Injection Of Diphtheria Toxin Research Articles

Engineered Deafness Reveals That Mouse Courtship Vocalizations Do Not Require Auditory Experience

Auditory experience during development is necessary for normal language acquisition in humans. Although songbirds, some cetaceans, and maybe bats may also be vocal learners, vocal learning has yet to be well established for a laboratory mammal. Mice are potentially an excellent model organism for studying mechanisms underlying vocal communication. Mice vocalize in different social contexts, yet whether they learn their vocalizations remains unresolved. To address this question, we compared ultrasonic courtship vocalizations emitted by chronically deaf and normal hearing adult male mice. We deafened CBA/CaJ male mice, engineered to express diphtheria toxin (DT) receptors in hair cells, by systemic injection of DT at postnatal day 2 (P2). By P9, almost all inner hair cells were absent and by P16 all inner and outer hair cells were absent in DTR mice. These mice did not show any auditory brainstem responses as adults. Wild-type littermates, also treated with DT at P2, had normal hair cells and normal auditory brainstem responses. We compared the temporal structure of vocalization bouts, the types of vocalizations, the patterns of syllables, and the acoustic features of each syllable type emitted by hearing and deaf males in the presence of a female. We found that almost all of the vocalization features we examined were similar in hearing and deaf animals. These findings indicate that mice do not need auditory experience during development to produce normal ultrasonic vocalizations in adulthood. We conclude that mouse courtship vocalizations are not acquired through auditory feedback-dependent learning.

Persistent Antigen and Germinal Center B Cells Sustain T Follicular Helper Cell Responses and Phenotype

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

Ablation of Neurons Expressing Melanin-Concentrating Hormone (MCH) in Adult Mice Improves Glucose Tolerance Independent of MCH Signaling

Melanin-concentrating hormone (MCH)-expressing neurons have been ascribed many roles based on studies of MCH-deficient mice. However, MCH neurons express other neurotransmitters, including GABA, nesfatin, and cocaine-amphetamine-regulated transcript. The importance of these other signaling molecules made by MCH neurons remains incompletely characterized. To determine the roles of MCH neurons in vivo, we targeted expression of the human diphtheria toxin receptor (DTR) to the gene for MCH (Pmch). Within 2 weeks of diphtheria toxin injection, heterozygous Pmch(DTR/+) mice lost 98% of their MCH neurons. These mice became lean but ate normally and were hyperactive, especially during a fast. They also responded abnormally to psychostimulants. For these phenotypes, ablation of MCH neurons recapitulated knock-out of MCH, so MCH appears to be the critical neuromodulator released by these neurons. In contrast, MCH-neuron-ablated mice showed improved glucose tolerance when compared with MCH-deficient mutant mice and wild-type mice. We conclude that MCH neurons regulate glucose tolerance through signaling molecules other than MCH.

The Role of Osteocytes in Bone Resorption during Orthodontic Tooth Movement

We investigated the roles of osteocytes in osteoclastic bone resorption during orthodontic tooth movement using the transgenic mice in which osteocytes can be specifically ablated. Because these transgenic mice express the receptor for diphtheria toxin on the cell surfaces of osteocytes, the injection of diphtheria toxin can ablate their osteocytes in vivo. Injection of diphtheria toxin into the transgenic mice significantly increased the number of ablated osteocytes in alveolar bone compared with that in wild-type mice with or without diphtheria toxin injection. Increased numbers of ablated osteocytes were observed from day 4 to day 12 after the injection in alveolar bones as well as in cortical bone of the tibiae. We applied the orthodontic force 4 days after the injection of diphtheria toxin, and the distance of tooth movement on day 12 was significantly smaller in transgenic mice than that in control mice. The numbers of osteoclasts and the quantity of eroded bone surface at the compression site were significantly reduced in the transgenic mice injected with diphtheria toxin than in control mice. These results provide in vivo demonstration of osteocyte involvement in osteoclastic bone resorption during orthodontic tooth movement.

Abstract B72: CD8αα+ langerin+ dendritic cells maintain the effector CD8 T cell population after adoptive transfer.

Abstract Adoptive cell transfer (ACT) is a promising new area of anti-cancer therapy. The process involves the isolation of T cells from a patient's blood, in vitro stimulation of the T cells against cancer antigens, then re-infusion of the activated cells back into the patient. This process provides a population of effector CD8 T cells capable of recognizing and killing cancer cells. ACT is currently limited by the ability of the transferred cells to survive in high numbers for an extended period of time and therefore overcoming this problem is a focus to improve the efficacy of the treatment. The activation of cancer-specific CD8 T cells is dependent on the ability of dendritic cells (DCs) to acquire and present exogenous antigens on major histocompatibility complex class I (a process known as cross-presentation) for the stimulation of CD8 T cells. We have previously found a subset of splenic DCs expressing the CD8αα homodimer and the c-type lectin receptor langerin (CD207), that have a strong ability to activate CD8 T cells by cross-presentation. This DC subset is found in the marginal zone of the spleen, is highly efficient at phagocytosing apoptotic cells from the blood, and is believed to play an important role in scanning the blood for antigens. We therefore hypothesized that CD8αα+ langerin+ DCs influence the survival and expansion of adoptively transferred effector CD8 T cells in the blood. Within this project we have used langerin-diphtheria toxin receptor mice, in which all langerin-expressing cells are ablated in vivo upon injection of diphtheria toxin, to address the role of the CD8αα+ langerin+ DC subset in maintaining the CD8 T cell population after ACT. For this purpose, we challenged mice intravenously with the T cell thymoma EG7.OVA and adoptively transferred in vitro activated OVA-specific CD8 T cells (OT-I T cells) 12 days later with or without prior langerin-DC depletion. We found a larger population of OT-I T cells when these were transferred into an EG7.OVA bearing host when compared to mice without tumors. Interestingly, mice that were depleted of their CD8αα+ langerin+ DCs did not develop this increase in OT-I T cell number and their absence resulted in reduced survival. Our data therefore suggest that antigen presentation by CD8αα+ langerin+ DCs play a role in sustaining high numbers of transferred CD8 T cells after ACT, resulting in increased protection against EG7.OVA. Manipulations that increase the stimulatory capacity of this DC subset could be a way to increase the efficacy of ACT therapy. Citation Format: John David Gibbins, Ian Hermans, Troels Petersen. CD8αα+ langerin+ dendritic cells maintain the effector CD8 T cell population after adoptive transfer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B72.

Advantages and limitations of mouse models to deplete dendritic cells

Dendritic cells (DCs) play a key role in regulating innate and adaptive immunity. Our understanding of DC biology has benefited from studies in CD11c.DTR and CD11c.DOG mouse models that use the CD11c promoter to express a diphtheria toxin (DT) receptor transgene to inducibly deplete CD11c(+) cells. Other models to inducibly deplete specific DC subsets upon administration of DT have also been generated. However, most models suffer from limitations such as depletion of additional cell types or the requirement to be used as radiation chimeras. Moreover, CD11c.DTR and CD11c.DOG mice have recently been reported to display neutrophilia and monocytosis upon DT injection. We discuss here some of the limitations that should be taken into consideration when interpreting results obtained with mouse models of DC ablation.

Impairment of podocyte function by diphtheria toxin—a new reversible proteinuria model in mice

Diphtheria toxin (DTx) receptor (DTR)-mediated conditional cell ablation in transgenic mice is a powerful tool to analyze cell function in vivo. Transgenic mice with cell-specific expression of the human DTR have been developed that allow conditional depletion of these cells in vivo through administration of the toxin. We have performed a careful analysis of mice after DTx injection and found an unexpected side effect. Treatment of wild-type C57BL/6 mice with DTx leads to a marked transient and completely reversible proteinuria, as a consequence of podocyte dysfunction that is morphologically characterized by foot process fusion and detachment from the glomerular basal membrane. In vitro analysis displayed that DTx-treated podocytes show diminished attachment to basal membrane proteins. Five to 9 days after DTx application the mice recover completely. Glomerular proteinuria is a hallmark of glomerular disease due to dysfunction of the filtration barrier. Rodents have been extensively used experimentally to better define the mechanisms of disease induction and progression. However, nongenetic mouse models of proteinuric glomerular damage are limited and display various shortcomings. We suggest DTx-induced transient kidney dysfunction as a new reversible model of experimental podocyte injury, which could be used as an additional approach to complement studies in human.

Use of FoxP3 Diptheria Toxin Receptor (Foxp3DTR) Donor T Cells to Define the Mechanism Involved in the Inhibition of GvHD by Azacytidine in Vivo

AbstractAbstract 4113Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired graft versus leukemia (GvL) effect is frequently accompanied by Graft versus Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression or,1 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells.2To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT).3 To induce GvHD, 1×107 panT cells obtained from Foxp3 DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16 +18 +20 (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery.Mice injected with PBS (n=5) or DT (n=5) (without azaC) developed severe GvHD and died by day 26 post HSCT. In sharp contrast, 100% of mice (n=5) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC +DT (n=5) resulted in significantly prolonged survival compared to the PBS treated mice (p=0.003) but in contrast to the azaC group, failed to survive beyond day 60 (p=0.003). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=0.001), CD3+ T cells (p=0.002), and B220+ B cells (p=0.02) than the azaC +DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. These results suggest that BOTH pharmacologic conversion of alloreactive FoxP3- donor T cells to FoxP3-expressing Tregs and direct toxicity to allogeneic T cells by azaC represent important mechanisms involved in the amelioration of GvHD by azaC in vivo. Disclosures:No relevant conflicts of interest to declare.

Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice

The strength of antiviral T cell responses correlates with clearance of hepatitis B virus (HBV) infection, but the immunological mechanisms mitigating or suppressing HBV-specific T cells are still poorly understood. In this study, we examined the role of CD4(+) Foxp3(+) regulatory T cells (Tregs) in a mouse model of acute HBV infection. We initiated HBV infection via an adenoviral vector transferring a 1.3-fold overlength HBV genome (AdHBV) into transgenic DEREG mice, where Tregs can be transiently but selectively depleted by injection of diphtheria toxin. The effect of Treg depletion on the outcome of HBV infection was characterized by detailed virological, immunological, and histopathological analysis. Numbers of Tregs increase in the liver rapidly after initiation of HBV replication. Initial depletion of Tregs revealed their complex regulatory function during acute infection. Tregs mitigated immunomediated liver damage by down-regulating the antiviral activity of effector T cells by limiting cytokine production and cytotoxicity, but did not influence development of HBV-specific CD8 T cells or development of memory T cells. Furthermore, Tregs controlled the recruitment of innate immune cells such as macrophages and dendritic cells to the infected liver. As a consequence, Tregs significantly delayed clearance of HBV from blood and infected hepatocytes. Tregs limit immunomediated liver damage early after an acute infection of the liver, thereby contributing to conservation of tissue integrity and organ function at the cost of prolonging virus clearance.

CX(3)CR1(+) macrophages support IL-22 production by innate lymphoid cells during infection with Citrobacter rodentium.

Innate immune cells, such as intestinal epithelial cells, dendritic cells (DCs), macrophages, granulocytes, and innate lymphoid cells provide a first line of defence to enteric pathogens. To study the role of CX3CR1+ DCs and macrophages in host defence, we infected CX3CR1-GFP animals with Citrobacter rodentium. When transgenic CX3CR1-GFP animals are infected with the natural mouse pathogen C. rodentium, CX3CR1−/− animals showed a delayed clearance of C. rodentium as compared with (age- and sex-matched) wild-type B6 animals. The delayed clearance of C. rodentium is associated with reduced interleukin (IL)-22 expression. In C. rodentium-infected CX3CR1-GFP animals, IL-22 producing lymphoid-tissue inducer cells (LTi cells) were selectively reduced in the absence of CX3CR1. The reduced IL-22 expression correlates with decreased expression of the antimicrobial peptides RegIIIβ and RegIIIγ. The depletion of CX3CR1+ cells by diphtheria toxin injection in CX3CR1-GFP × CD11c.DOG animals confirmed the role of CX3CR1+ phagocytes in establishing IL-22 production, supporting the clearance of a C. rodentium infection.

Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage

Classical dendritic cells (cDCs), monocytes, and plasmacytoid DCs (pDCs) arise from a common bone marrow precursor (macrophage and DC progenitors [MDPs]) and express many of the same surface markers, including CD11c. We describe a previously uncharacterized zinc finger transcription factor, zDC (Zbtb46, Btbd4), which is specifically expressed by cDCs and committed cDC precursors but not by monocytes, pDCs, or other immune cell populations. We inserted diphtheria toxin (DT) receptor (DTR) cDNA into the 3' UTR of the zDC locus to serve as an indicator of zDC expression and as a means to specifically deplete cDCs. Mice bearing this knockin express DTR in cDCs but not other immune cell populations, and DT injection into zDC-DTR bone marrow chimeras results in cDC depletion. In contrast to previously characterized CD11c-DTR mice, non-cDCs, including pDCs, monocytes, macrophages, and NK cells, were spared after DT injection in zDC-DTR mice. We compared immune responses to Toxoplasma gondii and MO4 melanoma in DT-treated zDC- and CD11c-DTR mice and found that immunity was only partially impaired in zDC-DTR mice. Our results indicate that CD11c-expressing non-cDCs make significant contributions to initiating immunity to parasites and tumors.

Effects of depletion of CD4+FoxP3+ Treg on development of spontaneous autoimmune thyroiditis (SAT) in WT and B cell deficient (B-/-) NOD.H-2h4 mice (123.10)

Abstract NOD.H-2h4 mice given NaI in their water develop SAT with chronic inflammation of the thyroid by T and B cells. B-/- mice are resistant to SAT, but develop SAT after transient depletion of Treg by anti-CD25. However, NOD.H-2h4 mice have significant numbers of CD4+FoxP3+ Treg that are CD25-. Generation of mice expressing the human diphtheria toxin receptor (DTR) under control of the FoxP3 promoter allows for selective depletion of Foxp3+ cells by diphtheria toxin (DT). Anti-CD25 depletion of Treg lasts 3-4 weeks, whereas most Treg return 4-5 days after DT treatment. In WT and B-/- FoxP3-DTR NOD.H-2h4 mice, injection of DT eliminates essentially all Treg in blood and spleen within 2 days. As with anti-CD25 depletion, DT treatment allows for SAT development in B-/- mice even though Treg are depleted for only a few days, and SAT in DT-treated WT mice is slightly increased. We established a transfer model in which splenocytes from CD28-/-B-/- mice (effector cells) were cultured with or without sorted Treg from FoxP3-DTR WT or B-/- mice. After transfer to TCRα-/- mice, recipients of Treg from B-/- mice developed significantly less severe SAT than did mice receiving Treg from WT mice. As there is no source of new FoxP3-DTR Treg, DT treatment 2 days after transfer to recipient mice resulted in permanent depletion of Treg. This abrogated the suppression, suggesting that suppression of SAT requires continued inhibition of effectors by Treg during disease development.

Ablation of Sim1 Neurons Causes Obesity through Hyperphagia and Reduced Energy Expenditure

Single-minded 1 (Sim1) is a transcription factor necessary for development of the paraventricular nucleus of the hypothalamus (PVH). This nucleus is a critical regulator of appetite, energy expenditure and body weight. Previously we showed that Sim1+/− mice and conditional postnatal Sim1−/− mice exhibit hyperphagia, obesity, increased linear growth and susceptibility to diet-induced obesity, but no decrease in energy expenditure. Bilateral ablation of the PVH causes obesity due to hyperphagia and reduced energy expenditure. It remains unknown whether Sim1 neurons regulate energy expenditure. In this study, Sim1cre mice were bred to homozygous inducible diphtheria toxin receptor (iDTR) mice to generate mice expressing the simian DTR in Sim1 cells. In these mice, Sim1 neuron ablation was performed by intracerebroventricular (ICV) injection of diphtheria toxin. Compared to controls, mice with Sim1 neuron ablation became obese (with increased fat mass) on a chow diet due to increased food intake and reduced energy expenditure. In post-injection mice, we observed a strong inverse correlation between the degree of obesity and hypothalamic Sim1 expression. The reduction in baseline energy expenditure observed in these mice was accompanied by a reduction in activity. This reduction in activity did not fully account for the reduced energy expenditure as these mice exhibited decreased resting energy expenditure, decreased body temperature, decreased brown adipose tissue temperature, and decreased UCP1 expression suggesting an impairment of thermogenesis. In injected mice, hypothalamic gene expression of Sim1, oxytocin (OXT) and thyrotropin releasing hormone (TRH) was reduced by about 50%. These results demonstrate that Sim1 neurons in adult mice regulate both food intake and energy expenditure. Based on the body of work in the field, feeding regulation by Sim1 neurons likely occurs in both the PVH and medial amygdala, in contrast to energy expenditure regulation by Sim1 neurons, which likely is localized to the PVH.

A limited role for regulatory T cells in post‐ischemic neovascularization

Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4+CD25+FoxP3+ T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro-coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)-2 with the IL-2 monoclonal antibody JES6–1, or depleted by anti-CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post-ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non-depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.

Bone Marrow Neuropathy Prevents Hematopoietic Regeneration

Abstract 139Chemotherapy regimens commonly produce long-term hematopoietic injury, possibly through a lesion to the hematopoietic stem cell (HSC) and/or its niche but the mechanisms have yet to be elucidated. Nestin-expressing mesenchymal stem cells, under regulation of the sympathetic nervous system (SNS), are a critical component of the HSC niche (Nature. 2010; 466:829). Since peripheral neuropathy is a common complication of chemotherapy, we hypothesized that chemotherapy-induced sympathetic bone marrow (BM) neuropathy could impair BM regeneration. To test this issue, we sympathectomized mice using 6-hydroxydopamine (6OHDA) or diphtheria toxin (DT) injection into TH-Cre-iDTR mice, which express the DT receptor exclusively on catecholaminergic neurons. In both models, SNS ablation led to reduced BM regeneration following 5-fluorouracil (5FU) challenge, as determined by the delayed recovery in BM cellularity (1.7-fold↓*), clonogenic progenitors (CFU-C; 2.5-fold↓*) and Lin−Sca-1+c-Kit+Flt3− cells (LSKF cells 2.5-fold↓*). Further, inhibition of β3 adrenergic signaling with the antagonist SR5920A impaired BM recovery to the same extent as 6OHDA sympathectomy: BM cells (1.6-fold↓*), CFU-C (1.6-fold↓*), LSKF (1.6-fold↓*). We then treated mice with the chemotherapy drug cisplatin, which induces neuropathies in mice (2.8-fold↓* in SNS fibers in BM). Cisplatin-treated mice exhibited reduced survival after BM transplantation (66% vs 100%* for cisplatin-treated and control, respectively) due to delayed regeneration: BM cells (4.6-fold↓*), CFU-C (8.8-fold↓*), LSKF (6.4-fold↓*). To evaluate the potential benefit of neuroprotection, we treated mice with 4-methylcatechol (4-MC), a powerful inducer of Nerve Growth Factor (NGF), together with cisplatin or 6OHDA, which led to the protection of SNS fibers (1.8-fold↑* and 11-fold↑* respectively). This prevented transplantation-associated death and increased BM recovery following transplantation or 5FU administration: BM cells (1.9- and 1.6-fold↑, respectively *), CFU-C (3.6- and 1.4-fold↑*), LSKF (4.5- and 2.4-fold↑*). To ascertain whether 4-MC/NGF acted specifically on the nervous system, rather than on hematopoietic or stromal cells, we used TrkAneo/neo mice, which are genetically deficient in the NGF receptor in non-neuronal tissues (Development. 2004;131:5185). Administration of 4-MC to sympathectomized TrkAneo/neo mice enhanced BM regeneration after 5FU: BM cells (2.0-fold↑*), CFU-C (2.0-fold↑*), LSKF (2.2-fold↑*), suggesting that 4-MC acts directly on neurons to rescue hematopoiesis. Since deletion of p53 promotes neuron survival after genotoxic damage (Neurodegeneration. 1996;5:233), we intercrossed mice expressing TH-Cre mice with p53flox/flox animals to delete p53 specifically in catecholaminergic neurons. SNS fibers of TH-Cre;p53flox/flox mice were protected after cisplatin treatment compared to TH-Cre;p53+/flox controls, and this was associated with improved BM recovery after transplantation: BM cells (2.4-fold↑*), CFU-C (2.9-fold↑*), LSKF (6.5-fold↑*) when compared to TH-Cre;p53+/flox animals. Thus, these various pharmacologic and genetic models strongly suggest that chemotherapy-induced sympathetic neuropathy prevents BM regeneration. To evaluate further the mechanisms, we investigated whether BM neuropathy might lead to dysfunctional HSC niche. SNS injury using 6OHDA or cisplatin treatment led to Nestin+ cell expansion and accumulation (1.7- and 3.8-fold↑*, respectively). Treatment with 4-MC reverted this phenotype (1.8-fold↓* for 6OHDA and 1.5-fold↓* for cisplatin), indicating that Nestin+ cell expansion is due to reduced SNS signaling. SNS injury also led to increased Nestin+ cell sensitivity to genotoxic insult as determined by increased Nestin+ cell death and apoptosis (1.4-fold↑*) 24h after 5FU injection, which reduced Nestin+ cell numbers during regeneration (2.1-fold↓* 12 days after 5FU). By contrast, the number of osteoblasts and endothelial cells were unaffected. These results indicate that adrenergic signals promote the quiescence of HSC niche cells and that sympathetic neuropathy renders the niche vulnerable to genotoxic insult, impairing hematopoietic regeneration. Neural protection may represent a novel strategy to maintain hematopoietic reserve and enhance transplantation efficiency in chemotherapy-treated patients. *P<0.05 Disclosures:No relevant conflicts of interest to declare.

Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Abstract 721Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures:No relevant conflicts of interest to declare.

Pancreatic Mesenchyme Regulates Epithelial Organogenesis throughout Development

Author SummaryEmbryonic development is a highly complex process that requires tight orchestration of cellular proliferation, differentiation, and migration as cells grow within loosely aggregated mesenchyme and more organized epithelial sheets to form organs and tissues. In addition to intrinsic cell-autonomous signals, these events are further regulated by environmental cues provided by neighboring cells. Prior work demonstrated a critical role for the surrounding mesenchyme in guiding epithelial growth during the early stages of pancreas development. However, it remained unclear whether the mesenchyme also guided the later stages of pancreas organogenesis when the functional exocrine and endocrine cells are formed. Here, we show that specific genetic ablation of the mesenchyme at distinct developmental stages in vivo results in the formation of a smaller, misshapen pancreas. Loss of the mesenchyme profoundly impairs the expansion of both endocrine and exocrine pancreatic progenitors, as well as the proliferative capacity of maturing cells, including insulin-producing beta-cells. Thus, our studies reveal unappreciated roles for the mesenchyme in guiding the formation of the epithelial pancreas throughout development. The results suggest that identifying the specific mesenchymal signals might help to optimize cell culture protocols that aim to achieve the differentiation of stem cells into insulin-producing beta cells.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

Targeted Ablation of Oligodendrocytes Triggers Axonal Damage

Glial dysfunction has been implicated in a number of neurodegenerative diseases. In this study we investigated the consequences of glial and oligodendrocyte ablation on neuronal integrity and survival in Drosophila and adult mice, respectively. Targeted genetic ablation of glia was achieved in the adult Drosophila nervous system using the GAL80-GAL4 system. In mice, oligodendrocytes were depleted by the injection of diphtheria toxin in MOGi-Cre/iDTR double transgenic animals. Acute depletion of oligodendrocytes induced axonal injury, but did not cause neuronal cell death in mice. Ablation of glia in adult flies triggered neuronal apoptosis and resulted in a marked reduction in motor performance and lifespan. Our study shows that the targeted depletion of glia triggers secondary neurotoxicity and underscores the central contribution of glia to neuronal homeostasis. The models used in this study provide valuable systems for the investigation of therapeutic strategies to prevent axonal or neuronal damage.