NEUTROPENIA AND PROGENITOR CELL MOBILIZATION IN AP3-Β1 MUTANT PEARL MICE MATTHEW VALLEJO

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1 NEUTROPENIA AND PROGENITOR CELL MOBILIZATION IN AP3-Β1 MUTANT PEARL MICE by MATTHEW VALLEJO CLINTON LOTHROP, JR., COMMITTEE CHAIR XU FENG CHRISTOPHER KLUG THOMAS RYAN GENE P. SIEGAL A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2013

2 Copyright by Matthew Vallejo 2013

3 NEUTROPENIA AND PROGENITOR CELL MOBILIZATION IN AP3-Β1 MUTANT PEARL MICE MATTHEW VALLEJO PATHOLOGY ABSTRACT Mutations in the AP3-β1 gene are known to cause Hermansky Pudlak syndrome type 2 (HPS2) in humans and canine cyclic hematopoiesis (CH) in gray collie dogs. HPS2 patients have severe congenital neutropenia (SCN), and dogs have cyclic neutropenia (CN). The mechanism behind neutropenia is uncertain. Pearl mice have an AP3-β1 mutation, but whether they actually have neutropenia has not yet been evaluated. This dissertation presents a discussion of the evaluation of neutropenia in pearl mice, hematopoietic progenitor cell mobilization defect in pearl mice and finally an evaluation of methods of dog-to-mouse xenotransplantation to establish a xenotransplant model of the dog hematopoietic system. Dog xenotransplant models could be used to model canine CN. Based on complete blood counts as part of this dissertation research, Pearl mice do not have SCN or CN. The number of colony forming units (CFU) that form colonies containing granulocytes and bone marrow lineage negative (lin - ), Sca + and c-kit + cells in the bone marrow were, in fact, shown to be moderately increased in pearl mice. Normal and pearl mice also had similar responses to cyclophosphamide and bortezomib. AMD3100 treatment resulted in similar numbers of peripheral blood CFUs and CBCs in Bl/6 and pearl mice. However, Bl/6 mice administered G-CSF had higher peripheral blood neutrophil counts and greater peripheral blood CFU numbers than pearl mice. Pearl mice did have decreased hematopoietic progenitor cell/granulocyte mobilization in v

4 response to G-CSF. Additional studies will be necessary to determine if CH dogs or HPS2 patients also have a mobilization defect. Dog-to-NSG mouse xenotransplant models could aid in the study of neutropenia in dogs. Four methods of cell delivery that have been used in human xenotransplant studies were tested: Tail-vein injections, intrafemoral injections, intrafemoral injections after flushing the femur, and neonatal liver injections. Mice that stably engrafted developed severe graft vs. host disease. Further studies are necessary to determine pre- and post-transplant conditions that will allow for improved survival and engraftment. Establishment of an optimal dog-to-mouse xenotransplant protocol will aid in the study of neutropenia in the CH dog. It will also allow for opportunities to establish dog xenotransplant models of other diseases. Keywords: xenotransplant; NSG; mobilization; AP3; neutropenia; pearl vi

5 ACKNOWLEDGMENTS I would like to give thanks to the lab members who gave me guidance and support. I want to thank Dr. Tommy Hock for teaching me lab techniques early in my training. I also want to thank Dr. Glenn Niemeyer for his invaluable advice and guidance on experiments. In addition Dr. Niemeyer s advice on writing the dissertation proved to be very valuable. A special thanks goes to Drs. Christopher Klug, Gene Siegal, Thomas Ryan and Xu Feng for their serving on my dissertation committee. Thanks also goes to Dr. Rakesh Patel for helping me with the graduation process. Finally, I want to thank Dr. Clint Lothrop, Jr. for giving me the opportunity to earn a Ph.D. in his lab. Outside of graduate school I want to thank my parents for never discouraging me and for their continuing sacrifices to help me in my pursuits. Also special thanks to my wife, Talita Araujo, for bearing with me as I pursued my degree. And finally thanks to my two-year-old son, Nathanael, for being a beacon of joy though he does not know it. vii

6 TABLE OF CONTENTS Page ABSTRACT...v ACKNOWLEDGMENTS... vii LIST OF TABLES...x LIST OF FIGURES... xii INTRODUCTION... 1 Neutrophils... 1 Hematopoietic Stem Cell Niche... 1 Granulocyte Colony Stimulating Factor... 2 Neutrophil Granules... 2 Neutrophil Elastase... 3 Elastase and Neutropenia... 3 AP3 Complex... 4 Adaptor Related Proteins... 4 AP3 Complex... 4 Neutropenias in Humans... 5 Severe Congenital Neutropenia... 5 Mutations Linked to SCN... 6 Cyclic Neutropenia... 7 G-CSF as a Treatment for Neutropenia... 7 G-CSF s Effects on the HSC Niche... 8 AP3-β1 Mutation and Hermansky-Pudlak Syndrome Type II... 8 Link Between Neutrophil Elastase and Beta3a in Neutropenia... 9 Animal Models of Neutropenia Related Mutations... 9 Drosophila AP3 Mutants CH Dog G-CSF Treatment of CH Dogs Pearl Mice Mocha Mice Beige Mice GFI1 Mutant and GFI1 Knockout Mice CSF3R Mutant and Knockout Mice SBDS Knockout Mice TAZ Knockdown Mice ELA2 Mutant and ELA2 Knockout Mice Animal Model Summary Immunodeficient Mice viii

7 The Non-Obese Diabetic Mouse The Severe-Combined Immunodeficient Mouse NOD/SCID Mouse NSG mice Human Xenotransplants NSG Xenotransplant Engraftment Models Xenotransplant Models of Infectious Diseases Xenotransplant Models of Cancer Xenotransplant Delivery Methods Dog Xenotransplants Dogs as Preclinical Models of Transplants Dogs as Models of Human Disease NOD/SCID Xenotransplant of Dog Cancers NOD/SCID Xenotransplant of Normal Cells Introduction Summary DECREASED HEMATOPOIETIC PROGENITOR CELL MOBILIZATIONIN AP3B1 DEFICIENT MICE TRANSPLANT OF CANINE BONE MARROW MONONUCLEAR CELLS IN NSG MICE CAUSES SEVERE GRAFT VS. HOST DISEASE CONCLUSION: SUMMARIES, DISCUSSION, AND FUTURE DIRECTIONS Summary of Neutropenia and Mobilization in Pearl Mice Future Studies: Mobilization in Neutropenia Models Mobilization in Pearl Mice Mobilization in Other Mice Models of Neutropenia Related Mutations.. 98 Mobilization in Larger Animals Summary of Dog-to-Mouse Xenotransplants Future Studies: Dog-to-Mouse Xenotransplants Using Xenotransplantation to Study Neutropenia Conclusion REFERENCES APPENDIX: IACUC APPROVAL LETTER ix

8 LIST OF TABLES Table Page DECREASED HEMATOPOIETIC PROGENITOR CELL MOBILIZATIONIN AP3B1 DEFICIENT MICE 1 Pearl Mice Have Significantly (p< 0.05) Higher WBC, Granulocyte, Lymphocyte and Monocyte Counts and Significantly Lower RBC, Hgb Level and Hematocrit Levels.50 2 Bone Marrow Differential Counts in Pearl and Bl/6 Mice Pearl Mice Have Significantly Increased (p < 0.05) Colony Forming Unit Granulocyte, Erythroid, Macrophage, and Megakaryocyte (CFU-GEMM) and Colony Forming Unit Granulocyte (CFU-G) but Decreased (p < 0.05) Colony Forming Unit Erythroid (CFU-E) from Bone Marrow Bl/6 and Pearl Mice Show Similar Engraftment Bone Marrow Differential Counts in Pearl and Bl/6 Mice Treated with G-CSF...54 TRANSPLANT OF CANINE BONE MARROW MONONUCLEAR CELLS IN NSG MICE CAUSES SEVERE GRAFT VS. HOST DISEASE 1 Survival Rate of Mice Receiving Dog Bone Marrow Transplants via Different Methods of Transplant Number of Surviving Mice with Dog Leukocyte Engraftment Percentage of Dog Leukocytes in Mice Transplanted with Dog Bone Marrow Mononuclear Cells Using Tail-vein Injections Percentage of Dog Leukocytes in Mice Transplanted with Dog Bone Marrow Mononuclear Cells Intrafemoral Injection Percentage of Dog Leukocytes in Mice Transplanted with Dog Bone Marrow Mononuclear Cells Intrafemoral Injection after Flushing of the Femur...91 x

9 6 Percentage of Dog Leukocytes in Mice Transplanted with Dog Bone Marrow Mononuclear Cells Hepatic Injection in Neonatal Mice Graft vs. Host Disease in NSG Mice...92 xi

10 LIST OF FIGURES Figures Page INTRODUCTION 1 Condensed Overview of the Hematopoietic Stem Cell Niche under Steady State Including Cells and Ligand/Receptor Pairs Involved in Niche Maintenance Condensed Overview of the Effects of G-CSF on the Hematopoietic Stem Cell Niche Under Steady State Including Cells and Ligand/Receptor Pairs Involved in Niche Maintenance...25 DECREASED HEMATOPOIETIC PROGENITOR CELL MOBILIZATIONIN AP3B1 DEFICIENT MICE 1 Pearl Mice Show Similar PMN Counts to Bl/6 Mice Early HSC Numbers are Increased In Pearl Mice Recovery Following Cyclophosphamide-Induced Myelosuppression is Similar in Bl/6 and Pearl Mice Granulopoiesis after Bortezomib Treatment is Similar in Pearl and Bl/ Elastase, MPO and Cathepsin G Activity Normal in Pearl Mice Elastase Activity of Pearl Mouse Neutrophils Distributed Broadly over Several Subcellular Fractions as Determined with Discontinuous Percoll Gradients Pearl Mice have Beta3a Granulocytes Counts are Higher in Bl/6 Mice than Pearl Mice after Mobilization with G-CSF CFU Number in Bl/6 Higher than Pearl in Peripheral Blood but not Higher in Bone Marrow after Mobilization with G-CSF CFU Number in Peripheral Blood after AMD3100 Mobilization Similar in Bl/6 and Pearl Mice...65 xii

11 TRANSPLANT OF CANINE BONE MARROW MONONUCLEAR CELLS IN NSG MICE CAUSES SEVERE GRAFT VS. HOST DISEASE 1 Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via tail-vein injection 7-9 weeks post-transplant Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrafemoral injection 7-9 weeks post-transplant Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrafemoral injection after flushing the left femur 7-9 weeks posttransplant Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrahepatic injection of neonatal mice 7-9 weeks post-transplant...95 xiii

12 1 INTRODUCTION Neutrophils Adult human beings generate more than 20 billion new white blood cells daily. White blood cells such as neutrophils arise from on the progressive differentiation of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) [1]. HPCs amplify and differentiate into myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, and then finally into mature neutrophils. Cytokines and growth factors such as interleukin-3, interleukin-6, stem cell factor, granulocyte/macrophage colony stimulating factor, and granulocyte colony stimulating factor (G-CSF) stimulate the development of neutrophils from HSCs/progenitor cells [1, 2]. Mature neutrophils are then released into the blood [3]. HSCs and progenitor cells in the bone marrow can greatly increase neutrophil production in response to stress or infection [4-6]. Neutrophils kill invading pathogens via phagocytosis and extracellular release of enzymes [2]. Hematopoietic Stem Cell Niche The HSC niche establishes an appropriate microenvironment for the orderly and progressive maturation of HSCs. The HSC niche may play an important role in neutropenia since in neutropenia progenitor cells that differentiate from HSCs may be lacking, have visible phenotypes, or have defects in function. In the HSC niche several

13 2 receptors on HSCs interact with ligands during quiescence. Some of these receptor/ligand interactions are very late antigen 4 (VLA4)/vascular cellular adhesion molecule 1 (VCAM1), C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12), c-kit/stem cell factor (SCF), c-mpl/thrombopoietin (TPO), TIE2/aginopoietin-1 (ANG1), and sphingosine-1-phosphate (S1P) receptor/s1p. These receptors/ligand interactions allow HSCs to attach to osteoblasts, mesenchymal stem cells (MSC), and CXCL12 abundant reticular cells (CARS). Besides the aforementioned cells, macrophages also play a role in HSC quiescence. Macrophages stimulate MSCs to maintain HSCs through a poorly defined mechanism(s) [7] (Figure 1). Granulocyte Colony Stimulating Factor Granulocyte colony stimulating factor (G-CSF) is produced by the endothelium, monocytes, and several other types of immune cells. The G-CSF receptor is expressed on HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, cells of the granulocyte lineage, monocytes, lymphocytes, and platelets [8-13]. The G-CSF receptor is also found on non-hematopoietic cells such as cardiomyocytes, endothelial cells, neurons, and neural stem cells [14-17]. Neutrophil Granules Neutrophil granules are closely related to lysosomes; they even share some contents. Based on the contents of the granules, size, density and extracellular secretion tendency, the granules are classified differently. The major classifications are secretory

14 3 vesicles, tertiary/gelatinase granules, specific secondary granules, and primary/azurophil granules. Primary granules store defensins, proteinase 3, cathepsin G, myeloperoxidase and elastase [18-21]. Though granules can be classified based on contents there is overlap of granule characteristics [22]. Based on the key functions these granules perform, alterations in the contents or formation of these granules may lead to hematopoietic and/or immune defects [23, 24]. Neutrophil Elastase Neutrophil elastase is synthesized during the myeloblast and promyelocyte stage of hematopoietic development and is stored in azurophil granules [25, 26]. The gene for elastase, ELA2, is located in a cluster on chromosome 19 in humans along with azurocidin and proteinase 3, two other serine proteases [26]. Elastase is 218 amino acids long and is glycosylated at two different sites [27]. The C and N terminals are cleaved within the granules to form mature elastase, which is then stored in the azurophil granules [28]. Neutrophil elastase is found in monocytes as well as in neutrophils [29-31]. Activated elastase is capable of digesting several substrates such as complement proteins, cytokines, proteoglycans, collagen, and elastin [32, 33]. This wide range of activity destroys bacteria both within the neutrophil via endocytoses and outside the neutrophil via secretion of elastase and other enzymes [34]. Elastase and Neutropenia Mutations in ELA2 have been linked to severe congenital neutropenia (SCN) and cyclic neutropenia (CN) [35-37]. The possibility that ELA2 mutations may cause

15 4 endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) has been suggested. Under this model, the ELA2 mutation causes a misfolding of elastase at the promyelocytes stage, which then triggers ER stress and UPR, causing apoptosis of the myeloid progenitor cells. The reduced neutrophil precursor expansion then leads to SCN or CN [35, 36, 38]. Mutant versions of ELA2 expressed in RBL-1 cells demonstrate normal expression of elastase and also elastase enzymatic activity that in one study varied between mutants but never was abolished [39]. Apparently the defect causing neutropenia is not caused by lack of expression of elastase or abolishment of enzyme activity. AP3 Complex Adaptor Related Proteins There are four known adaptor related proteins: AP-1, AP-2, AP-3, and AP-4. They each consist of 4 subunits with 2 large subunits, 1 medium subunit, and 1 small subunit. All four are believed to have two ears connected by hinges to a main structure. All the known adaptor related proteins have all been associated with vesicle biogenesis and protein trafficking. Adaptor related proteins shuttle cargo proteins to appropriate vesicles [40]. AP3 Complex The AP3 complex, which has relevance in cyclic hematopoietic (CH) dogs, pearl mice, and HPS2 patients consists of the subunits beta3a, delta, sigma, and mu encoded by the genes AP3-β1, AP3-δ, AP3-σ, and AP3-μ [41]. The beta3a and the delta subunits

16 5 are the two large subunits with mu and sigma being the medium and the small unit, respectively. Beta3b is found in some of the AP3 complexes in neuronal cells rather than beta3a [42]. Beta3a is expressed ubiquitously, including within neuronal cells [43, 44]. Mu also has two isoforms, one which is expressed ubiquitously and one which is only expressed in neuronal cells [45]. Sigma has two isoforms but both are expressed ubiquitously [41]. Ap3 associates with clathrin [46, 47], although pathways requiring AP3 do not always require clathrin [48]. Neutropenias in Humans Neutropenia is a lack of neutrophils. The lack of neutrophils results in an increased risk for severe infection due to neutrophils role in the immune system. Neutrophils release the proteases stored in the azurophil granules and reactive oxygen species which destroy invading pathogens. SCN and CN are the two most common forms of congenital neutropenia. Severe Congenital Neutropenia SCN is characterized by an absolute neutrophil count (ANC) chronically below 500/µL (normal count is 1,500-8,000/µL). Two to three people out of a million in the US have SCN. Most cases (38%-80%) of SCN are caused by mutations in the ELA2 gene which encodes neutrophil elastase [35-37], but several other mutations have been linked to SCN.

17 6 Mutations Linked to SCN SCN has been linked to several mutations in several disparate genes besides ELA2. Most other mutations associated with SCN are accompanied by other symptoms. The mechanisms causing neutropenia in patients with these aforementioned mutations remain largely unknown. Mutations associated with SCN have been found in the hematopoietic cell-specific Lyn substrate 1 (HCLS1)-associated protein X-1 (HAX1), Wiskott-Aldrich syndrome (WAS), growth factor independent 1 (GFI1), colony stimulating factor receptor (CSF3R), glucose-6-phosphatase catalytic subunit 3 (G6PC3), mitogen activated protein binding protein interacting protein (MAPBPIP), adenylate kinase 2 (AK2), glucose 6-phosphate translocase (G6PT1), Shwachman-Bodian- Diamond syndrome (SBDS), Tafazzin (TAZ), chemokine receptor 4 (CXCR4), and lysosomal trafficking regulator (LYST) genes[49-64]. The HAX1 gene encodes mitochondrial protein HAX1 which is also found in the endoplasmic reticulum and the nuclear envelope. HAX1 is believed to promote cell survival and interacts with lymphoid-enhancer binding factor 1. HCLS which is associated with HAX1 is essential for G-CSF induced granulopoiesis [50, 51]. The GFI1 gene encodes GFI1 protein, an oncoprotein transcriptional repressor [65]. GFI1 also represses expression of ELA2 [54]. The WAS gene encodes the WAS protein, an actin cytoskeleton regulator. The mutation related to SCN is in the gene region of WAS coding for the conserved GTPase binding domain [53]. Other mutations of WAS are not associated with neutropenia. The G6PC3 gene encodes the catalytic subunit 3 of glucose-6-phosphatase. The CSF3R gene encodes the G-CSF receptor. The MAPBPIP gene encodes MAPBPIP, also known as p14 an endosomal adaptor protein. The AK2 gene encodes the AK2 protein, an intermembrane

18 7 space mitochondrial protein that may control apoptosis via FADD and caspase 10 [66]. The G6PT1 gene encodes glucose 6-phosphate translocase that mediates transport of glucose 6-phosphate into the endoplasmic reticulum. The SBDS gene encodes a ribosomal protein. The TAZ gene most likely encodes a protein involved in cardiolipin transacylation in the mitochondria [67]. The CXCR4 gene encodes the chemokine receptor 4 protein. The LYST gene which encodes lysosomal trafficking regulator, a 425 kd protein that has been suggested to be a critical lysosomal trafficking regulator of vesicle fusion and/or fission [64]. The number and variety of gene mutations associated with SCN suggests that several different mechanisms may cause SCN. Cyclic Neutropenia The other form of congenital neutropenia is cyclic neutropenia (CN). In human CN, the ANC cycles from normal to low every three weeks. One out of every million in the United States has CN [37]. Virtually all known cases of CN in humans have been linked to ELA2 gene mutations. CN is inherited in an autosomal dominant manner. G-CSF as a Treatment for Neutropenia G-CSF has been cloned and expressed in E. coli. As a result, Filgrastim (human recombinant G-CSF) has been made commercially available for treatment of congenital neutropenia in humans. Side effects from Filgrastim include bone pain, headache, and rash. Neutropenia patients receiving Filgrastim show elevated levels of blood neutrophils

19 8 and a higher percentage of neutrophils in the bone marrow [68]. A peglyated form of Filgrastim with a longer half-life is also commercially available. G-CSF s Effects on the HSC Niche G-CSF is known to stimulate hematopoietic progenitor cell mobilization. G-CSF receptor expression only in monocytes is sufficient to induce HSC mobilization [69]. G- CSF functions by expanding the number of HSCs, depleting osteoblasts, inhibiting macrophage stimulation of MSCs, changing concentration of some of HSCs ligands, increasing sympathetic tone in the bone marrow microenvironment. G-CSF may also act through other mechanisms [7, 70] (Figure 2). AP3-β1 Mutation and Hermansky-Pudlak Syndrome Type II The recessively inherited Hermansky-Pudlak syndrome type II (HPS2) is associated with mutations in the AP3- β1 gene, which codes for the beta3a subunit of the AP3 complex. HPS2 patients have SCN. The other symptoms of HPS2 that accompanies the SCN are platelet storage pool deficiency, reduced lipid antigen presentation, reduced natural killer cells activity, and reduced pigmentation in hair, skin, and eyes. Neutrophils, platelets, natural killer cells, melanocytes and cytotoxic T cells, all cells with a large amount of intracellular granules or lysosome related organelles, typically have observable phenotypes [71-73]. These findings suggest AP3 is critical to normal myelopoiesis.

20 9 Link Between Neutrophil Elastase and Beta3a in Neutropenia It has been hypothesized that there is a link between neutrophil elastase and beta3a. Mutations in the genes encoding both proteins have been linked to SCN and/or CN. Elastase may bind directly or indirectly to AP3 via the beta3a domain. AP3 may then transport the elastase to the azurophil granules [74, 75]. Mutations in ELA2 and AP3-β1 may cause mistrafficking of elastase [74, 75]. Mistrafficked elastase has been proposed to cause CN and SCN via different mechanisms. Cleavage of G-CSF and its receptor has been suggested as a mechanism for neutropenia [32]. Elastase may activate cell death via the unfolded protein response (UPR) [35, 38]. However, a decrease in the half-life of neutrophils in dogs with CN has not been found [76]. Elastase may also serve as negative feedback on neutrophil production [77]. The exact roles of elastase and AP3 in controlling myelopoiesis in non-disease states and disease state remain unexplained. Animal Models of Neutropenia Related Mutations Several animal models have been used to study mutations directly or indirectly linked to neutropenia. These animal models include fruit flies, mice, and dogs. There are several different types of mouse models that represent a wide range of different mutations that are linked to neutropenia. Knockout models of some of the genes related to neutropenia also exist. Fly mutants have been used to study the various subunits of the AP3 complex.

21 10 Drosophila AP3 Mutants Drosophila mutants are known to have mutations in each of the subunits of AP3. Drosophila AP3-δ mutants have garnet eye color and reduced pigmentation of other organs [78, 79]. AP3-μ mutants have carmine eye color [81]. AP3-β1 mutants have ruby eye color [80, 81]. And AP3-σ mutants have orange eye color [81]. CH Dog The mutation associated with the CH dog phenotype is an adenine insertion in the AP3-β1 gene resulting in a frameshift and a premature termination [75]. CH dogs have CN instead of the SCN observed in HPS2 patients with an AP3-β1 mutation. The neutrophil count cycles approximately every 12 days in CH dogs[82]. The number of monocytes, lymphocytes, eosinophils, reticulocytes, and platelets cycle out of phase with neutrophils [83]. Along with the CN and cyclic hematopoiesis, CH dogs exhibit platelet storage pool deficiency, and reduced hair, skin, and eye pigmentation much like HPS2 patients [71, 75]. G-CSF Treatment of CH Dogs G-CSF treatment in CH dogs varies based on the individual dog, but in general CH dog neutrophil blood counts do rise. The neutrophil count still cycles, but at higher numbers at both the peaks and nadirs relative to CH dogs not receiving treatments [84-86]. CH dog bone marrow mononuclear cells (BMMC) have been observed to require an

22 11 elevated dose of G-CSF in vitro in order to achieve similar colony counts as normal dog BMMCs [87]. Pearl Mice AP3-β1 mice mutants, known as pearl mice, have platelet storage pool deficiency, reduced lipid antigen presentation, reduced natural killer cell activity, and reduced pigmentation in hair, skin, and eyes. Like the CH dog and HPS2 patients, pearl mice have abnormal melanosomes, natural killer cell cytotoxic granules, and platelet dense granules [88-92]. The mutation giving rise to pearl mice consists of a tandem duplication 793 base pairs long. The tandem duplication introduces a stop codon at the junction of the duplication [93]. The mutation appeared spontaneously in C3H/He mice. The C3H/He mice with the AP3-β1 mutation were then backcrossed into Black 6 mice for 10 generations resulting in the pearl mouse. Mocha Mice Mocha mice have a mutation in the AP3-δ gene that causes a truncation of the delta subunit. The entire AP3 complex is missing in mocha mice [94]. Mocha mice typically have hypopigmentation, platelet storage pool deficiency [95, 96], balance problems, renal lysosomal enzyme deficiency, deafness that develops later [95, 97], and abnormal cortical excitability [98]. Mocha mice have no known neutropenia even though dogs and humans with a mutation in AP3-β1 have CN and SCN respectively.

23 12 Beige Mice Beige mice have a mutation in LYST and are an animal model of Chediak Higashi syndrome (CHS). Beige mice have immune defects, platelet storage pool disease, and coat color dilution. Beige mice are characterized by the large, bizarre lysosomal granules in multiple cells including neutrophils [99-103]. In the absence of LYST there is a loss of granule integrity and leakage of granule contents. Elastase and cathepsin G activity in mature neutrophils is significantly decreased in beige mice [104]. Beige mice accumulate a 46 kd elastase precursor that is enzymatically inactive, but can be activated with proteolytic processing [105]. Unlike CHS patients, beige mice are not neutropenic [104]. GFI1 Mutant and GFI1 Knockout Mice GFI1 knockout mice exhibit SCN [106, 107]. Mice transduced with Gfi1N382S, a GFI1 mutation associated with SCN in humans, also exhibit neutropenia [108]. Neither the knockout nor the transgenic mouse produces neutrophils in response to treatment with G-CSF [ ]. Down regulation of the colony stem cell factor 1 and its receptor have been proposed as the mechanism leading to SCN [108]. CSF3R Mutant and Knockout Mice Mice transgenic for a CSF3R mutation associated with SCN in humans have not been shown to have neutropenia. The transgenic mice are hyper responsive to G-CSF treatment with the number of circulating neutrophils increasing much more than normal

24 13 mice treated with G-CSF [109]. In contrast humans with neutropenia associated CSF3R mutations are unresponsive to G-CSF treatment [49]. CSF3R knockout mice have decreased neutrophil counts [110]. SBDS Knockout Mice Mice have been generated with a targeted SBDS mutation that disrupts translation of the SBDS gene. The mice homozygous for the mutation are embryonic lethal. Mice that are heterozygous for the mutation have normal phenotypes consistent with the fact that humans heterozygous for mutation in the SBDS gene have normal phenotypes. In humans, patients homozygous for a mutation in the SBDS gene that causes early truncation have not been found, consistent with the fact that the homozygous knockout mice are embryonic lethal [111]. TAZ Knockdown Mice Mice with TAZ knockdown die as neonates due to cardiomyopathy [112]. Human patients with TAZ mutations also develop cardiomyopathy early. Whether TAZ knockdown mice have neutropenia or not is unknown. ELA2 Mutant and ELA2 Knockout Mice ELA2 knockout mice have not been associated with neutropenia [113]. Mice transgenic for the V72M ELA2 mutation and mice transgenic for the G193X ELA2 mutation do not exhibit SCN [114, 115]. In humans both the V72M and the G193X

25 14 mutation are associated with SCN. However, mice with the G193X ELA2 mutation do exhibit neutropenia when treated with bortezomib, a proteosome inhibitor which causes inhibition of the endoplasmic reticulum associated degradation pathway [115]. Animal Model Summary Many of the mutations linked to CN and SCN have species dependent effects. Humans, fruit flies, dogs, and mice do share hypopigmentation linked to mutations in genes coding for AP3 subunits. In humans, mutations in ELA2 are associated with both SCN and CN, and mutations in AP3-β1 are associated with SCN. CH dogs have CN and not SCN, though the CH dog shares many symptoms with HPS2 patients [71, 75]. Many of the mouse models with mutations in genes linked with neutropenia have not been shown to have neutropenia. Mice with mutations in ELA2, CSF3R, LYST, and AP3-β1 genes have not been shown to have neutropenia. ELA2 knockout mice have also not been shown to have neutropenia. Mice with a mutation in a gene for the delta subunit of AP3 do not have neutropenia but exhibit other pathology. Oddly, CSF3R mice mutants are hyperresponsive to G-CSF treatment whereas humans with the same mutation are not responsive to G-CSF. SBDS knockdown mice and TAZ knockout mice die before measurements of circulating neutrophils can be practically done. GFI1 mutant mice have SCN as do GFI1 knockout mice. Mice overall seem resistant to neutropenia although the GFI1 mutant and knockout mice do develop neutropenia.

26 15 Immunodeficient Mice Many diseases related to the immune system can be readily investigated using transplant studies. However, ethical concerns prevent using humans as transplant recipients for most studies. Instead, human cells are often transplanted into mice. Immunodeficient mice are often used to enhance engraftment of human HSCs. Immunodeficient mice have even been developed to provide better recipients for xenotransplantation studies. The Non-Obese Diabetic Mouse The non-obese diabetic (NOD) mouse is a mouse strain susceptible to development of Type I diabetes developed by Makino and colleagues [116]. NOD mice have a mutation in the CTLA-4 gene [117]. CTLA-4 encodes a protein that is an inhibitor of T-cell activation [118, 119]. The Severe-Combined Immunodeficient Mouse The severe-combined immunodeficient (SCID) mouse has a mutation in the SCID gene which originally occurred spontaneously in CB17 mice. The SCID mouse has few to no lymphocytes [120]. NOD/SCID Mouse The NOD/SCID mice were bred at Jackson Laboratories by backcrossing the SCID mutation into NOD mice. Unlike NOD mice the NOD/SCID mouse does not

27 16 develop diabetes. Unlike the SCID mouse the NOD/SCID mouse has severely reduced natural killer cell activity. The NOD/SCID mouse also has less mature macrophages and lack complement activity unlike SCID mice. Defects in both the adaptive and innate immune system make the NOD/SCID mouse a better candidate for human cell reconstitution than mice that have either the NOD or the SCID mutation alone and have been used in many transplant studies [121, 122]. NSG mice Since the development of NOD/SCID mice, several different NOD/SCID strains have been developed [122]. One of these NOD/SCID mice is the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. The added gamma chain knockout has made the NSG mouse especially useful in human-to-mouse xenotransplant studies [ ]. Human Xenotransplants Xenotransplantation has been used to study hematopoiesis, the immune system, and transplantation [ ]. NSG Xenotransplant Engraftment Models Several NSG mice xenotransplant models have been researched. NSG newborn mice injected with 1 X 10 5 CD34 + or CD human cord blood cells via facial vein showed 70% engraftment. Human myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes all developed in the mice after three months [125]. Adult NSG

28 17 mice injected with 1 X 10 7 human peripheral blood mononuclear cells via the tail vein also showed high engraftment of lymphocytes. Human peripheral blood mononuclear cells transplanted into NSG mice via the tail-vein showed high early engraftment. However, survival was 0% after 30 days [124]. High engraftment of B and T cells four months post-transplant has been shown [126]. Other studies have also shown engraftment of human hematopoietic cells. Human CD34 + cells (7 X 10 5 ) injected via tail-vein showed high engraftment of lymphoid cells and low myeloid engraftment [127]. NSG mice transplanted with CD34 + have good engraftment after several weeks [123, , 130]. NSG mice transplanted with human hematopoietic cells had functional human immune systems [126, 127, 130]. Even development of human red blood cells and human platelets after transplantation has been shown [125]. As a result of moderately efficient human hematopoietic cell engraftment in NSG mice, NSG mice have been used extensively for studies of human hematopoiesis [122]. Xenotransplant Models of Infectious Diseases NOD/SCID mice xenotransplant models of infectious disease have been developed [131]. NOD/SCID mice transplanted with human CD34 + cells had symptoms of Dengue fever when infected with the dengue virus [132]. NOD/SCID mice transplanted with human CD34 + cells had symptoms of Epstein-Barr virus (EBV)- induced lymphoproliferative disease when infected with EBV [133]. Research of a possible drug response model showed that Hepatitis C therapies in scid-alb-upa mice transplanted with human hepatocytes and subsequently infected with hepatitis C virus responded similarly to hepatitis C patients receiving the same therapies [134].

29 18 Xenotransplant models of malaria have also been developed. Mice bred to have both the SCID mutation and an albumin urokinase-type plasminogen activator mutation and transplanted with human hepatocytes were then infected with Plasmodium falciparum. The human hepatocytes within the mice were infected with P. falciparum and showed a similar phenotype to hepatocytes found in humans infected with P. falciparum [135]. NOD/LtSz-SCID mice engrafted with human erythrocytes were also infected with P. falciparum. All the mice in the study developed a P. falciparum infection [136]. Due to functional human T lymphocytes in NSG mice after engraftment of human cells, NSG mice have also been proposed to be useful in the study of HIV [130]. The use of mice to replicate the human immune system and then using the mice to study human infectious disease demonstrates that creating immunodeficient models of human immune responses is feasible. Xenotransplant Models of Cancer Besides infectious diseases, human leukemia and other cancers have been studied using NSG mice [ ]. NOD/SCID mice have proven to be excellent models to study leukemia and lymphomas [143]. Human skin grafts on SCID mice were used to study cancer induced by high exposure to UVB light [144]. Xenotransplants have also been used to specifically study cancer stem cells. Transplantation of juvenile myelomonocytic leukemia (JMML) cells into NSG mice gave evidence that JMML originate in HSCs [145]. Acute myeloid leukemia stem cells were investigated using NOD/SCID mice transplanted with human acute myeloid leukemia (AML) cells [146]. Multiple myeloma (MM) stem cells have also been investigated using NOD/SCID mice

30 19 transplanted with CD34 + cells from human MM patients [147]. A small population of breast cancer cells has been shown to cause tumors in NOD/SCID mice [148]. NOD/SCID mice were used to identify human colon cancer stem cells [149]. NOD/SCID mice have also been used to identify human brain tumor stem cells [150]. The fact that immunodeficient mice can be used to model the human immune system and several diseases demonstrates the versatility of immunodeficient mice as well the practicality of using them. Xenotransplant Delivery Methods Several different methods of xenotransplantation have been investigated in the NSG mouse xenograft model. One of the methods that has been used is tail-vein injection [123, 124, 126, 127]. Another method that has been used in xenotransplantation studies is intra-femoral injection [151, 152]. Metastatic prostate cancer cells from patients have successfully been engrafted into Rag2-/-γc-/- mice using intra-femoral transplantation [151]. A study using intra-femoral injection demonstrated excellent engraftment of human breast cancer cell lines [152]. Another method of transplantation that has been used is intrahepatic injection of neonatal mice. Intrahepatically transplanting human CD34 + cells from cord blood cells into Rag2-/-γc-/- mice on a BALB/c background showed high engraftment of human cells. It even showed development of a human adaptive immune system [129].

31 20 Dog Xenotransplants Obtaining human tissue samples for xenotransplants can be ethically and practically challenging. Dog tissue samples are an alterative sample source that could be used. Dogs are well established as preclinical models of transplantation as well as multiple genetic diseases. Dogs as Preclinical Models of Transplants Dogs are a well established preclinical models of human bone marrow transplantation [153, 154]. Dogs have been used to determine the survival effect of infusing bone marrow into dogs following high dose chemotherapy [155], immunocompetence of grafted bone marrow over time [156], transfer of immunity via transplant of immune memory cells [157], post-transplant infections [158], engraftment of CD34 + cells [159], pre-transplant irradiation [160], pre-transplant drug regimens [161] and post-transplant evaluation of chimerism [162]. Gene therapy via transplant of transfected hematopoietic cells has also been studied in dogs [163]. Dogs have also been used to study the effects of transplant on specific disease models. Some of the diseases that have been studied are X-linked severe combined immunodeficiency [164] and leukocyte adhesion deficiency [165]. Bone marrow transplant of pyruvate kinase deficient dogs has ameliorated hemolytic anemia secondary to the pyruvate kinase deficiency [166, 167]. Besides the study of bone marrow transplantation in dogs, transplantation of other cells from organs has been studied. An example of other cell type transplant studies is transplant of cryopreserved dog fetal liver cells [168, 169] as well as renal transplantation [170].

32 21 Dogs as Models of Human Disease Dogs have also been used as hematopoietic disease models in addition to preclinical models of transplant. Leukemias spontaneously develop in dogs much like humans [171]. Dog models of Hermansky-Pudlak syndrome type II, pyruvate kinase deficiency, Hemophilia A and B and type I Glanzmann's thrombasthenia have also been described [82, 86, ]. NOD/SCID Xenotransplant of Dog Cancers In addition to being used as preclinical transplant models and human disease models, dogs have also been used in studies employing xenotransplantation. Canine transmissible venereal tumor has been established in NOD/SCID mice via subcutaneous injection [180]. Another canine cancer, Spirocerca lupi-associated sarcoma, has been successfully transplanted into NOD/SCID mice [181]. Intraperitoneal injection of NOD/SCID mice with T-cell lymphomas has successfully been performed [182]. Canine mammary tumors have been shown to successfully form in NOD/SCID mice [183]. Ema, CLC, Nody-1 and UL-1 lymphoma dog cell lines have also formed tumors within NOD/SCID mice [184]. The NI-1 cell line, a canine mastocytoma cell line, has been shown to form mastocytoma lesions in NSG mice. The NSG model of the NI-1 cell line has been used to research mast cell activation and drug resistance [185]. The success of dog cancer xenotransplants has given promise for future dog xenotransplant studies.

33 22 NOD/SCID Xenotransplant of Normal Cells In addition to dog cancer cells, NOD/SCID mice have also been transplanted with other cell types. Bone marrow stromal cells (BMSC) from dogs were transplanted into the cranial ventricles of NSG mice. A few of the BMSCs from young dogs had characteristics of neural progenitor cells after ten days [186]. NOD/SCID mice have also been transplanted with dog bone marrow mononuclear cells (BMMCs) and lineage depleted BMMCs. Low levels of dog leukocyte engraftment occurred in the bone marrow and spleen of the recipient mice [187]. Dog CD34 + cells intravenously injected into NOD/SCID mice failed to show engraftment of dog cells, but transplant of human CD34+ cells showed consistent engraftment possibly indicating that successful engraftment of dog cells may be more challenging than successful engraftment of human cells in NOD/SCID mice [188]. Introduction Summary The mechanisms causing the pathology of neutropenia have not yet been pinpointed. Several animal models of mutations related to neutropenia exist that can help in the study of neutropenia. One large animal model of neutropenia is known. The CH dog has a mutation in the AP3-β1 gene which codes for the beta3a protein, a subunit of the AP3 complex. Humans with AP3-β1 mutations have HPS2 of which SCN is one of the symptoms. CH dogs also have neutropenia, but they have CN instead of SCN. Pearl mice are known to have an AP3-β1 mutation. Like the CH dog, pearl mice express many of the same symptoms as HPS2 patients, though the expression of neutropenia is uncertain in these mice. However, the similarity of the pearl mouse symptoms to HPS2

34 23 symptoms indicates that pearl mice are good models of HPS2. Study of the CH dog and pearl mouse together may help elucidate mechanisms behind neutropenia. The establishment of a CH dog hematopoietic system in NSG mice, one of the best mice for xenotransplants, may assist in the study of neutropenia by providing another method to study neutropenia. Immunodeficient mice have already been used extensively in several xenotransplant studies and hold promise for the study of neutropenia in dogs. However, the successful establishment of the dog hematopoietic system in mouse is necessary before a xenotransplant model can be used. Three obstacles to successful establishment of the dog hematopoietic system are: host vs. graft disease (HVGD), failure to engraft and graft vs. host disease (GVHD). The use of immunodeficient mice greatly decreases the likelihood of HVGD. Although successful engraftment may prove challenging, several different transplant methods have been used successfully in xenotransplant studies, greatly increasing the chance that an optimal method for the engraftment of dog cells will be found. The more successful engraftment is, the higher the chance GVHD will develop [189]. GVHD developed in mice given leukocytes if the mice had high engraftment [190]. The success of human xenotransplant studies does indicate that obstacles to successful establishment of the dog hematopoietic system can be overcome. The two main hypotheses tested in this dissertation was that pearl mice have altered hematopoiesis and that a dog xenotransplant model can be established in NSG mice.

35 24 Bone Macrophage Bone Sympathetic Nerve Nerve Macrophage HSC HSC MSC MSC CXCL12 BM Sinusiod CXCL12 BM Sinusoid Osteoblasts Osteoblasts CXCR4 CAR CXCR4 CAR Figure 1: Condensed overview of the hematopoietic stem cell niche under steady state including cells and ligand/receptor pairs involved in niche maintenance. HSC = hematopoietic stem cell; CXCL12 = Chemokine (C-X-C motif) ligand 12; CXCR4 = C- X-C chemokine receptor type 4; CAR = CXCL12 abundant reticular cells; mesenchymal stem cell; BM = bone marrow.

36 25 Bone Sympathetic Nerve Macrophage MSC HSC G-CSF MSC BM Sinusoid Osteoblasts CXCR4 CXCL12 CAR Figure 2: Condensed overview of the effects of G-CSF on the hematopoietic stem cell niche under steady state including cells and ligand/receptor pairs involved in niche maintenance. HSC = hematopoietic stem cell; CXCL12 = Chemokine (C-X-C motif) ligand 12; CXCR4 = C-X-C chemokine receptor type 4; CAR = CXCL12 abundant reticular cells; mesenchymal stem cell; BM = bone marrow.

37 26 DECREASED HEMATOPOIETIC PROGENITOR CELL MOBILIZATIONIN AP3B1 DEFICIENT MICE by MATTHEW O VALLEJO, GLENN P NIEMEYER, ALEX VAGLENOV, TOMMY HOCK, BRIDGET URIE, PETER CHRISTOPHERSON, AND CLINTON D LOTHROP JR Submitted to Experimental Hematology Format adapted for dissertation

38 27 Abstract Neutropenia is common to both Hermansky Pudlak syndrome type 2 (HPS2) and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if AP3B1 deficient pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin-), Sca+ and c-kit+ cells (LSK cells), bone marrow elastase, myeloperoxidase and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or G-CSF administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM and LSK cells were moderately increased in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers than pearl mice. Unlike HPS-2 patients and CH dogs, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell/granulocyte mobilization in response to G-CSF. Key Words: niche; hematopoiesis; neutropenia; AP3B1; mobilization

39 28 Introduction AP-3 is a heterotetrameric protein complex involved in intracellular vesicle transport and sorting of cargo molecules to lysosome-related organelles. AP-3 is a member of a family of adaptor protein (AP) complexes that includes AP-1 to AP-4. AP complexes are widely distributed and highly conserved among eukaryotes. Molecular analysis has shown that mutations in the AP3B1 gene, which encodes the Beta3a subunit of the AP-3 complex, cause Hermansky-Pudlak syndrome 2 (HPS-2) in humans, cyclic hematopoiesis (CH) in gray collie dogs and granule defects in pearl mice [1, 2]. Beta3a is ubiquitously expressed in most cells. A related gene AP3B2 which encodes Beta3b (B- NAP) is expressed in neuronal cells [3]. A mutation in the AP3B1 gene typically results in observable phenotypes in cells with abundant lysosome-related organelles or intracellular granules such as cytotoxic T lymphocytes, melanocytes, natural killer cells, neutrophils and platelets [4-6]. Because many of these cell types are derived from the hematopoietic stem cells (HSC) and because lysosome-related organelles have important housekeeping functions for maintaining cellular homeostasis, alterations in their contents or formation may have a deleterious effect on the hematopoietic or immune systems [7, 8] Cyclic neutropenia and approximately 50% of the cases of congenital neutropenia result from mutations in the ELANE (ELA2) gene which encodes neutrophil elastase [9]. Since Beta3a deficiency is associated with CH in gray collie dogs and congenital neutropenia in HPS-2 patients [1, 10], it has been hypothesized that AP-3 functions as a chaperone to shuttle elastase from the site of biosynthesis in the ER/Golgi complex to its storage depot, the primary granules [1, 11, 12].

40 29 Primary granule protein biosynthesis is tightly regulated and occurs primarily in neutrophilic promyelocytes. It has been proposed that ELA2 mutations result in elastase misfolding in promyelocytes causing increased ER stress, induction of the unfolded protein response (UPR) and apoptosis of myeloid progenitor cells which results in decreased expansion of neutrophil precursors and cyclic neutropenia or chronic neutropenia [13-15]. Abnormalities in platelet dense granules, melanosomes and NK cell cytotoxic granules have been well described in the pearl mouse, HPS-2 patients and CH dogs [2, 10, 16-19]. However, it was not known if cyclic neutropenia or chronic neutropenia occur in pearl mice with mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice have CH or chronic neutropenia. The results demonstrate that pearl mice do not have congenital neutropenia or cyclic neutropenia but do have decreased hematopoietic progenitor cell/granulocyte mobilization. Materials and Methods Animals C57BL/6J (Bl/6), B6Pin.C3-Ap3b1 pe /J (pearl) and NOD.CB17-Prkdcscid/J (NOD/SCID) mouse strains were purchased from Jackson Laboratories, (Bar Harbor, ME, USA). Mice were housed in ventilated racks in a barrier facility. Male and female 12- to 26-week old mice were used for experimental procedures. All animal procedures were approved by the Auburn University and University of Alabama at Birmingham Institutional Animal Care and Use Committees (IACUC).

41 30 Peripheral blood and bone marrow analysis Peripheral blood (50 to 100µl) from femoral and tail vein bleeds was collected with 1cc syringes with zero volume swaged 27 gauge needles that had been heparin preflushed into EDTA (ethylenediaminetetraacetic acid)-coated Eppendorf tubes. Complete blood counts and differential counts were performed using a Heska Veterinary Hematology Analyzer (Heska Corp., Denver, CO, USA). Bone marrow was harvested by flushing both femurs with phosphate-buffered saline containing 0.1% bovine serum albumin (BSA). Manual bone marrow differential counts were performed on Wrightstained cytospin preparations. A minimum of 500 cells was counted to determine the bone marrow differential count. Measurement of LSK cells Bone marrow was harvested from femurs by flushing as described above. Cells were then washed twice with PBS. Washed cells were resuspended in PBS supplemented with 0.5% BSA and 2 mm EDTA for antibody staining. Bone marrow cells were incubated with purified anti-cd16/cd32 mab 2.4G2 to block Fc binding (BD Bioscience, San Diego, CA, USA). The cells were then stained with FITC-conjugated anti-sca-1 Ab, phycoerythrin (PE)-conjugated anti-c-kit Ab, and APC-conjugated Abs to CD3e, CD11b, CD45R/B220, Ly-76 (erythroid), Ly-6G, Ly-6C (gr-1) or APCconjugated isotype-matched control antibodies for 20 minutes at 4 C in the dark (BD Bioscience). After washing twice with PBS, cells were fixed with BD cytofix buffer (BD Bioscience). Cells were analyzed on a MoFlo flow cytometer (Beckman Coulter, Inc.,

42 31 Fullerton, CA). For measurement of lin Sca-1 + c-kit + (LSK) cells, the percentage of c- kit + Sca-1 + cells were analyzed on electronically gated lin cells. Hematopoietic progenitor cell assay Bone marrow mononuclear cells (BMMC) were plated in 3.0 ml methylcellulose media (MethoCult M3334 for erythroid progenitors, M3534 for granulocyte and macrophage progenitors: Stem Cell Technologies, Vancouver, BC, Canada) supplemented with human erythropoietin for erythroid progenitors (CFU-E and BFU-E) or murine stem cell factor, murine interleukin-3 (IL-3) and human interleukin-6 (IL-6) for quantification of granulocyte and macrophage progenitors (CFU-GM, CFU-M, CFU-G). Cultures were plated, 2 X 10 5 cells/35mm dish for erythroid progenitors or 2 X 10 4 cells/ 35 mm dish for granulocytic progenitors, in duplicate and placed in a humidified chamber with 5% CO 2 at 37 o C. Colony-forming unit-erythroid (CFU-E) were enumerated after 2-3 days of culture, mature burst-forming unit-erythroid (BFU-E) were detected after 3-4 days of culture. Granulocyte and macrophage progenitor colony formation was counted after 12 days. Stress granulopoiesis assay Cyclophosphamide (Sigma, St. Louis, MO, USA) was reconstituted in sterile water and given as a single intraperitoneal injection at a dose of 200 mg/kg [20]. The CBCs were determined prior to injection and on 3, 5, 7 and 10 days after cyclophosphamide administration.

43 32 Bortezomib treatment Isogenic pearl and Bl/6 mice were administered bortezomib at 1 mg/kg, subcutaneously. The percent of bone marrow granulocytes and the percent of B cells in peripheral blood were determined by flow cytometry on days 2, 3, 4 and 5 after injection as described above. Bone marrow cells were incubated with anti-mouse Ly-6G PE ( BD Pharmingen, San Diego, CA, USA) for 20 minutes at 25 C. The percent of granulocytes was determined as the percent of Ly-6G positive cells. Peripheral blood ( µl) was incubated with anti-mouse CD19 Alexa Fluor 647 ( BD Pharmingen) for 30 minutes at 25 C. Blood was then incubated with RBC Lysis Buffer ( ebioscience, San Diego, CA, USA ) for 10 minutes at 25 C. Antibody and lysis Buffer was then discarded and cells washed once in 1% Rat Gamma Globulin in PBS. Cells were then resuspended in 500 µl of 1 % Rat Gamma Globulin in PBS. The % of B cells was determined as the percent of CD19 positive cells. Bone Marrow Transplantation Bone marrow cells were obtained from the long bones of 3-6 month old isogenic Bl/6 and pearl mice. Increasing numbers of bone marrow cells were administered by tail vein injection to 2 to 6 month old NOD/SCID recipient mice that received 3.0Gy total body irradiation 2 to 4 hours prior to transplantation. The % donor cell engraftment in spleen and bone marrow was determined after 4-5 weeks by flow cytometry. Bone marrow and spleen cells (1 X 10 6 cells) were placed in 400 µl of red blood cell lysis buffer (Purogene, Minneapolis, Minnesota, USA) for 5 minutes. Lysis buffer was then removed from the cells, and 100 µl of 10% Mouse Serum ( Jackson

44 33 Immunoresearch, West Grove, PA, USA) in PBS was added to the cells. Bone marrow and spleen cells were then incubated with anti-mouse CD45.2/Ly5.2 ( BD Pharmingen) and anti-mouse CD45.1/Ly5.1 ( BD Pharmingen) for 20 minutes at 25 C. The antibodies were then removed from the cells. The cells were resuspended in 500 µl of 10% mouse serum in PBS. The % of donor cells was determined as the number of Ly 5.2 positive cells. The % of recipient cells was determined as the number of Ly 5.1 positive cells. 7-AAD ( E BD Pharmingen) was added to exclude dead cells. Western Blots and Enzyme Assays BMMCs (1 X 10 6 ) were lysed in RIPA buffer containing protease inhibitors. Cell lysates were subfractionated on discontinuous percoll gradients and Western blots performed as previously described [21]. Anti-Beta3a (sc Santa Cruz, Santa Cruz, CA, USA) and anti-βnap ( BD, San Diego, CA, USA) were used to detect Beta3a and βnap respectively. Anti-Actin (sc Santa Cruz) was used as a loading control. Elastase, myeloperoxidase and cathepsin G enzyme activity were determined using labeled peptide substrates for elastase (N-Methoxysuccinyl-ala-ala-pro-val P- nitroanilide M4765 Sigma), for MPO (3,3,5,5 -Tetramethylbenzidine (TMB) 0440 Sigma) and for cathepsin G (N-succinyl-ala-ala-pro-phe pna S7388 Sigma). Specificity was determined using specific inhibitors for elastase, for MPO and for cathepsin G.

45 34 G-CSF and AMD3100 Administration 100 µg/kg of G-CSF (Filgrastim, Amgen, Thousand Oaks, CA) was administered via intraperitoneal injection into Bl/6 mice (n=8) and pearl mice (n=8) daily for 5 days. The mice were sacrificed 5 hours post-injection on day 5. CBCs were performed on all mice on day 0 and again on day 5 just prior to sacrifice. Peripheral blood and bone marrow cells were prepared as described above. Fifty µl of peripheral blood or 2 X 10 4 bone marrow cells were placed in methylcellulose medium (StemCell Technologies 03434) containing insulin, transferrin, stem cell factor, IL-3, IL-6, erythropoietin and after 7 days the number of CFUs were determined. Five mg/kg of AMD3100 (A5602 Sigma-Aldrich) was administered via subcutaneous injection to Bl/6 mice (n=3) and pearl mice (n=3) one hour prior to sacrifice. The number of peripheral blood CFUs was determined after 7 days of culture. Results Peripheral blood and bone marrow analysis The CBC was determined daily for 14 days to determine if pearl mice were neutropenic. A 2 nd group of mice were bled every other day for 30 days for CBC analysis. Based on peripheral cell counts neutropenia or cyclic neutropenia was not observed in the pearl mice (Figure 1 and Table 1). However, hematologic parameter analysis indicated a mild but significant increase (p<0.05) in the total WBC count, lymphocytes, monocytes, and granulocytes in pearl mice compared to BL/6 mice (Table 1). Bone marrow differential counts (500 cells) in pearl (n=7) and Bl/6 (n=8) mice demonstrated a significant (p < 0.05) increase in the number of lymphoid and lymphoid-like cells in the

46 35 pearl mice (65.1 ± 19.2) compared to the Bl/6 mice (41.2 ± 11.8) (Table 2). The myeloid and erythroid cell counts and the M: E ratio were not significantly different in the Bl/6 and pearl mice. Colony-forming assays Colony-forming assays were performed using total BMMCs from BL/6 and pearl mice to determine if there were differences in the number of committed hematopoietic progenitors in the bone marrow in pearl and BL6 mice (Table 3). The in vitro colonyforming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) was significantly (p<0.05) increased in the pearl (41.9 ± 6.0, n=6) compared to BL/6 (33.8 ± 4.3, n=8) mice. The colony forming unit-granulocyte (CFU-G) was also increased in pearl (13.6 ± 3.3, n=6) compared to BL/6 mice (7.0 ± 0.5, n=8). The number of CFU- GEMM and CFU-G in the spleen was similarly increased in the pearl mice compared to the Bl/6 mice The bone marrow colony forming unit-erythroid (CFU-E) was decreased in pearl mice (3.5 ± 1.1 to 6.4 ± 0.9, pearl to BL/6, respectively) but the reverse was observed in the spleen. The number of burst forming unit-erythroid (BFU-E) was similar in the pearl and Bl/6 mice. Lin - Sca-1 + c-kit + HSC population in bone marrow Flow cytometric analysis was performed to compare the number of early hematopoietic stem cells in pearl (n=4) and normal BL/6 (n=4) mice. Based on analysis of LSK cells (Lin-, Sca-1+, c-kit+) the percentage of early HSCs in pearl mice was

47 36 significantly (p=.04) increased in pearl (4.75 ± 0.95) compared to Bl/6 (3.18 ± 0.39) (Figure 2). Neutrophil recovery following cyclophosphamide treatment Stress granulopoiesis was compared in Bl/6 (n=8) and pearl mice (n=8) administered the myelosuppressive agent cyclophosphamide [20]. The recovery from neutropenia was indistinguishable in wild-type and pearl mice suggesting that granulopoiesis in pearl mice is similar to Bl/6 mice even with moderate hematopoietic stress (Figure 3). Granulopoiesis after bortezomib treatment To determine if granulopoiesis in pearl mice was more sensitive to ER stress, pearl and Bl/6 mice were administered 1 mg/kg of bortezomib. The percent of bone marrow granulocytes and peripheral blood B cells was determined by flow cytometry prior to and on days 2, 3, 4 and 5 after bortezomib administration. There was no statistically significant difference in bone marrow granulocytes between Bl/6 and pearl mice on any day (Figure 4A). The B cells percentage decreased in both Bl/6 and pearl mice (Figure 4B). There was no significant (p < 0.05) difference between Bl/6 and pearl mice B cells on any day except day 5.

48 37 Bone Marrow Transplantation Sublethally irradiated (3.0Gy) NOD/SCID mice were transplanted with increasing doses of bone marrow cells from Bl/6 or pearl mice. The % donor cell engraftment at 4 to 5 weeks was determined by flow cytometry by measuring the number of Ly5.2 (Bl/6 or pearl) positive cells in bone marrow and spleen of recipient mice. There was no significant (p < 0.05) difference in the % donor engraftment at 1 X 10 5 and 1 X 10 6 donor cell dose (Table 4). At 1 X 10 4 donor cells neither Bl/6 or pearl mice showed % donor engraftment above background. Secondary transplants with bone marrow (1 X 10 6 cells) obtained from primary recipients 4 weeks after transplantation demonstrated similar engraftment in normal and pearl mice (unpublished data, 2010). Granule Enzyme Assays Elastase, MPO and cathepsin G enzyme activity were determined in bone marrow lysates from Bl/6 (n=8) and pearl (n=8) mice (Figure 5). Elastase, MPO and cathepsin G enzyme activity was not significantly different in pearl mice compared to Bl/6 mice. Subcellular fractionazation using discontinuous percoll gradients demonstrated a single sharp peak of enzyme activity in fractions 6-8 in the normal mice (Figure 6A). In contrast in pearl mice the elastase enzyme activity peak was broadly distributed over fractions 4 to 8 similar to CH dogs (Figure 6B) [21].

49 38 Beta3a in pearl mice The presence of Beta3a protein in bone marrow was determined by western blot. Beta3a was present in the bone marrow of both pearl and Bl/6 mice (Figure 7A). The illustrated blot is representative in that other blots also showed large individual variability in the amount of Beta3a. In order to confirm that pearl mice did not compensate for a possible loss of functional Beta3a in myeloid cells by expressing βnap, a protein normally found in nervous tissue [22, 23], blots were probed for βnap. Bone marrow lysates from both Bl/6 and pearl mice were negative for βnap. Brain lysates from Bl/6 and pearl were positive for βnap (Figure 7B). G-CSF Stimulation G-CSF promotes differentiation of hematopoietic cells into granulocytes and stimulates the mobilization of HSCs and granulocytes into the blood [24, 25]. The number of granulocytes was significantly (p<0.05) greater in Bl/6 (n=8) than in pearl (n=8) mice following G-CSF administration (Figure 8). The number of peripheral blood CFU was also significantly (p<0.05) increased in Bl/6 compared to pearl mice (Figure 9A). The number of bone marrow CFUs was not significantly different between Bl/6 (n=4) and pearl (n=4) mice (Figure 9B). Bone marrow differential counts from Bl/6 (n=5) and pearl (n=6) mice after 5 days of G-CSF treatment were also compared. The number of band cells/neutrophils was significantly increased (Table 5) compared to untreated bone marrow (Table 2). Erythroid cell numbers were greatly decreased in the G-CSF treated bone marrow compared to untreated bone marrow. In the G-CSF treated bone marrow myeloid and erythroid cells

50 39 were not significantly different between Bl/6 and pearl mice although lymphoid cells were significantly higher in the Bl/6 mice. AMD3100 stimulation AMD3100 is known to promote release of HSCs and hematopoietic progenitor cells from the bone marrow by interacting with CXCR4 [26, 27]. To determine if pearl mice may have fewer HSC/progenitor cells than normal Bl/6 mice, AMD3100 was administered to Bl/6 (n=3) and pearl (n=3) mice. The CBCs (data not shown) and the number of peripheral blood CFUs were not significantly different between pearl and Bl/6 mice (Figure 10). Discussion The major inherited disorders of neutrophil production are severe congenital neutropenia and cyclic neutropenia. Mutations in the ELA2 gene encoding the primary granule protein elastase cause human cyclic neutropenia and most cases of congenital neutropenia [13, 28]. Mutations in the AP3B1 gene cause HPS-2 and canine CH, which are also characterized by congenital neutropenia and cyclic neutropenia, respectively [1, 5]. Yet, the exact role of elastase and AP-3 in controlling myelopoiesis in normal adults remains unexplained. Determining the molecular mechanisms for the neutropenia syndromes should provide an improved understanding into key hematopoiesis regulatory processes. We therefore wanted to determine if pearl mice, which also have an AP-3 deficiency, have abnormal myelopoiesis or neutropenia.

51 40 The CBC with differential count was determined daily for 14 days or every other day for 30 days in normal and pearl mice using an automated hematology instrument capable of differential analysis. In both studies there was a modest but significant increase in the neutrophil and monocyte counts in pearl compared to Bl/6 mice. Bone marrow myeloid differential counts were not significantly different in Bl/6 and pearl mice. In contrast both blood and bone marrow lymphocyte counts were significantly (p<0.05) increased in the pearl mice. The apparent lack of a neutropenic or cyclic phenotype may be due to a cycle so short that detection of cycle neutropenia would be extremely difficult. A mathematical model extrapolated from cyclic neutropenia in dogs and humans showed that mice would likely have a cycling period of 3 days and measurements of neutrophil number every 18 hours would have to be taken to determine a cycling period [29]. Comparison of CFU assays and putative HSC pools (LSK cells) revealed a modest increase in the number of progenitor cells in pearl mice. Furthermore, inducing proliferative stress with cyclophosphamide administration or bone marrow transplantation demonstrated similar responses in normal and pearl mice. Induction of ER stress by bortezomib administration did not reveal differences in granulopoiesis between Bl/6 and pearl mice. The failure to show a difference between Bl/6 and pearl mice granulocytes in response to bortezomib treatment differs from results with elastase mutant mice [30]. There is a marked accumulation of incompletely processed elastase precursor in myeloid cells and mature neutrophils from CH dogs. There are also variable amounts of elastase enzyme activity in neutrophils from CH dogs [21]. Surprisingly, there were no

52 41 differences in cathepsin G, MPO and elastase enzyme activity in bone marrow cell lysates from normal and pearl mice. Nevertheless the subcellular distribution of elastase was markedly different in pearl and normal mice. Similar to CH dogs [21], elastase was distributed over multiple fractions rather than a single sharp peak associated with the primary granules. This observation suggests that the structural integrity of the primary granules is altered in both CH dogs and pearl mice. These results indicate that elastase mislocalization per se is not sufficient to cause neutropenia in mice. Elastase undergoes several post translational modifications as it is processed and stored in neutrophilic primary granules. Serglycin, a highly charged proteoglycan that neutralizes the net negative charges on elastase is required for elastase packaging in the primary granules [31]. A 2 nd protein, CD63 (Lamp-1), is also required for elastase packaging in the primary granules [32]. The CD63 protein may function to bind AP-3 and elastase. Serglycin and CD63 deficient mice have decreased elastase enzyme activity and accumulate higher molecular weight elastase proforms. Yet, in spite of the total loss of elastase enzyme activity in the knockout mice, they are not neutropenic and do not have abnormalities in neutrophil production [33, 34]. Mice in which the elastase gene has been deleted have decreased bactericidal killing activity but no defects in hematopoiesis or neutrophil production [35]. Mice transgenic for the dominant V72M elastase mutation which cause severe congenital neutropenia in patients also did not have neutropenia [20]. Transgenic mice carrying the G192 elastase mutation found in congenital neutropenia patients did not have neutropenia [30]. It is not surprising then that we did not observe granulocyte production defects or CH in the pearl mice. These findings point to the difficulty in using mice to model disorders of granulopoiesis.

53 42 Western blots showed that Beta3a was present in both Bl/6 and pearl mice. This contrasts with another study that found the Beta3a protein absent in pearl mice [2]. The difference in results may be due to the antibodies used for detecting Beta3a. The antibody (sc anti-beta3a) used in this study recognizes a motif in the N-terminal region of Beta3a whereas the mutation occurs closer to the C-terminal [2]. Since βnap is absent in bone marrow as shown by western blotting, sc anti-beta3a is not recognizing βnap in lieu of Beta3a in pearl mouse bone marrow. The lack of abnormal myelopoiesis in pearl mice cannot be attributed to a compensating expression of βnap in the bone marrow since pearl mice do not express βnap in bone marrow to compensate for the loss of Beta3a activity. Though pearl mice appear to have normal myelopoiesis there is a difference in mobilization of progenitor cells and granulocytes following G-CSF administration. The fact that Bl/6 and pearl mice do not have a significant difference in number of HSC/progenitor cells in the bone marrow after G-CSF administration suggests that the number of HSC/progenitor cells in the bone marrow are the same between Bl/6 and pearl mice but that the difference is in the release of cells into the blood. Peripheral blood granulocyte counts and CFUs were similar in pearl and Bl/6 mice following AMD3100 administration. The lack of difference between Bl/6 and pearl mice in following AMD3100 administration suggests that the HSC/progenitor cell pools are similar in Bl/6 and pearl mice. The difference in G-CSF response in pearl mice suggests there may be a difference in the HSC niche in pearl and Bl\6 mice. AMD3100 and G-CSF mobilize HSCs via different mechanisms. AMD3100 directly interacts with HSCs via CXCR4 [24] whereas G-CSF interacts with multiple cellular targets in the bone marrow.

54 43 In light of the complexity of the HSC niche and multiple actions of G-CSF, several mechanisms may explain why pearl mice do not mobilize as well as normal mice following G-CSF administration. Since mutations in the G-CSF receptor are known to cause neutropenia [9], G-CSF signaling might be altered in the pearl niche. However, the fact that the number of band cells/neutrophils increases in pearl mice following G-CSF administration suggests that G-CSF signaling is normal. Furthermore, HSC/progenitor cell amplification in the bone marrow is also similar between Bl/6 and pearl mice based on the CFU assay. Secondly, it is possible that there is a functional difference in the macrophages of Bl/6 and pearl mice even though the macrophage number is similar. Thirdly, pearl mice osteoblasts may be affected by the AP3-B1 mutation leading to decreased mobilization of HSCs normally attached to osteoblasts. Osteoblasts in pearl mice have been poorly studied. Another possibility is that one or more of the receptors on HSCs or their ligands may interact with AP-3. More than one of the aforementioned mechanisms may be at play in causing the difference between Bl/6 and pearl mice mobilization. If there is indeed a HSC niche defect in pearl mice, the same may hold true for dogs and humans with AP3B1 mutations. The HSC niche defect may become more pronounced in larger organisms resulting in neutropenia or CH. Identifying the unique differences in hematopoiesis control genes and HSC niche function in mice and higher animals will improve our understanding of control mechanisms essential to normal steady state hematopoiesis and neutrophil production.

55 44 Acknowledgements The research was supported by NHLBI awards HL and HL to CDL and GPN. The authors are extremely grateful to Holly Bachus and the UAB Animal Research Program for excellent animal care. Authorship and Conflict of Interest Statement 1. Matthew Vallejo- performed research, analyzed data and helped write the paper. 2. Glenn Niemeyer-performed research, analyzed data and helped write the paper. 3. Alex Vaglenov- performed research and analyzed data. 4.Tommy Hock- performed research 5. Bridget Urie-performed research 6. Peter Christopherson-performed research and analyzed data 7. Clinton D. Lothrop Jr.- designed research, analyzed data and wrote paper The authors have no conflict of interest to report.

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61 50 Table 1: Pearl mice have significantly (p< 0.05) higher WBC, granulocyte, lymphocyte and monocyte counts and significantly lower RBC, Hgb level and hematocrit levels. Hematologic parameter Bl/6 Pearl WBC count, x ± ±0.3* Lymphocytes 9.97± ±1.90* Granulocytes 1.77± ±0.90* Monocytes 1.96± ±0.48* RBC count, x 10 6 / µl 10.6± ±1.1 * Plt count x 10 3 / µl 604.5± ±136.4 Hgb level, g/dl 16.1± ±0.2* Hct, % 47.3± ±4.60* Percentage of WBC count Lymphocytes, % 72.7± ±0.5 Monocytes, % 14.1± ±0.1 Granulocytes, % 13.2± ±0.4 Blood Samples were taken every other day for 30 days from individual mice. * P<0.05

62 51 Table 2: Bone marrow differential counts in pearl and Bl/6 mice. Mouse Normal Pearl n=8 n=7 AV SD AV SD Blasts/Promyelocytes % Blasts/Promyelocytes Myelocytes % Myelocytes Metamyelocytes % Metamyelocytes Bands/Neutrophils % Band/neutrophils Eosinophils % Eosinophils Erythroid % Erythroid Lymphoid % Lymphoid M:E ratio There were no significant differences in myeloid and erythroid progenitors between Bl/6 and pearl mice. The number of bone marrow lymphoid cells was significantly (P<0.05) increased in pearl mice.

63 52 Table 3: Pearl mice have significantly increased (p < 0.05) colony forming unit granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM) and colony forming unit granulocyte (CFU-G) but decreased (p < 0.05) colony forming unit erythroid (CFU-E) from bone marrow. Bone Marrow Spleen # C57Bl/6J # Ap3b1 pe /J # C57Bl/6J # Ap3b1 pe /J CFU-E 8 6.4± ±1.1* 8 4.6± ±1.1 BFU-E ± ± ± ±3.3 CFU-G 8 7.0± ±3.3* 7 2.5± ±0.7* CFU-M 8 7.6± ± ± ±0.5 CFU-GEMM ± ±6.0* 8 5.8± ±1.4* Total ± ±9.1* 8 9.7± ±1.1* Colony assays were done in vitro from total bone marrow mononuclear cells as described in the materials and methods section. 2 X 10 5 cells/35 mm dish were plated for erythroid progenitors, and 2 X 10 4 cells/35 mm dish were plated for granulocytic progenitors.

64 53 Table 4: Bl/6 and pearl mice show similar engraftment. Donor No. of No. of BM or Avg. % Donor Mice Cells Spleen Black X10 6 BM ± Pearl 15 1X10 6 BM ± Black X10 6 Spleen ± Pearl 14 1X10 6 Spleen ± Black 6 7 1X10 5 BM ± Pearl 12 1X10 5 BM ± Black 6 7 1X10 5 Spleen ± Pearl 12 1X10 5 Spleen ± Black X10 4 BM 1.1 ± 0.88 Pearl 5 1X10 4 BM 0.02 ± 0.04 Black X10 4 Spleen 1.8 ± 1.16 Pearl 5 1X10 4 Spleen 0 ± 0 Limiting dilution bone marrow transplantation into NOD/SCID mice show no significant difference (p < 0.05) between Bl6 and pearl mice in engraftment in bone marrow or spleen after 4 weeks.

65 54 Table 5: Bone marrow differential counts in pearl and Bl/6 mice treated with G-CSF. Mouse Normal Pearl n=5 n=6 AV SD AV SD Blasts/Promyelocytes % Blasts/Promyelocytes 1.60% % 0 Myelocytes % Myelocytes 4.76% % 0.01 Metamyelocytes % Metamyelocytes 17.96% % 0.06 Bands/Neutrophils % Band/neutrophils 54.76% % 0.06 Eosinophils % Eosinophils 3.48% % 0.02 Erythroid % Erythroid 1.52% % 0.06 Lymphoid % Lymphoid 15.92% % 0.02 M:E ratio The number of band cells/neutrophils were increased in both Bl/6 and pearl mice. There were no significant differences in myeloid and erythroid progenitors between pearl and Bl/6 mice. The lymphoid cells were significantly (P<0.05) increased in Bl/6 mice.

66 55 Figure 1: Pearl mice show similar PMN counts to Bl/6 mice. Blood samples were collected daily for 14 days and analyzed using a Heska Veterinary Hematology Analyzer.

67 56 Figure 2: Early HSC numbers are increased in pearl mice. The percentage of early HSCs was significantly greater in pearl mice than in Bl/6 mice. * = p<0.05

68 57 Figure 3: Recovery following cyclophosphamide-induced myelosuppression is similar in Bl/6 and pearl mice. Absolute neutrophil counts (ANC) from Bl/6 and pearl mice measured on days 0, 3, 5 and 10 after injection of 200 mg/kg of cyclophosphamide.

69 58 Figure 4: Granulopoiesis after bortezomib treatment is similar in pearl and Bl/6. (A) The percent of bone marrow cells in Bl/6 and pearl mice positive for Ly-6G (granulocytes) on days 0 thru 5 after administration of bortezomib. There was no significant difference between Bl/6 and pearl mice. (B) There was no difference in the percentage of blood B cells between Bl/6 and pearl mice except on day 5. *p = 0.007

70 59 Figure 5: Elastase, MPO and Cathepsin G activity normal in pearl mice. Elastase, MPO and Cathepsin G activity in Bl/6 mice was defined as one, and pearl mouse activity for each enzyme was adjusted accordingly. No significant difference was found in elastase, MPO, or Cathepsin G enzyme activity between Bl/6 and pearl mice.

71 60 Figure 6: Elastase activity of pearl mouse neutrophils distributed broadly over several subcellular fractions as determined with discontinuous percoll gradients. (A) Elastase activity in different subcellular fractions of Bl/6 and (B) pearl bone marrow cells. Elastase activity in Bl/6 is mostly confined to fraction 7 with lower activity in fractions 6 and 8 and minimal activity in other fractions. Elastase activity in pearl is broadly spread across fractions 5-7 with lower activity in fractions 4 and 8 and minimal activity in other fractions.

72 61 Figure 7: Pearl mice have Beta3a. (A) Western blot analysis shows Beta3a in both Bl/6 and pearl mice between 100 and 120 kd with large individual variability in the amount of Beta3a. (B) βnap was detected in Bl/6 and pearl mice cerebral lysates but not bone marrow lysates.

73 62 Figure 8: Granulocytes counts are higher in Bl/6 mice than pearl mice after mobilization with G-CSF. After 5 days of treatment with G-CSF, Bl/6 had a significantly greater neutrophil count than pearl mice. * = p<0.05

74 63 Figure 9: CFU number in Bl/6 higher than pearl in peripheral blood but not higher in bone marrow after mobilization with G-CSF. After 7 days of culture colonies were counted to evaluate the number of CFUs. (A) Bl/6 had significantly higher number of CFUs from the peripheral blood than pearl, (B) but CFUs from the bone marrow did not show the same difference. * = p<0.05

75 64 Figure 10: CFU number in peripheral blood after AMD3100 mobilization similar in Bl/6 and pearl mice. After 7 days of culture colonies were counted to evaluate the number of CFUs. There was no significant difference between Bl/6 and pearl mice.

76 65 TRANSPLANT OF CANINE BONE MARROW MONONUCLEAR CELLS IN NSG MICE CAUSES SEVERE GRAFT VS. HOST DISEASE by MATTHEW O VALLEJO, GLENN P NIEMEYER, HOLLY BACHUS, TRENTON R SCHOEB, MARGARET M JULIANA AND CLINTON D LOTHROP, Jr In preparation for Journal of Immunology Format adapted for dissertation

77 66 Abstract NSG mice are a well established model for study of human xenotransplantation. Dogs have served as preclinical models of human diseases. The purpose of this study was to evaluate multiple dog bone marrow transplant methods for canine bone marrow mononuclear cells (BMMC) in NSG mice. Canine BMMCs were administered by tailvein injection, intrafemoral injection, intrafemoral injection after flushing the femur and liver injection into neonatal NSG mice. The survival rate was 40% for tail-vein injection, 54% for intrafemoral injection, 7% for intrafemoral injection after femur flushing and 37% for neonate liver injection. Intrafemoral injections after flushing the femur had an engraftment rate of 67% but a survival rate of 7%. Neonatal liver injections had the best balance of engraftment and survival with a 37% survival rate and an 86% engraftment rate. Histopathological evaluation of the NSG mice showed development of severe GVHD in the groups of mice with high engraftment perhaps explaining low survival rates following xenotransplantation of canine BMMCs. Key Words: NSG; xenotransplant; canine; dog; mouse

78 67 Introduction Immunodeficient mice models are extensively used in studies of transplantation. The non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse was used in early studies of xenotransplantation of human tissue (1). In order to provide a better recipient of human tissue a NOD/SCID mouse with an IL2 receptor gamma chain knockout was developed. Known as the NSG mouse, the NOD/SCID gamma chain knockout mouse lacks mature lymphocytes and natural killer cells. NSG mice provide better engraftment of human hematopoietic stem cells (HSC), human acute myeloid leukemia cells and acute lymphoblastic leukemia cells than any of the NOD/SCID predecessors (1-4). Dogs are a well established preclinical model of human bone marrow transplantation. Mixed hematopoietic chimerism post-transplant has been studied in dogs (5). Survival effect of infusing bone marrow into dogs administered high dose of chemotherapy has been investigated (6). Immunocompetence of grafted bone marrow over time has been researched in dogs (7). The transfer of immunity via transplant of immune memory cells has been achieved in dogs (8). Transplant of cadaver dog bone marrow has also been done in dogs to study post-transplant infections (9). Dog CD34 + cells have been purified from bone marrow and successfully transplanted (10). Gene therapy via transplant of transfected hematopoietic cells has been another area studied in dogs (11-13). Pre-transplant irradiation has also been studied using dog models (14) and also pre-transplant drug regimens (15). Dogs have not only modeled transplants, but have also been used as models of human diseases. Dogs may offer a suitable replacement for a source of human tissue in a

79 68 variety of disease models. Dogs spontaneously develop cancers including leukemia much like humans (16). Human leukemia as well as other cancers have been studied in xenotransplant studies using NSG mice (3, 17-21). Besides dog cancer models there are dog models of other diseases. Some of these dog models of disease include models of human hematopoietic diseases. Dog models of Hermansky-Pudlak syndrome type II, pyruvate kinase deficiency, Hemophilia A and B and type I Glanzmann's thrombasthenia have been described (22-31). Canine bone marrow CD34 + cell transplant effects on a dog model of X-linked severe combined immunodeficiency has been studied (32). Effect of dog bone marrow transplantation on leukocyte adhesion deficiency, has also been studied in dogs (33). Amelioration of hemolytic anemia secondary to pyruvate kinase deficiency was achieved by bone marrow transplantation (34, 35). Besides study of bone marrow derived cell transplants in dogs, transplant of cryopreserved dog fetal liver cells that produce hematopoietic cells has been studied in dog (36, 37). Renal transplant has also been studied in dogs (38). Establishing hematopoietic systems of these dog disease models in NSG mice may prove valuable in the study of hematologic diseases. The purpose of this study was to determine if there was stable engraftment of dog BMMCs in NSG mice. The transplant methods used in this study, tail-vein injection, intrafemoral injection and hepatic injection into neonatal mice, have been used in humanto-mouse xenotransplant studies (2, 39-44). Mice were also evaluated for the presence of GVHD.

80 69 Materials and Methods Animals The NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ (NSG) mouse strain was purchased from Jackson Laboratories, (Bar Harbor, ME). Mice were housed in ventilated racks in a barrier facility. Male and female 0- to 20-week old mice were used for experimental procedures. Dogs used in this study were out bred collie dogs. The dogs were also housed in a barrier facility. All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committees (IACUC). Irradiation of NSG Mice NSG mice were irradiated (1.5 grays) via x-rays pre-transplant for the tail-vein and intra-femoral transplant methods. Mice were given sulfamethoxazole and trimethoprim via their drinking water supply until they were sacrificed or spontaneously died. Bone Marrow Mononuclear Cell Isolation from Dog Bone Marrow Bone marrow cells were obtained from the long bones of dogs. Bone marrow samples were carefully layered on top of 3 ml of Ficoll-Paque Plus ( GE Healthcare) in 15 ml conical tubes ( Fisher Scientific). Samples were then centrifuged for 25 minutes at 300 rcf. The middle bands in each tube were then siphoned up and placed in clean 15 ml conical tubes. Hank s balanced salt solution was added to the tube to fill the remaining volume left in the tube. Tubes were then centrifuged for 10

81 70 minutes at 300 rcf and the supernatant discarded. The cell pellets were resuspended in 2 ml of balanced salt solution. The cell suspension was then filtered through a 70 µm cell strainer ( BD Falcon). Bone marrow mononuclear cells (BMMC) were then counted on a hemacytometer (Hausser Scientific) and administered to the NSG mice within the hour. Bone Marrow Mononuclear Cells Transplantation Canine BMMCs were administered to NSG mice by 4 different methods. BMMCs (1 X 10 7 ) were administered by tail-vein injection to NSG recipient mice that received 1.5 grays total body irradiation prior to transplantation in the first method. For the second method BMMCs (1 X 10 7 ) were administered by directly injecting the BMMCs into the left bone marrow cavity of the left femur of NSG mice that received 1.5 Gy total body irradiation prior to transplantation. The bone marrow cavity of the left femur was flushed with 40 µl of Hank s Balanced Salt Solution ( Gibco) prior to the intrafemoral administration of the BMMCs (1 X 10 7 ) for the third method. The fourth method used 1 to 4 day old neonatal NSG mice. The neonatal mice were administered BMMCs (1 X 10 7 ) directly into the liver via a 30 guage needle at a single injection site. The % donor cell engraftment in spleen and bone marrow of the femur was determined by flow cytometry 7-9 weeks post-transplant. Bone marrow and spleen cells (1 X 10 7 ) were suspended in 100 µl of 10% Mouse Serum (Jackson Immunoresearch ) and 1% Rat Gamma Globulin (Pierce 31885) in PBS. Cells from the bone marrow of the femur and spleen were then incubated with anti-dog CD45/Ly5 (AbD Serotec MCA1781A647) and anti-mouse CD45.1/Ly5.1 (BD Pharmingen ) for 20

82 71 minutes at 25 C. The antibodies were then removed from the cells. For the intra-femoral injection methods, the contralateral femur bone marrow cells and the ipsilateral femur bone marrow cells were processed separately. The cells were resuspended in 500 µl of 10% mouse serum in PBS. Blood ( µl) from recipient mice was incubated with anti-dog CD45/Ly5 (AbD Serotec MCA1781A647) and anti-mouse CD45.1/Ly5.1 (BD Pharmingen ) for 30 minutes at 25 C. The blood sample was then incubated with red blood cell lysis Buffer ( ebioscience) for 10 minutes. The lysis buffer was then removed from the cells, and the cells washed with PBS. Blood cells were resuspended in 100 µl of 10% Mouse Serum (Jackson Immunoresearch ) and 1% Rat Gamma Globulin (Pierce 31885) in PBS. The % of donor cells was determined as the number of Dog CD45/Ly 5 positive cells. The % of recipient cells was determined as the number of Mouse CD45.1/Ly 5.1 positive cells. 7-AAD (BD Pharmingen E) was used to exclude dead cells. Histopathology Analysis Tissues were fixed for 48 hours in 10% neutral buffered formalin, processed routinely into paraffin, sectioned at 5 µm, and stained with HE. Histological slides were prepared in the Animal Resources Program Comparative Pathology Laboratory at the University of Alabama at Birmingham.

83 72 Results Tail-Vein Bone Marrow Mononuclear Cell Transplantation Sublethally irradiated (1.5Gy) NSG mice were transplanted with 1 X 10 7 BMMCs via the tail-vein. The % donor cell engraftment at 8 weeks was determined by flow cytometry measuring % of dog CD45 positive cells in the bone marrow and spleen. An example of a flow cytometry readout is shown in Figure 1. Forty % of the transplanted mice survived until analysis (Table 1). Twenty nine % of the surviving mice showed engraftment (Table 2). Positive engraftment was defined as 1% dog CD45 positive cells in the femur bone marrow. Average percentage of engraftment in the bone marrow and spleen of engrafted mice was 9.4% and 26.7% respectively (Table 3). The percentage of dog CD45 + cells in both the bone marrow and spleen of engrafted mice varied considerably from mouse to mouse. Left Intrafemoral Bone Marrow Mononuclear Cell Transplantation Sublethally irradiated (1.5Gy) NSG mice were transplanted with 1 X 10 7 BMMCs via injection directly into the left femur bone marrow cavity. The % donor cell engraftment at 8-9 weeks was determined by flow cytometry measuring the percentage of dog CD45 + cells in bone marrow and spleen of recipient mice. An example of a flow cytometry readout is shown in Figure 2. Fifty four % of the transplanted mice survived until analysis (Table 1). Twenty two % of the surviving mice showed engraftment (Table 2). Average percentage of engraftment in the bone marrow of the left femur, bone marrow of the right marrow and spleen was 17.3%, 17.2% and 16.4% respectively (Table

84 73 4).The percentage of dog CD45 + cells in the bone marrow and spleen of engrafted mice also varied considerably from mouse to mouse with this method. Left Intrafemoral Bone Marrow Mononuclear Cell Transplantation with Flushing Sublethally irradiated (1.5Gy) NSG mice were transplanted with 1 X 10 7 BMMCs via injection directly into the left femur bone marrow cavity after the cavity had been flushed with Hank s balanced saline solution. The % donor cell engraftment at 8-9 weeks was determined by flow cytometry measuring the % CD45 + dog cells in bone marrow, spleen and blood of recipient mice. An example of a flow cytometry readout is shown in Figure 3. Seven % of the transplanted mice survived until analysis (Table 1). Sixty seven % of the surviving mice showed engraftment (Table 2). The average percentage of engraftment in the bone marrow of the left femur, bone marrow of the right marrow, spleen and blood was 8.3%, 12.5%, 19.4% and 2.9% respectively (Table 5). Engraftment in the bone marrow and spleen of engrafted mice varied considerably between the mice. Neonate Liver Bone Marrow Mononuclear Cell Transplantation Neonatal (1-4 day-old) NSG mice were transplanted with 1 X 10 7 BMMCs via injection of the BMMCs directly into the liver. The % donor cell engraftment at 7-9 weeks was determined by flow cytometry measuring the percentage of dog CD45 + cells in bone marrow, spleen and peripheral blood of recipient mice. An example of a flow cytometry readout is shown in Figure 4. Thirty seven % of the transplanted mice survived until analysis (Table 1). Eighty six % of the surviving mice showed engraftment (Table

85 74 2). The average percentage of engraftment in the bone marrow, spleen and blood was 6.6%, 5.8% and 14.0% respectively (Table 6). Engraftment varied between individual mice the greatest in the blood. Histopathology Analysis Multifocal necrosis and accumulation of large reactive lymphocytes and plasma cells around vessels were seen in the kidney, pancreas, spleen, liver, lung, bone marrow and/or adrenal gland in 50% of the NSG mice transplanted with dog BMMC that were analyzed (Table 7). Only mice transplanted using intra-femoral injection after flushing and neonatal intrahepatic injection were analyzed. Both methods yielded high engraftment rates of surviving mice. One of the four analyzed mice transplanted via intrafemoral injection after flushing had graft vs. host disease. Five of the eight analyzed mice transplanted via intrafemoral injection after flushing had graft vs. host disease. Discussion Neonatal NSG mice transplanted with canine BMMCs by intrahepatic injection had the highest engraftment. Left intrafemoral transplantation after the left femur had been flushed led to relatively high engraftment, but the survival rate was extremely low. Left intrafemoral transplantation without pre-flushing the femur had the highest survival rate but also the lowest rate of engraftment. The results of this study indicate a generally inverse relationship between survival and engraftment. The seemingly inverse relationship of survival and engraftment is likely due to graft versus host disease

86 75 (GVHD). GVHD was found in several of the mice with stable engraftment GVHD was characterized by necrosis in the kidney, spleen, liver, lung, bone marrow and adrenal gland along with other symptoms. Sepsis can also result in widespread multifocal necrosis. However, GVHD, unlike sepsis, is also associated with accumulation of large reactive lymphocytes and plasma cells around vessels and in the spleen (45-47). GVHD often occurs when immunocompetent lymphocytes are transplanted into a host and the host is not able to reject the immunocompetent cells (48). NSG mice without mature lymphocytes or natural killer cells likely cannot reject any of the dog s immunocompetent cells. GVHD occurrence in xenotransplants has been shown in several studies. SCID/beige mice given dog leukocytes developed GVHD (45). In human xenotransplant studies using NOD/SCID mice, high engraftment invariably led to GVHD (46, 47). Human peripheral blood mononuclear cell (PBMC) xenotransplant using NSG and BALB/c-Rag2(null) IL-2Rγ(null) mice showed higher engraftment and higher rate of GVHD in NSG mice. NSG mice given 1 X 10 7 PBMCs had a 0% survival rate after 50 days. Virtually all mice died due to GVHD. All transplanted mice had human leukocyte engraftment (39). Human PBMCs (1 X 10 7 ) given to NSG mice via intraperitoneal injection had a 0% survival rate by 30 days post-transplant (49). Both studies resulted in lower survival rates than this study, but all the mice engrafted in one of the studies supporting the hypothesis that mice that consistently engraft also consistently develop GVHD and have high mortality rates. In one study all NSG mice developed GVHD when given as few as 5 X 10 6 human PBMCs (50). Steps to improve survival of dog xenotransplants will be necessary. The fetal liver transplanted neonatal mice had the highest engraftment rate but not the lowest survival

87 76 rate. The neonatal mice may be a better option than older mice for transplant studies. Human xenotransplant studies that have shown human cell engraftment several weeks post-transplant in NSG mice used human cord blood purified CD34 + cells (40, 51, 52). Using dog CD34 + cells especially ones purified from cord blood may be an option to help prevent GVHD development in NSG mice. T cell depletion prior to transplant or even post-transplant is another step that could be taken to prevent GVHD (53). ATG-Fresenius and cyclosporine are both anti-t cell compounds that have been used to ameliorate GVHD and increase the success of transplantation (54-56). Bortezomib and cyclophosphamide have also been used to improve the success of transplants and prevent GVHD (57, 58). Another drug that has been used in the treatment of GVHD is methotrexate. In one clinical study, methotrexate treated patients demonstrated less incidence of GVHD than patients treated with cyclosporine (59). Prednisone has also clinically been used to successfully treat GVDH (60). A single high dose of azathioprine has been shown to effectively abolish GVHD in mouse recipients of parental spleen cells (61). The aforementioned drugs or a combination of the drugs may be useful for improving the success of dog-to-mouse transplants. In one study azathioprine and prednisone were used in combination with good success in treating GVHD in humans that had received bone marrow transplants(62). Sirolimus and tacrolimus are another set of drugs that have been used in tandem to prevent GVHD in study of patients who had received hematopoietic stem cell transplants. Sirolimus and tacrolimus achieved similar results to cyclosporine in preventing GVHD (63). Other available drugs may also be useful in preventing GVHD. Ideally any treatment to prevent GVHD will not negatively affect engraftment. Further studies are needed to determine what treatment regimens and

88 77 conditions pre- and post-transplant are necessary to allow for optimal survival and engraftment of dog bone marrow cells.

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100 89 Acknowledgements The authors are extremely grateful to the UAB Animal Research Program for excellent animal care. Authorship and Conflict of Interest Statement 1. Matthew Vallejo- wrote paper, designed research, performed research and analyzed data. 2. Glenn Niemeyer- designed research, analyzed data, performed research. 3. Holly Bachus- performed research. 4. Trenton Schoeb- performed analysis. 5. Margaret Juliana- performed analysis. 6. Clinton D. Lothrop Jr.- designed research, analyzed data, wrote paper. The authors have no conflict of interest to report.

101 90 Table 1: Survival rate of mice receiving dog bone marrow transplants via different methods of transplant. Method # of mice # of Mice Surviving Survival Transplanted to Analysis % Tail-Vein Femur Femur w/ Flush Neonate Intrafemoral injections had the highest survival rate, but intrafemoral transplants that preflushed the left femur had the lowest survival rate. Table 2: Number of surviving mice with dog leukocyte engraftment. Method # of Mice % of Mice # of Mice Engrafted Analyzed Engrafted Tail-Vein Femur Femur w/ Flush Neonate Transplants done via neonatal liver injection had the highest level of engraftment for mice surviving 7-9 weeks posttransplant. Table 3: Percentage of dog leukocytes in mice transplanted with dog bone marrow mononuclear cells using tail-vein injections. Source of Cells % Dog CD45 + % Dog CD45 + Cells - Cells - Average Standard Deviation Femur Spleen Spleen had a higher variation of the percentage of engraftment than the femurs.

102 91 Table 4: Percentage of dog leukocytes in mice transplanted with dog bone morrow mononuclear cells using intrafemoral injection. Source of Cells % Dog CD45 + % Dog CD45 + Cells - Cells - Average Standard Deviation Left Femur Right Femur Spleen Intrafemoral injection was done in left femur. Engraftment was virtually equal in both the left and right femur. Table 5: Percentage of dog leukocytes in mice transplanted with dog bone morrow mononuclear cells using intrafemoral injection after the flushing of the femur. Source of Cells % Dog CD45 + % Dog CD45 + Cells - Cells - Average Standard Deviation Left Femur Right Femur Spleen Blood The difference between engraftment in the left femur and right femur was not statistically significant. Table 6: Percentage of dog leukocytes in mice transplanted with dog bone morrow mononuclear cells using hepatic injection in neonatal mice. Source of Cells % Dog CD45 + % Dog CD45 + Cells - Cells - Average Standard Deviation Femur Spleen Blood The percentage of dog leukocytes in the peripheral blood varied greatly between individual mice.

103 92 Table 7: Graft vs. host disease in NSG mice transplanted with dog bone marrow mononuclear cells. Method Weeks Post- Femur % Spleen % Blood % Transplant Dog CD45+ Dog CD45+ Dog CD45+ GVHD Femur + Flush 4 N/A N/A N/A Yes Femur + Flush 8 0.0% 0.0% 0.0% No Femur + Flush 8 1.5% 29.8% 0.0% No Femur + Flush % 9.0% 5.8% No Liver % 9.4% 74.5% Yes Liver % 7.8% 0.2% Yes Liver 7 5.8% 9.2% 78.3% Yes Liver 7 5.0% 0.0% 0.0% No Liver 8 0.7% 0.5% 3.9% No Liver 8 3.8% 2.0% 1.7% Yes Liver % 17.2% 2.2% Yes Liver 9 6.4% 2.7% 0.1% No The incidence of graft vs. host varied in mice transplanted with bone marrow mononuclear cells.

104 93 Donor CD 45 + cells Left Femur 1.33% 0.78% 84.86% 13.04% Right Femur 1.02% 1.47% 75.62% 21.90% Spleen 3.15% 0.95% 95.20% 0.71% Recipient CD 45 + cells Figure 1: Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via tail-vein injection 7-9 weeks post-transplant. Donor cells are shown in the upper two quadrants. Recipient cells are shown in the right two quadrants. Why some cells appeared positive for both donor and recipient CD45 + cells is unknown. Addition of the two upper right quadrants gave similar numbers of donor cells to samples treated only with anti-bodies to dog CD45 + cells. Donor CD 45 + cells Left Femur 11.11% 14.53% 55.22% 17.79% Right Femur 9.00% 11.09% 62.74% 15.83% Spleen 12.91% 18.47% 62.04% 4.47% Recipient CD 45 + cells Figure 2: Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrafemoral injection 7-9 weeks post-transplant.

105 94 Left Femur 9.36% 5.57% 5.57% Donor CD 45 + cells 79.76% 4.82% 79.76% 4.82% Right Femur 16.00% 16.00% 7.81% 7.81% 70.21% 5.17% 70.21% 5.17% Spleen 8.05% 8.05% 0.98% 0.98% 87.81% 87.81% 2.18% 2.18% Recipient CD CD cells Figure 3: Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrafemoral injection after flushing the left femur 7-9 weeks post-transplant. Left Femur 9.36% 4.04% 1.63% 5.57% Donor CD 45 + cells 92.41% 0.33% 79.76% 92.41% 4.82% 0.33% Right Femur 4.96% 16.00% 0.92% 7.81% 93.35% 0.55% 70.21% 93.35% 0.55% 5.17% Spleen 7.51% 8.05% 1.69% 0.98% 87.81% 90.32% 2.18% 0.21% Recipient CD CD cells Figure 4 Example of flow cytometry readout of donor and recipient leukocytes in NSG mice transplanted via intrahepatic injection of neonatal mice 7-9 weeks posttransplant.