Wounding creates a breach in the otherwise intact physical and chemical cutaneous barrier against the outer environment. Shortly after a wound is created, epithelial cells become activated, produce multiple factors to protect against microbial invasion, and trigger recruitment of leukocytes to further defend the wound. These epithelially derived molecules include AMPs, enzymes, cytokines, and growth factors that initiate a complex and incompletely understood process of repair. Only after several hours to days do keratinocytes from the edge of the wound begin to migrate and reepithelialize the injury, reestablishing an effective physical barrier. Prior to final formation of this barrier, it is essential for the unique wound micromilieu to protect the host from infection. Cathelicidin AMPs have been shown to be essential to this process (18
), but to our knowledge the elements responsible for control of expression during injury were not previously known.
Recently, 1,25D3 was found to induce human CAMP
expression in keratinocytes in vitro and in vivo (6
), and an observed increase in cathelicidin expression after UVB irradiation was proposed to be caused by an increase in 1,25D3 (30
). These observations led us to examine whether the regulation of vitamin D3
is involved in the wound repair response. We found multiple genes under the influence of vitamin D3
, and TLR2, which to our knowledge was not previously known to be inducible by 1,25D3, was induced in skin after injury. Our results suggest that soluble factors in the wound such as TGF-β1
stimulate keratinocytes to increase the metabolic conversion of 25D3 to 1,25D3, thus driving the expression and function of human cathelicidin and a TLR2 complex. The increase in TLR2 enabled cells to respond to microbial stimulation and further enhance the AMP response if necessary, while also amplifying the generation of active vitamin D3
. This elegant system of control of innate immunity by vitamin D3
was to our knowledge previously unknown and adds a new element to the understanding of innate immune defense during wound repair.
Keratinocytes possess the complete enzymatic machinery to produce active 1,25D3 from stored inactive proforms (31
). The final step of endogenous activation of vitamin D3
is hydroxylation of 25D3 by CYP27B1. Cytokines such as IFN-γ and TNF-α were found over a decade ago to induce 1,25D3 production in nondifferentiated keratinocytes (27
), but the relevance of this was unclear. Our results suggest that the induction of TLR2, CD14, and cathelicidin is a direct consequence of increased vitamin D3
metabolism. Furthermore, TGF-β1
was found to increase CYP27B1 and subsequently induce local 1,25D3. Lacking 25D3 as a precursor substrate, inhibiting CYP27B1 enzymatic activity, and blocking the VDR all inhibited the capacity of TGF-β1
to act in vitro. In vivo, mice lacking the CYP27B1 enzyme failed to respond to skin injury with an increase in 1,25D3-regulated CD14. Further investigations to confirm in vivo that inhibition of CYP27B1 also blocks the AMP response would be helpful, but are hindered by the lack of a VDRE in murine Camp
). This distinct difference between humans and mice remains to be explained, but may reflect an evolutionary adaptation to the nocturnal nature of mice compared with humans. However, data derived from the results of topical administration of excess 1,25D3 to human volunteers did confirm that 1,25D3 can act in vivo to induce both TLR2 and cathelicidin (Figure D).
Our findings also show that activation of CYP27B1, and subsequent generation of 1,25D3, enables keratinocytes to respond to microbial components such as Malp-2 or zymosan. These observations complement, but are distinct from, recent work in monocytes showing that activation of TLR2 leads to an increase in 1,25D3 (4
). One sees that an amplification loop is possible in skin that is not apparent in monocytes (Figure ). 1,25D3 leads to increased TLR2 in keratinocytes, and increased signaling by TLR2 leads to elevated 1,25D3. This does not appear to occur in monocytes and keratinocytes are specific to each cell type. In the epidermis, factors involved in the wound repair process such as TGF-β1
trigger 1,25D3-dependent immune responses in keratinocytes. In monocytes, TLR2/1 activation increases 1,25D3-dependent antimicrobial activity (4
), but vitamin D3
inhibits TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns (32
). Thus, distinct effects on innate immune recognition and response are operative during infection of skin or when microbes come in contact with monocytes.
Schematic model for 1,25D3-regulated innate immune functions in keratinocytes and monocytes.
Future studies of TLR and AMP expression in keratinocytes must include constitutively present elements of the epidermal micromilieu such as precursor forms of vitamin D3 not typically present in keratinocyte culture systems. Without inclusion of 25D3, the function of CYP27B1 to generate 1,25D3 would not have been detected, and the capacity of TGF-β1 or TLR2 to influence cathelicidin expression would have been overlooked.
Our results show for the first time to our knowledge that vitamin D3
signaling is important to microbial recognition and the antimicrobial response during injury. Activated vitamin D3
generated in the healing process initiates an antimicrobial response and increased sensitivity to microbial challenge. The recognition receptors TLR2 and CD14, which are induced by 1,25D3 are important for defense against a wide range of flora relevant to wound infections because they recognize elements of both Gram-negative and Gram-positive infections such as peptidoglycan and lipoteichoic acid (19
), and heterodimers of TLR2 with TLR1 and TLR6 detect triacetylated and diacetylated lipopeptides, respectively (33
). In addition, TLR2/6 recognizes fungal wall and mycoplasma components. This is consistent with clinical observations that a large proportion of acute wounds show fungal colonization, yet do not show overt signs of fungal infection (35
), and that cathelicidin is fungicidal (36
). It will be important to investigate whether disturbed vitamin D3
metabolism following injury contributes to abnormal wound repair or infection, or whether other disorders characterized by altered innate immune responses may benefit from modification of vitamin D3
metabolism in skin.