Targeting NETs using dual-active DNase1 variants

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states

Hematopoietic stem and progenitor recovery in sex steroid ablation-mediated immune regeneration

Lifelong hematopoiesis is supported by hematopoietic stem cells (HSCs) differentiating within the bone marrow (BM) down all blood cell lineages including erythrocytes, platelets, myeloid and lymphoid cells. Although most blood cells are generated entirely within the BM, T cells complete their development in the thymus, a primary lymphoid organ within the mediastinal cavity. Together, B and T cells represent the lymphoid arm of the immune system and are vital in the adaptive protection against foreign pathogens. While hematopoiesis is continuous for the lifespan of the organism, it is currently well established that, paradoxical to its fundamental importance for establishing and maintaining good health, the adaptive immune system degenerates very early with age; a phenomenon temporally linked to sex steroid exposure. Beginning from birth, but accelerated at the onset of puberty, both the primary lymphoid organs – the BM and thymus, gradually deteriorate, resulting in a decline in the generation of new naïve B and T cells. In healthy adults this does not pose a major threat, however in patients who are immunocompromised following cytoreductive treatments, such as chemo- or radiation- therapy or chronic infections best exemplified by acquired immunodeficiency syndrome (AIDS), immune recovery is considerably delayed leaving these individuals susceptible to opportunistic infections and malignant relapses. As a corollary to the hypothesized underlying cause of immune atrophy, our laboratory and others have previously demonstrated that removing the negative effects of sex steroids by either surgical or chemical (reversible) castration (sex steroid ablation; SSA) facilitates significant immune regeneration in aged mice and can enhance lymphoid recovery post HSC transplantation (HSCT) or treatment with chemotherapy.   3 This thesis aimed to elucidate the mechanisms underlying SSA-mediated lymphoid recovery by comprehensively assessing the impact of SSA on (1) the function of hematopoietic stem and progenitor cells in the BM, (2) the ability of the supporting BM stromal microenvironment to support hematopoiesis, and (3) the function of the earliest T-lineage progenitors in the thymus. Consistent with previous reports, we observed an age-associated accumulation of HSCs that demonstrated inferior self-renewal capacity and lymphoid differentiation potential when compared to young HSCs. We further demonstrated both a numerical and functional enhancement of the primitive long-term HSC (LT-HSC) population following SSA. Not only did these LT-HSCs show enhanced self-renewal, they were also more efficient at differentiating into downstream lymphoid cells with SSA. Detailed molecular analysis revealed important cell intrinsic changes pertaining to quiescence, self-renewal, lymphoid differentiation and DNA replication processes occurring within these primitive HSCs, collectively suggesting their role in establishing SSA-mediated immune regeneration. Since HSCs rely heavily on their stromal cell-based microenvironmental “niches” for signals governing their differentiation and survival, the effects of SSA on the the endosteal and vascular (central marrow) compartments were profiled. Interestingly, a population of osteoblasts (OBLs) with high Runx2 expression was revealed within the vascular niche of the BM that numerically increased with age; this correlated with an accumulation of LT- HSCs seen with age in the BM. These Runx2 –rich OBLs also expressed hematopoietic supporting molecules, albeit to a lesser degree than the canonical LT-HSC-associated endosteal osteoblasts. These age-related changes were not observed within the endosteal osteoblasts compartment however. While SSA was unable to reverse the age-associated increase in the vascular OBL population, there was increased expression of genes linked to promoting HSC quiescence, self-renewal, lymphoid differentiation and cell adhesion. This   4   clearly indicates the important synergisms between the hematopoietic and stromal compartments of the bone marrow – with both contributing to the SSA-induced rejuvenation of the blood system. With this evidence suggesting that enhanced HSC and BM niche function led to downstream improvements in lymphopoiesis, the final section of this thesis focused on the effects of SSA on the lymphoid progenitors: their ability to seed the thymus and subsequently differentiate into T cells. While there has been considerable insight into the developmental hierarchy of HSCs and the downstream progenitors within the BM, little is known about the identity of the thymic-bound progenitor that egresses from the BM. Increases in the CD27+CD62L+ lymphoid primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLPs) within the BM, and the early thymic progenitor (ETP) and CLP-2 within the thymus were evident following SSA. The earliest T cell progenitor within the thymus is the ETP, which has proficient T differentiation potential. However the earliest cellular increase in the thymus post SSA was by the CLP2 population at day 2. The CLP and its downstream CLP-2 produced in the BM, are by default a B cell progenitors, yet have a very efficient capacity for thymic entry and the propensity to form T cells once influenced by Notch signaling within the thymus. Several thymic homing molecules, CCR-7, CCR-9 and PSGL-1 have been identified on various circulating thymic progenitors (CTPs), however the expression of these molecules was unaltered following SSA. This would suggest the increased ability of these BM derived CLP-2s to migrate into the aged regenerating thymus, is possibly due to a noncanonical “stress” or “damage” associated pathway. The data are thus consistent with the downstream improvements observed in lymphopoiesis being the result of an increased supply of lymphoid progenitors from the BM, subsequently entering the thymus coupled with an enhanced ability of the intrathymic microenvironment in supporting thymopoiesis, rather than an enhanced intrinsic ability of these progenitors to differentiate into T cells, on a per cell level. Collectively, the work presented here provides a tantalizing glimpse into the mechanisms underlying SSA-mediated lymphoid regeneration, and hence a means of improving transplant outcomes, perhaps by either conditioning donor HSCs or the recipient BM niche, or by targeted acceleration of the regeneration process to reduce the time required for immune recovery

Hematopoietic stem and progenitor recovery in sex steroid ablation-mediated immune regeneration

Lifelong hematopoiesis is supported by hematopoietic stem cells (HSCs) differentiating within the bone marrow (BM) down all blood cell lineages including erythrocytes, platelets, myeloid and lymphoid cells. Although most blood cells are generated entirely within the BM, T cells complete their development in the thymus, a primary lymphoid organ within the mediastinal cavity. Together, B and T cells represent the lymphoid arm of the immune system and are vital in the adaptive protection against foreign pathogens. While hematopoiesis is continuous for the lifespan of the organism, it is currently well established that, paradoxical to its fundamental importance for establishing and maintaining good health, the adaptive immune system degenerates very early with age; a phenomenon temporally linked to sex steroid exposure. Beginning from birth, but accelerated at the onset of puberty, both the primary lymphoid organs – the BM and thymus, gradually deteriorate, resulting in a decline in the generation of new naïve B and T cells. In healthy adults this does not pose a major threat, however in patients who are immunocompromised following cytoreductive treatments, such as chemo- or radiation- therapy or chronic infections best exemplified by acquired immunodeficiency syndrome (AIDS), immune recovery is considerably delayed leaving these individuals susceptible to opportunistic infections and malignant relapses. As a corollary to the hypothesized underlying cause of immune atrophy, our laboratory and others have previously demonstrated that removing the negative effects of sex steroids by either surgical or chemical (reversible) castration (sex steroid ablation; SSA) facilitates significant immune regeneration in aged mice and can enhance lymphoid recovery post HSC transplantation (HSCT) or treatment with chemotherapy.   3 This thesis aimed to elucidate the mechanisms underlying SSA-mediated lymphoid recovery by comprehensively assessing the impact of SSA on (1) the function of hematopoietic stem and progenitor cells in the BM, (2) the ability of the supporting BM stromal microenvironment to support hematopoiesis, and (3) the function of the earliest T-lineage progenitors in the thymus. Consistent with previous reports, we observed an age-associated accumulation of HSCs that demonstrated inferior self-renewal capacity and lymphoid differentiation potential when compared to young HSCs. We further demonstrated both a numerical and functional enhancement of the primitive long-term HSC (LT-HSC) population following SSA. Not only did these LT-HSCs show enhanced self-renewal, they were also more efficient at differentiating into downstream lymphoid cells with SSA. Detailed molecular analysis revealed important cell intrinsic changes pertaining to quiescence, self-renewal, lymphoid differentiation and DNA replication processes occurring within these primitive HSCs, collectively suggesting their role in establishing SSA-mediated immune regeneration. Since HSCs rely heavily on their stromal cell-based microenvironmental “niches” for signals governing their differentiation and survival, the effects of SSA on the the endosteal and vascular (central marrow) compartments were profiled. Interestingly, a population of osteoblasts (OBLs) with high Runx2 expression was revealed within the vascular niche of the BM that numerically increased with age; this correlated with an accumulation of LT- HSCs seen with age in the BM. These Runx2 –rich OBLs also expressed hematopoietic supporting molecules, albeit to a lesser degree than the canonical LT-HSC-associated endosteal osteoblasts. These age-related changes were not observed within the endosteal osteoblasts compartment however. While SSA was unable to reverse the age-associated increase in the vascular OBL population, there was increased expression of genes linked to promoting HSC quiescence, self-renewal, lymphoid differentiation and cell adhesion. This   4   clearly indicates the important synergisms between the hematopoietic and stromal compartments of the bone marrow – with both contributing to the SSA-induced rejuvenation of the blood system. With this evidence suggesting that enhanced HSC and BM niche function led to downstream improvements in lymphopoiesis, the final section of this thesis focused on the effects of SSA on the lymphoid progenitors: their ability to seed the thymus and subsequently differentiate into T cells. While there has been considerable insight into the developmental hierarchy of HSCs and the downstream progenitors within the BM, little is known about the identity of the thymic-bound progenitor that egresses from the BM. Increases in the CD27+CD62L+ lymphoid primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLPs) within the BM, and the early thymic progenitor (ETP) and CLP-2 within the thymus were evident following SSA. The earliest T cell progenitor within the thymus is the ETP, which has proficient T differentiation potential. However the earliest cellular increase in the thymus post SSA was by the CLP2 population at day 2. The CLP and its downstream CLP-2 produced in the BM, are by default a B cell progenitors, yet have a very efficient capacity for thymic entry and the propensity to form T cells once influenced by Notch signaling within the thymus. Several thymic homing molecules, CCR-7, CCR-9 and PSGL-1 have been identified on various circulating thymic progenitors (CTPs), however the expression of these molecules was unaltered following SSA. This would suggest the increased ability of these BM derived CLP-2s to migrate into the aged regenerating thymus, is possibly due to a noncanonical “stress” or “damage” associated pathway. The data are thus consistent with the downstream improvements observed in lymphopoiesis being the result of an increased supply of lymphoid progenitors from the BM, subsequently entering the thymus coupled with an enhanced ability of the intrathymic microenvironment in supporting thymopoiesis, rather than an enhanced intrinsic ability of these progenitors to differentiate into T cells, on a per cell level. Collectively, the work presented here provides a tantalizing glimpse into the mechanisms underlying SSA-mediated lymphoid regeneration, and hence a means of improving transplant outcomes, perhaps by either conditioning donor HSCs or the recipient BM niche, or by targeted acceleration of the regeneration process to reduce the time required for immune recovery

Enhanced hematopoietic stem cell function mediates immune regeneration following sex steroid blockade

Mechanisms underlying age-related defects within lymphoid-lineages remain poorly understood. We previously reported that sex steroid ablation (SSA) induced lymphoid rejuvenation and enhanced recovery from hematopoietic stem cell (HSC) transplantation (HSCT). We herein show that, mechanistically, SSA induces hematopoietic and lymphoid recovery by functionally enhancing both HSC self-renewal and propensity for lymphoid differentiation through intrinsic molecular changes. Our transcriptome analysis revealed further hematopoietic support through rejuvenation of the bone marrow (BM) microenvironment, with upregulation of key hematopoietic factors and master regulatory factors associated with aging such as Foxo1. These studies provide important cellular and molecular insights into understanding how SSA-induced regeneration of the hematopoietic compartment can underpin recovery of the immune system following damaging cytoablative treatments. These findings support a short-term strategy for clinical use of SSA to enhance the production of lymphoid cells and HSC engraftment, leading to improved outcomes in adult patients undergoing HSCT and immune depletion in general

Multilineage Potential and Self-Renewal Define an Epithelial Progenitor Cell Population in the Adult Thymus

Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration

Biomanufacturing for clinically advanced cell therapies

The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use

Restoring Systemic GDF11 Levels Reverses Age-Related Dysfunction in Mouse Skeletal Muscle

Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral “rejuvenating” factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.Human Evolutionary Biolog

Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-b superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.Stem Cell and Regenerative Biolog

Targeting NETs using dual-active DNase1 variants

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.</p