Kelly Marshall McNagny

The immune system evolved to protect the host from external pathogens as well as manage host internal processes including tissue repair after damage. This thesis explores different aspects of immune responses in the context of viral infection, allergic disease, and internal, sterile muscle injury. Firstly, the characterization of peripheral blood immune cells in patients admitted to the intensive care unit (ICU) due to an infection with SARS-CoV-2, the virus that causes COVID-19, uncovered immune biomarkers, specifically serum IL-10 and CD11cˡºʷ classical monocytes, that are predictive of disease outcome. The unprecedented and ongoing burden caused by the COVID-19 pandemic makes this research timely and critical to assist with ICU patient management. Secondly, the characterization of immune cells in archived umbilical cord blood revealed biomarkers predictive of the development of childhood allergic disease, which occurs commonly and severely and for which disease origin is unclear. Our findings suggest that alterations in naive CD8 T cells and monocytes are already established prior to birth in children who, by the age of 5, develop a combined wheeze and atopy phenotype. The stages of prenatal immune development are rarely explored in the context of allergic disease development and may be a new avenue for early disease detection and prevention. Lastly, the characterization and manipulations of type-2 immune responses in mouse models of sterile acute and chronic skeletal muscle injury showed that type-2 immune cells are largely expendable in normal tissue regeneration but accelerate muscle pathology. Specifically, IL-33- induced type-2 immunity promoted prominent influx of ILC2s and eosinophils in acutely injured skeletal muscle with no discernable impact on muscle regeneration but accelerated fibrosis deposition in mdx mice – a model of Duchenne muscular dystrophy. These findings add to our understanding of how the immune system supports normal tissue function and how tissue is damaged in disease, which is fundamental for future development of disease treatments and preventative measures.

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Dominant and recessive mutations in podocalyxin (PODXL/Podxl) are associated with human kidney disease. Some PODXL/Podxl mutations manifest as anuria while others are associated with proteinuria. Homozygous loss-of-function mutations in the gene are associated with early-onset congenital nephrotic syndrome. By contrast, PODXL heterozygosity is associated with adult-onset kidney disease and podocalyxin shedding into the urine is a common biomarker of podocyte injury in various nephrotic contexts. It is unknown, however, how different lesions in PODXL/Podxl contribute to these disparate disease pathologies. Here we generated three mutant mouse stains: one that lacks Podxl in all tissues (Podxl-/-), a second that selectively deletes the gene in mature (capillary loop stage) podocytes (Podxl∆ᴾᵒᵈ), and a third that is heterozygous for Podxl in all tissues (Podxl⁺/-). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to Podxl-/- mice, which have previously been shown to die 24 hours after birth from anuria and hypertension, we show that Podxl∆ᴾᵒᵈ mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis and severe proteinuria. Podxl⁺/- mice have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6J mice are resistant to chemically-induced nephrosis, Podxl⁺/- mice are highly susceptible, as administration of puromycin aminonucleoside induces collapsing FSGS and nephrotic syndrome. The severity of kidney injury in Podxl⁺/- mice in response to puromycin aminonucleoside was optimized through dose-response experiments, and we used this model to evaluate the anti-proteinuric effects of the two calcineurin inhibitors, cyclosporine and voclosporin. Calcineurin inhibition by either voclosporin or cyclosporine did not attenuate proteinuria in Podxl⁺/- mice induced with puromycin aminonucleoside. In conclusion, we find that the developmental timepoint at which Podxl expression is ablated (immature vs. mature podocytes) has a profound impact on the renal disease phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆ᴾᵒᵈ and Podxl⁺/- mice offer powerful new tools to evaluate early biomarkers of proteinuric kidney disease, to define the function of genes during kidney injury, and to test new therapies.

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The advent of targeted therapies has vastly improved cancer diagnostics and treatments over the last three decades, however, cancer still remains the second leading cause of death worldwide. Importantly, the majority of cancer-related deaths are the result of metastatic disease. This highlights the need to identify biomarkers of tumors at high risk of metastasizing, and to generate targeted therapies against them. Previous studies have demonstrated a strong association between high expression of podocalyxin and decreased patient survival, however, little is known about the role of podocalyxin in promoting cancer progression or its potential as a therapeutic target.Here we perform an in-depth characterization of PODO83, an anti-podocalyxin antibody that we previously demonstrated delays primary tumor growth in murine tumor models. We show that despite its ability to delay primary tumor growth, its primary effect is actually the prevention of metastasis to the lung. We identified the binding epitope of PODO83 on the extracellular juxtamembrane domain of podocalyxin and showed that the antibody recognizes the core podocalyxin protein in both tumors and healthy tissue. Further, using urothelial, breast and ovarian carcinomas tissue microarrays, we present PODO83 as a promising diagnostic tool.Podocalyxin is normally readily expressed in the vascular endothelia and kidney podocytes, representing a source of concern surrounding the use of podocalyxin-based therapies. Here, we identified a novel tumor-restricted glycoepitope on podocalyxin and generated and characterized an antibody (PODO447) to target it. We found that while unconjugated PODO447 does not exert any inhibitory or toxic effect on tumor cells, when used as the targeting arm in a Vedotin antibody-drug conjugate (ADC), the PODO447-ADC specifically targets cancerous cells and increases the survival in pre-clinical models. The work presented in this thesis contributes directly to scientific understanding of the role of podocalyxin in tumor growth and metastasis. Further, we provide pre-clinical evidence supporting the furthered development of the novel podocalyxin antibodies PODO83, and PODO447 as diagnostic and targeted immunotherapies, respectively, to be used in the fight against cancer and to improve patient outcome.

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Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. Type 2 immunity has long been associated with fibrotic scarring because of its role in wound healing and parasite-initiated tissue remodeling. Our objective was to examine two components of this inflammatory pathway that could potentially be modulated to limit fibrosis, namely RORα, a nuclear receptor required for ILC2 development, and CD34, a sialomucin involved in trafficking of eosinophils and mast cells to peripheral tissues. Using a model of infection-induced chronic gut inflammation, we demonstrate that Rora-deficient mice are protected from fibrosis; infected intestinal tissues displayed diminished pathology and attenuated collagen deposition. Although Rora is known for its role in ILC2s, we found that Salmonella-induced fibrosis was independent of eosinophils, STAT6 signaling and Th2 cytokines arguing that ILC2s are dispensable in this disease model. Instead, we observed reduced levels of ILC3- and T cell-derived IL-17A and IL-22 in infected tissues. Furthermore, using Rorasg/sg/Rag1-/- bone marrow chimeric mice, we found that restoring ILC function was sufficient to re-establish IL-17A and IL-22 production and a profibrotic phenotype. Our findings suggest that RORα-dependent ILC3 functions are pivotal in mediating gut fibrosis and they offer an avenue for therapeutic intervention in Crohn’s-like diseases. CD34 has been shown to drive lung inflammation and colitis by coordinating immune cell recruitment. However CD34 is also expressed by multiple non-hematopoietic subsets including endothelial and mesenchymal cells. To assess CD34 function in pulmonary repair, we induced lung injury by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared to WT controls. CD34-deficient animals developed severe interstitial edema and endothelial delamination, indicating impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared to WT mice reconstituted with Cd34-/- cells thus confirming this to be a non-hematopoietic defect. Lastly, CD34-deficient mice were more sensitive to lung damage caused by influenza infection, displaying greater weight loss and more extensive pulmonary remodeling. These results suggest that CD34 plays a protective role in maintaining vascular integrity in response to lung damage.

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Mucosal surfaces present an important barrier between the host and environment. Maintenance of barrier function requires intricate cross-talk between a diverse array of immune cells and the epithelia, acting synergistically to respond to harmful antigens and maintain tolerance to innocuous antigens. In this thesis I utilized an array of transgenic animals to explore the cellular and molecular mechanisms that initiate adaptive immune responses in the lung and gut mucosa.Recently, innate lymphoid cells have been characterized for their role in maintaining barrier immunity. Group 2 innate lymphoid cells (ILC2s) colonize the lung and provide a rapid source of IL-5 and IL-13 in a T and B cell independent manner in response to protease antigens. Using ILC2-deficient mice, I examined the role of these cells in mucosal inflammation using mouse models of allergic asthma and hypersensitivity pneumonitis (HP). ILC2s were critical in initiation of a Th2 response to locally, but not systemically delivered allergens and were completely dispensable for Th1 and Th17 dependent responses. The PI3K pathway plays an important role in regulating leukocyte activation, survival, migration and cytokine release. It is negatively regulated by the lipid phosphatase Ship1, and Ship1-/- mice develop a wide array of hematological disorders leading to a reduced lifespan. The severe phenotype associated with loss of Ship1 throughout the immune systems masks subtler roles it plays in specific leukocyte subsets. Using a conditional deletion approach, I examined the role of Ship1 in T cells, B cells and dendritic cells (DCs) in mouse models of allergic asthma and helminth infection. While loss of Ship1 in B cells did not influence susceptibility to a HDM model of allergic asthma, loss of Ship1 in either the T cells or DCs protected from disease development due to an immune skewing to a Th1 response. Additionally, loss of Ship1 in DCs rendered mice susceptible to infection with the intestinal helminth Trichuris muris, further highlighting this Th1 immune skewing.

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Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin (Podxl) andendoglycan) as vascular endothelial cell (EC) markers, there is remarkably little known oftheir role in vascular development and functions with the exception of vessel lumenformation in the developing mouse embryo (Podxl) and vessel patency during tumorangiogenesis and inflammation (CD34). Because germ-line deletion of Podxl in mice causesperinatal death, we generated mice that conditionally delete Podxl in vascular endothelialcells (PodxlΔEC mice) to study the role of podocalyxin in adult mouse vessels. AlthoughPodxlΔEC adult mice are viable and thrive, we discovered increased basal and inflammationinducedpulmonary vascular permeability. Furthermore, PodxlΔEC mouse adult lungs displayairspace enlargement with increased collagen deposition and exhibit a gene expressionprofile similar to regenerating lung. To study whether endothelial cell morphology influencesthe defective lung architecture and the vascular permeability phenotype in PodxlΔEC mice, weisolated primary vascular ECs from lung tissue. PodxlΔEC ECs display enhanced adhesion tofibronectin (FN) in a static adhesion assay. When plated on matrix-coated transwells,PodxlΔEC EC spread normally on FN but display defective spreading on laminin and collagenI. Thus, expression of Podxl in EC is required for normal lung architecture and function inadult mice and adhesion of EC to extracellular matrix components.Although its expression has not been well characterized, in humans, endoglycan expressionhas been reported in vascularized tissues. In mouse blood vessels, we showed that vascularsmooth muscle cells (vSMC), but not EC, express the highest levels of endoglycan. Using amouse aortic smooth muscle line (MOVAS-1) we found that forced expression ofendoglycan enhances basal but not platelet derived growth factor (PDGF)ββ-dependentvSMC migration in vitro. Further studies to understand the role of endoglycan in primaryvSMC showed that endoglycan is upregulated with differentiation to a contractile phenotype,but is not influenced by inflammatory stimuli or mitogenic factors. The findings of this thesis suggest that the CD34 family regulates vascular development andfunction with a role for podocalyxin in EC-matrix adhesion relevant to normal lung functionand a role for endoglycan in SMC differentiation and migration.

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No abstract available.

Overall, we aimed to discover more about mast cell physiology, focusing on their homeostatic regulation in vivo, their activation in vitro and in allergic disease, their gene expression patterns, and their surface antigens. In our first study, our objective was to establish the function of Src homology 2-containing inositol 5’-phosphatase (SHIPI), in mast cells in vivo. SHIP1 inhibits immune receptor signaling throughhydrolysis of the phosphatidylinositol-3 kinase (P13K) product Pl-3,4,5-P₃ ,forming P1-3,4-P₂. In mast cells, SHIPI represses FcεRI- and cytokine-mediated activation in vitro, but little is known regarding the function of SHIPI in mast cells in vivo, or the susceptibility of Shipl⁻/⁻ mice to mast cell-associated diseases. We found that ⁻ mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and a heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1⁺/⁺ or Shipl⁻/⁻ mast cells, we found that the above defects were due to loss of SHIPI in mast cells. Additionally, we found that micereconstituted with Shipl⁻/⁻ mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1⁺/⁺ mast cells. In summary, our data show that SHIPI represses allergic inflammation and mast cell hyperplasia in vivo, and that SHIPI exerts these effects specifically in mast cells. In our second study we compared Lin⁻Sca-1⁺c-kit⁺ (LSK) cells, which are highlyenriched for hematopoietic stem cells (HSC), and mast cells, using microarray expression analysis, and identified prion protein (PrPC) as a potentially novel marker of mast cells. Upon further investigation, we found that PrPC (1) is expressed on the surface of human and mouse mast cells, both in vitro and in vivo; (2) is not required for mast cell differentiation or tissue homeostasis; (3) is released by mast cells atsteady state and rapidly upon activation; and (4) is released in response to mast cell dependent allergic inflammation in vivo. Since mast cells are long-lived and known to traffic to the brain and central nervous system (CNS), our observations could have important implications for the transmission and pathology of prion diseases. Further, mast cells could be a unique system to investigate PrPc’s normal function.

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CD34 and its relatives, Podocalyxin and Endoglycan, comprise of a family ofsurface sialomucins expressed by hematopoietic stem/progenitor cells, and vascularendothelia. Recent data suggest that they serve as either pro- or anti-adhesion moleculesdepending on their cellular context and their post-translational modifications. We wereinterested in identifying Podocalyxin ligands and their cellular distribution andunderstanding the role of these factors in signaling, adhesion and migration. Using both alambda phage screen assay and mass spectrometry, we identified the Na⁺/H⁺ exchangerregulatory factor-i (NHERF-l) as a selective ligand for Podocalyxin and Endoglycan butnot for the closely related CD34. Furthermore, we showed that NHERF-1 is expressedby all, lineage⁻, Sca-1⁺ and c-kit⁺ (LSK) cells, which are known to express Podocalyxinand have long-term repopulating characteristics of hematopoietic stem cells. In addition,upon IL-3 stimulation of a factor dependent cell line (FDC-P 1) these proteins re-localizeand co-localize in an asymmetrical pattern. By using a lentiviral based shRNA system tosilence Podocalyxin and NHERF- i proteins, we observed that migration across stromalmonolayer towards a CXCL12 and SCF gradient is significantly impeded in cells thatlack Podocalyxin but not NHERF-1. Following in vitro stimulation with a combinationof CXCL12 and SCF we observed that Podocalyxin co-associates with CXCR4.Furthermore, cells lacking Podocalyxin have decreased phospho-AKT, a key signalingmolecule downstream of c-kit and CXCR4 receptors. Taken together, our data supportsthe conclusion that Podocalyxin co-association with CXCR4 modulates downstreamsignaling to efficiently regulate HSC homing.

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