Vitamin D3 reduces the expression of M1 and M2 macrophage markers in breast cancer patients.

Vitamin D3 (VD) is known for its immunomodulatory and anticancer effects. This study aimed to characterize tumor-associated macrophages (TAMs) in breast cancer (BC) and assess the influence of VD and its active metabolite, calcitriol, on their polarization. TAMs were isolated from BC patients and characterized. Monocytes were differentiated into macrophage classes (M0, M1, M2a, M2c) and treated ex vivo with calcitriol. The expression of VD-related proteins in tumor tissue was correlated with TAMs and monocyte-derived macrophages (MDMs) characteristics. TAM expression of CD200R, CD204, CD80, HLA-DR, and CD44 was negatively correlated with CYP27B1 in selected patient groups. Patients with high CYP27B1 tumor expression showed significantly lower CD200R, CD204, and CD44 expression. In patients with normal VD levels and premenopausal, CD80 expression in M2a and M2c MDMs (control, untreated ex vivo with calcitriol) was negatively correlated with plasma VD. Calcitriol reduced HLA-DR during MDM differentiation in all patients; CD80 decrease significantly except in patients with normal VD levels or metastasis. Calcitriol also decreased CD163 expression. The decrease in both M1 and M2 macrophage markers by calcitriol or their negative correlation with CYP27B1 indicate the modulatory, but rather anticancer activity of VD. The intensity of these effects was the strongest in postmenopausal patients and those without metastases.

Skewed epithelial cell differentiation and premature aging of the thymus in the absence of vitamin D signaling.

Central tolerance of thymocytes to self-antigen depends on the medullary thymic epithelial cell (mTEC) transcription factor autoimmune regulator (Aire), which drives tissue-restricted antigen (TRA) gene expression. Vitamin D signaling regulates Aire and TRA expression in mTECs, providing a basis for links between vitamin D deficiency and autoimmunity. We find that mice lacking Cyp27b1, which cannot produce hormonally active vitamin D, display profoundly reduced thymic cellularity, with a reduced proportion of Aire+ mTECs, attenuated TRA expression, and poorly defined cortical-medullary boundaries. Markers of T cell negative selection are diminished, and organ-specific autoantibodies are present in knockout (KO) mice. Single-cell RNA sequencing revealed that loss of Cyp27b1 skews mTEC differentiation toward Ccl21+ intertypical TECs and generates a gene expression profile consistent with premature aging. KO thymi display accelerated involution and reduced expression of thymic longevity factors. Thus, loss of thymic vitamin D signaling disrupts normal mTEC differentiation and function and accelerates thymic aging.

Effects of High-Dose Vitamin D Supplementation on Placental Vitamin D Metabolism and Neonatal Vitamin D Status.

Vitamin D (vitD) deficiency (25-hydroxy-vitamin D < 50 nmol/L) is common in pregnancy and associated with an increased risk of adverse pregnancy outcomes. High-dose vitD supplementation is suggested to improve pregnancy health, but there is limited knowledge about the effects on placental vitD transport and metabolism and the vitD status of newborns. Comparing the current standard maternal supplementation, 10 µg/day to a 90 µg vitD supplement, we investigated placental gene expression, maternal vitD transport and neonatal vitD status. Biological material was obtained from pregnant women randomized to 10 µg or 90 µg vitD supplements from week 11-16 onwards. Possible associations between maternal exposure, neonatal vitD status and placental expression of the vitD receptor (VDR), the transporters (Cubilin, CUBN and Megalin, LRP2) and the vitD-activating and -degrading enzymes (CYP24A1, CYP27B1) were investigated. Maternal vitD-binding protein (VDBP) was determined before and after supplementation. Overall, 51% of neonates in the 10 µg vitD group were vitD-deficient in contrast to 11% in the 90 µg group. High-dose vitD supplementation did not significantly affect VDBP or placental gene expression. However, the descriptive analyses indicate that maternal obesity may lead to the differential expression of CUBN, CYP24A1 and CYP27B1 and a changed VDBP response. High-dose vitD improves neonatal vitD status without affecting placental vitD regulation.

Exploring the role of vitamin D-VDR pathway in pemphigus foliaceous: a novel perspective on disease pathogenesis.

Several auto-immune diseases have been linked to vitamin D deficiency as a contributing environmental factor. Its pleiotropic effects on the immune system, especially its essential role in maintaining immune tolerance, make the vitamin D pathway of great interest. In this study, we focused on Pemphigus foliaceous (PF) in Tunisian population. we aimed to quantify the Serum 25[OH]D levels using chemiluminescence assay and to analyze the differential expression of the VDR, CYP27B1 and CYP24A1 genes in the circulating blood cells and lesional skin tissue of PF patients using Q-PCR. A genetic explanation was then sought to explore any direct relationship between tag polymorphisms and the inherited features of PF. Results confirmed a vitamin D hypovitaminosis in Tunisian PF patients. Interestingly, a differential gene expression correlated to the disease stratification was noted. Indeed, at the systemic level, an upregulation of VDR and CYP27B1 genes was observed in healthy controls compared to PF patients. Notably, in lesional skin tissue, the clinical and serological remission phase was correlated with high transcriptional levels of the VDR gene and conversely a drop in expression of the CYP24A1 gene. Genetic analysis indicated the involvement of the most appealing polymorphisms, rs2228570 and poly (A) microsatellite, in PF etiopathogenesis. Indeed, CAC13 haplotype was associated with a higher risk of PF development. Our findings suggest that alterations in the vitamin D-VDR pathway may influence PF physiopathology, making this pathway a potential target for pharmacological modulation, especially for cortico-resistant PF patients.

Unraveling the role of the vitamin D-VDR pathway in pemphigus vulgaris from Tunisian patients.

Vitamin D dysregulation has been recognized as a factor that may cause or aggravate autoimmunity. Vitamin D deficiency was found to be common in pemphigus vulgaris (PV) in different populations. This study aimed to investigate the vitamin D-VDR pathway in PV in the Tunisian population. A serological study was carried out to determine the vitamin D status in newly diagnosed PV patients. CYP27B1, CYP24A1 and VDR mRNA expression was assessed using quantitative real-time PCR in peripheral blood mononuclear cells (PBMC) from untreated newly diagnosed and treated PV patients. In addition, a genetic study was accomplished on VDR polymorphisms to investigate the changes in VDR gene expression. Overall, the serological study confirmed the hypovitaminosis D in newly diagnosed PV patients. Vitamin D-VDR pathway gene expression showed downregulation of CYP27B1 and CYP24A1 mRNA in first-discovery patients compared to healthy controls, while VDR mRNA was highly expressed in newly diagnosed PV patients. Moreover, CYP27B1, CYP24A1 and VDR mRNA were significantly upregulated in chronic disease severity groups compared to mild disease groups. The genetic study showed low VDR gene expression in carriers of FokI > CC genotype, which was more frequent among PV patients, and FokI > C-TaqI > C-ApaI > A-polyA > A16 haplotype, suggesting that the VDR gene polymorphisms testing can provide useful information for PV treatment decision-making. In conclusion, our findings underline the impact of vitamin D-VDR pathway disruption in the PV pathophysiology in Tunisian patients.

Association of CYP27B1 gene polymorphisms with pulmonary tuberculosis and vitamin D levels.

BACKGROUND AND OBJECTIVES: Genetic factors are reported to be connected with tuberculosis (TB) infection. Studies have shown that genetic variations in genes involved in the vitamin D pathway influence the levels of vitamin D found in the bloodstream (serum). Cyp27b1 (1α-hydroxylase) is an enzyme that activates the synthesis of bioactive vitamin D3 by hydroxylation of 25(OH)D3.The in vitro studies reported rare gene variants of Cyp27b1 such as rs118204011 and rs118204012, associated with loss of Cyp27b1 function and lower serum vitamin D levels. Globally, a critical gap exists in understanding the link between these gene variants with TB and vitamin D levels. Hence, the study objective is to comprehend the association of Cyp27b1 rs118204009 (G/A), rs118204011 (C/T), and rs118204012 (A/G) with tuberculosis susceptibility/protection and to assess the influence of gene variants on vitamin D levels in both healthy controls (HCs) and those with pulmonary tuberculosis (PTB) in South India. METHODS: Genomic DNA extraction was performed by salting-out procedure and subsequently genotyped through polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Vitamin D level was measured by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: In rs118204012 (A/G), a substantial association was found with PTB susceptibility in allele 'A' [Odds Ratio (OR): 1.52 (1.02-2.26); p = 0.044] and 'AA' genotype [OR: 1.69 (1.02-2.81); p = 0.040] through the dominant model. Allele 'G' [OR: 0.66 (0.44-0.98); p = 0.044) was found to be associated with protection against TB. Males were associated with increased susceptibility towards TB compared to females in the rs118204011 "CC" [OR: 3.94 (1.94-7.98); p = 0.002] and rs118204012 'AA' [OR: 4.57 (2.13-9.79); p = 0.0001] genotypes. Vitamin D insufficiency (<30 ng/ml) was more prevalent in PTB patients (66.67 %) with the rs118201012 'AA' genotype compared with healthy controls (57.14 %). This genotype was associated with disease susceptible odds ratio of 1.5. CONCLUSION: Cyp27b1 rs118204012 'AA' genotype was found to have association with vitamin D insufficiency and TB susceptibility. In terms of gender, our findings suggest that male individuals are correlated with a higher TB risk. This suggest that the gene variants may be involved in the downstream processing of serum Vitamin D levels and its association with the disease.

Siblings with vitamin D-dependent rickets type 1A: Importance of genetic testing and a review of genotype-phenotype correlations.

Vitamin D-dependent rickets type 1A (VDDR1A) is a rare condition caused by biallelic pathogenic variants in CYP27B1, which encodes 25-hydroxyvitamin D3-1-α-hydroxylase. Inadequate activity of this enzyme results in deficient 1α-hydroxylation of inactive 25-hydroxyvitamin D to biologically active 1,25-dihydroxyvitamin D, with consequent adverse effects on calcium and phosphate metabolism. A female child was clinically diagnosed at 18 months old with hypophosphatemic rickets based on phenotype and biochemical testing, with neither parent affected. A subsequent affected male sibling led to the reconsideration of the diagnosis. Exome sequencing showed a homozygous CYP27B1 c.1040T>A (p.Ile347Asn) variant for both children. No variants were found in genes associated with hypophosphatemic rickets. A review of published cases of VDDR1A with homozygous CYP27B1 variants indicates variable clinical presentation, lack of genotype-phenotype correlation, and low serum phosphate at diagnosis in most cases. These findings emphasize the clinical importance of molecular testing as part of the diagnostic evaluation for cases of non-nutritional rickets.

Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging.

The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in nonrenal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these 2 sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in nonrenal tissues (unlike global Cyp27b1-KO) but no expression within the kidney. Here, utilizing on-tissue chemical derivatization and matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in nonrenal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this, we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate nonrenal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and nonrenal 1,25(OH)2D3 production in cytokine changes.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Vitamin D Metabolites Before and After Kidney Transplantation in Patients Who Are Anephric.

RATIONALE & OBJECTIVE: Kidneys are vital for vitamin D metabolism, and disruptions in both production and catabolism occur in chronic kidney disease. Although vitamin D activation occurs in numerous tissues, the kidneys are the most relevant source of circulating active vitamin D. This study investigates extrarenal vitamin D activation and the impact of kidney transplantation on vitamin D metabolism in patients who are anephric. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Adult patients with previous bilateral nephrectomy (anephric) not receiving active vitamin D therapy evaluated at the time of (N=38) and 1 year after (n=25) kidney transplantation. ANALYTICAL APPROACH: Chromatography with tandem mass spectrometry was used to measure vitamin D metabolites. Activity of CYP24A1 [24,25(OH)2D/25(OH)D] and CYP27B1 [1α,25(OH)2D/25(OH)D] is expressed as metabolic ratios. Differences between time points were evaluated by paired t-test or Wilcoxon matched-pairs signed-rank test. RESULTS: At time of transplantation, 1α,25(OH)2D was detectable in all patients (4-36pg/mL). There was a linear relationship between 25(OH)D and 1α,25(OH)2D levels (r=0.58, P<0.001), with 25(OH)D explaining 34% of the variation in 1α,25(OH)2D levels. There were no associations between 1α,25(OH)2D and biointact parathyroid hormone (PTH) or fibroblast growth factor 23 (FGF-23). One year after transplantation, 1α,25(OH)2D levels recovered (+205%), and CYP27B1 activity increased (+352%). Measures of vitamin D catabolism, 24,25(OH)2D and CYP24A1 activity increased 3- to 5-fold. Also, at 12 months after transplantation, 1α,25(OH)2D was positively correlated with PTH (ρ=0.603, P=0.04) but not with levels of 25(OH)D or FGF-23. LIMITATIONS: Retrospective, observational study design with a small cohort size. CONCLUSIONS: Low-normal levels of 1α,25(OH)2D was demonstrated in anephric patients, indicating production outside the kidneys. This extrarenal CYP27B1 activity may be more substrate driven than hormonally regulated. Kidney transplantation seems to restore kidney CYP27B1 and CYP24A1 activity, as evaluated by vitamin D metabolic ratios, resulting in both increased vitamin D production and catabolism. These findings may have implications for vitamin D supplementation strategies in the setting of kidney failure and transplantation. PLAIN-LANGUAGE SUMMARY: Vitamin D activation occurs in multiple tissues, but the kidneys are considered the only relevant source of circulating levels. This study investigates vitamin D activation outside the kidneys by measuring vitamin D metabolites in 38 patients without kidneys. Active vitamin D was detectable in all patients, indicating production outside of the kidneys. There was a strong relationship between active and precursor vitamin D levels, but no association with mineral metabolism hormones, indicating that vitamin D production was more substrate dependent than hormonally regulated. One year after kidney transplantation, active vitamin D levels increased 2-fold and breakdown products increased 3-fold, indicating that production and degradation of the hormone recovers after kidney transplantation. These findings are relevant for future research into vitamin D supplementation in kidney failure.

Accumulated LPS induced by colitis altered the activities of vitamin D-metabolizing hydroxylases and decreased the generation of 25-hydroxyvitamin D.

It is generally accepted that low vitamin D (VD) levels are associated with a high prevalence factor for Inflammatory bowel disease (IBD). IBD patients have observed higher levels of lipopolysaccharide (LPS), ALT, and AST than healthy people. Gut-derived LPS causes inflammatory injury in the liver and kidney. The VD-metabolizing mechanism is involved in the liver and kidney, which means IBD might impact VD metabolism. However, whether IBD affects VD metabolism has not been studied. In vitro LPS resulted in decreased CYP2R1 in liver cells as well as decreased CYP27B1 and increased CYP24A1 in kidney cells, revealing that LPS changed the activities of several hydroxylases. Mice with acute colitis had an increased LPS in serum and liver with mild hepatic injuries, while mice with chronic colitis had a significant elevation of LPS in serum, liver, and kidney with hepatorenal injuries. Thus, the liver hydroxylase for VD metabolism would be the first to be affected in IBD. Consequently, serum 25-hydroxyvitamin D declined dramatically with a significant elevation of 24,25-dihydroxyvitamin D and 1,24,25-trihydroxyvitamin D. Unchanged serum levels of 1,25-dihydroxyvitamin D might be the result of other factors in vivo. In acute colitis, a small dosage (4 IU/day) of cholecalciferol could protect the colon, decrease the serum level of LPS, and finally increase serum 25-hydroxyvitamin D. However, this improvement of cholecalciferol was fading in chronic colitis. These results suggested that VD supplementations for preventing and curing IBD in the clinic should consider hepatorenal hydroxylases and be employed as soon as possible for a better outcome.

Loss of maternal calcitriol reversibly alters early offspring growth and skeletal development in mice.

Ablation of Cyp27b1 eliminates calcitriol but does not disturb fetal mineral homeostasis or skeletal development. However, independent of fetal genotypes, maternal loss of Cyp27b1 altered fetal mineral and hormonal levels compared to offspring of WT dams. We hypothesized that these maternal influences would alter postnatal skeletal development. Cyp27b1 null and WT females were mated to bear only Cyp27b1+/- offspring. Forty-eight hours after birth, pups were cross-fostered to dams of the same or opposite genotype that bore them. Maternal and offspring samples were collected on days 21 (weaning) and 42. Offspring measurements included minerals and hormones, BMC by DXA, ash weight and mineral content, gene expression, 3-point bending tests, and microCT. Maternal lactational behavior was evaluated. Milk was analyzed for nutritional content. At day 21, offspring fostered by nulls, independent of birth dam, had ~20% lower weight, BMC, ash weight, and ash calcium than pups fostered by WT dams. Adjustment for body weight accounted for the lower BMC but not the lower ash weight and ash calcium. Hormones and serum/urine minerals did not differ across offspring groups. Offspring fostered by nulls had shorter femurs and lower cortical thickness, mean polar moment of inertia, cortical area, trabecular bone volume, and trabecular number. Dam lactational behaviors and milk nutritional content did not differ between groups. At day 42, body weight, ash weight, lengths, BMC, and tibial bone strength were no longer different between pups fostered by null vs WT dams. In summary, pups fostered by Cyp27b1 nulls, regardless of birth dam, have proportionately smaller skeletons at 21 d, impaired microstructure, but normal mineral homeostasis. The skeletal effects are largely recovered by day 42 (3 wk after weaning). In conclusion, maternal loss of calcitriol impairs early postnatal cortical bone growth and trabecular bone mass, but affected offspring catch up after weaning.

The Distinct Regulation of the Vitamin D and Aryl Hydrocarbon Receptors in COVID-19.

(1) Background: SARS-CoV-2 affects several immune pathways, including the vitamin D (VDR) and the aryl hydrocarbon receptor pathways (AhR). The aim of the study was the evaluation of the VDR and AhR pathways in the blood of COVID-19 patients with regard to the severity of disease. (2) Methods: Observational, single-center, case-control design. A total of 240 samples were selected for exploration. Patients who tested negative for SARS-CoV-2 but suffered from other respiratory infections (ORIs) served as a control group. (3) Results: VDR-specific mRNA in the blood of patients with mild symptoms (131.2 ± 198.6) was significantly upregulated relative to the VDR expression of the ORI group (23.24 ± 42.60; p < 0.0001); however, VDR expression of critically ill patients showed an impaired upregulation (54.73 ± 68.34; p < 0.001). CYP27B1 expression was not significantly regulated during SARS-CoV-2 infection. There was a downregulation of VDR and CYP27B1 compared to survivors. There was no significant difference in 25(OH)-vitamin D3 levels between critically ill patients with regard to survival (24.3 ± 9.4 vs. 27.1 ± 11.3; p = 0.433). (4) Conclusion: The VDR and AhR pathways are distinctively regulated in patients suffering from COVID-19 depending on the severity of disease. A combination treatment of antiviral drugs and vitamin D substitution should be evaluated for potentially improved prognosis in COVID-19.

Oleanolic acid exerts bone anabolic effects via activation of osteoblastic 25-hydroxyvitamin D 1-alpha hydroxylase.

Oleanolic acid (OA) is previously shown to exert bone protective effects in aged animals. However, its role in regulating osteoblastic vitamin D bioactivation, which is one of major causes of age-related bone loss, remains unclear. Our results revealed that treatment of OA significantly increased skeletal CYP27B1 expression and circulating 1,25(OH)2D3 in ovariectomized mice (p <0.01). Moreover, OA upregulated CYP27B1 protein expression and activity, as well as the vitamin D-responsive bone markers alkaline phosphatase (ALP) activity and osteopontin (OPN) protein expression, in human osteoblast-like MG-63 cells (p<0.05). CYP27B1 expression increased along with the osteoblastic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). CYP27B1 expression and cellular 1,25(OH)2D3 production were further potentiated by OA in cells at mature osteogenic stages. Notably, our study suggested that the osteogenic actions of OA were CYP27B1 dependent. In summary, the bone protective effects of OA were associated with the induction of CYP27B1 activity and expression in bone tissues and osteoblastic lineages. Hence, OA might be a potential approach for management of age-related bone loss.

Decreased vitamin D bio-availability with altered DNA methylation of its metabolism genes in association with the metabolic disorders among the school-aged children with degree I, II, and III obesity.

Obesity is strongly associated with disturbances of vitamin D (VD) metabolites in the animal models. However, the related epidemiological evidence is still controversial, especially the different degrees of obesity children. Hence, in this present representative case-control study, 106 obesity school-age children aged 7-12 years were included and divided into different subgroups as degree I (the age- and sex-specific BMI≥95th percentile, n=45), II (BMI ≥120% percentile, n=34) and III (BMI ≥140% percentile, n=27) obesity groups across the ranges of body mass index (BMI). While the age- and sex-matched subjects without obesity were as the control group. Notably, it was significantly different of body composition, anthropological and clinical characteristics among the above four subgroups with the dose-response relationships (P<.05). Moreover, comparing with the control group, the serum VD concentrations were higher, VD metabolites like 25(OH)D, 25(OH)D3 and 1,25(OH)2D, and related hydroxylases as CYP27A1, CYP2R1 and CYP27B1 were lower in the degree I, II, and III obesity subgroups (P<.05), which were more disorder with the anthropological and clinical characteristics as the obesity was worsen in a BMI-independent manner (P<.05). However, there was a significant increase of CYP27B1 in the degree III obesity group than those in the degree I and II obesity subgroups. Furthermore, the methylation patterns on the genome-wide (Methylation/Hydroxymethylation) and VD metabolism genes (CYP27A1, CYP2R1 and CYP27B1) were negatively correlated with the worse obesity and their related expressions (P<.05). In summary, these results indicated that obesity could affect the homeostasis of VD metabolism related genes such as CYP27A1, CYP2R1, CYP27B1 and etc through abnormal DNA methylation, resulting in the disorders of VD related metabolites to decrease VD bio-availability with the BMI-independent manner. In turn, the lower levels of VD metabolites would affect the liver function to exacerbate the progression of obesity, as the Degree II and III obesity subgroups.

Novel Vitamin D3 Hydroxymetabolites Require Involvement of the Vitamin D Receptor or Retinoic Acid-Related Orphan Receptors for Their Antifibrogenic Activities in Human Fibroblasts.

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 and 1,20,23(OH)3D3 were compared to those of classical 1,25(OH)2D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR, RORs, or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)2D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required.

Vitamin D status alters genes involved in ovarian steroidogenesis in muskrat granulosa cells.

This study aims to explore the relationship between altered vitamin D (VitD3) status and ovarian steroidogenesis in muskrats during the breeding and non-breeding seasons. During the breeding season, the ovaries of muskrats were observably enlarged and increased in weight, accompanied by elevated serum and ovarian VitD3 status. Vitamin D receptor (VDR), VitD3 metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes were immunolocalized in the ovarian cells of muskrats. The mRNA levels of VDR, CYP2R1, CYP27B1, and steroidogenic enzymes were considerably higher during the breeding season compared to the non-breeding season. RNA-seq analysis revealed a prominent enrichment of vitamin-related and ovarian steroidogenesis pathways. Furthermore, the addition of 1,25(OH)2D3 to the muskrat granulosa cells in vitro increased VDR and steroidogenic enzymes mRNA levels and enhanced the 17ß-estradiol level. Overall, these findings supported that VitD3 promotes the secretion of steroid hormones, thereby affecting seasonal changes in ovarian function in the muskrats.

Impaired local Vitamin D3 metabolism contributes to IL-36g overproduction in epithelial cells in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation and function of VD3 in CRSwNP. METHODS: 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR) and cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzyme that converts 25VD3 to the active 1,25-hydroxyvitamin D3 (1,25VD3), and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence, and flow cytometry. RNA sequencing was performed to identify genes regulated by 1,25VD3 in HNECs. HNECs and polyp tissue explants were treated with 1,25VD3, 25VD3, and dexamethasone. RESULTS: 25VD3 levels in serum and nasal tissue lysates were decreased in patients with eosinophilic and noneosinophilic CRSwNP than control subjects. The expression of VDR and CYP27B1 were reduced in eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells. VDR and CYP27B1 expression in HNECs were downregulated by interferon y and poly (I:C). Polyp-derived epithelial cells demonstrated an impaired ability to convert 25VD3 to 1,25VD3 than control tissues. 1,25VD3 and 25VD3 suppressed IL-36y production in HNECs and polyp tissues, and the effect of 25VD3 was abolished by siCYP27B1 treatment. Tissue 25VD3 levels negatively correlated with IL-36y expression and neutrophilic inflammation in CRSwNP. CONCLUSION: Reduced systemic 25VD3 level, local 1,25VD3 generation and VDR expression result in impaired VD3 signaling activation in nasal epithelial cells, thereby exaggerating IL-36y production and neutrophilic inflammation in CRSwNP.

A PAI-1 antagonist ameliorates hypophosphatemia in the Hyp vitamin D-resistant rickets model mouse.

Congenital fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets/osteomalacia is a rare bone metabolism disorder characterized by hypophosphatemia and caused by genetic abnormalities that result in excessive secretion of FGF23. Hyp mice are a model of X-linked hypophosphatemia (XLH) caused by deletion of the PHEX gene and excessive production of FGF23. The purpose of this study was to investigate the potential of TM5614 as a therapeutic agent for the treatment of congenital FGF23-related hypophosphatemic rickets and osteomalacia in humans by administering TM5614 to Hyp mice and examining its curative effect on hypophosphatemia. After a single oral administration of TM5614 10 mg·kg-1 to female Hyp mice starting at 17 weeks of age, the serum phosphate concentration increased with a peak at 6 h after administration. ELISA confirmed that TM5614 administration decreased the intact FGF23 concentration in the blood. Expression of 25-hydroxyvitamin D-1α-hydroxylase protein encoded by Cyp27b1 mRNA in the kidney was suppressed in Hyp mice, and treatment with 10 mg·kg-1 of TM5614 normalized the expression of 25-hydroxyvitamin D-1α-hydroxylase protein and Cyp27b1 mRNA in the kidneys of these mice. Our data indicate that oral administration of TM5614 ameliorates hypophosphatemia in Hyp mice, suggesting that TM5614 may be an effective treatment for congenital FGF23-related hypophosphatemic rickets and osteomalacia.