More Than Skin Deep: Autophagy Is Vital for Skin Barrier Function

The skin is a highly organized first line of defense that stretches up to 1.8 m2 and is home to more than a million commensal bacteria. The microenvironment of skin is driven by factors such as pH, temperature, moisture, sebum level, oxidative stress, diet, resident immune cells, and infectious exposure. The skin has a high turnover of cells as it continually bares itself to environmental stresses. Notwithstanding these limitations, it has devised strategies to adapt as a nutrient-scarce site. To perform its protective function efficiently, it relies on mechanisms to continuously remove dead cells without alarming the immune system, actively purging the dying/senescent cells by immunotolerant efferocytosis. Both canonical (starvation-induced, reactive oxygen species, stress, and environmental insults) and non-canonical (selective) autophagy in the skin have evolved to perform astute due-diligence and housekeeping in a quiescent fashion for survival, cellular functioning, homeostasis, and immune tolerance. The autophagic “homeostatic rheostat” works tirelessly to uphold the delicate balance in immunoregulation and tolerance. If this equilibrium is upset, the immune system can wreak havoc and initiate pathogenesis. Out of all the organs, the skin remains under-studied in the context of autophagy. Here, we touch upon some of the salient features of autophagy active in the skin.


FigURe 1 | Panel (A) depicts the resting and activated state of the skin. In a normal or resting state, epidermis and dermis have circulating immune cells [DETCs, αβ T cells, γd T cells, macrophages, neutrophils, LCs, dermal DCs (dDCs), NK cells, B cells, innate lymphoid cells (ILCs)] and non-immune cells (melanocytes, keratinocytes, and merkel cells)
. Upon exposure to pathogens, chemicals, UV, or reactive oxygen species (ROS), the immune cells infiltrate at the site of infliction to defend the host and finally to resolve the inflammation after damage. Panel (B) shows the process of autophagy. mTOR inhibition triggers the activation of AMPK and initiates an autophagy-inducing signals during a low energy state such as starvation, ROS, exercise, infection, drugs, and hypoxic stress. This initiates the formation of pre-initiation complex (ULK1/2, ATG13, and FIP200) in the presence of unwanted cargo (such as, mitochondria, pathogens, protein aggregates, and intracellular components). This will, in turn activates the Class III phosphatidylinositol-3-kinase (PI3K) complex, composed of ATG14 (UVRAG)-VPS15-VPS34-Beclin1. The Class III PI3K complex completes the autophagosome formation by producing PI3P which recruits downstream ubiquitin-like conjugation systems (ATG5-12) and converts LC3-I to form LC3-PE. Finally, lysosome fuses with the autophagosome to form the autolysosome to degrade the enclosed cargo. The degraded cargo is finally assimilated and recycled. foreign particles. Autophagy participates in various physiological activities to ensure the smooth and quiescent operation of the immunotolerant environment and to maintain skin integrity. These activities include maintaining homeostasis, performing efferocytosis, as well as determining skin color, host defense, longevity, antigen presentation, and survival (11).

AUTOPHAgY AS A CeLL SURvivAL MeCHAniSM
Autophagy means self (auto) eating (phagy) and is a highly conserved cellular process across eukaryotes, which allows cells to recycle cytoplasmic materials via the lysosome and survive periods of nutrient deprivation (11). The term autophagy is derived from ancient Greek, but the word first garnered attention when Christian de Duve not only coined it but also won the Nobel prize in Physiology or Medicine in 1974 for his work on lysosomes (12)(13)(14)(15)(16). More recently Dr. Yoshinori Oshumi, described the autophagy-related genes (ATG) in yeast in 1993 and received the Nobel prize in 2016 (11,12,17). His pioneering work led to the discovery of other ATG genes and its human orthologs. Autophagy pathways include macroautophagy (canonical autophagy/autophagy), microautophagy, and chaperone-mediated autophagy (CMA) (11,13,18). Traditionally, autophagy is orchestrated by the group of ATG proteins, which precisely control the autophagic process (11,19). The process kickstarts the formation of the pre-initiation complex, followed by generation of the phagophore, autophagosome, and autolysosome, leading to cargo degradation ( Figure 1B) (11,19,20). Mammalian target of rapamycin complex 1 inhibition leads to the induction of autophagy and assembles ULK1/2, ATG13, and FIP200 to form the pre-initiation complex at the phagophore ( Figure 1B). Once activated, it targets the Class III phosphatidylinositol-3-kinase (PI3K) complex (Beclin1, VPS34, VPS15, and ATG14) which recruits downstream conjugation ATG proteins. During autophagosome elongation, E3(Ubiquitin)-ligase ATG7 mediates ATG5-ATG12-ATG16L1 complex formation and is recruited to the autophagosome membrane. Ubiquitinconjugating/E2-like enzyme ATG10 mediates covalent conjugation of the ubiquitin-like ATG12-ATG5 (21). E2-like enzyme ATG3 forms ATG12-ATG3 conjugate, controls mitochondrial homeostasis (21). ATG7 can recruit ATG3 and ATG10 forming ATG7-ATG3 and ATG10-ATG3, respectively (22). Mice lacking ULK1/2, ATG3, ATG5, ATG7, ATG12, or ATG16L1 are embryonic lethal mutations (23). ATG12-conjugation is essential for the formation of preautophagosomes (24). ATG3 aids in conjugation of LC3-I with phosphatidylethanolaminie (PE) required for the formation of autophagosomes (21,24). This facilitates the LC3 lipidation with PE and forms LC3-PE (or LC3-II). LC3-PE embeds into the mature autophagosome which finally fuses with the lysosome, wherein the cargo is degraded and recycled. The autophagy pathway is not only limited to the processes of degradation and survival during starvation but is also active in regulating other cellular functions (11). This bolsters the need for investigating autophagy's widespread influence on different biological mechanisms.

AUTOPHAgY in SKin iMMUne CeLLS
Skin inflammation induced by environmental irritants and pathogens requires autophagy as well as a crosstalk between immune and non-immune skin cells (listed below and in Table 1) to effectively alleviate the damage (4).

non-immune Components
• Melanocytes reside in the epidermis between keratinocytes, and they contain melanin or biochrome, a natural pigment found in hair, skin, and eyes (49). The process of producing melanin pigment continuously is called melanogenesis (87). Hair follicular melanogenesis (FM), unlike epidermal melanogenesis, is cyclic (88). FM starts at the anagen (active growth phase) stage of the hair cycle and is turned off by catagen (transition stage) and telogen (shedding stage) (88). Upon aging, the number of melanocytes in hair follicles is reduced and dendritic cells (including Langerhans cells) move from the upper to lower hair follicles as a response to age-related degradation of melanocytes (88). Stress can stimulate melanocyte to activate neurotransmitters, neutropeptides, and hormones which aids in regulating skin homeostasis (89). Neuroendocrine has been shown to govern the production and secretion of l-tyrosine and l-dihydroxyphenylalanine (l-DOPA), or its derivatives, during melanogenisis (89). Melanocytes are often called "neurons of the skin" and possesses melanocytes-stimulating hormone (MSH) receptors (89,90). Certain substrates of melanogenesis like l-tyrosine and l-DOPA can also regulate cell (melanocyte) functions and cellular metabolism through non-receptormediated processes (91).
• Merkel cells populate broadly across the epidermis. They have high turnover and differentiate terminally (2,99). Merkel cells have a longer life span than keratinocytes and require autophagy for differentiation (66). Atg7-deficient Merkel cells show accumulation of p62 (66,67).

immune Components
• Neutrophils are a most abundant granulocytes and are considered the foot soldiers of the immune system (68). Autophagydeficient neutrophils show impaired degranulation, NADPHoxidase-mediated reactive oxygen species (ROS) production, and inflammatory responses (68). Neutrophils from the skin of leprosy patients release TNF-α, show increased autophagy and exhibit accelerated apoptosis in vitro (69). • Macrophages are skin resident phagocytes surveilling the epidermis and dermis (2). They help in maintaining the immunotolerant environment of the skin (2,92). Specialized macrophages in the skin called melanophages can engulf melanocyte fragments and melanin. These melanocyte fragments and melanin are processed by autophagy (70)(71)(72). In leprosy patients, skin-derived macrophages have significant upregulation of autophagy genes, including Beclin1 and Atg14 (73).
• Epidermal DCs, also known as Langerhans cells (LCs) are dendritic cells of the skin and are found among the keratinocytes in the epidermis (2,104). The LCs surveil the epidermis and promote tolerance to environmental stressors (104)(105)(106).
Once activated, LCs migrate to the draining lymph nodes and aid in T cell polarization via antigen presentation to define the adaptive immune environment (106). Cytosolic and endocytosed antigens are processing and presentation (or cross presentation) by DCs to either CD4 + or CD8 + T cells via MHC class II and MHC class I molecules, respectively, and this process requires the autophagy machinery (74 Both LCs and dDCs can induce interferon (IFN) and pro-inflammatory responses (107). Type I IFN can induce autophagy during viral infection, autoimmune disorders, and certain cancers (108,109). ATG, Map1lc3b and Atg3 have been shown to be important in the production of hapten 2,4-dinitrofluorobenzene-induced (DNFB) ROS in skin DCs (76).
• Mast cells are granulocytes crucial for allergic inflammatory reactions (77). In the skin, they are triggered by UV or ROSinducing irritants and undergo degranulation (77,78). In addition, the degranulation process requires ATG7 (77). • NK cells are cytotoxic innate immune lymphocytes present in the dermis. A recent study showed that targeting autophagy by inhibiting Beclin1 can increase NK cells resulting in melanoma tumor (79). Beclin1 inhibition reduces degradation of NK-derived Granzyme B as a result of which melanoma cells can thrive even in the presence of NK cells driven by CCL5 (79). • ILCs are a new addition to the family of innate immune cells, and they are involved in shaping the immune system (110,111). There are three types of ILCs defined by their cytokine profile: ILC1 produces IFN-γ, ILC2 produce IL-5 and IL-13, and ILC3 produces IL-17A and IL-22 (110). In human and murine skins, ILC2 are the most prelevant. The role of ILCs in skin autophagy is not well reported. • Human skin contains approximately 20 billion skin T cells or T lymphocytes (112). T cells define the adaptive immune responses and are differentiated from other lymphocytes by the presence of surface T cell receptor (TCR) (112). TCR engagement activates autophagy, induces ROS, and causes activation of nuclear factor of activated T-cells (NFATC1) and lysosome-associated membrane protein 2 (LAMP-2) (113). Autophagy can alter the T cell repertoire through selection and aid in cross presentation by DCs (113)(114)(115)(116). ✓ Alpha beta (αβ) T cells (CD8 + T cells and CD4 + T cells) survive and persist in the skin long after the immune reac-tion is over (113,(117)(118)(119)(120)(121)(122)(123). This ensures rapid protection from future exposures to pathogens or antigens (112,124). Skin resident-memory Treg cells mitigate inflammation and regulate immune response via autophagy (81,82). Tregs (20-60% of CD4 + T cells) have also been shown to alleviate autoimmune disorder by suppressing DC autophagy (81,83). Atg7-deficient Tregs cannot establish skin homeostasis (84,85). ✓ Gamma delta (γd) T cells comprise 1-5% of the circulating T cells found in both mice and humans (125).
However, in skin about 50% of γd T cells are present among the T cells (126). They populate the skin epidermis and dermis and are named dendritic epidermal γd T cells (DETCs) and dermal γd T cells, respectively (125,127). Like αβ T cells, they possess effector functions as well as mediate tissue repair (86,125). γd T cells are the first responders to skin damage and are responsible for maintaining homeostasis, wound repair, and production of cytokines like insulin-like growth factor 1, TNF-α, and KGF-1 (86). DETCs are in close contact with keratinocytes and aid in wound healing by releasing KGF-7 and KGF-10 (128,129). Skin γd T cells are adaptable under immunosuppression by rapamycin (inhibits mTOR pathway) or roseotoxin B (suppresses activated T cells but does not effect naïve T cells), and they undergo autophagy to survive in the absence of cytokines (70,86). • B cells are a component of the adaptive immune system populating the dermal layers and produces antibodies. In B cells, ATG5 seems to play an important role in differentiation and survival (80). Autoantibodies induce autophagy and can sometimes lead to autoimmune disorders (80).

SKin eXPOSUReS AnD AUTOPHAgY
When the skin encounters pathogens, injury, or UVR, it deploys various defense mechanisms. Autophagy responds to these unwanted encounters to ensure inflammation resolution. Defects in autophagy can cause a hyperinflammatory skin reaction due to inflammasome activation (as seen in human keratinocytes), unpredicted ROS activation via UVR, and aberrant proinflammatory cytokine release ( Figure 1A) (4,113,(130)(131)(132)(133).

Pathogens
Evolutionarily, epidermal pathogenic bacteria have devised multiple evasion mechanisms to avoid autophagic clearance (134,135). Numerous instances of the autophagy machinery engaging skin pathogens during cutaneous infection have been described ( Table 2). Autophagy is critical for the clearance of group A streptococci (GAS) which evades endosomal capture (13,136). A recent study showed that the elimination of GAS is severely reduced in Atg5-deficient cells elucidating its vital role in pathogen clearance (13,136). Streptococcus pyogenes is responsible for causing impetigo, a common skin infection in children (137,138). Similarly, autophagy is essential in preventing infectious skin diseases such as Staphylococcus epidermidis infection, leprosy, and sepsis ( Table 1).
The skin is susceptible to fungal infections, such as cutaneous candidiasis and Candida intertrigo caused by Candida albicans (168). Studies have shown that the intracellular clearance of C. albicans depends on both autophagy (ATG5) and LAP (168,177). Autophagy in skin also plays a unique role in anti-viral function ( Table 2). Herpes simplex virus (HSV) is a double-stranded DNA virus. It is categorized into HSV-1 and HSV-2, which causes oral herpes and genital herpes (13). Autophagy aids in processing and presentation of HSV-1 antigens on MHC class I molecule for effective viral elimination (178)(179)(180). HSV-2 is more susceptible to the host ATG5 (13,179,181). Furthermore, skin-related viruses, including human papillomavirus, varicella zoster virus, and Zika virus can induce autophagy to degrade viral capsid proteins in the skin cells and keratinocytes (172,182).

wound Healing
During wound healing after an injury or pathogen invasion, skin immune responses halt the ongoing inflammation to initiate the restoration process (2). In rats, autophagy heals burnt hair follicle epithelium (183). A recent study shows that the use of mesenchymal stem cells (MSCs) in skin repair requires autophagy (184). Rapamycin-induced autophagy in MSCs causes secretion of vascular endothelial growth factor (VEGF) and improves VEGFmediated blood circulation which in turn promotes skin wound healing and tissue regeneration (184).

Ultraviolet Radiation
Sun-generated UVR is a mixture of UVA and UVB (185). Upon exposure to UVR, basal autophagy increases in keratinocytes (28) and causes epidermal thickening or hyperkeratosis (28). This in turn, leads to epidermal hyperplasia which prevents the UVR (UVA or UVB, depending on the wavelength) to penetrate the skin (28,186). UVR-induced cell death in the skin can promote autoimmunity due to defective clearance of apoptotic keratinocytes (5,108). Patients with systemic lupus erythematosus, an autophagy-related autoimmune disorder, are unable to clear dead cells and suffer from severe cutaneous lesions upon exposure to UVR as a result (187)(188)(189)(190)(191). UVR also inhibits antigen presentation by LCs and Treg migration, as seen in cutaneous T cell-mediated dermatitis (70,(192)(193)(194)(195)(196).
UV stimulates the central stress response center via hypothalamic-pituitary-adrenal axis, however, the mechanism is not well understood (204). UVR-induced β-endorphin and corticotropin-releasing hormone release from the skin causes soluble neuro-endocrine-immune factors to seep into the systemic circulation (204,210). The skin immune cells stimulated by UV act as "second messengers" allowing crosstalk between neuroendocrine system and immune system (210). Certain neuropeptides, such as calcitonin gene-related peptide (CGRP), mediate anti-inflammatory environment by UVR (211). Steriods like proopiomelanocortin produced from α-melanocyte-stimulating hormone (α-MSH) of UVRtriggered epidermal, dermal cells and macrophages induce immunosuppression (210,211).

SKin CAnCeR AnD AUTOiMMUne DiSORDeRS
Autoimmune disorders are the result of the adaptive immune system generating autoreactive lymphocytes (T and B cells) that target self-antigens. Skin disorders often arise as a secondary complication, as reported in diabetes, cancer, and dermatitis. The role of autophagy reported in skin-related disorders ( Table 1) is limited, yet nonetheless deserves recognition.
Atg5-deficiency, Atg7-deficiency, and Beclin-1 partial deletion can spur spontaneous tumor growth commonly seen in most cancers (152,(212)(213)(214). Contrary to that, targeting Beclin-1 inhibits autophagy, overexpresses CCL5 and aids in recruit NK cells to the melanoma tumor (79). Melanoma is fatal in its aggressive form and is highly metastatic (153). Atg5-deficient melanoma cells in vitro have diminished survival (152,154). Interestingly, in advanced stages, melanoma cells promote a tumor-suppressive environment by hijacking the autophagy machinery to ease stress induced by drugs (215). This suggests that cancer cells can manipulate the autophagy machinery to resist treatment (154,216).
Diabetes mellitus (both type 1 and type 2) patients have defective insulin signaling in the keratinocytes and often suffer from skin lesions and foot ulcers (145)(146)(147)217). In diabetic patients, IRF8 (an autophagy regulator) activation induces autophagy, poises the macrophages to permit inflammation and thus, impairs cutaneous wound healing (145,217). Chronic hyperglycemia and hyperlipidemia disrupt ER homeostasis and resulted in increased unfolded protein burden (147,217). Both autophagy and mitophagy are defective in diabetic patients and inhibit keratinocyte proliferation and migration that are requisite for wound healing (145)(146)(147).
Psoriasis is a chronic, polygenic autoimmune disease characterized by epidermal hyperplasia, defective keratinization, and infiltration of immune cells within the skin, causing dermatitis and thickened plaques formation. Genetic screening of psoriasis patients of Estonian origin reveals several single-nucleotide polymorphisms (SNPs) associated with ATG16L1, though the functional role of ATG16L1 variants in skin biology is unclear (139,218). In pustular psoriasis cases, mutations in AP1S3, a gene encoding an autophagosome trafficking protein, result in the disruption of autophagy in keratinocytes and drive them to produce pro-inflammatory cytokines, including IL-1β, IL-8, and IL-36A (140). Furthermore, a recent study demonstrated that autophagy inhibition in vivo shows aberrations in keratinocyte differentiation, resulting in dysregulation of autophagy in psoriatic epidermis (219).
Vitiligo is a pigmentation disorder characterized by sharp demarcated white macules on skin due to the CD8 + T cellmediated destruction of melanocytes (220,221). This results in localized (segmental vitiligo) and/or generalized (non-segmental vitiligo) partial loss of melanin. In addition, melanocytes show increased autophagy due to misfolding of tyrosinase (tyr) and X-box binding protein 1 (Xbp1) in the ER (220,221). Individuals suffering from vitiligo may have defective epidermal permeability barrier functions which can be alleviated by the use of topical histamine treatment (222,223). Vitiligo patients from Chinese Han population show abnormal antioxidant Nrf2 [nuclear factor (erythroid-derived 2)-like 2] gene expressions in autophagy and associated pathways (142,224,225). Compared to normal melanocytes, vitiligo melanocytes in vitro show greater similarity with Atg7-deficient melanocytes (57, 60). Both exhibit impaired redox-sensitive Nrf2 activation and decreased activation of the antioxidant enzyme system in response to oxidative challenges induced by environmental stress and ROS (93,226,227). Multiple evidences indicate that autophagy controls melanosome degradation (92,228). Furthermore, a Korean cohort study demonstrated an association between two UVRAG gene SNPs and non-segmental vitiligo (vitiligo vulgaris) (229). Skin autoimmune disorders are overly complicated, involve many aspects of the immune system and arise due to defects in autophagy machinery. Accumulating evidence displaying the importance of autophagy in skin disorders demonstrates the need for further research.

COnCLUDing ReMARKS
Being at the forefront of the environmental defense system, a plethora of specialized cells reinforces the skin. It is a unique surface that is highly specialized in preserving immunotolerance. Therefore, autophagic clearance of senescent and damaged cells is necessary for the maintenance of specialized cellular machinery to effectively keep inflammatory triggers at bay. Several research groups have implicated the role of ATG5, ATG7, and ATG16L1 in keratinocytes, melanocytes, and immune cells in autoimmunity and cancer. Skin in the context of autophagy remains an uncharted territory and warrants further investigation. Autophagy dictates the immune response and resolution in skin cells to neutralize pathogens, clear senescent cells, or heal wounds. However, the autophagy machinery can either protect or cause autoimmune disorders. The physiological circumstances that govern this "balancing act" are not well understood. The cytokine microenvironment from dying immune cells, pathogens, or senescent cells can potentially direct the autophagic response in skin. In addition, there are evidences suggesting potential communication between autophagic machinery and skin cells (both immune and non-immune cells) in an inflammatory state that resonates in the literature; however, the mechanism is not yet reported (230). Adequate tools to examine the molecular response are lacking.
In addition, skin microbiota has been shown to shape the immune response by producing AMPs, complement, IL-1, and IL-17 as well as modulating the local T cell response (3,231). The interaction with skin cells and microbiota may reveal underlying mechanisms to aid in conscientious operation of the autophagic machinery as seen in Crohn's disease and colorectal cancer (232)(233)(234). Overall, further research investigating the role of autophagy in barrier function will pave the way forward for therapeutic advancements in the field of skin inflammaging and dermatology.   (14)