the initial metabolism of vitamin d3 occurs in the skin where 7-dehydrocholesterol is converted to previtamin d3 after exposure to uvb radiation . after isomerization , vitamin d3 is metabolized by hepatic 25-hydroxylase ( cyp2r1 ) to form 25-hydroxyvitamin d3 ( 25ohd3 ) , which is the major circulating form of vitamin d3 . 25ohd3 is metabolized by 1-hydroxylase ( cyp27b1 ) that is present in the kidney and many other target tissues including the skin . the active form of vitamin d3 , 1,25 dihydroxyvitamin d3 ( 1,25d3 ) translocates to the nucleus of target cells , where it binds to the vitamin d receptor ( vdr ) , a member of the nuclear receptor supergene family . the vdr heterodimerizes with the retinoid x receptor and this complex recognizes vitamin d response elements in the promoters of many genes to modulate their transcription . 1,25d3 is well characterized for its function in maintaining appropriate serum calcium concentrations as well as its critical requirement for proper bone formation . in addition to these roles , 1,25d3 is important in the regulation of immune responses . vitamin d has important roles in leukocyte differentiation , dendritic cell maturation , and modulation of the t - helper cell dichotomy [ 15 ] . symptoms of th-1-mediated autoimmune diseases can be reduced or eliminated by treatment with 1,25d3 [ 68 ] . in addition to its role in autoimmune disease , recent studies have explored a role for vitamin d - mediated signaling in infectious disease resistance . for example , mycobacterium tuberculosis infection induces both vdr and 1- hydroxylase , which are necessary for production of cathelicidin as part of an antimicrobial peptide response against the m. tuberculosis infection in vitro .    here , we investigated the role of vdr in immunity to murine experimental cutaneous leishmaniasis ( cl ) , the prototypical in vivo model of the th-1/th-2 dichotomy . parasites from the genus leishmania are responsible for a spectrum of diseases ranging from self - healing cutaneous disease to the potentially fatal visceral disease . immunity to l. major , a parasite responsible for cutaneous leishmaniasis , is highly dependent on a strong ifn-mediated t - helper 1 ( th-1 ) response . this th-1 response induces production of nitric oxide ( no ) which is critical in elimination of parasites . we report that vdrko mice exhibit decreased lesion development as well as decreased parasite loads at the height of l. major infection . additionally , we demonstrate that resistant mice that are rendered vitamin d - deficient by ablation of the 1- hydroxylase enzyme in cyp27b1 ko mice mimic vdrko mice with regards to decreased lesion development , suggesting that the effect is dependent on the presence of the ligand . surprisingly , deficiency of vitamin d signaling in genetically susceptible mice has no effect on l. major infection . vdrko , cyp27b1ko , and wt mice , on both balb / c and c57bl/6 backgrounds , were maintained at friemann life sciences center at the university of notre dame ( notre dame , ind , usa ) .   to generate the balb / c vdrko strain , the female offspring from this mating were genotyped by pcr   and the mice identified as heterozygous for the vdrko allele were backcrossed to wt male balb / c mice . this process was repeated 7 times resulting in mice with a greater than 99.2% balb / c background . male and female heterozygous mice were bred to each other to generate offspring that were homozygous for either the vdrko allele or for the wt allele . these homozygous mice were then used to establish a breeding colony of both vdrko and wt mice on a balb / c background . vdrko and wt mice were maintained from weaning on a high - calcium , high - lactose rescue diet ( td96348 ; teklad , madison , wis , usa ) to prevent hypocalcemia associated with vdr deficiency . all animal protocols were approved and reviewed by the university of notre dame institutional animal care and use committee ( iacuc ) . all mice used in the experiments described below were females between 1 and 4 months of age . l. major nih friedlin v1 strain ( mhom / il/80/fn ) was used in this study . the parasites were cultivated and infective stage , metacyclic promastigotes were generated as previously described .   for infections , 1    10 metacyclic promastigotes in 20  l of pbs were injected intradermally into the outside surface of the ear . lesion diameter and thickness and ulcer diameter was measured weekly with a digital vernier caliper . a lesion  score  was calculated by adding the values obtained for lesion diameter , ulcer diameter , and lesion thickness . infected ear tissue was homogenized to determine relative parasite load by performing a limiting dilution assay as previously described . segments of ear tissue were embedded in tissue - tek oct freezing media ( fisher scientific , pittsburgh , pa ) , sectioned and stained with mayer 's hematoxylin and eosin to distinguish cytoplasm and nuclei , respectively . images were obtained using a nikon e400 light microscope ( nikon , melville , ny , usa ) . igg subclass specific secondary antibodies ( southern biotech , birmingham , ala , usa ) were used to determine igg subclass levels . absorbances were recorded using a spectramax m2 plate reader ( molecular devices , sunnyvale , calif , usa ) . sera from mice 12 weeks after post - infection were pooled and run as a normalizing sample on each plate .   for each isotype , the absorbance for each normalizing sample was used to generate a mean absorbance ( abs ) . plate to plate variation was eliminated by using the equation ( mean abs / plate abs )  ( plate abs ) = normalized abs . total rna was isolated from pulverized ears using an rneasy mini kit ( qiagen , valencia , calif , usa ) according to the manufacturer 's instructions and contaminating dna was removed via dnase i treatment ( invitrogen , carlsbad , calif , usa ) . reverse transcription was performed using 1  g of dna - free total rna , 250  ng random primers ( invitrogen ) , and superscript iii kit ( invitrogen ) . real - time pcr was performed on an abi 7900ht sequence detection analyzer ( applied biosystems , foster city , calif , usa ) using the 2x sybr green kit ( applied biosystems ) .    the primers ( 300  nm ; idt , coralville , iolua , usa ) used were hprt , ifn , il-4 , il-12p40 , arg1 ,   inos , il-10 : 5-cac aaa gca gcc ttg cag aa-3/ 5-ctg gcc cct gct gat cct-3 , tnf : 5-gaa aca caa gat gct ggg aca gt-3/ 5-cat tcg agg ctc cag tga att c-3.   for foxp3 , gene expression was determined using a premade gene expression assay ( applied biosystems ) according to manufacturer 's instructions . lymph nodes from infected mice were disrupted using a syringe plunger and a cell strainer ( bd biosciences , san jose , calif , usa ) and cells from 4 mice per condition were pooled and stimulated with 50  g of sla . cell supernatant was harvested at 120  hr after treatment and was analyzed on a luminex 200 instrument using a multiplex biomarker immunoassay ( millipore , billerica , mass , usa ) . two biological replicates were assessed , the first in quadruplicate on two separate luminex plates and the second in duplicate on one plate . fcreceptors were blocked with 10% normal mouse serum and stained using 1% bsa in pbs . cell surface labeling was performed using the following antimouse antibodies :   -cd45 , -cd11b , -cd11c , -ly-6c / g , -cd4 , -cd8 , -tcr ( bd biosciences ) , -f4/80 ( invitrogen ) , and -foxp3 ( ebioscience , san diego , calif , usa ) . flow cytometry was performed using an fc-500 flow cytometer ( beckman coulter , fullerton , ca ) and analyzed with mxp analysis software ( beckman coulter ) . for vitamin d deficiency experiments , mice were fed td.04179 , a vitamin d - deficient diet ( harlan teklad ) from birth prior to mating with male mice from the same background .   once weaned , the offspring from these mice were fed solely using the vitamin d - deficient diet throughout the study . vitamin d analysis of serum was performed using both a 25(oh)d3 elisa and a 1,25d3 elisa ( ids , fountain hills , az , usa ) following the manufacturer 's instructions . red blood cells were lysed using ice - cold sterile ack lysis buffer ( 0.15 m nh4cl , 10  mm khco3 , 0.1  mm na2edta ) . for the generation of dc , progenitor cells were counted and resuspended at 2    10/ml in rpmi - c containing 20  ng / ml granulocyte macrophage colony - stimulating factor ( gm - csf ) ( peprotech , rocky hill , nj , usa ) .   the cells were supplemented with fresh rpmi - c containing 20  ng / ml gm - csf on days 3 , 6 , 8 , and 10 .   on day 12 , the cells were transferred to 24 well plates for the duration of the experiment . macrophages or dc were treated with ifn- ( 500  u / ml ) and/or 1,25d3 ( biomol , plymouth meeting , pa , usa ) ( 40  nm ) for 24  hr or left untreated .     cells were infected at a ratio of 5  :  1 with v1 strain l. major metacyclic promastigotes . parasites were opsonized with 5% normal mouse serum for 30 minutes , washed , then coincubated with the macrophages or dendritic cells for 1 , 2 , 4 , and 24  hr .   coverslips were stained with diff - quick ( fisher scientific ) . levels of il-12 were analyzed using anti - il-12p40 elisa ( pierce , rockford , ill , usa ) . no production was assessed by griess reaction according to the manufacturer 's instructions ( promega , madison , wis , usa ) . for statistical analysis of lesion size , analysis of variance ( anova ) between wt and ko strains was utilized with a subsequent bonferonni 's post - test to determine at what time points the strains were different . paired t - tests were performed to determine differences between wt and ko populations for parasite burdens , igg levels , cytokine regulation , and all in vitro analyses . all statistical analyses were performed using graphpad prism 4.0 software .   in all cases , to investigate the role of vdr in immunity to l. major infection , c57bl/6 vdrko and wt mice were infected in the ear dermis with 10  l. major metacyclic promastigotes . both groups of mice developed lesions that eventually healed ; however , vdrko mice developed lesions that were significantly smaller and healed 3 weeks faster than their wt counterparts ( figure 1(a ) ) .   upon reinfection , vdrko mice did not develop lesions at the site of secondary infection whereas the wt mice developed small lesions that resolved within 3 weeks ( figure 1(b ) ) . these results indicate that vdrko mice have an increased resistance to l. major infection compared to wt mice as demonstrated by decreased lesion development . additionally , vdrko mice have an intact and possibly heightened memory response to secondary infection . because vdrko mice had decreased lesion development , we compared the parasite load at the site of infection between vdrko and wt mice using a limiting dilution assay .   at 4 weeks after infection , both groups of mice had similar levels of parasites at the site of infection and at week 8 , the vdrko mice had significantly fewer parasites compared to the wt mice ( figure 1(c ) ) . however , by 12 weeks after infection , vdrko mice harbored significantly elevated parasite loads , suggesting that wound healing and parasite killing may be occurring via different mechanisms . cyp27b1ko mice , which lack the 1- hydroxylase enzyme required to produce 1,25d3 , displayed prototypical lesion development that is observed in resistant ( c57bl/6 ) strains of mice ; that is , lesions develop after a few weeks and eventually resolve . similar to vdrko mice , cyp27b1ko mice developed significantly smaller lesions compared to their wt counterparts throughout the first 6 weeks of infection ( figure 2(a ) ) . cyp27b1ko mice resolved their infections at a similar rate as wt mice and both groups were completely healed by week 11 after infection . parasite quantification of infected ears from these groups of mice demonstrated that that cyp27b1ko and wt mice possess similar parasite burdens throughout the infection ( figure 2(b ) ) . c57bl/6 mice have the propensity to completely heal after leishmania infection , whereas balb / c mice are unable to heal or resolve l. major induced lesions . unlike c57bl/6 vdrko mice , vdrko mice on a susceptible balb / c background did not develop smaller lesions compared to their wt counterparts ( figure 3 ) . to investigate the causes of reduced lesions and parasite burden in c57bl/6 vdrko mice , we performed further analyses on lesions and immune responses to l. major infection . vdrko mice exhibit alopecia , develop dermal cysts , and do not recycle epidermal layers properly [ 21 , 22 ] .   to assess if these skin differences were involved in the differential lesion development , we performed a histological study to explore lesion architecture . uninfected c57bl/6 vdrko and wt mice do not have any differences in their skin architecture and displayed no characteristics indicative of inflammation ( figure 4 ) .   at 4 weeks after infection , both vdrko and wt mice exhibit similar levels of inflammation at the site of infection . although numbers of infiltrating cells were similar in the two mouse strains through 8 weeks of infection ( data not shown ) , vdrko mice appeared to display decreased inflammation by 8 weeks after infection ( figure 4 ) . inflammation continued to decrease in both groups of mice , and by 12 weeks after infection lesions from vdrko mice exhibited little signs of inflammation . inflammation was still observed in the wt mice at 12 weeks after infection although greatly reduced . there were no significant differences between wt and vdrko mice in terms of macrophage ( cd11b+/f480 + ) , neutrophil ( ly-6c / g+/f480- ) ,   t cell ( cd4 + or cd8 + ) , or dendritic cell ( cd11c+ ) cell infiltration ( data not shown ) . wt mice produced more ifn and il-10 mrna at the infection site 2 weeks after l. major infection ( figure 5 ) . in addition , il-4 and il-12 mrna was elevated in wt mice compared to vdrko mice . inducible nitric oxide synthase ( inos ) was upregulated during l. major infection , however no differences between wt and vdrko were detected . as previously reported , wt mice express more arginase mrna than vdrko mice ( figure 5 ) . flow cytometry analysis of the draining lymph nodes indicated that t - helper cells ( cd4 + ) , cytotoxic t - cells ( cd8 + ) , and foxp3 + treg ( cd4+/cd25 + ) cells are elevated in vdrko mice at most time points relative to wt animals ( suppl . the number of each of these cell types increased as the lesions progressed and then decreased during healing . no obvious differences in other cell types such as macrophages ( cd11b+/f480 + ) , dendritic cells ( cd11c+ ) , or neutrophils ( ly-6c / g+/f480- ) were observed in the draining lymph nodes ( data not shown ) . vdrko lymph node cells produced significantly lower amounts of inflammatory cytokines ifn , tnf- , gm - csf , and mip-1 at the height of infection than the cells from wt mice ( suppl . conversely , more mcp-1 was produced by lymph node cells from vdrko mice compared to the cells from wt mice . early after infection ( 2 weeks ) , cells from both mouse strains up regulated il-4 , il-5 , and il-13 , production that decreased by 12 wks post - infection ( suppl . furthermore , vdrko and wt lymph nodes generated equivalent amounts of il-1 , il-6 , il-10 , rantes , and kc at all time points . total igg serum levels increased in both groups of mice as lesion development progressed and remained elevated even after the mice had healed ( figure 6 ) . significantly elevated levels of igg2a / c were detected in vdrko mice beginning at week 4 after infection and remained significantly elevated throughout infection ( figure 6 ) . no differences in igg1 titers were observed between the vdrko and wt mice at any time point ( data not shown ) . no production is a potent mechanism used by antigen presenting cells to eliminate l. major parasites . dc from both groups of mice were either left untreated or preincubated with combinations of ifn- and/or 1,25d3 prior to l. major infection and 48 hours after infection , cell culture supernatant was analyzed for levels of no production . dc from vdrko generate significantly more no upon stimulation with ifn or ifn and 1,25d3 ( figure 7(a ) ) . preincubation of ifn or a combination of ifn and 1,25d3 resulted in significantly more no production as compared to untreated cells in both cell types . we did not observe an inhibition of no production by the dc upon treatment with 1,25d3 in either the vdrko or wt derived cells . in addition , there is no effect of either the vdr or 1,25d3 on no production in macrophages ( suppl . regardless of the ifn and/or 1,25d3 treatment , il-12p40 production by vdrko dc is significantly increased compared to wt dc , suggesting that vdr may contribute to regulation of il-12p40 production by dc ( figure 7(b ) ) . leishmania are obligate intracellular parasites that are eliminated by a strong th-1 host response .   vitamin d treatment reduces inappropriate th-1 responses thus decreasing or eliminating symptoms of autoimmune diseases [ 68 , 23 ] . as vitamin d exerts these effects through the vdr , we hypothesized that ablation of the vdr would tilt the th-1/th-2 balance towards a th-1 bias and lead to enhanced resistance to l. major infection .   using a mouse model of cutaneous leishmaniasis , in which the parasites were injected into the ear dermis , we observed that vdrko mice developed significantly smaller lesions and have fewer parasites at the site of infection during the height of infection than their wt counterparts . these results are similar to those observed by previous studies using a foot pad model of cutaneous leishmaniasis . as vdrko mice healed earlier than wt mice , we anticipated that vdrko mice would exhibit decreased parasite loads compared to wt mice , because vdrko mice resolve their lesions more quickly than wt mice . however , at 12 weeks after infection , vdrko mice had elevated levels of parasites at the infection site even though they have decreased lesion size . this disparity may indicate that different processes are contributing to wound healing and parasite killing . the ability of vdrko mice to produce increased dermal depositions of collagen may contribute to the increased healing phenotype we observed in vdrko mice , as research indicates that orderly collagen fiber deposition in the skin is one factor that may contribute to increased healing in l. major infected mice . successful clearance of a l. major infection depends on initiation of a robust th-1 response that leads to parasite killing via production of no . vdrko mice produce significantly lower levels of inflammatory cytokines locally at the infection site and following restimulation in vitro suggesting that vdrko mice generate a decreased th-1 response to l. major infection . we expected to detect upregulated transcription of inos in vdrko mice as this enzyme leads to production of the no necessary for killing of l. major ; however , inos is not upregulated at any point post l. major infection in vdrko compared to wt mice .   rather , arginase transcript levels are higher in wt compared to vdrko mice , supporting the suggestion by others that upregulation of arginase antagonizes the metabolism of no by competing with inos for a common substrate , l - arginine . the lack of such competition would allow vdrko mice to generate more no than wt mice , leading to increased parasite killing . this hypothesis is further supported by studies demonstrating that inhibition of arginase by n - hydroxy - l - arginine , a precursor of no , results in increased macrophage killing of l. major parasites in vitro , while induction of arginase contributes to the growth of l. major by providing the parasites with the polyamines required for replication [ 24 , 25 ] . toll like receptor activation initiates upregulation of vdr and cyp27b1 leading to an antimicrobial peptide response against mycobacteria [ 10 , 26 ] , which contributes to killing of m. tuberculosis in human macrophages . this pathway is unlikely to play a role in resistance to l. major as antimicrobial peptides have little effect in killing of leishmania [ 28 , 29 ] . vdr expression , treatment with ifn and/or vitamin d does not affect the ability of macrophage and dc to phagocytose l. major ( data not shown and ) . furthermore , 1,25d3 treatment alone had no effect on ifn-induced no production in infected macrophage and dc in vitro . this contrasts with other published data suggesting that no production by l. major infected macrophages is inhibited by 1,25d3 . these authors suggest that enhanced production of arginase competes with inos for a common substrate , ultimately resulting in decreased production of no in macrophage . stimulation of vdrko macrophage with ifn and lps resulted in significantly less no than similarly treated wt macrophage ( data not shown ) suggesting that vdr may play a role in modulation of no production in macrophage . conversely , ifn-induced no production by infected vdrko dc was significantly higher than wt dc implying that vdr ablation increases rather than decreases no production in dc . our data suggests that vitamin d and the vdr differentially regulate no production in macrophage versus dc . other studies demonstrate that 1,25d3 inhibits ifn signaling in both t cells , macrophages and dc [ 17 , 3033 ] . indirect inhibition of dc produced no then could result via vdr inhibition of ifn activated stat1 signaling . the ablation of the vdr results in elimination of this inhibition resulting in upregulation of no and il-12p40 production .   as a similar increase of il-12p40 and no production was not observed in vdrko macrophage this implies that macrophage differ from dc in their ifn induced production of il-12p40 . vdrko mice generate significantly more antigen specific igg2a / c antibodies , that serve as an indicator of th-1 biased immune responses [ 34 , 35 ] than wt mice . the elevated igg2a / c production in vdrko mice is not surprising as treatment with 1,25d3 inhibits igg2a production in mice , in cattle , and in pigs . these data suggest that elimination of inhibitory effects of 1,25d3 skews antibody response towards production of igg2a / c . the higher levels of igg2a / c possibly explain the increased resistance to secondary infection with l. major observed in the vdrko mice ( figure 1(b ) ) . indeed , elevated antigen specific and nonspecific induction of igg2a / c has been observed in older vdrko mice infected with listeria monocytogenes . however , the exact relationship between igg2a / c production and vitamin d signaling remains to be elucidated . we demonstrate that neither genetic removal of the vdr or depletion of vitamin d from the diet ( data not shown ) of mice on the balb / c background does not alter susceptibility to l. major infection .   susceptibility in wt balb / c mice is attributed to their inability to mount a th-1 response against infection . our results indicate that the effect of the vdr may depend on the nature of the host immune response . additional mechanisms , such as wound healing and genetic background , have also been shown to be important in resistance to l. major infection [ 20 , 40 ] . our data clearly show that the absence of vitamin d signaling can not overcome the susceptibility traits of balb / c mice . contrary to the results observed in vdrko or cyp27b1ko mice , dietary deficiency of vitamin d in resistant c57bl/6 mice did not reduce the severity of l. major lesions ( data not shown ) . a similar disparity has been observed in studies investigating the role of vdr deletion versus dietary vitamin d deficiency in the development of diabetes [ 23 , 41 ] and ms [ 8 , 42 ] .   our data may indicate that some of the effects of vdr on the course of l. major infection are ligand independent , as has been shown in the case of alopecia , or that sufficient 1,25d3 persists in mice maintained on the deficient diet to activate the receptor .     in summary , we have demonstrated that vdrko mice on the c57bl/6 background develop smaller lesions than wt mice upon infection with l. major , but this phenotype is not observed on the balb / c genetic background . enzymatic depletion of 1,25d3 also enhances resistance to l. major infection in c57bl/6 mice . the data further suggest that increased il-12p40 and no production by vdrko dendritic cells may contribute to increased resistance to l. major . these studies indicate that ablation of vdr or elimination of its ligand , 1,25d3 , is able to increase resistance to l. major infection , but only in a host that is predisposed for th-1 immune responses . our data confirm an important role for vitamin d for regulating immune responses that depend on th-1 cells .
vitamin d signaling modulates a variety of immune responses .   here , we assessed the role of vitamin d in immunity to experimental leishmaniasis infection in vitamin d receptor - deficient mice ( vdrko ) . 
 we observed that vdrko mice on a genetically resistant background have decreased leishmania major - induced lesion development compared to wild - type ( wt ) mice ; additionally , parasite loads in infected dermis were significantly lower at the height of infection . 
 enzymatic depletion of the active form of vitamin d mimics the ablation of vdr resulting in an increased resistance to l. major . 
 conversely , vdrko or vitamin d - deficient mice on the susceptible th2-biased background had no change in susceptibility . 
 these studies indicate vitamin d deficiency , either through the ablation of vdr or elimination of its ligand , 1,25d3 , leads to an increase resistance to l. major infection but only in a host that is predisposed for th-1 immune responses .