Autophagy is the key to making chronic wounds acute in skin wound healing

ABSTRACT

As a highly regulated and dynamically balanced intracellular degradation mechanism, macroautophagy/autophagy plays an essential housekeeping role in different successive stages of skin wound healing; from the homeostasis and inflammatory stages to the proliferative and remodeling stages. Under both progressive and defective skin wound healing conditions, autophagy operates at different levels with a precise extent of activity, at the interface of inflammation, stress signaling and cell metabolism through a complex spatiotemporal cascade of molecular and cellular events. Depending on the wound healing conditions autophagic activity is fine-tuned and differentially modulated at each stage of skin wound healing in order to cope with stage-specific requirements. Here, we postulate that under favorable conditions autophagy may act as the key modulator of skin wound healing by making chronic wounds acute. Enhancing autophagy through the topical application of pro-autophagy biologics in an appropriate hydrating vehicle/moisturizing base such as hydrogels, onto a chronic skin wound may provide moisture and immune modulation, thus contributing to rapid and efficient skin wound healing. A moist environment is more conducive to skin wound healing as it helps to not only accelerate cell proliferation and migration, and extracellular matrix reorganization, but also promotes autophagy and reduces the incidence of inflammation.

Abbreviation: AKT: AKT serine/threonine protein kinase; ECM: extracellular matrix; FN1: fibronectin 1; LAM: laminin; MMPs: matrix metallopeptidases; MMP2: matrix metallopeptidase 2; MRSA: methicillin-resistant Staphylococcus aureus; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; TNF/TNF-α: tumor necrosis factor.

Background

Healthy human skin is composed of three primary layers that are structurally distinct but functionally interdependent: (1) the outermost epidermis; (2) the middle dermis; and (3) the innermost hypodermis [1,2]. Their individual positioning within the skin, as well as their complex and delicate layer-specific composition, undoubtedly determine their contribution to the skin’s overall function and integrity [1–4]. Such contributions enable the skin to perform several vital physiological functions, synergistically manifested through protection, thermoregulation and sensation [1]. When the skin encounters any disruption or imbalance in its structural and functional integrity [1,3,4] either by pathogen infiltration, ultraviolet (UV) radiation or injury, it can rapidly lead to damage of the protective barrier, thus impairing sensation and thermoregulation, and resulting in extensive water loss and microbial infection [5].

Wound healing is a physiological process that involves three distinct but overlapping sequential stages: (1) homeostasis and inflammation; (2) cell proliferation and tissue replacement; and (3) remodeling, maturation and resolution [6–10]. When skin wounding occurs, the skin recruits and ignites a cascade of endogenous defense mechanisms. Of these diverse mechanisms, autophagy is essential for homeostasis, degradation and recycling of the skin and its components [11–13] as it contributes significantly to the overall wound healing process [7] responsible for the regeneration of injured and damaged skin [8,10,14]. Explicitly, in the initial stage of wound healing, the homeostatic/inflammatory stage, autophagy has an anti-infection role, and exhibits an anti-inflammatory response, thus preventing excessive inflammation and infection from causing tissue damage [10]. In the proliferative stage, inadequate oxygen levels (hypoxia) in the wound can induce autophagy, which plays a role in anti-oxidative stress and promotes cell survival by reducing apoptotic cell death. In the final stage of wound healing, the remodeling stage, autophagy promotes wound angiogenesis of endothelial cells, and differentiation, proliferation and migration of keratinocytes and fibroblasts, which is conducive to the completion of wound repair and reconstruction [10].

Autophagy is the key to turning chronic wounds into acute ones

The process of wound healing is governed by a spectrum of complex and highly dynamic interactions between different cell types (e.g., macrophages, immune cells, fibroblasts, keratinocytes) [8,10,14], growth factors [8,10,14], the extracellular matrix (ECM) proteins (e.g., FN1 [fibronectin 1], LAM [laminin], COL [collagen], ELN [elastin], and MMPs [matrix metallopeptidases]) [15,16] and inflammatory mediators (e.g., cytokines) [8,10,14], involving numerous signaling pathways [8,10,14]. Under optimal wound healing conditions (e.g., appropriate moisture control, presence and balance of required factors and mediators) [17,18], wounds will completely heal in a timely and organized manner, both functionally and anatomically, usually within three months after the initial injury [9]. This type of wound is known as an acute or non-chronic wound [9]. A delay of such orderly spatiotemporal wound healing progression in any shape or form, in any of the healing stages, especially in the inflammatory stage, causes the skin to lose some of its functional and structural properties as it leads to the development of a non-healing, chronic wound [9] such as pressure ulcers, and diabetic leg and foot ulcers [9,19]. The development of a chronic wound is usually caused by a range of unfavorable wound healing parameters: (1) a lack of moisture [17,18]; (2) an overabundance of inflammatory cytokines such as tumor necrosis factor (TNF/TNF-α) [14]; (3) elevated accumulation of MMPs (e.g., MMP2) responsible for the degradation of the ECM components [14]; (4) restricted angiogenesis [14]; and (5) impaired autophagy modulation [10,20]. Such parameters can influence wound healing by preventing the commencement of the proliferative stage [14], which in turn can result in a persistent, self-perpetuating and aberrant inflammation and prolonged healing time, usually associated with pain [6,9]. Therefore, it is plausible to suggest that addressing the two main limiting factors of chronic wound healing, namely, (1) lack of moisture and (2) excessive inflammation, by applying an appropriate moisturizing base (e.g., hydrogel) containing anti-inflammatory biologics (e.g., corticosteroids such as mometasone furoate) and/or antimicrobials, can potentially initiate a positive response in terms of autophagy-mediated modulation, thus resulting in healing of a previously chronic wound (Figure 1).

Figure 1.

Appropriate hydration and immunomodulation are hypothesized to modulate autophagy, which in turn acts as a “switch” to turn a chronic wound to an acute one, facilitating healing and recovery, and ultimately leading to wound resolution. Conversely, a lack of hydration and immunomodulation at the wound site prevents the modulated autophagy-induced switch from chronic to acute wounds [5,6,10,17,18,20–25]. Schematic diagram of progression steps of autophagy-mediated modulation in skin wound healing was created with BioRender (https://Biorender.com/).

Autophagy is a ubiquitously conserved, mechanistically diverse, multistage process (Figure 1) involved in the degradation and recycling of superfluous and potentially dangerous cargoes (e.g., harmful protein aggregates, damaged proteins and organelles, aberrant cellular components, invading pathogens) [26–28]. Under “stress-free” physiological conditions, autophagy plays an important role in maintaining stable cellular homeostasis. However, under adverse conditions, autophagy provides a coping defense mechanism that is indispensable in the prevention of nutritional, metabolic and infection-mediated stresses [10,28,29]. Crucially, autophagy regulates wound healing at all three stages [10,20] through its pleiotropic roles [10,20,30]. During the transient inflammatory stage, autophagy exerts anti-inflammatory and anti-infective effects by enhancing activity of neutrophils and by contributing to the conversion of pro-inflammatory macrophages (M1 type) into tissue repair macrophages (M2 type) [10].

For example, at the inflammatory stage of thermal burn wound healing, autophagic activity influenced by different levels of cytokines (notably IL1, IL6, IL10, and IL18), TNF and IFNG/IFN-γ (interferon gamma) begins to increase after 24 h but it does not reach normal levels until up to 72 h after the burn injury occurs [31]. At this stage, autophagy acts as a molecular pro-survival mechanism – as demonstrated by increased levels of biomarkers of autophagic activity, namely MAP1LC3/LC3 (microtubule associated protein 1 light chain 3; specifically membrane-bound LC3-II) and BECN1 (beclin 1) – that protects cells against stress effects, and even cell necrosis, caused by strong pro-inflammatory cytokines and/or triggered by microbial load [31,32].

At the inflammatory stage, autophagy also directly participates in the degradation of invading pathogens. Key autophagy receptors, such as SQSTM1/p62 (sequestosome 1), CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) and OPTN (optineurin) recognize and recruit ubiquitinated pathogens for degradation [10,26]. In terms of managing bacterial infections in chronic wounds, the interaction between autophagy or more specifically xenophagy (a type of selective autophagy involved in degradation of invading pathogens), their associated molecular machinery and antimicrobial agents (e.g., antibiotics), is more complicated and complex than may be expected. For example, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) requires the continuous development of new antibiotics to overcome the fact that once a pathogen finds its niche and becomes intracellular, newly developed antibiotics may not achieve the expected effect and may have an undesired impact on cell function. Therefore, development of new antibiotics must be explored in order to “defeat” S. aureus [33,34]. S. aureus strains with high “escape system” activity are usually able to escape and replicate intracellularly using autophagy, while strains lacking those escape systems are usually unable to evade the autophagosomes and are eventually degraded through sequential autophagy/xenophagy steps: (1) initiation and phagophore formation; (2) phagophore nucleation and elongation, and invading pathogen engulfment; (3) autophagosome fusion with lysosome; (4) autolysosome formation and (5) cargo degradation and recycling [34] (Figure 1). It seems that autophagy/xenophagy inducers might be beneficial for treating S. aureus infections, but in turn might simultaneously facilitate other bacterial infections [33,34]. Additionally, the ability of S. aureus to escape and survive autophagic/xenophagic degradation pathways is dependent on both the strain and cell type. Treatment with autophagy inhibitors has been shown to reduce S. aureus load, and the induction of autophagy by an autophagy-inducer (rapamycin) restored replication of S. aureus. Therefore, the use of autophagy modulators including antibiotics should be done with the utmost caution [34].

During the proliferative stage, autophagy promotes the differentiation, proliferation and migration of the skin cells, namely fibroblasts and keratinocytes during wound re-epithelization and repair, concomitantly suppressing oxidative damage and apoptotic cell death, and promoting angiogenesis and cell survival [10,20]. A hypoxic microenvironment formed during the early stage of injuries induces the production of abundant reactive oxygen species (ROS), which activates MAPK/p38 (mitogen-activated protein kinase) and MAPK/JNK signaling cascade and upregulates BNIP3 (BCL2 interacting protein 3)-mediated autophagy to promote keratinocyte migration [10]. A recent study suggested that hypoxia-induced autophagy serves not only as a protective mechanism for endothelial cells but also as an inducer of angiogenesis during the recovery of heat-denatured endothelial cells [10,35]. During the remodeling stage, autophagy participates in the fine-tuning of the ECM reorganization by maintaining its deposition and turnover rate, thus allowing the wound to “fully mature” and eventually close with minimal or no hindrance [10,20]. For example, autophagy downregulation (rather than autophagy activation) in fibroblasts induces apoptosis by degrading excessive ECM components, improving overall deposition of ECM at the wound site and inhibiting hypertrophic scarring [10].

To once again look at the effects of autophagy in thermal burn wound healing, keratinocyte growth factor-controlled differentiation at the proliferative and remodeling stages of wound healing triggers LC3 expression and autophagy initiation via the phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase)-MTOR (mechanistic target of rapamycin kinase) signaling pathway, lysosomal enzyme activation and downstream autophagic machinery – as Atg5-deficient keratinocytes are unable to undergo differentiation [31]. Additionally, autophagy levels increase during heat-denatured endothelial cell recovery, depending on intracellular reactive oxygen species generation and overall oxidative stress as well as subsequent initiation of autophagy through AMP-activated protein kinase/AMPK and MTOR signaling pathways resulting in enhanced angiogenesis [31,32].

Recently, multiple transcriptomic data of patients experiencing trauma from different early time points of wound healing (0-, 7- and 14-days post treatment) were systematically investigated [36]. Results showed that a large number of differentially expressed genes (DEGs) were present at 7- and 14-days post treatment compared to day 0, with a total of 226 genes undergoing significant upregulation or downregulation. Those 226 DEGs were intersected with autophagy-related genes to obtain wound healing-related autophagy genes. It was found that a serine/threonine kinase gene, EIF2AK2, and RB1 (RB transcriptional corepressor 1) could be key genes involved in autophagy in trauma healing; (1) EIF2AK2 can play a dual role in inflammation as it can activate the pro-inflammatory pathway and trigger anti-inflammatory activity, whereas (2) RB1 regulates innate immune responses and determines the fate of immune progenitor cells. A further analysis by western blotting validation of blood samples from patients at 0-, 7- and 11-days after treatment revealed that both EIF2AK2 and RB1 were gradually increased with wound healing progression (at day 7 and day 11), suggesting that both of those genes can positively regulate wound healing [36]. Therefore, there is a possibility that similar observations can be true for chronic wound healing progression where autophagy can reduce the production of inflammatory factors, induce wound angiogenesis, re-epithelialization and scar formation, and can also improve the impaired cell activity [10,20].

On the downside, any subtle changes or alterations in the relative balance of autophagic activity (basal vs downregulated vs activated) in an ill-timed manner can lead to a functionally and anatomically “imperfect” wound healing, characterized by two extreme outcomes: (1) persistent chronic wounds or (2) excessive ECM deposition and hypertrophic scarring [10,20]. Nevertheless, it seems that under appropriate conditions (e.g., a moist environment, controlled inflammation and infection – see below) autophagy can act as a modulator that monitors wound healing status and progression in a timely and proper manner. Therefore, autophagy is not only a crucial part of wound healing (as discussed above) but also it seems that impaired autophagy modulation is one of the crucial factors that prevent the switch from chronic to acute wounds (Figure 1).

Improving hydration and reducing inflammation at the wound site is crucial for proper autophagy modulation

Maintaining adequate skin hydration is crucial for the normal functioning of healthy skin as any disturbance of the hydration balance will result in impairment of the skin’s structural and functional integrity [18]. Once skin integrity is compromised, for example by wounding, the mechanisms responsible for maintaining the appropriate levels of skin hydration are significantly challenged and will result in increased dryness and delayed wound healing [17,18]. A moist environment actively supports the wound healing response when compared with a dry environment [17,18,21]; therefore, it is obvious that there is a need for any form of treatment to include a moisturizing base that can provide optimal hydration levels during wound healing. Such optimal hydration levels can provide and promote the following benefits: (1) growth factor retention at the wound site [17,18]; (2) cell proliferation and migration, and tissue re-epithelialization [17,18]; (3) ECM reorganization [17,18,20]; (4) lower infection rate [17,18]; (5) removal of damaging, unwanted and excess skin components [10,17,18]; and (6) reduced pain perception [17,18]. Overall, the ultimate goal is to maintain and safeguard optimal hydration of the skin for the purpose of maximizing wound healing. In practical terms, this can be successfully achieved by topically applying a hydrating vehicle (e.g., cream, ointment, hydrogel) to the wound site to support a hydrated environment and to protect the injury site from additional stress, thus allowing the damaged tissue to heal expeditiously [5,22].

However, keeping the wound moist during the treatment and healing process [17,18,21] is also a balancing act given that moisture can often provide fertile conditions for microbial (both bacterial and fungal) growth. Innumerable Gram-positive, Gram-negative, aerobic and anaerobic bacteria [37,38] as well as different species of fungi (e.g., Candida and Aspergillus species) have been isolated from wound infections [39]. The most common bacterial pathogens implicated in wound infections include S. aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa [37–39]. The pathogenicity and heterogeneity of microorganisms that colonize a wound depend on a variety of host factors (e.g., wound etiology characterized by wound site, size, depth, severity) as well as microbial factors (e.g., virulence, load) [40]. At a critical colonization threshold at which microbial colonization crosses into infection, many pathogenic microorganisms produce virulence factors, toxins, proteolytic enzymes and even superantigens that damage and destroy viable tissues by depleting nutrient-rich substances and killing immune cells. If not mitigated in a timely and proper manner (e.g., by antibiotics), uncontrolled microbial colonization and subsequent infection usually results in an increase in wound-related pain and potentially systemic issues (e.g., sepsis). Importantly in the context of this Commentary, infection can also lead to the formation of persistent chronic wounds. At wound sites where pathogenic microorganisms actively strive for niche possession, these microorganisms may also form persistent biofilms through microbial clustering that firmly attach to the wound surface and form a protective and almost impenetrable extracellular matrix, making them more resistant to active biomolecules including antibiotics [39], thereby exacerbating the chronicity of wounds with impaired healing [38].

Due to the heterogeneous nature of pathogenic microorganisms found in infected chronic wounds [38], hydrogels that can deliver active biomolecules are an attractive delivery vehicle for the treatment of chronic wounds. For instance, hydrogels can be tailored to allow the slow release of active biomolecules such as topical antibiotics over extended periods of time, which could decrease treatment frequency and limit exposure to sub-inhibitory concentrations of those actives [39]. The delivery of antibiotics in tailorable hydrogels may be an effective strategy in keeping the wound area uninfected and reducing the frequency and severity of wound-related pain [41] as well as potentially enabling the autophagy-mediated modulation from a chronic to an acute wound. It is well-known that topical antibiotics are most widely used to treat S. aureus infection. However, S. aureus has rapidly and quite successfully developed resistance to antibiotics, where approximately 90% of S. aureus strains show resistance to multiple antibiotics (e.g., MRSA) resulting in decreased antibiotic application and reduced antibiotic effectiveness. Invasive MRSA strains possess a series of virulence factors and toxins, allowing them to spread rapidly [34], thus impeding wound healing [38]. Therefore, as an alternative way to keep in check “rebellious” S. aureus infection, new targets, including autophagy, have gradually become the prime focus in order to manipulate and enhance host immune defenses. Autophagy, especially its selective type xenophagy, specifically recognizes ubiquitin-coated S. aureus and associates it with its adaptor-receptor degradation machinery, effectively limiting S. aureus growth by fusion with the lysosome. However, some S. aureus strains have evolved self-defense mechanisms against xenophagy, and even evade the xenophagy pathway altogether (as discussed earlier) [34].

This double-edged sword concept regarding xenophagic elimination of invading pathogens is supported by the fact that antibiotics such as isoniazid and pyrazinamide are widely and extensively used against Mycobacterium tuberculosis in non-wound related infections. Both antibiotics, although able to kill the bacteria directly in vitro, have been recently shown to induce autophagy in the host cell promoting mycobacterial clearance. On the other hand, azithromycin was shown to prevent lysosomal function (more specifically acidification), resulting in impaired autophagic/xenophagic degradation of mycobacteria. This suggests that continuous use of this antibiotic may predispose the host to mycobacterial disease. Therefore, it seems that during bacterial infection and its treatment with antibiotics, there exists a delicate balance between a protective immune system-induced autophagy/xenophagy, and a deleterious metabolism-induced autophagy by invading bacteria [33].

The importance of topical vehicle formulations for autophagy-induced chronic wound healing

Topical vehicle formulations such as creams and ointments have their places, but for chronic wounds, especially those that produce excessive exudate, hydrogels [22] may be the most beneficial hydrating vehicles due to their unique properties (see below). The correct combination of base components and bioactives (e.g., specific bioactives, mediators) allows for a wide range of topical vehicle formulations characterized by distinct physicochemical properties resulting in some advantages but also some specific drawbacks for wound healing [42,43], especially when applying creams [43] or ointments [42]. For example, occlusive ointments, which often have a fatty/hydrophobic base, are considered rather unfavorable in chronic wound treatment, and are primarily suitable for use on irritated, unwounded skin [42]. They can protect against dehydration but tend to not contain water [42]. Meanwhile, the common ineffectiveness of cream formulations on exuding wounds has to do with the dilution of the cream in exude, thus altering the physicochemical properties and wound healing activity of the cream [43]. Furthermore, topical creams usually exhibit slow absorption rate and can only be used for a specific medication [43]. To avoid such drawbacks and to promote optimal conditions for an effective wound healing process, hydrogels have an ability to provide elementary parameters (e.g., moisture, bioactives) during the entire wound healing process [43].

Hydrogels offer great advantages for chronic wound healing due to their inherent smart and self-adapting properties encompassing extensive hydrophilic networks and high water-holding capacity, biocompatibility, versatility and ability to incorporate multifarious bioactive molecules [5,6,9,23,24,44]. Together, moisture and added bioactive molecules can then be delivered with precise temporal and spatial control, which represents an edge in relation to drug delivery when compared to other topical delivery vehicles (e.g., creams, lotions, ointments) [5,23]. Depending on the application, hydrogel properties such as scaffold nature and composition, ability to mimic the biochemical and mechanical properties of target tissues and sensitivity to wound stimuli can be tailored to deliver specific drugs and mediators such as antioxidants [5], anti-inflammatories [23,25] and antibiotics [39,41] to match the time scale for distinct stages of wound healing [5]. As a result, these unique hydrogel properties may support wound healing efficiency and successfully prevent the development or even persistence of chronic wounds [5].

In vivo data [22] from a rat pressure ulcer model demonstrate that a non-fouling, zwitterionic poly sulfobetaine methacrylate/SBMA hydrogel, when compared to placebo treatment; (1) enhances ECM remodeling by upregulating FN1 and LAM expression [22] (two key ECM-related protein components required for epithelial migration and cellular adhesion during the initial homeostasis and tissue formation) [15]; and (2) decreases zinc-dependent MMP2 enzyme expression [22] involved in the degradation of FN1 and LAM – excessive expression of MMPs in chronic wounds, especially MMP2, may inhibit wound closure [45]. Crucially, decreased MMP2 expression is due to the activation of autophagy through the inhibited PI3K-AKT-MTOR signaling pathway and significantly reduces inflammation by downregulating the expression of pro-inflammatory TNF [22]. Under normal physiological conditions, PI3K first phosphorylates and activates AKT, which in turn leads to phosphorylation and activation of MTOR, thus inhibiting autophagy [22,26–28]. Zwitterionic poly sulfobetaine methacrylate hydrogel treatment inhibits the PI3K-AKT-MTOR signaling pathway (without altering the expression of the unphosphorylated form of these proteins) and upregulates autophagy that leads to downregulation of both MMP2 and TNF [22]. Another investigation in a variety of chronic wounds displaying abnormal and excessive inflammation showed that a direct application of a steroid plus antibiotic and antifungal-containing agent can improve healing rates, presumably by reducing inflammation [41]. Unfortunately, here, the authors did not investigate autophagy-inflammation-wound healing interrelationship. Nevertheless, from these studies it is plausible to suggest that the interplay between topical drug-delivery hydrating vehicles (specifically hydrogels), inflammation and autophagy may be a crucial tool in optimizing autophagy modulation in chronic wounds.

Conclusions and future work

In conclusion, we propose the hypothesis that autophagy in a moist, non-healing wound with an underlying immune etiology is a crucial process through which such a chronic wound becomes acute, leading to eventual resolution. While the precise spatiotemporal molecular mechanism(s) underlying the regulation of autophagy by moisture conditions in the context of chronic wound healing is unknown, our analysis of the literature suggests that autophagy is indeed crucial to the process, not least due to the pleiotropic roles of autophagy demonstrated in ECM remodeling, regulation of zinc-dependent MMP levels and levels of pro-inflammatory factors, as well as degradation of invading pathogens, on which the evolution and successful completion of the wound healing process relies. We hope that his Commentary will provide a starting point from which to elucidate the precise involvement of autophagy in the healing of chronic wounds. Undoubtedly, deciphering such a highly complex physiologic process with multicellular and spatiotemporal considerations will help clinicians to establish novel therapeutic approaches to chronic wound care that are more accurate, effective and even situation specific.

Funding Statement

The work was supported by the National Institute of General Medical Sciences [GM131919]

Disclosure statement

Dalibor Mijaljica, Fabrizio Spada and Ian P Harrison are full-time employees of Ego Pharmaceuticals Pty Ltd.