Genetic and mechanistic analyses of type I interferon-driven lyme arthritis

B6 and C3H mice develop opposite Lyme arthritis phenotypes in response to the same Borrelia burgdorferi infection, providing a unique opportunity to use unbiased genetic approaches to identify host genes modulating pathogenic responses. Previously, gene expression profiling in joint tissue revealed a robust type I IFN profile in C3H mice that was formally linked to arthritis severity through IFNAR1 mAb blockade and genetic ablation. Independently, forward genetics identified a QTL, termed Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1), which regulates Lyme arthritis severity and includes the type I IFN gene cluster. Here, interval specific congenic lines on B6 and C3H backgrounds were generated to mechanistically analyze the role of Bbaa1 as a regulator of Lyme arthritis severity. B6 mice congenic for the C3H Bbaa1 allele (B6.C3-Bbaa1) developed more severe Lyme arthritis than parental B6, which was correctable by IFNAR1 mAb blockade. Bbaa1 also regulated the magnitude of interferon-stimulated gene expression in BMDMs. Extensive analysis on phagocytic uptake, bacterial sensing and trafficking pathways, and IFN-responsive states further established that genes within Bbaa1 intrinsically control the differential IFN response. B6.C3-Bbaa1 mice also developed more severe K/BÃ-N serum transfer arthritis through dysregulated type I IFN, establishing shared pathological processes in models of Lyme and rheumatoid arthritis. Refined, interval specific recombinant congenic lines further highlighted thecontribution of C3H type I IFN genes to Lyme arthritis. Specific mAb blockade identified IFN-β (and not IFN-α) as the proarthritogenic type I IFN in B6.C3-Bbaa1 mice, and IFN-β was solely responsible for interferon-stimulated gene expression and feed-forward amplification in BMDMs. Reciprocal radiation chimeras between B6.C3-Bbaa1 and B6 mice illuminated a critical “pass off” in joint tissue, where radiation-sensitive cells initiate arthritis (likely through internalization-dependent initiation of IFN-β) and radiation-resistant joint resident cells choreograph arthritis development through myostatin upregulation. Together, these findings suggest tantalizing new options for therapeutic intervention in Lyme arthritic patients: (1) blockade of IFN-β to only partially suppress the antiviral response, and (2) blockade of myostatin to correct dysregulated inflammation without interfering with conventional inflammatory pathways.

GENETIC AND MECHANISTIC ANALYSES OF TYPE I INTERFERON-DRIVEN LYME ARTHRITIS by Jacqueline Kaylah Paquette A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Pathology The University of Utah August 2017 Copyright © Jacqueline Kaylah Paquette 2017 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Jacqueline Kaylah Paquette has been approved by the following supervisory committee members: Janis J. Weis , Chair 05/23/2017 Xiao He , Member 05/23/2017 Daniel Leung , Member 05/23/2017 Ryan O'Connell , Member 05/23/2017 Matthew Williams , Member 05/23/2017 and by Peter E. Jensen the Department/College/School of and by David B. Kieda, Dean of The Graduate School. Date Approved Date Approved Date Approved Date Approved Date Approved , Chair/Dean of Pathology ABSTRACT B6 and C3H mice develop opposite Lyme arthritis phenotypes in response to the same Borrelia burgdorferi infection, providing a unique opportunity to use unbiased genetic approaches to identify host genes modulating pathogenic responses. Previously, gene expression profiling in joint tissue revealed a robust type I IFN profile in C3H mice that was formally linked to arthritis severity through IFNAR1 mAb blockade and genetic ablation. Independently, forward genetics identified a QTL, termed Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1), which regulates Lyme arthritis severity and includes the type I IFN gene cluster. Here, interval specific congenic lines on B6 and C3H backgrounds were generated to mechanistically analyze the role of Bbaa1 as a regulator of Lyme arthritis severity. B6 mice congenic for the C3H Bbaa1 allele (B6.C3-Bbaa1) developed more severe Lyme arthritis than parental B6, which was correctable by IFNAR1 mAb blockade. Bbaa1 also regulated the magnitude of interferon-stimulated gene expression in BMDMs. Extensive analysis on phagocytic uptake, bacterial sensing and trafficking pathways, and IFN-responsive states further established that genes within Bbaa1 intrinsically control the differential IFN response. B6.C3-Bbaa1 mice also developed more severe K/B×N serum transfer arthritis through dysregulated type I IFN, establishing shared pathological processes in models of Lyme and rheumatoid arthritis. Refined, interval specific recombinant congenic lines further highlighted the contribution of C3H type I IFN genes to Lyme arthritis. Specific mAb blockade identified IFN-β (and not IFN-α) as the proarthritogenic type I IFN in B6.C3-Bbaa1 mice, and IFNβ was solely responsible for interferon-stimulated gene expression and feed-forward amplification in BMDMs. Reciprocal radiation chimeras between B6.C3-Bbaa1 and B6 mice illuminated a critical "pass off" in joint tissue, where radiation-sensitive cells initiate arthritis (likely through internalization-dependent initiation of IFN-β) and radiation-resistant joint resident cells choreograph arthritis development through myostatin upregulation. Together, these findings suggest tantalizing new options for therapeutic intervention in Lyme arthritic patients: (1) blockade of IFN-β to only partially suppress the antiviral response, and (2) blockade of myostatin to correct dysregulated inflammation without interfering with conventional inflammatory pathways. iv To Andrew and Isabelle TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii LIST OF FIGURES ......................................................................................................... viii ACKNOWLEDGEMENTS .................................................................................................x Chapters 1. INTRODUCTION .........................................................................................................1 Lyme Disease ...........................................................................................................2 Lyme Arthritis..........................................................................................................3 The Interferon Family ..............................................................................................4 Pathologic Type I Interferon in Lyme Disease ........................................................5 Pathologic Type I Interferon in Other Diseases .......................................................6 Preview of Dissertation Research ............................................................................7 References ................................................................................................................8 2. BORRELIA BURGDORFERI ARTHRITIS-ASSOCIATED LOCUS BBAA1 REGULATES LYME ARTHRITIS AND K/B×N SERUM TRANSFER ARTHRITIS THROUGH INTRINSIC CONTROL OF TYPE I IFN PRODUCTION ...........................................................................................................14 Abstract ..................................................................................................................15 Introduction ............................................................................................................15 Materials and Methods ...........................................................................................16 Results ....................................................................................................................16 Discussion ..............................................................................................................22 References ..............................................................................................................23 3. DYSREGULATED PRODUCTION OF IFN-β BY BORRELIA BURGDORFERI ARTHRITIS-ASSOCIATED LOCUS 1 (BBAA1) DRIVES LYME ARTHRITIS THROUGH MYOSTATIN UPREGULATION .........................................................26 Abstract ..................................................................................................................27 Introduction ............................................................................................................28 Materials and Methods ...........................................................................................30 Results ....................................................................................................................35 Discussion ..............................................................................................................43 References ..............................................................................................................45 4. DISCUSSION ..............................................................................................................79 Overview ................................................................................................................80 Possible Mechanism of IFN-β Dysregulation ........................................................81 Investigations in Bbaa1 Mice Will Transcend Lyme Disease ...............................82 References ..............................................................................................................83 APPENDIX: RNA-SEQ IDENTIFICATION OF BBAA1 CANDIDATES THAT ARE EXPRESSED IN BONE MARROW-DERIVED MACROPHAGES FROM B6, ISRCL3, AND ISRCL4 MICE ....................................................................................89 vii LIST OF FIGURES 2.1 Interval-specific congenic mice reveal contribution of C3H allele of Bbaa1 to Lyme arthritis severity ...........................................................................................17 2.2 mAb blocking of type I IFN signaling prevents the Bbaa1-dependent increase in arthritis in B. burgdorferi-infected mice ............................................................17 2.3 BMDMs reveal Bbaa1 regulates the magnitude of the IFN response to B. burgdorferi .............................................................................................................18 2.4 Phagocytic capacity of macrophages from B6 and C3H mice ..............................19 2.5 Bbaa1 regulates BMDM responses to poly(I:C) ...................................................19 2.6 Bbaa1 regulation of IFN profile is dependent on IFNAR feed forward ................20 2.7 Responses of BMDMs to increasing doses of IFN-β ............................................21 2.8 Bbaa1 regulated levels of expression of M1 and M2 markers in resting and activated macrophages ...........................................................................................21 2.9 Impact of Bbaa1 on severity of K/B×N serum transfer arthritis ...........................22 2.10 Enhanced RA in B6.C3-Bbaa1 mice is dependent on type I IFN .........................22 3.1 mAb blocking of IFN-β suppresses Bbaa1-Lyme arthritis to the same extent as IFNAR1-blockade mice .....................................................................................53 3.2 IFN-β drives the type I IFN profile in B6.C3-Bbaa1 BMDMs in response to B. burgdorferi ........................................................................................................55 3.3 Reciprocal radiation chimeras between B6 and B6.C3-Bbaa1 mice .....................57 3.4 Physical boundaries of Bbaa1 congenic intervals and Lyme arthritis reveal that mice with C3H-derived genes spanning the type I IFN locus have increased arthritis severity compared to B6 mice ..................................................................59 3.5 BMDMs reveal that Bbaa1 genes regulating the magnitude of the IFN response to B. burgdorferi are retained in the genetic intervals of ISRCL3 and ISRCL4 mice ........................................................................................................................61 3.6 RNA-seq identification of myostatin (Mstn) as the strongest candidate for Bbaa1-directed Lyme arthritis development .........................................................63 3.7 IFN-β and B. burgdorferi are both required for the expression of myostatin by CD45- joint cells during infection and ex vivo .......................................................65 3.8 Myostatin inhibition suppresses the development of Lyme arthritis in Bbaa1 congenic mice ........................................................................................................67 3.S1 Isotype control does not impact the expression of IFN-inducible genes in BMDMs .................................................................................................................69 3.S2 Confirmation of anti-IFN-α functionality ..............................................................71 3.S3 Radiation chimera reconstitution efficiency and impact on host defense..............73 3.S4 Confirmation of cell viability in CD45- cells isolated from naïve B6 mouse joint and stimulated ex vivo....................................................................................75 3.S5 Volcano plot of genes identified in ISRCL3 vs. B6 RNA-seq comparisons .........77 ix ACKNOWLEDGEMENTS The work presented in this dissertation would not have been possible without the support of many individuals. First, I would like to thank Dr. Janis Weis for investing in me as a student in her lab. In addition to fostering my scientific development, she continually encouraged me to persevere through graduate school, allowed me to explore postgraduate career options, and accommodated my childcare needs. I will always be grateful for her unwavering commitment to my personal and professional success. Next, I thank Ying Ma for her endless help with experiments and friendship. It has been a privilege to learn from and work with the legendary Ying, and her camaraderie was critical to my success. Thank you also to Colleen Fisher and Jinze Li for being reliable, motivated lab mates, and for providing critical assistance with various aspects of my project. Dr. John Weis was also a major inspiration to me, and his authenticity and passion for science will always be in my memory. I would also like to thank the members of my thesis committee: Drs. Xiao He, Daniel Leung, Ryan O'Connell, and Matthew Williams. Each of these brilliant scientists provided thoughtful suggestions and guidance that inspired me and helped shape the direction of this work. And thank you to all of the faculty in the Pathology department for asking stimulating questions during presentations that helped sharpen my critical thinking and communication skills. It has been a blessing to be a part of this elite, collaborative, and friendly community. Most of all, I am thankful to my husband, Andrew, for being my ultimate supporter, teammate, and best friend. We have had the privilege and challenge of working toward our PhD degrees at the same time, and I could not have asked for a better life partner. We also grew our family during graduate school, and I am thankful to our precious daughter, Isabelle, for continually being a breath of fresh air. It is because of my beloved family, friends, and Savior that I have completed this journey. xi CHAPTER 1 INTRODUCTION 2 Lyme Disease Lyme disease is a multistage, multisystem inflammatory disorder triggered by infection with a tick-borne bacteria belonging to the genus Borrelia (1, 2). Lyme disease is rampant in the United States, Europe, and Asia, specifically in regions amenable to the life cycle of the Ixodes sps tick vector (3). The three primary disease-causing species of Borrelia are B. burgdorferi, B. afzelii, and B. garnii, but B. bavariensis has also been identified in a small percentage of cases (4-6). Borrelia burgdorferi is the only species found in the United States and causes approximately 300,000 cases of Lyme disease each year, making this the most common vector-borne disease in the U.S. (7). Unfortunately, Lyme disease incidence is expected to increase internationally as Ixodes populations are rapidly expanding due to many man-made changes to the environment (8). Thus, Lyme disease is a major global public health concern with an urgent need for preventative measures. Lyme disease begins with a localized dermal infection at the site of the tick bite. Infection with B. burgdorferi usually manifests as a painless bulls-eye rash called an erythema migrans (which is diagnostic of infection) accompanied by flu-like symptoms including fever, headache, and fatigue (2, 9). Weeks to months after infection, B. burgdorferi disseminates (possibly through the bloodstream, lymphatic system, or direct penetration of tissues (10)) to distal sites of the body, typically the joint synovial fluid, nervous system, or heart (2). Thus, patients with disseminated infection may experience intermittent attacks of arthritis, neuropathies, or carditis, the latter of which can be deadly (7, 9, 11, 12). Early administration of oral antibiotics, including doxycycline, amoxicillin, or cefuroxime azetil, usually promotes a full recovery, while more severe neurological 3 and cardiac forms of illness require intravenous treatment with ceftriaxone or penicillin and have a worse prognosis (7). A subset of patients experience post-treatment Lyme disease syndrome (PTLDS) with continued fatigue, pain, or joint and muscle aches after antibiotic therapy (7). Although the exact cause of PTLDS is unknown (with hypotheses of infection-induced autoimmunity (13), persistence of nonviable bacterial antigens (14) or drug-tolerant persister cells (15)), long-term antibiotic therapy is clearly not effective (7) and these patients are currently underserved. Lyme Arthritis Lyme arthritis is the most common late disease manifestation, affecting up to 60% of untreated patients and persisting in 10% of patients after antibiotic therapy (16, 17). Lyme arthritis is characterized by edema, synovial hyperplasia, and neutrophil infiltration, and usually develops asymmetrically in the knee joint, though multiple joints may be affected throughout the course of infection (16, 18). A spectrum of Lyme arthritis severity exists among patients, ranging from acute-episodic to chronic-persistent (16, 19), and has been broadly attributed to the genetic composition of the host (involving genes of the immune response) as well as the infecting B. burgdorferi isolate (involving genes aiding dissemination and immune evasion) (20). The mouse model of Lyme arthritis has provided invaluable insight into all aspects of the host-pathogen interplay, particularly with regard to the genetic contribution of the host. Mice are reservoir hosts for B. burgdorferi infection in the wild (21), and inbred laboratory strains display a reproducible spectrum of arthritis severity in their rear ankle joints, with clinical features similar to human Lyme arthritis (22). Thus, in addition 4 to the plethora of laboratory tools and reagents available for mouse studies, the inherent genetic differences among mice has allowed the impact of host genes on pathogenic responses to be systematically explored in a natural model of B. burgdorferi infection. Accordingly, Weis et al. initiated a forward genetics approach over twenty years ago whereby C3H mice (which develop severe Lyme arthritis) were bred with C57BL/6 (B6) mice (which develop mild Lyme arthritis), and identified six quantitative trait loci (QTL) associated with Borrelia burgdorferi arthritis development, termed Bbaa1-6 (23, 24). Further analysis of the Bbaa2 mouse congenic line led to the identification of Gusb as a novel arthritis susceptibility gene (25) and validated the utility of this rigorous and unbiased approach. Importantly, the Weis group also conducted an unbiased screen using microarray technology to identify gene expression differences in C3H and B6 joint tissue that promote arthritis development (26). This thesis shows that these two independent approaches have converged on the identification of a pathologic type I IFN response, which is further elaborated below. The Interferon Family Interferons (IFNs) are a major family of cytokines first identified for their ability to interfere with viral replication (27). IFNs are divided into three classes (type I, II, or III) based on the receptors in which they interact (IFNAR1/2, IFNGR1/2, or IFNLR1/IL10R2, respectively) (28, 29). Type I IFN is the largest class, consisting of IFN-α, β, ε, κ, ω, or ζ (aka limitin) proteins, and is further expanded by numerous IFN-α subtypes (13 in humans and 14 in mice, not including pseudogenes) (30-32). Type II IFN consists of a single IFN-γ protein (33), and type III IFN is represented by four IFN-λ subtypes (29). 5 Although the IFN classes (as well as members) have distinct upstream triggers, downstream IFN-stimulated gene (ISG) profiles, and may be cell type or tissue-restricted, each plays a vital role in immunomodulation-with balanced regulation being key to their antimicrobial success (29, 33-35). Pathologic Type I Interferon in Lyme Disease Soon after the protective role of IFNs was established, their dichotomous ability to promote pathogenesis became evident (28, 33, 36). In the case of Borrelia burgdorferi infection, IFN pathogenicity was first suspected when an unbiased microarray analysis identified a preclinical IFN profile in the joint tissue of Lyme arthritis susceptible C3H mice that was absent in Lyme arthritis resistant B6 mice (26). Although the ISGs identified by the microarray could have been induced by type I or II IFNs (26), IFN-γ had already been shown to be dispensable for Lyme arthritis development in C3H mice (37). Thus, the contribution of type I IFN was assessed by IFNAR1 mAb blockade (38) and genetic ablation (39) and revealed a significant (and equivalent) reduction in Lyme arthritis severity in C3H mice, formally linking pathologic production of type I IFN to arthritogenesis. Intriguingly, an independent forward genetics approach also revealed the type I IFN gene cluster in the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1), a QTL regulating arthritis severity (23, 24). Further analysis of Bbaa1 is the subject of Chapters 2 and 3. The surprising finding that B. burgdorferi, an extracellular pathogen, induces type I IFN production in mice has also been observed in humans (40, 41), and multiple studies have been conducted in both human and murine cells in order to better understand the 6 mechanism of B. burgdorferi-triggered IFN-induction. Specifically, B. burgdorferi factors capable of inducing a type I IFN profile include the surface lipoprotein OspA, various secreted non-nucleic acid ligands, RNA, linear plasmid 36, and other dissemination factors (42-46). Host pattern recognition receptors (PRRs) involved in IFN-induction in response to B. burgdorferi include NOD2 (47), TLR2 (48, 49), TLR8 (in human monocytes) (43, 50), TLR7 and TLR9 (in human peripheral blood mononuclear cells (51) and dendritic cells (44)), and there seems to be a MyD88- and TRIF-independent pathway (52) but details remain to be elucidated. In regard to the initiating cell type, there is a unique requirement for phagocytosis and degradation of B. burgdorferi (38, 47, 49, 50). This was corroborated by the finding that CD45+ cells harvested from a naïve mouse joint are capable of generating an IFN profile upon B. burgdorferi stimulation, whereas CD45- cells are only capable of responding to exogenous IFN-β protein (39). Undoubtedly, IFN-induction in response to B. burgdorferi is complex, but the empirical identification of a differential type I IFN response between B6 and C3H mice (26, 38, 39) that was also suggested by forward genetics (23, 24) has provided a unique platform to uncover novel genetic mechanisms of type I IFN regulation. Pathologic Type I Interferon in Other Diseases There are additional links between arthritis and type I IFN in other diseases. Specifically, arthritis is a documented side effect of hepatitis C (53) and multiple sclerosis (54) patients treated with type I IFN. Arthritis is also a common symptom of systemic lupus erythematosus patients, who, for still unclear etiological reasons, 7 aberrantly produce type I IFN (55). And, a subset of rheumatoid arthritis patients, particularly those who fail to respond to TNF-α blockade, display an IFN signature (56) and will require novel treatment strategies (57). Moreover, a number of viral infections including human immunodeficiency virus (58), hepatitis C virus (59), and persistent lymphocytic choriomeningitis virus (60) induce a type I IFN profile that has been correlated with disease severity. Other bacteria that have been reported to use type I IFN to their advantage include Listeria monocytogenes, Francisella tularensis, and Mycobacterium tuberculosis (28). Other autoimmune diseases that have been associated with dysregulated type I IFN include Sjögren's syndrome (61), scleroderma (62), type I diabetes (63), and systemic sclerosis (64). Thus, pathologic production of type I IFN is highly relevant to a plethora of human diseases, and insights gleaned from our model may transcend Lyme disease. Preview of Dissertation Research This dissertation focuses on examining the role of Bbaa1 (the QTL that harbors the type I IFN locus (23, 24)) in modulating Lyme arthritis severity. In Chapter 2, we developed Bbaa1 interval specific congenic lines on the B6 and C3H backgrounds and tested the hypothesis that type I IFN within Bbaa1 drives Lyme arthritis severity. In Chapter 3, we assessed the specific contributions of IFN-α and IFN-β to Lyme arthritis severity, refined the Bbaa1 congenic line, and utilized RNA-seq to elucidate the mechanism of Bbaa1-directed Lyme arthritis development. In addition to the novel findings presented herein, this work will provide the foundation for uncovering novel genetic mechanisms of type I IFN regulation, with vast potential to impact human 8 disease. References 1. Burgdorfer, W., A. Barbour, S. 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A. Todd, E. Bonifacio, C. Wallace, and A.-G. Ziegler. 2014. Transcriptional signature precedes autoimmunity in children genetically at risk for type 1 diabetes. Diabetes 63: 2538- 2550. 64. Guo, X., B. W. Higgs, A. C. Bay-jensen, M. A. Karsdal, Y. Yao, L. K. Roskos, and W. I. White. 2015. Suppression of T cell activation and collagen accumulation by an anti-IFNAR1 mAb, anifrolumab, in adult patients with systemic sclerosis. J Invest Dermatol 135: 2402-2409. CHAPTER 2 BORRELIA BURGDORFERI ARTHRITIS-ASSOCIATED LOCUS BBAA1 REGULATES LYME ARTHRITIS AND K/B×N SERUM TRANSFER ARTHRITIS THROUGH INTRINSIC CONTROL OF TYPE I IFN PRODUCTION Originally published in The Journal of Immunology. Ying Ma, Kenneth K.C. Bramwell, Robert B. Lochhead, Jackie K. Paquette, James F. Zachary, John H. Weis, Cory Teuscher and Janis J. Weis. 2014. Borrelia burgdorferi Arthritis-Associated Locus Bbaa1 Regulates Lyme Arthritis and K/B×N Serum Transfer Arthritis through Intrinsic Control of Type I IFN Production. J Immunol 193:6050-6060. Copyright © 2014 by The American Association of Immunologists, Inc. Reprinted with permission from publisher. 15 16 17 18 19 20 21 22 23 24 25 CHAPTER 3 DYSREGULATED PRODUCTION OF IFN-β BY BORRELIA BURGDORFERI ARTHRITIS-ASSOCIATED LOCUS 1 (BBAA1) DRIVES LYME ARTHRITIS THROUGH MYOSTATIN UPREGULATION 27 Abstract Forward genetics previously identified a genetic locus, denoted B. burgdorferi arthritis-associated locus 1 (Bbaa1), which regulates Lyme arthritis through heightened production of type I IFN and which physically encompasses the type I IFN gene cluster. In this study, mAb blockade of IFN-β, but not IFN-α, suppressed Lyme arthritis development in B6.C3-Bbaa1 mice. Bbaa1 regulation of IFN-β was also identified in bone marrow-derived macrophages stimulated with B. burgdorferi, and was responsible for feed-forward amplification of interferon-stimulated genes. Reciprocal radiation chimeras between B6.C3-Bbaa1 and B6 mice revealed that arthritis is initiated by radiation-sensitive cells, but orchestrated by radiation-resistant components of the joint tissue. Advanced congenic lines were developed in order to reduce the physical size of the Bbaa1 interval, and confirmed the contribution of type I IFN genes to Lyme arthritis. RNA-seq of the resident CD45- joint cellular fraction from advanced interval specific recombinant congenic lines (ISRCL4 and ISRCL3) identified myostatin as uniquely upregulated in association with Bbaa1 arthritis development, and demonstrated myostatin expression to be linked to IFN-β production. Furthermore, in vivo inhibition of myostatin suppressed Lyme arthritis in Bbaa1 (ISRCL4) congenic mice, formally implicating myostatin as a downstream mediator of joint-specific inflammatory response to B. burgdorferi. These findings suggest a previously unappreciated role for myostatin in Lyme arthritis development. 28 Introduction Lyme disease, caused by infection with the bacteria Borrelia burgdorferi, affects 300,000 Americans each year (1). Disease outcomes range from acute to chronic, sometimes resulting in irreversible damage to the nervous (2) and cardiovascular (3, 4) systems. Arthritis is the most common late disease manifestation (5), affecting up to 60% of patients and persisting in 10% of patients despite antibiotic therapy (6). Lyme arthritis is frequently characterized by synovitis at one or both knee joints and may persist for years after infection (6, 7). While both bacterial and host factors contribute to the spectrum of Lyme disease severity (8), many studies have shown that Lyme arthritis results from a genetically determined dysregulated immune response (9-17). The spectrum of disease severity in humans can be modeled in inbred strains of mice, which display different disease outcomes upon infection with B. burgdorferi (13). Specifically, C57BL/6 (B6) mice develop mild arthritis and C3H mice develop severe arthritis in response to the same B. burgdorferi inoculum. We have successfully employed these mice during empirical (18, 19) and forward genetic (20-22) approaches to identify genetic determinants of Lyme arthritis severity. Empirical approaches comparing the global gene expression profiles of B6 and C3H joint tissue revealed a type I IFN signature in C3H mice that is absent from B6 mice (18), and a receptor-blocking antibody and receptor ablation formally linked type I IFN expression to Lyme arthritis severity. Independently, forward genetic approaches identified a quantitative trait locus (named Bbaa1) that contains the type I IFN gene cluster and controls Lyme arthritis severity (20). An interval specific congenic line in which the C3H Bbaa1 allele was introgressed onto the B6 background (B6.C3-Bbaa1) displays increased Lyme arthritis 29 relative to B6 mice (20) unless pretreated with the type I IFN receptor-blocking antibody (21), formally linking the expression of type I IFN within Bbaa1 to increased Lyme arthritis. Interestingly, Bbaa1 was also found to regulate rheumatoid arthritis (RA) severity through type I IFN production (21), indicating that insight gleaned from our model extends to a distinct inflammatory arthritis. Our finding of a pathologic type I IFN profile is consistent with several publications that have shown a type I IFN signature in the serum of human Lyme patients at stages of active disease (2, 23) and in the synovial fluid of treatment-refractory RA patients (24). Arthritis is also a transient side effect in hepatitis C and multiple sclerosis patients treated with IFN-α/β (25, 26), further supporting the linkage between elevated levels of type I IFN and arthritis. However, type I IFN is a key element in the innate and adaptive response against a variety of microbial infections, posing an inherent problem to treatment blockade. Because type I IFN regulation requires a sensitive balance, and because IFN-α and IFN-β members play unique roles in various infections, we sought to identify the specific type I IFN driving Lyme arthritis pathogenesis. Our unique genetic tools have allowed us to separate type I IFN production from the IFN response (which is triggered immediately upon cytokine sensing), and to look at type I IFN production in isolation from other QTL contributing to Lyme arthritis severity (20). Herein, we used the B6.C3-Bbaa1 mouse to identify the specific type I IFN member that causes Bbaa1 Lyme arthritis, segregated cells that initiate type I IFN in response to B. burgdorferi from those that respond to it, and identified myostatin as an IFN-regulated mediator of Lyme arthritis development. 30 Materials and Methods Mice B6 and C3H mice were obtained from Jackson Laboratories. B6.C3-Bbaa1 mice (Chr4: 9.32-94.97 Mbp) were previously generated (20) and maintained as a colony in our Animal Research Center. Interval specific recombinant congenic lines (ISRCL1-4) with Chr4 Bbaa1 intervals 11.6 - 77.8 Mbp, 76.48 - 93.46 Mbp, 83.7 - 93.46 Mbp, and 88.3 - 93.46 Mbp were generated through repeated backcrosses of B6.C3-Bbaa1 to the parental B6 line. Filial offspring were selected based on SNP identification by highresolution melting analysis as described (27), and homozygous lines were fixed by mating littermates. All mice used in this study were housed in the University of Utah Animal Research Center (Salt Lake City, UT) and handled in accordance with protocols approved by the institutional review committee. B. burgdorferi cultures, infections, and arthritis assessments B. burgdorferi strain N40 was cultured for 4 days in Barbour-Stoenner-Kelly II medium containing 6% rabbit serum (Sigma-Aldrich). Mice were infected with 2×104 live spirochetes intradermally into the skin of the back (28). Arthritis was assessed by rear ankle joint measurements obtained with a metric caliper at days 0 and 28 post infection, and by histopathological analysis of the most severely swollen rear ankle joint following fixation, decalcification, and H&E staining as described previously (29). Histopathological scores were determined blindly and ranged from 0 to 5 for various aspects of disease, including severity and extent of the lesion, PMN leukocyte and mononuclear cell (e.g. monocyte, macrophage) infiltration, tendon sheath thickening (e.g. 31 synoviocyte and fibroblast hyperplasia), and reactive/reparative responses (e.g. periosteal hyperplasia and new bone formation and remodeling), with 5 representing the most severe lesion and 0 representing no lesion. Infection was confirmed in mice euthanized 28 days post infection by ELISA quantification of B. burgdorferi specific IgG concentrations in serum (30) and 16S rRNA B. burgdorferi transcripts in joints (31). Inhibitory reagents and treatments Monoclonal Abs were used to neutralize murine IFNAR1 (MAR1-5A3), IFN-α (TIF-3C5), or IFN-β (HDβ-4A7) (Leinco Technologies, Inc.). TIF-3C5 is considered a "pan-IFN-α" mAb because it neutralized all IFN-α subtypes available for testing: αA, α1, α4, α5, α11, and α13 (32). Mice received mAbs or isotype controls by intraperitoneal injection following a dosage regimen described previously (32, 33). Briefly, antiIFNAR1 was administered in a single 2.5mg dose the day before infection with B. burgdorferi (19, 21, 33), anti-IFN-β was administered in two doses (300µg each, 600µg total) the day before and 2 days following infection, and anti-IFN-α was administered in three doses (333µg each, 1mg total) the day before and days 1 and 3 post infection. Boosts were determined based on pharmacokinetics previously reported (32) and in consideration with the IFN profile peak 7 days post infection with B. burgdorferi (18). For BMDM experiments, 10µg/ml of each mAb was administered in combination with the stimulation. The myostatin inhibitor (MBP-fMSTNpro45-100-Fc) is a recombinant peptide derived from the prodomain of full-length myostatin protein, as described (34). Mice received four intraperitoneal injections of vehicle (PBS) or myostatin inhibitor (400µg each, 1.6mg total) once weekly beginning the day after infection. 32 Generation of reciprocal radiation chimeras Chimeras were generated in all pairwise combinations between B6 (CD45.1), B6 (CD45.2), and B6.C3-Bbaa1 (CD45.2) using a rapid reconstitution protocol as described (35, 36). Rapid reconstitution involves transplanting donor splenocytes into irradiated recipient mice and allows for the infection of young mice <8 wk old, necessary for a reliable arthritis phenotype. Chimerism was evaluated by flow cytometric analysis of peripheral blood leukocytes 25 days post transplant (Figure 3.S3A). Isolation of CD45- cells from joint tissue Mouse rear ankle joints were gently digested into single-cell suspensions as described (36). Briefly, skin was removed and tibiotarsel tissue was teased away from bone using 20-gauge syringe needles followed by 1 hr incubation at 37oC in RPMI 1640 containing 0.2mg/ml endotoxin-free Liberase TM (Roche) and 100µg/ml DNase I (Sigma-Aldrich). Single-cell suspensions were filtered through a 100µm cell strainer, RBCs were lysed in ammonium-chloride-potassium buffer, and cells were labeled with biotinylated anti-CD45.2 (BioLegend) followed by streptavidin magnetic bead labeling (Miltenyi Biotec). Magnetic bead separation was performed on MS columns (Miltenyi Biotec) according to the manufacturer's instructions. Flow cytometric analysis revealed >85% purity in the CD45- fraction, as previously reported (36). CD45- cells from both rear ankle joints of two mice were pooled for each n sample in order to increase RNA concentration for transcript analysis (Figures 3.6 and 3.7A). For ex vivo stimulation, CD45- cells from both rear ankle joints of >8 mice were pooled, plated in RPMI 1640 containing 2% FBS (36), and stimulated with live B. burgdorferi (6×106/ml) or 100U/ml 33 IFN-β (PBL Laboratories) for 3 hr (Figure 3.7B). Cell culture Bone marrow-derived macrophages (BMDMs) were prepared by culturing bone marrow isolated from the femurs and tibias of mice for 7 days in L929 cell-conditioned media as a source of M-CSF, as previously described (37). Harvested macrophages were then replated in 24-well dishes at a density of 6×105/ml in media containing 1% of the serum replacement Nutridoma (Roche). Cultures were stimulated with live B. burgdorferi (6×106/ml) for 6hr at 37oC with 5% CO2. Flow cytometric analysis of P-Stat 1 Phosphorylated Stat1 was stained as described (32, 38) following 15hr incubation with B. burgdorferi and type I IFN blocking mAbs. Briefly, cells were incubated in 0.05% trypsin (Fisher Scientific) and scraped to detach from plate. Cells were fixed using 1.5% paraformaldehyde and permeabilized with 100% methanol. P-Stat1 was stained using an unconjugated Phospho-Stat1 mAb (pY701, Cell Signaling) at a 1:200 dilution and incubated for 1 hr at RT, followed by a secondary stain with Goat anti-Rabbit IgG conjugated to Alexa Fluor 647 (Fisher Scientific) at a 1:500 dilution and incubated for 30 min at RT. Data were collected on a FACSCanto II (BD Biosciences) flow cytometer and analyzed using FlowJo v.10.0.8 software. 34 Gene expression analysis Total RNA was recovered using TRIzol reagent (Invitrogen) and purified using the Direct-zol RNA MiniPrep kit (Zymo Research). For RNA-sequencing, libraries were prepared using PolyA enrichment and sequenced with Illumina HiSeq 50 Cycle SingleRead Sequencing version 4 at the University of Utah High Throughput Genomics Core Facility (Salt Lake City, UT). Sequences were aligned and annotated with help from the University of Utah Bioinformatics Core Facility (Salt Lake City, UT). For qRT-PCR analyses, RNA was reverse transcribed, and transcripts were quantified using a Roche LC-480 according to our previously described protocols (20). Primer sequences used in this study for β-actin, Iigp (18), Oasl2, Cxcl10, Tyki (19), Gbp2 (39), Tnfa, and Ifnb (40) can be found at the indicated citations. Mstn primer sequences designed in this study were as follows: forward (5'-GATCTTGCTGTAACCTTCCC-3') and reverse (5'CTCCTGAGCAGTAATTGGC-3'). Data and statistical analyses All graphical data depict the mean + SEM. Statistical analyses were performed using GraphPad Prism 7.0b software. Multiple-sample data sets were analyzed by oneway ANOVA with Dunnet's post hoc test for pairwise comparisons. Two-sample data sets were analyzed by Student t test (2-tailed). Categorical histopathology scores were assessed by the Mann-Whitney U test. Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) is indicated. 35 Results Determination of the proarthritogenic type I IFN cytokine in B6.C3-Bbaa1 mice Both empirical and forward genetic studies from our lab have converged on the finding that a pathologic type I IFN profile drives Lyme arthritis in C3H mice (18-21, 36). We identified the type I IFN gene cluster in Bbaa1, a QTL regulating Lyme arthritis severity, and generated a congenic line, denoted B6.C3-Bbaa1 (Chr4: 9.32-94.97 Mbp), wherein we established that dysregulated production of type I IFN within Bbaa1 drives Lyme arthritis (21). Nevertheless, there are multiple members of the type I IFN family, with the IFN-α subtypes and IFN-β playing the largest roles in pathogenesis to date, and although each signals through a shared receptor heterodimer, their unique roles in host protection and pathogenesis are becoming increasingly appreciated (32, 41). To assess the specific contribution of IFN-α and IFN-β to Lyme arthritis development, B6.C3-Bbaa1 mice were treated with a pan-acting mAb that blocks multiple IFN-α subtypes (TIF-3C5) or a mAb that targets IFN-β specifically (HDβ-4A7). We previously showed that the anti-IFNAR1 mAb (MAR1-5A3) suppresses arthritis development to the baseline of B6 (21). Because blocking IFN-β resulted in the same reduction in Lyme arthritis as blocking IFNAR1 (Figure 3.1A), we conclude that IFN-β and not IFN-α is the proarthritic cytokine generated in B6.C3-Bbaa1 mice. Analysis of anti- B. burgdorferi IgG in the serum and B. burgdorferi 16S rRNA transcripts in the joint confirm that blocking type I IFN does not impact the ability of the host to generate a Bcell response or to control infection (Figure 3.1B). These findings underscore the fact that Bbaa1 intrinsically regulates pathologic production of IFN-β in vivo, and IFN-β does not 36 protect the host from B. burgdorferi expansion. Effect of blocking IFN-α or IFN-β on type I IFN activation in BMDMs We previously showed that BMDMs from B6.C3-Bbaa1 mice express higher levels of IFN-inducible transcripts in response to B. burgdorferi than do BMDMs from B6 mice (21). Thus, B6.C3-Bbaa1 BMDMs were used as a surrogate for myeloid cells in joint tissue to assess the specific contribution IFN-α or IFN-β to the Bbaa1-directed induction of type I IFN. BMDMs were stimulated with B. burgdorferi and treated with each mAb for 6 hr. Treatment with anti-IFN-β resulted in complete suppression of IFN inducible genes, indistinguishable from levels of macrophages treated with IFN receptor blockade (Figure 3.2A). In contrast, treatment with anti-IFN-α had no effect on IFNinduction, indicating that IFN-β, and not IFN-α, is regulated by Bbaa1 in response to B. burgdorferi. Isotype control mAbs had no effect (Figure 3.S1). Importantly, none of these treatments impacted the expression of Tnfa (Figure 3.2A), which is downstream of the critical MyD88-dependent host defense pathway (42, 43). This highlights the in vivo observation that type I IFN is not required for host defense in Lyme arthritis (Figure 3.1B) and its suppression does not influence the expression of initial MyD88-dependent cytokines (21, 36) Flow cytometric analysis of phosphorylated Stat1 protein was used as a complementary approach to determine the impact of IFN-α and IFN-β protein on the feed-forward IFN amplification. Stat1 is rapidly phosphorylated when IFN-α and IFN-β proteins bind the receptor (32, 44), but stimulation with B. burgdorferi requires time for transcription and translation of type I IFNs. Therefore, Stat1 phosphorylation was 37 assessed in BMDMs 15 hr post stimulation with B. burgdorferi and mAb treatment. Strikingly, IFN-β neutralization completely inhibited Stat1 phosphorylation, similar to levels in macrophages treated with receptor blocking mAb, while blocking IFN-α had no effect (Figure 3.2B). Because anti-IFN-α did not impact arthritis severity or macrophage responses to B. burgdorferi, mAb functionality was assessed (Figure 3.S2). Confirmation of anti-IFN-α activity supports the conclusion that IFN-β (but not IFN-α) is the type I IFN made in response to B. burgdorferi both in vivo and in vitro, and that low levels of IFN-α do not contribute to response during the feed-forward amplification. Does Bbaa1 initiate arthritis through hematopoietic or resident cells? Multiple lines of evidence point to the fact that IFN-β is pathologic in Bbaa1 congenic mice, leading to the question of whether Bbaa1 exerts its proarthritic effect through hematopoietic or resident cells. The Bbaa1 congenic mouse is a unique tool that allows the interrogation of IFN signaling independent of feed-forward IFN amplification because Bbaa1 initiates IFN-β production. MHC compatibility between mildly arthritic B6 mice and more severely arthritic B6.C3-Bbaa1 mice allowed reciprocal radiation chimeras to be generated to specifically determine if Bbaa1 exerts its proarthritic effect through radiosensitive-hematopoietic cells or radioresistant-resident cells in the joint (Figure 3.3A). Chimeras were generated using a rapid reconstitution protocol (35, 36), and reconstitution was determined to be adequate for host defense based on the similar abilities of irradiated mice to generate a B-cell dependent antibody response and sufficient myeloid cells to control infection (Figure 3.S3). As expected, B6 mice reconstituted with autologous splenocytes (B6→B6) 38 developed mild arthritis and B6.C3-Bbaa1 mice reconstituted with autologous splenocytes (Bbaa1→Bbaa1) developed more severe arthritis 4 wk post infection with B. burgdorferi (Figure 3.3B). Notably, B6.C3-Bbaa1 mice reconstituted with B6 cells (B6→Bbaa1) developed mild arthritis similar to B6→B6 chimeras, whereas B6 mice reconstituted with B6.C3-Bbaa1 cells (Bbaa1→B6) developed severe arthritis indistinguishable from Bbaa1→Bbaa1 chimeras (Figure 3.3B). Thus, Bbaa1 regulates Lyme arthritis severity through the radiosensitive, hematopoietic cellular constituents of the joint. Considering that Bbaa1 controls the magnitude of B. burgdorferi-triggered IFNβ production, we can further deduce that radiosensitive cells in the joint, which are likely myeloid (36), initiate the production of proarthritic IFN-β (Figure 3.3C). This is consistent with our previous findings that CD45+ cells harvested from a naïve mouse joint possess the unique ability to generate an IFN profile upon ex vivo stimulation with B. burgdorferi, whereas CD45- cells are capable of responding to but not initiating type I IFN in response to B. burgdorferi (Figure 3.S4 and Lochhead et al. (36)). Another striking finding is that Bbaa1→B6 chimeras develop the full arthritis phenotype, strongly implicating that regardless of genotype, radioresistant-resident cells are fully competent to choreograph arthritis manifestation in the joint (Figure 3.3, B and C). This is consistent with the previous finding that endothelial cells and fibroblasts, both of which are radioresistant cell types in the joint, are major responders to and amplifiers of type I IFN resulting in production of chemokines in infected C3H mice (36). This provides the first in vivo evidence for the "pass off" that occurs between cells that initiate IFN-β in response to B. burgdorferi and cells that respond to IFN-β to direct arthritis development (Figure 3.3C). 39 C3H-derived Bbaa1 genes spanning the type I IFN locus confer increased arthritis on B6 background Because the original B6.C3-Bbaa1 congenic interval was very large (Chr4: 9.32 - 94.97 Mbp) with >450 genes in addition to Ifnb, it was necessary to reduce the physical interval by backcrossing B6.C3-Bbaa1 to the parental B6 line. This led to the generation of four new Bbaa1 interval specific recombinant congenic lines (denoted ISRCL1-4) harboring various subintervals of Bbaa1 (Figure 3.3). ISRCL1 contains the largest portion of C3H Chr4 but excludes the C3H type I IFN locus, while ISRCL2-4 retains the C3H type I IFN gene cluster with further reduction in the amount of C3H donor sequence (Figure 3.4). After infection with B. burgdorferi, congenic lines retaining C3H-derived Bbaa1 genes spanning the type I IFN locus displayed more severe arthritis compared to B6 mice (Figure 3.4). Importantly, ISRCL3 mice exhibit the same arthritis phenotype as B6.C3-Bbaa1 mice, indicating that all of the genes required for maximal Bbaa1 arthritis are contained within Chr4: 83.7 - 93.46 Mbp. Interestingly, ISRCL4 mice displayed a submaximal phenotype that was still significantly increased compared to B6 mice. This indicates that there are 2 loci regulating maximal Bbaa1 arthritis: the first locus is shared by ISRCL3 and ISRCL4 mice and spans the type I IFN gene cluster, while the second locus is only contained in ISRCL3 mice and is outside of type I IFN genes. Nevertheless, because blocking IFN-β in the full-length B6.C3-Bbaa1 congenic line completely suppresses Lyme arthritis (Figure 3.1A and Ma et al. (21)), we can infer that the phenotype of both of these loci is dependent on IFN-β production. Next we assessed the magnitude of the IFN response to B. burgdorferi in BMDMs from ISRCL3 and ISRCL4 mice, which both possess the C3H allele for the type I IFN 40 locus. Importantly, BMDMs from ISRCL3 and ISRCL4 mice displayed greater IFN transcriptional responses than BMDMs from B6 mice, with magnitudes similar to full length B6.C3-Bbaa1 BMDMs (Figure 3.5). This indicates that genes responsible for IFNβ dysregulation are retained in the ISRCL4 Bbaa1 locus (Chr4: 88.3 - 93.46 Mbp). As shown, Ifnb expression is low, but none of the 14 IFN-α transcripts were detectable, further supporting the role of IFN-β in driving Lyme arthritis pathogenesis. The fact that ISRCL3 & ISRCL4 mice display greater arthritis severity and their macrophages express more Ifnb compared to B6 macrophages provides a refined tool to study Bbaa1 Lyme arthritis. How does Bbaa1 change the transcriptome of the radioresistant-resident responders? Now that we have established the role of radioresistant-resident cells in directing Bbaa1 Lyme arthritis downstream of IFN-β, we pursued a better understanding of the mechanism of arthritis development through transcriptome analysis of CD45- (resident) joint cells. Newly developed ISRCL3 and ISRCL4 lines were compared to B6 mice in order to capture all genes related to IFN-β production and arthritis development with a minimal physical interval. To capture the stage of active arthritis development (as opposed to full-blown arthritis with confounding wound repair pathways), joint cells were harvested from mice 22 days post infection with B. burgdorferi. Following recovery of single-cell suspensions, the CD45- population was isolated by magnetic bead separation and RNA-seq was performed. Not surprisingly, comparing the transcriptomes of these highly similar genotypes 41 at a time point submaximal to arthritis development revealed very few differences in gene expression profiles (Figure 3.6A). Of those differences, only five genes were independently identified in both ISRCL4 vs. B6 and ISRCL3 vs. B6 comparisons with >1.5-fold change and p-adj <0.05 (Figure 3.6A & Figure 3.S5), supporting their involvement in Bbaa1-directed arthritis. Importantly, myostatin (Mstn) had the greatest induction and achieved the highest level of significance in both analyses (Figure 3.6A & Figure 3.S5), illuminating it as a strong candidate for Bbaa1 Lyme arthritis development. Quantitative RT-PCR analysis of Mstn expression in CD45- cells from uninfected animals revealed similar expression among uninfected mice of all three genotypes (data not shown), and revealed that Bbaa1 specifically regulates the induction of myostatin following infection with B. burgdorferi (Figure 3.6B). This was a striking finding for Bbaa1 Lyme arthritis development in light of recent evidence that myostatin is involved in RA development in humans and mice (45). Impact of IFN-β on myostatin expression in CD45- cells isolated from joint tissue To assess the connection between IFN-β production and myostatin expression in Bbaa1 Lyme arthritis, IFN-β was blocked in ISRCL4 and ISRCL3 mice infected with B. burgdorferi (as described in Figure 3.1). CD45- cells were isolated from joint tissue 22 days post infection, and myostatin expression was assessed by qRT-PCR. Importantly, CD45- cells from ISRCL4 and ISRCL3 mice infected with B. burgdorferi and treated with anti-IFN-β mAb expressed nearly 2-fold fewer Mstn transcripts compared to mice treated with isotype control (Figure 3.7A). The fold suppression by blocking IFN-β is 42 internally consistent with the level of induction found in our RNA-seq (Figure 3.6). This directly connects the Bbaa1-dependent production of IFN-β to Mstn upregulation in vivo. Several proinflammatory cytokines, TNF-α, IL-1α, and IL-17, were previously shown to directly induce expression of myostatin in the context of RA, but the impact of IFN-β was not addressed (45). To test whether IFN-β directly induces expression of myostatin, CD45- cells were isolated from the joints of naïve B6 mice and stimulated ex vivo with IFN-β, B. burgdorferi, or IFN-β and B. burgdorferi in combination. Mstn expression assessed by qRT-PCR revealed that IFN-β works synergistically with B. burgdorferi to directly induce Mstn expression (Figure 3.7B). To our knowledge, this is the first report of IFN-β induction of myostatin. Inhibition of myostatin suppresses Lyme arthritis in Bbaa1 congenic mice In order to determine whether myostatin is a marker or a mediator of Bbaa1 Lyme arthritis, infected ISRCL4 mice were treated with a highly effective myostatin propeptide (34) to inhibit myostatin protein activity during arthritis development. Consistent with the dual requirement of B. burgdorferi and IFN-β for myostatin upregulation (Figure 3.7, A and B), myostatin inhibition did not impact ankle swelling in uninfected animals (Figure 3.8). However, myostatin inhibition in infected ISRCL4 congenic mice led to a remarkable suppression in arthritis 4 wk post infection with B. burgdorferi (Figure 3.8), similar to the low level normally seen in infected B6 mice (Figure 3.4). These findings demonstrate a direct effect of myostatin on joint-specific inflammatory responses. 43 Discussion In this study, we established IFN-β as the Lyme arthritis-driving type I IFN that is regulated by a locus, Bbaa1, previously identified by forward genetics. This culminates a decade of our work wherein two parallel pathways of investigation revealed a pathologic type I IFN profile in C3H mice infected with B. burgdorferi that is suppressed by IFNAR1 blockade (14, 18-21, 36). Others have corroborated the association between pathologic production of type I IFN and Lyme disease pathogenesis in murine studies (46, 47) as well as in human patients at various stages of disease (2, 23), and many investigators have examined the type I IFN response to B. burgdorferi in murine and human cells (19, 36, 39, 47-56). But the specific culprit of B. burgdorferi-induced type I IFN pathogenicity has remained elusive due to the transient expression of IFN-α/β (57, 58), overlapping interferon-stimulated gene pathways induced by IFN-α/β (59), and differing contexts of B. burgdorferi infection in which IFN-α/β transcripts have been detected. The strength of this study centers on the employment of B6.C3-Bbaa1 mice, which allowed the impact of type I IFN dysregulation to be assessed in isolation from the five other B. burgdorferi arthritis-associated QTL contained within the C3H genome (20), coupled with the use of newly available mAbs to assess IFN-α and IFN-β individually. Multiple mAb experiments along with the transcriptional detection of Ifnb in BMDMs from refined congenic lines (and lack of evidence for Ifna transcripts) revealed that IFN-β is the sole contributor to the type I IFN profile and arthritogenesis in Bbaa1directed Lyme arthritis. Another major finding was that IFN-β is intrinsically controlled within the Bbaa1 locus. The intrinsic regulation of IFN-β within Bbaa1 was surprising 44 due to the lack of coding or regulatory SNPs within 10,000 flanking base pairs of the Ifnb genetic sequence (60, 61) and suggests a novel mechanism for IFN-β regulation, which is a topic of future investigation. Here, the congenic mice provided a unique tool to segregate IFN-β initiation from feed-forward amplification, and radiation chimeras demonstrated that the Bbaa1 arthritis-initiating lineage is radiation-sensitive in the joint. This finding is in contrast to our previous publication on the Bbaa2 QTL, in which the hypomorphic allele of Gusb drives arthritis through the radiation-resistant resident joint population (22). Thus, our forward genetic approach has identified two distinct QTLs that regulate Lyme arthritis through independent mechanisms and responsible initiating tissues. In the case of Bbaa1, the discrimination among cell types along with refined congenic lines was critical to understanding the mechanism of arthritis development. An unbiased RNA-seq on resident (CD45-) joint cells led to the third major finding that Bbaa1-directed production of IFN-β causes myostatin upregulation. This was surprising given that myostatin is widely appreciated for its role as a negative regulator of skeletal muscle growth and regeneration (62), and intensely investigated for its positive impact on food production in the animal agriculture industry. Nevertheless, we were able to utilize newly developed reagents in the field of muscle development to inhibit myostatin protein activity in vivo and discovered that myostatin is a direct mediator of Bbaa1 Lyme arthritis development. Strikingly, myostatin was recently found to be a mediator of a distinct inflammatory arthritis using the TNF-α overexpression model of RA in mice, and found to be expressed by synovial fibroblasts from patients with RA (45). Although we do not completely understand the mechanism of myostatin-mediated 45 inflammation in Lyme arthritis development, myostatin promotes RA in TNF-α overexpressing mice by activating osteoclast differentiation (45). Although bone pathologies have not been widely investigated in Lyme arthritis patients or mice, Tang et al. recently identified B. burgdorferi infection in mice bone, but implicated osteoblast inhibition in bone destruction (63). Interestingly, Hodzic et al. have also identified B. burgdorferi in quadriceps muscle (64, 65), suggesting that a more global dysregulation of bone and muscle homeostasis could be involved in Lyme arthritis development than previously appreciated. Future studies are needed to investigate the role of myostatin in osteoclasts and other tissues of the joint. Herein, we modeled the power of forward genetics in allowing the impact of genetics on pathogenic responses to be directly assessed, and this is the second report of forward genetic identification of novel genes associated with Lyme arthritis development (22). Identifying IFN-β as the pathologic type I IFN in Bbaa1 Lyme arthritis parallels the finding that IFN-β is more pathologic than IFN-α in RA patients (66), and underscores the need to assess IFN-α/β ratios in serum from Lyme patients as an indicator of disease severity. The more selective blockade of a single IFN may also be more amendable to clinical outcomes where the entire type I IFN response has not been ablated, and myostatin may be a tantalizing target for therapeutic intervention that is outside of the conventional inflammatory host defense (67). 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B6.C3-Bbaa1 mice were infected with 2 x 104 B. burgdorferi and treated with anti-IFNAR1 (MAR1-5A3), anti-IFN-α (TIF-3C5) or anti-IFN-β (HDβ-4A7) as described in Materials and Methods (n = 5 to 6 mice per group). (A) Arthritis was assessed 4 wk post infection, and shown for change in ankle measurement and overall lesion scores. (B) Host defense was assessed by anti-B. burgdorferi IgG in the serum and B. burgdorferi 16S rRNA transcripts in the joint. Statistical significance for ankle swelling, serum IgG, and bacterial numbers in the joint were determined by 1-way ANOVA followed by Dunnett's multiple comparison test versus isotype, and MannWhitney U test was used for overall lesion. *p < 0.05. 54 A. Arthritis severity Arthritis severity isotypes * isotypes * anti-IFNAR1 anti-IFNAR1 * p= 0.06 anti-IFNα anti-IFNα anti-IFNβ anti-IFNβ 0.0 0.1 0.2 0.3 0.4 0.0 Ankle Swelling (mm) B. anti-B. burgdorferi IgG (Serum) isotypes anti-IFNAR1 anti-IFNAR1 anti-IFNα anti-IFNα anti-IFNβ anti-IFNβ 1 2 3 mg/ml (ELISA) 4 1.0 1.5 2.0 B. burgdorferi 16S rRNA (Joint Tissue) isotypes 0 0.5 Overall Lesion Score 0.0 0.1 0.2 0.3 0.4 Copies/1000 β-actin 0.5 55 Figure 3.2. IFN-β drives the type I IFN profile in B6.C3-Bbaa1 BMDMs in response to B. burgdorferi. (A) qRT-PCR analysis of transcripts after 6 hr stimulation with live B. burgdorferi (10:1 MOI) and treatment with anti-IFNAR1, anti-IFN-α, or anti-IFN-β (10µg/ml each). Transcript levels for Oasl2, Gbp2, Cxcl10, Tyki, Iigp, and Tnfa were normalized to β-actin. Data are pooled from two experiments conducted on separate days (n = 4 per group). Significance determined by 1-way ANOVA followed by Dunnett's multiple comparison test versus media. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Flow cytometric analysis of Stat-1 phosphorylation after 15 hr stimulation with B. burgdorferi and treatment with blocking antibodies. Data are representative of three independent experiments. 56 Gbp2 Oasl2 **** 5 0 ** 50 10 40 30 20 10 0 media anti-IFNAR1 anti-IFNα anti-IFNβ anti-IFNAR1 anti-IFNα Tyki Copies / 1000 β-actin Copies / 1000 β-actin 100 50 0 media anti-IFNAR1 anti-IFNα anti-IFNβ B. 150 3 2 1 0 media anti-IFNAR1 anti-IFNα media anti-IFNAR1 anti-IFNα anti-IFNβ Tnfa ** Unstimulated B. burgdorferi alone + anti-IFNAR1 + anti-IFNα + anti-IFNβ P-Stat1 100 anti-IFNβ ** 4 150 200 Iigp **** **** **** 300 0 media **** 200 400 Copies / 1000 β-actin **** Copies / 1000 β-actin Copies / 1000 β-actin 15 Cxcl10 ** Copies / 1000 β-actin A. anti-IFNβ 100 50 0 media anti-IFNAR1 anti-IFNα anti-IFNβ 57 Figure 3.3. Reciprocal radiation chimeras between B6 and B6.C3-Bbaa1 mice. (A) Experimental design: following a lethal dose of irradiation, B6 mice were reconstituted with B6.C3-Bbaa1 splenocytes (Bbaa1→B6) and B6.C3-Bbaa1 mice were reconstituted with B6 splenocytes (B6→Bbaa1). Autologous transplants (B6→B6 and Bbaa1→Bbaa1) were also generated for impact of myeloablative radiation. Arrows indicate direction of transplantation from donor to recipient. (B) Bbaa1 influences arthritis severity through the radiosensitive hematopoietic lineage. Notably, Bbaa1→B6 mice developed the full Lyme arthritis phenotype while B6→Bbaa1 mice were resistant. Arthritis measurements were taken 4 wk post infection with B. burgdorferi (n = 8 to 19 mice per group). Statistical significance was assessed between mice of the same recipient genotype by Student t test for ankle swelling and Mann-Whitney U test for overall lesion. *p < 0.05. (C) Model depicting the cellular "pass off" between radiosensitive myeloid (CD45+) cells that initiate IFN-β in response to B. burgdorferi and radioresistant resident (CD45-) cells that respond to IFN-β and drive arthritis development. 58 A. B6.C3&Bbaa1% or'B6' B6'or## B6.C3&Bbaa1' B6.C3'Bbaa1% B6# B. Bbaa1 → Bbaa1 Bbaa1 → Bbaa1 * p= 0.06 B6 → Bbaa1 B6 → Bbaa1 Bbaa1 → B6 Bbaa1 → B6 * * B6 → B6 B6 → B6 0.0 0.2 0.4 0.6 0.8 1.0 0 Ini,ators) Responders) CD45+% Endothelial% B6.C3+Bbaa1% (CD45+)% Myeloid% ! IFNβ% 2 3 Overall Lesion Score Ankle Swelling (mm) C. 1 C D 4 5 +% Fibroblast% Other%CD45+% Chondrocytes% Muscle%cells% Epithelial%cells% !!! ArthriFs% !!! % 59 Figure 3.4. Physical boundaries of Bbaa1 congenic intervals (left) and Lyme arthritis (right) reveal that mice with C3H-derived genes spanning the type I IFN locus have increased arthritis severity compared to B6 mice. Parentheses indicate the exact interval of each congenic line, and rows represent the genetic composition across Chr4, with C3H-derived regions shaded black and B6-derived regions in white. Arthritis shown for ankle swelling measured 4 wk after B. burgdorferi infection (n = 10 to 35 mice per group). Significance assessed by 1-way ANOVA followed by Dunnett's multiple comparison test versus B6. *p < 0.05, ****p < 0.0001. 60 Strain (Bbaa1 interval) C3H-derived B6-derived Type I IFN locus C3H **** B6 B6.C3-Bbaa1 (11.6 - 94.92) **** ISRCL1 (11.6 - 77.8) ISRCL2 (76.48 - 93.46) **** **** ISRCL3 (83.7 - 93.46) ISRCL4 (88.3 - 93.46) * 0 20 40 60 80 100 Chromosome 4 position (megabases) 0.0 0.2 0.4 0.6 Arthritis severity 0.8 61 Figure 3.5. BMDMs reveal that Bbaa1 genes regulating the magnitude of the IFN response to B. burgdorferi are retained in the genetic intervals of ISRCL3 and ISRCL4 mice. qRT-PCR analysis of transcripts in BMDMs from B6, B6.C3-Bbaa1, ISRCL3, ICRCL4, and C3H mice stimulated with live B. burgdorferi for 6 hr (n = 3 to 4 per group). Transcript levels for Ifnb, Gbp2, Cxcl10, Iigp, and Tyki were normalized to βactin. Significant difference from B6 mice is shown and was determined by Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 0 150 3H 200 C ** L4 2 Tyki B 6. C * **** ** *** 100 50 0 C L4 3H C L3 0 C 400 IS R 50 300 a1 ** ba ** 6 *** B *** Copies / 1000 β-actin Gbp2 IS R 3B L4 L3 a1 3H C C C IS R IS R 6 **** C 4 ba 150 L3 * C 6 B Copies / 1000 β-actin Ifnb IS R *** a1 3B 0.0 100 IS R C L4 L3 3H C C IS R C 6 a1 B ba IS R 3B 0.2 ba 6. Iigp B C 0.4 6 ** Copies / 1000 β-actin 6. * B 3H L4 B * 3B C C L3 a1 6 10 IS R C ba B 8 IS R 3B 0.6 C C ** 6. 6. Copies / 1000 β-actin 0.8 B B Copies / 1000 β-actin 62 Cxcl10 ** ** ** * 200 100 0 63 Figure 3.6. RNA-seq identification of myostatin (Mstn) as the strongest candidate for Bbaa1-directed Lyme arthritis development. (A) Volcano plot depicts log2 fold change (x-axis) and -log10 adjusted p-value (y-axis) of genes identified by ISRCL4 vs. B6 RNAseq comparison. Single genes are plotted as dots, with those achieving significance (p-adj <0.05) colored black (n = 5 per group). Red and blue dashed lines mark 1.5-fold increase in the congenic or B6, respectively. Circled genes also had >1.5-fold change and p-adj <0.05 in the ISRCL3 vs. B6 RNA-seq comparison. (B) qRT-PCR confirmation of Mstn expression in CD45- cells isolated from B6, ISRCL4, and ISRCL3 mice that were infected with B. burgdorferi for 22 days (n = 5 to 6 per group). Mstn transcripts were normalized to β-actin and fold change relative to uninfected levels were calculated for each strain. Significance assessed by 1-way ANOVA followed by Dunnett's multiple comparison test versus B6. **p < 0.01, ***p < 0.001 64 A. − Log10 adjusted p−value 20 Mstn 15 10 5 0 −1.0 −0.5 0.0 0.5 ISRCL4 vs. B6 Log2 fold change Fold change relative to uninfected B. Mstn 3 *** ** 2 1 0 B6 ISRCL4 ISRCL3 CD45- cells from mouse joints (22 dpi with B. burgdorferi) 1.0 65 Figure 3.7. IFN-β and B. burgdorferi are both required for the expression of myostatin by CD45- joint cells during infection and ex vivo. (A.) In vivo mAb blocking of IFN-β (600µg total) prevents transcriptional upregulation of Mstn in CD45- joint cells from ISRCL4 and ISRCL3 mice 22 days post infection with B. burgdorferi (n = 3 to 4 per group). Mstn transcripts were normalized to β-actin and fold change relative to isotype control was calculated for each strain. Significance determined by unpaired Student t test. *p < 0.05, **p < 0.01. (B.) Ex vivo administration of exogenous IFN-β (100U/ml) in combination with B. burgdorferi (10:1 MOI) for 3 hr caused transcriptional upregulation of Mstn in CD45- cells isolated from a naïve B6 mouse joint. Transcripts were normalized to β-actin and fold change was calculated relative to media control. Results are pooled data from two experiments using CD45- cells from 8 or more mice done on separate days (n = 5 wells per group). Significance determined by 1-way ANOVA followed by Dunnett's multiple comparison test versus media. *p < 0.05 66 Fold change relative to isotype A. Mstn 1.5 isotype anti-IFNβ * ** 1.0 0.5 0.0 ISRCL4 ISRCL3 CD45- cells from mouse joints (22 dpi with B. burgdorferi) Mstn * 3 2 1 IF N β ri fe r i+ fe .b ur gd or .b ur gd or B B m IF N β ia 0 ed Fold change relative to media B. CD45- cells from naïve B6 mouse joints stimulated ex vivo 67 Figure 3.8. Myostatin inhibition suppresses the development of Lyme arthritis in Bbaa1 congenic mice. ISRCL4 mice were infected with 2 x 104 B. burgdorferi and treated with a myostatin inhibitor as described in Materials and Methods (n = 3 to 4 mice per group). Arthritis was assessed 4 wk post infection, and shown for change in ankle measurement. Significance was determined by Student t test. ***p < 0.001 68 Ankle Swelling (mm) Arthritis severity 0.5 0.4 PBS Mstn inhibitor *** 0.3 0.2 0.1 0.0 BSK B. burgdorferi 69 Figure 3.S1. Isotype control does not impact the expression of IFN-inducible genes in BMDMs. qRT-PCR analysis of transcripts 6 hr post stimulation with live B. burgdorferi (10:1 MOI) and treatment with anti-IFN-β or isotype control (10µg/ml each). Transcript levels for Tyki, Oasl2, Gbp2, and Iigp were normalized to β-actin. Significance was determined by Student t test comparison to isotype control. **p < 0.01, ****p < 0.0001. 70 Tyki 400 300 200 100 0 media isotype 30 20 10 0 anti-IFNβ Copies / 1000 β-actin Copies / 1000 β-actin 100 50 isotype anti-IFNβ 6 hr post stim with B. burgdorferi isotype anti-IFNβ Iigp 15 ** media media 6 hr post stim with B. burgdorferi Gbp2 150 0 **** 40 6 hr post stim with B. burgdorferi 200 Oasl2 50 **** Copies / 1000 β-actin Copies / 1000 β-actin 500 **** 10 5 0 media isotype anti-IFNβ 6 hr post stim with B. burgdorferi 71 Figure 3.S2. Confirmation of anti-IFN-α functionality. Flow cytometric analysis of Stat1 phosphorylation in BMDMs 15 min post stimulation with exogenous murine IFN-αA alone (3.3ng/ml) or in combination with anti-IFN-α or anti-IFNAR1 (10µg/ml each). Exogenous IFN-αA potently induced Stat1 phosphorylation (black line), isotype control had no effect on induction (dashed line), and anti-IFN-α or anti-IFNAR1 completely blocked phosphorylation of Stat1. 72 73 Figure 3.S3. Radiation chimera reconstitution efficiency and impact on host defense. (A) The percentages of donor-derived and recipient-derived cells in mice that were lethally irradiated and reconstituted with splenocytes, 25 days post transplant. Myeloid compartments were sufficiently reconstituted by donor-derived cells (~80%) prior to infection. B and T cells were about 50% donor-derived, but are dispensable for Lyme arthritis development. Statistical significance assessed by Student t test. ****p < 0.0001. (B) Reconstitution of irradiated mice was adequate for host defense, as measured by antiB. burgdorferi IgG in the serum and B. burgdorferi 16S rRNA transcripts in the joint. 74 donor recipient 100 Percent cells A. **** 80 60 40 20 0 B cells (B220) T cells (CD3e) Møs (F4/80) 25 days post-transplant B. anti-B. burgdorferi IgG (Serum) B. burgdorferi 16S rRNA (Joint Tissue) Bbaa1 →Bbaa1 Bbaa1 →Bbaa1 B6 →Bbaa1 B6 →Bbaa1 Bbaa1 → B6 Bbaa1 → B6 B6 → B6 B6 → B6 0 10 20 mg/ml (ELISA) 30 0.0 0.5 1.0 1.5 Copies/1000 β-actin 2.0 75 Figure 3.S4. Confirmation of cell viability in CD45- cells isolated from naïve B6 mouse joint and stimulated ex vivo. Administration of exogenous IFN-β (100U/ml) for 3 hr caused transcriptional upregulation of IFN-inducible genes (Tyki, Cxcl10, and Gbp2), and addition of B. burgdorferi (10:1 MOI) for 3 hr induced expression of Tnfa. Transcripts were normalized to β-actin. Results are pooled data from two experiments using CD45cells from 8 or more mice done on separate days (n = 5 wells per group). Significance determined by 1-way ANOVA followed by Dunnett's multiple comparison test versus media. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IF N β 0 + 200 ri 80 fe 400 60 i Cxcl10 fe r *** B fe ri + ia fe β ri IF N .b ur gd or .b ur gd or B ed IF N β m β 0 .b ur gd or *** Copies / 1000 β-actin 100 IF N β 800 Copies / 1000 β-actin IF N 25 ia β + 50 ed IF N ri ri 75 m + fe fe ** .b ur gd or ri ri .b ur gd or .b ur gd or Tnfa B fe fe B ia IF N β ed * B .b ur gd or .b ur gd or B m 125 B ia Copies / 1000 β-actin 100 B ed 600 IF N β m Copies / 1000 β-actin 76 Tyki 150 *** **** 50 0 Gbp2 *** **** 40 20 0 77 Figure 3.S5. Volcano plot of genes identified in ISRCL3 vs. B6 RNA-seq comparisons. Plot depicts log2 fold change (x-axis) and -log10 adjusted p-value (y-axis). Single genes are plotted as dots, with those achieving significance (p-adj <0.05) colored black (n = 5 per group). Red and blue dashed lines mark 1.5-fold increase in the congenic or B6, respectively. Circled genes also had >1.5-fold change and p-adj <0.05 in the ISRCL3 vs. B6 RNA-seq comparison. Myostatin (Mstn) was again identified as the most highly induced gene in ISRCL3 mice. 78 − Log10 adjusted p−value 20 Mstn 15 10 5 0 −1.0 −0.5 0.0 0.5 ISRCL3 vs. B6 Log2 fold change 1.0 CHAPTER 4 DISCUSSION 80 Overview Harnessing the power of forward genetics, B6.C3-Bbaa1 mice allowed for mechanistic interrogation of the Bbaa1 QTL, independent from the other 5 QTL regulating Lyme arthritis severity on the C3H background (1, 2). The finding that B6.C3Bbaa1 mice have increased Lyme arthritis compared to B6 mice, but intermediate to C3H mice, confirmed that Bbaa1 is one of multiple Lyme arthritis regulators, and is consistent with our previous identification of another major regulator, Gusb in the Bbaa2 QTL (3). Complete suppression of Lyme arthritis in B6.C3-Bbaa1 mice by anti-IFNAR1 mAb blockade further revealed that their intermediate phenotype is entirely due to pathologic type I IFN production, which is complementary to our previous publications showing that type I IFN drives ~50% of the arthritis phenotype in C3H mice (4, 5). Refined, interval specific recombinant congenic lines (ISRCL1-4) further supported that Lyme arthritis severity is uniquely linked to the C3H allele for the type I IFN locus, and precise treatment of B6.C3-Bbaa1 mice with anti-IFN-α or anti-IFN-β mAbs established that IFN-β is the sole arthritis-driving factor. Thus, our Bbaa1 congenic mouse model has provided a unique opportunity to explore all aspects of IFN-β dysregulation on a highly genetically similar B6 background. Because Bbaa1 intrinsically controls IFN-β production, reciprocal radiation chimeras between B6.C3-Bbaa1 and B6 mice allowed in vivo identification of the cellular source of pathologic IFN-β, independent from feed-forward amplification. Previously, reciprocal radiation chimeras between C3H and C3H-IFNAR1-/- mice revealed that both radiation-sensitive and radiation-resistant cells in the joint contribute to the proarthritic type I IFN response (5). Now, B6 and B6.C3-Bbaa1 mice specified that 81 radiation-sensitive (likely myeloid) cells initiate IFN-β, and radiation-resistant cells responding to IFN-β manifest arthritis in the joint. Identifying this segregation of roles among cell types in the joint milieu, coupled with the generation of refined congenic lines (in which the original 85 Mbp C3-Bbaa1 locus was reduced to 10 and 5 Mbp in ISRCL3 and ISRCL4 mice, respectively), prompted us to design cell-appropriate RNA-seq experiments to better understand: the mechanism of arthritis development (wherein myostatin was identified, Chapter 3), and the mechanism of IFN-β dysregulation (wherein expressed Bbaa1 candidates were identified, Appendix). In summary, our utility of unbiased genetic tools throughout this dissertation has allowed us to distinguish key players in Bbaa1-directed Lyme arthritis development from start to finish. Possible Mechanism of IFN-β Dysregulation It is reasonable to suggest that another gene in the 5-10 Mbp Bbaa1 interval regulates IFN-β expression because Ifnb is identical between B6 and C3H mice (including the well-characterized enhanceosome (6)), and there are only two noncoding SNPs (positioned at 3,000 bps upstream and 1,000 bps downstream) within 10,000 flanking base pairs of the coding sequence (7, 8), neither of which has any connection to IFN-β regulation in the literature. Interestingly, this observation is consistent with the fact that genome-wide association studies (GWASs) and other genetic analyses for systemic lupus erythematosus (a prototypic autoimmune disease with a type I IFN signature) have never identified susceptibility polymorphisms in the actual IFN-α/β genetic sequences, but have identified many genes involved in the IFN signaling pathway (9-13). Although there is not another gene in the Bbaa1 interval that has been linked to IFN signaling 82 before, novel mechanisms of IFN-β regulation are continually being discovered (14). Thus, a Bbaa1 gene that is (1) expressed in the same (myeloid) cell type that initiates Ifnb, (2) expressed during or prior to Ifnb expression, and (3) polymorphic between B6 and C3H sequences (to explain subsequently differential Ifnb expression) may be responsible for this novel function. Based on the aforementioned criteria, an unbiased RNA-seq experiment was performed, and a list of Bbaa1 candidates was generated to serve as a starting point for future studies defining this novel mechanism (Appendix). Intriguingly, the list encompasses enzymes (which, as a broad category, are often identified in GWAS studies and majorly impact cell signaling pathways (15)), lincRNAs (which play important gene regulatory roles (16, 17), especially in myeloid cells (18-26), with lincRNA-Cox2 exemplifying a transcriptional repressor of type I IFN signaling in resting macrophages (26)), and unannotated genes (which is consistent with this being a novel gene function). In summary, the progression of this dissertation has led to a manageable number of exciting Bbaa1 candidates to be tested in future studies. Investigations in Bbaa1 Mice Will Transcend Lyme Disease Understanding the genetic mechanism of Bbaa1-directed IFN-β dysregulation will be highly impactful because a number of human diseases are associated with a pathologic type I IFN signature (27-31), and Bbaa1 mice provide a natural model for this genetic phenomenon. Although there has not yet been a GWAS for Lyme disease, GWASs for many autoimmune diseases with a type I IFN signature (including systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, Sjögren's syndrome, and systemic 83 sclerosis) have identified common susceptibility genes (notably Irf5 and Ifih1) involved in type I IFN signaling (15, 32). This highlights that shared genetic polymorphisms (especially those impacting the type I IFN pathway) can manifest as different diseases, and underscores the widespread importance of characterizing the novel Bbaa1 susceptibility gene. Additional similarities between our murine model of Lyme arthritis and several human autoimmune diseases further illuminate the power of our Bbaa1 mouse tool. Specifically, type I diabetes patients display a transient but robust type I IFN profile that precedes active disease (31), similar to the kinetics we observe during Lyme arthritis development in C3H and B6.C3-Bbaa1 mice (Crandall et al. (33) and Chapter 2). IFN-β also seems to be more pathologic than IFN-α in rheumatoid arthritis patients (34), similar to our finding that IFN-β is the proarthritogenic factor in B6.C3-Bbaa1 mice (Chapter 3). And, patients with idiopathic inflammatory myopathies display a type I IFN signature (35-38) and share susceptibility genes with other autoimmune disorders (39, 40), suggesting that myostatin upregulation in B6.C3-Bbaa1 mice (Chapter 3) occurs via a shared pathological pathway downstream of a common type I IFN trigger. In conclusion, future studies in Bbaa1 mice will likely contribute to a global understanding of pathological processes shared among many human diseases. References 1. Weis, J. J., B. A. McCracken, Y. Ma, D. Fairbairn, R. J. Roper, T. B. Morrison, J. H. Weis, J. F. Zachary, R. W. Doerge, and C. Teuscher. 1999. Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease. J Immunol 162: 948-956. 2. 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Because bone marrow-derived macrophages are an appropriate model for myeloid cells in joint tissue, macrophages from B6, ISRCL3, and ISRCL4 mice were cultured as described previously (Chapters 2 and 3, Materials and Methods) and stimulated with live B. burgdorferi for 0, 3, or 6 hr (n = 3 to 4 mice per genotype). RNAseq libraries were prepared using a Ribo-Zero rRNA removal kit in order to retain the maximal number of both coding and noncoding RNA transcripts, and sequenced as described previously (Chapter 3, Materials and Methods). Shown is a list of candidate genes arranged in ascending order along Chr4. Genes with SNPs between B6 and C3H sequences are noted, and will be the subject of future investigations determining the consequence of structural changes on IFN-β regulation. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Gene$ Ccdc171 Gm11415 Gm12416 Bnc2 Gm12421 Gm12420 Cntln1 Rraga Haus6 Scarna8 Gm12551 Plin2 Dennd4c Rps6 Acer2 Mllt3 Focad Hacd4 Ifnb1 Mrpl48Eps Klhl9 Mtap Gm12606 Cdkn2a Cdkn2b Gm12609 Gm12610 Gm12634 Gm12632 Gm12669 Gm12641 Tusc1 * * * * * * * * * * * * * * * * * * * * * * SNPs$ Gene$title coiled)coil*domain*containing*171 predicted*gene*11415 predicted*gene*12416 basonuclin*2 predicted*gene*12421 predicted*gene*12420 centlein,*centrosomal*protein Ras)related*GTP*binding*A HAUS*augmin)like*complex,*subunit*6 small*Cajal*body)specific*RNA*8 predicted*gene*12551 perilipin*2 DENN/MADD*domain*containing*4C ribosomal*protein*S6 alkaline*ceramidase*2 myeloid/lymphoid*or*mixed)lineage*leukemia;*translocated*to,*3 focadhesin 3)hydroxyacyl)CoA*dehydratase*4 interferon$beta$1 mitochondrial*ribosomal*protein*L48*pseudogene kelch)like*9 methylthioadenosine*phosphorylase predicted*gene*12606 cyclin)dependent*kinase*inhibitor*2A cyclin)dependent*kinase*inhibitor*2B predicted*gene*12609 predicted*gene*12610 predicted*gene*12634 predicted*gene*12632 predicted*gene*12669 predicted*gene*12641 tumor*suppressor*candidate*1 Biotype ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) = ) ) ) ) ) ) ) ) ) ) ) ) ) 83,864,731 83,888,858 84,105,902 84,675,275 84,429,124 84,698,910 85,131,921 86,577,281 86,612,055 86,586,570 86,631,251 86,670,060 86,850,603 86,857,412 86,934,822 88,033,364 88,411,011 88,438,928 88,522,794 88,599,810 88,722,465 89,181,081 89,273,403 89,294,653 89,311,032 89,399,880 89,463,487 90,369,722 90,857,377 91,806,561 93,109,914 93,335,511 Chr4$position protein*coding ******83,525,545 lincRNA ******83,884,738 processed*pseudogene ******84,104,895 protein*coding ******84,275,095 processed*pseudogene ******84,428,818 processed*pseudogene ******84,697,863 protein*coding ******84,884,309 protein*coding ******86,575,668 protein*coding ******86,578,855 scaRNA ******86,586,453 unprocessed*pseudogene ******86,627,075 protein*coding ******86,648,386 protein*coding ******86,748,555 protein*coding ******86,854,660 protein*coding ******86,874,396 protein*coding ******87,769,925 protein*coding ******88,094,629 protein*coding ******88,396,144 protein$coding $$$$$$88,522,025 processed*pseudogene ******88,599,175 protein*coding ******88,718,292 protein*coding ******89,137,122 lincRNA ******89,235,699 protein*coding ******89,274,471 protein*coding ******89,306,289 processed*transcript ******89,381,957 processed*transcript ******89,421,848 processed*pseudogene ******90,368,731 processed*pseudogene ******90,856,883 processed*pseudogene ******91,805,547 processed*pseudogene ******93,106,988 protein*coding ******93,334,138 91