Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice

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Sheena Rose Baratono University of Pennsylvania, [email protected]

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Recommended Citation Baratono, Sheena Rose, "Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice" (2014). Publicly Accessible Penn Dissertations. 1204. https://repository.upenn.edu/edissertations/1204

This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1204 For more information, please contact [email protected]. Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice

Abstract TOLL-LIKE RECEPTOR 9 AND INTFERON-GAMMA RECEPTOR SIGNALING SUPPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE

Sheena Baratono

Edward M. Behrens

Hyperinflammatory syndromes come in a variety of flavors with different immunogens and cytokines leading to variations in organ pathology. This inflammation can be driven by viral, autoimmune, or neoplastic conditions leading to profound and widespread organ pathology including hepatitis, fever, splenomegaly, cytokinemia and pan-cytopenias, including T and B lymphopenias. The developing immune cells in the bone marrow are known to express a variety of cytokine and inflammatory receptors yet the effects of signaling through these receptors on hematopoiesis is incomplete. Since many infectious and autoimmune syndromes result in sustained TLR9 stimulation, understanding the effects of TLR9 driven inflammation on hematopoiesis is important for both questions of pathogenesis as well as possible therapeutic interventions that might help to restore homeostasis. Utilizing in vitro and in vivo techniques, we demonstrate that B lymphopoiesis is inhibited during TLR9 driven inflammation from the Ly-6D+ CLP stage onwards with different effects inhibiting development at multiple stages across B cell development. We show that TLR9 signaling directly decreases in vitro B cell yields from CLPs, Pro and Pre B cells yet increases T cell yields. While TLR9 can inhibit B cell growth in vitro, using mixed bone marrow chimeras, we show in vivo that this TLR9 intrinsic effect is masked, likely due to other TLR9 induced inflammatory factors that also mediate decreases in B lymphopoiesis. This led us to demonstrate that IFN-γ also directly inhibits B cell differentiation in vitro as well as when induced by TLR9 in vivo. Microarray and RT- PCR analysis of Ly-6D- CLPs points to HOXa9 as an early B-cell directing transcription factor that is altered by TLR9 induced inflammation, as it and many of its known targets are decreased in Ly-6D- CLPs. Additionally EBF-1, another factor essential for initiation of the B cell program was transcriptionally decreased in Ly-6D- and Ly-6D+ CLPs. Our work demonstrates both cellular and molecular targets that lead to altered B-lymphopoiesis in sustained TLR9 driven inflammation that may be relevant in a number of infectious and autoimmune/inflammatory settings.

Degree Type Dissertation

Degree Name Doctor of Philosophy (PhD)

Graduate Group Immunology

First Advisor Edward M. Behrens

Keywords B cell, inflammation, interferon, lymphopoiesis, T cell, TLR9 Subject Categories Allergy and Immunology | Developmental Biology | Immunology and Infectious Disease | Medical Immunology | Medicine and Health Sciences

This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1204 TOLL-LIKE RECEPTOR 9 AND INTERFERON-GAMMA RECEPTOR SIGNALING SUPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE

Sheena Baratono

A DISSERTATION

Immunology

Presented to the Faculties of the University of Pennsylvania

Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

2014

Supervisor of Dissertation

______

Edward M. Behrens Associate Professor of Pediatrics

Graduate Group Chairperson

______

David M. Allman Associate Professor of Pathology and Laboratory Medicine

Dissertation Committee:

David M. Allman, Associate Professor of Pathology and Laboratory Medicine Avinash Bhandoola, Senior Investigator National Cancer Institute Igor E. Brodsky, (Chair) Assistant Professor of Micorbiology Kathleen Sullivan, Professor of Pediatrics ACKNOWLEDGMENTS

I have had an extremely rewarding learning experience throughout my graduate career. To start, I would like to thank my mentor, Edward Behrens, for all of his advice, guidance, and expertise. His flexibility to explore my interests and follow where the data leads, even if it’s not where you were expecting, have been invaluable to my development as a scientist.

I would also like to thank all of the members of the Behrens and Koretzky labs for their help, support, and scientific advice over the past few years. I am especially grateful to Niansheng Chu and Lee Richardson for help with some of the experiments in this manuscript. I would like to thank Michael P. Cancro and David M. Allman for their critical review of our manuscript and Martha Jordan, Taku Kambayashi, Avinash

Bhandoola, and Gary Koretzky for their support and helpful discussions. I am grateful for the scientific discussions and advice from various lab meeting groups I was a part of including the Cancro, Allman, and Laufer labs.

Thank you to Igor Brodsky for chairing my thesis committee, and to the other members Dave Allman, Avinash Bhandoola, and Kate Sullivan. I would also like to thank Gary Koretzky for his guidance and support throughout the PhD. These individuals have allowed me to get the most out of my committee meetings and have guided my development as a young scientist. I would also like to thank the Immunology Graduate

Group including the countless faculty and staff that have facilitated my training during my graduate career.

I would also thank the NapCore at the Children’s Hospital of Philadelphia for their help running the microarray and technical support and the flow cytometry core at the University of Pennsylvania for technical help.

Lastly, I would like to thank my family and friends for their support throughout graduate school. Their patience and support have enabled me to get through the roughest times of my training. I would like to give a special thanks to Dustin Shilling for his support and council throughout the PhD process, as well as for his encouragement and patience through the trails and excitements of graduate school.

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ABSTRACT

TOLL-LIKE RECEPTOR 9 AND INTFERON-GAMMA RECEPTOR SIGNALING

SUPPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE

Sheena Baratono

Edward M. Behrens

B lymphopoiesis is inhibited during TLR9 driven inflammation from the Ly-6D+ CLP stage onwards with different effects inhibiting development at multiple stages across B cell development. We show that TLR9 signaling directly decreases in vitro B cell yields from CLPs, Pro and Pre B cells yet increases T cell yields. While TLR9 can inhibit B cell growth in vitro, using mixed bone marrow chimeras, we show in vivo that this TLR9 iv

intrinsic effect is masked, likely due to other TLR9 induced inflammatory factors that also mediate decreases in B lymphopoiesis. This led us to demonstrate that IFN-γ also directly inhibits B cell differentiation in vitro as well as when induced by TLR9 in vivo.

Microarray and RT-PCR analysis of Ly-6D- CLPs points to HOXa9 as an early B-cell directing transcription factor that is altered by TLR9 induced inflammation, as it and many of its known targets are decreased in Ly-6D- CLPs. Additionally EBF-1, another factor essential for initiation of the B cell program was transcriptionally decreased in Ly-

6D- and Ly-6D+ CLPs. Our work demonstrates both cellular and molecular targets that lead to altered B-lymphopoiesis in sustained TLR9 driven inflammation that may be relevant in a number of infectious and autoimmune/inflammatory settings.

TABLE OF CONTENTS

ACKNOWLEDGMENTS …………………………..….…………………………..….. ii

ABSTRACT ……………………………………………….……………...……….…… iv

TABLE OF CONTENTS ………………………………………………………...... …. vi

LIST OF TABLES ……………………………………………………….….……...... ix

LIST OF FIGURES ……………………….….………….……………….…..…...... x

CHAPTER 1 – Introduction……..……………………………….……….………...... 1

Diseases of Hyperinflammation ………………………………………….…….... 1

Normal B and T Lymphopoiesis ……………………..…………….……….….... 5

B and T lymphopoiesis during inflammation ………………………….……..…. 8

Our model of systemic inflammation …………………..…………….………... 13

CHAPTER 2 – Materials and Methods …………………………….…….………..... 15

Mice ………………………………………..……….……………….…………. 15

Antibodies and flow cytometry ……………………..……….….….…………... 15

Mouse models and isolation of samples ……………………..….…………...… 16

in vitro culture assays ……………………..…………….…………….………... 16

Statistical analyses ……………………..……….….….……………………….. 17

Affymetrix microarray ……………………..………………………….……..… 17

Quantitative realtime PCR ……………………..…………….…….……..…..... 18

CHAPTER 3 – Systemic inflammation impairs lymphopoiesis …………...... ….. 19

Introduction ………………………………………..……………….…..………. 19

Results ……………………..……….….…………………………….……..…....21

Discussion ……………………..……………………………………….……..... 25

Figures ……………………..………….…………………………….…...……... 29

CHAPTER 4 – TLR9 and IFNγR signaling directly impair B lymphopoiesis...... 34 Introduction …………………………………………………….………….….... 34 Results ……………………..……………………….……………………...... …. 36 Discussion ……………………..…………………….………………….……… 41 Figures ……………………..……………………….…………….…………...... 43

CHAPTER 5 – Inflammation skews CLP potentials away from the B cell fate...... 49

Introduction ………………………………………..………….…………...….... 49

Results ……………………..……….….………………….…………….…..….. 53

Discussion ……………………..………………………….………………….… 57

Figures ……………………..………………………………….…………….….. 59

CHAPTER 6 – Discussion and future directions……………….………………...…. 64

Future directions …………………………………………...………………..…. 64

vii

Discussion ……………………..……….….…………….……………...…….... 67

Appendix ………….………………………….……….………………………...……... 73

Abbreviations……………………………………….………….…………...…... 73

Bibliography ……………………………………..………………….…………...……. 77

viii

List of Tables

Chapter 5

Table 1: Ingenuity Pathway Analysis of Ly-6D- CLPs from CpG and PBS treated

mice.….…………………………………………..……………………………..63

List of Figures

Chapter 1

Figure 1: Repeated CpG injection model …………………………………....…..14

Chapter 3

Figure 2: Repeated 10-day CpG induced inflammation impairs lymphopoiesis...29

Figure 3: Repeated 10-day CpG induced inflammation impairs lymphopoiesis...29

Figure 4: Repeated 4-day CpG induced inflammation impairs

lymphopoiesis…..……...... 30

Figure 5: 48 hours post single dose CpG lymphopoiesis is normal………...... ….31

Figure 6: LCMV infection of perforin null mice impairs lymphopoiesis…...…...32

Chapter 4

Figure 7: CpG and IFN-γ differentially inhibit B lymphopoiesis in vitro…...... 43

Figure 8: TLR9 signaling directly skews lymphocyte development in vitro …....44

Figure 9: Direct TLR9 signaling effects on lymphocyte development are masked

in vivo……………………………………………………………………..45

Figure 10: IFN-γR but not IFN-αR signaling inhibits B cell development in

vivo……………………………………………………..………..………..46

Figure 11: IFN-γ directly inhibits Ly-6D+ CLP development in vitro...... 47

Figure 12: IFN-γ and CpG independently decrease lymphopoiesis….……..…...48

Chapter 5

Figure 13: CpG induced inflammation skews CLPs away form the B cell lineage

and towards the T cell fate………….………..….………………………..59

Figure 14: CpG induced inflammation does not skew CLPs towards the DC

fate………….………..….…………………………………...………..…..60

Figure 15: Ly-6D expression and CpG treatment induce transcriptional profile

changes……………………………………………………………………61

Figure 16: Ly-6D expression and CpG treatment induce transcriptional profile

changes……………………………………………………………………61

Chapter 1

Introduction

Systemic hyperinflammation comes in a variety of flavors with different immunogens and cytokines leading to variations in organ pathology. This inflammation can be initiated by infectious, genetic, traumatic, autoimmune or neoplastic conditions, all leading to profound and widespread organ damage. This pathology includes hepatitis, fever, splenomegaly, cytokinemia and pan-cytopenias, specifically T and B lymphopenias and in severe cases, hemodynamic insufficiency and death. (Behrens et al., 2011; Canna et al., 2013; Hinze et al., 2010; Zhang et al., 2014a) The widespread inflammation affects a variety of organ systems including the bone marrow, which is the site of hematopoiesis.

While bone marrow hematopoietic progenitors express several cytokine and inflammatory receptors, our knowledge regarding the effects of signaling through these receptors on hematopoiesis is incomplete. Phenotypic variations in the inflammation depend on the inciting pathogenic factors and the cytokine networks they induce. One common thread across various causes of inflammation is that lymphopoiesis is diminished in the bone marrow, specifically leading to drastic reductions in B and T lymphopoiesis.

Diseases of hyperinflammation

Bacterial mediated sepsis induces profound inflammation through stimulation of

Toll-Like Receptors (TLR), primarily TLR4, which recognizes the microbial

1 lipopolysaccharide moiety. Stimulation of TLR4 on myeloid cells releases high levels of tumor necrosis factor α (TNFα) and interleukin (IL) -1 leading to widespread inflammation and hemodynamic instability.(Beutler and Poltorak, 2001) In mice, chronic low dose lipopolysaccharide stimulation through TLR4 and the resulting cytokine release inhibit hematopoietic stem cell quiescence inducing them to differentiate. (Esplin et al.,

2011) Serial bone marrow transplants of stem cells from inflamed mice show that the inflammation skews stem cells away from the lymphoid lineages. (Esplin et al., 2011;

Nagai et al., 2006) In this model, myelopoiesis in the bone marrow was also decreased while B cell numbers in the spleen were increased suggesting that inflammation alters multiple hematopoietic lineages.

Superantigens produce sudden and profound inflammation by non-specific activation of T cells caused by cross-linking T cell receptors or their ligands, the major histocompatibility complex. This non-discriminate activation leads to T cell release of interferon-γ (IFN-γ), which activates macrophages to produce IL-1, IL-6 and TNFα.

(Edwards et al., 1996) These cytokines lead to fever, splenomegaly and shock or hemodynamic instability. In studies using human and canine cells, antibodies that cross- link the major histocompatibility complex molecule HLA-DR mimicking superantigens severely diminish hematopoiesis, in part due to increased apoptosis and decreased levels of stem cell factor. (Hong et al., 1995; Lee et al., 1997; Yamaguchi et al., 1999) In these mice lymphoid progenitors were decreased in the bone marrow but increased in the blood, while myeloid progenitors were increased in the bone marrow. This suggests that inflammation may drive hematopoiesis towards certain lineages at the expense of others.

Patients with primary Hemophagocytic Lymphohistiocytosis (HLH) have defects in the machinery associated with cytotoxicity of functions of T and natural killer (NK) cells. They develop profound cytokine storm induced inflammation upon infection with various pathologic agents. (Zhang et al., 2014a) By being unable to clear the infection, the immune system is constantly bombarded with increasing levels of pathogenic stimuli leading to release of high levels of IFN-γ, TNFα and other inflammatory cytokines.

Macrophage activation syndrome is a form of HLH that associates with rheumatologic disorders including systemic lupus erythematosus and juvenile idiopathic arthritis.

(Zhang et al., 2014a) While it is still unclear why some lupus and arthritis patients develop macrophage activation syndrome, lupus in particular is associated with the immune system responding to apoptotic debris in a pro-inflammatory manner. Apoptotic debris engulfed by phagocytic cells can stimulate TLR9 and in abnormal cases, drive an adaptive immune response. (Biermann et al., 2014; Munoz et al., 2010; Zhang et al.,

2010) Plasmacytoid and traditional dendritic cells respond to the apoptotic debris releasing both type 1 and type 2 interferons which drive further inflammation. (Biermann et al., 2014) Many HLH patients have decreased peripheral B and T cell counts as well as poor rates of bone marrow engraftment upon transplantation, suggesting that the hematopoietic compartment is altered during inflammation. (Hinze et al., 2010; Sumegi et al., 2011)

A variety of viral infections are linked to induction of aplastic anemia with decreases in erythropoiesis, leukopoiesis, and thrombopoiesis. The viruses associated with aplastic anemia in humans are heterogeneous and include parvovirus B19, Epstein-

Barr virus, cytomegalovirus, human immunodeficiency virus, dengue, measles virus and hepatitis C. (Nakao et al., 1989; Ozawa et al., 1987) While the patterns of infection among these viruses vary, they all induce high levels of IFN-γ, an inverted cluster of differentiation (CD) 4:8 ratio and increased CD8 cytotoxicity. (Nakao et al., 1989; Ozawa et al., 1987) Some of these viruses directly infect bone marrow progenitor cells leading to disruption of normal hematopoiesis. Others alter the ability of bone marrow stromal cells to support normal hematopoietic development in response to various inflammatory cytokines and stimuli. (Manchester et al., 2002; Rosenfeld and Young, 1991; Simmons et al., 1990; Young et al., 1987)

Multicentric Castelman’s disease is a lymphoproliferative, cytokine storm disorder sometimes associated with human herpes virus-8. Idiopathic, or non-human herpes virus-8 associated, multicentric Castelman’s disease is a diagnosis of exclusion when other autoimmune, infectious, and neoplastic disorders are ruled out. (Fajgenbaum et al., 2014) In this disorder, polyclonal lymphocytes become highly activated and produce high quantities of various cytokines. The most common dominating cytokines are IL-6 and type 1 interferons. (Muskardin et al., 2012) The cytokine storm and side effects of the various chemotherapy and immuno-regulatory therapies severely alter the hematopoietic compartment leaving patients immuno-suppressed post resolution of the inflammatory periods.

A variety of infectious, autoimmune, and neoplastic conditions can induce hyperinflammation. Patients with these inflammatory disorders display widespread organ damage with variations depending on the inciting factor and the dominating cytokines

4 within the inflammatory cascade. Many of them also have alterations in hematopoiesis and develop prolonged lymphopenias leaving the patients immuno-suppressed even after recovery from the initiating incident. How inflammation alters hematopoiesis and the long-term ramifications of these changes are still unclear.

Normal murine B and T lymphopoiesis

The progression from undifferentiated hematopoietic stem cell to mature lymphocyte has been well characterized in murine models. During non-inflammatory conditions, hematopoietic stem cells differentiate towards the lymphoid lineage losing long-term self-renewal capabilities. These cells are termed multipotent progenitors.

(Beaudin et al., 2014; Luc et al., 2008) A subset of these multipotent progenitors upregulate FMS-like tyrosine kinase 3 (Flt3) and are termed lymphoid primed multipotent progenitors (LMPP). These LMPPS have no erythroid and megakaryocyte potentials and limited myeloid potential.(Beaudin et al., 2014) Some of these LMPPs migrate further towards the lymphoid lineages, upregulating IL-7Rα and are termed common lymphoid progenitors (CLPs). These CLPs are defined as being Lineage-

Sca1intcKitintIL-7Rα+Flt3+. (Harman et al., 2008) They express high levels of the transcription factors homeobox A9 (Hoxa9) and transcription factor 3 (E2A), which contribute to upregulation of the cell surface receptors Flt3 and IL-7 receptor α (IL-7Rα) whose ligands further support lymphoid development.(Bain et al., 1994; Gwin et al.,

2013a; Rolink et al., 1999; Seet et al., 2004) CLPs however are a heterogeneous population; CLPs expressing Ly-6D+, are exclusively committed to the B cell lineage in

5 vivo, while the Ly-6D- CLPs can give rise to B, T, NK, or dendritic cells. (Allman et al.,

1999; Inlay et al., 2009; Mansson et al., 2010) These Ly-6D+ CLPs retain the ability to generate T cells in vitro on OP9-dl1 cultures but do not generate T cells in vivo by intrathymic injection. (Inlay et al., 2009) The Ly-6D+ CLPs begin to express high levels of early B cell factor 1 (EBF-1), which specifies the B cell fate and turns on paired box 5

(PAX5) and other B cell factors that are important for initiating and maintaining the B cell program. (Lin and Grosschedl, 1995; Pongubala et al., 2008; Rolink et al., 1999; Seet et al., 2004; Tsapogas et al., 2011) As these B cell promoting transcription factors are turned on, the progenitors also turn on recombination activating gene (RAG) and rearrange the heavy chain of the B cell receptor (BCR). (Allman et al., 1999) Pro B cells are committed to the B cell lineage and express CD19, CD93, protein tyrosine phosphatase receptor type C (B220), and CD43. (Hardy et al., 1991) Upon successful rearrangement of the heavy chain, these cells downregulate CD43 and undergo several rounds of proliferation as Pre B cells. After successful rearrangement of the light chain, the progenitors express surface IgM and are considered Immature B cells. (Hardy et al.,

1991) These cells can then leave the bone marrow and finish development in the spleen.

These peripheral developing B cells are termed Transitional B cells and are differentiated by their expression of surface IgM and CD23. Stimulation of Transitional 1 or 2 B cells through their B cell receptor leads to apoptosis while stimulation of the more mature

Transitional 3 cells leads to proliferation and further maturation, suggesting that downstream signaling fates are changed as cells mature. (Carsetti et al., 1995)

Unlike B cell development, potential T ell progenitors must leave the bone

6 marrow and travel through the circulation to the thymus where they receive instructing signals that drive the T cell fate. These thymic settling progenitors can arise from the Ly-

6D- CLPs or straight from the LMPPs. (Chi et al., 2009) Of these early progenitors, those that express the chemokine (C-C motif) receptors (CCR) CCR7 and CCR9 are able to successfully home to the thymus where high levels of notch ligands on the thymic stroma initiate the T cell transcriptional program while also repressing factors important for B cell development. (Pui et al., 1999; Zhang et al., 2014b; Zlotoff et al., 2010) These thymic settling progenitors are positive for stem cell growth factor receptor (cKit) but negative for CD25, CD4 and CD8a and are termed early thymic progenitors (ETP).

(Bhandoola et al., 2007) ETPs retain NK and dendritic cell (DC) potential. The ETPs then begin to express CD25 and are termed double negative (DN) 2 cells. As cKit expression decreases, the cells lose DC potential. (Germain, 2002) The CD25+ cKit- cells are the DN3 thymocytes, which undergo selection for the T cell receptor. These cells can mature into two broadly different T cell lineages, the gamma-delta T cells and alpha-beta

T cells, named by their T cell receptor (TCR) expression. DN3 cells that successfully rearrange the gamma-delta TCR go on to become mature gamma-delta T cells.

(Rothenberg, 2000) DN3 cells that successfully rearrange the beta chain progress to the

DN4 stage where they downregulate CD25 and prepare to upregulate CD4 and CD8a.

(Bhandoola et al., 2007; Germain, 2002) The CD4 and CD8a double positive cells (DP) then undergo rearrangement of the alpha chain and express the full alpha-beta TCR on their surface. Cells successfully rearranging TCRs enter positive selection where the affinity of the newly arranged TCR for self-peptide-major histocompatibility complexes

7 is assessed. These DP cells are highly susceptible to apoptosis as they become refractive to IL-7, a pro-survival cytokine, due in part to decreased IL-7 receptor expression.

(Ribeiro et al., 2013) DPs are therefore sensitive to glucocorticoids and other external stimuli that induce apoptosis. More than 90% of these DP thymocytes undergo death by neglect due to non-appropriate TCR rearrangements. (Brewer et al., 2002; Lu et al., 2000)

DP cells that successfully undergo positive selection progress to the CD4 and CD8a single positive T cell lineages where they undergo negative selection for auto-reactive

TCRs and exit the thymus.

B and T lymphopoiesis during inflammation

Depending on the inflammatory model and which cytokines are produced, different components of hematopoiesis are impacted. In a model where mice are immunized with nitro phenol-chicken gamma globulin in incomplete Freund’s adjuvant, both bone marrow B lymphopoiesis and myelopoiesis are altered. The B cell defect is due in part by migration of some B cell precursors to the spleen. (Borrow et al., 2005; Ueda et al., 2005; Ueda et al., 2004) The mobilization was revealed to be due to TNFα mediated decreases in C-X-C motif chemokine ligand (CXCL) 12, which is important for maintaining the B cell niche in the bone marrow. (Ueda et al., 2005; Ueda et al., 2004)

While CXCL12 levels recover within 8 days post-immunization, it is unclear how repeated exposure to adjuvant-induced inflammation would alter TNFα and CXCL12 levels. In this same model, IL-1 or treatment with pertussis toxin, a broad chemokine receptor blocker, increases granulopoiesis, leading to a net shift in production of

8 granulocytes. (Ueda et al., 2005; Ueda et al., 2004) This may be due to the decrease in B lymphopoiesis opening up space for increased granulopoiesis in the marrow or to independent actions of differing cytokines. In T cell development, TNFα and IL-1 have been shown to increase expression of CD25 in fetal thymic organ cultures. Blockade of

TNFα and IL-1 leads to decreased CD25 expression and impaired development of DP thymocytes. (Zuniga-Pflucker et al., 1995) These data suggest that TNFα and IL-1 have differing affects on B and T lymphopoiesis.

Studies looking at cultures of multipotent progenitors in vitro found that addition of recombinant IL-6 to single cell cultures inhibited B cell potential but increased myeloid potential. (Jenkins et al., 2007; Maeda et al., 2010) However, when single RAG+ early lymphoid progenitors were plated, addition of IL-6 increased B cell yields. (Maeda et al., 2005) This suggests that IL-6 acts differentially on multipotent progenitors to inhibit lymphoid fates, but once the cell is already committed to the lymphoid lineages, it may enhance differentiation or proliferation. In an in vivo mouse model of lupus with high levels of serum IL-6, mice had significantly decreased bone marrow B cell and CLP numbers, which were restored when the mice were crossed to an IL-6 null background.

(Maeda et al., 2009) In a different mouse model with increased IL-6 receptor signaling, thymocytes expressed higher levels of IL-6 transcripts and proteins than WT controls.

(Jenkins et al., 2007) The thymocytes also expressed higher levels of IL-6Rα suggesting that they are both making and responding to IL-6 in an enhanced manner. Surprisingly, these mice had increased thymic cellularity yet decreased percentages of DPs suggesting that different thymocyte subsets also react differentially to IL-6. (Jenkins et al., 2007)

These studies suggest that IL-6, like BCR and TCR signaling, has varying effects depending on the stage of development of the precursor.

Type 1 interferons significantly alter hematopoiesis in a lymphocytic choriomeningitis virus (LCMV)-WE model of infection. Three days after infection with

LCMV-WE, wildtype (WT) and IFN-γR knockout (IFN-γRKO) mice had <1% normal bone marrow cellularity, with impaired abilities to generate granulocytes, erythrocytes, and megakaryocytes. IFNα-RKO mice, however, mimic uninfected controls with normal rates of hematopoiesis. (Binder et al., 1997) In another study looking at exogenous treatment of IFNα2/α1 in neonatal mice, B lymphopoiesis was severely impaired and Pre

B cells exhibited decreased proliferation in response to IL-7. (Lin et al., 1998)

Thymopoiesis was also affected in this model, with the largest thymocyte reductions occurring in the DP compartment. (Lin et al., 1998) However, it is unclear if these decreases are a direct effect of the interferons on the precursor cells. Administration of type 1 interferons to fetal thymic organ cultures lead to decreased thymic cellularity with the largest decrease occurring at the DN3 stage, suggesting a block during beta chain rearrangement. (Baron et al., 2008) However, administration of recombinant type 1 interferons to OP9-dl1 fetal derived hematopoietic stem cell co-cultures lead to a block in

T cell development at the DN1 stages with increased annexin-V staining cells. Together, these data suggest that high levels of type 1 interferons impair lymphopoiesis at multiple stages of T and B cell development in vivo and in vitro.

IFN-γ also inhibits lymphopoiesis in in vivo and in vitro models. B lymphopoiesis was diminished in T cell protein tyrosine phosphatase null mice due to aberrant stromal

10 cell secretion of IFN-γ. The Pre B cells from these mice also demonstrated impaired proliferation in response to IL-7, suggesting IFN-γ may be inhibiting responses to pro- survival cytokines. (Bourdeau et al., 2007) However, Pre B cells kept in culture with IL-7 and IFN-γ did not show increased rates of apoptosis within the first 48 hours in culture.

(Bourdeau et al., 2007) Transgenic mice expressing IFN-γ under the Ig-lambda enhancer had varying phenotypes, with some mice becoming moribund while others remained stable. These mice have severe defects in B lymphopoiesis and severe B cell loss in the periphery. (Young et al., 1997) They also have increased expression of IFN-γ in the thymus and increased thymic cellularity with higher numbers of both CD4 and CD8 single positive thymocytes but significantly fewer numbers of DPs. (Young et al., 1997)

The increase in single positive thymocytes may be due to proliferation of precursors in response to IFN-γ or increased homing of mature T cells via the circulation.

Histologically, thymii from healthy transgenic mice were normal while those from moribund transgenic mice showed severe atrophy, possibly due in part to stress and glucocorticoid release. (Young et al., 1997) Together these data suggest that IFN-γ inhibits IL-7 responsiveness and B and T lymphopoiesis, but it is unclear whether it is acting directly on the precursors.

Another report looking at the effects of microbial products on B lymphopoiesis found that stimulation through TLR9 with CpG or acute herpes simplex virus infection leads to lower Pre B cells but increased bone marrow plasmacytoid dendritic cell numbers. (Welner et al., 2008) While the reduction in B cells was dependent on TLR9 signaling, TNFα also played a partial role in mediating the decrease in B cell precursors

11 in the bone marrow. However it but did not contribute to the increase in dendritic cells, suggesting that they are not entirely reciprocal processes. In addition to decreased B lymphopoiesis in vivo, CpG or herpes simplex virus administration appeared to alter the lineage potential of CLPs in these models when they were placed in in vitro cultures.

(Esplin et al., 2011) However, CLPs are a heterogeneous population and it is unclear if the inverse effects on B and dendritic cells are due to skewing of progenitors from one fate to another, or if the Ly-6D+ CLPs are decreased, thereby biasing the inflammatory

CLP population towards Ly-6D- CLPs with DC potential. Together these findings suggest that multiple inflammatory factors, including TLR9 and TNFα, reduce bone marrow B lymphopoiesis, yet they may not be the same factors directly leading to the increase in dendritic cells. In the thymus, both TLR3 and TLR4 high dose stimulation is associated with decreased thymic output and decreased DP thymocytes. However, these models are associated with very high levels of pro-inflammatory cytokines and glucocorticoids, which can also affect thymopoiesis. The direct effect of TLR signaling on thymopoiesis remains unclear. (Baron et al., 2008; Billard et al., 2011)

In summary, prior work in the field has elucidated roles for a variety of cytokines in inhibiting murine lymphopoiesis. However, for most cytokines, it remains unclear whether they are acting directly on the precursors or on other immune cells. Specific roles for TNFα and IL-1 in mediating migration of B cell precursors from the bone marrow and inducing myelopoiesis respectively are known to inhibit normal bone marrow lymphopoiesis. IL-6 appears to have opposing affects on B lymphopoiesis depending on the stage of precursors exposed to IL-6 signaling. Whether or not this is

12 due to changes in the downstream signaling is unclear. Type 1 and 2 interferons are also known to affect lymphopoiesis although it is not known which cell type they are acting on and whether physiologic amounts that could be seen during inflammation would have similar inhibitory effects. TLR signaling also impairs lymphopoiesis but it is not clear at which stages of development these inflammatory mediators are acting or whether they are acting directly on B and T cell progenitors. In many of these cases it is also uncertain how the inflammation is preventing normal B lymphopoiesis, whether by skewing or progenitors away from lymphoid lineages, or by inducing apoptosis or migration from the bone marrow.

Our model of systemic inflammation

In our model of systemic inflammation, we inject mice repeatedly with CpG, the

TLR9 agonist, five times over the course of ten days (Figure 1). These mice develop systemic inflammation including splenomegaly, hepatitis, weight loss and pancytopenias, specifically including decreased lymphocyte counts. By using genetically modified knockout mice, we are able to characterize the direct effects of TLR9 and IFN-γ on B and

T lymphopoiesis at multiple stages of development in vitro and in our in vivo model of cytokine storm. Since many infectious and autoimmune syndromes result in sustained

TLR9 stimulation and IFN-γ production, understanding the effects of hyperinflammation on hematopoiesis is important for questions of pathogenesis as well as development of possible therapeutic interventions to restore homeostasis.

Chapter 2

Materials and Methods

Mice.

6 week to 12 week old mice were used throughout these studies. No differences in sex or age were noted across experiments. C57BL6, C57BL/6.SJL IFN-γKO and IFN-

γRKO mice were obtained form Jackson Laboratories. TLR9KO and IFN-αRKO mice were used as previously described and bred in our colony. (Hemmi et al., 2000; Leitner et al., 2006) Bone marrow chimeras were administered 950 greys of gamma irradiation followed by intravenous injection of 4-7 million bone marrow cells. Chimeric mice were allowed 10 weeks to rest before use in experiments. Mice were maintained at the

University of Pennsylvania and Children’s Hospital of Philadelphia animal facilities. All procedures were performed with consent from institutional ethics boards.

Antibodies and flow cytometry.

The lineage panel included antibodies against TCRβ, CD3, CD19, B220, NK1.1,

CD11c, CD11b, GR-1 (Ly-6C and Ly-6G), Ter119, FcεR, and CD8α. Aqua live dead was used to visualize dead cells. All populations were first gated on forward and size scatter to limit dead cells and doublets. Samples were then gated as Aqua low for live cells. Antibodies were obtained from Biolegend, eBioscience, and BD Biosciences. Cells were run on LSRII/II or FACS Aria sorters at the University of Pennsylvania flow

15 cytometry core. FACS data were analyzed using FlowJo software.

Mouse models and isolation of samples.

Mice were injected intraperitoneally on days 0, 2, 4, 7, and 9 with phosphate buffered saline (PBS), 50µg CpG 1826 (synthesized by IDT), or 10ηg IFN-γ (peprotech) in 200 or 100µl of volume. On day 10 mice were euthanized and organs were taken for analysis. In the 4-day model, mice were injected with PBS or 50µg CpG on days 0, 1, 2 and 3 and taken down on day 4. In the single dose model, mice were injected with PBS or

100µg CpG and taken down 48hrs later. In the LCMV-Armstrong model, mice were infected with 2x105 plaque forming units and taken down 8 days later. All injections were performed intraperitoneally. Two tibias and femurs per mouse were flushed using a 27.5 gauge needle with cold PBS. To ensure full and equal removal of cells, bones were dissected open and any remaining cells were flushed out. Red blood cells were lysed using ammonium chloride-potassium bicarbonate lysis buffer. Thymi were removed by careful dissection and dissociated using fine-tipped forceps and vigorous pipetting with cold PBS.

in vitro culture assays.

OP9 and OP9-dl1 stromal cells were used essentially as described. (Cho et al.,

1999; Schmitt and Zuniga-Pflucker, 2006) Both B and T cell cultures were supplemented with 10 ηg/ml of IL-7, Flt3l, and stem cell factor (SCF) (peprotech). For dendritic cell

16 cultures, wells were supplemented with 1 ηg/ml IL-7, 100ηg/ml Flt3l, and 10ηg/ml SCF.

OP9 and OP9-dl1 cells were plated at 20,00 cells/ml on day -2 and irradiated with 25 grey gamma irradiation on day 0. Stromal cells rested for 2-6 hours before media was changed and 500 CLPs or 1000 Pro or Pre B cells were plated per well. Ly-6D+, Pro, and

Pre B cell co-cultures were allowed to grow untouched for 7-9 days and Ly-6D- co- cultures for 13-15 days. Dendritic cell cultures were taken down on day 8. When used,

CpG was administered at 0.6µg/ml and IFN-γ at 100ρg/ml to individual cultures. Cell numbers from OP9 experiments represent the number of events collected per volume per time for a given well to allow for comparison.

Statistical Analyses.

Student’s t-tests were performed for all single comparisons. For data with multiple comparisons, 1-way ANOVAs were performed with post-hoc analyses. For data comparing two independent variables, 2-way ANOVAs were performed using Prism software.

Affymetrix Microarray.

Mice were treated with PBS (n=3) of CpG (n=3) as described above. On day 10

Ly-6D- and Ly-6D+ CLPs were sorted separately from each mouse directly into lysis buffer from the Qiagen RNAeasy Micro kit Plus using a FACS Aria. Ribonucleic acid

(RNA) isolation was performed in accordance with the Qiagen protocol. RNA was purified using the (deoxyribonucleic acid (DNA)se treatment kit from Qiagen. Further

17 analysis and processing of the RNA for quality, amplification and hybridization to the mouse Affymetrix ST 2.0 Chip were performed by the NAPcore at the Children’s

Hospital of Philadelphia as previously described. (Canna et al., 2014) Microarray data have been uploaded to the Gene Expression Omnibus repository (GEO accession number will be provided upon acceptance). The array dataset was modeled using a 2-way comparison of the Ly-6D- versus Ly-6D+ and PBS versus CpG. Principle component analysis was performed using Partek Genomics Suite. Genome-wide significant genes from the Ly-6D- PBS vs. CpG comparison were analyzed with the Ingenuity Pathway

Analysis program using a cutoff of a p value less than 0.00001 and an absolute value Z score greater than or equal to 2.

Quantitative real-time polymerase chain reaction (qRTPCR)

Mice were treated with PBS (n=3) or CpG (n=3) and RNA was isolated from sorted CLPs as described above. cDNA was made according to manufacturers instructions (Superscript III First-Strand Synthesis System, Invitrogen). qRTPCR was performed using the LifeTechnologies protocol. Primers were obtained from Applied

Biosciences. Real time PCR reactions were done using an ABI700 sequence detection system and the 2XSYBR green master mix according to the manufacturer’s specifications. The delta-delta CT method was used to quantify results. Values for each gene were normalized to β -actin and one of the PBS treated samples for fold change comparison.

Chapter 3.

Systemic inflammation impairs lymphopoiesis

Introduction

While it is known that hematopoiesis is altered during severe inflammation, we wanted to pursue how IFN-γ and TLR9 alter B and T lymphopoiesis. Our lab has two models of systemic inflammation. One involves TLR9 stimulation by CpG, and the other involves LCMV-Armstrong infection of perforin null mice.

In our primary model, mice are stimulated with CpG 5 times over the course of 10 days. These mice develop the signs and symptoms of cytokine storm disorders including peripheral pancytopenias, splenomegaly, hepatitis, and weight loss but do not become moribund or require euthanasia. (Behrens et al., 2011; Canna et al., 2014; Canna et al.,

2013) Prior work from our lab has characterized the serum cytokine levels and found that

IFN-γ, IL-12, IL-6, and type 1 interferons are produced as well as the anti-inflammatory cytokine IL-10.34, 36 We also wanted to know if shorter courses of CpG could result in similar alterations in hematopoiesis, as the longer model may produce excess inflammation thereby complicating the roles individual factors play in directly mediating decreased lymphopoiesis. The comparison of shorter and longer models may also reveal whether inflammation is acting at multiple stages across development since it takes time for early progenitors to move through lymphoid developmental stages. We therefore also gave doses of CpG every day for 4 days as well as a larger, single dose to different cohorts of mice.

The second model involves infection of perforin null mice with LCMV-

Armstrong. While normal WT mice can clear this infection, these knockout mice are unable to kill infected cells and therefore are cannot shut down the immune response.

These mice develop many of the same signs and symptoms as repeated CpG administered mice, yet to a larger degree. In fact, many of these mice lose too much weight or become moribund and require euthanasia. While LCMV is an RNA virus and therefore does not signal through TLR9, it stimulates other TLRs resulting in very high levels of type 1 and

2 interferons. (Pien et al., 2002; Wherry et al., 2007) Because LCMV is a live viral infection; it induces inflammation through multiple immunologic receptors in addition to

TLRs. (Jung et al., 2008) Since LCMV infection results in fulminant inflammation, the stress response is activated producing glucocorticoids, which contribute to alterations in hematopoiesis, specifically reductions in lymphopoiesis.

Chapter 3

Results

Repeated CpG inhibits the developing B and T cell compartments.

We first induced sustained TLR9 driven inflammation in mice over a 10-day period as previously described. (Behrens et al., 2011; Canna et al., 2014; Canna et al.,

2013) On day 10, we examined the bone marrow and found decreased total cellularity

(Figure 2A). Interestingly, this was driven primarily by a reduction in the CD19+ and erythroblast compartments while other lineages were grossly intact (Figure 2B and data not shown). This suggests that repeated CpG induced inflammation is not affecting gross hematopoiesis, but that it is acting directly on the B cell lineage. As the B cell compartment in the bone marrow includes both mature recirculating B cells as well as immature developing cells, we further analyzed the B cell compartment and found that

Pre, Pro, and Immature B cells were all decreased approximately 10-fold in the mice receiving CpG compared to PBS treated controls (Figure 2C). As all stages of committed

B cell precursors were down, we looked one stage earlier at the CLPs and found that the lymphocyte antigen 6 complex locus D (Ly-6D) negative, or uncommitted CLPs were relatively normal in number, but the Ly-6D+, or B cell committed CLPs were significantly decreased (Figure 2C). We therefore also wanted to know if there would be a similar effect in T lymphopoiesis since B and T cells develop from the same early progenitors and are susceptible to many of the same insults. We found that thymic cellularity was also significantly decreased (Figure 3A). Since many stressors specifically

21 alter the DP stage of thymic development, we characterized thymocyte numbers based on

CD4, CD8, cKit, and CD25 expression and found that all stages were decreased without any obvious blocks in development (Figure 3B). Concurrent decreases in T and B lymphopoiesis could be due to alterations in early lymphoid development that are carried through both lineages, or it could be due to inflammatory factors acting separately on both lineages.

4 day CpG diminishes B and T cell lymphopoiesis.

In our 10-day model it is possible for decreases in early stages of hematopoiesis to carry through into later stages. We therefore wanted to look in a shorter model of CpG induced inflammation for similar or lessened changes in B and T cell development. First, we wanted to ensure that total bone marrow changes mimicked those found in the 10-day model. To test this we injected mice with CpG 4 on consecutive days and took down the mice on day 5. We found similar reductions in total bone marrow cellularity to the 10-day model (Figure 4A), which was also primarily driven by decreases in the B cell compartment (Figure 4B). Within 4 days, a lone defect in the CLP compartment should not lead to 10 fold decreases in Immature B cells like in the 10 day model. To test if this was the case, we looked at B cell precursor numbers in these mice and found that unlike the 10-day model, only Pre and Immature B cells were significantly decreased (Figure

4C). This suggests that the CpG induced inflammatory environment can act at the later stages of bone marrow B cell development. Thymic cell counts were also similarly decreased (Figure 4D), suggesting that the factors driving decreased Pre and Immature B

22 cells and T cells in the 10 day model are induced within 4 days and do not require protracted dosing to mediate their effects. The factors affecting the CLPs, however, do require a longer dosing protocol.

Single dose CpG diminishes myeloid, not lymphoid compartments.

As four consecutive doses sufficiently impaired the lymphoid compartments, we wanted to know if a single dose would have similar effects. We therefore gave one larger dose of CpG and looked 48 hours later at the bone marrow and thymus. While total bone marrow cellularity was still decreased (Figure 5A), surprisingly this was due to diminished myeloid compartments, as the number of B cells did not change (Figure 5B).

Thymic cellularity was also not significantly decreased (Figure 5C). Together this suggests that the single dose of CpG was not sufficient to alter lymphopoiesis in the same manner as in a sustained CpG model.

LCMV induced cytokine storm severely impairs the bone marrow and thymic compartments.

We then wanted to test whether the bone marrow and thymic compartments were altered in the more severe inflammatory environment of uncontrolled infection. Perforin null mice were infected with LCMV-Armstrong and taken down on day 8 corresponding to the peak of the immune response in LCMV infected WT mice. Unfortunately, one infected mouse was too sick and did not survive until day 8 preventing statistical analysis for significance. When we looked at the bone marrow counts in the surviving mice, we

23 found that bone marrow cellularity was decreased and that the B cell compartment was severely inhibited (Figures 6A and 6B). We again looked at the thymus for reductions in thymopoiesis and found severely decreased thymic cellularity (Figure 6C). These findings suggest that LCMV induced cytokine storm inflammation impairs thymic development and inhibits the B cell compartment in the bone marrow.

Chapter 3

Discussion

Using various models and dosing methods, we have shown that lymphopoiesis is inhibited during systemic inflammation. Interestingly, these data suggest that the inflammatory environment is acting at multiple stages of B lymphopoiesis. It is reducing numbers of Ly-6D+ CLPs, the earliest known stage of B cell commitment as well as acting at the Pre and Immature B cell stages as evidenced in our 4 day CpG model.

Insults at each stage of development can also account for why Ly-6D+ CLPs decrease 2- fold while Pro, Pre, and Immature B cells decrease 10-fold. Generally, small decreases in early progenitors can be compensated for by the proliferative burst during later stages of development. Our data suggest that the inflammatory environment is inhibiting lineage development, blocking proliferation, or inducing apoptosis of progenitor cells.

Surprisingly, single dose CpG lead to significant reductions in myeloid populations but not lymphoid populations in our model. This suggests that repeated TLR9 stimulation induces unique factors that mediate lymphopoiesis.

As developing T cells are also reduced, factors could be acting early in lymphoid development inhibiting both lineages. However, similar to B cells, reductions in early T cell progenitors can be compensated for by the large proliferative capacity of the cells that reach the thymus. (Zlotoff et al., 2010) While homing may also be affected during inflammation, decreases across development suggest that there are alterations in the thymic stromal compartments, such as is seen during systemic irradiation. (Zhang et al.,

2014b; Zlotoff et al., 2011)

TLR9 stimulation produces a number of cytokines and it is possible that different factors are acting at the various stages of B lymphopoiesis. The roles of TLR9 and IFN-γ will be discussed in more depth in chapter 2. It is possible however, that type 1 interferons, IL-6, and TNFα could be contributing to the decreases seen in our model.

One possibility is that TNFα is reducing levels of CXCL12 that retains progenitors in the bone marrow, thereby allowing them to migrate to the spleen. We however see decreased total numbers of splenic B cells (data not shown), suggesting that this is not the only explanation for the decrease in bone marrow B lymphopoiesis. Additionally, as thymic subsets are equally decreased across development, it would suggest that TNFα is not directly inhibiting CD25 expression or DP thymocyte development. Type 1 interferons are also produced in our model and can diminish erythropoiesis as well as myelopoiesis and lymphopoiesis. (Binder et al., 1997; Buechler et al., 2013) While we do see reduced erythrocyte progenitors in the bone marrow of mice receiving repeated CpG, myeloid populations are stable. Even though erythropoiesis is reduced in the bone marrow, it is increased in the spleen and liver. Most of the extra-medullary hematopoiesis seen in the spleens of our mice is in fact due to increased erythroblasts (data not shown). These findings suggest that high dose type 1 interferons are not the sole factors mediating the decrease in our models of inflammation. IL-6 is also produced in our model and may be acting at the early stages of lymphopoiesis where it can diminish lymphoid potential.

(Maeda et al., 2005; Maeda et al., 2009; Maeda et al., 2010) It is unlikely the main contributing factor, however, as it would increase later B progenitors in contrast to our

26 data.

Severe inflammation acts as a stressor to the body producing steroid hormones called glucocorticoids. At high doses, these steroid hormones have profound anti- lymphopoiesis effects. In B lymphopoiesis they are known to act primarily at the Pro and

Pre B cell stages as the cells undergo selection. (Andreau et al., 1998; Gruver-Yates et al.,

2014; Igarashi et al., 2005) This is also true in the thymus, where steroids have potent effects inhibiting DP thymocytes that are undergoing positive selection. (Brewer et al.,

2002; Lu et al., 2000) While it is possible that steroids are contributing to the effects we see in our CpG model, the decrease in CLPs and thymocytes across development suggest they are not the sole factors blocking lymphopoiesis.

It is possible that TLR9 signaling alone drives these reductions in B lymphopoiesis as all progenitors express TLR9. (Welner et al., 2008) However, we see similar decreases in B cell precursors in perforin null mice infected with LCMV suggesting that other TLRs and the broad inflammatory environment can also inhibit lymphopoiesis. As LCMV is an RNA virus, it stimulates TLR3, TLR7 and TLR8, of which TLR7 is expressed on lymphoid progenitors. (Sioud et al., 2006) LCMV infection does produce high levels of IFN-γ, which is similar to our CpG model. Since LCMV infection is a much stronger stressor than repeated CpG dosing, it is likely that steroids are also playing a larger inhibitory role in this model.

Overall, these data suggest that CpG induced inflammation acts at multiple stages of B cell development while also inhibiting T cell development. This may be occurring by multiple mechanisms, including skewing of early progenitors away from the B

27 lineage, decreased proliferation at the Pre B cell and DP thymocyte stages and increased apoptosis. As thymopoiesis is also impaired, it suggests that global lymphoid development may be diminished or that inflammatory factors act independently after progenitors commit to separate lineages. Most likely, inflammation is doing both because it acts at early and later stages of development. The decreases at multiple stages of development are likely due to various inflammatory factors, including TLR9 signaling itself, IFN-γ, type 1 interferons, IL-6, and TNFα.

Chapter 4

TLR9 and IFNγR signaling directly impair B lymphopoiesis

Introduction

Early hematopoietic cells including CLPs and developing B cells are known to express high levels of TLR9. (Welner et al., 2008) Data from Paul Kincaide’s lab suggests that TLR9 stimulation of CLPs may divert these progenitors away from the B cell lineage and towards the dendritic cell lineage. (Welner et al., 2008) While the CLP population used in that paper is heterogeneous including both Ly-6D- and B cell committed Ly-6D+ CLPs, it shows that CLPs can receive instructive signals through their

TLRs. We do know that the decrease in lymphopoiesis is dependent on global CpG signaling through TLR9, as TLR9 null mice do not develop the signs of systemic inflammation nor do they have decreased B or T lymphopoiesis (data not shown). It is unclear however, whether TLR9 must signal on the lymphoid progenitors themselves or whether it is signaling on other cells to release secondary factors that then mediate the decreases in lymphopoiesis.

One candidate factor is IFN-γ. Our lab has also shown that IFN-γ is essential for the full spectrum of disease to develop, as IFN-γR knockout (IFN-γRKO) mice are protected from developing widespread inflammation and do not produce the array of cytokines listed above.34, 36 The IFN-γ receptor is a heterodimer of two subunits, IFN-γR1 and IFN-γR2. (Bernabei et al., 2001; Jung et al., 1987) IFN-γR1 is expressed almost

34 ubiquitously in the immune system, while IFN-γR2 is expressed on subsets of myeloid cells, B cells, stromal cells and certain populations of T cells. (Gazit et al., 2013) Both subunits are expressed on CLPs and B precursors. Early thymocyte precursors also express IFN-γR, but decrease expression during thymic development until cells reach the single positive stages. (Mingueneau et al., 2013) As stromal cells also express the IFN-

γR, it is possible that IFN-γ is directly acting to inhibit stromal cell release of cytokines necessary to promote development. (Stagg et al., 2006)

In myeloid cells, it is well documented that TLR and IFN-γR signals can interact and enhance the cellular response to either stimuli. In macrophages, IFN-γ exposure primes cells to respond to TLR4 signaling. (Chen and Ivashkiv, 2010; Franklin et al.,

2009; Hu et al., 2008) IFN-γ priming causes less production of the anti-inflammatory factor IL-10 and excess production of TNFα, IL-6, and IL-12. This occurs by a variety of mechanisms, including increased expression of TLRs, enhanced downstream signaling, as well as epigenetic modulation leading to increased and prolonged recruitment of TLR signaling molecules to the promoters of inflammatory genes. Perhaps sustained TLR signaling and IFN-γ production are required to drive the decreases in B lymphopoiesis, which is why a single dose is insufficient compared to 4 or 10 days of dosing. It is possible that IFN-γR and TLR9 signaling are acting synergistically directly at the level of the lymphoid progenitors or on peripheral cells to produce a secondary mediator that decreases B and T lymphopoiesis. We therefore assayed the roles of TLR9 and IFN-γR signaling play in inhibiting B lymphopoiesis by using in vitro and in vivo models.

Chapter 4

Results

CpG and IFN-γ stimulation differentially affect B and T cell growth in vitro.

As B cell precursors express both TLR9 and IFN-γR, we investigated which B cell progenitors were affected by CpG and IFN-γ treatment using the established OP9 co- culture system to eliminate confounding factors that may be present in vivo. (Cho et al.,

1999; Schmitt and Zuniga-Pflucker, 2006) Ly-6D- CLPs, Ly-6D+ CLPs, Pro, or Pre B cells were cultured in B cell promoting conditions and administered PBS, CpG, or IFN-γ.

We found significant reductions in the number of B cells generated from Ly-6D+ CLPs,

Pro and Pre B cells that received CpG or IFN-γ compared to PBS (Figure 7A). To test if treatment effects were specific to the B cell lineage, CLPs were also cultured in T cell promoting conditions. Surprisingly, Ly-6D- CLPs receiving CpG yielded more T cell precursors while IFN-γ still severely decreased T cell yields (Figure 7B). This suggests that CpG and IFN-γ are acting via different pathways to alter CLP development.

TLR9 signaling directly inhibits B cell differentiation but increases T cell differentiation in vitro.

To interrogate whether the CpG induced inhibition of B lymphopoiesis was acting in a TLR9 intrinsic manner, we performed similar in vitro experiments using mixed cultures of equal numbers of TLR9 sufficient and TLR9 deficient (TLR9KO) CLPs. In the B cell promoting conditions, we found that only the TLR9 sufficient progenitors

36 produced lower B cell yields while the TLR9KO cells yielded similar numbers in the

PBS and CpG treated groups (Figure 8A). We performed the same experiment in T cell promoting conditions and found that only the TLR9 sufficient CLPs led to increased T cell yields with CpG treatment, suggesting that TLR9 signaling directly alters the lineage potential of CLPs (Figure 8B).

TLR9 extrinsic factors contribute to the decrease of B lymphopoiesis in vivo.

To determine if TLR9 signaling was the primary cause of the lymphopoiesis defects in vivo, we made mixed bone marrow chimeras. We injected lethally irradiated

TLR9KO hosts with mixed 50:50 congenically marked WT/TLR9KO bone marrow and waited 10 weeks for reconstitution before administering the 10-day CpG regimen. In contrast to the in vitro data, we found that the TLR9KO B cell progenitors were not spared, as Pre B cell numbers from mixed chimeras receiving CpG demonstrated similar decreases in numbers of both wild type (WT) and TLR9KO precursors (Figure 9A). Pro and Immature B cells yielded similar results to the Pre B cells (data not shown). This suggests that in vivo, TLR9 intrinsic signaling does not decrease lymphopoiesis or that the contribution of other TLR9 extrinsic factors mask the role of TLR9. While the thymocyte data demonstrated an upward trend in numbers of cells seeing TLR9 stimulation, the changes were not significant suggesting that other factors are also acting on thymopoiesis in vivo. These factors, including IFN-γ, may be inhibiting thymopoiesis thereby masking a TLR9 intrinsic effect (Figure 9B).

Type 2 but not type 1 interferons mediate decreases in lymphopoiesis.

Because IFN-γ is induced by in vivo TLR9 stimulation and it inhibited CLP differentiation in vitro (Figure 7), we investigated whether IFN-γ was contributing to B and T lymphopoiesis defects in vivo. We injected WT or IFN-γRKO mice with CpG or

PBS and found that the IFN-γRKO mice were protected from inflammation as they had significantly smaller decreases in B cell progenitors compared to WT mice (Figure 10A).

This suggests that IFN-γ is one of the main mediators leading to decreased B cell progenitors in vivo. It may be the confounding factor responsible for masking the TLR9 intrinsic effect. This is not true of all TLR9 induced cytokines, as mice lacking the IFN-α receptor gene (IFN-αRKO) were not protected compared to the WT mice (Figure 10B).

The restoration of B cell precursors in IFN-γRKO mice was not complete, suggesting that other signals also contribute to the defects in B lymphopoiesis. Thymic cellularity, in contrast to B lymphopoiesis, was not dependent on type 1 or 2 interferons since both

IFN-αRKO and IFN-γRKO mutant mice had statistically similar decreases compared to

PBS controls, and the interaction coefficient was not significant (Figures 10C and D).

This suggests that B and T lymphopoiesis are differentially regulated by interferons.

Interferon gamma receptor signaling directly inhibits Ly-6D+ CLP development in vitro.

To test if the decreases we saw in vitro with IFN-γ administration were IFN-γR

38 precursor cell intrinsic, we used the same mixed culture strategy as before but with WT and IFN-γRKO cells and IFN-γ administration. In these mixed cultures, we found that only the WT cells yielded fewer B cells in response to IFN-γ, as the IFN-γR KO cell yields were similar to cultures receiving PBS (Figure 11A). Since IFN-γ decreased T cell yields when added to OP9-dl1 cultures, we wanted to test whether IFN-γ is acting directly on the T cell precursors like it was acting on the B cell precursors. Using the same mixed strategy as for B cells, we found that only the Ly-6D+ cultures demonstrated a IFN-γR intrinsic decrease in T cell yields while both IFN-γRKO and WT cells decreased in the Ly-6D- cultures (Figure 11B). Even though Ly-6D+ CLPs do not generate T cells in vivo, they can in vitro. These data suggest that Ly-6D+ CLPs are more sensitive to IFN-γ than Ly-6D-, and that the OP9-dl1 cells or T cell precursors produce factors in response to IFN-γ that also decrease T lymphopoiesis.

Interferon gamma receptor signaling directly inhibits B cell development in vivo.

We then investigated whether a cell intrinsic IFN-γR response is the dominant force leading to decreased B cell progenitors in vivo by making mixed IFN-γRKO and

WT congenically marked bone marrow chimeras. We used a 90% WT and 10% IFN-

γRKO mix since mice generated with fewer IFN-γ responsive cells do not generate the full panel of cytokines normally seen in the sustained TLR9 inflammation model

(unpublished observations). We found that there was a statistically significant differential in Pre B cell numbers in the CpG treated mice such that the IFN-γRKO B cells were less affected than WT (Figure 12A). This suggests that there is a measurable cell intrinsic

IFN-γR signaling event leading to decreased B lymphopoiesis during sustained TLR9 inflammation in vivo. Consistent with the results from the IFN-γRKO experiments

(Figure 10A), the IFN-γRKO cells in the chimeras also trended towards a smaller decrease, emphasizing that IFN-yR signaling is not the only factor leading to decreased B lymphopoiesis and perhaps unmasking a TLR9 effect.

IFN-γ and CpG act via distinct pathways to inhibit B lymphopoiesis in vivo.

Given that there appear to be different TLR9 and IFN-γR effects on B cell development in vitro, we next investigated whether these two pathways interact in vivo, particularly since previous work in innate cells found that IFN-γR and TLR9 signaling can synergize at the epigenetic level. (Chen and Ivashkiv, 2010; Hu et al., 2008; Qiao et al., 2013) To address this question, we used IFN-γ deficient mice (IFN-γKO), that are capable of responding to IFN-γ but are unable to produce it, and examined how CpG,

IFN-γ or the combination of both stimuli affect B and T lymphopoiesis. The dose of IFN-

γ was selected to replicate serum levels of IFN-γ seen in WT mice treated with the repeated CpG protocol. (Behrens et al., 2011) We observed that both CpG and IFN-γ are capable of to reducing Pre B cell and thymocyte numbers (Figure 12B). However, statistical modeling did not reveal a significant interaction effect, suggesting that they are acting independently via additive and not synergistic pathways, corroborating our in vitro data.

Chapter 4

Discussion

Our data demonstrate that TLR9 has disparate effects on the B and T cell lineages, impairing early progenitors from becoming B cells, yet enhancing their ability to become T cells in vitro. One possibility is that TLR9 is acting at the level of the CLP to skew precursors away from the B cell lineage, and perhaps towards the T cell lineage.

This may be intrinsic to each CLP or a result of general skewing of the population towards CLPs with greater T cell potential since those with greater B cell potential are decreased. As CpG also had inhibitory effects on plated Pre B cells, it suggests that it also induces apoptosis or inhibits proliferation in later progenitors. This is in contrast to TLR9 signaling on mature B cells, which promotes maturation and induces proliferation.

(Guerrier et al., 2012) The differential effects of TLR9 on immature versus mature B cells may be due to alterations in downstream signaling molecules or epigenetic changes allowing TLR9 signaling factors to promote different outcomes. This differential signaling is not unheard of, as immature B cells receiving signals through their BCR undergo apoptosis while mature B cells are activated and undergo proliferation.

IFN-γ in contrast, had devastating effects on both B and T cell cultures of CLPs suggesting that it is inducing apoptosis or preventing proliferation regardless of exposure to B or T instructive signals. It had similar results on plated Pre B cells suggesting that

IFN-γ signaling effects do not change as the progenitors mature. Perhaps this strong IFN-

γ effect overwhelms the ability to detect an intrinsic TLR9 role in vivo in the mixed bone

41 marrow chimera system. Surprisingly, even though type 2 interferons directly reduced B lymphopoiesis, type 1 interferons did not. While type 1 and 2 interferons share similar downstream signaling factors, IFN-γ is much more highly expressed in our model, which may account for its dominating effects in vivo. (Canna et al., 2014; Canna et al., 2013)

IFN-αRKO null mice do however show slightly improved measures of systemic disease including weight loss and anemia. (Canna et al., 2013) This suggests that Type 1 interferons are important for some aspects of inflammation, (Canna et al., 2014; Canna et al., 2013) but may play a redundant role with IFN-γ in mediating decreased B lymphopoiesis.

Interestingly, while TLR9 and IFN-γ are known to act via synergistic pathways in macrophages, our statistical modeling data suggest that they are acting independently to inhibit B lymphopoiesis. This is supported by our in vitro data where TLR9 signaling encourages T cell development but IFN-γ inhibits both T and B cell development. TLR9 signaling appears to skew CLPs away from the B cell lineage while IFN-γ solely acts to induce apoptosis or block proliferation. It may be possible that the common sites where synergism is observed in myeloid cells, promoters of TNFα and IL-6 for example, are not transcriptionally active in these early progenitors or are not main contributors responsible for decreasing lymphopoiesis.

Chapter 5

Introduction

The ability of TLR9 to enhance T cell potential but inhibit B cell potential suggests that transcriptional changes may be are occurring within these progenitors altering their lineage potential. Early hematopoiesis relies on complex interactions of various transcription factors important for directing cells towards specific lineages.

Hoxa9 is a homeobox transcription factor important for early hematopoiesis. Hoxa9 null mice exhibit lower numbers of peripheral myeloid and lymphoid cells. They also have reduced clonogenic potential for erythroid and myeloid colony types with even larger reductions in B cell outputs from lymphoid colonies. (Lawrence et al., 1997) One key aspect of Hoxa9’s regulation of lymphopoiesis is control of Flt3 expression. (Gwin et al.,

2010) Hoxa9 null mice have very low levels of FLt3 expression, which accounts for part of the reductions in lymphoid cells. (Gwin et al., 2013b) Hoxa9 is highly expressed at the

Ly-6D- CLP stage where it turns on genes important for B cell specification before turning off allowing for further development. (Gwin et al., 2013a; Gwin et al., 2010;

Gwin et al., 2013b) Spleen focus forming virus pro-viral integration oncogene (Pu.1) is another transcription factor important in hematopoiesis. Pu.1 mutant mice have normal numbers of megakaryocytes and erythroid progenitors but display a multilineage defect in myeloid and lymphoid lineages. (Scott et al., 1994) Pu.1 also controls expression of

IL-7Rα and is required for the generation of Pro B cells. (DeKoter et al., 2002) Another transcription factor expressed early during development is Ikaros. Ikaros proteins act as

49 transcriptional repressors and activators and in lymphoid progenitors contribute to upregulation of Flt3 and IL-7Rα. (Georgopoulos et al., 1994) Ikaros mutant mice have normal myeloid erythroid formation yet lack T, B, and NK cells as well as common lymphoid progenitors .(Georgopoulos et al., 1994)

Once these early regulating transcription factors upregulate expression of Flt3 and

IL-7Rα, cells are directed towards the lymphoid fates retaining minimal myeloid potential. Expression of E12 and E47, collectively known as E2A, is essential for B cell development as E2A mutant mice have intact peripheral myeloid and T cell populations but absent peripheral B cells and low numbers of CLPs. (Bain et al., 1994) E2A is also an important regulator of IL-7Rα, which together turn on the transcription factor EBF-1.

(Seet et al., 2004; Tsapogas et al., 2011) EBF-1 plays a central role in B lineage specification, regulating many of the genes associated with the B cell receptor and immunoglobulin heavy chain recombination. (Lin and Grosschedl, 1995) While EBF-1 specifies the B cell fate, its induction of the transcription factor Pax5 solidifies B cell commitment as Pax5 over-expressing cells can no longer generate T cells or macrophages, (Souabni et al., 2002) and Pax5 mutant Pro B cells maintain multilineage potential. (Nutt et al., 1999; Rolink et al., 1999)

Many of the same early genes important for B lymphoid development are important for T cell development including Hoxa9, Pu.1, and Ikaros. One unique issue during T cell development is the migration of uncommitted lymphoid precursors from the bone marrow to the thymus. (Carlyle and Zuniga-Pflucker, 1998) Successful thymic settling requires expression of chemokine receptors including CCR7 and CCR9. (Zlotoff

50 et al., 2010) While loss of CCR7 and CCR9 reduces the numbers of early thymic progenitors, total cellularity is near normal due to the large proliferative capacity of cells that do successfully enter the thymic environment. (Zlotoff et al., 2010) Once these progenitors settle the thymus, high levels of notch signaling instruct the T cell fate and block factors important for the B cell and dendritic cell fates including E2A, Pax5, and

CCAAT/enhancer binding protein α. (De Obaldia et al., 2013; Pongubala et al., 2008; Pui et al., 1999; Radtke et al., 1999), 66 Notch1 also induces the transcription factors hairy and enhancer of split 1 and T cell factor -1 (TCF1), which silence other cell fates further committing precursors to the T cell fate. (De Obaldia et al., 2013; Rapino et al., 2013;

Tomita et al., 1999; Weber et al., 2011)

Differentiation into the various subsets of dendritic cells requires either granulocyte-macrophage colony stimulating factor or Flt3. Common lymphoid progenitors that become DCs primarily become pDCs or lymphoid resident DCs. (Carotta et al., 2010) High levels of Flt3 drive dendritic cell differentiation from lymphoid precursors. Mice deficient in Flt3 or its downstream signaling molecule signal transducer and activator of transcription 3 demonstrate markedly lower lymphoid resident and plasmacytoid dendritic cells (pDC). (Kingston et al., 2009) Similar to T and B cells, Pu.1 and Ikaros play roles in dendritic cell differentiation from lymphoid progenitors. Induced deletion of Pu.1 leads to decreased pDCs and conventional DCs (cDC) in part through decreased expression of Flt3. (Carotta et al., 2010) Mice expressing low levels of Ikaros have decreased peripheral pDCs but normal cDCs also due to an inability to respond to

Flt3 ligand. (Carotta et al., 2010) Expression of the E protein E2-2 specifies and

51 differentiates dendritic cell precursors to the pDC lineage as E2-2 mutant mice have a specific defect in the pDC compartment with normal cDC and B cell numbers. (Cisse et al., 2008) Correspondingly, pDCs require lower levels of the protein inhibitor of DNA binding 2 (Id2), which is a direct inhibitor of E proteins. Other DC lineages express high levels of Id2, corresponding to their low levels of E2-2. (Jackson et al., 2011) E2-2 also acts to turn on interferon regulatory factor 8, which induces expression of TLR9 and type

1 interferons. (Schroder et al., 2007; Tailor et al., 2007) Type 1 interferons in turn further promote pDC differentiation from CLPs. (Chen et al., 2013)

The ability of TLR9 to enhance T cell potential but inhibit B cell potential in our model suggests that transcriptional changes may be are occurring within early lymphoid progenitors thereby altering their lineage potential. Early hematopoiesis relies on complex interactions of various transcription factors important for directing cells towards specific lineages. We wanted to look for alterations in lineage potential of CLPs in our

CpG treated mice.

Chapter 5

Results

Exposure to CpG induced inflammation in vivo skews CLPs away from the B cell lineage and enhances T cell differentiation in vitro.

To determine if the complex in vivo TLR9 induced inflammatory milieu causes lineage skewing, we sorted Ly-6D+ and Ly-6D- CLPs from mice receiving repeated CpG or PBS injections and placed them in the OP9 culture system under B, T or dendritic cell promoting conditions. No further CpG or inflammatory cytokines were given during the in vitro culture process. We found that Ly-6D+ CLPs isolated from CpG treated mice yielded significantly fewer B cells than those from the PBS treated mice in B cell conditions (Figure 13A). In contrast, we found that the CLPs exposed to the hyperinflammatory environment demonstrated enhanced T cell potential (Figure 13B).

This may occur in a cell intrinsic manner or by skewing of the population towards CLPs that are predisposed to generate T cells. CLPs destined to generate T cells may express higher levels of the thymic homing receptors CCR9 and CCR7. To test this we stained for

CCR9 levels but found lower CCR9 expression on Ly-6D- CLPs form CpG treated mice

(Figure 13C). To test if the increase in T cell yields was unique to the T cell lineage, we also tested DC potential. Ly-6D+ CLPs, while able to differentiate into T cells in vitro generated minimal dendritic cells even under dendritic cell promoting conditions. They did generate B cells however, which showed similar results to the B cell conditions

(Figure 14A). Ly-6D- CLPs gave rise to very few B cells but did generate both pDCs and

53 cDCs (Figure 14B). Surprisingly, exposure to the CpG inflammatory environment did not alter DC yields compared to PBS treated mice.

CpG induced inflammation alters the B cell transcriptional program.

We next assayed the transcriptional program of CLPs during inflammation for factors that might explain the skewing away from the B cell lineage. To get a global transcriptional view, we analyzed mRNA expression from Ly-6D- and Ly-6D+ CLPs from repeated CpG or PBS treated mice by microarray. 3 mice were used per group and samples were run individually. Principle component analysis across all 4 groups of samples demonstrated that the four groups cluster independently, suggesting different transcriptional programs based on both stage of development and the presence of inflammation (Figure 15A). In order to look for differentially expressed genes between the different groups, we performed a two-way ANOVA. Using a false discovery rate of

20% (Benjamini-Hochberg), we found 2734 genes differentially expressed in Ly-6D- versus Ly-6D+ CLPs and 382 genes differentially expressed between PBS and CpG treatments, 225 of which were in both lists (Figure 15B). Thus, approximately 60%

(225/382) of the genes altered by sustained TLR9 induced inflammation are also genes that appeared to be altered during the transition from the Ly-6D- to Ly-6D+ stage of B cell development.

Ingenuity Pathway Analysis suggests Hoxa9 is down-regulated in Ly-6D- CLPs during CpG induced inflammation.

Focusing on the Ly-6D- CLPs, which have in vivo multi-lineage potential, we explored potential factors that could account for the pattern of differentially expressed genes between PBS and CpG exposures using the upstream analysis module of the

Ingenuity Pathway Analysis (IPA) program. We found that the transcription factor Hoxa9 was predicted to have the most down-regulated activity (Table 1). This was of particular interest, as Hoxa9 is described to be an important B cell lineage coordination factor.

(Gwin et al., 2013a; Gwin et al., 2010; Gwin et al., 2013b; Izon et al., 1998) In contrast, we did not find significant evidence suggesting changes in Pu.1, E2A, Ikaros, or E2-2.

Additionally, in concert with what would be expected from our mouse model, many of the predicted up-regulated factors were related to inflammatory cytokines produced in our model.

Hoxa9 and EBF-1 are down-regulated in CLPs exposed to CpG induced inflammation.

We then interrogated transcriptional levels of Hoxa9 and some of its targets by qRTPCR. We found that Ly-6D- CLPs from CpG treated mice had lower levels of

Hoxa9 than their PBS treated counterparts. Known Hoxa9 targets important for B cell specification, Flt3, IL-7Rα and RAG1, (Gwin et al., 2013a; Gwin et al., 2010; Gwin et al., 2013b; Izon et al., 1998) were also decreased (Figure 16A). As many of these targets are also known to be mediated by the transcription factor E2A, we looked at E2A and its

55 unique target FOXO1 but found that neither were significantly decreased transcriptionally, suggesting that alterations in Hoxa9 may preferentially account for the decreases in Flt3, RAG1, and IL-7Rα in CpG treated mice (Figure 16B). While E2A is primarily regulated at the protein level, lack of change in FOXO1 suggests that it is not decreased. EBF-1, a target of IL-7Rα and an important factor for initiating the B cell program, (Tsapogas et al., 2011) was also decreased in Ly-6D- CLPs, (Figure 16A).

Together these data suggest that Hoxa9 and EBF-1 are decreased in CpG treated CLPs and perhaps account for the skewing away from the B cell lineage.

Chapter 5

Discussion

The complex inflammatory environment induced by sustained TLR9 activation leads to distinct transcriptional programs in CLP populations. Via both a bioinformatics approach and by examining transcript levels, we identified loss of Hoxa9 and its indirect target EBF-1 as potential pathways by which inflammation suppresses B lymphopoiesis.

It is interesting to note that NFkB activation suppresses Hoxa9 transcription in a number of different cell types, (Trivedi et al., 2008) consistent with a role for inflammation in this process. While Hoxa9 is important for development of other lineages, Hoxa9 mutant mice have more profound effects in the B cell pathway. (Lawrence et al., 1997) Since

EBF-1 is one of the orchestrating factors of the B cell program, its decrease could allow for progenitors to remain open to other lineage fates although our data suggest this is specific to the T cell and not dendritic cell fates. EBF-1 is normally expressed at high levels during the Ly-6D+ stage of B cell differentiation, which may be why it did not come out of the pathway analysis program, which was focused on Ly-6D- CLPs.

Our data surprisingly suggested that Ly-6D- CLPs exposed to the CpG induced inflammatory environment are impaired in their ability to become B cells, enhanced to become T cells, and indifferent to become DCs. This may be due to yet unknown regulation of genes important for the T cell fate by CpG induced inflammation. In fact, when we look at the raw values from the microarray, TCF1 transcripts are significantly increased in both Ly-6D- and Ly-6D+ CLPs exposed to CpG induced inflammation (data

57 not shown). This is surprising as Notch1 transcripts were significantly decreased in Ly-

6D- CpG CLPs (data not shown), and Notch induces TCF1 under homeostatic conditions.

This suggests that there may be factors biasing progenitors towards or against the T and

B cell fates that are yet unclear and that there is a connection between inflammation and induction of TCF1. Perhaps inflammation serves to augment the low level of Notch signaling present in the bone marrow. Previous studies have suggested that TLR9 signaling drives CLPs towards the dendritic cell fate. (Welner et al., 2008) However, these studies examined bulk CLPs, and thus the loss of Ly-6D+ CLPs during inflammation would skew the bulk CLP population towards the Ly-6D- CLPs that have broader differentiation capabilities. This would make it appear as if bulk CLPs have greater DC potential. In our report, we separate the Ly-6D- and Ly-6D+ populations revealing their true B and DC potentials. This may be due to skewing of individual cells away from the B cell fate or from further heterogeneity within the CLP compartment.

Consistent with our cell culture findings, levels of E2-2 are not significantly changed in our array data (data not shown).

Table I. Ingenuity Pathway Analysis of Ly-6D- CLPs from CpG and PBS treated mice.

Upstream Regulator Activation p-value of overlap Z-score HOXA9 -3.08 5.45e-06

POR -2.813 1.65e-04

HOXA7 -2.789 1.49e-07 RB1 -2.262 3.17e-04 CD3 -2.076 8.76e-06

IFNγ 3.857 3.71e-07 CD38 3.536 3.67e-07 IL5 3.019 5.71e-07 STAT3 2.954 2.74e-05 IL6 2.691 1.82e-08 IL1B 2.673 1.56e-04

CSF2 2.670 4.23e-05

IL4 2.638 1.84e-08 IFN alpha/beta 2.191 3.84e-05 Ifnar 2.135 3.79e-04 IKBKB 2.122 1.74e-05 APP 2.087 1.72e-05

Chapter 6

Future Directions and Discussion

Apoptosis versus proliferation

While it is known that inflammation alters hematopoiesis, it is unclear if individual factors act on the progenitors or on the environment and whether they act at multiple stages. In this thesis, we have shown that B and T lymphopoiesis are diminished in a cell intrinsic manner by both TLR9 and IFN-γR signaling, and that they act independently at multiple stages of development. TLR9 skews early progenitors away from the B cell fate towards the T cell as well as decreases Pre B cell numbers in vitro.

IFN-γ acts independently of CpG, directly decreasing both T and B cells likely at multiple stages of development. While CpG does affect lineage determination, it is still unclear whether TLR9 and IFN-γR signaling induce apoptosis or reduce proliferation of developing cells. In another mouse model, high dose CpG stimulation inhibits Pro B cell proliferation by inducing apoptosis. (Lalanne et al., 2010) Preliminary data from our lab found similar rates of apoptotic B cell progenitors by annexin V staining (data not shown). While this could be because apoptosis is not a primary factor leading to decreased lymphopoiesis, it is also possible that apoptotic cells are cleared too quickly in vivo and therefore changes in rates are undetectable. Future work employing other methods to detect apoptosis including TUNEL staining for DNA fragmentation may be useful in vivo and in in vitro co-cultures. Additionally, if the main cause of decreased lymphopoiesis is due to apoptosis, blocking apoptosis should allow cells to overcome this

64 defect. Use of B cell lymphoma (BCL)-xL or BCL-2 transgenic mice the transgene under control of lymphoid restricted promoters should shed light on whether apoptosis is a main contributor. Proliferation can be tested in vitro by labeling CLPs with carboxyfluorescein succinimidyl ester (CFSE) before plating them on OP9 cells. Dilution of CFSE based on the number of cell divisions can then be compared between CpG and PBS conditions.

This method will require optimization as we attempted this once with in vitro CpG administration but had poor survival of CLPs after CFSE labeling. Analysis of the few surviving cells showed full CFSE dilution between both CpG and PBS treated samples suggesting that the surviving cells have normal proliferative capacities (data not shown).

Feeding mice bromodeoxyuridine (BrdU), a synthetic nucleoside analog of thymidine that is incorporated into proliferating cells, would allow for detection of proliferating cells in vivo. Like CFSE, Brdu levels would dilute out every time the cells proliferate and can be assayed by flow cytometry using antibodies against BrdU. If CLPs, Pro, and

Pre B cells have reduced levels of proliferation in CpG treated mice, one would expect to see fewer rounds of BrdU dilution. Single cell or liming dilution assays could be used to asses changes in cell intrinsic capabilities to become B versus T cells with CpG administration in vitro. We attempted these studies but had poor survival of CLPs at lower concentrations. Therefore these methods will require further optimization.

Decreased thymic cellularity

How inflammatory factors mediate the decrease in thymopoiesis in our model is unclear. While we see decreased levels of CCR9 on Ly-6D- CLPs from CpG treated

65 mice, a homing defect would not fully account for decreases in thymic subsets across development. Homing defects have minimal effects on thymic cellularity, but profound effects on early thymic progenitor numbers. (Zlotoff et al., 2010) Therefore, the primary contributor to the decrease in total thymic cellularity is likely due to an effect of the inflammation on the thymus itself. Recent work looking at the effects of irradiation on thymic development has shown that irradiation leads to decreased thymic epithelial cell numbers. These thymic epithelial cells are important for producing the chemokines that recruit thymic settling progenitors and provide the space required for T cell development.

Decreased epithelial cells would then lead to decreased homing in addition to downsizing the thymic niche so it cannot support normal levels of thymopoiesis. (Zhang et al., 2014b;

Zlotoff et al., 2011) Decreased epithelial cells could account for the decreased thymocyte numbers across all stages of thymopoiesis seen in our model. Future studies characterizing the number and transcriptional profiles of thymic epithelial cells during inflammation may be useful in determining how thymic cellularity is decreasing and whether epithelial cell production of thymic growth factors are altered.

Roles of EBF-1 and Hoxa9 in mediating decreased B lymphopoiesis

Our work identified the important B cell lineage factors EBF-1 and Hoxa9 as being decreased in early progenitors from CpG treated mice. It is unclear which inflammatory factors directly mediate the decreases in Hoxa9 and EBF-1. Because TLR9 signaling had similar effects in vitro, we sorted CLPs from treated TLR9 mixed bone marrow chimeras with the intention of looking at EBF-1 and Hoxa9 levels. If TLR9

66 signaling directly leads to these decreases, then only the TLR9 sufficient CLPs in the

CpG treated mice would have defects in expression. Unfortunately, we are yet unable to perform transcriptional analysis due to recovery of low numbers of CLPs. To assess whether EBF-1 and Hoxa9 can rescue defects in B lymphopoiesis, future studies transfecting bone marrow with EBF-1 or Hoxa9 expressing constructs would be useful.

These cells could then be used in vitro or to make bone marrow chimeras to assess whether forced expression of EBF-1 or HOXa9 restore B lymphopoiesis. EBF-1 turns on early in B cell commitment and stays on throughout development. It specifies the B cell fate, which may rescue defects associated with potential TLR9 skewing of early progenitors away from the B cell lineage. Additionally, as EBF-1 interacts with IL-7, it is possible that restoration of EBF-1 levels could improve survival and differentiation of B cells and mitigate the effects of inflammation. Control of Hoxa9 expression during development is more nuanced, with its peak expression occurring during the Ly-6D- stage before turning back off. In fact, over-expression of Hoxa9 leads to suppression of B lymphopoiesis. (Thorsteinsdottir et al., 2002) Forced expression of Hoxa9 in CpG and

PBS treated mice would be difficult but may still be useful when comparing numbers of

Ly-6D+ CLPs and Pro B cells.

Discussion

A multitude of potential factors can induce hyperinflammation leading to alterations in hematopoiesis. Depending on the instigating signals and dominating cytokines, the resulting inflammation can lead to mobilization of precursors, increased or

67 decreased myelo- or lymphopoiesis, and skewing of progenitors towards different populations. While it is known that certain cytokines such as TNFα and IL-1 impact lymphopoiesis, our work provides novel insights into how TLR9 driven cytokine storm and IFN-γ are acting directly on hematopoietic progenitors at multiple stages of development to impair lymphopoiesis. Sustained TLR9 mediated inflammation occurs in a number of clinical scenarios such as viral or bacterial sepsis and auto-inflammatory disorders including lupus or rheumatoid arthritis. Immuno-paralysis in sepsis, and B cell autoimmunity in lupus may in part arise from dysfunctional B lymphopoiesis. Other hyperinflammatory scenarios where IFN-γ is a dominant inflammatory cytokine, such as

HLH, are associated with B and T lymphopenias and suppression of genes important for

B lymphopoiesis. (Hinze et al., 2010; Sumegi et al., 2011) As the effects of TLRs and inflammatory cytokines signaling on hematopoiesis are better understood, specific blocking agents may serve as effective therapeutics to improve bone marrow homeostasis in patients during hyperinflammation.

It is interesting to speculate why lymphopoiesis is suppressed during inflammation. Emergency myelopoiesis at the expense of lymphopoiesis is well described. (Behrens et al., 2011) Mice over-expressing TLR7 develop a lupus-like syndrome with high levels of type 1 interferons and increased myelopoiesis in the bone marrow and periphery. (Buechler et al., 2013) LPS stimulation of TLR4 causes increased myelopoiesis in a stromal cell dependent manner due to high production of granulocyte colony-stimulating factor. (Boettcher et al., 2012; Quinton et al., 2002) However, our data does not provide evidence of increased bone marrow myelopoiesis. Since we see

68 decreased lymphopoiesis without an increase in myeloid or dendritic cell development, it suggests that these are not simply reciprocal functions, but that each is driven by separate factors. This is supported by data from other models that show TNFα induces migration of B lymphoid progenitors from the marrow whereas IL-1 is required for increased myelopoiesis. (Cain et al., 2011; Ueda et al., 2005; Ueda et al., 2004) Further characterization of myeloid precursors in the marrow and periphery may shed light on whether CpG induced inflammation affects myelopoiesis.

If reduced lymphopoiesis is not due to skewing of progenitors towards myeloid cell fates, an alternative hypothesis is that shutting down lymphopoiesis is the immune system’s attempt to prevent development of autoreactive B and T cells. This may especially be relevant with DNA based TLR9 stimulation that leads to nuclear antigen reactive B cells. In fact, lupus patients with anti-nuclear antibodies have higher percentages of peripheral blood mononuclear cells that express TLR9. TLR9 expression also correlates with disease severity. (Klonowska-Szymczyk et al., 2014) TLR9 is important for development of anti-DNA antibodies in mice. (Christensen et al., 2005)

MRL/MPlpr/lpr mice, which are predisposed to developing a lupus-like syndrome due to a mutation in Fas, produce significantly less anti-DNA and anti-chromatin antibodies when crossed to TLR9 mutant mice. (Christensen et al., 2005) Similar effects are observed in lupus mice crossed to TLR7 deficient mice, which develop significantly fewer anti-RNA antibodies and produce less type 1 interferons. (Jackson et al., 2014) The role of TLR7 and TLR9 in promoting development of anti-nucleotide antibodies was found to be B cell and not myeloid cells intrinsic expression of TLR7 as conditional TLR knockouts in B

69 cells replicated the findings of straight knockouts crossed to the lupus MRL/MPlpr/lpr background. (Jackson et al., 2014) This suggests that preventing B lymphopoiesis during

TLR stimulation causes the developing population to be skewed away from DNA reactive B cells. Long-term experiments monitoring for development of auto-immunity or auto-inflammation after exposure to hyperinflammation while open ended, may shed light on the consequences of decreased lymphopoiesis during inflammation. Studies testing T and B cell repertoires after inflammation may also prove useful in understanding whether decreased lymphopoiesis, or prevention by using TLR9 or IFN-γ blocking antibodies, alters the repertoires and skews developing cells towards known heavy chain usages associated with higher rates of autoimmunity.

Because of DNA’s immuno-stimulatory role, CpG is considered a vaccine adjuvant. However, its use has yielded mixed results, potentially because of its inhibitory roles in lymphopoiesis. Our work suggests that repeated dosing of CpG leads to abnormal lymphopoiesis, which may alter the immuno-repertoire. Sustained decreased in lymphopoiesis may compromise the immune system thereby potentially supporting tumor growth or infection. In a human phase 1 study using TLR9 agonism to activate cells against cutaneous T cell lymphoma, 54% of patients exhibited lymphopenias. (Kim et al.,

2010) Response rates varied depending on the dose of TL9 agonist given with no clear correlation between dose and clinical response. In another phase 1 study of CpG as an adjuvant for a tumor specific peptide, human telomerase reverse transcriptase, patients developed lymphopenia with very low circulating B, NK, and T cell numbers. (Haining et al., 2008) Clinical responses in this trial were also varied with few patients exhibiting

70 clear responses to treatment. As mature B cells are tolerant of self-antigens, impaired lymphopoiesis due to adjuvant induced inflammation could compromise generation of new B cells reactive against the tumor peptide. CpG may act as an appropriate adjuvant for certain cancers and infections, yet its actions on lymphopoiesis and ability to induce auto-reactive B cells may undermine its effectiveness compared to other adjuvants currently in use.

CpG treatment is effective in mediating B cell acute lymphoblastic leukemia (B-

ALL) in mouse models. Mice injected with syngeneic B-ALL cells normally die within

40 days due to tumor burden. However, mice given repeated CpG doses 7 days after tumor injection are able to clear the tumor and have near 100% survival at 140 days.

(Fujii et al., 2007; Seif et al., 2009) This increased survival is in part due to CpG acting on innate and lymphoid cells as mice lacking certain arms of the innate immune system impaired tumor clearance. (Fujii et al., 2007; Seif et al., 2009) One hypothesis is that

CpG is acting as an adjuvant for tumor peptides, however, it is also possible that the inflammation induced by repeated CpG injections, including type 1 and 2 interferons contribute to inhibiting B-ALL growth.

Not all hyperinflammation is equal. Depending on the cytokines that control the inflammatory network, the resulting pathologies can lead to excess stimulation of certain arms of the immune system. In particular, certain cytokines decrease hematopoiesis of various lineages while some increase production of other lineages or lead to mobilization of hematopoiesis from the bone marrow. Prior to the work within this thesis, the roles of

TLR9 and IFN-γR signaling on lymphopoiesis were unclear. We have shown that

71 hyperinflammation downstream of TLR9 stimulation impairs B and T lymphopoiesis at multiple stages of development in part via distinct IFN-γR and TLR9 pathways. TLR9 acts at the CLP stages skewing progenitors away from the B cell lineage. In contrast IFN-

γ acts on both T and B cell fates diminishing lymphopoiesis. Additionally, our data suggest that CLPs exposed to the in vivo inflammatory environment have diminished capability to differentiate into B cells even when removed from the inflammation, This is possibly due to reduced levels of transcription factors important for B cell development.

These CLPs are skewed towards the T cell lineage rather than the DC lineage, which suggests that the DC lineage is more disparate from the B cell lineage compared to the T cell lineage. Together our findings provide insights into the mechanisms that allow inflammation and hypercytokinemia to inhibit B and T cell development. As specific cytokine blocking therapeutics continue to have expanded clinical applications, an enhanced understanding of how cytokines regulate B cell development in disease will be important for predicting efficacy and toxicity of these agents.

Appendix

Abbreviations

B220, protein tyrosine phosphatase receptor type c

B-ALL, B cell acute lymphoblastic leukemia

BCL, B cell lymphoma

BCR, B cell receptor

CD, cluster of differentiation

cDC, conventional dendritic cell

CFSE, carboxyfluorescein succinimidyl ester

cKit, stem cell growth factor receptor

CLP, common lymphoid progenitor

CXCL, C-X-C motif chemokine ligand

DC, dendritic cell

Dl1, delta-like 1

DN, double negative

DNA, deoxyribonucleic acid

DP, double positive

E2A, transcription factor 3

EBF-1, early B cell factor 1

ETP, early thymic progenitor

Flt3, fms related tyrosine kinase 3

Flt3l, fms related tyrosine kinase 3 ligand

HLH, hemophagocytic lymphohistiocytosis

HOXa9, homeobox A9

Id2, inhibitor of DNA binding 2

IFN, interferon

IFNα-R, interferon-alpha receptor

IFN γ -R, interferon-gamma receptor

Ig, immunoglobulin

IL-7Rα, Interleukin 7 receptor alpha

IPA, ingenuity pathway analysis

LMPP, lymphoid primed multipotent progenitor

Ly-6D, lymphocyte antigen 6 complex, locus D

MHC, major histocompatibility complex

NK, natural killer

PAX5, paired box gene 5

PBS, phosphate buffered saline

pDC, plasmacytoid dendritic cell

Pu-1, spleen focus forming virus pro-viral integration oncogene

qRTPCR, quantitative real-time polymerase chain reaction

RAG, recombination activating gene

RNA, ribonucleic acid

Sca-1, lymphocyte antigen 6 complex, locus A/E

SCF, stem cell factor

TCF1, T cell factor-1

TCR, T cell receptor

TLR, toll-like receptor

TNF, tumor necrosis factor

WT, wild type

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