Plasmacytoid DCs (pDCs) are a rare population of circulating cells specialized in the production of large amounts of type I IFNs (IFN-α/β) in response to viral infections (Cella et al., 1999; Siegal et al., 1999). This ability is linked to their unique intracellular expression of toll-like receptor (TLR) 7 and TLR9 (Kadowaki et al., 2001), which recognize single-stranded (ss) viral RNA and DNA transported into endosomal compartments by the virus infecting the cell (Hemmi et al., 2000, 2002; Diebold et al., 2004; Heil et al., 2004). IFN-α/β produced by pDCs was shown to be critical in inhibiting viral replication but also to contribute to the induction and expansion of an antiviral immune response by activating memory T cells, B cells, and NK cells (Theofilopoulos et al., 2005; Gilliet et al., 2008). Under steady-state conditions, pDCs are present in the blood stream and secondary lymphoid organs but are normally absent from most peripheral tissues including the skin (Wollenberg et al., 2002; Gilliet et al., 2004). pDCs can, however, infiltrate the skin infected by viruses including varicella zoster virus (Gerlini et al., 2006), human papillomavirus (Vanbervliet et al., 2003), and herpes simplex virus (Donaghy et al., 2009). These skin-infiltrating pDCs were found to produce IFN-α/β, which is consistent with the ability of viruses to infect pDCs and deliver their nucleic acid cargo into intracellular TLR7/9 compartments of pDCs. Surprisingly, large numbers of pDCs have also been found in the skin of patients with psoriasis (Wollenberg et al., 2002; Gilliet et al., 2004; Nestle et al., 2005), a chronic inflammatory disease of the skin mediated by autoimmune T cells. In psoriatic skin, pDCs are chronically activated to produce IFN-α/β, a process which triggers the activation and expansion of autoimmune T cells leading to the epidermal hyperproliferation and the formation of psoriasis (Nestle et al., 2005). We have recently found that pDC activation in psoriatic skin is driven by the human cathelicidin antimicrobial peptide known as LL37 (Lande et al., 2007). LL37 was found to convert otherwise inert extracellular host-derived (self) nucleic acids, into a potent trigger of pDC activation by forming a complex with the self-RNA and self-DNA and by transporting them into intracellular TLR7 and TLR9 compartments (Lande et al., 2007; Ganguly et al., 2009). The cathelicidin peptide is usually not expressed in healthy skin but was found to be continuously overexpressed by keratinocytes of psoriatic skin, providing an explanation for the chronic activation of pDCs in psoriasis (Lande et al., 2007). Interestingly, the expression of cathelicidin peptides can be transiently induced in keratinocytes by common skin injury (Dorschner et al., 2001; Schauber et al., 2007). However, whether skin injury is associated with pDC infiltration and activation to produce IFN-α/β is not known. In this paper, we found that skin injury induces an early and short-lived infiltration of pDCs into skin wounds. These pDCs were activated to produce IFN-α/β through TLR7 and TLR9, indicating that they recognize self-nucleic acids released by damaged cells in skin wounds. Cathelicidin gene expression closely paralleled pDC activation and cathelicidin peptides were found to be sufficient to induce IFN-α/β production by pDC in the skin. However, cathelicidins were not required to induce IFN-α/β expression, suggesting a redundancy of this pathway for pDC activation in injured skin. Depletion of pDCs or inhibition of IFN-α/β receptor signaling significantly impaired the acute inflammatory cytokine response and delayed reepithelization of skin wounds. These data uncover a novel role of pDCs in sensing nucleic acids in wounded skin and demonstrate their involvement in the acute inflammatory response and wound healing through their production of IFN-α/β. RESULTS Skin injury induces a rapid infiltration of pDCs To determine whether injury of normal skin induces infiltration and activation of pDCs, we adopted a mechanical skin injury model based on tape stripping of shaved murine skin (Sano et al., 2005; Jin et al., 2009). This procedure mechanically removes the upper epidermal layers and injures the basal layer, leading to an acute inflammatory response and reepithelization of the skin (Wojcik et al., 2000). We found that tape stripping induced a robust dermal influx of leukocytes reaching a peak between 24 and 48 h after skin injury (Fig. 1, A and B). Analysis of single cell suspensions revealed that, 24 h after skin injury, the majority of cells infiltrating the dermis were Gr-1+CD11b+ neutrophils (mean, 37.8%; range, 31–42.9%; Fig. 1 C). Interestingly, a large number of pDCs, detected as PDCA1+B220+ cells, also infiltrated the dermis 24 h after injury (mean, 14.4%; range, 3.7–28%; Fig. 1, C and D). The pDC identity was confirmed by showing that these cells coexpressed CD11c, MHC class II molecules, and the pDC-specific marker Siglec-H (Zhang et al., 2006; Fig. 1 E) and lacked common lineage markers CD3, CD19, and CD11b (not depicted). Furthermore, immunohistochemistry for Siglec-H showed that these cells had typical lymphocytic morphology (Fig. 1 F). The accumulation of pDCs and neutrophils in skin wounds was rapid and transient, as they accumulated at 24 h but returned to preinjury levels after 48 h. In contrast, CD3+ T cells were constitutively present in uninjured skin, and increased in number at a later time point (48 h after injury; Fig. 1, C and D). Conventional DCs in the dermal compartment of injured skin were detected as CD11c+PDCA1− cells, and their number did not increase but, rather, showed a tendency toward a decrease (Fig. S1), potentially reflecting their activation and migration to lymph nodes as previously reported (Holzmann et al., 2004). Thus, skin injury induces a rapid and robust infiltration of pDCs that parallels the early wound infiltration by neutrophils. Figure 1. Rapid infiltration of pDCs into injured skin. (A) Representative image of shaved murine back skin 24 h after tape stripping. (B) Dermal cell suspensions isolated from skin at various times after injury and viable cells were counted. The mean number of cells ± SEM per cm2 of injured skin is given and represent data from five mice over a 3-d time course. (C) The percentage of pDCs (B220+PDCA-1+), neutrophils (Gr-1+CD11b+), and T cells (CD3+) in dermal single cell suspensions isolated from injured skin was measured by flow cytometry. Data are the mean ± SEM of five mice per each time point. (D) Flow cytometry plot of pDC, neutrophils, and T cells in injured skin over a 3-d time course. Data are representative of five mice. The percentage of each population is shown in the plots. (E) Flow cytometry for CD11c, IA/IE, and Siglec H surface expression on B220+PDCA-1+ pDC 24 h after skin injury. (F) Representative immunohistochemical staining for Siglec H in injured skin collected 24 h after tape stripping. Bars: (main image) 50 µm; (inset) 10 µm. Data in A–F are representative of two independent experiments. Skin injury induces a transient activation of pDCs to produce type I IFNs Because pDCs are specialized producers of IFN-α/β, producing 100–1,000× more than any other cell type (Siegal et al., 1999), we sought to investigate whether pDCs infiltrating injured skin are activated to produce IFN-α/β. First, we isolated total skin from mice at different time points after injury and performed gene expression analysis. mRNA expression levels of both IFN-α2 (Fig. 2 A) and IFN-β (not depicted) was undetectable in normal skin before injury but rapidly induced by skin injury. Both IFN-α2 and IFN-β (unpublished data) mRNA expression levels reached a peak 24 h after injury and rapidly declined thereafter. This expression profile closely paralleled the presence of pDCs, suggesting that pDCs are the main source of IFN-α/β in injured skin (Fig. 1 C and Fig. 2 A). In contrast, the expression of the inflammatory cytokines IL-6 and TNF showed a distinct time course. IL-6 mRNA expression reached a peak 6 h after injury (Fig. 2 A), which is consistent with injured keratinocytes as an early source of this cytokine (Sehgal, 1990). TNF was induced 24 h after injury but its expression was sustained for up to 72 h (Fig. 2 A), suggesting that cell types other than pDCs are a major source of this cytokine. To confirm the role of pDCs as principal producers of IFN-α/β in injured skin, we depleted pDCs by treating mice with antibodies recognizing BST-2, a transmembrane protein specifically expressed on resting mouse pDCs (Blasius et al., 2006). Two injections of these antibodies at 48 and 24 h before skin injury efficiently depleted pDCs (Fig. S2), as previously reported (Krug et al., 2004; Yoneyama et al., 2005; Blasius et al., 2006; Kuwajima et al., 2006). pDC depletion completely inhibited the accumulation of pDC in injured skin (not depicted) and abrogated the induction of IFN-α2 (Fig. 2 B) and IFN-β (not depicted) expression at 24 h, confirming that pDCs are the principal source of IFN-α/β in injured skin. Interestingly, pDC depletion partially affected the expression of IL-6, whereas the expression of TNF was not affected at 24 h after injury. Because the infiltration of pDCs into injured skin is paralleled by the infiltration of neutrophils, we next sought to determine the role of neutrophils in the expression of these cytokines. Neutrophil depletion using a Ly6G-specific antibody did not show significant effect on the expression of IFN-α/β and IL-6 (Fig. 2 B) but significantly decreased the expression of TNF (Fig. 2 B), which is consistent with neutrophils being a main early source of this cytokine (Dubravec et al., 1990; Hübner et al., 1996). These data demonstrate that skin injury induces early infiltration of pDCs and their local activation to produce IFN-α/β. Figure 2. pDCs are transiently activated to produce type I IFNs in injured skin. (A) Time course analysis of IFN-α, IL-6, and TNF mRNA tissue expression in injured skin. The data are given as fold induction over time 0 and represent the mean ± SEM of five mice per time point. (B) Relative IFN-α, IL-6, and TNF mRNA tissue expression of uninjured skin or injured skin collected 24 h after tape stripping of pDC-depleted, neutrophil-depleted, or control IgG-treated mice. Data represent the mean ± SEM of five mice per group. *, P = 0.001; **, P = 0.02; ***, P 90%. 50,000 cells were cultured overnight in RPMI-based media (10% FCS, 50 µM β-mercaptoethanol, pen/strep, glutamine, sodium pyruvate, and Hepes). The supernatants were used to determine IFN-α protein secretion using a commercial Elisa kit (PBL). As a source of DNA, we used synthetic phosphodiesteric CpG-containing oligonucleotides hybridized to its complementary strand to mimick natural mammalian DNA fragments. 0.3 µM dsDNA was mixed with 30 µM cathelicidin peptides in a volume of 20 µl for 30 min at room temperature. Online supplemental material. Fig. S1 shows the kinetics of mDCs in the skin after skin injury. Fig. S2 shows the efficiency of pDC depletion in the spleen over a 5-d period using BST-specific monoclonal antibodies. Fig. S3 shows the role of TLR7 and TLR9 in the expression of type I IFN, IL-6, and TNF induced by skin injury. Fig. S4 shows the role of MyD88 and TLR7/9 in the induction of IL-6 and TNF in injured skin. Fig. S5 shows the role of MyD88 in the induction of T cell–derived IL-17A, IL-22, and IFN-γ in injured skin. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20101102/DC1.
