roy Min, Kevin Shenderov, 89

Takeshi Egawa, David E Levy, Warren J Leonard, and Dan R Littman. 2007. “IL-6 Programs T(H)-17 Cell Differentiation by Promoting Sequential Engagement of the IL-21 and IL-23 Pathways..” Nature Immunology 8 (9) (September): 967–974. doi:10.1038/ni1488.