Autocrine vitamin D signaling switches off pro-inflammatory programs of TH1 cells.

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.

Vitamin D Switches BAF Complexes to Protect ß Cells.

A primary cause of disease progression in type 2 diabetes (T2D) is ß cell dysfunction due to inflammatory stress and insulin resistance. However, preventing ß cell exhaustion under diabetic conditions is a major therapeutic challenge. Here, we identify the vitamin D receptor (VDR) as a key modulator of inflammation and ß cell survival. Alternative recognition of an acetylated lysine in VDR by bromodomain proteins BRD7 and BRD9 directs association to PBAF and BAF chromatin remodeling complexes, respectively. Mechanistically, ligand promotes VDR association with PBAF to effect genome-wide changes in chromatin accessibility and enhancer landscape, resulting in an anti-inflammatory response. Importantly, pharmacological inhibition of BRD9 promotes PBAF-VDR association to restore ß cell function and ameliorate hyperglycemia in murine T2D models. These studies reveal an unrecognized VDR-dependent transcriptional program underpinning ß cell survival and identifies the VDR:PBAF/BAF association as a potential therapeutic target for T2D.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

Evidence supports a causal association between allele-specific vitamin D receptor binding and multiple sclerosis among Europeans.

Although evidence exists for a causal association between 25-hydroxyvitamin D (25(OH)D) serum levels, and multiple sclerosis (MS), the role of variation in vitamin D receptor (VDR) binding in MS is unknown. Here, we leveraged previously identified variants associated with allele imbalance in VDR binding (VDR-binding variant; VDR-BV) in ChIP-exo data from calcitriol-stimulated lymphoblastoid cell lines and 25(OH)D serum levels from genome-wide association studies to construct genetic instrumental variables (GIVs). GIVs are composed of one or more genetic variants that serve as proxies for exposures of interest. Here, GIVs for both VDR-BVs and 25(OH)D were used in a two-sample Mendelian Randomization study to investigate the relationship between VDR binding at a locus, 25(OH)D serum levels, and MS risk. Data for 13,598 MS cases and 38,887 controls of European ancestry from Kaiser Permanente Northern California, Swedish MS studies, and the UK Biobank were included. We estimated the association between each VDR-BV GIV and MS. Significant interaction between a VDR-BV GIV and a GIV for serum 25OH(D) was evidence for a causal association between VDR-BVs and MS unbiased by pleiotropy. We observed evidence for associations between two VDR-BVs (rs2881514, rs2531804) and MS after correction for multiple tests. There was evidence of interaction between rs2881514 and a 25(OH)D GIV, providing evidence of a causal association between rs2881514 and MS. This study is the first to demonstrate evidence that variation in VDR binding at a locus contributes to MS risk. Our results are relevant to other autoimmune diseases in which vitamin D plays a role.

Gene-vegetarianism interactions in calcium, estimated glomerular filtration rate, and testosterone identified in genome-wide analysis across 30 biomarkers.

We examined the associations of vegetarianism with metabolic biomarkers using traditional and genetic epidemiology. First, we addressed inconsistencies in self-reported vegetarianism among UK Biobank participants by utilizing data from two dietary surveys to find a cohort of strict European vegetarians (N = 2,312). Vegetarians were matched 1:4 with nonvegetarians for non-genetic association analyses, revealing significant effects of vegetarianism in 15 of 30 biomarkers. Cholesterol measures plus vitamin D were significantly lower in vegetarians, while triglycerides were higher. A genome-wide association study revealed no genome-wide significant (GWS; 5×10-8) associations with vegetarian behavior. We performed genome-wide gene-vegetarianism interaction analyses for the biomarkers, and detected a GWS interaction impacting calcium at rs72952628 (P = 4.47×10-8). rs72952628 is in MMAA, a B12 metabolic pathway gene; B12 has major deficiency potential in vegetarians. Gene-based interaction tests revealed two significant genes, RNF168 in testosterone (P = 1.45×10-6) and DOCK4 in estimated glomerular filtration rate (eGFR) (P = 6.76×10-7), which have previously been associated with testicular and renal traits, respectively. These nutrigenetic findings indicate genotype can modify the associations between vegetarianism and health outcomes.

Obesity and dysregulated innate immune responses: impact of micronutrient deficiencies.

Obesity is associated with the development of various complications, including diabetes, atherosclerosis, and an increased risk for infections, driven by dysfunctional innate immune responses. Recent insights have revealed that the availability of nutrients is a key determinant of innate immune cell function. Although the presence of obesity is associated with overnutrition of macronutrients, several micronutrient deficiencies, including Vitamin D and zinc, are often present. Micronutrients have been attributed important immunomodulatory roles. In this review, we summarize current knowledge of the immunomodulatory effects of Vitamin D and zinc. We also suggest future lines of research to further improve our understanding of these micronutrients; this may serve as a stepping-stone to explore micronutrient supplementation to improve innate immune cell function during obesity.

Cross-ancestry analyses identify new genetic loci associated with 25-hydroxyvitamin D.

Vitamin D status-a complex trait influenced by environmental and genetic factors-is tightly associated with skin colour and ancestry. Yet very few studies have investigated the genetic underpinnings of vitamin D levels across diverse ancestries, and the ones that have, relied on small sample sizes, resulting in inconclusive results. Here, we conduct genome-wide association studies (GWAS) of 25 hydroxyvitamin D (25OHD)-the main circulating form of vitamin D-in 442,435 individuals from four broad genetically-determined ancestry groups represented in the UK Biobank: European (N = 421,867), South Asian (N = 9,983), African (N = 8,306) and East Asian (N = 2,279). We identify a new genetic determinant of 25OHD (rs146759773) in individuals of African ancestry, which was not detected in previous analysis of much larger European cohorts due to low minor allele frequency. We show genome-wide significant evidence of dominance effects in 25OHD that protect against vitamin D deficiency. Given that key events in the synthesis of 25OHD occur in the skin and are affected by pigmentation levels, we conduct GWAS of 25OHD stratified by skin colour and identify new associations. Lastly, we test the interaction between skin colour and variants associated with variance in 25OHD levels and identify two loci (rs10832254 and rs1352846) whose association with 25OHD differs in individuals of distinct complexions. Collectively, our results provide new insights into the complex relationship between 25OHD and skin colour and highlight the importance of diversity in genomic studies. Despite the much larger rates of vitamin D deficiency that we and others report for ancestry groups with dark skin (e.g., South Asian), our study highlights the importance of considering ancestral background and/or skin colour when assessing the implications of low vitamin D.

Melatonin and vitamin D, two sides of the same coin, better to land on its edge to improve multiple sclerosis.

Previous studies revealed a latitudinal gradient of multiple sclerosis (MS) prevalence, increasing by moving from the equator to the poles. The duration and quality of an individual's exposure to sunlight vary with latitude. Skin exposure to sunlight activates vitamin D synthesis, while light absence, as perceived by the eyes, activates melatonin synthesis in the pineal gland. Vitamin D or melatonin deficiency/insufficiency or overdose can occur at any latitude due to specific lifestyles and diets. Moving away from the equator, especially beyond 37°, decreases vitamin D while raising melatonin. Furthermore, melatonin synthesis increases in cold habitats like northern countries. Since melatonin's beneficial role was shown in MS, it is expected that northern countries whose individuals have higher endogenous melatonin should show a lower MS prevalence; however, these are ranked with the highest scores. In addition, countries like the United States and Canada have uncontrolled over-the-counter usage. In high latitudes, vitamin D deficiency and a higher MS prevalence persist even though vitamin D is typically compensated for by supplementation and not sunlight. Recently, we found that prolonged darkness increased MS melatonin levels, mimicking the long-term increase in northern countries. This caused a reduction in cortisol and increased infiltration, inflammation, and demyelination, which were all rescued by constant light therapy. In this review, we explain melatonin and vitamin D's possible roles in MS prevalence. The possible causes in northern countries are then discussed. Finally, we suggest strategies to treat MS by manipulating vitamin D and melatonin, preferably with sunlight or darkness, not supplements.

Scribble scrambles parathyroid hormone receptor interactions to regulate phosphate and vitamin D homeostasis.

G protein-coupled receptors, including PTHR, are pivotal for controlling metabolic processes ranging from serum phosphate and vitamin D levels to glucose uptake, and cytoplasmic interactors may modulate their signaling, trafficking, and function. We now show that direct interaction with Scribble, a cell polarity-regulating adaptor protein, modulates PTHR activity. Scribble is a crucial regulator for establishing and developing tissue architecture, and its dysregulation is involved in various disease conditions, including tumor expansion and viral infections. Scribble co-localizes with PTHR at basal and lateral surfaces in polarized cells. Using X-ray crystallography, we show that colocalization is mediated by engaging a short sequence motif at the PTHR C-terminus using Scribble PDZ1 and PDZ3 domain, with binding affinities of 31.7 and 13.4 µM, respectively. Since PTHR controls metabolic functions by actions on renal proximal tubules, we engineered mice to selectively knockout Scribble in proximal tubules. The loss of Scribble impacted serum phosphate and vitamin D levels and caused significant plasma phosphate elevation and increased aggregate vitamin D3 levels, whereas blood glucose levels remained unchanged. Collectively these results identify Scribble as a vital regulator of PTHR-mediated signaling and function. Our findings reveal an unexpected link between renal metabolism and cell polarity signaling.

Loss of the glycosyltransferase Galnt11 affects vitamin D homeostasis and bone composition.

O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.

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.

Cutting Edge: Aire Is a Coactivator of the Vitamin D Receptor.

Vitamin D deficiency is associated with the development of autoimmunity, which arises from defects in T cell tolerance to self-antigens. Interactions of developing T cells with medullary thymic epithelial cells, which express tissue-restricted Ags, are essential for the establishment of central tolerance. However, vitamin D signaling in the thymus is poorly characterized. We find that stromal and hematopoietic cells in the mouse thymus express the vitamin D receptor (Vdr) and Cyp27b1, the enzyme that produces hormonal 1,25-dihydroxyvitamin D (1,25D). Treatment of cultured thymic slices with 1,25D enhances expression of the critical medullary thymic epithelial cell transcription factor autoimmune regulator (Aire), its colocalization with the Vdr, and enhances tissue-restricted Ag gene expression. Moreover, the Vdr interacts with Aire in a 1,25D-dependent manner and recruits Aire to DNA at vitamin D response elements, where it acts as a Vdr coactivator. These data link vitamin D signaling directly to critical transcriptional events necessary for central tolerance.

Vitamin D did not reduce multiple sclerosis disease activity after a clinically isolated syndrome.

Low serum levels of 25-hydroxyvitamin D [25(OH)D] and low sunlight exposure are known risk factors for the development of multiple sclerosis. Add-on vitamin D supplementation trials in established multiple sclerosis have been inconclusive. The effects of vitamin D supplementation to prevent multiple sclerosis is unknown. We aimed to test the hypothesis that oral vitamin D3 supplementation in high-risk clinically isolated syndrome (abnormal MRI, at least three T2 brain and/or spinal cord lesions), delays time to conversion to definite multiple sclerosis, that the therapeutic effect is dose-dependent, and that all doses are safe and well tolerated. We conducted a double-blind trial in Australia and New Zealand. Eligible participants were randomized 1:1:1:1 to placebo, 1000, 5000 or 10 000 international units (IU) of oral vitamin D3 daily within each study centre (n = 23) and followed for up to 48 weeks. Between 2013 and 2021, we enrolled 204 participants. Brain MRI scans were performed at baseline, 24 and 48 weeks. The main study outcome was conversion to clinically definite multiple sclerosis based on the 2010 McDonald criteria defined as either a clinical relapse or new brain MRI T2 lesion development. We included 199 cases in the intention-to-treat analysis based on assigned dose. Of these, 116 converted to multiple sclerosis by 48 weeks (58%). Compared to placebo, the hazard ratios (95% confidence interval) for conversion were 1000 IU 0.87 (0.50, 1.50); 5000 IU 1.37 (0.82, 2.29); and 10 000 IU 1.28 (0.76, 2.14). In an adjusted model including age, sex, latitude, study centre and baseline symptom number, clinically isolated syndrome onset site, presence of infratentorial lesions and use of steroids, the hazard ratios (versus placebo) were 1000 IU 0.80 (0.45, 1.44); 5000 IU 1.36 (0.78, 2.38); and 10 000 IU 1.07 (0.62, 1.85). Vitamin D3 supplementation was safe and well tolerated. We did not demonstrate reduction in multiple sclerosis disease activity by vitamin D3 supplementation after a high-risk clinically isolated syndrome.

Eldecalcitol ameliorates diabetic osteoporosis and glucolipid metabolic disorder by promoting Treg cell differentiation through SOCE.

Active vitamin D, known for its role in promoting osteoporosis, has immunomodulatory effects according to the latest evidence. Eldecalcitol (ED-71) is a representative of the third-generation novel active vitamin D analogs, and its specific immunological mechanisms in ameliorating diabetic osteoporosis remain unclear. We herein evaluated the therapeutic effects of ED-71 in the context of type 2 diabetes mellitus (T2DM), delving into its underlying mechanisms. In a T2DM mouse model, ED-71 attenuated bone loss and marrow adiposity. Simultaneously, it rectified imbalanced glucose homeostasis and dyslipidemia, ameliorated pancreatic ß-cell damage and hepatic glycolipid metabolism disorder. Subsequently, in mice injected with the Treg cell-depleting agent CD25, we observed that the beneficial effects of ED-71 mentioned earlier were partially contingent on the Treg subsets ratio. Mechanistically, ED-71 promoted the differentiation of CD4+ T cells into Treg subsets, facilitating Ca2+ influx and the expression of ORAI1 and STIM1, pivotal proteins in store-operated Ca2+ entry (SOCE). The SOCE inhibitor, 2-APB, partially attenuated the positive effects of ED-71 observed in the above results. Overall, ED-71 regulates SOCE-mediated Treg cell differentiation, accomplishing the dual purpose of simultaneously ameliorating diabetic osteoporosis and glucolipid metabolic disorders, showcasing its potential in osteoimmunity therapy and interventions for diseases involving SOCE.

Convergent total synthesis of (+)-calcipotriol: A scalable, modular approach to vitamin D analogs.

A convergent approach for the total synthesis of calcipotriol (brand name: Dovonex), a proven vitamin D analog used for the treatment of psoriasis, and medicinally relevant synthetic analogs is described. A complete approach, not wedded to semisynthesis, toward both the A-ring and CD-ring is reported. From a retrosynthetic standpoint, hidden symmetry within the decorated A-ring is disclosed, which allowed for scalable quantities of this advanced intermediate. In addition, a radical retrosynthetic approach is described, which highlights an electrochemical reductive coupling as well as an intramolecular hydrogen atom transfer Giese addition to establish the 6,5-transcarbon skeleton found in the vitamin D family. Finally, a late-stage decarboxylative cross-coupling approach allowed for the facile preparation of various C20-arylated derivatives that show promising biological activity in an initial bioassay.

Long-Term Effect of Randomization to Calcium and Vitamin D Supplementation on Health in Older Women : Postintervention Follow-up of a Randomized Clinical Trial.

BACKGROUND: Although calcium and vitamin D (CaD) supplementation may affect chronic disease in older women, evidence of long-term effects on health outcomes is limited. OBJECTIVE: To evaluate long-term health outcomes among postmenopausal women in the Women's Health Initiative CaD trial. DESIGN: Post hoc analysis of long-term postintervention follow-up of the 7-year randomized intervention trial of CaD. (ClinicalTrials.gov: NCT00000611). SETTING: A multicenter (n = 40) trial across the United States. PARTICIPANTS: 36 282 postmenopausal women with no history of breast or colorectal cancer. INTERVENTION: Random 1:1 assignment to 1000 mg of calcium carbonate (400 mg of elemental calcium) with 400 IU of vitamin D3 daily or placebo. MEASUREMENTS: Incidence of colorectal, invasive breast, and total cancer; disease-specific and all-cause mortality; total cardiovascular disease (CVD); and hip fracture by randomization assignment (through December 2020). Analyses were stratified on personal supplement use. RESULTS: For women randomly assigned to CaD versus placebo, a 7% reduction in cancer mortality was observed after a median cumulative follow-up of 22.3 years (1817 vs. 1943 deaths; hazard ratio [HR], 0.93 [95% CI, 0.87 to 0.99]), along with a 6% increase in CVD mortality (2621 vs. 2420 deaths; HR, 1.06 [CI, 1.01 to 1.12]). There was no overall effect on other measures, including all-cause mortality (7834 vs. 7748 deaths; HR, 1.00 [CI, 0.97 to 1.03]). Estimates for cancer incidence varied widely when stratified by whether participants reported supplement use before randomization, whereas estimates on mortality did not vary, except for CVD mortality. LIMITATION: Hip fracture and CVD outcomes were available on only a subset of participants, and effects of calcium versus vitamin D versus joint supplementation could not be disentangled. CONCLUSION: Calcium and vitamin D supplements seemed to reduce cancer mortality and increase CVD mortality after more than 20 years of follow-up among postmenopausal women, with no effect on all-cause mortality. PRIMARY FUNDING SOURCE: National Heart, Lung, and Blood Institute of the National Institutes of Health.

Prenatal vitamin D supplementation to prevent childhood asthma: 15-year results from the Vitamin D Antenatal Asthma Reduction Trial (VDAART).

This article provides an overview of the findings obtained from the Vitamin D Antenatal Asthma Reduction Trial (VDAART) spanning a period of 15 years. The review covers various aspects, including the trial's rationale, study design, and initial intent-to-treat analyses, as well as an explanation of why those analyses did not achieve statistical significance. Additionally, the article delves into the post hoc results obtained from stratified intent-to-treat analyses based on maternal vitamin D baseline levels and genotype-stratified analyses. These results demonstrate a statistically significant reduction in asthma among offspring aged 3 and 6 years when comparing vitamin D supplementation (4400 IU/d) to the standard prenatal multivitamin with vitamin D (400 IU/d). Furthermore, these post hoc analyses found that vitamin D supplementation led to a decrease in total serum IgE levels and improved lung function in children compared to those whose mothers received a placebo alongside the standard prenatal multivitamin with vitamin D. Last, the article concludes with recommendations regarding the optimal dosing of vitamin D for pregnant women to prevent childhood asthma as well as suggestions for future trials in this field.

Aberrant follicular regulatory T cells associate with immunoglobulin hyperproduction in nasal polyps with ectopic lymphoid tissues.

BACKGROUND: Ectopic lymphoid tissues (eLTs) and associated follicular helper T (TFH) cells contribute to local immunoglobulin hyperproduction in nasal polyps (NPs). Follicular regulatory T (TFR) cells in secondary lymphoid organs counteract TFH cells and suppress immunoglobulin production; however, the presence and function of TFR cells in eLTs in peripheral diseased tissues remain poorly understood. OBJECTIVE: We sought to investigate the presence, phenotype, and function of TFR cells in NPs. METHODS: The presence, abundance, and phenotype of TFR cells in NPs were examined using single-cell RNA sequencing, immunofluorescence staining, and flow cytometry. Sorted polyp and circulating T-cell subsets were cocultured with autologous circulating naïve B cells, and cytokine and immunoglobulin production were measured by ELISA. RESULTS: TFR cells were primarily localized within eLTs in NPs. TFR cell frequency and TFR cell/TFH cell ratio were decreased in NPs with eLTs compared with NPs without eLTs and control inferior turbinate tissues. TFR cells displayed an overlapping phenotype with TFH cells and FOXP3+ regulatory T cells in NPs. Polyp TFR cells had reduced CTLA-4 expression and decreased capacity to inhibit TFH cell-induced immunoglobulin production compared with their counterpart in blood and tonsils. Blocking CTLA-4 abolished the suppressive effect of TFR cells. Lower vitamin D receptor expression was observed on polyp TFR cells compared with TFR cells in blood and tonsils. Vitamin D treatment upregulated CTLA-4 expression on polyp TFR cells and restored their suppressive function in vitro. CONCLUSIONS: Polyp TFR cells in eLTs have decreased CLTA-4 and vitamin D receptor expression and impaired capacity to suppress TFH cell-induced immunoglobulin production, which can be reversed by vitamin D treatment in vitro.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.