Enhancing Human Treg Cell Induction through Engineered Dendritic Cells and Zinc Supplementation.

Regulatory T (Treg) cells hold promise for the ultimate cure of immune-mediated diseases. However, how to effectively restore Treg function in patients remains unknown. Previous reports suggest that activated dendritic cells (DCs) de novo synthesize locally high concentrations of 1,25-dihydroxy vitamin D, i.e., the active vitamin D or 1,25(OH)2D by upregulating the expression of 25-hydroxy vitamin D 1α-hydroxylase. Although 1,25(OH)2D has been shown to induce Treg cells, DC-derived 1,25(OH)2D only serves as a checkpoint to ensure well-balanced immune responses. Our animal studies have shown that 1,25(OH)2D requires high concentrations to generate Treg cells, which can cause severe side effects. In addition, our animal studies have also demonstrated that dendritic cells (DCs) overexpressing the 1α-hydroxylase de novo synthesize the effective Treg-inducing 1,25(OH)2D concentrations without causing the primary side effect of hypercalcemia (i.e., high blood calcium levels). This study furthers our previous animal studies and explores the efficacy of the la-hydroxylase-overexpressing DCs in inducing human CD4+FOXP3+regulatory T (Treg) cells. We discovered that the effective Treg-inducing doses of 1,25(OH)2D were within a range. Additionally, our data corroborated that the 1α-hydroxylase-overexpressing DCs synthesized 1,25(OH)2D within this concentration range in vivo, thus facilitating effective Treg cell induction. Moreover, this study demonstrated that 1α-hydroxylase expression levels were pivotal for DCs to induce Treg cells because physiological 25(OH)D levels were sufficient for the engineered but not parental DCs to enhance Treg cell induction. Interestingly, adding non-toxic zinc concentrations significantly augmented the Treg-inducing capacity of the engineered DCs. Our new findings offer a novel therapeutic avenue for immune-mediated human diseases, such as inflammatory bowel disease, type 1 diabetes, and multiple sclerosis, by integrating zinc with the 1α-hydroxylase-overexpressing DCs.

FBP1-Altered Carbohydrate Metabolism Reduces Leukemic Viability through Activating P53 and Modulating the Mitochondrial Quality Control System In Vitro.

Acute myeloid leukemia (AML)-the most frequent form of adult blood cancer-is characterized by heterogeneous mechanisms and disease progression. Developing an effective therapeutic strategy that targets metabolic homeostasis and energy production in immature leukemic cells (blasts) is essential for overcoming relapse and improving the prognosis of AML patients with different subtypes. With respect to metabolic regulation, fructose-1,6-bisphosphatase 1 (FBP1) is a gluconeogenic enzyme that is vital to carbohydrate metabolism, since gluconeogenesis is the central pathway for the production of important metabolites and energy necessary to maintain normal cellular activities. Beyond its catalytic activity, FBP1 inhibits aerobic glycolysis-known as the "Warburg effect"-in cancer cells. Importantly, while downregulation of FBP1 is associated with carcinogenesis in major human organs, restoration of FBP1 in cancer cells promotes apoptosis and prevents disease progression in solid tumors. Recently, our large-scale sequencing analyses revealed FBP1 as a novel inducible therapeutic target among 17,757 vitamin-D-responsive genes in MV4-11 or MOLM-14 blasts in vitro, both of which were derived from AML patients with FLT3 mutations. To investigate FBP1's anti-leukemic function in this study, we generated a new AML cell line through lentiviral overexpression of an FBP1 transgene in vitro (named FBP1-MV4-11). Results showed that FBP1-MV4-11 blasts are more prone to apoptosis than MV4-11 blasts. Mechanistically, FBP1-MV4-11 blasts have significantly increased gene and protein expression of P53, as confirmed by the P53 promoter assay in vitro. However, enhanced cell death and reduced proliferation of FBP1-MV4-11 blasts could be reversed by supplementation with post-glycolytic metabolites in vitro. Additionally, FBP1-MV4-11 blasts were found to have impaired mitochondrial homeostasis through reduced cytochrome c oxidase subunit 2 (COX2 or MT-CO2) and upregulated PTEN-induced kinase (PINK1) expressions. In summary, this is the first in vitro evidence that FBP1-altered carbohydrate metabolism and FBP1-activated P53 can initiate leukemic death by activating mitochondrial reprogramming in AML blasts, supporting the clinical potential of FBP1-based therapies for AML-like cancers.

The importance of vitamin d metabolism as a potential prophylactic, immunoregulatory and neuroprotective treatment for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has led to a declaration of a Public Health Emergency of International Concern by the World Health Organization. As of May 18, 2020, there have been more than 4.7 million cases and over 316,000 deaths worldwide. COVID-19 is caused by a highly infectious novel coronavirus known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leading to an acute infectious disease with mild-to-severe clinical symptoms such as flu-like symptoms, fever, headache, dry cough, muscle pain, loss of smell and taste, increased shortness of breath, bilateral viral pneumonia, conjunctivitis, acute respiratory distress syndromes, respiratory failure, cytokine release syndrome (CRS), sepsis, etc. While physicians and scientists have yet to discover a treatment, it is imperative that we urgently address 2 questions: how to prevent infection in immunologically naive individuals and how to treat severe symptoms such as CRS, acute respiratory failure, and the loss of somatosensation. Previous studies from the 1918 influenza pandemic have suggested vitamin D's non-classical role in reducing lethal pneumonia and case fatality rates. Recent clinical trials also reported that vitamin D supplementation can reduce incidence of acute respiratory infection and the severity of respiratory tract diseases in adults and children. According to our literature search, there are no similar findings of clinical trials that have been published as of July 1st, 2020, in relation to the supplementation of vitamin D in the potential prevention and treatment for COVID-19. In this review, we summarize the potential role of vitamin D extra-renal metabolism in the prevention and treatment of the SARS-CoV-2 infection, helping to bring us slightly closer to fulfilling that goal. We will focus on 3 major topics here: 1. Vitamin D might aid in preventing SARS-CoV-2 infection: Vitamin D: Overview of Renal and Extra-renal metabolism and regulation. Vitamin D: Overview of molecular mechanism and multifaceted functions beyond skeletal homeostasis. Vitamin D: Overview of local immunomodulation in human infectious diseases. Anti-viral infection. Anti-malaria and anti-systemic lupus erythematosus (SLE). 2. Vitamin D might act as a strong immunosuppressant inhibiting cytokine release syndrome in COVID-19: Vitamin D: Suppression of key pro-inflammatory pathways including nuclear factor kappa B (NF-kB), interleukin-6 (IL-6), and tumor necrosis factor (TNF). 3. Vitamin D might prevent loss of neural sensation in COVID-19 by stimulating expression of neurotrophins like Nerve Growth Factor (NGF): Vitamin D: Induction of key neurotrophic factors. .

Vitamin D as a Potential Therapy for Multiple Sclerosis: Where Are We?

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and is caused by an aberrant immune response to myelin sheath. Disease-modifying medications, which mainly aim to suppress such aberrant immune response, have significantly improved MS treatment. However, the disease severity continues to worsen. In contrast, progressively more data suggest that 1,25-dihydroxyvitamin D or 1,25(OH)2D, i.e., the active vitamin D, suppresses the differentiation of potentially pathogenic T cells associated with MS, enhances the differentiation of regulatory T cells that suppress the pathogenic T cells, and promotes remyelination. These novel 1,25(OH)2D functions have encouraged investigators to develop vitamin D as a potential therapy for MS. However, because of the hypercalcemia that is associated with high 1,25(OH)2D concentrations, supplementation of native vitamin D has been a major focus in clinical trials for the treatment of MS, but such trials have produced mixed data. In this article, we will review current progress in the supplementation of different vitamin D forms for the treatment of experimental autoimmune encephalomyelitis (i.e., an MS animal model) as well as MS. Furthermore, we will review alternative strategies that our laboratory and others are pursuing in an attempt to circumvent the hurdles that are hampering the effective use of vitamin D as a potential therapy for MS.

Mechanistic study of the cause of decreased blood 1,25-Dihydroxyvitamin D in sepsis.

BACKGROUND: Vitamin D deficiency, determined by blood levels of 25-hydroxyvitamin D [25(OH) D, i.e. the major vitamin D form in blood], has been shown to associate with all-cause mortalities. We recently demonstrated that blood levels of 1,25-dihydroxyvitamin D [1,25(OH)2D, i.e. the active vitamin D] were significantly lower in non-survivors compared to survivors among sepsis patients. Unexpectedly, despite the well documented roles of 1,25(OH)2D in multiple biological functions such as regulation of immune responses, stimulation of antimicrobials, and maintenance of barrier function, 1,25(OH)2D supplementation failed to improve disease outcomes. These previous findings suggest that, in addition to 1,25(OH)2D deficiency, disorders leading to the 1,25(OH)2D deficiency also contribute to mortality among sepsis patients. Therefore, this study investigated the mechanisms leading to sepsis-associated 1,25(OH)2D deficiency. METHODS: We studied mechanisms known to regulate kidney 25-hydroxylvitamin D 1α-hydroxylase which physiologically catalyzes the conversion of 25(OH) D into 1,25(OH)2D. Such mechanisms included parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), fibroblast growth factor 23 (FGF-23), and kidney function. RESULTS: We demonstrated in both human subjects and mice that sepsis-associated 1,25(OH)2D deficiency could not be overcome by increased production of PTH which stimulates 1α-hydroxylase. Further studies showed that this failure of PTH to maintain blood 1,25(OH)2D levels was associated with decreased blood levels of IGF-1, increased blood levels of FGF-23, and kidney failure. Since the increase in blood levels of FGF-23 is known to associate with kidney failure, we further investigated the mechanisms leading to sepsis-induced decrease in blood levels of IGF-1. Our data showed that blood levels of growth hormone, which stimulates IGF-1 production in liver, were increased but could not overcome the IGF-1 deficiency. Additionally, we found that the inability of growth hormone to restore the IGF-1 deficiency was associated with suppressed expression and signaling of growth hormone receptor in liver. CONCLUSIONS: Because FGF-23 and IGF-1 have multiple biological functions besides their role in regulating kidney 1α-hydroxylase, our data suggest that FGF-23 and IGF-1 are warranted for further investigation as potential agents for the correction of 1,25(OH)2D deficiency and for the improvement of survival among sepsis patients.

Application of vitamin D and vitamin D analogs in acute myelogenous leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant, transformed immature hematopoietic myeloid precursors that have lost their ability to differentiate and proliferate normally. Current treatment for AML requires intensive cytotoxic chemotherapy and results in significant morbidity and mortality, especially in older patients. Effective and better-tolerated treatment is urgently needed. Studies have shown that 1α,25-dihydroxyvitamin D3 (1,25-D3, active VD3) or vitamin D analogs (VDAs) can potently differentiate AML cells in vitro and ex vivo, which led to early clinical trials in AML and myelodysplastic syndrome patients. However, one major limiting factor in the clinical application of active VD3 or VDAs is the supraphysiologic dose required, which results in systemic hypercalcemia. Several important questions (i.e., dosage, method of delivery, metabolism of 1,25-D3 in situ, systemic hypercalcemia, and mechanisms of action of combination treatment) have to be addressed before vitamin D treatment can be applied to the clinical setting. This review focuses on 1,25-D3's mechanism of action in AML, preclinical data, and clinical trial outcomes, with an emphasis on major roadblocks to successful trials and suggestions for future directions.

Characterization of an animal model of pregnancy-induced vitamin D deficiency due to metabolic gene dysregulation.

Vitamin D deficiency has been associated with pregnancy complications such as preeclampsia, gestational diabetes, and recurrent miscarriage. Therefore, we hypothesized differences in vitamin D status between healthy [Sprague-Dawley (SD) and Lewis (LW)] and complicated [Brown Norway (BN)] rat pregnancies. In SD, LW, and BN rats, we analyzed the maternal plasma levels of the vitamin D metabolites 25-OH-D and 1,25-(OH)2-D at prepregnancy, pregnancy, and postpartum. Analysis of the active metabolite 1,25-(OH)2-D showed a twofold increase in pregnant SD and LW rats but a nearly 10-fold decrease in pregnant BN rats compared with nonpregnant controls. BN rats had a pregnancy-dependent upregulation of CYP24a1 expression, a key enzyme that inactivates vitamin D metabolites. In contrast, the maternal renal expression of CYP24a1 in SD and LW rats remained constant throughout pregnancy. Analysis of the vitamin D receptor (VDR) indicated that LW and SD but not BN rats experience a pregnancy-induced 10-fold decrease in maternal renal VDR protein levels. Further analysis of bisulfite-converted and genomic DNA indicated that the observed differences in maternal renal regulation of CYP24a1 during pregnancy and lactation are not due to differences in CYP24a1 promoter methylation or single-nucleotide polymorphisms. Finally, supplementation with 1,25-(OH)2-D significantly improved the reproductive phenotype of BN rats by increasing litter size and maternal-fetal weight outcomes. We conclude that BN rats represent a novel animal model of pregnancy-specific vitamin D deficiency that is linked to pregnancy complications. Vitamin D deficiency in BN rats correlates with maternal renal CYP24a1 upregulation followed by CYP27b1 upregulation.

Prevention of colorectal cancer with vitamin D.

The fact that colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States emphasizes the need for more effective preventive and therapeutic modalities. There is growing evidence that vitamin D may reduce the incidence of CRC. Results of epidemiologic, in vitro, in vivo animal and clinical studies suggest that a low serum vitamin D level may be a serious risk factor for CRC and a high serum vitamin D level may reduce the risk of CRC. On a molecular level, vitamin D suppresses CRC development and growth by affecting cell proliferation, differentiation, apoptosis, and angiogenesis. Vitamin D insufficiency and CRC are common in the elderly population. Vitamin D insufficiency is simple to screen for and treatable with vitamin D supplementation. Serum 25-hydroxyvitamin D (calcidiol) is the best measure of vitamin D status and should be checked routinely for individuals with risk factors for CRC. Maintaining serum concentrations of calcidiol above 32 ng/ml (80 nmol/l) in individuals whose serum calcidiol level is low may help prevent CRC as well as osteoporosis, fractures, infections, and cardiovascular disease. Daily calcidiol intake of 1000 International Units can increase serum vitamin D to sufficient levels in most elderly persons and, based on available data, may substantially lower the incidence of CRC with minimal risks.

A chemical mutagenesis screen to identify modifier genes that interact with growth hormone and TGF-beta signaling pathways.

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as >3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p<0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.

Spontaneous fractures in the mouse mutant sfx are caused by deletion of the gulonolactone oxidase gene, causing vitamin C deficiency.

UNLABELLED: Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULO gene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures. INTRODUCTION: A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age. MATERIALS AND METHODS: Skeletal phenotype of the sfx phenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfx gene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real-time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay. RESULTS: Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfx mice at 5 weeks of age. Histomorphometric analyses of bones from sfx mice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfx gene was fine mapped to a 2 MB region containing approximately 30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase (GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfx phenotype. We established that ascorbic acid deficiency caused by deletion of the GULO gene (38,146-bp region) contributes to fractures and premature death because the sfx phenotype can be corrected in vivo by treating sfx mice with ascorbic acid and because osteoblasts derived from sfx mice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfx mice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several-fold. CONCLUSION: The sfx is a mutation of the GULO gene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.

Expression of a structurally unique osteoclastic protein-tyrosine phosphatase is driven by an alternative intronic, cell type-specific promoter.

An osteoclastic protein-tyrosine phosphatase (PTP-oc), essential for osteoclast activity, shows sequence identity with the intracellular domain of GLEPP1, a renal receptor-like transmembrane PTP. PTP-oc has been assumed to be a truncated variant of GLEPP1, resulting from alternative splicing. However, the 5'-untranslated region sequence of PTP-oc mRNA contains 217 bp from an intron of GLEPP1. There are no splicing acceptor sites at the PTP-oc transcription site. The intronic sequence flanking the 5' end of the PTP-oc transcription start site contains potential promoter elements essential for transcriptional initiation. To test the hypothesis that the PTP-oc gene has an alternative, tissue-specific, intronic promoter, the promoter activity of a 1.3-kb PCR fragment covering the 5'-flanking region of the PTP-oc gene was measured. The putative PTP-oc promoter fragment showed strong promoter activity in U937 cells. Mutation of the putative TATA box within the PTP-oc promoter abolished 60-90% of its promoter activity. The PTP-oc promoter fragment showed strong promoter activity in cells that express PTP-oc (U937 cells and RAW264.7 cells) but not in cells that do not express the enzyme (skin fibroblasts, TE85 cells, and HEK293 cells). These findings strongly support the conclusion that the 1.3-kb intronic fragment contains the tissue-specific, PTP-oc proximal promoter. Deletion and functional analyses indicate that the proximal 5' sequence flanking the TATA box of the PTP-oc contains potential repressor elements. The removal of the putative repressor elements led to the apparent loss of tissue specificity. In summary, we conclude that an intronic promoter within the GLEPP1 gene drives the expression of the PTP-oc in a cell type-specific manner. This GLEPP1/PTP-oc gene system is one of the very few systems in which two important tissue-specific enzymes are derived from the same gene by the use of alternative intronic promoters.

Gene expression between a congenic strain that contains a quantitative trait locus of high bone density from CAST/EiJ and its wild-type strain C57BL/6J.

Peak bone density is an important determining factor of future osteoporosis risk. We previously identified a quantitative trait locus (QTL) that contributes significantly to high bone density on mouse chromosome 1 from a cross between C57BL/6J (B6) and CAST/EiJ (CAST) mouse strains. We then generated a congenic strain, B6.CAST-1T, in which the chromosomal fragment containing this QTL had been transferred from CAST to the B6 background. The congenic mice have a significantly higher bone density than the B6 mice. In this study we performed cDNA microarray analysis to evaluate the gene expression profile that might yield insights into the mechanisms controlling the high bone density by this QTL. This study led to several interesting observations. First, approximately 60% of 8,734 gene accessions on GEM I chips were expressed in the femur of B6 mice. The expression and function of two-thirds of these expressed genes and ESTs have not been documented previously. Second, expression levels of genes related to bone formation were lower in congenic than in B6 mice. These data are consistent with a low bone formation in the congenic mice, a possibility that is confirmed by reduced skeletal alkaline phosphatase activity in serum compared with B6 mice. Third, expression levels of genes that might have negative regulatory action on bone resorption were higher in congenic than in B6 mice. Together these findings suggest that the congenic mice might have a lower bone turnover rate than B6 mice and raise the possibility that the high bone density in the congenic mice could be due to reduced bone resorption rather than increased bone formation.