Skin is the largest organ and acts as a protective barrier separating the body from the outside environment (1). Epidermis, dermis, and subcutaneous fat together with skin appendages, sweat glands, sebaceous glands, and hair follicles, form an integrated structure that enables proper skin function (2). The foremost physical barrier of the skin consists of the epidermis, which in its outermost layer is composed of brick and mortar-like stratum corneum and tight junctions that regulate the inward and outward passage of fluid and electrolytes in and out of the skin (1, 3). The skin has developed complex protective functions against constant exposure to various environmental insults, such as solar radiation, pollutants, a broad range of microbial pathogens, and allergens (4, 5). The immune system contributes to this first line of defense against microbial pathogens and chemical insults (2).

In the epidermis, Langerhans cells (LCs) are epidermal-resident antigen-presenting cells (APCs), which play a sentinel role as the first professional immune cells confronting the environmental insults (6, 7). Activated LCs capture foreign antigens by extending their dendrites through epidermal tight junctions. Next, they migrate to the lymph nodes to initiate cutaneous adaptive immunity (8, 9). In addition, tissue-resident memory T cells (T_RM cells), which are noncirculating lymphocytes in peripheral tissues, persist in the epidermis to provide long-lasting protective defense against future immunological challenges at the most probable sites of invasion (10–12). The dermis is the stromal layer below the epidermis that encompasses an active immunological microenvironment (2). The superficial papillary dermis is composed of a relatively loose connective tissue and contains vessels and sensory nerves (13). A diverse range of immune cell types, including several T cell subsets, macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and mast cells, is localized around vasculature and extracellular matrix proteins in the dermis (14–16). This complex network of dermal immune cells helps in many aspects of host physiology, including protection against pathogens, wound healing (17), sebum production (18), and hair follicle homeostasis (19).

Cytokine-mediated communication allows immune cells to achieve a context-appropriate response, as the homeostasis of a multi-cellular organism is made possible by the proper cell-cell communication across different cell types (20). The cytokine superfamily consists of many ligands and receptors that mediate key interactions between immune cells and non-immune cells, including keratinocytes, fibroblasts, and endothelial cells, in the skin microenvironment (21–23). As the primary barrier, which is constantly exposed to environmental insults, the epidermis functions as a key sensor and integrator of environmental cues to regulate immunity in the skin (24, 25). As such, epidermis-derived alarmin cytokines mediate an intricate intercellular communication between epidermal keratinocytes and immune cells to regulate cutaneous immune surveillance. Alarmins are endogenous molecules that function as danger signals and are rapidly released to the extracellular milieu in response to tissue damage to trigger defensive immune responses (26). Among them, the proper functions of type 2 alarmin cytokines, thymic stromal lymphopoietin (TSLP), interleukin (IL)-25 and IL-33, as central orchestrators of T helper 2 (Th2) immunity, are paramount to the skin homeostasis and their dysregulation is commonly associated with chronic allergic inflammation (27–29).

In this review, we discuss recent findings on the complex functions of type 2 alarmin cytokines in regulating epithelium-immune cell communication that governs host defense, chronic inflammation, and cancer.

TSLP is a member of the IL-2 family of cytokines, and its receptor is a heterodimer that consists of the IL-7 receptor α chain (IL-7Rα), which is shared with IL-7, and the TSLP receptor (TSLPR) (30–32). TSLP is mainly expressed by the epithelial cells of the gut, lung, and skin. Other cell types including DCs, basophils, and mast cells can express TSLP (33, 34). At the organ level, TSLP is widely distributed in several organs including the heart, liver, testis, spleen, prostate, skin, lung, kidney, ovary, small intestine, and colon (35). A variety of endogenous and environmental factors, such as pro-inflammatory cytokines, tryptase, invading pathogens, allergens, irritants, pollutants, and cigarette smoke, stimulate epithelial cells to release TSLP at barrier surfaces (36–45). Activation of protease-activated receptor 2 (PAR2), Toll-like receptor 4 (TLR4), and a member of transient receptor potential vanilloid (TRPV) channel family, including TRPV1, on the cell membrane mediate the production of TSLP through transcription factors, nuclear factor of activated T cells (NFAT), nuclear factor-kappa B (NF-κB), and interferon regulatory factor 3 (IRF-3) (46–48). TSLP is post-translationally modified by endogenous proteases, and cleaved TSLP has an increased biological activity (49). Despite poor sequence homology of TSLP (43% amino acid identity) and TSLPR (39% amino acid identity) between humans and mice, TSLP has been shown to exhibit similar biological functions in humans and mice (50).

Hematopoietic cell populations and sensory neurons express TSLPR. TSLP first interacts with the cognate TSLPR, then IL-7Rα can be recruited to the TSLP/TSLPR assembly to form the extracellular ternary complex. This leads to the activation of an intricate network of signaling pathways, including Janus kinase/signal transducer activator of transcription (JAK/STAT) and phosphatidylinositol-3 kinase (PI3K) pathways (51). Similar to IL-7, this signaling plays a critical role in the activation and differentiation of immune cells, such as B cells and T cells (52). In contrast, TSLP also mediates Th2 immunity associated with protection from helminth parasites and the pathogenesis of allergic inflammation at barrier surfaces. TSLP strongly induces the expression of major histocompatibility complex (MHC) class I and II molecules and costimulatory molecules on DCs. TSLP-activated DCs produce Th2-attracting chemokines, such as CCL17 and CCL22 (53), and induce Th2 differentiation through OX40 ligand upregulation on these cells (54, 55). Furthermore, TSLP can directly activate naïve CD4⁺ T cells to differentiate and promote Th2 effector function in a TCR-dependent manner (56–58), indicating that TSLP is a key driver of Th2 immunity. In addition, TSLP acts on CD4⁺ T cells to promote T helper 9 (Th9) differentiation and function through STAT5 activation in airway inflammation (59). Th9 cells, IL-9 producing CD4⁺ T cells, are closely related to Th2 cells and contribute to allergic inflammation and anti-tumor immunity (60, 61). It remains unclear whether these cells represent a truly unique Th cell subset. TSLP also acts on basophils and group 2 ILCs (ILC2s) to induce Th2 responses (62, 63). Thus, TSLP orchestrates type 2 immune responses by innate and adaptive immune cells at barrier sites.

IL-25 (also known as IL-17E) belongs to the IL-17 cytokine family, which consists of six members, and shares relatively low sequence similarity to the prototype member, IL-17 (alternative name IL-17A) (107–109). IL-25 receptor (IL-25R) is a heterodimer of the IL-17RA chain, which is shared with other IL-17 family members, and the IL-25-specific IL-17RB chain (108). Therefore, IL-25 exhibits a distinct function from other members of the IL-17 cytokine family and has been implicated as a type 2 cytokine that induces the production of IL-4, IL-5, and IL-13, which in turn inhibit the IL-17-dependent autoimmune diseases (110). Furthermore, IL-25 enhances Th9 cell response to prevent parasitic helminths infection (111, 112). IL-25 is produced by epithelial and immune cells including Th2 cells, macrophages, ILC2s, mast cells, basophils, and eosinophils (109, 113). In the extracellular space, IL-25 has been reported to be a substrate for proteolytic cleavage by matrix metalloproteinase-7 (MMP-7) from airway epithelial cells during inflammation, and cleaved IL-25 increases its activity to induce type 2 cytokines (114). Expression of IL-25R has been reported on non-immune cells, including fibroblasts and endothelial cells, and immune cells, such as Th2 cells, natural killer T cells (NKT cells), DCs, macrophages, ILC2s, mast cells, basophils, and eosinophils in the inflammatory state (115, 116).

IL-33 is the most recently discovered member of type 2 alarmin cytokines (132). IL-33 was first described in 2005. It belongs to the IL-1 family of cytokines, which includes IL-1α, IL-1β, IL-18, IL-36α, IL-36β, IL-36γ, and IL-37, and the receptor antagonists IL-1Ra, IL-36Ra, and IL-38 (133). In contrast to its other family members, IL-1 and IL-18, IL-33 has been shown to promote Th2 cytokine responses in helminth infection and allergic inflammation (132). IL-33 is mainly expressed by non-immune cells, including epithelial cells, endothelial cells, and fibroblasts. It can also be expressed by immune cells, including macrophages and mast cells, at the barrier sites, where it functions as an alarmin following tissue damage (134, 135). IL-33 is localized in the cell nucleus, and its N-terminal domain, which includes a chromatin-binding motif, is required for its nuclear localization (136). Unlike IL-1β and IL-18, the N-terminal portion of IL-33 does not require inflammasome-mediated cleavage by caspase-1 for extracellular release of the active form. Apoptosis-associated caspase-3 and caspase-7 cleave and inactivate IL-33 at a conserved residue, Asp¹⁷⁸ (Asp¹⁷⁵ in mouse), within the IL-1-like cytokine domain (137). On the other hand, N-terminal processing of extracellular full-length IL-33 can occur in the central domain between the nuclear domain and the IL-1-like cytokine domain. These residues are targeted by extracellular proteases in the inflammatory microenvironment, including neutrophil cathepsin G, neutrophil elastase, and mast cell serine proteases, and the resulting 18-21 kDa cytokine forms of IL-33 exhibit higher biological activity (138, 139). Full-length IL-33 can be rapidly cleaved in its central sensor domain by extracellular environmental allergens-derived proteases within 10-20 minutes (140). In contrast, cysteine oxidation of extracellular IL-33 diminishes its biological activity (141). Thus, following IL-33 release, the impact of IL-33 is tightly regulated by post-translational modifications in the extracellular milieu. Although IL-33 lacks a secretion sequence and is sequestered in the nucleus via chromatin binding, IL-33 seems to be released into extracellular space through an unconventional secretion pathway following various stimuli. Environmental allergens, including fungi and mites, and mechanical stress trigger the release of IL-33 (142–145). Two primary scenarios for IL-33 release have been proposed: passive release as alarmin from necrotic cells during tissue damage and unconventional secretion from living cells. A recent study suggests that IL-33 is secreted through the extracellular vesicles pathway, commonly referred to as exosomes, as surface-bound cargo, from living airway epithelial cells (146). However, the molecular mechanisms and pathways of IL-33 secretion in living cells remain unclear.

IL-33 binds to its receptor, suppressor of tumorigenesis 2 (ST2), on target cells (147, 148). ST2 is classified as a member of the IL-1 receptor superfamily, which has a common intracellular domain, known as the Toll/Interleukin-1 receptor (TIR) domain (147, 148). IL-33 signals via its cognate receptor ST2, which is highly expressed on Th2 cells, ILC2s, and mast cells, and induces Th2-skewed immunity to help with the removal of invading pathogens and helminths (29, 149). However, the detrimental effects of its chronic expression in response to environmental insults cause allergic inflammation. In addition, inappropriate activation of the IL-33/ST2 axis after tissue injury can lead to impaired wound healing and tissue remodeling (150–152). In contrast, IL-33 can also support tissue homeostasis and repair mediated by Tregs, which express ST2 predominantly in nonlymphoid tissue (153, 154).

TSLP, IL-25, and IL-33 alert the immune system in the skin to respond to environmental insults, and their chronic overexpression triggers allergic inflammation at barrier sites ( Figure 1 ). Importantly, the function of type 2 alarmin cytokines extends beyond their physiological function in host defense and pathological function in allergic inflammation and involves other critical roles including skin cancer regulation and sebum secretion ( Figure 2 ). Keratinocyte-derived TSLP protects the skin from carcinogenesis (181, 182). TSLP exerts its dominant anti-tumor effects through the induction of CD4⁺ Th2 cell immunity in the early stages of keratinocyte cancer development (181). In contrast, TSLP induces proliferation of the malignant CD4⁺ T cells in CTCL lesions, which are marked by a Th2 cell-dominant phenotype in advanced stages (183). It has also been shown that TSLP can recruit IgE-bearing basophils into inflamed skin, and IgE promotes inflammation-driven tumor growth during chronic tissue inflammation in a cutaneous squamous cell carcinoma model (184). Baseline TSLP expression in breast and pancreatic cancer has been linked to a pro-tumorigenic function (185–188). A tumor-myeloid cell axis independent of T cell response may mediate this tumor-promoting function of TSLP (189). However, systemic TSLP induction from the skin causes an effective CD4⁺ T cell-mediated anti-tumor immune response at the site of developing cancer in the breast (185). Topical treatment of calcipotriol, a TSLP inducer (75), blocks skin cancer development in mice in a TSLP-dependent manner, and synergistically with 5-fluorouracil (5-FU) induces an effective CD4⁺ T cell-mediated immunity against actinic keratosis, which is a precursor to cutaneous squamous cell carcinoma in humans (190). IL-25 also has potent anti-tumor effects against several tumor types, including melanoma, through an increase in eosinophils recruitment into the tumor (191, 192). In other models, IL-25 itself exhibits anti-tumor activity through the induction of apoptosis in cancer cells without affecting nonmalignant cells (193). In contrast, epidermal IL-33 contributes to a microenvironment that supports tumor growth and progression in murine skin. Nuclear IL-33 mediates focal adhesion kinase (FAK)-dependent secretion of soluble ST2, a decoy receptor, and CCL5 from squamous cell carcinoma cells, which stimulates immunosuppressive Tregs leading to cancer immune evasion (194). Continuous IL-33-driven stimulation of Tregs shapes a tumor-promoting immune environment associated with chronic inflammation in the murine skin, and an increase in IL-33 expression and Treg accumulation are observed in the perilesional skin of patients with cancer-prone chronic inflammation (178). Furthermore, IL-33 induces the recruitment of a subset of tumor-associated macrophages, which express ST2 and high-affinity IgE receptor, FcϵRIα, and produce TGF-β, in the tumor microenvironment, which results in tumor progression in a mouse model of squamous cell carcinoma (195). Nuclear IL-33 in keratinocytes also promotes intrinsic TGF-β signaling through the SMAD signaling pathway, which constitutes a cell-autonomous tumor promotion mechanism in chronic inflammation (196). IL-33-stimulated macrophages highly produce MMP-9, which proteolytically trims activating receptor natural killer group 2, member D (NKG2D) and its ligands MHC class I polypeptide-related sequence A/B (MICA/B) on the surface of tumor-infiltrating lymphocytes and melanoma cells, and thus impedes the immune surveillance of tumor-infiltrating lymphocytes (197). These findings indicate that type 2 alarmin cytokines have distinct mechanisms for the regulation of cutaneous malignancies.

Endogenous TSLP controls the steady-state level of sebum secretion and sebum-associated antimicrobial peptide expression through the activation of T cells in murine skin. TSLP overexpression results in loss of white adipose tissue in conjunction with sebum hypersecretion (18). TSLP helps to maintain skin-resident RORγt⁺ ILCs within hair follicles, and, by the virtue of their location, ILCs negatively regulate surrounding sebaceous gland size and lipid content to regulate commensal bacteria equilibrium and fine-tune the skin barrier surface in mice (198). These findings suggest that TSLP-elicited ILCs and T cells play opposing functions in sebum secretion. Finally, TSLP is found to activate neutrophils to protect the skin from infection by methicillin-resistant Staphylococcus aureus (MRSA) (199).

Epidermis-derived TSLP, IL-25, and IL-33 act as alarmins to instigate cutaneous type 2 immunity through a complex and pleiotropic network of innate and adaptive immune cells. Accumulating evidence indicates that these type 2 alarmin cytokines not only function in concert but also have distinct physiological functions. Thus, the panoramic view of the communications between keratinocytes and immune cells through type 2 alarmin cytokines is required to fully understand how these cytokines regulate cutaneous immunity. The function of type 2 alarmin cytokines partly depends on the nature of the inflammatory stimulus, the presence of supporting cytokines and chemokines, and the surrounding microenvironment. Recent studies indicate that type 2 alarmin cytokines exhibit not only conventional type 2 inflammatory properties but also pro-homeostatic properties in the skin. Therefore, further understanding of the spectrum of biologic processes regulated by type 2 alarmin cytokines will provide new insights for the development of effective therapeutic approaches to combat allergic inflammation while utilizing the beneficial effects of these cytokines in the skin.

All authors contributed to the writing of the manuscript and approved the submitted version.

TH is an employee of Shiseido Co. Ltd.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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