Neutrophils in Atopic Dermatitis

Abstract

Neutrophils have a critical role in inflammation. Recent studies have identified their distinctive presence in certain types of atopic dermatitis (AD), yet their exact function remains unclear. This review aims to compile studies elucidating the role of neutrophils in AD pathophysiology. Proteins released by neutrophils, including myeloperoxidase, elastase, and lipocalin, contribute to pruritus progression in AD. Neutrophilic oxidative stress and the formation of neutrophil extracellular traps may further worsen AD. Elevated neutrophil elastase and high-mobility group box 1 protein expression in AD patients' skin exacerbates epidermal barrier defects. Neutrophil-mast cell interactions in allergic inflammation steer the immunological response toward Th2 imbalance and activate the Th17 pathway, particularly in response to allergens or infections linked to AD. Notably, drugs alleviating pruritic symptoms in AD inhibit neutrophilic inflammation. In conclusion, these findings underscore that neutrophils may be therapeutic targets for AD symptoms, emphasizing their inclusion in AD treatment strategies.

Introduction

The immune response is a cascade of defense systems that protect the body from pathogens and foreign substances. It triggers extravasation to alert and recruit nearby immune cells to arrive at a localized area and prevent the further spread of an infection. Neutrophils migrate to the inflammatory tissue in a cascade-like manner. These cells are the most abundant leukocytes in circulation and are capable of protecting the body through phagocytosis, formation of neutrophil extracellular traps (NETs), generating reactive oxygen species (ROS), and/or release of granules [1, 2]. Neutrophils play an essential role in skin inflammation, migrating rapidly from the bloodstream to the inflamed site via chemotaxis. In the tissue, they move in an amoeboid fashion, responding to various chemokines and chemoattractants. The migration involves a complex interaction with other cells and the microenvironment. Neutrophils can also form swarms, enhancing their recruitment to the affected area [3]. Although neutrophils protect our body against infection or abnormal cells, they are also considered a double-edged sword. When immune cells are deregulated, confusion in the body causes uncontrolled inflammation, which leads to severe tissue damage [4]. Neutrophils play a role in acute and chronic diseases and autoimmune diseases [5, 6]. Uncontrolled neutrophils are also the culprit for allergic sensitization and allergic inflammation [7]. Allergic inflammation, such as atopic dermatitis (AD), is usually accompanied by itching, swelling, burning sensation, redness, mucus production, and pain [8]. The global prevalence of AD is estimated at 2.6%, impacting a total of 204.05 million individuals. This includes 101.27 million adults and 102.78 million children worldwide, with prevalence rates of 2.0% and 4.0%, respectively [9]. Notably, females exhibit a higher susceptibility, with a prevalence of 2.8%, affecting 108.29 million individuals, compared to males, with a prevalence of 2.4%, impacting 95.76 million individuals [10]. The exact cause of AD is unknown, but several risk factors such as family history of hypersensitivity, genetics (disruption of skin barrier function), exposure to environmental pollutants or irritants, hypersensitivity to food, or excessive hygiene are found to contribute to the development of this condition [11]. Furthermore, pruritogens from leukocyte degranulation were also reported to play a significant role in the pathology of AD inflammation [12].

Early studies suggested that neutrophils were not involved in the pathophysiology of AD causing their potential role in AD pathogenesis to be underestimated and unexplored for years. However, emerging evidence has shown that neutrophils, even in a small number, could exacerbate symptoms in AD [13–15]. Of importance, translational studies have demonstrated a significant presence of neutrophils in skin lesions of Asian AD when compared to those of the European American (EA) AD [16]. Furthermore, the intrinsic type of AD shows an elevated level of neutrophil elastase (NE) in skin lesions, whereas the extrinsic AD shows an elevated eosinophil and plasmacytoid dendritic cells (DCs) [17]. Despite recent findings, the connection between neutrophils and AD has yet to be thoroughly analyzed and summarized. Thus, the primary objective of this study is to systematically review and explore the available literature on the mechanism of neutrophils in AD pathology and their clinical impact. We hope this review can shed light on and encourage future researchers to study neutrophils' importance in AD further.

Atopic Dermatitis and Its Endotypes

The pathogenesis of AD involves complicated interactions of genes, immunity, and environment, although their relationship still needs to be fully understood [18]. A notable immune feature of AD is the presence of Th2 cells (expressing cytokines such as IL-4 and IL-13) in skin lesions and elevated concentrations of immunoglobulin E (IgE) in serum [19, 20]. The majority of affected patients exhibit increased IgE antibodies that specifically target allergens in the air, such as house dust mites, pollen, and animal dander [21]. AD is commonly found in early childhood but can manifest later in life. Symptoms may be resolved before adulthood; However, a certain percentage of the population will continue to experience this condition or may develop other atopic conditions, such as asthma and allergic rhinitis, in adulthood [22].

Clinical manifestations of AD vary among different age groups. Typically, infants develop lesions on their face, extensor sites of limbs, and trunk, but the diaper area is usually spared. In adolescents and adults, AD lesions are mainly on the flexural sites, wrists, ankles, hands, upper trunk, shoulders, neck, and head [23]. A study on early-onset pediatric AD showed that children with AD exhibited more significant epidermal hyperplasia and higher expression of Th17-related cytokines and interleukin-8 (IL-8) compared to adults with AD. Furthermore, filaggrin downregulation, a characteristic of AD in adults, was absent in pediatric AD. Another feature of adult AD that differs from that of pediatric AD is the reduction of antimicrobial peptide (AMP) levels. Pediatric AD is characterized by increased AMP levels (LL37, DEFB4B, and lipocalin 2) in both non-lesion and lesion skin, which is even higher than that observed in patients with psoriasis [24]. Moreover, while pediatric AD is clustered around patients with psoriasis, adult AD is not [25]. These findings suggest that pediatric AD's skin phenotype significantly differs from adult AD.

The differentiation between extrinsic AD and intrinsic AD is based on IgE-mediated sensitization. Intrinsic AD, or non-allergic atopic eczema/dermatitis syndrome, has normal IgE levels and lacks other atopic conditions unrelated to allergens [26]. These two endotypes exhibit nearly identical Th1 and Th2 responses, however, intrinsic AD is distinguished by a markedly elevated Th17 and Th22 immune response. Furthermore, the mRNA expression of IL-17-regulated CCL20 and elafin, along with an increased number of neutrophils in intrinsic AD, indicates higher IL-17 activation in intrinsic lesions. On the other hand, extrinsic AD involves elevated serum IgE and an identifiable external trigger or allergen contributing to the development or exacerbation of the condition [17]. It has been reported that the incidence of intrinsic AD is higher among children than adults [27]. However, severe cases of pediatric AD show an increase in the total and specific IgE levels [28]. It is important to note that the level of IgE is associated with the degree of severity of AD [29] but is not the sole factor contributing to AD progression.

The onset of acute skin lesions is associated with significant increases in the expression of genes linked to Th2 and Th22 cytokines with minor increases in Th17-related cytokines, along with notable elevations in IL-17 and IL-22 cytokines. In chronic lesions, there is a significant increase in the expression of products associated with the Th2 response, such as IL-5, IL-13, IL-10, IL-31, CCL5, CCL13, and CCL18. At the same time, there is an elevation in Th1 response products, including IFN-γ, MX1, and CXCL9-11, without a notable increase in Th17 response products [30]. Interestingly, IL-4 and its receptor levels decrease from acute to chronic lesions [31]. The transition to chronic AD is characterized by the heightened activation of immune pathways initially upregulated in acute lesions, notably Th22 and Th2. While IL-17 cytokine expression is less pronounced in chronic AD, several IL-17-regulated genes (CCL20, PI3, and LCN) are elevated in both acute and chronic AD lesions. Increased IL-17 levels are observed in the skin lesions of AD patients, with IL-17-producing cells mainly located in the upper papillary dermis and epidermis [32, 33].

This complicated cocktail of Th pathways interaction ultimately damages the skin barrier, causing dryness and itching, which is a perfect recipe for pruritus. Scratching exacerbates damage, perpetuating the cycle of allergy-induced inflammation, dry skin, and itching in AD. Chronic condition boosts cytokine expression, fueling more inflammation and immune activity. Notably, Th17, Th1, and Th22 cells are also involved in the development of some subtypes of AD, including intrinsic AD, Asian AD, and pediatric AD [34].

The Role of Neutrophils in Atopic Dermatitis

The notable elevation of skin neutrophils is not typically prominent in AD compared to psoriasis. However, neutrophils may contribute to the inflammatory process, especially during acute disease if a secondary infection occurs [35]. Moreover, neutrophil infiltration is significantly increased, and genes encoding neutrophil chemoattractants are highly expressed in the AD lesional skin [36]. In addition, the rapid infiltration of neutrophils into irritated skin was observed [37]. Recent studies also demonstrate the apparent increase of neutrophil infiltration in the epidermis of AD, particularly in the Chinese, Japanese, and Korean AD [16].

The neutrophil-to-lymphocyte ratio (NLR), a serum inflammatory parameter, is a prognostic factor in numerous diseases such as COVID-19 [38], sepsis [39], and chronic obstructive pulmonary disease (COPD) [40]. NLR in patients with AD presents a higher level than that of an average individual [13]. Furthermore, NLR correlates with the severity of skin inflammation and lesional skin area [14]. NLR in children with severe AD is higher than that in children with mild AD [15]. Patients with AD had significantly higher neutrophil counts and NLR [41]. Therefore, elevated circulating neutrophils may be associated with AD severity, and a high NLR may serve as a parameter for AD severity.

Increasing evidence implicates neutrophils in AD's pathogenesis (Fig. 1). AD involves Th1/Th2 imbalance, with Th17 and Th22 participation. The Th2 cytokines (IL-4 and IL-5) are required for IgE synthesis but suppress Th1. Th2 exposure increases Type 2 cytokines (IL-4, IL-5, IL-9, and IL-13), vital to initial inflammation [42]. Th2 leads to decreased protective type 1 immunity against a wide range of viral, bacterial, and protozoan pathogens and facilitates uncontrolled or persistent infection [43]. Th2 cytokines have been shown to affect the mobilization of neutrophils via the Th2-STAT6-C3 complement-NETs cascade [44]. Mast cells, which are key players in AD pathogenesis, release pro-inflammatory mediators like histamine, cytokines, and proteases, that contribute to AD symptoms [29]. They also interact with T cells and dendritic cells, modulating skin inflammation [45]. Tumor Necrosis Factor-alpha (TNF-α) released by mast cells can directly prime circulating neutrophils, enhancing their ability to attach and migrate to the surrounding tissues [46]. Furthermore, these immune cells contribute to S. aureus-induced skin inflammation, leading to spongiosis, parakeratosis, and neutrophil infiltration. This suggests that S. aureus-stimulated mast cells worsen AD conditions [47]. Several mechanisms of mast cell-regulated neutrophil accumulation in tissues were described, involving the defensive response of the host against microbial infection [48]. Neutrophils prominent feature NET formation was regulated by mast cell tryptase in vivo, indicating an intimate functional communication bridge between mast cells and neutrophils may contribute to the pathogenesis of AD [49].

Fig. 1.

Neutrophils in atopic dermatitis. AD originates from a complex interplay of both internal and external factors, which include allergic reactions, exposure to antigens, physical trauma, infections, and emotional stress. Immune responses are orchestrated by neutrophils, B cells, mast cells, and T cells, which release various chemokines and cytokines, such as IgE, IL-4 and IL-13, CXCL1, CXCL2, CXCL8, TNF-α, or LTB4. These substances mediate the recruitment and activation of neutrophils, a crucial component of inflammation. Neutrophils utilize multiple mechanisms during inflammation, including respiratory burst, degranulation, and the formation of neutrophil extracellular traps (NETs). In patients with AD, enhanced neutrophil activity is observed during flare-ups and colonization by S. aureus, leading to excessive oxidative stress. The respiratory burst involves the activation of NADPH oxidase (NOX2) and myeloperoxidase (MPO), which generate superoxide anion (O₂^•−) and reactive oxygen species (ROS). Upon activation, neutrophils release granular compounds, including neutrophil elastase (NE) and MPO, through a process known as degranulation. NETs, composed of extruded chromatin and antimicrobial compounds such as MPO and NE, contribute to inflammatory processes and are implicated in the pathogenesis of AD. Continued production of IL-17 by NETs and the subsequent stimulation of Th17 cells to release more IL-17 is a crucial process. This plays a pivotal role in initiating and enhancing neutrophil infiltration, which is a fundamental aspect of the immune response in AD. During the transition from acute to chronic lesions, there is an elevation in products related to the Th1 (including IFN-γ) and Th17 responses, which amplify the migration of neutrophils in the skin. IL-4, a regulator of neutrophil migration, is decreased in chronic AD. NETs continuously supply IL-17 and induce Th17 cells to release more IL-17, leading to T cell polarization in AD. This shift from a Th2-predominant acute phase to a Th1-characterized chronic phase facilitates increased skin neutrophil infiltration and pruritus lichenification. Lastly, neutrophils trigger itch by activating sensory neurons via CXCR3 signaling

Several cytokines are known to play a crucial role in AD development. IL-33, part of the Interleukin-1 family, is linked to genetic and functional activation of neutrophils. NE and cathepsin G activate IL-33. IL-8 plays a role in the generation of leukotriene B4 (LTB4) and cysteinyl leukotriene (LTC4) by neutrophils in patients with AD. IL-8 enhances the responsiveness of neutrophils to receptor-specific stimuli, contributing to the pathogenesis of AD. Both acute and chronic inflammatory responses in AD patients are influenced by IL-3 and IL-8. The skin levels of IL-8 in AD patients indicate the severity of inflammation, with a reduction in IL-8 levels observed in recovering patients [50]. Furthermore, IL-8 levels were significantly elevated in patients with severe AD compared to those with mild or moderate AD [51]. Interestingly, the anti-IL-33 monoclonal antibody etokimab has shown unanticipated CXCR1-dependent impacts on IL-8-driven neutrophil migration in AD [35]. Another marker, claudin-1 (CLDN1), a crucial component of the epidermal tight junction barrier, plays a significant role in human skin diseases, particularly AD. Reduced CLDN1 expression is linked to AD pathogenesis, as evidenced by AD-like skin features and increased neutrophil and macrophage recruitment in CLDN1-deficient mice. This underscores CLDN1 potential impact on AD disease development [52]. High-mobility group box 1 protein (HMGB1) has a proinflammatory function in various skin inflammation-related pathological conditions. Recent studies have found a correlation between elevated serum levels of HMGB1 in AD patients and the severity of the disease [53]. HMGB1 facilitates the recruitment of neutrophils and the formation of NETs in skin wounds. Furthermore, extracellular HMGB1 can cause tissue damage by triggering NET formation in inflammatory conditions [54, 55]. The prolonged presence of neutrophils and NETs in inflamed skin triggers oxidative stress in keratinocytes of AD patients. This stress results in the release of damage-associated molecular patterns like HMGB1, causing dysfunction in the epidermal barrier. Consequently, this dysfunction facilitates the colonization of S. aureus on the skin. The heightened S. aureus colonization, in turn, amplifies skin inflammation, thus promoting a vicious cycle in AD [56]. The nuclear HMGB1 has been described as a critical “gatekeeper” of dermal homeostasis. It suppresses skin inflammation by site-specific chromatin remodeling for the promoter region of the Il24 gene in the nucleus of keratinocytes. IL-24 stimulates acanthosis and the release of chemokines, leading to an influx of neutrophils. Interestingly, conflicting evidence shows that HMGB1 deficiency in keratinocytes led to elevated expression of CXCL1, resulting in infiltration of neutrophils observed in the ear of transgenic HMGB1 knockout mice using a 2,4-dinitrofluorobenzene-induced allergic contact dermatitis model [57]. β-Galactoside-binding protein galectin-1 (Gal-1), a class of carbohydrate-binding proteins, has elevated expression in the lesional skin of AD patients. This protein is distributed in nearly every tissue and exhibits robust and differential modulatory effects on neutrophils and T cells in AD [58]. Recombinant Gal-1 (rGal-1) treatment has been shown to reduce Th1 cytokines such as IL-1β, IFN-γ, and TNF-α, which leads to decreased neutrophil influx. Consequently, this reduction results in diminished clinical signs and skin thickness associated with AD [59].

The interplay between basophils and neutrophils is pivotal in the pathophysiology of AD. Basophils, stimulated by thymic stromal lymphopoietin (TSLP), interact with sensory neurons and T cells, leading to chronic inflammation and itch in AD skin [60]. Conversely, neutrophils are early recruited in the development of AD lesions through CXCR3 signaling, which is initiated by inflammatory cytokines such as CXCL1 and CXCL10 [61]. These neutrophils then release additional cytokines that amplify the inflammatory response. The combined actions of basophils and neutrophils drive the progression and severity of AD [60]. The reduction of basophils significantly decreases the infiltration of eosinophils and neutrophils, as well as skin thickness [62].

Neutrophils are recruited in the upper dermis and epidermis of lesional skin [36] and trigger itch in the early stages of AD [61]. It was observed that neutrophil-derived ROS and leukotrienes promote pain and/or itch in inflammatory conditions [63, 64]. Filaggrin is a crucial protein in the skin, playing a significant role in maintaining the skin barrier and hydration. In the context of AD, filaggrin dysfunction or deficiency has been strongly implicated [65]. In the AD mouse model, it was found that lesional skin has significantly increased epidermal thickness, dermal infiltration with eosinophils and neutrophils, and Il17a [66]. The enhanced presence of NETs in the skin promotes dysfunction of the skin barrier by downregulating FLG, which in return promotes S. aureus skin colonization [56].

Neutrophil Chemoattractant in Atopic Dermatitis

In the skin lesions of patients with AD, the increased expression of genes that attract neutrophils is similar to that seen in the skin lesions of psoriasis patients. Additionally, the number of neutrophils in AD skin lesions is comparable to those found in psoriasis skin lesions [36]. CXCL1 was reported to be associated with AD [67]. Th17-related biomarker CXCL1 was detected in the lesional and non-lesional skin of AD patients [68]. An analysis of the chemokine profiles from human skin biopsies revealed an increased concentration of CXCL1 in patients with AD, showing neutrophil infiltration [69]. The CXCL1 gene is highly expressed in MC903 mice with similar characteristics to human AD, suggesting that neutrophils are the first immune effectors infiltrating the lesional skin [61]. Administration of CXCL1 induces robust scratching and promotes neutrophil infiltration in mouse models. Neutrophil depletion using an anti-Gr1 antibody significantly alleviates itch-evoked scratching on the skin, attenuates CXCL10 induction, and deactivates sensory neurons via CXCR3, suggesting that neutrophils are required for the expression of itch-inducing chemokine CXCL10 [61, 70]. CXCL10 exerts pruritus on the skin by directly exciting sensory neurons through its receptor CXCR3 or by activating and recruiting immune cells such as T cells, eosinophils, monocytes, and NK cells to release inflammatory mediators that target sensory neurons [71, 72]. The participation of markers like CXCL1, CXCL2, IL-8, and CSF2 (GM-CSF) suggests a shared mechanism of neutrophilic inflammation in psoriasis and AD. A potential contributing factor in AD may involve tissue damage, resulting in the release of neutrophil-chemotactic factors. The potent chemokine IL-8, also known as CXCL8, was identified in the lesional stratum corneum of AD patients and showed a correlation with skin-barrier dysfunction. This chemokine can activate multiple signaling pathways by binding to CXCR1 and CXCR2 receptors expressed on human neutrophils [73]. Furthermore, CD4 + TRM cells enhance the expression of INF-γ and TNF-α, potentially leading to the upregulation of CXCL1 level. This, in turn, attracts neutrophils and contributes to the chronic recurrent inflammation observed in AD [37].

Leukotrienes (LTs) recruit and activate an array of leukocytes, including neutrophils, in several acute and chronic inflammatory reactions [74, 75]. LTs are pruritogens abundantly expressed in skin lesions of patients with AD [76]. These lipid mediators belong to the eicosanoid inflammatory mediators released by several immune cells, such as basophils and mast cells. They are known to induce pruritus in AD [77–79]. Patients with atopic disease have significant LT levels such as LTB4 in the skin lesions [80] and serum [81] and LTE4 in urine [82]. Repeated mechanical abrasion of the skin in AD resulted in robust neutrophil infiltration and increased levels of LTB4 and its receptor, BLT1, in the dermis. In an AD mouse model, LTB4 was derived from neutrophils and critical for establishing allergic skin inflammation [63].

Degranulation and Atopic Dermatitis

Neutrophilic granules are essential in different inflammatory diseases, including psoriasis [1]; however, their function in AD is still unclear. A proteomic study has found that myeloperoxidase (MPO) is upregulated in the serum of young and adult AD patients [83, 84]. It has been suggested that MPO and its ability to induce the overproduction of ROS could contribute to inflammation and pruritus in several diseases [84, 85]. The levels of NE and lipocalin-2 detected in the papillary epidermis of patients with AD were comparable to those observed in psoriasis [36]. Elevated NE activity has also been found in the lesional epidermis of AD patients [86]. NE, a neutral serine protease, can activate protease-activated receptor 2 (PAR2) and transient receptor potential vanilloid 4, thus leading to inflammation and pain [87]. PAR2 has been identified to be widely distributed throughout the human body and is expressed by several cell types present in the skin, including fibroblasts, endothelial cells, and keratinocytes [88]. PAR2 plays an essential role in altered epidermal barrier function, inflammation, and pruritic sensation of the skin, which are hallmarks of AD [89, 90]. Patients with AD exhibit higher levels of PAR-2 receptor expression than healthy donors. The percentage of PAR2⁺ neutrophils increases with the severity of AD [91]. Activation of PAR2 in keratinocytes via serine proteases induces the expression of ICAM-1, thymic stromal lymphopoietin, and several cytokine inflammatory mediators, which increase immune cell infiltration [90, 92]. This series of leukocyte recruitment (particularly Th2) leads to skin inflammation and pruritus [93]. These findings indicate that neutrophil granule proteases, such as MPO and NE, may have a role in the onset of AD. However, this mechanism is inferred from indirect observations, necessitating further studies to substantiate this claim.

Oxidative Stress, NETs, and Atopic Dermatitis

Oxidative stress caused by oxidants, such as ROS and superoxide anions, contributes to chronic skin inflammation. Oxidative stress significantly increases when patients with AD experience a flare-up [94]. Activated neutrophils could lead to oxidative stress due to respiratory bursts, producing ROS and superoxide anions [95]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and MPO are pivotal enzymes in the respiratory burst of neutrophils. Serum MPO level is significantly increased in AD patients [84].

To date, many studies have revealed that NETs play an essential role in numerous diseases such as COVID-19 [96], thrombotic disorders [97], COPD [98], cardiovascular diseases [99], cancers [100], systemic lupus erythematosus [101], rheumatoid arthritis [102], and psoriasis [1]. NETs are marginally increased in the isolated neutrophils from AD patients, and the NETs level is significantly lower than those in the neutrophils from psoriasis patients [103]. Detection of NETs in the stratum corneum of individuals with AD was confirmed through immunofluorescence staining for MPO and the enzyme 8-oxoguanine glycosylase. In vitro studies revealed that neutrophils and NETs can induce ROS production in primary human keratinocytes. The prolonged presence of neutrophils and NETs causes oxidative stress, leading to the release of HMGB1 and the activation of nuclear factor κB (NF-κB) signaling in primary human keratinocytes. This disrupts the skin barrier and promotes S. aureus colonization [56]. There is adequate evidence of the increased ROS in AD [104].

Bacterial Infection in Atopic Dermatitis Associated with Neutrophils

AD condition is associated with impaired barrier function and low levels of AMPs which could contribute to an increased susceptibility to skin infections with fungi, viruses, and bacteria (i.e., S. aureus) [105, 106]. On the other hand, although psoriasis exhibits similarly low AMP levels, it does not show an increased infection rate. Furthermore, AD has a lower level of IL-17 compared to psoriasis which is essential for stimulating AMPs and chemicals that attract neutrophils [107]. The presence of higher neutrophil counts in S. aureus-infected AD lesions, compared to uninfected AD lesions, underscores the complex immune responses in AD [108].

S. aureus is often isolated from AD lesional skin, with persistent overgrowth during a flare-up of AD [109]. Migration and phagocytosis of neutrophils were poor in patients with AD, which led patients to become more susceptible to infection [110]. Staphylococcus species colonize AD skin lesions, such as S. aureus and S. epidermidis. Bacteria form biofilms, which serve as a protection film against innate immune cells, especially macrophages and neutrophils. Biofilm formation leads to high resistance to antibiotics. Previous studies have shown that neutrophilic lysins produced by S. aureus, namely α-toxin, inhibit neutrophilic activity [111]. In contrast, other research studies have shown that neutrophil levels are enhanced in S. aureus-infected skin of AD patients. S. aureus skin infection leads to neutrophil recruitment in patients with AD [112]. A recent study indicated that the serine protease-like protein A (SplA) from S. aureus works in synergy with IL-17A to enhance IL-8 release and promote neutrophil migration in human keratinocytes of STAT3-HIES patients with clinical aspects similar to a Th2-dominated AD [113]. S. aureus can evade neutrophils-induced oxidative stress by sensing oxidants, utilizing antioxidant components, and repairing oxidative damage [114]. Moreover, inhibition of NETs formation by pretreatment of neutrophils with PAD4 inhibitors or DNase impairs the survival of S. aureus in the co-culture model with keratinocytes, suggesting NETs released by neutrophils could be responsible for enhanced S. aureus colonization in skin [115]. These contrasting findings highlight the complexity and heterogeneity of AD. The variability in immune responses emphasizes the multifaceted nature of AD, making it a challenging condition to fully understand.

Human neutrophil α-defensins (HNP) and dermcidin are microbicidal peptides that can modulate innate immunity. In patients with AD, there is an increase in the levels of antimicrobial peptide dermcidin, HNP, and Th2 chemokines (CCL17, CCL22, and CCL27) in the bloodstream. Compromised innate immunity against S. aureus in the skin may allow bacteria to enter the bloodstream, triggering systemic innate immunity, particularly involving neutrophils, which leads to higher levels of HNP and dermcidin in the circulation [116]. Furthermore, Helicobacter pylori neutrophil-activating protein (HP-NAP) can regulate Th1 and Th2 immune responses. When administered through intraperitoneal injection, HP-NAP has been shown to alleviate symptoms associated with AD, including erythema and swelling. HP-NAP reduces the infiltration of lymphocytes and mast cells and the secretion of IgE, IL-4, and various inflammatory cytokines, such as IL-1β, IL-5, IL-6, and TNF-α [117]. As previously stated, neutrophils show a pro-neutrophilic response in the pathogenesis of AD. The neutrophilic inflammatory response comprises the synthesis of cytokines, the degranulation of vesicles, and the formation of ROS.

TSLP, a cytokine derived from epithelial cells, is generated in reaction to pro-inflammatory signals. The level of TSLP expression in AD skin is linked to both the severity of the disease and the extent of epidermal barrier disruption [118]. TSLP directly stimulates dendritic cells to polarize naive T cells into Th2 cells, which secrete IL-4, IL-5, and IL-13, further promoting TSLP release [119]. Activation of TSLP mediates neutrophil killing of methicillin-resistant S. aureus [120].

Drugs Used in Atopic Dermatitis Affecting Neutrophilic Function

As the incidence of AD increases, the discovery and development of new treatments are increasingly important [121]. According to the American Academy of Family Physicians (AFP) 2022, corticosteroids, particularly topical corticosteroids (TCSs), have been and remain the first-line treatment of choice for AD flare-ups [122, 123]. TCSs were first introduced in 1952 by Sulzberger and Witten and are currently available in various concentrations and potencies [124, 125]. Corticosteroids reduce skin inflammation by modifying the function of dermal and epidermal cells and leukocytes involved in skin inflammation [126]. Glucocorticoids exert their effects by binding to the classic glucocorticoid receptor (GR), with neutrophils primarily expressing the GRβ variant, unlike other tissues that predominantly express the GRα variant [127]. This elevated GRβ expression (effect of splicing factor SRp30c) in neutrophils is linked to glucocorticoid resistance observed in allergic and inflammatory diseases [128]. Ligand-activated GR modulates the transcription of thousands of genes through direct binding to DNA response elements, affecting other transcription factors and causing a wide range of effects [128]. Chronic corticosteroid use raises concerns about side effects and rebound flares, especially in pediatric patients [129]. This has driven the development of new agents with fewer adverse effects, better tolerated by young patients [130–132].

The limited efficacy and significant side effects of traditional AD treatments have accelerated research into AD pathogenesis, leading to a translational revolution and rapid expansion in treatment strategies in the last decade [133]. It is important to clarify the role of each cytokine and immune pathway in AD that will lead the path to personalized medicine in the future. This review summarizes potential AD drugs and market-available products targeting neutrophils, supported by evidence from in vivo studies, human trials, and clinical research (Table 1, Fig. 2).

Table 1.

Pharmacological therapies for atopic dermatitis affecting neutrophils

Name	Neutrophil target	Neutrophil function	Route	Clinical Stage in AD	Clinical Trial related to AD and neutrophils	Reference
Crisaborole	PDE4	Decrease chemotaxis and activation	Topical	FDA-approved		[134]
Roflumilast		Inhibit neutrophil recruitmentb	Topical	Clinical trial	NCT04845620 (phase 3)	[135, 136]
Apremilast		Decrease the accumulation of neutrophilsb	Oral	Clinical trial	NCT02087943 (phase 2)	[137, 138]
Delgocitinib	Pan-JAK	Decrease respiratory burst and NETs	Topical or oral	Approved in Japan	NCT03725722 (phase 2)	[139]
Tofacitinib		Decrease neutrophil counts	Topical or oral	Clinical trial	NCT02001181 (phase 2)	[140]
Abrocitinib	JAK1	Decrease neutrophil counts	Oral	FDA-approved		[141, 142]
Upadacitinib		Decrease neutrophil counts	Oral	FDA-approved		[143]
Baricitinib	JAK1/2	Decrease neutrophil counts	Oral	Approved in Europe and Japan	NCT03435081 (phase 3)	[144]
Ruxolitinib	JAK1/JAK2	Decrease neutrophil counts	Topical	FDA-approved		[145]
Tacrolimus	Calcineurin inhibitor	Decrease MPO and chemotaxis	Topical	FDA-approved		[146, 147]
Pimecrolimus		Decrease chemotaxisc	Topical	FDA-approved		[148]
Cyclosporine		Inhibit neutrophil migration and secretion of ROSb	Oral	FDA-approved (off-label)	NCT00445081 (phase 4)	[149–151]
Azathioprine	Immunosuppressant (unknown)	Decrease neutrophil infiltration	Oral	Clinical trial (recruiting)	NCT05078294	[150, 152, 153]
Dupilumaba	Anti-IL-4 and IL-13 mAb	Decrease neutrophil counts	Subcutaneous injection	FDA-approved		[154]
Zileuton	Leukotriene pathway	Inhibit adhesion and chemotaxis of neutrophils	Topical or oral	Clinical trial	NCT03571620 (phase 2)	[155, 156]
Montelukast			Oral	Clinical trial	NCT00557284, NCT00903357 (phase 2)	[157]
Etokimab	Anti-IL-33 mAb (first-in-class)	Decrease neutrophil migration	Subcutaneous, intravenous i.v	Clinical trial	NCT03533751 (phase 2, terminated)	[35]

FDA Food and Drug Administration, IL interleukin, JAK Janus kinase, LOX lipoxygenase, NETs neutrophil extracellular traps; PDE phosphodiesterase; mAB, monoclonal antibody, AD atopic dermatitis

^aDupilumab is the only approved mAb for the treatment of AD in patients six years and older. Dupilomab has completed many Clinical Trials in AD patients, such as NCT05680298 (phase 4), NCT04345367 (phase 3), etc.

^bThe neutrophil function was reported in another disease model

^cThe function was derived from a study on T cells

Fig. 2.

Drugs targeting neutrophils in atopic dermatitis. Several FDA-approved AD drugs or under clinical trials exhibit anti-neutrophilic inflammatory activity. PDE4 inhibitors such as crisaborole and roflumilast inhibit neutrophil chemotaxis. Pan-JAK inhibitors such as delgocitinib, tofacitinib, abrocitinib, ruxolitinib, and baricitinib attenuate respiratory burst and NETs formation in activated neutrophils. Calcineurin inhibitors such as cyclosporine and tacrolimus reduce NETs formation and release of various proinflammatory cytokines. 5-Lipoxygenase inhibitors such as zileuton inhibit adhesion and chemotaxis of neutrophils. Apart from exhibiting antioxidant effects, N-acetylcysteine also ameliorates adhesion and respiratory burst of neutrophils. Vitamin E may inhibit neutrophil chemotaxis

Phosphodiesterase 4 Inhibitors

Phosphodiesterase 4 (PDE4), an enzyme regulating cyclic adenosine monophosphate (cAMP) levels, is present in immune cells like neutrophils and plays a key role in the inflammatory responses in AD [158]. PDE4 inhibitors effectively control neutrophilic inflammation by preventing neutrophil migration and activation [159, 160]. Crisaborole, a nonsteroidal topical PDE4 inhibitor approved by the FDA in 2016 [161] for the treatment of mild-to-moderate AD from 2 years of age and older [162–164] (Table 1), has demonstrated efficacy in reducing itching in patients with AD and reducing spontaneous itch-related responses in mice with chronic atopy-like dermatitis [165, 166]. By inhibiting cAMP degradation, crisaborole reduces the production of inflammatory cytokines and chemokines leading to reduced neutrophil infiltration in AD [167]. The accumulation of intracellular cAMP inhibits the inflammatory reactions of innate and adaptive immune cells, thereby reducing the production of various inflammatory cytokines and AD-related pathways (Th1, Th2, Th17, and Th22) [159]. Crisaborole prevented infiltration of neutrophils in the skin and neutrophilic chemokine expressions such as CXCL1, CXCL2, and CXCL5, thus suppressing itch in a mouse model of AD. Despite its benefits, crisaborole did not significantly affect the density of epidermal nerve fibers even though previous findings demonstrating an increase in animal models of persistent itch [134] and AD patients [168]. Thus, the success story of crisaborole highlights the potential of PDE4 inhibitors in targeting neutrophil-related inflammation in AD.

Roflumilast (Table 1), another PDE4 inhibitor, is currently in phase 3 clinical trial for treating AD. It has already been approved for treating psoriasis by reducing neutrophil infiltration and treating COPD by impeding the migration of neutrophils in the lungs [135]. Topical application of a PDE4 inhibitor attenuates the expression of inflammatory cytokines and chemokines and prevents neutrophil infiltration in mouse models of psoriasis, though similar data for AD is still pending [169]. Apremilast (Table 1), an FDA-approved PDE4 inhibitor for psoriasis and ulcers in Behçet’s disease, is currently in phase 2 clinical trials for AD. Its potential to modulate neutrophil activity makes it a promising candidate for AD treatment [137]. Apremilast reduced neutrophil activation, including NETosis in Behçet’s disease, an autoimmune disease with unclear onset [170]. PDE4 inhibitors, particularly crisaborole, significantly modulate neutrophil function and show considerable promise in treating AD, making them a key target for further therapeutic development.

Janus Kinase Inhibitors

Janus kinase (JAK) inhibitors are novel drugs for treating autoimmune diseases such as rheumatoid or psoriatic arthritis [171]. This type of drug regulates the activity of JAKs and disrupts the receptor-mediated signaling of various cytokines with proinflammatory activity [172]. Together with signal transducer and activator of transcription proteins (STATs) they modulate various key pathways (cytokines) in AD, including Th1 (IFN-γ, IL-2, TNF-β), Th2 (IL-5, IL-4, IL-13), Th17 (IL-17A, IL-17F, IL-21), and Th22 (IL-22) [132]. There are four members of JAK-STAT family, JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase 2). Their phosphorylation activates STAT1 to STAT6, which after translocation to the nucleus, leads to downstream activation of the above-mentioned cytokines. For instance, Th1 differentiation, IL-13 and IL-31 signaling is mediated by JAK1/2, while IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 is mediated by JAK1 and JAK3. IL-4 is mediated by STAT3, STAT5, and STAT6 [173]. JAK1 inhibition shows potential in controlling itch, with substantial antipruritic effects observed. Proposed mechanisms include the activation of JAK1 via IL-4Rα stimulation on sensory neurons, which may drive itch transmission [174]. Importantly, pan-JAK inhibitors inhibited respiratory bursts and NET formation in neutrophils [175].

Topical delgocitinib (Table 1), a pan-JAK inhibitor, has been approved for the treatment of AD (Corectim®) in Japan [139] and completed clinical trials in the USA (NCT03725722, phase 2). In addition, a pan-JAK inhibitor, tofacitinib, was found to decrease neutrophil counts [140]. Furthermore, several other topical and oral pan-JAK inhibitors were found effective in treatment and reducing the severity of AD symptoms, showing rapid and pro-longed anti-pruritic effects (Table 1) [132, 176].

Oral selective JAK1 inhibitor abrocitinib (Table 1) was approved by the FDA in 2022 for moderate to severe AD treatment in adults and was effective at 100 mg to 200 mg and well tolerated in patients with moderate to severe AD [177]. Abrocitinib decreased neutrophil counts, probably due to IL‐6 signaling inhibition via JAK1 [141, 142]. Another JAK1 inhibitor, upadacitinib, is an FDA-approved oral drug for treating AD for patients older than 12 years old [143], that is still undergoing over 20 clinical trials (NCT03661138, NCT03738397) [178].

Ruxolitinib, an FDA-approved topical JAK1/JAK2 inhibitor for treating mild to moderate AD, was proven to affect neutrophil counts [145]. Baricitinib (LY3009104) is an oral JAK-1/2 inhibitor approved in Europe and Japan for treating moderate to severe AD in adult patients with positive results in many clinical trials (e.g., NCT03435081 phase 3) [144]. Combining baricitinib with corticosteroids could improve the signs and symptoms of moderate-to-severe AD in both pediatric and adult patients [149, 179]. To summarize, topical pan-JAK and oral JAK1 inhibitors may alleviate AD symptoms due to their anti-neutrophil-inflammatory effects.

Calcineurin Inhibitors

Calcineurin inhibitors can inhibit T cell activation and proliferation as well as the production and release of various proinflammatory cytokines [180]. Tacrolimus (FK506) [181] and pimecrolimus [148] are available topical calcineurin inhibitors that the FDA approved to treat moderate-to-severe AD. Studies have shown that tacrolimus has an impact on neutrophil activity in AD, leading to various effects such as reduced numbers of neutrophil granulocytes, decreased production of MPO, impaired chemotaxis of neutrophils, as well as dysregulation of CXCR4 and IL-4 [146]. Furthermore, tacrolimus attenuates NETs formation in allogeneic hematopoietic stem cell transplant recipients, thus impairing neutrophil function [182]. Numerous clinical trials have demonstrated the effectiveness of pimecrolimus (Table 1), an immunosuppressant drug of the calcineurin inhibitor in treating AD [148]. However, no direct evidence exists regarding its impact on neutrophils in AD models. Oral cyclosporine (Table 1), a systemic calcineurin inhibitor, effectively treats severe AD [183]. In addition to its effects on T cells, cyclosporine influences innate immune cells like neutrophils. It can inhibit various neutrophil processes, including the production of ROS and formation of NETs, and even demonstrated anti-fungal properties [184, 185]. However, there is currently no direct evidence available regarding the impact of cyclosporine on neutrophils in AD models. Methotrexate and azathioprine showed improvement in clinical trials with AD patients, whereas azathioprine showed better evidence [150]. In a case study involving an AD patient, treatment with azathioprine resulted in an improvement of AD symptoms accompanied by a reduction of 40% in both leucocyte and neutrophil counts. The reductions observed in leucocyte and neutrophil counts were reversed upon ceasing the treatment [152].

Thymodepressin is an immunosuppressive agent that is used to treat autoimmune and allergic conditions caused by lymphocyte-mediated hyperimmune reactions. In skin biopsy samples obtained from patients with AD, thymodepressin was observed to reduce the count of Ki-67-positive nuclei in keratinocytes and alleviate symptoms such as sleep disturbances, itching, erythema, and desquamation [104]. The mechanisms by which oral and topical calcineurin inhibitors treat AD might be related to their anti-neutrophilic effects and deserve more attention and research in the future.

Leukotriene Pathway Inhibitors

The LTs pathway is crucial in AD [186]. Montelukast, an oral cysteinyl-leukotriene-1 receptor antagonist, inhibits neutrophilic inflammation by regulating the cAMP pathway [187]. Montelukast has been approved by the FDA for treating asthma [188] but showed only a weak efficacy in children with AD in a Korean clinical trial (NCT00557284) and insufficient results in US and Australian clinical trials in AD (NCT00903357 NCT00903357) [189]. Other studies also supported the off-label use of montelukast in treating AD [157]. 5-Lipoxygenase (5-LOX), an enzyme that oxidizes arachidonic acid to LTs such as LTB₄, is necessary for the chemotaxis and activation of neutrophils [190]. LTB4 from activated neutrophils is crucial for allergic skin inflammation [63, 155]. In addition, in an animal study, Q301 (zileuton) cream, a 5-LOX inhibitor, could ameliorate AD skin symptoms in phase 2 clinical trials for AD (NCT03571620, NCT02426359, both completed) [156, 191]. Treatment with docosahexaenoic acid and eicosapentaenoic acid plus tacrolimus decreased infiltration of T cells, B cells, eosinophils, neutrophils, and serum IgE level via inhibition of LTB₄ [155]. Moreover, dihomo-γ-linolenic acid (DGLA, DS107), which can be easily obtained by endogenous conversion from γ-linolenic acid (GLA)-rich vegetable oils, completed phase 2 clinical trials on safety and efficacy in moderate to severe AD patients (NCT02211417, NCT02864498, NCT02925793). The unsaturated fatty acids are well known for their inhibitory effects on human neutrophil function [192, 193], emphasizing the need for balanced arachidonic and unsaturated fatty acid intake [194]. Thus, drugs and nutrients targeting the LTs pathways might exert an anti-neutrophilic inflammation effect and are worthy of further development for AD treatment.

Monoclonal Antibodies

Several monoclonal antibodies (mAbs) targeting the Th2 axis have been developed for treating AD, reflecting the critical role of this pathway. Notably, IL-4 and IL-13, key Th2 cytokines, inhibit neutrophil effector functions via type I and type II IL-4Rs on the neutrophil surface [195]. Clinical improvements have been observed with fezakinumab (anti-IL-22 mAb) in severe AD cases. Other mAbs, including nemolizumab (anti-IL-31R), dupilumab (anti-IL-4R/IL-13), tralokinumab (anti-IL-13), and lebrikizumab (anti-IL-13), have also shown promising efficacy. However, their impact on neutrophil functions and broader AD subtypes warrants further investigation. Dupilumab is the first monoclonal antibody approved by the FDA in 2017 for treating moderate to severe AD with acceptable side effects [196]. Dupilumab partially decreased neutrophil count in treated patients [154] and reduced the risk of skin infection in adults with moderate-to-severe AD [197]. In 2021, dupilumab was further approved for children (6–11 years). Also in 2021, tralokinumab was approved by the FDA for the treatment of moderate to severe AD in adults. Both dupilumab and tralokinumab suppress the immune responses mediated by type 2 cytokines [198].

Bristol-Myers’s Squibb-981164 (anti-IL-31 mAb), tezepelumab (anti-TSLP mAb), and MK-8226 (anti-TSLP receptor mAb) have shown promise as potential therapies for AD [199]. However, while tezepelumab, a humanized anti-TSLP antibody, demonstrated safety in a randomized phase 2 trial (NCT00757042), it failed to significantly reduce pruritus and clinical scores (SCORAD, IGA) in AD patients (NCT03809663) [200]. This raises questions about its efficacy in this context despite its success in asthma trials [201]. Further investigation is warranted to clarify the therapeutic outcome of these monoclonal antibodies for AD treatment.

N-Acetylcysteine

N-acetylcysteine (NAC) is an antidote to acetaminophen overdose, which exerts mucolytic, antioxidant, and anti-inflammatory effects [202]. NAC inhibits chemotaxis, adhesion, and oxidative burst in human neutrophils [203]. In addition, topical application of NAC solution increased skin hydration and ameliorated transepidermal water loss in patients with AD [204]. NAC treatment enhances the formation of the skin barrier by increasing the expression of epidermal growth factor receptor, E-cadherin, and occludin (regulator of neutrophils transepithelial migration) in the skin of Flaky tail mice [205]. Therefore, topical treatment with NAC may help AD patients via its antioxidant, anti-neutrophilic, and skin-protective effects.

Nutrients and Vitamins

Nutrients, vitamins, and body homeostasis, including adequate sleep, are vital for immune system function. While genetic factors contribute to AD and other atopic conditions, nutritional deficiencies can disrupt immune responses. Long-term deficits in iron, zinc, vitamin A, and vitamin D are associated with increased Th2 response, antigen presentation, and activation of immune cells, including neutrophils and mast cells, leading to higher IgE production [206].

Zinc [207] and iron [208] supplementation has shown potential in improving AD symptoms by modulating neutrophil function, including degranulation and formation of NETs and ROS in mice [209]. Vitamin C [210], collagen [211], and probiotics [211] enhance skin hydration in AD patients, while vitamin D (clinical studies, such as NCT00789880 [212] and NCT02058186) supplementation has been linked to symptom improvement through mechanisms such as enhanced neutrophilic antimicrobial peptide production, which promotes the Th17 axis [143]. Vitamin E may help manage AD by reducing neutrophil-driven inflammation [213] and skin barrier protection [214].

Atopic Dermatitis Drugs Meta-analyses

According to the meta-analyses from 50 randomized controlled trials on AD drugs, dupilumab, azathioprine, baricitinib, and cyclosporine A, all the drugs that were reported to affect neutrophilic function (Table 1) received robust trial evidence for the efficacy and safety in adults [150]. Among topical AD treatments, meta-analyses from 10 randomized controlled trials showed tofacitinib, ruxolitinib, and delgocitinib with superior efficacy over other JAK and PDE4 inhibitors and much better than corticosteroids [215]. Another 5 randomized controlled trials showed tofacitinib with the best effect on pruritus [216]. All these drugs affected neutrophil counts in patients or suppressed the pathological effects of neutrophils in AD models (Table 1), highlighting their impact on the treatment of AD and the role of neutrophilic inflammation in AD, worth further development.

Study Limitations

Literature is scarce on where AD treatment drugs interfere with neutrophilic activity in AD samples. Nevertheless, the authors have identified several drugs in Table 1 that have been correlated with neutrophil function in AD. By highlighting these drugs, the aim is to inspire future researchers to explore the possibilities of these drugs in managing neutrophilic activities in AD.

Conclusion

Classically, AD is a chronic immune-mediated inflammatory skin disease that is mainly orchestrated by Th2 cells. The strong correlation between type 2 inflammation and AD has led to a scarcity of information regarding the potential involvement of neutrophils in the immunopathogenesis of AD. However, most of the presented data are from animal models, and only a small amount of data is derived from human AD patients. The emerging evidence offers insights into the potential roles of neutrophils in AD. Several key points have been identified: first, NLR in patients with AD is higher than that in normal individuals. Second, repeated skin scratching can promote neutrophil infiltration in AD. Third, high CXCL1 chemoattractant levels were detected in the lesional skin of patients with AD. Fourth, AD patients demonstrate increased expression of MPO, predominantly found in neutrophils and associated with oxidative stress. Fifth, NE, a serine protease released by neutrophils, activates PAR2 in keratinocytes, contributing to immune effector infiltration in AD. Sixth, neutrophil-derived oxidative stress and NETs are involved in the flare-up of AD caused by S. aureus. Lastly, certain drugs used to treat AD could affect neutrophilic inflammation. Enhancing our understanding of the pathogenic role of neutrophils in AD could facilitate the development of novel therapeutic strategies for the condition.

Materials and Methods

The PubMed and SciFinder databases were used as search engines for this literature review. Authors used the keywords “atopic dermatitis,” “atopic eczema,” ‘Th1,” and “Th2,” “neutrophils,” “neutrophil chemoattractant,” “neutrophilic degranulation,” “pruritus,” “NETs,” “oxidative stress,” and “skin inflammation” to find the articles that could potentially associate neutrophils and atopic dermatitis. There was no discrimination when selecting an article, whether the samples were human or animal models or the experiment was done in-vivo or ex-vivo. Figures 1 and 2 were created with BioRender.com.

The authors thoroughly read the abstract, methodology, results, discussion, and the article’s conclusion to gather information. EndNote ver. 20 was the citation app used to generate and manage the library reference.

Abbreviations

AD

Atopic dermatitis

Th cells

T helper cells

COVID-2

Coronavirus disease 2019

FDA

Food and Drug Administration

LT

Leukotriene

IL

Interleukin

MPO

Myeloperoxidase

JAK

Janus kinase

LOX

Lipoxygenase

NE

Neutrophil elastase

NETs

Neutrophil extracellular traps

PDE

Phosphodiesterase

Authors’ Contributions

CCC and JRMSDC wrote the paper. CWJ drew the figures. NLW, TR and MK consulted and revised the treatment schemes and the manuscript. HTL initiated the concept and supervised the writing. All authors read and approved the final manuscript.

Funding

This review was supported by grants from the National Science and Technology Council (NSTC), Taiwan (NSTC 113-2320-B-037-023; 113-2321-B-255-001; 112-2320-B-037-012; 111-2320-B-255-006-MY3), and in part by a grant from the Kaohsiung Medical University Research Foundation (KMU-Q113011). The funders had no role in the study design, data collection, data analysis, manuscript preparation, and/or publication decisions.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of Interest

The authors declare no competing interests.