Vitamin D deficiency and lower TGF-β/IL-17 ratio in a North Indian cohort of pemphigus vulgaris

Pemphigus vulgaris is an autoimmune disease which manifests with the blistering of skin and/or mucosa and may have fatal outcome. Autoantibodies against the desmosomal component desmoglein-3 are implicated in acantholysis of keratinocytes, leading to formation of clefts or bullae [1]. However, these autoantibodies are sometimes found to be absent in active disease. Moreover, they have also been at times reported in individuals without apparent disease [2]. Hence, it is postulated that various other factors such as genetic makeup and environmental triggers may contribute to disease onset. The disease pathogenesis clearly has an immunological basis. However, the exact immune mechanisms underlying the disease onset and progression are still not well understood.

T-helper (Th) cells are an essential components of host adaptive immune response, beneficial in protecting the host against infection. However, these cells can also mediate various immunological responses harmful to host such as autoimmune disorders and transplant rejection [3]. Th cells have been classified on the basis of signature cytokines secreted by them: IFNγ-secreting Th1, IL-4- and IL-5-secreting Th2 and IL-17- producing Th17 cells. Classically, the pathogenesis of autoimmune diseases were explained using Th1/Th2 paradigm, according to which some diseases manifested due to Th1 cells like Crohn’s disease, Hashimotos disease, Psoriasis, where others like SLE were thought to have a Th2 basis [4]. However, recent studies suggests that IL-17 producing Th17 cells play a crucial role in pathogenesis of various autoimmune diseases such as multiple sclerosis (MS) [5], rheumatoid arthritis (RA) [6], systemic lupus erythematosus (SLE) [7], psoriasis [8], and chronic inflammatory bowel disease [9]. Another cytokine, the role of which has gained importance in the pathogenesis of autoimmune disorders in recent times is TGF-β. It is found to be an essential differentiation factor in the homeostasis as well as in development of T regulatory (Tregs), which further inhibit inflammation and autoimmunity by counteracting the effects of other T helper cells [10]. Although there are various cytokine studies describing the Th1/Th2 cytokine levels in PV [11–13], the data on IL-17 and TGF-β is still scanty.

Vitamin D is a secosteroid, typically known for its role as a hormone involved in enhancing intestinal absorption of calcium and phosphate. In recent years, however, vitamin D has been found to have a much broader range of actions, including regulation of cell differentiation, proliferation, and apoptosis [14]. The presence of vitamin D Receptor (VDR) in human lymphocytes was one of the first observations implicating vitamin D in non calciotropic responses, and the receptor has now been described in a variety of immune cells [15]. The role of vitamin D in immune system is complex and diverse. The systemic or locally produced active form of vitamin D, 1, 25-dihydroxyvitamin D [1, 25(OH) 2D3], can exercise its effects on several immune cells, including macrophages, dendritic cells, and T and B cells. A key immuno modulatory property of 1, 25(OH) 2D3 is its ability to inhibit expression of Th1 cytokines, whilst augmenting Th2 cytokines, with 1, 25 (OH) 2D3 acting either directly via effects on T lymphocytes or indirectly via effects on antigen-presenting cells (APCs). Moreover, elevated VDR expression is also found on differentiated Th17 cells. Vitamin D provides protection against autoimmune inflammatory diseases, such as multiple sclerosis, type 1 diabetes, and IBD, partially due to its inhibitory effects on Th17 cells [16].

In recent years, a number of studies have emerged that link between vitamin D availability either from sunshine or from diet and the prevalence of autoimmune diseases. The use of supplemental vitamin D has been implicated in reducing the risk of developing multiple sclerosis [17]. Conversely, vitamin D deficiency is common in patients with autoimmune diseases. Therefore, the present study was designed to evaluate role of vitamin D deficiency and IL-17 levels in serum of PV patients in order to establish the role of these two molecules in the immune mechanisms underlying the pathogenesis of PV. TGF-β levels and TGF- β/IL-17 ratio were also analyzed to see if Treg/IL-17 balance was dysregulated in PV patients.

The present study demonstrates that the disease manifests at a younger age in our study group, with the youngest patient being 9 years old. Moreover, majority of patients (67%) were younger than 40 years of age. This finding is consistent with the findings complied by Kanwar et al. in their review stating that the epidemiology of pemphigus in India is in contrast to that from western studies that usually reported the first onset of pemphigus after 40 years of age [13]. This fact may be of significance in deciding the first line therapy, since cyclophosphamide, which has been used successfully in the management of pemphigus in India since 1986 [18], may have adverse effect on gonads, and hence, may not be suitable for adolescent and young adults. Moreover, our study indicated a male predominance. However, existing literature is abundant in studies indicating contrasting results on gender predisposition of the disease. Therefore, overall it may be reasonable to conclude that both the sexes are predisposed to this autoimmune disorder.

The present study also indicates that vitamin D deficiency is common in the North Indian population. This is in agreement with various other published reports that have evaluated serum vitamin D levels in general population of similar geographical locations of North India and Pakistan [19–21]. This indicates that despite being a sunshine abundant area, Vitamin D deficiency is common in North India. This may be attributed to a change in lifestyle that involves more indoor stay during the daytime hours. It is to be noted that all the healthy controls recruited in our study were the hospital staff, who also spent their daytime hours indoors. This explanation is further supported by the study published by Kochupillai et al. showing that all study groups from North India, except the one with maximum direct sunlight exposure had sub-normal concentrations of 25 (OH)vitamin D [19]. Several autoimmune diseases, including inflammatory bowel disease, MS, and type I diabetes and RA are more prevalent in population prone to Vitamin D deficiency due to inadequate sun exposure. Thus, Vitamin D deficiency may be a risk factor predisposing the North Indian population to PV, since it is established that Vitamin D can modulate the functions of a variety of immune cells, including Th-17 cells, and its role in pathogenesis of a various other autoimmune disorders is well established. Similar observations regarding vitamin D deficiency in PV patients have been reported by El-Komy et al.[22]. This is further strengthened by the observation that all our patients were deficient in Vitamin D levels during the active phase of disease as well as exhibited an increased activity of Th-17 subset, as indicated by an elevated serum of IL-17.

Until last decade, the autoimmune disorders were classified as either Th1 or Th2. However, since the discovery of IL-17, more and more studies implicating the role of Th-17 subset in autoimmune disorders have been reported. In our study, we report an increased serum IL-17(203.7 ± 104.7 pg/ml) levels as compared to controls. The levels in controls is in agreement with various other published reports that also indicate that in healthy individuals, the serum levels are usually maintained at levels <5 pg/ml [8, 23]. Thus, increased levels of IL-17 may be of significance in pathogenesis of PV, since IL-17 exhibits a variety of biological activities leading to tissue destruction during inflammation. It stimulates macrophages to produce various inflammatory cytokines, such as IL-1β and TNF-α. Expression of many genes involved in cellular adhesion, has been reported to decrease upon stimulation of keratinocytes by IL-17. Moreover IL-17 also induces the production of inflammatory mediators like nitric oxide and MMPs by keratinocytes [23]. The study also indicates a decrease in serum TGF-β levels, suggesting a compromised tolerance due to impaired function of T regulatory cells. However, the most remarkable differences were observed when we compared the TGF-β/IL-17 ratios in patients and controls, thus highlighting that the T regulatory and T effector axis has an important role in pathogenesis. These results are in agreement with findings of Xu et al. reporting higher Th17 cell numbers in peripheral blood as well as in lesions and lower Treg cells in pemphigus patients [24]. The hypothesis that IL-17 plays an important role in disease pathogenesis is further strengthened by the fact that the levels were also found to be high in patients on DCP therapy (data not shown) who relapsed, irrespective of their autoantibody levels as indicated by DIF. This also suggests that autoantibodies to desmosomal components may have only an initial role in inducing acantholysis, and the disease is perpetuated through other immune mediators such as cytokines. Thus, the present study firmly establishes the hypothesis that besides the classic Th1/Th2 axis, dysregulation of The Treg/Th17 axis is observed during the active form of the disease. However, to further validate this hypothesis, systematic studies in patients during various stages of disease activity are required. These studies should be designed to measure the various T cell subpopulations in lesions as well as circulation, as well as molecular studies focusing on expression of genes such as FOXP3 and RORγT during various stages of disease activity. Once well established, the gene expression ratio or cytokine levels can prove as important serological markers for monitoring disease progression, as well as to predict relapse. There is an urgent need of develop such markers, as the currently available methods such as DIF are invasive and have proved insufficient in predicting the relapse.

In the present study, since no significant difference is observed in serum vitamin D levels in patients and controls, thus, it is clear that serum vitamin D levels alone cannot account for the disease pathogenesis, and is likely to be one of the many factors underlying the complex immune mechanism operating in disease pathogenesis. This is expected since autoimmune disorders usually have a complex etiology, with various factors such as environmental as well as genetic factors contributing to disease onset. Thus, future research pertaining to role of vitamin D in PV should be designed to address the effect of vitamin D supplementation on disease outcome, effect of vitamin D treatment on various immune cells, VDR receptor expression and its polymorphism in patients of PV.

Most importantly, this study demonstrated the deranged TGF-β/IL-17 balance in PV. This paves the way for instituting anti IL-17 therapy with/without Treg modulation in future, to help the patient achieve a relapse free state.

This study reaffirms that hypovitaminosis is common in North Indian population and may be a predisposing factor for autoimmune diseases onset. The study also firmly establishes that TGF-B/IL-17 axis is skewed during disease manifestation, and hence immune dysregulation plays an important role in disease pathogenesis. However, further studies including a larger patient number, and more extensive cytokine and T cell profiling are required to fully understand the exact immune mechanisms operating in disease pathogenesis.