Expression Of Diphtheria Toxin Research Articles

Distinct effects of dipeptidyl peptidase-4 inhibitor and glucagon-like peptide-1 receptor agonist on islet morphology and function.

Although the two anti-diabetic drugs, dipeptidyl peptidase-4 inhibitors (DPP4is) and glucagon-like peptide-1 (GLP-1) receptor agonists (GLP1RAs), have distinct effects on the dynamics of circulating incretins, little is known of the difference in their consequences on morphology and function of pancreatic islets. We examined these in a mouse model of β cell injury/regeneration. The model mice were generated so as to express diphtheria toxin (DT) receptor and a fluorescent protein (Tomato) specifically in β cells. The mice were treated with a DPP4i (MK-0626) and a GLP1RA (liraglutide), singly or doubly, and the morphology and function of the islets were compared. Prior administration of MK-0626 and/or liraglutide similarly protected β cells from DT-induced cell death, indicating that enhanced GLP-1 signaling can account for the cytoprotection. However, 2-week intervention of MK-0626 and/or liraglutide in DT-injected mice resulted in different islet morphology and function: β cell proliferation and glucose-stimulated insulin secretion (GSIS) were increased by MK-0626 but not by liraglutide; α cell mass was decreased by liraglutide but not by MK-0626. Although liraglutide administration nullified MK-0626-induced β cell proliferation, their co-administration resulted in increased GSIS, decreased α cell mass, and improved glucose tolerance. The pro-proliferative effect of MK-0626 was lost by co-administration of the GLP-1 receptor antagonist exendin-(9-39), indicating that GLP-1 signaling is required for this effect. Comparison of the effects of DPP4is and/or GLP1RAs treatment in a single mouse model shows that the two anti-diabetic drugs have distinct consequences on islet morphology and function.

Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A.

As an immunotoxin, diphtheria toxin has been widely used in gene therapy and gene function assays for its roles in protein synthesis inhibition, and the aim of our study is to set up a nonintegrating lentiviral system for specific expression of diphtheria toxin A (DTA) used in cancer gene therapy.Here, we established a lentiviral system that could coordinately express fluorescent protein and DTA driven by the cytomegalovirus (CMV) promoter, which is convenient for us to precisely trace the expression of DTA and monitor the process of lentivirus packaging. To achieve safer cancer therapy, we replaced the CMV promoter with the Survivin promoter, a specific promoter that is dramatically activated in cancer tissues and cells, but not in normal tissues and cells, and that will impose greater therapeutic potential because a significant expression difference occurred between these 2 groups. Meanwhile, we obtained integrase-deficient lentivirus (IDLV) after packaging with the integrase mutant, which expresses defective integrase RRK262263264AAH, to minimize the side effects that derived from the insertional mutagenesis of the host genome.Our results suggest that the IDLV system that we generated possesses therapeutic potential in cancers in vitro and in vivo.

A Peptide Nucleic Acid Targeting Nuclear Rad51 Sensitizes Myeloma Cells to Melphalan Chemotoxicity Both in Vitro and in Vivo

Multiple myeloma (MM) cells are characterized by extensive genomic heterogeneity, which contributes to patient differences in prognosis and response to treatment. We previously reported that MM cells have elevated homologous recombination (HR) rates and expression of RAD51 and its paralogs, promoting genomic instability and disease progression that are reversed by RAD51 siRNA. We now examine the roles of HR and RAD51 in resistance to melphalan, one of the most widely used drugs for MM chemotherapy. The drug induces a variety of DNA lesions, with DNA interstrand crosslinks (ICL) accounting for most of the drug’s cytotoxicity. RAD51 is a central protein in the HR pathway and its overexpression may contribute to chemoresistance by enabling repair of DNA lesions induced by DNA damaging agents such as melphalan. MM cell sensitivity to melphalan correlates directly with melphalan-induced RAD51 foci, and high RAD51 expression predicts poor event-free and overall survival of MM patients. Activity of the Rad51 promoter increases >850-fold in cancer cells compared to normal cells, and tumor cells are selectively killed by a construct in which PRad51 drives expression of diphtheria toxin. In this study, we tested whether inhibiting RAD51 expression with a peptide nucleic acid (PNA) would inhibit MM cell growth and/or sensitize MM cells to melphalan. PNAs are DNA or RNA mimics in which a polymer of (2-amino­ethyl) glycine replaces the nucleic acid’s sugar-phosphate backbone. PNAs are highly specific, binding DNA with higher affinity than RNA or DNA, and they are quite stable to degradation both in vitro and in vivo.We designed a PNA to target the promoter region of the RAD51 gene (PNArad51), encompassing the transcription start site. To enhance cellular uptake and nuclear delivery without transfection, we conjugated the PNA to a nuclear localization signal rich in basic residues (PKKKRKVR). As a control we employed a scrambled PNA (PNAmt) with the same nucleotide composition but not targeting any genomic sequences. We used qRT-PCR to assess the effect of PNA on RAD51 mRNA expression and that of melphalan on mRNA levels of RAD51 and its paralogs (RAD51B, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and BRCA1. Propidium iodide staining and flow cytometry were used to examine the cell-cycle effects of melphalan. γH2AX and RAD51 foci were quantitated using confocal immuno­fluorescence microscopy, and MM cell viability was assessed with the WST-1 assay. To examine the in vivo consequences of PNA ± melphalan for tumor growth, we injected H929 MM cells expressing luciferase into rabbit bone fragments implanted in SCID-rab mice, as previously described by us,. Total RNA extracted from cells recovered from the rabbit bones was analyzed by qRT-PCR to determine the in vivo effect of PNA on expression of RAD51.Melphalan treatment (10 µM) significantly induced expression of RAD51 and its paralogs, particularly RAD51 and XRCC3 (p≤0.01). Melphalan caused cell-cycle arrest, predominantly in the S-phase (55%, significantly elevated over vehicle alone, 17%; p<0.0001), the period in which HR is most active, and during which ICLs are converted into double strand breaks (DSBs) on encountering DNA replication forks. PNArad51 (10 µM) significantly reduced expression of RAD51 (~60%, p<0.001) relative to PNAmt. Pretreatment with PNArad51 inhibited melphalan-induced RAD51 focus formation, far more than PNAmt pretreatment (21% compared to 66%, p<0.0001) whereas the number of γH2AX foci increased (66%) relative to PNAmt (39%; p<0.0001). Consequently, pretreatment with PNArad51 produced synergistic synthetic lethality with melphalan, reducing the IC50 of melphalan by 4.5-fold. PNArad51 alone caused significant cytotoxicity compared to PNAmt (p<0.05). In the SCID-rab mouse model, a two-week treatment with PNArad51 alone or in combination with melphalan resulted in significant inhibition of tumor volume (p≈0.01 and p<0.05, respectively) compared to PNAmt, although the combination of PNAmt plus melphalan was ineffectual. Prolonged treatment (4 weeks) with PNArad51 ± melphalan (but not PNAmt + melphalan) reduced tumor growth compared to PNAmt treatment, although this was not statistically significant (p>0.05).These results highlight the importance of RAD51 in the response of MM cells to melphalan, and indicate for the first time the potential for RAD51-targeted PNA in tumor chemosensitization. DisclosuresNo relevant conflicts of interest to declare.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells.

BackgroundWe previously reported that the peripheral regulatory T cells (pTregs) generated ‘on-demand’ in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo.MethodsTransgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (βgal) as retinal neo-self antigen and βgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination.ResultsLocal depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in βgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal βgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local βgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after βgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation.ConclusionsOur results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Diphtheria toxin treatment of Pet-1-Cre floxed diphtheria toxin receptor mice disrupts thermoregulation without affecting respiratory chemoreception

In genetically-modified Lmx1bf/f/p mice, selective deletion of LMX1B in Pet-1 expressing cells leads to failure of embryonic development of serotonin (5-HT) neurons. As adults, these mice have a decreased hypercapnic ventilatory response and abnormal thermoregulation. This mouse model has been valuable in defining the normal role of 5-HT neurons, but it is possible that developmental compensation reduces the severity of observed deficits. Here we studied mice genetically modified to express diphtheria toxin receptors (DTR) on Pet-1 expressing neurons (Pet-1-Cre/floxed DTR or Pet1/DTR mice). These mice developed with a normal complement of 5-HT neurons. As adults, systemic treatment with 2–35μg of diphtheria toxin (DT) reduced the number of tryptophan hydroxylase-immunoreactive (TpOH-ir) neurons in the raphe nuclei and ventrolateral medulla by 80%. There were no effects of DT on minute ventilation (VE) or the ventilatory response to hypercapnia or hypoxia. At an ambient temperature (TA) of 24°C, all Pet1/DTR mice dropped their body temperature (TB) below 35°C after DT treatment, but the latency was shorter in males than females (3.0±0.37 vs. 4.57±0.29days, respectively; p<0.001). One week after DT treatment, mice were challenged by dropping TA from 37°C to 24°C, which caused TB to decrease more in males than in females (29.7±0.31°C vs. 33.0±1.3°C, p<0.01). We conclude that the 20% of 5-HT neurons that remain after DT treatment in Pet1/DTR mice are sufficient to maintain normal baseline breathing and a normal response to CO2, while those affected include some essential for thermoregulation, in males more than females. In comparison to models with deficient embryonic development of 5-HT neurons, acute deletion of 5-HT neurons in adults leads to a greater defect in thermoregulation, suggesting that significant developmental compensation can occur.

Macrophage depletion disrupts immune balance and energy homeostasis.

Increased macrophage infiltration in tissues including white adipose tissue and skeletal muscle has been recognized as a pro-inflammatory factor that impairs insulin sensitivity in obesity. However, the relationship between tissue macrophages and energy metabolism under non-obese physiological conditions is not clear. To study a homeostatic role of macrophages in energy homeostasis, we depleted tissue macrophages in adult mice through conditional expression of diphtheria toxin (DT) receptor and DT-induced apoptosis. Macrophage depletion robustly reduced body fat mass due to reduced energy intake. These phenotypes were reversed after macrophage recovery. As a potential mechanism, severe hypothalamic and systemic inflammation was induced by neutrophil (NE) infiltration in the absence of macrophages. In addition, macrophage depletion dramatically increased circulating granulocyte colony-stimulating factor (G-CSF) which is indispensable for NE production and tissue infiltration. Our in vitro study further revealed that macrophages directly suppress G-CSF gene expression. Therefore, our study indicates that macrophages may play a critical role in integrating immune balance and energy homeostasis under physiological conditions.

Optogenetic manipulation of activity and temporally controlled cell-specific ablation reveal a role for MCH neurons in sleep/wake regulation.

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.

Analysis of CNS tissue-specific Tregs and dendritic cells in T cell responses and autoimmunity (MUC7P.757)

Abstract We assessed locally generated, locally acting peripheral regulatory T cells (pTregs) in retinal immune homeostasis and ability of retinal dendritic cells (DC) to support T cell activation/expansion within retina. We used transgenic (Tg) mice expressing beta-galactosidase (bgal mice) as retinal neo-self antigen, TCR Tg mice (BG2) specific for bgal, and mice with labeled, depletable Tregs or DC, based on expression of diphtheria toxin (DTx) receptor and/or GFP controlled by FoxP3 or CD11c promoter (FDG or CDG mice). Treg and DC depletion from retina was done by DTx injection into the eye. T cells and DC were assayed by FACS of retina or blood. Retinal autoimmunity was assessed by histological analysis. After bgal (FDG/bgal mice) or IRBP (FDG mice) immunization, Treg depleted retinas showed increased incidence and severity of autoimmunity. We found local depletion of Tregs from retina sufficient to induce spontaneous autoimmunity in FDG/bgal/BG2 mice but not in single or double Tg mice. Dose of DTx used to eliminate retinal Tregs did not induce retinal autoimmunity if given systemically. DC depletion from the retina prevented Treg and Teff generation within retina after bgal injection. Retinal microglia remaining after DC depletion did not make up for the loss of DC-dependent antigen presentation. We conclude local Tregs within the retina protect against spontaneous organ-specific autoimmunity and local DC must be present within the retina for antigen presentation to T cells.

Cell Depletion in Mice That Express Diphtheria Toxin Receptor under the Control of SiglecH Encompasses More Than Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDC) produce IFN-I in response to viruses and are routinely identified in mice by SiglecH expression. SiglecH is a sialic acid-binding Ig-like lectin that has an immunomodulatory role during viral infections. In this study, we evaluated the impact of SiglecH deficiency on cytokine responses in the presence and absence of pDC. We found that lack of SiglecH enhanced IFN-I responses to viral infection, regardless of whether pDC were depleted. We also examined the expression pattern of SiglecH and observed that it was expressed by specialized macrophages and progenitors of classical dendritic cells and pDC. Accordingly, marginal zone macrophages and pDC precursors were eliminated in newly generated SiglecH-diphtheria toxin receptor (DTR)-transgenic (Tg) mice but not in CLEC4C-DTR-Tg mice after diphtheria toxin (DT) treatment. Using two bacterial models, we found that SiglecH-DTR-Tg mice injected with DT had altered bacterial uptake and were more susceptible to lethal Listeria monocytogenes infection than were DT-treated CLEC4C-DTR-Tg mice. Taken together, our findings suggest that lack of SiglecH may affect cytokine responses by cell types other than pDC during viral infections, perhaps by altering viral distribution or burden, and that cell depletion in SiglecH-DTR-Tg mice encompasses more than pDC.

Opposing Effects of Acute versus Chronic Blockade of Frontal Cortex Somatostatin-Positive Inhibitory Neurons on Behavioral Emotionality in Mice

Reduced expression of somatostatin (SST) is reported across chronic brain conditions including major depression and normal aging. SST is a signaling neuropeptide and marker of gamma-amino butyric acid (GABA) neurons, which specifically inhibit pyramidal neuron dendrites. Studies in auditory cortex suggest that chronic reduction in dendritic inhibition induces compensatory homeostatic adaptations that oppose the effects of acute inhibition. Whether such mechanisms occur in frontal cortex (FC) and affect behavioral outcome is not known. Here, we used two complementary viral vector strategies to examine the effects of acute vs chronic inhibition of SST-positive neurons on behavioral emotionality in adult mice. SST-IRES-Cre mice were injected in FC (prelimbic/precingulate) with CRE-dependent adeno-associated viral (AAV) vector encoding the engineered Gi/o-coupled human muscarinic M4 designer receptor exclusively activated by a designer drug (DREADD-hM4Di) or a control reporter (AAV-DIO-mCherry) for acute or chronic cellular inhibition. A separate cohort was injected with CRE-dependent AAV vectors expressing diphtheria toxin (DTA) to selectively ablate FC SST neurons. Mice were assessed for anxiety- and depressive-like behaviors (defined as emotionality). Results indicate that acute inhibition of FC SST neurons increased behavioral emotionality, whereas chronic inhibition decreased behavioral emotionality. Furthermore, ablation of FC SST neurons also decreased behavioral emotionality under baseline condition and after chronic stress. Together, our results reveal opposite effects of acute and chronic inhibition of FC SST neurons on behavioral emotionality and suggest the recruitment of homeostatic plasticity mechanisms that have implications for understanding the neurobiology of chronic brain conditions affecting dendritic-targeting inhibitory neurons.

Cover Picture: Eur. J. Immunol. 1'14

The cover features an immunofluorescent image of an ear explant from a Kit+/+ mouse, which was stained for avidin (green) and laminin (red). The image is taken from the article by Heger et al. (pp. 296‐306) in which the authors describe the generation of KitCreERT2/+ mice by the introduction of a tamoxifen‐inducible version of the Cre recombinase within the c‐Kit locus. The authors also show that crossing the KitCreERT2/+ mouse with the R26 strain, in which expression of diphtheria toxin A (DTA) depends on removal of a loxP‐flanked STOP cassette, allows selective and effective ablation of mast cells at precise anatomical sites via DTA expression. The KitCreERT2/+ mouse would be a valuable tool in the further study of mast cell development, homeostasis and function.

New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates

Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

In vivo optical signatures of neuronal death in a mouse model of Alzheimer's disease

BackgroundThere currently is a need for cost-effective, quantitative techniques to evaluate the gradual progression of Alzheimer's disease (AD). Measurement techniques based on diffuse optical spectroscopy can detect blood perfusion and brain cellular composition changes, through measuring the absorption (µa) and reduced scattering (µs′) coefficients, respectively, using non-ionizing near-infrared light. Previous work has shown that brain perfusion deficits in an AD mouse model can be detected. The objective of this study was to determine if µs′ is sensitive to the inflammation and neuron death found in AD.MethodsWe used spatial frequency domain imaging (SFDI) to form quantitative maps of µa and µs′ in 3-month old male CaM/Tet-DTA mice harboring transgenes for the doxycyline-regulated neuronal expression of diphtheria toxin. When doxycycline is removed from the diet, CaM/Tet-DTA mice develop progressive neuronal loss in forebrain neurons. Mice (n = 5) were imaged longitudinally immediately prior to and after 23 days of lesion induction, and µa and µs′ (30 wavelengths, 650–970 nm) were compared to properties obtained from Tet-DTA controls (n = 5). Neuron death and infiltration of inflammatory cells in brain cortical slices was confirmed with immunohistochemistry.ResultsNo significant difference in baseline scattering and absorption were measured between CaM/Tet-DTA mice and controls. After 23 days of lesion induction, brain cortical µs′ was 11–16% higher in the CaM/Tet-DTA mice than in controls (P < 0.03). Longitudinal imaging showed no significant difference in µs′ between the first and 23rd day of imaging in controls. Removing doxycycline from the diet was associated with a significant decrease in total hemoglobin concentrations (119 ± 9 µM vs. 91 ± 8 µM) (P < 0.05) in controls, but not in CaM/Tet-DTA mice.ConclusionsNeuronal death and brain inflammation are associated with increased tissue scattering (µs′) and this optical biomarker may be useful in pre-clinical AD therapy evaluation or monitoring of disease progression in AD patients.Lasers Surg. Med. 46:27–33, 2014. © 2013 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Perforin Positive Regulatory DCs In Immune Tolerance: Novel Anti-Inflammatory Players In Metabolic Syndrome

Immature dendritic cells (imDCs) can have a tolerizing effect in the steady state or following transplantation. However, due to the significant heterogeneity of this cell population it is difficult to study the mechanisms of their tolerance induction. We previously described the generation of a highly defined population of imDCs expressing perforin and granzyme A (Perf-DCs) from hematopoietic progenitors; using TCR transgenic T cells we monitored their ability to delete cognate CD4 and CD8 T cells. While the former are deleted via an MHC-independent mechanism through the nitric oxide system, CD8+ T cell deletion occurs through a unique MHC-dependent perforin-based killing mechanism. This involves activation of Toll-like receptors 7, and signaling through Triggering Receptor Expressed on Myeloid cells -1. Importantly, this novel subpopulation of Perf-DCs was also detected in various lymphoid tissues in normal animals, and its frequency is markedly enhanced upon GM-CSF administration (Zangi et al, Blood 2012).Here, we investigated the potential regulatory role of Perf-DCs in steady state in-vivo by selectively knocking out the expression of perforin in these cells. To this end, we generated BM chimeras using a 1:1 mixture of BM from perforin KO mice and from BM of mice ablated of CD11chigh DCs using diphtheria toxin expression under the CD11c promoter (Birnberg et al, Immunity 2008). In the resulting PKO-DTA chimeras, perforin expression was completely lost in conventional CD11c+ DCs, while 50% of the T and NK cell populations still expressed perforin. At 6 months post transplant, DTA-PKO chimeric mice spontaneously gained more weight than chimeras created using a mixture of normal BM with BM from perforin KO mice (WT-PKO). The increased weight gain observed in DTA-PKO mice prompted us to test whether this phenomenon was accompanied by other metabolic alterations. Indeed, DTA-PKO mice exhibited elevated serum cholesterol and triglyceride levels compared to control WT-PKO chimeras (140±3.5 vs. 115±8.6, 125±31vs. 88±9.8 mg/dl, N≥5). Total body fat percent as measured by body composition MRI was significantly higher in DTA-PKO mice (30.3%±2.2 vs. 14.5%±2.3), along with highly elevated levels of leptin (37±10.5 vs. 9.8±3 ng/ml).In addition, DTA-PKO chimeric mice exhibited glucose intolerance (p=0.034) and reduced insulin sensitivity (p=6.07x10-6). Immunohistological evaluation revealed a significant reduction in the percentage of insulin expressing pancreatic β cell- tissue (2.2%±0.54 vs. 5.75%±1.98). Importantly, the visceral adipose tissue (VAT) of DTA-PKO chimeras contained more crown-like structures typically formed when macrophages within inflamed AT surround dead adipocytes.Based on these characteristics of metabolic syndrome that develop in DTA-PKO chimeras over 6 months, we tested whether high-fat diet (HFD) enhances the rate of disease development. Indeed, DTA-PKO chimeras maintained on HFD displayed more pronounced weight gain compared to their HFD-maintained WT counterparts when tested 6 weeks after HFD initiation. Likewise, cholesterol and triglycerides as well as leptin and IL-1b in the serum, and TNF-α and IL-6 in the AT were elevated in DTA-PKO mice compared to the WT-PKO animals. Importantly, analysis of immune cell populations in collagenase-digested VAT revealed more CD8+ and CD4+ T cells in DTA-PKO mice compared to control chimeras (78.3x103±17.5x103 vs. 24.9x103±3.2x103 and 113x103±21x103 vs. 43x103±4.4x103respectively). Thus, triggering of inflammation in the AT previously shown to be mediated by T cells (Winer et al, Nat.Med 2009; Nishimura et al, Nat.Med 2009), is not effectively regulated in mice lacking Perf-DCs. Interestingly, a similar enhanced rate of metabolic imbalance was found in regular diet-fed DTA-PKO chimeras using RIP-mOVA mice expressing ovalbumin in the thymus, pancreas and kidneys, and known to be more prone to autoimmunity. Moreover, a significantly larger fraction of dividing cells was observed when CD8 T cells, isolated from AT of DTA-PKO chimeric RIP-mOVA mice were stimulated against splenocytes of mice expressing ovalbumin in all tissues (wOVA mice). Taken together, our results demonstrate that Perf-DCs have a unique immune regulatory role under steady state, controlling unwanted inflammatory processes in adipose tissue. Further studies of the role of Perf-DCs in other models of autoimmunity are warranted.▪ Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Microbiological changes and diversity in autochthonous non-toxigenic Corynebacterium diphtheriae isolated in France

AbstractAutochtonous toxigenic Corynebacterium diphtheriae have disappeared in mainland France, but non-toxigenic C. diphtheriae are still circulating. Using phenotypic and molecular tools, we retrospectively characterized 103 non-toxigenic C. diphtheriae collected in mainland France and highlight several changes. The proportion of C. diphtheriae belfanti increased between 1977 and 2011 and it is the most frequent biotype recovered in recent years. Resistance to ciprofloxacin has increased and most isolates with decreased sensitivity belong to the belfanti biotype. Using multilocus sequence typing, we demonstrate that French isolates are distributed in a large number of sequence types and identify three distinct lineages. C. diphtheriae mitis and gravis form lineage I while C. diphtheriae belfanti forms lineages II and III. Almost all isolates of lineage II are part of a unique clonal complex or are very close to it. Most French isolates have a dtxR sequence homologous to that of toxigenic isolates, suggesting that if lyzogenised by a corynephage, they can express diphtheria toxin.

Abstract 23: Foxd1+ Stromal Cells, the Required Progenitors for Kidney Vascular Development

The mechanisms underlying the establishment, assembly and maintenance of the renal blood vessels are poorly understood. We have previously suggested using detailed lineage tracing that renal stromal cells, characterized by their early and transient expression of the transcription factor Foxd1 , give rise to the entirety of the mural cell layer of the renal arterial tree and mesangial cells. Mural cells as defined here exclude endothelial cells, which we identified as having a separate precursor, the renal hemangioblast. To define whether Foxd1 cells are the required essential progenitor or whether their role as such could be assumed by other cell types, we used the cre-lox system to generate mice expressing diphtheria toxin subunit A in Foxd1+ cells ( Foxd1-DTA mice ) which resulted in animals with selective ablation of Foxd1+ cells. Kidneys from Foxd1-DTA embryos had a significantly reduced complement of arterial mural cells, lacking smooth muscle cells, perivascular fibroblasts, renin cells and mesangial cells. Interestingly, the few vessels that remained were also abnormal: they originated underneath the kidney capsule and elongated towards the center of the kidney rather than radiating outward from the center of the kidney. In addition, ablation of Foxd1 cells resulted in significantly delayed nephrogenesis and reduction in glomerular number. In conjunction with our previous data showing a similar phenotype upon global deletion of the Foxd1 ,gene, this illustrates the central role of Foxd1 and the cells that express it during early development. We conclude that Foxd1 stromal cells are the required progenitors for the establishment of the mural cells of the kidney arterioles and (via Foxd1 expression) for the proper origin and orientation of the kidney vessels.

165 : Critical regulation of type 2 innate lymphoid cells by mast cells during allergic airway inflammation

Mast cells and basophils mediate tissue inflammation during allergy and anaphylaxis by secreting granules and cytokines; however, their role in the development of Th2 cells and type 2 innate lymphoid cells (ILC2) is not fully understood. Bas-TRECK and Mas-TRECK mice have been recently described to express diphtheria toxin receptor exclusively on basophils, or on both basophils and mast cells, respectively. Here, by using these genetic tools, we show that depletion of basophil minimally affected the size of pulmonary Th2 cells in response to fungal protease allergens, whereas additional depletion of mast cells almost completely abolished pulmonary Th2 responses as well as infiltration of inflammatory cells into airway. Of note, diphtheria toxin-treated Mas-TRECK mice exhibited a significant decrease in the frequency and number of Lin−CD45+CD127+ IL-13-producing ILC2 in the lung compared to wild-type and Bas-TRECK mice that received same treatment. Moreover, lungs of the toxin-treated Mas-TRECK mice showed significantly decreased expression of Il33 transcript than those of wild-type. Our findings together unveil a critical contribution of mast cells rather than basophils to the induction of ILC2 as well as Th2 cells in the airway in response to fungal allergen in vivo.

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Conditional ablation of Langerhans cells exacerbates autoimmune corneal inflammation (P5134)

Abstract Ocular surface inflammation is common and sometimes severe in autoimmune diseases. Little is known about the role of cornea-resident dendritic cells, including Langerhans cells (LC), in ocular surface autoimmunity. Here, we analyzed corneal LC in autoimmune-prone MRL mice. LC (CD11c+ CD207+) were more abundant and activated (CD86+ CD40+) in the corneal epithelium of MRL mice than of B6 mice. However, LC were ~5-fold lower in the corneal stroma of MRL mice than of B6 mice. LC in cervical lymph nodes were also lower in MRL mice than in B6 mice. Ongoing studies will examine whether the increase of activated LC in corneal epithelium, but their reduction in corneal stroma and eye-draining lymph nodes in autoimmune mice is due to a defect in the migration of LC from the cornea to eye-draining lymph nodes. Since LC are believed to carry antigens from tissues to their respective draining lymph nodes to maintain tolerance in a tissue-specific manner, the reduced LC in eye-draining lymph nodes may lead to the breakdown of tolerance to eye antigens. To directly test the role of LC in ocular autoimmunity, we introgressed the Lang-eGFP.DTR knockin mutation from the stock B6 mice that express diphtheria toxin receptor (DTR) driven by Langerin promoter (Malissen, 2005) onto the MRL background. Diphtheria toxin injections depleted LC in Lang-eGFP.DTR MRL mice and accelerated corneal inflammation. Taken together, these data suggest a protective role of LC in corneal inflammation.

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.