Diphtheria Toxin Receptor Research Articles

Islet cell replacement and transplantation immunology in a mouse strain with inducible diabetes

Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet β-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.

Exposed and Sequestered Antigens in Testes and Their Protection by Regulatory T Cell-Dependent Systemic Tolerance.

Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.

Loss of γδ intraepithelial lymphocytes and reduced immunosurveillance of the epithelial barrier precedes the onset of Crohn’s disease‐like ileitis

Inflammatory bowel disease, including Crohn’s disease (CD), affects over 3 million Americans. Recently, single cell sequencing of resected ileal tissue from CD patients revealed a loss of γδ intraepithelial lymphocytes (IELs). IELs expressing the γδ T cell receptor (γδ IEL) bridge innate and adaptive immunity and migrate dynamically within the intestinal epithelium as a means of immunosurveillance. However, the contribution of γδ IELs to the initiation of ileitis remains unknown. We and others have found that the onset of histopathology in TNFΔARE/+mice, which develop a chronic CD‐like ileitis, occurs at 8 weeks of age. We profiled the IEL populations of TNFΔARE/+mice prior to and during the initial stages of ileal inflammation (4, 6, 8 and 10 weeks). In agreement with CD patient data, we observed about a 2‐fold reduction in the γδ IEL population in ilea of 8‐week‐old TNFΔARE/+mice compared to TNF+/+ (WT) littermates (p=0.05). Seeding of γδ IELs within the epithelial compartment is regulated in part by expression of epithelial butyrophilin‐like genes Btnl1 and Btnl6. We find that the expression of both Btnl1 and Btnl6is reduced in 4‐ and 6‐week‐old TNFΔARE/+mice (p=0.05). Furthermore, the frequency of EdU+ γδ IELs is decreased in TNFΔARE/+mice compared to WT littermates at 6 weeks of age (p=0.05). γδ IEL motility and function contribute to maintenance of an intact epithelial barrier, as we have recently demonstrated that γδ IELs facilitate shedding of apoptotic epithelial cells. Cleaved caspase 3 staining in fixed ileal tissue revealed increased shedding of apoptotic enterocytes in 6‐week‐old TNFΔARE/+mice compared to WTlittermates (p=0.05). Since γδ IEL surveillance behavior is altered during the shedding of apoptotic enterocyte, we next assessed γδ IEL motility in 6‐week‐old WT and TNFΔARE/+ mice using intravital microscopy. γδ IEL migratory behavior is significantly impaired in TNFΔARE/+ mice, as reflected by reduced track speed (7.9 vs 2.8 μm/min, p=0.005) and increased arrest coefficient (p=0.001) relative to WT mice. To determine the contribution of γδ IELs in the development of CD‐like ileitis, we generated TNFΔARE/+ mice expressing diphtheria toxin (DT) receptor driven by a γδ T‐cell‐specific promoter (TNFΔARE/+ TcrdGDL). Our preliminary findings indicate that DT‐treated 3‐week‐old TNFΔARE/+ TcrdGDL mice exhibit reduced survival compared to TNFΔARE/+ littermates treated with vehicle control (Mantel‐Cox log‐rank test, p=0.155). In summary, we report that the onset of ileal inflammation coincides with the reduction of γδ IELs, which may be attributed to decreased epithelial Btnl expression and impaired γδ IEL proliferation. Reduced survival of TNFΔARE/+ mice following γδ T cell depletion suggests that γδ IEL‐mediated maintenance and surveillance of the barrier is critical to maintain mucosal tolerance prior to disease onset. These studies suggest that restoring γδ IEL number or migratory behavior may be effective therapeutic strategies to maintain remission in CD patients.

Microglia depletion elicits neuroprotective effects to alleviate vascular damage and neuronal cell loss in the diabetic retina

Abstract Diabetic retinopathy (DR), an incurable eye disease, is a common complication of diabetes mellitus and the leading cause of blindness amongst working age adults. Prolonged high glucose levels damage retinal blood vessels leading to hemorrhages, ischemia and ultimately vision loss. Microglia, the resident macrophages of the central nervous system, are rapidly activated and respond to transient hyperglycemia, and reset the homeostatic threshold of the retina. As hyperglycemia persists, capillary damage occurs resulting in serum proteins and DAMPs from the periphery leaking into the retina perpetuating microgliosis, pro-inflammatory cytokine production and vascular damage that correlates with neuronal loss. Therefore, we transiently depleted microglia to determine the mechanism by which resident retinal macrophages regulate neuronal and vascular damage at early stages of diabetes. We hypothesize that transient depletion of CX3CR1CreER:R26iDTR microglia during the course of disease when microglia receive the early-stage environmental cues of retinal inflammation due to hyperglycemia will induce neuroprotective cues. Utilizing a genetic model to express diphtheria toxin receptor under the tamoxifen inducible CX3CR1-promoter, microglia become susceptible to the effects of diphtheria toxin. Our findings revealed that transient depletion of microglia induced neuroprotective cues to upregulate TUJ1+ neurons and ameliorate vascular damage in the diabetic retina. In conclusion these studies suggest that early transient microglia depletion is neuroprotective by inducing the proliferation of a homeostatic microglia cell population that induce axonal regeneration of TUJ1+ neurons and vascular repair. Supported by grants from NIH (R01EY029913) and NIH RISE Grant (GM061655).

Stress Induced Eosinophilpoiesis Contributes to Mortality after Pulmonary Resection

Abstract Factors affecting mortality after lung surgery remain poorly explored. We performed pulmonary lobar resections(removing □ 1/5 of the total pulmonary volume) from C57BL/6 mice and evaluated quantitative changes in various leucocytes in the remaining lung, blood and spleen. A nearly four-fold increase in eosinophils was evident by day-7 post resection with a gradual return to baseline by day 30. Evaluation of the bone marrow demonstrated no change in common myeloid, granulocyte-monocyte or eosinophil progenitors. However the number of early stage and mature eosinophils doubled after lobar resection suggesting that pulmonary surgery augmented maturation of eosinophils. Neutralization of IL-5 decreased overall eosinophil numbers but the relative differences between resting and post-resection mice remained. Further evaluation of bone marrow demonstrated an increase in IL-25, IL-6, IL-9, KC and MCP-1 after pulmonary resection. We next performed right pneumonectomies in saline or diphtheria toxin-treated iPHIL mice, where diphtheria toxin receptor expression is limited to eosinophils, and noted a 70% mortality in eosinophil sufficient vs. 20% mortality in eosinophil depleted mice (p=.005). Severe hypoxia was noted in eosinophils sufficient vs. deficient mice (pO2 of 54.3+/−5.7 vs. 83.6+/−11.63; p=.04) four hours prior to resection. Taken together out data demonstrate that pulmonary surgery leads to bone marrow-specific maturation of eosinophils and systemic eosinophilia which directly contributes to perioperative mortality. Thus defining factors which may disrupt this process offers the opportunity to improve outcomes after lung surgery. Supported by PO1 AI116501, RO1 AI145108-01

Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato+ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.

Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart.

Macrophages play an important role in cardiac repair after myocardial infarction (MI). In addition to the resident macrophages and blood-derived monocytes, Gata6+ cavity macrophages located in the pericardial space were recently reported to relocate to the injured myocardium and prevent cardiac fibrosis. However, there is no direct genetic evidence to support it. We used dual recombinases (Cre and Dre) to specifically label Gata6+ pericardial macrophages (GPCMs) in vivo. For functional study, we generated genetic systems to specifically ablate GPCMs by induced expression of DTR (diphtheria toxin receptor) or knockout of Gata6 (GATA binding protein 6) gene in GPCMs. We used these genetic systems to study GPCMs in pericardium intact MI model. Dual recombinases-mediated genetic system targeted GPCMs specifically and efficiently. Lineage tracing study revealed accumulation of GPCMs on the surface of MI heart without deep penetration into the myocardium. We did not detect significant change of cardiac fibrosis or function of MI hearts after cell ablation or Gata6 knockout in GPCMs. GPCMs minimally invade the injured heart after MI. Nor do they prevent cardiac fibrosis and exhibit reparative function on injured heart. This study also underlines the importance of using specific genetic tool for studying in vivo cell fates and functions.

Partial Ablation of Astrocytes Exacerbates Cerebral Infiltration of Monocytes and Neuronal Loss After Brain Stab Injury in Mice.

In traumatic brain injury (TBI), mechanical injury results in instantaneous tissue damages accompanied by subsequent pro-inflammatory cascades composed of microgliosis and astrogliosis. However, the interactive roles between microglia and astrocytes during the pathogenesis of TBI remain unclear and sometimes debatable. In this study, we used a forebrain stab injury mouse model to investigate the pathological role of reactive astrocytes in cellular and molecular changes of inflammatory response following TBI. In the ipsilateral hemisphere of stab-injured brain, monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and inflammatory cytokines were observed. To verify the role of reactive astrocytes in TBI, local and partial ablation of astrocytes was achieved by stereotactic injection of diphtheria toxin in the forebrain of Aldh1l1-CreERT2::Ai9::iDTR transgenic mice which expressed diphtheria toxin receptor (DTR) in astrocytes after tamoxifen induction. This strategy achieved about 20% of astrocytes reduction at the stab site as validated by immunofluorescence co-staining of GFAP with tdTomato-positive astrocytes. Interestingly, reduction of astrocytes showed increased microglia activation and monocyte infiltration, accompanied with increased severity in stab injury-induced neuronal loss when compared with DTR-/- mice, together with elevation of inflammatory chemokines such as CCL2, CCL5 and CXCL10 in astrogliosis-reduced mice. Collectively, our data verified the interactive role of astrocytes as an immune modulator in suppressing inflammatory responses in the injured brain. Schematic diagram shows monocyte infiltration and neuronal loss, as well as increased elevated astrogliosis, microglia activation and chemokines were observed in the injured site after stab injury. Local and partial ablation of astrocytes led to increased microglia activation and monocyte infiltration, accompanied with increased severity in neuronal loss together with elevation of inflammatory chemokines as compared with control mice subjected stab injury.

A bioluminescence reporter mouse model for visualizing and quantifying CD8+ T cells in vivo.

Cytotoxic CD8+ T cells are the primary effector cells mediating anti-tumor responses. In vivo monitoring of CD8+ T cells has broad implications for the development of novel cancer therapies. Here we describe the development of a genetically engineered mouse model (GEMM) in which CD8+ T cells are labeled with an optical reporter, enabling in vivo, longitudinal monitoring using bioluminescence imaging (BLI). Firefly luciferase (Luc2), human diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) cDNAs are engineered under the CD8α promoter to generate a transgenic mouse line. Luciferase mRNA and CD8α mRNA were generally correlated in various tissues from these mice. Sorted splenic CD8+ T cells, CD4+ T cells and CD3- non-T cells verified that the luciferase signal is specific to CD8+ T cells. In vivo imaging showed that luciferase signal was detected in various immune organs, such as lymph nodes, thymus, and spleen, and the detection was confirmed by ex vivo examination. Administration of diphtheria toxin markedly reduced luciferase signal systemically, confirming the function of the DTR. In the MC38 mouse syngeneic model, we observed significant increases in CD8+ T cells with mDX400 treatment, an anti PD-1 mouse monoclonal antibody that correlated with tumor growth inhibition. This novel reporter GEMM is a valuable drug discovery tool for profiling compounds and understanding mechanisms of action in immunotherapy of cancer.

Modification of Neurogenic Colonic Motor Behaviours by Chemogenetic Ablation of Calretinin Neurons.

How the enteric nervous system determines the pacing and propagation direction of neurogenic contractions along the colon remains largely unknown. We used a chemogenetic strategy to ablate enteric neurons expressing calretinin (CAL). Mice expressing human diphtheria toxin receptor (DTR) in CAL neurons were generated by crossing CAL-ires-Cre mice with Cre-dependent ROSA26-DTR mice. Immunohistochemical analysis revealed treatment with diphtheria toxin incurred a 42% reduction in counts of Hu-expressing colonic myenteric neurons (P = 0.036), and 57% loss of CAL neurons (comprising ∼25% of all Hu neurons; P = 0.004) compared to control. As proportions of Hu-expressing neurons, CAL neurons that contained nitric oxide synthase (NOS) were relatively spared (control: 15 ± 2%, CAL-DTR: 13 ± 1%; P = 0.145), while calretinin neurons lacking NOS were significantly reduced (control: 26 ± 2%, CAL-DTR: 18 ± 5%; P = 0.010). Colonic length and pellet sizes were significantly reduced without overt inflammation or changes in ganglionic density. Interestingly, colonic motor complexes (CMCs) persisted with increased frequency (mid-colon interval 111 ± 19 vs. 189 ± 24 s, CAL-DTR vs. control, respectively, P < 0.001), decreased contraction size (mid-colon AUC 26 ± 24 vs. 59 ± 13 gram/seconds, CAL-DTR vs. control, respectively, P < 0.001), and lacked preferential anterograde migration (P < 0.001). The functional effects of modest calretinin neuron ablation, particularly increased neurogenic motor activity frequencies, differ from models that incur general enteric neuron loss, and suggest calretinin neurons may contribute to pacing, force, and polarity of CMCs in the large bowel.

Different syngeneic tumors show distinctive intrinsic tumor-immunity and mechanisms of actions (MOA) of anti-PD-1 treatment

Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8+-T-cells, CD4+-T-cells, Treg, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8+ cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4+ Teff TILs played a key role. TIL-Treg also played a critical role in supporting tumor growth in all four syngeneic models as well as M2-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.

New transgenic mouse models enabling pan-hematopoietic or selective hematopoietic stem cell depletion in vivo

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in “Vav-DTR” mice. Crossing these mice to Flk2-Cre mice results in “HSC-DTR” mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis.

Abstract TP140: Loss Of Fibroblasts Aggravates Hemorrhagic Brain Injury By Diminishing Timp2 Secretion

Although Col1a1 + fibroblast-like cells have been identified in large blood vessels in the CNS, their function remains largely unknown. To answer this important question, we generated mutant mice (Col1a1-DTR) with ablated fibroblast-like cells by crossing the Col1a1-Cre mice with DTR mice, which contain a loxP-flanked STOP sequence before diphtheria toxin receptor. The Col1a1-DTR mice were grossly normal after diphtheria toxin injection, suggesting that Col1a1 + fibroblast-like cells are dispensable under homeostatic conditions. After hemorrhagic stroke, however, these mice demonstrated exacerbated brain injury, including enhanced blood-brain barrier (BBB) leakage and aggravated injury volume. In addition, the expression of Collagen-1 and PDGFRα, two fibroblast markers, was significantly reduced. These findings highlight a beneficial role of Col1a1 + fibroblast-like cells in hemorrhagic stroke. Consistent with these results, similar results were also observed in vitro. Specifically, fibroblast-conditioned medium (FCM) attenuated endothelial damage (increased TEER and decreased FITC-Dextran leakage) in an in vitro model of hemorrhagic stroke. Subsequent mechanistic study revealed that FCM up-regulated the expression of various tight junction proteins (occludin, ZO-1, and claudin-5) without affecting that of caveolin-1, a key protein in transcytosis. These results indicate that fibroblasts protect BBB via paracellular rather than transcellular mechanism. To further investigate how exactly fibroblasts exert this beneficial role in hemorrhagic stroke, we performed mass spectrometry analysis, and identified TIMP2 (a protein with known BBB-protecting function) in the FCM. Using both pharmacological and genetic approaches, we further showed that ablating TIMP2 abolished the neuroprotective effect of FCM. Altogether, these findings suggest that Col1a1 + fibroblasts secrete TIMP2, which inhibits tight junction protein loss and paracellular leakage, promoting BBB repair and hemorrhagic stroke recovery. These results suggest that fibroblasts and TIMP2 may be targeted in the therapy of stroke.

3168 – DEATH MODELS OF HEMATOPOIESIS: NEW TRANSGENIC MOUSE MODELS ENABLING PAN-HEMATOPOIETIC OR SELECTIVE HEMATOPOIETIC STEM CELL DEPLETION IN VIVO

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in "Vav-DTR" mice. Crossing these mice to Flk2-Cre mice results in "HSC-DTR" mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis.

The role of lumbosacral innervating noradrenergic neurons in micturition control

A long-standing observation is that the micturition reflex receives supraspinal descending control. Although one supraspinal nucleus (Barrington’s nucleus) is identified as the pontine micturition center, it remains largely unknown whether and how other supraspinal tracts are involved in micturition control. Here, we focused on the role of lumbosacral projecting neurons located in the Locus Coeruleus (LC) in modulating micturition, since previous studies indicated that the LC is involved in controlling bladder contraction. First, by performing an AAV mediated retrograde labeling using a TH-iCre mouse line, we demonstrated specific targeting of LC noradrenergic neurons innervating the lumbosacral spinal cord with high efficiency. Next, by lumbosacral injection of a retro-AAV carrying Cre-dependent human diphtheria toxin receptors (DTR), we achieved specific ablation of LC NA+ neurons with lumbosacral projections upon the administration of diphtheria toxin. Our results showed that specific ablation of theseneurons led to overflow incontinence leaks and lower void efficiency. Mechanistically, by performing the urodynamics analysis, we showed that ablation of lumbosacral innervating NAneurons resulted in detrusor-sphincter dyssynergia. Taken together, our study provided novel insights into the underlying mechanisms of supraspinal control of micturition reflex and thus shed light on developing novel treatment to improve micturition control in patients with SCI or lower urinary tract symptoms.

Tumor-propagating side population cells are a dynamic subpopulation in undifferentiated pleomorphic sarcoma.

Sarcomas contain a subpopulation of tumor-propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-TPCs. In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-SP cells. By cotransplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor-propagating potential in vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the diphtheria toxin receptor is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin did not impede tumor growth or self-renewal. Altogether, we show that the sarcoma SP represent a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.

Abstract 10460: Urinary Fatty Acid Binding Protein 1 is a Biomarker for Impaired Proximal Tubular Protein Reabsorption and is Synergistically Enhanced by Concurrent Liver Injury

Introduction: Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Methods and Results: Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or PTEC-specific manner, or both. Upon diphtheria toxin (DT) administration, massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Conclusions: Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function.

Abstract 12456: Apelin Expressing Endothelial Stem-Like Cells Orchestrate Lung Microvascular Repair in a Model of Lung-Targeted Endothelial Cell Ablation

Introduction: Endothelial damage plays a central role in acute lung injury (ALI) contributing to alveolar leak and inflammatory cell trafficking. In preclinical models of ALI, repair and regeneration of lung vascular endothelium is required for its resolution. Therefore, we sought to define the cellular and molecular mechanisms underlying lung microvascular regeneration in acute lung injury induced by lung endothelial cell (EC) ablation. Methods and Results: Transgenic mice were created expressing endothelial-targeted human diphtheria toxin receptor. Intratracheal instillation of diphtheria toxin (DT) resulted in the ablation of ~70% of lung endothelial cells, producing severe acute lung injury, with complete resolution by 7 days. Changes in global lung cell populations and gene expression profiles were determined using single-cell RNA sequencing of dissociated lung cells (10x Genomics) at baseline (day 0), 3, 5 and 7 days after lung EC ablation. Single cell analysis revealed 8 distinct EC clusters, including type-A capillary ECs which were characterized by the unique expression of apelin at baseline. DT-induced EC ablation resulted in the emergence of novel endothelial stem-like cells within a transient population of ‘general’ capillary ECs, characterized by the de novo expression of apelin at day 3 post injury. As well, this population exhibited unique expression of the vascular endothelial stem cell marker, protein C receptor, together with the progenitor cell marker, CD34. This was followed by the appearance of proliferative ECs at day 5 expressing apelin receptor and the transcription factor, FoxM1, and these cells were responsible for replenishment of all depleted EC populations. Finally, treatment with an apelin receptor antagonist, ML221, prevented lung microvascular recovery and resulted in death at 4-5 days post DT. Conclusion: Targeted EC ablation revealed a remarkable regenerative capacity of the lung microvasculature. Using single cell transcriptomics, we have demonstrated that lung microvascular repair was orchestrated by newly emergent apelin-expressing endothelial stem-like cells.

Establishment of a pig CRISPR/Cas9 knockout library for functional gene screening in pig cells.

As an important farm animal, pig functional genomic study can help understand the molecular mechanism related to the key economic traits of pig, such as growth, reproduction, or disease. The genome-scale library based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system facilitates discovery of key genes involved in a specific function or phenotype, allowing for an effective "phenotype-to-genotype" strategy for functional genomic study. We designed and constructed a pig genome-scale CRISPR/Cas9 knockout library targeting 16,888 genes with 970,001 unique sgRNAs. The library is a single-vector system including both Cas9 and sgRNA, and packaged into lentivirus for an easy cell delivery for screening. To establish a screening method in pig cells, we used diphtheria toxin (DT)-induced cell death as a model to screen the host genes critical for DT toxicity in pig PK-15 cells. After lentiviral transduction and two sequential screening with DT treatment, the highest-ranking candidates we identified were previously validated genes, HBEGF, DPH1, DPH2, DPH3, DPH5, DNAJC24, and ZBTB17, which are DT receptor and the key factors involved in biosynthesis of diphthamide, the target of DT action. The function and gene essentiality of candidates were further confirmed by gene knockout and DT toxicity assay in PK-15 cells. Our CRISPR knockout library targeting pig whole genome establishes a promising platform for pig functional genomic analysis.

Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin‐synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin‐synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin‐synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin‐immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin‐immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1‐deamino‐8‐d‐arginine vasopressin via an osmotic mini‐pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin‐immunoreactive neurons, but not the number of oxytocin‐immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin‐synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.