Regulatory T cells in skin injury: At the crossroads of tolerance and tissue repair

Structure and function of skin

Mammalian skin is composed of two layers, the epidermis and dermis, situated above a layer of adipose tissue and fascia, known as the subcutis, hypodermis, and/or dermal white adipose tissue. The skin forms a physical barrier between the host and the external environment, which protects against mechanical and chemical insults, pathogens, and regulates insensible water loss. The primary cellular constituent of the epidermis is keratinocytes, which undergo sequential differentiation and upward migration from basal layer stem cells, terminating in an anucleate cornified layer that interfaces with the external environment (3). The epidermal surface is studded with hair follicles that undergo homeostatic cycling through resting and growth phases. The latter is mediated by hair follicle stem cells (HFSCs) residing within a region of the follicle known as the bulge (4).

The dermis is a collagen-rich tissue that houses a plethora of stromal and immune cell populations. Dermal fibroblasts produce collagen, elastin, and other components of the extracellular matrix (ECM) that are critical to maintain the structural integrity of skin in the steady state and during skin repair. The subcutis is largely composed of mature adipocytes that have thermoregulatory and mechanoprotective functions. Both the dermis and subcutis are lined with a dense network of blood vessels, neurons, and lymphatics, which form migratory conduits for immune cells and play important roles in skin homeostasis, inflammation, and repair.

Cutaneous wounds and repair

Human skin is subject to a wide spectrum of traumatic injury, ranging from superficial abrasions of the epidermis to blunt/crush injury to deep penetrating wounds that damage both the skin and underlying tissue. In addition, a variety of chronic wounds are associated with vasculopathy and diabetes; however, little is known about the role of the immune system in many of these injuries. We therefore focus on the acute wound healing response, which is most commonly studied in animal models by excising circular skin biopsies to create full-thickness wounds. These wounds initiate a repair response that results in epidermal regeneration and dermal scarring, with a relative loss of adnexal structures (e.g., hair follicles and sebaceous glands) (5).

The process of wound repair is classically divided into four phases: (i) hemostasis, (ii) inflammation, (iii) proliferation, and (iv) remodeling (see Fig. 1) (6). In the first phase, traumatic damage results in endothelial cell injury and activation, leading to platelet activation, initiation of the coagulation cascade, and formation of a fibrin clot over the wounded area. This sequence of events prevents excessive red blood cell loss from the circulation and provides a scaffold for wound repair. The second phase is dominated by neutrophils, which rapidly traffic into the wound bed and reach maximal numbers at 24 to 48 hours after injury (7). Molecules released by degenerating cells immediately after injury known as damage-associated molecular patterns (DAMPs) result in the secretion of neutrophilic and macrophage/monocyte chemoattractants (8). Neutrophilic inflammation is a double-edged sword: Although necessary to eliminate offending microbes, it has the potential to inflict collateral damage to the tissue through production of reactive oxygen species, nitric oxide, and neutrophilic extracellular traps (9). During the inflammatory phase, tissue-resident macrophages and monocytes become activated by DAMPs and alarmin cytokines such as the interleukin-1 (IL-1) family members IL-1α/β. The quantity and quality of this response can have lasting impacts on proliferative and remodeling phases that follow (10). Macrophages prevent infection of the wound environment by engulfing pathogens and apoptotic/necrotic cells. They influence tissue repair by modulating the functions of other immune cells and promoting angiogenesis and fibroblast activation (11). Historically, tissue macrophages have been split into classically (pro-inflammatory/M1) and alternatively activated (reparative/M2) subsets; however, it is increasingly appreciated that this binary classification is oversimplified and that much greater heterogeneity and plasticity exist within the macrophage lineage in vivo. “Pro-inflammatory” macrophages can be activated by a variety of DAMPs and alarmins, which result in the expression of pro-inflammatory mediators including tumor necrosis factor–α (TNF-α), IL-1β, IL-6, IL-8, and IL-12. “Reparative” macrophages are polarized by the cytokines IL-4, IL-13, and IL-10, resulting in the expression of transforming growth factor–β (TGF-β), IL-10, and various matrix remodeling mediators (12). Distinct macrophage subsets fluctuate depending on the different phases of the wound healing response, and perturbation of these subsets can have profound consequences on the quality of the repaired skin (13).

• Download high-res image
• Open in new tab
• Download Powerpoint

During the proliferative phase, keratinocytes, fibroblasts, and endothelial cells increase in number and differentiate and migrate to cover the wound site. Keratinocytes re-epithelialize the wound to replace fibrin clot with regenerated epidermis. Lineage-tracing experiments in mice have shown that several epithelial populations, including stem cells and committed progenitors, can contribute to patch the regenerating epidermis. Interfollicular keratinocytes are the first responders to injury and the primary contributors to re-epithelialization (14). HFSCs that reside in the isthmic/infundibulum region and bulge also undergo differentiation and migration in response to skin wounding at later time points (15). Beneath the regenerating epithelium, fibroblasts from the reticular dermis migrate into the wound bed and deposit collagen and other ECM components to plug the wound (16), initially forming a scaffold of granulation tissue. This phase is also marked by the accumulation of myofibroblasts, which express α-smooth muscle actin (α-SMA) and contract to draw the wound together and decrease its surface area (17). Recent evidence suggests that in deep, full-thickness skin wounds, a subset of fibroblasts within the fascia of the subcutis migrates upward and produces ECM components that act as a provisional scar-like matrix, after which some of these cells differentiate into myofibroblasts (18).

Tissue remodeling is the final phase of the wound healing response and is an attempt to restore the skin’s architecture to the original pre-wounded state, which can last for weeks to years. It is characterized by a diminution in myofibroblast numbers, regression of blood vessels, and a decreased recruitment of immune cells. There is also a notable shift in the production of type 3 to type 1 collagen by fibroblasts (19). The ECM undergoes extensive remodeling by production of growth factors and matrix metalloproteinases, in part by wound-associated macrophages (20). The end result is collagenous scar formation and increased overall strength of the repaired skin. This process comes at the cost of the tissue’s original composition and function, as key components of skin such as hair follicles, sweat glands, and adipocytes are infrequently regenerated (5).

The type 2 immune module: Orchestrator of full-thickness wound healing

Type 2 immunity is tightly integrated with organ homeostasis and directs reparative programs after tissue damage caused by metazoan parasites, venoms, toxins, and mechanical wounding (24). Soon after injury, initial type 2 immune responses are coordinated by tissue-resident cells such as ILC2s and mast cells at the site of damage. Additional effector cells associated with type 2 immune responses, including eosinophils, basophils, CD4⁺ T_H2 cells, and GATA-3⁺ T_regs, are recruited from circulation in subsequent waves (25). This sequence of type 2 immune activation has largely been studied in helminth and allergic models of inflammation and is likely conserved to some degree in wound healing, although further studies are required to fully elucidate this. Healthy skin of naïve C57BL/6 mice contains a population of resident ILC2s that expand after wounding (26, 27); however, T_H2 cells are absent in this tissue at steady state (28), and their accumulation after cutaneous injury has not been quantified. After wounding, a large influx of type 2 immune cells enters the wound bed from circulation, including eosinophils, additional ILC2s, and type 2–polarized T_regs (27, 29, 30). The bolstering of type 2–polarized lymphocytes and myeloid cells promotes both the regeneration of lost tissue and the deposition of fibrotic scar. The net result is to expediently restore the structural integrity of skin at the expense of full regeneration (Table 1).

Type 2 immune cells fulfill their tissue-reparative functions via extensive cytokine-mediated cross-talk with most of the major parenchymal and stromal cell types in the tissue (Fig. 2). Type 2 immune responses are initiated and propagated by the release of cytokines such as TSLP (thymic stromal lymphopoietin), IL-18, IL-25, and IL-33, which act as damage signals and are collectively referred to as “alarmins.” Epithelial cells, fibroblasts, endothelial cells, neurons, and lymphatics are all capable of producing alarmins to varying degrees in different tissues (31). These cytokines are present in basal amounts in healthy tissues, where they support tissue-resident cells like ILC2s (32). However, tissue damage induces a marked increase in alarmin release, which activates tissue-resident sentinels such as ILC2s, licenses the maturation of effector T_H2 cells, and drives bone marrow production and tissue recruitment of myeloid cells such as basophils and eosinophils (33, 34). Many parenchymal cell types, in turn, express receptors for the type 2 signature cytokines IL-4 and IL-13, which drive further release of inducing cytokines in an inflammatory feedback loop and effect profound changes in the cell state of nonhematopoietic cells. After barrier disruption in skin, keratinocyte-derived TSLP and IL-33 induce T_H2 and ILC2 responses (35, 36). In return, amphiregulin (AREG), IL-4, and IL-13 are all capable of promoting keratinocyte proliferation (37, 38), although the necessity of each these cytokines for re-epithelialization during physiologic wound healing is not clear. Stromal cells are likewise capable of activating and responding to type 2 immune cells during homeostasis and inflammation (32). In skin, dermal fibroblasts express the IL-13 receptor complex (39), and IL-13 potently drives myofibroblast activation and increased collagen deposition (25). Wound healing exemplifies the multifaceted effects of type 2 cytokines on skin: After excisional wounding, IL-33 drives both re-epithelialization and dermal collagen production to facilitate scarring, perhaps in part by activating ILC2 cells (27, 40). Although type 2 cytokine signaling is important for optimal wound healing, the relevant cellular sources of type 2 cytokines during wound healing have not been elucidated. Reparative cross-talk between type 2 immune cells and tissues has been best studied in the ILC2 literature, but neither ILC2s nor T_H2 cells have been definitively shown to influence the progression of skin wound healing. Basophils, eosinophils, and mast cells can produce IL-4, are abundant in wounds, and affect tissue repair and fibrosis in other organs (41, 42). The contribution of these cell types to the global type 2 immune response after cutaneous injury merits further study.

• Download high-res image
• Open in new tab
• Download Powerpoint

Alternatively activated macrophages (AAMs; or reparative/M2 macrophages) are the foremost effectors of the type 2 immune response in full-thickness cutaneous wounding. These cells differentiate in response to IL-4 and IL-13, which induce the up-regulation of effector molecules like RELM-α/FIZZ1, arginase-1, and Ym1/2 (43). Ablation of AAMs by deleting IL-4Rα in macrophages causes a variety of defects in wound healing, including delayed wound closure, impaired formation of granulation tissue, and aberrant collagen deposition (44). Macrophages interact extensively with fibroblasts during the wound healing process by driving the proliferation and differentiation of specific skin fibroblast subsets into myofibroblasts (45). AAM-derived RELM-α signals to fibroblasts to direct the proper assembly and cross-linking of collagen fibers in the scar (44). Deletion of the AAM effector molecule arginase-1 likewise impairs wound healing (46). Given the critical role of AAMs in wound healing, a principal function of type 2 lymphocytes is likely to serve as sources of IL-4 and IL-13 to drive AAM differentiation. The relative importance of T_H2 cytokines in promoting wound healing by macrophage polarization versus direct signaling to nonhematopoietic cells remains to be elucidated.

The type 3 immune module: Preserving the integrity of the epidermal barrier

Type 3 immunity evolved to combat extracellular microbial pathogens and maintain homeostasis with commensal microbiota. Type 3 immune responses in skin are thus focused at the interface between the host epidermis and the overlying microbiome. Skin is home to a rich array of specialized tissue-resident lymphocytes expressing the master transcriptional regulator RORγt (retinoic acid–related orphan receptor γt) and capable of producing the type 3 signature cytokines IL-17 and IL-22. These include dermal γδ T cells (47), ILC3s (48), MAIT cells (49), as well as T_H17 and IL-17⁺ CD8⁺ T cells specific for microbial antigens (50, 51). Imaging studies have demonstrated that these cell types predominantly localize within the epidermis or at the dermal-epidermal interface, with high densities near microbiota-rich hair follicles (48, 52). Several different populations of type 3 lymphocytes have been shown to play a role in wound healing by accelerating re-epithelialization and wound closure (49, 51, 53, 54), although the individual role of each subset may be partially compensated for by other cell types.

As with type 2 immunity, signaling between parenchymal cells and immune cells maintains and/or drives activation of type 3 immune responses during the steady state and after tissue injury (Fig. 2). However, the best-characterized mechanisms in skin are largely confined to epidermal-immune cell cross-talk. Epidermal damage exposes the underlying dermis to microbes, resulting in the release of inflammatory cytokines and the activation of type 3 immune cells. Recognition of microbial molecules by Toll-like receptors (TLRs) on keratinocytes and myeloid cells induces secretion of cytokines such as IL-1β and IL-23, which activate local tissue-resident type 3 lymphocytes and prime T_H17 cells in skin-draining lymph nodes (50). After full-thickness wounding or epidermal abrasion by tape stripping, IL-17 from lymphocytes drives the production of neutrophil chemoattractants by keratinocytes to promote neutrophil influx during the inflammatory phase of wounding (55, 56). IL-22 acts on keratinocytes to increase proliferation and inhibit differentiation after epidermal damage (57, 58). Both cytokines are further capable of inducing the production of numerous inflammatory cytokines and antimicrobial peptides (59, 60).

Although type 3 cytokines promote rapid reconstitution of the epidermal barrier, they hinder the formation of healthy epidermis and instead drive acanthosis (keratinocyte hyperplasia) and parakeratosis (abnormal retention of nucleated keratinocytes in the cornified layer) (61). The effect of uncontrolled type 3 immunity on the epidermis is exemplified by topical application of the TLR7 ligand imiquimod, a model of human psoriasis that triggers IL-17 production primarily by dermal γδ T cells (47). This results in a pronounced neutrophilic infiltrate and epidermal acanthosis resembling that of the immature, reparative epidermis observed in wounded skin. Because the psoriatic plaques have highly acanthotic epidermis and can be triggered by physical trauma, some have characterized this disease as an “exaggerated wound healing response” (62). Together, the net effect of type 3 immune activation after skin injury is thus to bolster antimicrobial defense and to rapidly repair the epidermal barrier by strongly promoting epidermal proliferation at the expense of maturation (Table 1). It should be noted that although both type 2 and type 3 immune responses affect the epidermis after cutaneous injury, their cross-regulation is poorly understood.

Participation of other arms of adaptive immunity in wound repair

Type 1 immunity contributes most prominently to the inflammatory stage of tissue repair by promoting antimicrobial defense at the expense of tissue regeneration (Table 1). The production of the type 1 signature cytokine IFN-γ increases for the first several days after full-thickness wounding (63), although the relevant cellular sources of IFN-γ have not been documented. IFN-γ activates macrophages toward an inflammatory phenotype analogous to classically activated (“M1”) macrophages described in vitro (64). IFN-γ–activated macrophages produce elevated levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6, which, in combination with IFN-γ, inhibit angiogenesis and re-epithelialization [possibly in part by sensitizing keratinocytes to apoptosis (65)]. Signaling of IFN-γ to fibroblasts antagonizes profibrotic TGF-β signaling and thus inhibits collagen deposition (63, 66). Wounds of IFN-γ knockout mice therefore exhibit enhanced wound closure, increased granulation tissue, and reduced monocyte infiltration (63).

Dendritic epidermal T cells (DETCs) are another lymphocyte population found in the epidermis of mice (but not humans) that play a well-established role in cutaneous tissue repair (54). These cells express a semi-invariant T cell receptor (TCR) that recognizes an unidentified ligand released from damaged keratinocytes, prompting them to produce reparative factors such as keratinocyte growth factor (KGF/FGF7) and insulin-like growth factor 1 (IGF-1) when activated (54). The hard-wired ability of DETCs to sense damage and directly enact tissue repair is instructive for understanding innate-like reparative functions of conventional lymphocytes in skin, including T_regs.

Skin T_reg suppressive and homeostatic functions

Constant T_reg-mediated immune tolerance to tissue autoantigens is critical for preventing systemic autoimmunity (75). The autoimmune manifestations of T_reg deficiency in skin are particularly severe: Dermatitis driven by massive infiltration of T cells is one of the most salient features of genetic T_reg deficiency (76). Tissue-resident T_regs express higher levels of suppressive molecules than lymphoid T_regs and are believed to be necessary for maintaining immune regulation within tissues. However, the precise mechanisms by which T_regs suppress autoimmunity in specific organs independent of their lymph node–resident counterparts are not fully understood. In skin, secretion of the anti-inflammatory cytokine IL-10 is likely to play a role, because lymph node T_regs express little IL-10, and T_reg-specific deletion of IL-10 predisposes to allergic dermatitis (68). However, the skin phenotype of mice lacking IL-10 in T_regs is considerably milder than that seen in the gut or after systemic T_reg ablation (68, 75), suggesting that these cells may use multiple suppressive mechanisms that can be relatively tissue-specific. The contribution of other mechanisms [e.g., CTLA-4 (cytotoxic T lymphocyte–associated protein 4) expression and IL-2 competition] that skin-resident T_regs may use remains to be elucidated, largely because of the lack of tools for deleting genes specifically in skin T_regs without affecting T_regs in lymphoid organs or other tissues.

In addition to preventing spontaneous autoimmunity, T_reg suppressive function is also critical for mitigating the extent of tissue inflammation after an inciting stimulus. T_regs accumulate alongside effector T cells at sites of infection and contribute to the resolution of inflammation, although this can lead to prolonged pathogen persistence (77, 78). In some cases, the antigen specificity of T_regs during infection has been traced to specific microbial peptides, as in the case of Mycobacterium tuberculosis infections (79). Impairment of T_reg differentiation or suppressive function during infections leads to increased production of effector T cell cytokines and more severe tissue pathology: For example, T_regs that lack the TNF superfamily members CD27 and OX40 develop more severe skin disease after cutaneous infection with Candida albicans (80, 81). Infectious models thus highlight the ability of T_regs to limit collateral tissue damage in situations when some degree of inflammation is desirable. T_regs reprise this role during tissue repair after sterile or noninfectious injury as well, although the antigens that drive T_reg accumulation in these contexts have not been identified (Box 1).

As with other tissue-resident T cell populations, T_regs have nonclassical functions that enable them to participate in organ homeostasis (67). In contrast to T_reg suppressive functions, which are primarily adaptive (i.e., triggered by TCR recognition of cognate antigen), many nonclassical T_reg functions are innate (precipitated by nonspecific tissue signals independent of TCR stimulation), although this division is not absolute. A prominent homeostatic function of skin T_regs is to promote hair regeneration after depilation and during the natural hair follicle cycle (71). This occurs, in part, by direct paracrine signaling of the skin T_reg–expressed Notch ligand Jagged-1 to HFSCs, facilitating their proliferation and differentiation without requiring classical T_reg suppressive capabilities. In many cases, T_regs share tissue adaptations with effector lymphocyte populations to enable joint participation in tissue homeostasis. For instance, in visceral adipose tissue, T_regs, ILC2s, and AAMs are all necessary for maintaining metabolic homeostasis and preventing the development of glucose intolerance, a role that is enabled, in part, by expression of the transcription factor PPAR-γ (peroxisome proliferator–activated receptor–γ) in both T_regs and AAMs (86). Several lines of evidence suggest that reparative functions of skin T_regs are shared with other resident lymphocytes and primarily function innately, whereas suppressive T_reg functions are predominantly (although not always) adaptive and are unique to T_regs during tissue repair. This will be discussed below.

Skin-resident T_regs are polarized toward a type 2 tissue-reparative phenotype

Activated T_regs are highly heterogeneous and are able to differentiate into subsets that mirror the helper T cell subsets T_H1, T_H2, and T_H17. “Type 1” T_regs express the T_H1 lineage master transcription factor T-bet and are required for suppressing T_H1 immune responses; “type 2” T_regs express IRF4 and GATA-3 to suppress T_H2 immunity; and “type 3” T_regs express RORγt, c-MAF, and STAT-3 to suppress T_H17 cell responses (69, 87). By expressing T helper lineage transcription factors, T_regs co-opt portions of T helper transcriptional modules, likely enabling them to adopt localization and tissue-specific adaptations similar to helper T cell and ILC subsets. T_reg polarization is thus necessary both for suppressing inflammation caused by specific T helper subsets and for participating in organ homeostasis alongside helper T cells and ILCs.

Among all reported organs, skin bears the highest proportion of T_regs expressing the type 2 lineage defining transcription factor GATA-3. Different publications report that 50 to 80% of skin T_regs are GATA-3⁺ at baseline (52, 88, 89). GATA-3 and other coexpressed type 2–associated transcription factors are necessary both for T_reg-restraint of type 2 inflammation in skin and for participation in tissue repair. Mice with a T_reg-specific deletion of GATA-3 gradually develop a lymphoproliferative disease characterized by T_H2-driven inflammation in skin and gut, although disease severity varies somewhat by mouse facility (52, 90, 91). Notably, patients who lack functional T_regs because of genetic mutations in FoxP3 also develop severe atopic dermatitis (AD), underscoring the role of T_regs in suppressing type 2 skin inflammation (76). GATA-3 promotes T_reg suppressive function in part by binding to the CNS2 (conserved non-coding DNA sequence 2) regulatory region of FoxP3 and stabilizing FoxP3 expression, which up-regulates T_reg signature genes such as ICOS (inducible T cell costimulator) without inducing T_H2 cytokine expression (90, 91). GATA-3 also represses T_reg polarization toward other T helper–like fates by repressing non–type 2 lineage transcription factors like T-bet and RORγt (92). Accordingly, loss of GATA-3 in T_regs results in T_reg instability and IL-17 production (90, 93). GATA-3 likely also promotes the expression of T_H2 chemokine receptors that facilitate localization of GATA-3⁺ T_regs near T_H2 cells at sites of inflammation, as has been observed in T-bet⁺ type 1 T_regs (94).

In addition to suppressing type 2 skin inflammation, GATA-3⁺ T_regs acquire innate tissue-reparative functions that are shared by T_H2 and ILC2 cells. These cells, which also require GATA-3 for their transcriptional programs, are able to sense tissue damage in a variety of organs by expressing receptors for the alarmins TSLP, IL-18, IL-25, and IL-33 released from nonhematopoietic cells upon stress or damage (31). In skin (89), wounded muscle (84), visceral adipose tissue (95), and several other organs, GATA-3⁺ T_regs express the IL-33 receptor ST2, which enables them to enact reparative functions in response to IL-33 released by stressed and dying cells. This method of T_reg activation is independent of TCR stimulation (Fig. 3) (96). In addition to activating innate T_reg functions, IL-33 is also a T_reg trophic factor that is necessary for maximal T_reg accumulation in organs like visceral fat (95). TSLP is similarly released from keratinocytes during inflammation and has been shown to promote skin T_reg activation (97). IL-18 may be another relevant alarmin in skin, as skin ILC2s and CD8 T cells were recently shown to be activated by this cytokine (26, 52). T_reg tissue repair can be innately triggered by sensing of IL-18 in vitro (96), although T_reg expression of the IL-18 receptor complex in skin has not yet been reported. The type 2 T_regs that predominate in skin are thus rewired to use tissue-reparative elements of the type 2 immune module.

Provenance and accumulation of T_regs in wounded skin

Wounding is a highly inflammatory process that results in the recruitment of many circulating immune cells into injured tissue (as outlined for skin above). Even in tissues that normally do not contain resident T_reg populations such as the brain and muscle, injury causes a marked influx of T_regs necessary for optimal organ repair (84, 98). T_regs accumulate in skin after either epidermal abrasion or full-thickness wounding, reaching their maximal levels ~1 week after injury (30, 55). The relative contributions of T_reg recruitment from circulation versus local proliferation of the resident pool are currently unknown. In other settings, inflammation drives lymphocyte recruitment from circulation, local proliferation of tissue-resident cells, and egress of tissue-derived cells into the lymph (99). T_reg accumulation in wounded skin is likely a combination of all these factors, although it is unclear whether there are functional differences between T_regs derived from these different sources. T_regs recruited into injured muscle share many of the same reparative transcriptional signatures as nonrecirculating tissue-resident T_regs in visceral adipose tissue, suggesting that regardless of ontogeny, T_regs in wounded tissues can ultimately achieve similar reparative capacities (84, 100). However, there may be temporal or spatial differences between tissue-resident and tissue-infiltrating T_regs. Skin-resident T_regs are already present in the tissue and poised to respond quickly to injury, whereas T_regs derived from circulation likely require time to enable their activation, differentiation, and migration to the injury site. Thus, T_regs that are recruited to skin after injury may play more of a reinforcing role.

Skin repair by direct T_reg–parenchymal cell interactions

Secretion of AREG, a ligand for the epidermal growth factor receptor (EGFR), is a major mechanism of tissue repair shared by many type 2 immune cells including GATA-3⁺ T_regs (38). T_reg expression of AREG is driven innately in response to IL-18 and IL-33 (96), and binding of AREG to EGFR induces proliferation and differentiation of target cells to regenerate tissue lost after injury. For instance, in wounded muscle, AREG expressed by reparative T_regs drives the regeneration of myofibers from satellite cell progenitors (84). Although AREG is expressed by many cell types including T_H2 cells, ILC2s, macrophages, and basophils (Fig. 3), T_reg expression of AREG plays a nonredundant role in some contexts, as mice with a T_reg-specific ablation of AREG exhibit more severe lung pathology and higher mortality rates after influenza infection (96). Although T_reg expression of AREG has not been functionally dissected in skin injury models, several lines of evidence argue for a role. Skin-resident T_regs express AREG (89), and AREG drives proliferation of keratinocytes (38), consistent with its effect on other epithelial tissues. In addition to its effects on the epithelium, AREG has been shown to restore vascular integrity by augmenting pericyte activation of TGF-β, suggesting that AREG-mediated signaling through EGFR may promote reparative programs in stromal compartments after skin wounding (101). T_regs in wounded skin express the AREG receptor EGFR, and T_reg-specific ablation of EGFR results in decreased T_reg numbers and slower wound closure, hinting at an autocrine function of AREG in promoting T_reg accumulation during injury (Fig. 3) (30).

• Download high-res image
• Open in new tab
• Download Powerpoint

In addition to AREG, T_regs have been reported to express a variety of other growth factors that act directly on nonhematopoietic cells to promote repair after injury. T_regs express KGF to promote alveolar regeneration by type 2 alveolar epithelial cells after lung injury (102), and colonic T_regs perform a similar function by secreting FGF2 to increase proliferation of the intestinal epithelium during colitis (103). In zebrafish, T_regs facilitate regeneration of a variety of organs after injury, with one predominant growth factor driving repair in each organ [NTF in spinal cord, NRG1 in heart, and IGF-1 in retina] (104). Notably, KGF and IGF-1 are among the factors secreted by DETCs to promote epidermal regeneration after skin wounding (54). Thus, it is quite possible that skin T_regs also use these mechanisms to promote repair after skin injury.

Regulation of skin repair by T_reg suppressive functions

Although inflammation early in the wound healing process is necessary to prevent infection, excessive inflammation is counterproductive to wound healing. Therefore, the classical function of T_regs—suppressing inflammation—is critical in regulating the repair process. Early during wound healing, T_regs are necessary for the transition from the inflammatory to the reparative stages of healing. T_reg depletion immediately after wounding delays wound closure and increases the amount of granulation tissue and eschar formed in the wound bed (30). This effect is due to the accumulation of IFN-γ⁺ T effector cells in T_reg-depleted skin, which increases the accumulation of pro-inflammatory Ly6C⁺ monocytes (Fig. 4). T_reg suppression of type 1 immunity facilitates the transition from the inflammatory phase to the reparative phase of wound healing, during which AAMs predominate and drive tissue remodeling. Some reports further suggest that T_regs can direct AAM polarization by secreting IL-4 and IL-13 (105). T_reg-derived IL-13 has also been proposed to increase macrophage efferocytosis (106), a key feature of AAMs that enables clearance of debris (107). However, in vivo secretion of T helper cytokines by T_regs is controversial and may only occur in pathologic situations that destabilize T_reg cell state (108). Further studies using in vivo cytokine reporters and T_reg-specific cytokine knockouts are therefore needed to validate T_reg cytokine production as a mechanism of tissue repair.

Skin T_regs were recently shown to suppress myofibroblast activation at steady state and during experimental models of cutaneous fibrosis (88), suggesting that these cells may prevent excessive scarring by restraining type 2 immunity during later stages of wound healing (Fig. 4). GATA-3 expression by T_regs is necessary to prevent excessive myofibroblast activation and collagen deposition, and T_reg-specific deletion of GATA-3 leads to the accumulation of T_H2 cells in skin that robustly produce the profibrotic cytokines IL-4 and IL-13 (88). Because fibrosis results from dysregulation of normal wound healing programs, GATA-3⁺ Treg–mediated suppression of T_H2 cells is likely to regulate physiologic wound healing as well as pathologic fibrosis.

T_reg suppression of type 3 immunity is important for re-epithelialization during the initial stages of tissue repair. In a tape-stripping model of epidermal abrasion, T_reg depletion causes delayed recovery of barrier function (55). In the absence of T_regs, T_H17 cells accumulate in skin and cause increased expression of the neutrophil chemoattractant CXCL5 (55). Barrier damage normally prompts Lgr5⁺ stem cells within the hair follicle bulge to proliferate and migrate into the interfollicular epidermis to regenerate the epithelium (109). However, the exuberant CXCL5–IL-17 inflammatory response to tape stripping in T_reg-depleted mice impedes bulge HFSC differentiation and migration into the interfollicular epidermis, possibly accounting for the defect in barrier function recovery after T_reg depletion (55). These results demonstrate an indirect role for T_regs in promoting normal epidermal regeneration from stem cells by attenuating excessive type 3 immunity (Fig. 4).

In addition to T_reg suppression of subset-specific helper T cell responses, two broadly anti-inflammatory and highly pleiotropic cytokines secreted by T_regs—IL-10 and TGF-β—have been extensively implicated in wound healing (110). IL-10 is a potent suppressor of both inflammation and fibrosis, and global loss of IL-10 results in both faster wound closure and increased scar formation due to elevated myofibroblast accumulation (111). T_reg expression of IL-10 is substantially higher in skin than in lymphoid organs (89), and T_reg-derived IL-10 is crucial to preventing excessive inflammation in other contexts (68). It is therefore plausible that T_reg-derived IL-10 contributes to the suppression of excessive fibrosis after cutaneous wounding.

TGF-β is perhaps the most potent profibrotic cytokine and is a master regulator of numerous reparative processes including physiologic scar formation and pathologic fibrosis (110). T_reg involvement in TGF-β–mediated reparative processes can be conceptually divided into three aspects: TGF-β signaling to T_regs via TGF-β receptors, T_reg secretion of TGF-β, and T_reg activation of latent TGF-β. TGF-β is required for the generation of both thymic and peripherally generated T_regs and helps maintain T_regs (along with numerous other immune cells) in tissues (112). In line with these data, skin T_regs express higher levels of TGF-β receptors than lymph node T_regs. Thus, TGF-β may play a role in maintaining skin T_regs and/or promoting their suppressive activity during wound healing. T_regs themselves produce TGF-β in many organs, but it is important to note that this alone does not constitute evidence of a profibrotic role for T_regs. Many cells secrete TGF-β, and T_reg contribution to the tissue pool of TGF-β is likely to be small (Fig. 3). It is further possible that TGF-β secreted by T_regs primarily acts to maintain T_regs in cis. The difficulty in elucidating the function of T_reg production of TGF-β is exemplified by studies of this cytokine’s role in T_reg suppressive function, which conflict as to whether T_reg-derived TGF-β is required to suppress inflammation in vivo (112). An additional complexity is that TGF-β can be presented on the cell surface or deposited on ECM in a latent form and must be activated by proteases or integrins expressed in trans (113). The function of TGF-β from any given cell type is thus dependent on the mode of presentation, the timing of cytokine activation, and the recipient cell identity. T_regs additionally express the TGF-β–activating integrin α_vβ₈, and T_reg expression of β₈ integrin is necessary for optimal suppression of inflammation in the intestine (114). It is therefore possible that T_regs activate latent TGF-β after tissue injury. Thus, although T_regs sense, secrete, and activate TGF-β in a variety of inflammatory contexts, the in vivo significance of all three functions during tissue repair requires further investigation.

• Download high-res image
• Open in new tab
• Download Powerpoint