A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Psoriasis is a chronic inflammatory skin disease featuring abnormal keratinocyte proliferation and differentiation. A genetic risk factor for psoriasis (PSORS4) is a deletion of LCE3B and LCE3C genes encoding structural proteins in differentiated keratinocytes. Because analogs of 1,25-dihydroxyvitamin D3 (1,25D) are used in psoriasis treatment, we hypothesized that 1,25D and other VDR ligands act via the vitamin D receptor (VDR) to upregulate expression of LCE 3A/3D/3E genes, potentially mitigating the absence of LCE3B/LCE3C gene products. This hypothesis was pursued using cultured keratinocytes, quantitative real time PCR (qPCR) to assess LCE gene expression, competition binding assays to assess direct ligand binding to VDR, and reporter gene assays to assess the ability of VDR ligands to activate transcription in a VDR- and VDR response element-dependent fashion. qPCR results in a human keratinocyte line, HaCaT, suggested that 1,25D and selected alternate or candidate VDR ligands might upregulate LCE transcripts. Further experiments in primary human keratinocytes confirmed that 1,25D and 10 µM delphinidin do indeed upregulate all five LCE3 genes (LCE3A–E), especially in calcium-differentiated cultures. Further, competition binding assays revealed that delphinidin does in fact bind VDR, but only weakly (IC50 approximately 1 mM). Finally, 20 µM delphinidin was shown to be capable of upregulating a luciferase reporter gene linked to a vitamin D responsive element found near the LCE3 gene cluster. Taken together, these results are consistent with delphinidin (or a metabolite) stimulating LCE3 transcription in a VDR/VDRE-dependent manner. We propose that upregulation of LCE genes may be part of the therapeutic effect of 1,25D to ameliorate psoriasis by providing sufficient LCE proteins, especially in individuals missing the LCE3B and 3C genes. Results with delphinidin further suggest that this compound or its metabolite(s) might offer an alternative to 1,25D in psoriasis therapy.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] plays an important role in the regulation of musculoskeletal growth and differentiation. 1α,25(OH)2D3 mediates its effects on cells, including chondrocytes and osteoblasts, through the classical nuclear 1α,25(OH)2D3 receptor. Additionally, recent evidence indicates that several cellular responses to 1α,25(OH)2D3 are mediated via a rapid, calcium-dependent membrane-mediated pathway. These actions of 1α,25(OH)2D3 can be blocked by antibodies to protein-disulfide isomerase family A, member 3 (Pdia3), indicating that it is part of the receptor complex; however, the pathway which is activated by this receptor is not fully understood. The overall goal of this thesis was to examine the roles of phospholipase A2 activating protein (PLAA) and calcium calmodulin-dependent kinase II (CaMKII) in 1α,25(OH)2D3 rapid membrane-mediated signaling. We further investigated the interaction between two pathways regulating growth plate cartilage and endochondral bone formation, 1α,25(OH)2D3 and Wnt5a, at the receptor complex level. Results indicated that PLAA was required for mediating 1α,25(OH)2D3 signal from Pdia3. Furthermore, CaM and CaMKII were identified as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral bone formation. This study demonstrated that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components in osteoblasts and chondrocytes suggesting that these pathways may interact.

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

Epidemiological evidence from season of birth and migrant studies has led to the proposal that developmental vitamin D3 (DVD) deficiency may be a risk factor for schizophrenia. Concurrently, there has been recent intense interest in vitamin D3 as a modifying factor in the development of immune responses. However, the effects of DVD deficiency on immune function remains unknown. Thus, a model of DVD deficiency has been developed in Sprague-Dawley rats. Briefly, female rats were maintained on a vitamin D3 deficient diet prior to and during gestation. At birth dams were returned to a vitamin D3 replete diet. Post-weaning their offspring, the DVD-deficient rats, were maintained on a vitamin D3 replete diet. Therefore, this model represents a transient, prenatal vitamin D3 deficiency. A number of structural and cellular changes have been described in the DVD-deficient rat brain, including ventriculomegaly, decreased growth factor expression, and increased rates of mitosis. These changes are correlated with altered adult behaviour in the DVD-deficient rat, including a locomotor sensitivity to novelty. This thesis will focus on extending these findings and characterising immune function in the DVD-deficient rat. Vitamin D3 can affect cellular differentiation and is anti-proliferative and pro-apoptotic in many tissues including the brain. The effect of DVD deficiency on brain development has been restricted to neuronal cells in vivo and in vitro. However, the effect of this exposure on glial cell maturation and phenotype was unknown. The experiments reported in this thesis demonstrated that primary cortical glial cell cultures from DVD-deficient rat neonates displayed a similar phenotype and maturational status compared with cells from control rats. Learning and memory was examined in this model by exploring the phenomenon of latent inhibition. DVD-deficient rats displayed normal latent inhibition, however, they exhibited a subtle performance acquisition deficit during the early stages of the conditioned avoidance learning task. Extended handling and pre-exposure were able to ameliorate this deficit, though with this treatment normal latent inhibition was also abolished in both control and DVD-deficient rats. Ultrasonic vocalization and nociceptive threshold testing confirmed that alterations in peripheral sensation could not explain this performance acquisition deficit. The results suggested that anxiety or attentional mechanisms may have contributed to this rate of learning deficit. Finally, as vitamin D3 is a powerful immunoregulator, there is the potential for a transient DVD deficiency to induce a persistent alteration in the development and function of the immune system. This hypothesis was supported by findings that showed DVD deficiency resulted in a primed immune system, as indicated by an enlarged spleen, thymus and peripheral lymph tissue as well as increased pro-inflammatory cytokine production in response to an in vitro stimulation. However, these findings did not lead to an alteration in cell mediated immune response in vivo. The results from the research reported in this thesis indicate that a transient, prenatal vitamin D3 deficiency had a subtle yet significant effect on the immune system and behaviour of the adult rat. These findings add further weight to the body of evidence that link prenatal vitamin D3 status to various adverse health outcomes. The DVD-deficient rat model is an integral step in understanding prenatal vitamin D3 deficiency as a potential environment risk factor in the development of immune and psychiatric disorders.