The current study demonstrates that PTH/PTHrP and vitamin D both influence the expression of cathelicidin antimicrobial peptide and the capacity of mice to resist infection by group A Streptococcus
. Previously, cell culture has suggested a link between infection and vitamin D (26
), but animal model data have been less compelling. A lack of a useful mouse model for dietary vitamin D deficiency has been explained by the difficulty in generating severe vitamin D deficiency in this species (15
). Here, the action of PTH, which compensates for vitamin D deficiency by maintaining calcium homeostasis, was found to be a potent mechanism for inducing the expression of cathelicidin in both mice and human cells. These observations advance our understanding of how vitamin D3
influences immune defense by suggesting that the elevation of PTH in response to dietary vitamin D3
restriction may compensate for the loss of vitamin D and its role in immune functions.
1,25-D3 and PTH/PTHrP appear to act together in human keratinocytes. Several interrelationships exist in the innate immune response between metabolic activation of vitamin D and regulation of PTH/PTHrP. An overview of the interactions between PTH/PTHrP, PTH1R, vitamin D, and cathelicidin expression is shown in . We found that 1,25-D3 induced PTH1R expression, thus enabling a response to PTH/PTHrP. 25-D3 enabled keratinocytes to respond to PTH/PTHrP because of the endogenous capacity of keratinocytes to hydroxylate 25-D3 to 1,25-D3. Inhibition of this activity by either addition of itraconazole in culture or targeted deletion of CYP27B1 in mice blocked the function of PTH/PTHrP. Furthermore, in the absence of 1,25-D3, the expression of PTH1R was low. Therefore, this result supported previous reports that 1,25-D3 acts as a stimulus of CAMP
) and also suggests that it enables action of PTH/PTHrP. PTH can also increase 1,25-D3 levels in keratinocytes (28
), suggesting a positive feedback system. We also observed that PTH/PTHrP and PTH1R also had an effect on keratinocyte CAMP
expression in the absence of exogenous sources of vitamin D, suggesting that the PTH system has the capacity to act by itself or amplify other stimuli that regulate cathelicidin expression. Alternative pathways that induce cathelicidin expression exist in the mouse, where a vitamin D receptor element is absent from the Camp
promoter. Therefore, the PTH/PTHrP enhancer system is active in the control of cathelicidin in mice even if the mouse promoter lacks a vitamin D receptor element.
Fig. 6 Schematic showing how cathelicidin expression, PTH/PTHrP, and vitamin D may be connected. The human cathelicidin gene CAMP is induced by vitamin D, and its expression is further enhanced by the action of PTH/PTHrP. (1) PTH levels are elevated in response (more ...)
Our current findings provide evidence that DNA methylation is involved in the control of cathelicidin. Previous studies have shown that transcriptional suppression through DNA methylation is derepressed upon PTH1R activation (25
). We find that the cathelicidin promoter in human keratinocytes is methylated and that chemical inhibition of methylation can increase CAMP
expression to a degree similar to that induced by PTH/PTHrP. These associations, as well as the role of PKC, support the notion that activation of PTH1R can induce CAMP
. Future work is needed to investigate this system and expand on the hypothesis that PTH1R acts on CAMP
through epigenetic mechanisms involving DNA methylation.
The exact functions of PTH/PTHrP in skin are poorly understood. PTHrP may have a role in the hair growth cycle (29
), angiogenesis (30
), epidermal cell proliferation, and differentiation (31
). PTHrP modulates intracellular calcium in a number of cell types, and calcium has been implicated as an early signal in the differentiation of keratinocytes (32
). Given that keratinocytes under high-calcium conditions show enhanced expression of skin antimicrobial peptides and their processing enzymes (33
), this may be an alternate mechanism of action. Furthermore, a recent study has shown that expression of CAMP
can be induced by CREB (cyclic adenosine monophosphate response element–binding protein) (35
), a downstream transcriptional regulator activated by engagement of the PTH1R (36
). Thus, several cellular signaling mechanisms may enable PTH/PTHrP to influence innate immunity.
The relevance of PTH/PTHrP in vivo was demonstrated by observations that mice injected with PTH increase their resistance to invasive group A Streptococcus infection. Although the decrease induced by PTH in the absolute number of group A Streptococcus colony-forming units (CFU) measured in the skin was relatively modest, a large decrease in absolute lesion size was observed, therefore suggesting that a local increase in PTH is important to enhance resistance. Furthermore, the potential consequences of diet were illustrated in mice on dietary vitamin D restriction. Mice with intact 1,25-D3 levels but restricted dietary vitamin D had elevated PTH and Camp, and maintained normal protection against infection. On the basis of our results in culture, it is tempting to speculate that the increase in Camp was a result of the increase in PTH. Supporting this were additional observations that mice lacking CYP27B1 expression did not increase Camp despite elevated PTH. Only this group showed increased susceptibility to infection by group A Streptococcus. This is consistent with our findings in culture that response to PTH is optimal in the presence of 1,25-D3 and that mice injected with mPTH do not increase resistance to infection if they lack CYP27B1.
The antimicrobial responses of mice on vitamin D restriction support the notion of a role for PTH in innate immunity. Wild-type mice on dietary vitamin D3 restriction had a 600-fold increase in cathelicidin that accompanied the rise in PTH. We believe that this increase may have been induced by the elevated PTH and protected these mice against increased infection. The unexpected response in this system was that CYP27B1−/− mice on a normal diet showed a large increase in mBD4 and also did not have enhanced skin infection. This finding suggests that 25-D3, in the absence of 1,25-D3, has other influences on innate immunity beyond cathelicidin. Given that vitamin D3 and PTH induce CD14, one may speculate that enhanced pattern recognition through CD14 may result in enhanced mBD4 and thus offer protection in the absence of cathelicidin. Future work is required to better understand this potential alternate system.
We have shown that either PTH or PTHrP can induce an antimicrobial peptide response and enhance immune defense. The current study also shows a role of dietary vitamin D3 in the prevention of group A Streptococcus skin infection. We find that multiple factors can cooperate with dietary vitamin D3 to influence innate immune responses in the skin: vitamin D3 metabolism, PTH1R expression, and pattern receptor activation. These observations provide a new understanding of the relationship between vitamin D3 status and infection.