Immunology of Wound Healing

Neutrophils represent the most abundant inflammatory cells to infiltrate a new wound and function mainly to remove debris and prevent infection [22, 23]. Their influx is mediated by a number of chemical signals, including IL-8, or CXCL8, as mentioned above, and neutrophils have over 30 different receptors that mitigate their migration and activation response [24]. It is clear that neutrophils do function in debris removal early in wound healing, but their persistence, as will be discussed in detail below, has been associated with delayed wound healing and chronic wounds. Moreover, mouse models of wound healing have shown that in non-aged, non-impaired models, neutrophil depletion does not negatively affect wound healing as profoundly as macrophage deletion [25‐27]. In impaired models of wound healing, such as diabetes, where infection risk is higher, neutrophils are clearly required [28].

DAMPs, released from necrotic cells, are thought to be the first signals to recruit neutrophils to the wound bed [29]. These danger signal molecules can activate neutrophils directly by binding various neutrophil surface receptors, in addition to signaling tissue-resident cells to produce neutrophil chemoattractants [23, 30].

One of the most well-described chemoattractants produced by tissue-resident macrophages and fibroblasts is CXCL8 (IL-8) [31]. CXCL8 binds and stimulates neutrophil surface receptors CXCR1 and CXCR2, leading to avid recruitment of neutrophils to the site of tissue injury [32, 33]. Interestingly, once neutrophils migrate into the wound they are also able to secrete CXCL8, creating a pro-inflammatory feedback loop [34]. Endothelial permeability is also increased by CXCL8, further encouraging inflammatory cell influx into the wound site [35]. Other CXCL8 family chemokines, such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7, have also been shown to play a role in neutrophil chemotaxis [18, 34, 36]. By binding glycosaminoglycans on tissue cell walls and in the extracellular matrix, these chemokines, including CXCL8, create a signaling gradient to allow for clear directional migration of neutrophils towards the injury [18, 34, 37, 38]. Additional DAMP-induced cellular byproducts, such as hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄), also form gradients to drive focused migration of neutrophils [23].

Although neutrophils are not considered an essential cell type in non-impaired wound healing, they do carry out a variety of functions that support the process [27, 39]. First and foremost, neutrophils defend against wound infection by phagocytosing pathogens then killing them through release of reactive oxygen species, proteases, or antimicrobial proteins [28]. With degranulation, antimicrobial proteins can also be released into the surrounding milieu to destroy extracellular organisms [40]. More recent evidence indicates that neutrophils can also eliminate organisms residing in the extracellular environment through the deployment of neutrophil extracellular traps (NETs). NETs are web-like structures comprised of strands of decondensed chromatin bound to neutrophil-produced bactericidal proteins. They work by either directly killing microorganisms or via immobilizing pathogens to facilitate phagocytosis [41, 42].

In addition to clearing pathogens, neutrophils also regulate inflammation and generate growth factors and cytokines to induce wound healing. In the wound environment, neutrophils have exhibited the ability to upregulate gene expression of chemokines that are key recruiters of macrophages, T cells, and additional neutrophils, such as TNF-α, IL-1β, IL-6, CXCL8, CXCL2, and monocyte chemoattractant protein-1 (MCP-1) [16, 34, 43]. Neutrophils also show increased expression of cytokines that promote angiogenesis [e.g., vascular endothelial growth factor (VEGF), CXCL3, and MCP-1], proliferation of fibroblasts and keratinocytes (IL-8, IL-1 β, and MCP-1), adhesion of keratinocytes to the dermal layer (laminin 5 β-3), and tissue remodeling [urokinase-type plasminogen activator (uPA)] [34, 43‐45].

While neutrophils do play an important role in propagating the inflammatory response in the early stages of wound healing, they also serve as a signal to inactivate the inflammatory phase [46]. In physiologic wound repair, neutrophils undergo apoptosis after carrying out their various functions at the site of injury. Local macrophage uptake of apoptotic neutrophils then triggers a transition out of inflammatory phase [47‐49]. More recent studies also indicate that some neutrophils may actually undergo reverse migration, away from the site of injury and back into circulation. This is called reverse transendothelial migration (rTEM), and serves two potential functions: a mechanism to resolve local inflammation and/or a mechanism to redistribute activated neutrophils to other locations in the body, leading to inflammation at other sites [23].

Although the recruitment of neutrophils is crucial in host protection, the associated robust inflammatory response may also be detrimental to proper wound healing [28, 50, 51]. Many studies suggest that the prolonged presence of neutrophils and their associated inflammatory mediators in the wound milieu contributes to the formation and persistence of chronic wounds. For example, neutrophil-derived proteases, such as elastase and matrix metalloproteinases (MMPs), can degrade healthy extracellular matrix (ECM), and increased levels of these proteases have been repeatedly detected in chronic wounds [52‐56]. Neutrophils can also generate deleterious levels of reactive oxygen species in chronic wounds, damaging cell membranes and causing additional destruction of the ECM. This destruction encourages additional production of inflammatory mediators (e.g., IL-1, TNF-α) and proteolytic enzymes (e.g., MMPs), propagating a cycle of inflammation amplification [28, 57]. Thus, it comes as no surprise that chronic wounds demonstrate significantly increased levels of the potent neutrophil chemoattractant, CXCL8 [58].

NETs have also been detected in excess in diabetic foot wounds and have been shown to delay healing, and inhibition of NETosis and NET function in mouse models of delayed wound healing improves outcomes [59•]. In sum, the sustained and inappropriate presence of neutrophils at the site of injury is a major contributing factor in non-healing wounds.

Macrophages play a critical role in wound healing, and their roles in angiogenesis, fibroplasia, cell proliferation, and transition out of the inflammatory phase is clear (Fig. 2) [25‐27, 60]. At baseline, macrophages are phagocytic monocyte-derived cells that constitutively scavenge and remove dead cells, necrotic tissue, and toxic metabolites from the tissues [61]. However, after injury, these homeostatic functions are amplified by a variety of stimuli in order to facilitate tissue repair.

With cutaneous injury, local, skin-resident macrophages become activated via danger signals and other injury-by-product molecules (e.g., H₂O₂), while monocytes in circulation exit blood vessels and enter the wound site. It was previously thought that neutrophils were the only inflammatory cells infiltrating a wound immediately after injury; however, a recent study demonstrated that a surge of monocytes enter the wound bed simultaneously, traveling through sites of vascular leakage [62, 63].

DAMPs and PAMPs (released from necrotic tissue and pathogens, respectively), as well as interferon-γ [(IFN-γ), released from natural killer cells] polarize macrophages into a pro-inflammatory phenotype [64]. These inflammatory macrophages are often referred to as classically activated macrophages, or the M1 phenotype [65]. This phenotype secretes pro-inflammatory cytokines, such as IL-1β, IL-6, IL-12, IL-23, and TNF-α, as well as chemokines that induce increased natural killer cell, macrophage, and helper T cell responses [65‐67]. These inflammatory amplification molecules are essential, because prior to injury, the skin has relatively few resident macrophages, and the majority of wound-related macrophages are derived from intravascular monocytes [68, 69]. In addition to signaling recruitment of leukocytes to the wound bed, these M1 macrophages demonstrate an increased capacity to destroy and phagocytose microbes and cellular debris to keep the site of injury clean [70‐72].

Macrophages in the wound site are responsible for phagocytosis of apoptotic neutrophils, a process known as efferocytosis [73••]. This action itself induces macrophages to transition from a pro-inflammatory phenotype, to an anti-inflammatory phenotype, often referred to as M2 or “alternatively activated” macrophages [47]. Rather than be considered distinct M1 and M2 cell types, it is better to characterize these macrophages as existing on a spectrum of activation between pro-inflammatory and anti-inflammatory [74]. Other mediators that induce this transition include glucocorticoids, IL-10, prostaglandins, the IL-4/IL-13 pathway, and engagement of specific toll-like receptors (TLRs) [26, 60, 75]. Interestingly, while IL-4/13 are the primary signals to induce this phenotype in vitro, recent studies have shown that they are not required in vivo [26, 76]. More recent work has demonstrated roles for regulatory T cells (Tregs), adenosine signaling, and microRNAs (miRNAs) [10••, 77, 78]. Anti-inflammatory macrophages represent a more heterogeneous cell population, and are comprised of all macrophages that do not have the pro-inflammatory phenotype [79]. These cells tamper down inflammation and stimulate tissue repair by generating anti-inflammatory molecules such as IL-1 receptor antagonist and IL-10, as well as growth factors that promote ECM synthesis, angiogenesis, and fibroblast proliferation, such as transforming growth factor-β (TGF-β) and VEGF [80]. Transitioning from a pro-inflammatory macrophage-dominant wound to an anti-inflammatory macrophage-dominant milieu is essential in resolving inflammation and preparing the wound for effective repair [72].

When the transition from a pro- to an anti-inflammatory macrophage phenotype is impaired, wound healing stalls in the inflammatory phase and a chronic wound results [10••]. Certain signals are known to prolong the presence of pro-inflammatory macrophages, including iron overload, which is commonly seen in the skin in the setting of venous stasis [81, 82].

In fact, in a mouse model of iron overload, local wound macrophages persisted in a pro-inflammatory state with excess production of inducible nitric oxide synthase (iNOS), IL-12, and TNF-α, in addition to free radicals, leading to impaired wound healing [81]. In addition to increased pro-inflammatory molecule expression, iron-overloaded macrophages showed decreased levels of anti-inflammatory markers such as IL-4, IL-10, and CD206 [81]. Macrophages from patients with chronic, non-healing, wounds have been shown to have higher levels of iron, based on Prussian blue staining [82, 83].

Additional signals, such as hyperglycemia in the setting of diabetes, hypoxia in the setting of arterial or venous insufficiency, or secondary infection likely also contribute to the persistence of pro-inflammatory macrophages.

Remodeling starts several weeks after wounding, and continues for up to 1 year. It marks the transition from granulation tissue to scar, which involves the slowing of angiogenesis and replacing type III collagen in granulation tissue with stronger type I collagen. It should be noted that fully mature scars return to only 80% of their initial tensile strength [10••]. This remodeling phase is largely driven by myofibroblasts, which develop from fibroblasts in response to both mechanical tension and TGF-β signaling and are responsible for contraction of the wound [94].

Myofibroblasts express smooth muscle actin (SMA), which is responsible for generating the contractile force attributed to this cell type [94, 95]. In addition to contraction of wound beds and generation of collagen, myofibroblasts contribute to remodeling by release of MMPs that degrade collagen laid down during granulation tissue formation [96, 97]. Conventional dogma states that myofibroblasts are terminally differentiated and undergo apoptosis following wound remodeling. However, exciting new research indicates that these wound bed myofibroblasts may further differentiate into fat cells, replenishing subcutaneous adipose tissue. This process is dependent upon neogenic hair follicles, which lead to bone morphogenic protein (BMP) signaling and activation of adipocyte transcription factors [98••].

When scar formation is excessive, the scar itself can lead to pruritus, pain, or a disfiguring appearance. While excess inflammation may lead to either keloid or hypertrophic scar formation, there are several key clinical differences. Hypertrophic scars arise within 1–2 months of injury, and tend to arise in areas of high tension. Keloid scars can occur at any point post-injury, do not tend to occur in areas of high tension, and may grow beyond the borders of the initial scar [99].

Normally, myofibroblasts carefully coordinate the breakdown of granulation tissue and replacement with long-lasting type I collagen [100]. Recent evidence indicates that myofibroblast-induced fibrosis can be over-activated, not only in the setting of signaling through TGF-β, but also in the setting of Th2-derived cytokines IL-4 and IL-13; Th1 cytokines, such as IFN-γ, attenuate excessive scar formation [100, 101]. In fact, clinical trials are currently underway to explore anti-IL-4/IL-13 therapies in pulmonary fibrosis [101]. A role for these therapies in cutaneous fibrosis remains to be explored, though in vitro studies have shown that blocking signaling of the related cytokine IL-10 in fibroblasts may decrease keloid scar formation [102].