Two lineages of immune cells that differentially express the vitamin D receptor.

Since 1983 it has been known that monocytes and activated T and B cells expressed the vitamin D receptor (VDR) and are therefore vitamin D targets. New data identified two lineages of immune cells that can be differentiated by the expression of the VDR. Monocytes, macrophages, neutrophils, and hematopoietic stem cells were mostly from VDR positive lineages. T cells, ILC1 and ILC3 were also largely VDR positive, which is consistent with the known effects of vitamin D as regulators of type-1 and type-3 immunity. Activation of the VDR negative T cells did not induce the expression of the VDR reporter, suggesting that perhaps only a subset of the T cells in the periphery express the VDR. When activated, the VDR negative T cells responded as if they were VDR knockout T cells in that they made more IFN-γ and proliferated faster than the VDR positive T cells. The ability of vitamin D to regulate immune function will depend on which cells express the VDR and a better understanding of the signals that regulate VDR expression in immune cells.

Vitamin D and the Ability to Produce 1,25(OH)2D Are Critical for Protection from Viral Infection of the Lungs.

Vitamin D supplementation is linked to improved outcomes from respiratory virus infection, and the COVID-19 pandemic renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of vitamin D using animal models of pandemic H1N1 influenza and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In mice, dietary-induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D- Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication, as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of SARS-CoV-2 infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficiency/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

Novel insight into the role of the vitamin D receptor in the development and function of the immune system.

Immune cells express the vitamin D receptor (VDR) and are therefore vitamin D targets. The Vdr protein can be readily measured in the kidney using antibodies to the Vdr and western blot. It is much more difficult to measure Vdr protein in the spleen because of the low level of VDR expression in resting immune cells. In order to more sensitively measure VDR expression, the Cre enzyme was inserted in the 3rd exon of the VDR gene and a reporter mouse that irreversibly expresses tdTomato was made. Mice that express one copy of the VDRCre gene were confirmed to be VDR +/- and mice that express two copies were confirmed to be VDR -/-. Initial characterization of the immune cells from the VDR +/-/VDRtdTomato+ mice, compared to VDR+/+ wildtype (WT) littermates, showed no effect of being hemizygous for the VDR on immune cell frequencies. High tdTomato expression was shown to be present in the bone marrow (BM) and thymus immune cell precursors. In the periphery, monocytes, neutrophils and macrophages had very high tdTomato+ (88-98%) expression while lymphocytes ranged from 60% to 70% tdTomato+. Tissue resident innate lymphoid cell (ILC) 1 and 3 cells were about 60-80% tdTomoto+, while ILC2 cells had very low tdTomato expression. Stimulation of VDRtdTomato+ splenocytes showed that the tdTomato- CD4+ and CD8+ T cells proliferated more than their tdTomato+ counterparts. T cells were sorted for tdTomato+ and tdTomato- and then activated for 72 h. Sorted tdTomato+ T cells expressed the VDR protein only after 72 h post-activation. The sorted tdTomato- T cells proliferated more than the sorted tdTomato+ T cells. Interestingly, activation of the tdTomato- T cells failed to induce new tdTomato expression. The data suggest that an early immune precursor expresses the VDR. In the periphery, neutrophils and monocytes are almost all tdTomato+, while some immune cells (ILC2 and some T cells) may never express the VDR.

Retinoid Signaling in Intestinal Epithelial Cells Is Essential for Early Survival From Gastrointestinal Infection.

Vitamin A deficiency (A-) increases morbidity and mortality to gastrointestinal (GI) infection. Blocking retinoid signaling (dominant negative retinoic acid receptor, dnRAR) in intestinal epithelial cells (IEC, IECdnRAR) had no effect on vitamin A absorption, the expression of tight junction proteins or the integrity of the barrier. Immune cells in the gut were present in normal frequencies in the IECdnRAR mice, with the exception of the T cell receptor (TCR)αß+/CD8αα cells, which were significantly lower than in wildtype littermates. Challenging the IECdnRAR mice with dextran sodium sulfate to induce colitis or Citrobacter rodentium infection resulted in similar disease to wildtype littermates. Feeding mice vitamin A deficient diets reduced vitamin A status and the A- IECdnRAR mice developed more severe colitis and C. rodentium infection. In particular, retinoid signaling in the IEC was crucial for the A- host to survive early infection following C. rodentium. Treating A- mice with retinoic acid (RA) beginning on the day of infection protects most mice from early lethality. However, RA treatment of the A- IECdnRAR mice was ineffective for preventing lethality following C. rodentium infection. Retionid signaling in IEC is critical, especially when there are reduced levels of dietary vitamin A. IEC are direct targets of vitamin A for mounting early defense against infection.

Vitamin D Regulates the Microbiota to Control the Numbers of RORγt/FoxP3+ Regulatory T Cells in the Colon.

The active form of vitamin D (1,25(OH)2D) suppresses experimental models of inflammatory bowel disease in part by regulating the microbiota. In this study, the role of vitamin D in the regulation of microbe induced RORγt/FoxP3+ T regulatory (reg) cells in the colon was determined. Vitamin D sufficient (D+) mice had significantly higher frequencies of FoxP3+ and RORγt/FoxP3+ T reg cells in the colon compared to vitamin D deficient (D-) mice. The higher frequency of RORγt/FoxP3+ T reg cells in D+ colon correlated with higher numbers of bacteria from the Clostridium XIVa and Bacteroides in D+ compared to D- cecum. D- mice with fewer RORγt/FoxP3+ T reg cells were significantly more susceptible to colitis than D+ mice. Transfer of the cecal bacteria from D+ or D- mice to germfree recipients phenocopied the higher numbers of RORγt/FoxP3+ cells and reduced susceptibility to colitis in D+ vs. D- recipient mice. 1,25(OH)2D treatment of the D- mice beginning at 3 weeks of age did not completely recover RORγt/FoxP3+ T reg cells or the Bacteriodes, Bacteriodes thetaiotaomicron, and Clostridium XIVa numbers to D+ values. Early vitamin D status shapes the microbiota to optimize the population of colonic RORγt/FoxP3+ T reg cells important for resistance to colitis.

Vitamin A and vitamin D regulate the microbial complexity, barrier function, and the mucosal immune responses to ensure intestinal homeostasis.

Diet is an important regulator of the gastrointestinal microbiota. Vitamin A and vitamin D deficiencies result in less diverse, dysbiotic microbial communities and increased susceptibility to infection or injury of the gastrointestinal tract. The vitamin A and vitamin D receptors are nuclear receptors expressed by the host, but not the microbiota. Vitamin A- and vitamin D-mediated regulation of the intestinal epithelium and mucosal immune cells underlies the effects of these nutrients on the microbiota. Vitamin A and vitamin D regulate the expression of tight junction proteins on intestinal epithelial cells that are critical for barrier function in the gut. Other shared functions of vitamin A and vitamin D include the support of innate lymphoid cells that produce IL-22, suppression of IFN-γ and IL-17 by T cells, and induction of regulatory T cells in the mucosal tissues. There are some unique functions of vitamin A and D; for example, vitamin A induces gut homing receptors on T cells, while vitamin D suppresses gut homing receptors on T cells. Together, vitamin A- and vitamin D-mediated regulation of the intestinal epithelium and mucosal immune system shape the microbial communities in the gut to maintain homeostasis.

Aligning the Paradoxical Role of Vitamin D in Gastrointestinal Immunity.

Inflammatory bowel disease (IBD) is a chronic disorder characterized by inflammation of the gastrointestinal tract and an immune-mediated attack against the commensal microbiota. Vitamin D is an essential vitamin that not only promotes calcium and phosphate absorption but also regulates immune function. The active form of vitamin D [1,25(OH)2D] has been shown to suppress symptoms of IBD by inhibiting T cell responses. Host protection from gastrointestinal infection depends on T cells. Paradoxically, vitamin D deficiency increases susceptibility to IBD and gastrointestinal infection. Here we review the roles of vitamin D in immune cells using a kinetic model of the vitamin D-mediated effects on infection to explain the sometimes paradoxical effects of vitamin D on gastrointestinal immunity.

Vitamin D Is Required for ILC3 Derived IL-22 and Protection From Citrobacter rodentium Infection.

Citrobacter rodentium is a gastrointestinal infection that requires early IL-22 from group 3 innate lymphoid cells (ILC3) for resistance. The role of vitamin D in the clearance of C. rodentium infection was tested in vitamin D sufficient (D+) and vitamin D deficient (D-) wildtype (WT) and Cyp27B1 (Cyp) KO mice (unable to produce the high affinity vitamin D ligand 1,25(OH)2D, 1,25D). Feeding Cyp KO mice D- diets reduced vitamin D levels and prevented synthesis of 1,25D. D- (WT and Cyp KO) mice had fewer ILC3 cells and less IL-22 than D+ mice. D- Cyp KO mice developed a severe infection that resulted in the lethality of the mice by d14 post-infection. T and B cell deficient D- Rag KO mice also developed a severe and lethal infection with C. rodentium compared to D+ Rag KO mice. D- WT mice survived the infection but took significantly longer to clear the C. rodentium infection than D+ WT or D+ Cyp KO mice. Treating infected D- Cyp KO mice with IL-22 protected the mice from lethality. Treating the D- WT mice with 1,25D reconstituted the ILC3 cells in the colon and protected the mice from C. rodentium. IL-22 treatment of D- WT mice eliminated the need for vitamin D to clear the C. rodentium infection. Vitamin D is required for early IL-22 production from ILC3 cells and protection from enteric infection with C. rodentium.

Clinical relevance of cyclooxygenase 2 and peroxisome proliferator-activated receptor γ in eyelid sebaceous gland carcinoma.

AIMS: Sebaceous gland carcinoma (SGC) is a malignancy associated with the pilosebaceous unit, and occurs at ocular or non-ocular sites. Cyclooxygenases (COXs) are enzymes that are crucial for lipid metabolism. COX-2 is overexpressed in various cancers, and its inhibition by non-steroidal anti-inflammatory drugs is known to reduce the risk of many cancers. Peroxisome proliferator-activated receptor (PPAR)-γ is a transcription factor involved in adipogenesis. PPAR-γ is a potential therapeutic target for the treatment of malignant tumours, including colon carcinoma. The aim of this study was to explore the status of COX-2 and PPAR-γ as prognostic markers in human eyelid SGC. METHODS AND RESULTS: The immunohistochemical expression of COX-2 and PPAR-γ was evaluated in 31 SGC cases. Cytoplasmic expression of COX-2 was detected in 80% of the SGC cases, and nuclear expression of PPAR-γ in 87%. There were significant correlations of PPAR-γ expression with well-differentiated SGC [19/21 (90%)] and of COX-2 overexpression with reduced disease-free survival (P = 0.0441, log rank analysis). COX-2 expression [odds ratio (OR) 3.82, 95% confidence interval (CI) 1.02-14.33, P = 0.046] and lymph node metastasis (OR 0.17, 95% CI 0.04-0.65, P = 0.009) emerged as significant risk factors in the univariate analysis. However, COX-2 expression did not emerge as a significant independent prognostic factor in multivariate analysis. CONCLUSIONS: COX-2 is a potential marker for identifying high-risk SGC patients. Expression of PPAR-γ in eyelid SGC cases reflects terminal sebaceous differentiation. Inhibitors of COX-2 signalling and PPAR-γ agonists are both prospective novel therapeutic targets in the management of eyelid SGC patients.