Expression Of Diphtheria Toxin Research Articles

Tat-dependent conditionally replicating adenoviruses expressing diphtheria toxin A for specifically killing HIV-1-infected cells

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effect and drive the emergence of drug resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1 infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1 infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1 infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1 negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. Findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

The Evaluation of the N-cadherin Promoter's ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines.

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-β and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and β-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of β-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-β in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus.

Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.

Genomic Epidemiology of Corynebacterium diphtheriae in New Caledonia.

An increasing number of isolations of Corynebacterium diphtheriae has been observed in recent years in the archipelago of New Caledonia. We aimed to analyze the clinical and microbiological features of samples with C. diphtheriae. All C. diphtheriae isolates identified in New Caledonia from May 2015 to May 2019 were included. For each case, a retrospective consultation of the patient files was conducted. Antimicrobial susceptibility phenotypes, tox gene and diphtheria toxin expression, biovar, and the genomic sequence were determined. Core genome multilocus sequence typing (cgMLST), 7-gene MLST, and search of genes of interest were performed from genomic assemblies. Fifty-eight isolates were included, with a median age of patients of 28 years (range: 9 days to 78 years). Cutaneous origin accounted for 51 of 58 (87.9%) isolates, and C. diphtheriae was associated with Staphylococcus aureus and/or Streptococcus pyogenes in three-quarters of cases. Half of cases came either from the main city Noumea (24%, 14/58) or from the sparsely populated island of Lifou (26%, 15/58). Six tox-positive isolates were identified, associated with recent travel to Vanuatu; 5 of these cases were linked and cgMLST confirmed recent transmission. Two cases of endocarditis in young female patients with a history of rheumatic fever involved tox-negative isolates. The 58 isolates were mostly susceptible to commonly used antibiotics. In particular, no isolate was resistant to the first-line molecules amoxicillin or erythromycin. Resistance to tetracycline was found in a genomic cluster of 17 (29%) isolates, 16 of which carried the tetO gene. There were 13 cgMLST sublineages, most of which were also observed in the neighboring country Australia. Cutaneous infections may harbor nontoxigenic C. diphtheriae isolates, which circulate largely silently in nonspecific wounds. The possible introduction of tox-positive strains from a neighboring island illustrates that diphtheria surveillance should be maintained in New Caledonia, and that immunization in neighboring islands must be improved. Genomic sequencing uncovers how genotypes circulate locally and across neighboring countries. IMPORTANCE The analysis of C. diphtheriae from the tropical archipelago of New Caledonia revealed a high genetic diversity with sublineages that may be linked to Polynesia, Australia, or metropolitan France. Genomic typing allowed confirming or excluding suspected transmission events among cases and contacts. A highly prevalent tetracycline-resistant sublineage harboring the tetO gene was uncovered. Toxigenic isolates were observed from patients returning from Vanuatu, showing the importance of improving vaccination coverage in settings where it is insufficient. This study also illustrates the importance for diphtheria surveillance of the inclusion of isolates from cutaneous sources in addition to respiratory cases, in order to provide a more complete epidemiological picture of the diversity and transmission of C. diphtheriae.

OFF Starburst Amacrine Cells in the Retina Trigger Looming-evoked Fear Responses in Mice.

A rapidly approaching dark object evokes an evolutionarily conserved fear response in both vertebrates and invertebrates, young to old. A looming visual stimulus mimics an approaching object and triggers a similarly robust fear response in mice, resulting in freeze and flight. However, the retinal neural pathway responsible for this innate response has not been fully understood. We first explored a variety of visual stimuli that reliably induced these innate responses, and found that a looming stimulus with two-day acclimation consistently evoked fear responses. Because the fear responses were triggered by the looming stimulus with moving edges, but not by a screen flipping from light to dark, we targeted the starburst amacrine cells (SACs), crucial neurons for retinal motion detection. We utilized intraocular injection of diphtheria toxin (DT) in mutant mice expressing diphtheria toxin receptors (DTR) in SACs. The looming-evoked fear responses disappeared in half of the DT-injected mice, and the other mice still exhibited the fear responses. The optomotor responses were reduced or eliminated, which occurred independent of the disappearance of the fear responses. A histological examination revealed that ON SACs were reduced in both mouse groups preserved or absent fear responses. In contrast, the number of OFF SACs was different among two groups. The OFF SACs were relatively preserved in mice exhibiting continued fear responses, whereas they were ablated in mice lacking fear response to looming stimulation. These results indicate that OFF SACs and the direction-selective pathway in the retina play a role in looming-induced fear behaviors.Significance StatementIn response to a suddenly approaching dark object, mice exhibit a defensive response: either flight to a refuge or freeze in the same spot. How the visual system evokes this response in the mouse has not been fully understood. We focused on the initial neural component of the visual system, the retina, and examined a type of neuron known for sensing object motion: the starburst amacrine cell. We found that ablation of OFF starburst amacrine cells removed the dark object-evoked defensive responses. We believe that our findings will contribute to understanding the neural network connecting the visual system and the brain circuit regarding fear and emotion.

Spatiotemporally controllable diphtherin transgene system and neoantigen immunotherapy.

Individualized immunotherapy has attracted great attention due to its high specificity, effectiveness, and safety. We used an exogenous antigen to label tumor cells with MHC I molecules, which allowed neoantigen-specific T cells to recognize and kill tumor cells. A neoantigen vaccine alone cannot achieve complete tumor clearance due to a tumor immunosuppressive microenvironment. The LightOn system was developed to effectively eliminate tumor cells through the spatiotemporally controllable expression of diphtheria toxin A fragment, leading to antigen release in the tumor region. These antigens stimulated and enhanced immunological function and thus, recruited neoantigen-specific T cells to infiltrate tumor tissue. Using the nanoparticle delivery system, neoantigens produced higher delivery efficiency to lymph nodes and improved tumor targeting ability for tumor cell labelling. Good tumor inhibition and prolonged survival were achieved, while eliciting a strong immune response. The combination of a spatiotemporally controllable transgene system with tumor neoantigen labeling has great potential for tumor immunotherapy.

Analysis of Cre lines for targeting embryonic airway smooth muscle

During development of the embryonic mouse lung, the pulmonary mesenchyme differentiates into smooth muscle that wraps around the airway epithelium. Inhibiting smooth muscle differentiation leads to cystic airways, while enhancing it stunts epithelial branching. These findings support a conceptual model wherein the differentiation of smooth muscle sculpts the growing epithelium into branches at precise positions and with stereotyped morphologies. Unfortunately, most approaches to manipulate the differentiation of airway smooth muscle rely on pharmacological or physical perturbations that are conducted ex vivo. Here, we explored the use of diphtheria toxin-based genetic ablation strategies to eliminate airway smooth muscle in the embryonic mouse lung. Surprisingly, neither airway smooth muscle wrapping nor epithelial branching were affected in embryos in which the expression of diphtheria toxin or its receptor were driven by several different smooth muscle-specific Cre lines. Close examination of spatial patterns of Cre activity in the embryonic lung revealed that none of these commonly used Cre lines target embryonic airway smooth muscle robustly or specifically. Our findings demonstrate the need for airway smooth muscle-specific Cre lines that are active in the embryonic lung, and serve as a resource for researchers contemplating the use of these commonly used Cre lines for studying embryonic airway smooth muscle.

Constitutive bone marrow adipocytes suppress local bone formation.

BM adipocytes (BMAd) are a unique cell population derived from BM mesenchymal progenitors and marrow adipogenic lineage precursors. Although they have long been considered to be a space filler within bone cavities, recent studies have revealed important physiological roles in hematopoiesis and bone metabolism. To date, the approaches used to study BMAd function have been confounded by contributions by nonmarrow adipocytes or by BM stromal cells. To address this gap in the field, we have developed a BMAd-specific Cre mouse model to deplete BMAds by expression of diphtheria toxin A (DTA) or by deletion of peroxisome proliferator-activated receptor gamma (Pparg). We found that DTA-induced loss of BMAds results in decreased hematopoietic stem and progenitor cell numbers and increased bone mass in BMAd-enriched locations, including the distal tibiae and caudal vertebrae. Elevated bone mass appears to be secondary to enhanced endosteal bone formation, suggesting a local effect caused by depletion of BMAd. Augmented bone formation with BMAd depletion protects mice from bone loss induced by caloric restriction or ovariectomy, and it facilitates the bone-healing process after fracture. Finally, ablation of Pparg also reduces BMAd numbers and largely recapitulates high-bone mass phenotypes observed with DTA-induced BMAd depletion.

New Corynebacterium Species with the Potential to Produce Diphtheria Toxin

Only three Corynebacterium species are known to produce a lethal exotoxin called diphtheria toxin. These are C. diphtheriae, C. ulcerans and C. pseudotuberculosis. The diphtheria toxin gene (tox) is carried in a family of closely related corynebacteriophages and therefore the toxin can be produced only through lysogenisation, in which the corynephage encoding tox is stably inserted into the chromosome. However, 'nontoxigenic tox gene-bearing' (NTTB) strains, which are genotypically tox-positive but do not express the protein, have been described. The emergence of NTTB strains was first observed during the 1990s diphtheria epidemic in Eastern Europe and nowadays such isolates have been detected in many countries in the world. Recently, novel species of Corynebacterium genus have been described which might have the potential of producing the diphtheria toxin due to the possession of the diphtheria toxin gene but it has not produced toxin in laboratory tests. The circulation of NTTB strains could be related to the increased risk for diphtheria disease arising from the risk of re-emerging toxin expression. The article presents the mechanism of diphtheria toxin expression and action, recently described novel species of NTTB corynebacteria as well as the taxonomic changes within the C. diphtheriae group.

Abstract 090: Cells Of The Renin Lineage Promote Kidney Regeneration Post - Release Of Ureteral Obstruction In Neonatal Mice.

Renin, the rate-limiting enzyme of the renin-angiotensin system, is increased in the kidneys subjected to ureteral obstruction. We elucidated the changes and contribution of renin-producing cells (RPCs) and the cells of renin lineage (CoRL) during kidney damage and regeneration using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice. Renin cells were ablated by cell-specific expression of diphtheria toxin A chain (DTA) and labeled with GFP. We compared the cell fate changes between kidneys from the wild-type (WT) and the DTA-expressing animals, using the pUUO model. The kidneys of 3W old DTA-positive Ren1 dDTA/+ ;R26R DTA/+ and Ren1 dCre/+ ;R26R DTA/+ genotypes were grossly normal. We observed no significant differences in the Myh11-positive renal vascular area between the WT and the DTA-positive mice (WT-3W: 1.72±0.10 % (n=6), Ren1 dDTA/+ ;R26R DTA/+ -3W: 1.94 ±0.123 % (n=7), Ren1 dCre/+ ;R26R DTA/+ -3W: 2.017±0.16% (n=4)). In the kidneys of the WT group, RPCs and the CoRL significantly increased at 3W post-obstruction and the distribution was similar to the sham-operated kidneys at 2W post-relief of obstruction (Renin-positive area-WT: Sham-3W: 0.57±0.02% (n=6), Obstructed-3W: 1.59±0.44% (n=4), Post-release-2W: 0.71±0.12% (n=3)). There was a significant decrease in RPCs in the DTA animals, and the reduction of RPCs and CoRL was markedly higher in Ren1 dCre/+ ;R26R DTA/+ mice in comparison to Ren1 dDTA/+ ;R26R DTA/+ group. Also, the DTA-positive animals did not respond to pUUO with a significant increase in renin as in the WT (Renin-positive area: Ren1 dDTA/+ ;R26R DTA/+ Sham-3W: 0.13±0.048% (n=5), Obstructed-3W: 0.23±0.10% (n=5), Post-release-2W: 0.14±0.04% (n=9) Ren1 dCre/+ ;R26R DTA/+ Sham: 0.010±0.004% (n=4), Obstructed: 0.07±0.004% (n=4), Post-release: 0.03±0.004% (n=3)). The renal fibrotic area and Tgfb1 mRNA levels post-obstruction and relief of obstruction were significantly high in Ren1 dCre/+ ;R26R DTA/+ group in comparison to Ren1 dDTA/+ ;R26R DTA/+ genotype due to the depletion of CoRL. Thus, our current study reveals that CoRL undergoes cell fate changes in response to obstruction and the release of obstruction, and these changes play a critical role in promoting kidney regeneration post-relief of obstruction.

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.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation

BackgroundCardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. Methods and resultsWe evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70–80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. ConclusionsThese studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Skeletal Muscle Injury is Sufficient for Angiogenesis but Myofiber Regeneration is Required for Restoring Microvascular Integrity

Acute injury of skeletal muscle damages myofibers, microvessels, and supporting tissue components. To restore the structural and functional integrity of intact muscle, each tissue component must regenerate. In adult skeletal muscle, resident muscle stem cells (satellite cells) efficiently regenerate myofibers within 10 days post injury (dpi) and microvascular integrity recovers by 21 dpi. However, the requirement for crosstalk between regenerating myofibers and microvessels is poorly understood. We tested the hypothesis that myofiber regeneration is necessary for revascularization and restoration of microvascular integrity. To ablate satellite cells and impair myofiber regeneration, we studied male and female mice (age, ~4 mo) in which tamoxifen‐dependent Cre recombination induced the expression of diphtheria toxin in quiescent satellite cells, killing them prior to injury. For acute muscle injury, the gluteus maximus muscle (GM) was injected with BaCl2 (1.2%, 75 µL). Microvascular perfusion and permeability were evaluated by injecting fluorescent dextrans (70 & 155 kDa) into the circulation during intravital imaging of the GM at defined timepoints following injury. Despite the absence of myofiber regeneration, sprouting angiogenesis was robust and capillary perfusion was restored within 7 dpi, as observed in wild‐type mice. However, the permeability barrier of nascent microvessels was compromised as shown by profuse leakage of both fluorescent dextrans. At 21 dpi, the absence of regenerated myofibers was accompanied by sustained inflammation and myosteatosis. Areas of ectopic adipocyte accumulation were hypervascular and localized around venules. Adjacent areas contained empty myofiber “sheaths” which were underperfused; residual microvascular networks exhibited pruning. These findings suggest that, following acute injury of adult skeletal muscle, revascularization occurs in the absence of myogenesis but is defective and nascent vessels are unstable. We conclude that myofiber regeneration is essential to recovering a functionally intact microcirculation following muscle injury.

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).

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.

Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo.Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII.CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively.Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals.We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. DisclosuresNo relevant conflicts of interest to declare.

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

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). 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 in a PTEC‐specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. 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. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Ninjin'yoeito, a traditional Japanese Kampo medicine, suppresses the onset of anhedonia induced by dysfunction in the striatal dopamine receptor type 2-expressing medium spiny neurons.

ObjectiveRecent studies have suggested that ninjin’yoeito (NYT), a traditional Japanese Kampo medicine, improves diminished motivation in humans and animals, rendering it a novel therapeutic option for impaired motivation. To better characterize the effect of NYT on motivation, we examined its effect on motivated behaviors in mice.MethodsMouse models of neurodegeneration-related apathy, in which striatal dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) were progressively damaged by diphtheria toxin expression, were chosen.ResultsThe decrease in effort-based operant responding for rewards (sucrose pellets), indicative of the mouse’s motivated behavior, in the affected mice was not suppressed by chronic treatment with NYT suspended in drinking water at 1% (w/v). Mice were then subjected to a sucrose preference test, wherein they freely chose to ingest tap water and a sucrose solution without being required to exert effort. The affected mice showed a decline in preference for sucrose over tap water, relative to nonaffected controls, indicating anhedonia-like traits. In contrast to the diminished operant behavior, the anhedonic behavior in the affected mice was prevented by the NYT administration. Furthermore, NYT did not affect the size of Drd2 mRNA disappearance in the striatum of affected mice, suggesting that the NYT effect was unrelated to DTA-mediated neurodegeneration.ConclusionThese results demonstrate that the beneficial effect of NYT on motivation is mediated, at least in part, through the potentiation of hedonic capacity by certain neuromodulatory pathways.