Foxp3-expressing regulatory T cells (Tregs) reside in tissues where they control inflammation and mediate tissue-specific functions. The skin of mice and humans contain a large number of Tregs; however, the mechanisms of how these cells function in skin remain largely unknown. In this article, we show that Tregs facilitate cutaneous wound healing. Highly activated Tregs accumulated in skin early after wounding, and specific ablation of these cells resulted in delayed wound re-epithelialization and kinetics of wound closure. Tregs in wounded skin attenuated IFN-γ production and proinflammatory macrophage accumulation. Upon wounding, Tregs induce expression of the epidermal growth factor receptor (EGFR). Lineage-specific deletion of EGFR in Tregs resulted in reduced Treg accumulation and activation in wounded skin, delayed wound closure, and increased proinflammatory macrophage accumulation. Taken together, our results reveal a novel role for Tregs in facilitating skin wound repair and suggest that they use the EGFR pathway to mediate these effects.
Forkhead box protein 3–expressing regulatory T cells (Tregs) play an indispensable role in establishing and maintaining immune homeostasis. Although previously thought to be a relatively homogenous population, it has become increasingly accepted that Tregs residing in peripheral tissues possess tissue-specific functions. Both mouse and human skin contain a large number of tissue-resident Tregs (1, 2). However, the molecular mechanisms used by Tregs in skin are largely unknown. In addition, it is unknown whether Tregs in skin play important roles in tissue-specific functions.
The skin is highly susceptible to traumatic injury. As such, wound healing is an extremely common and vital process coordinately mediated by multiple cell types and molecular pathways. The epidermal growth factor receptor (EGFR) pathway plays a major role in skin wound healing through stimulating epidermal and dermal regeneration (3). Interestingly, this pathway was also shown to play a role in immune cell function. The EGFR ligand, AREG, is expressed by Tregs, where it enhances muscle and lung tissue repair after injury (4, 5). In addition, EGFR was shown to be expressed on Tregs, where it plays a role in augmenting their suppressive capacity in vitro (6).
Because Tregs play a major role in mediating skin immune homeostasis, we set out to determine whether these cells play a role in attenuating wound-associated inflammation. In addition, we set out to determine whether Tregs in skin use the EGFR pathway to facilitate wound repair. We show that, upon full-thickness wounding, highly activated Tregs accumulate in skin and play a major role in limiting IFN-γ production and proinflammatory macrophage accumulation. Specific ablation of Tregs early after wounding resulted in delayed wound re-epithelialization and kinetics of wound closure. Tregs in skin induce expression of EGFR early after wounding and used this pathway to attenuate wound-associated inflammation and facilitate normal wound repair.
Materials and Methods
All animal studies were performed in compliance with institutional guidelines in a specific pathogen–free facility. C57BL/6 wild-type (WT) mice were purchased from Simonsen Laboratories (Gilroy, CA). Foxp3-DTR mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Foxp3cre and EGFRfl/fl mice on C57BL/6 background were kindly provided by Dr. Jeffrey Bluestone (University of California, San Francisco) and Dr. David Threadgill (University of North Carolina at Chapel Hill, Chapel Hill, NC), respectively.
Skin-wounding assays and analysis
Six full-thickness excisional wounds were generated with a 4-mm sterile punch (Stiefel Laboratories, Research Triangle Park, NC) after depilation. Wounds were photographed and wound area was measured with image analysis software (ImageJ 1.47v). The surface area of wound defects was expressed as a percentage of closure, relative to the initial surface of each wound.
In vivo Treg ablation
Foxp3-DTR and control mice were injected i.p. with diphtheria toxin (DT; Sigma), at 30 ng/g body weight, according to three regimens. For prolonged Treg depletion, mice were injected with DT on days −2 and −1 prior to wounding, as well as every other day after wounding until day 11 postwounding. For “early” and “late” Treg depletion, mice received a total of five injections, starting either 2 d before (days −2–5) or 5 d after (days 5–10) wounding.
Results and Discussion
Tregs facilitate normal skin repair
Restoration of barrier integrity following trauma is a critical and highly conserved function of skin. Response to skin injury occurs in three overlapping stages: inflammatory, new tissue formation, and remodeling (7). The inflammatory phase occurs early after wounding and is characterized by an abrupt activation of the innate and adaptive immune systems. Because Tregs constitute a large percentage of lymphocytes that reside in skin (1, 2), and because these cells play a major role in regulating tissue inflammation, we set out to determine whether Tregs play a role in cutaneous wound healing. Mice transgenic for the DT receptor (DTR) under the control of the Foxp3 promoter (Foxp3-DTR) allow for robust deletion of Tregs following administration of DT (8). In initial experiments, Foxp3-DTR or WT mice were treated with DT for 2 d to deplete Tregs prior to full-thickness wounding on dorsal skin. After wounding, mice were continuously treated with DT (every 2 d) to maintain Treg depletion, and the size of the skin defect was measured clinically and histologically over time. Wounded mice depleted of Tregs had significantly delayed kinetics of wound closure compared with WT mice treated with DT (Fig. 1A, 1B). Although all wounds in control animals were completely closed (100% closure) between 9 and 11 d after wounding, only 75% closure was observed at this time in Treg-depleted mice, with some wounds never completely closing during the study period (Fig. 1B).
To determine when Tregs are required to facilitate wound repair, we depleted these cells “early” after wounding (during the inflammatory phase of the response) or “late” after wounding (after resolution of the inflammatory phase, when new tissue formation predominates). Depletion of Tregs during the first 5 d after wounding resulted in a significant attenuation of wound closure during the days that followed (days 5–11) (Fig. 1C), with the most pronounced difference occurring 7 d after wounding (Fig. 1D). In contrast, depleting Tregs after the inflammatory phase (i.e., days 5–12) had no effect on the kinetics of wound closure (Fig. 1E). The effects of early Treg depletion were most pronounced upon histologic examination of wounded tissue. Although control mice showed complete re-epithelialization of the skin defect by 7 d after wounding, mice depleted of Tregs had markedly reduced kinetics of wound re-epithelialization, with small areas of keratinocyte hyperplasia present only at the wound edges (Fig. 1F). Mice depleted of Tregs also showed a trend toward increased granulation tissue and size of the overlying eschar (Fig. 1F). These results demonstrate that Tregs play a role in facilitating skin wound healing. In addition, they suggest that these cells act early after wounding, during the inflammatory phase.
Activated Tregs accumulate in wounded skin
Given that Tregs facilitate skin wound repair and mediate their effects early after wounding, we set out to functionally characterize these cells in skin of wounded mice. Consistent with previous reports, we observed a marked accumulation of CD4+ T cells in wounded skin, peaking ∼7 d after wounding (data not shown) (9). Interestingly, the majority of CD4+ T cells that accumulate in wounded skin are highly activated Tregs (Fig. 2). Although Foxp3-expressing Tregs make up 30–50% of total CD4+ T cells in adult mouse skin in the steady-state, the percentage of these cells progressively increased with time after wounding, peaking at 7 d, where they accounted for ∼70% of the total CD4+ population (Fig. 2A). In addition, the density of Tregs increased ∼20-fold in skin by 7 d after wounding (12 ± 2 Tregs/cm2 of skin at baseline versus 279 ± 65 at 7 d after wounding, p = 0.0003). Treg accumulation in wounded skin is primarily a result of migration from secondary lymphoid organs, because treatment of wounded mice with FTY720 (which blocks lymphocyte egress from lymphoid organs) resulted in a significant reduction in these cells in skin at days 3 and 7 after wounding (Supplemental Fig. 1). Tregs accumulating in wounded skin had a highly activated phenotype, with increased percentages of cells expressing high levels of CD25, CTLA-4, and ICOS with time after wounding (Fig. 2B–D). Increased expression of CD25 preceded increases in CTLA-4 and ICOS expression, suggesting that IL-2 may play a role in Treg activation early after injury. Taken together, these results demonstrate that Tregs are activated and preferentially accumulate in skin during the inflammatory phase of wound healing and that depleting these cells during this phase attenuates normal wound closure.
Tregs suppress IFN-γ production and proinflammatory macrophage accumulation in wounded skin
Quantitative and/or qualitative defects in macrophages significantly inhibit skin wound healing. Macrophage depletion early after wounding results in impaired granulation tissue formation and wound re-epithelialization (10). In addition, the retention and persistence of proinflammatory macrophages in skin significantly attenuate wound closure in mouse models and are hallmarks of nonhealing skin ulcers in humans (11). Interestingly, Tregs were shown to influence macrophage function in muscle (6, 12). Thus, we hypothesized that a major role for Tregs in wounded skin is to regulate macrophage polarization during the inflammatory phase of wound repair. To test this, we depleted Tregs early after wounding and assessed macrophage polarization. Proinflammatory macrophages (defined as CD45+CD11bhighF4/80+Ly-6ChighLy-6GlowCD206low) accumulated in skin early after wounding, peaking at 24 h and gradually declining to background levels by 14 d (Fig. 3A). Depletion of Tregs augmented accumulation of proinflammatory macrophages in wounded skin (Fig. 3B). There were significantly higher percentages (Fig. 3B) and absolute numbers (data not shown) of proinflammatory macrophages in the skin of Treg-depleted mice early after wounding (day 3), which translated to increased persistence of these cells during the entire course of wound closure.
IFN-γ is a major driver of proinflammatory macrophage differentiation and accumulation (13). In addition, Tregs suppress IFN-γ production from T cells in skin (14). Thus, we speculated that increased proinflammatory macrophages observed in Treg-depleted mice would correlate with high levels of IFN-γ–producing T cells infiltrating wounded skin. To test this, we treated Foxp3-DTR or WT mice with DT and quantified skin T cell subsets and relative cytokine production from these cells after wounding. Depletion of Tregs resulted in a marked increase in CD4+ and CD8+ T cells infiltrating wounded skin, with little change in dermal γδTCR+ cells, a T cell population resident in normal mouse skin (15) (data not shown). Low numbers of cytokine-producing T cells were observed in wounded skin of WT mice; however, there was a pronounced increase in cytokine-producing cells upon depletion of Tregs, with the overwhelming majority of these cells producing IFN-γ, relative to IL-17A and IL-13 (Fig. 3C). Consistent with a role for IFN-γ in driving proinflammatory macrophage accumulation, in vivo neutralization of IFN-γ resulted in a significant reduction in the accumulation of these cells in skin of Treg-depleted mice after wounding (Supplemental Fig. 2)
These results suggest that a major function of Tregs in the context of skin injury is to limit the accumulation of IFN-γ–producing T cells and proinflammatory macrophages in wounded skin. This is consistent with studies showing accelerated wound healing in mice genetically deficient in IFN-γ (16). We speculate that the persistence of proinflammatory macrophages in Treg-depleted mice is a major contributor to the delay in wound healing observed in these animals. Our findings add to a growing body of literature defining the role of Tregs in regulating macrophage function in tissues.
The EGFR pathway is required for Treg function in wounded skin
Given that the EGFR pathway plays a role in both wound healing and T cell function (3–5), we set out to determine whether this pathway participates in the ability of Tregs to attenuate wound-associated inflammation and facilitate skin repair. To this end, we first characterized EGFR expression on skin-draining lymph nodes (SDLNs) and skin Tregs purified from Foxp3-reporter (Foxp3-DTR) mice. Because robust Abs to murine EGFR are not available for flow cytometry, we performed quantitative RT-PCR for EGFR expression on cells isolated from SDLNs and skin before and after wounding. Expression of EGFR was not detected in Foxp3+ or Foxp3− CD4+ T cells isolated from skin or SDLNs prior to wounding (Fig. 4A). However, a marked induction of EGFR expression was detected in skin Tregs 3 d after wounding (Fig. 4A). Interestingly, EGFR expression was not observed in SDLN Tregs at any time postwounding, suggesting that induction of EGFR occurs preferentially on Tregs in inflamed skin in our model.
To determine whether EGFR expression on Tregs plays a functional role in their ability to facilitate wound repair, we bred Foxp3cre mice (17) with EGFRfl/fl mice (18) to generate animals with a conditional deletion of EGFR only in Tregs (Foxp3creEGFRfl/fl). Consistent with the absence of EGFR expression on Tregs in the steady-state, adult Foxp3creEGFRfl/fl mice had similar percentages of Tregs in the skin and SDLNs compared with age- and gender-matched WT mice (data not shown). In addition, the basal activation of Tregs was similar between Foxp3creEGFRfl/fl mice and WT mice in the steady-state, and Foxp3creEGFRfl/fl mice do not develop de novo skin inflammation (data not shown). Interestingly, Foxp3creEGFRfl/fl mice had significantly delayed kinetics of wound closure compared with Foxp3creEGFRwt/wt mice or WT controls (Fig. 4B). The effects of the EGFR pathway in Tregs were observed early after wounding, with attenuated wound closure seen only on days 3 and 5 postwounding (Fig. 4B). Mechanistically, deletion of the EGFR pathway in Tregs resulted in reduced percentages and absolute numbers of these cells in skin early after wounding (Fig. 4C). In addition, Tregs in skin of wounded Foxp3creEGFRfl/fl mice were less activated, with reduced percentages of cells expressing high levels of CD25 and CTLA-4 (Fig. 4C). Consistent with a role for Tregs in attenuating inflammatory macrophages after wounding, Foxp3creEGFRfl/fl mice had increased percentages of proinflammatory macrophages early after wounding (Fig. 4E).
Taken together, our data support a role for Tregs in attenuating wound-associated inflammation and facilitating skin wound healing. These cells primarily mediate their effects during the inflammatory phase of wounding, and EGFR expression on Tregs plays a role, at least in part, in their regulatory effects in this context. Preliminary data from our laboratory suggest that EGFR expression on Tregs may enhance Treg survival in inflamed tissues (data not shown). Our results add to an emerging body of work showing that the EGFR pathway is important for Treg function in tissues. The EGFR ligand, AREG, is produced by Tregs that infiltrate injured muscle and lung, where it plays a role in enhancing tissue repair (4, 5). In contrast, activated Tregs in mice and humans can express EGFR, and signaling through this receptor enhances their suppressive capacity in vitro (6). Our data are consistent with the notion that Tregs increase EGFR expression in vivo upon induction of tissue inflammation and that signaling through this receptor enhances their function. However, the fact that EGFR deletion in Tregs has a lesser effect than specific ablation of the entire cell subset (i.e., Foxp3-DTR mice) suggests that Tregs utilize more than just the EGFR pathway to mediate their functions in facilitating wound repair.
In skin wound healing, current dogma suggests that the EGFR pathway mediates its effects on nonlymphoid parenchymal cells, such as fibroblasts, keratinocytes, and endothelial cells. However, our data suggest that this pathway also augments Treg function. It is interesting to speculate that the adaptive immune system has co-opted this highly conserved and vital pathway in skin to regulate tissue inflammation, in an attempt to facilitate proper repair. Therapeutic approaches to treat chronic skin ulcers have focused on augmentation of EGFR signaling (3). Our data suggest that these strategies may work, in part, by augmenting cutaneous Treg function. Conversely, ineffective Tregs may predispose to chronic nonhealing ulcers. Thus, local therapeutic manipulation of Tregs may be a novel strategy to treat wound-associated inflammation with the potential to expedite healing.
We thank C. Benetiz for assistance with animal husbandry.
This work was supported primarily by a Scleroderma Research Foundation Grant, National Institutes of Health Grant K08-AR062064, Burroughs Wellcome Fund CAMS-1010934, National Institutes of Health Grant R21-AR066821, National Institutes of Health Grant DP2-AR068130, and a National Psoriasis Foundation Translational Grant (all to M.D.R.). A.N. was supported by the René Touraine Foundation, the Philippe Foundation, and the Santé Immunologie Vaccination Association. Flow cytometry data were generated in the University of California, San Francisco Parnassus Flow Cytometry Core, which is supported by a Diabetes Research Center grant, National Institutes of Health Grant P30 DK063720. Histology was performed with assistance from the University of California, San Francisco Mouse Pathology Core, which is supported by National Institutes of Health Grant 5P30CA082103-15.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.