The Roles of Innate Immune Cells in Atopic Dermatitis

Atopic dermatitis (AD) is one of the most common chronic and recurrent inflammatory skin conditions, characterized by recurrent eczematous lesions and intense itching [1]. Early-onset AD usually occurs in childhood, typically at 3–6 months, whereas AD manifests as persistent and new onset forms in adults [2, 3]. The pathogenesis of AD is complex and multifactorial, including immune dysregulation, genetic disorders, epidermal barrier dysfunction, microbial dysbiosis of skin, and environmental factors [4, 5].

Immune dysregulation plays an essential role in the disorder of AD. One hallmark of AD is the accumulation of inflammatory cells within the lesion, including T cells, macrophages, eosinophils, and mast cells. On the contrary, non-lesional atopic skin has a sparse perivascular T-cell infiltrate in the dermis [6]. T helper 1 (Th1)- and T helper 2 (Th2)-cell imbalances and IgE-mediated hypersensitivity have been reported as essential factors in AD pathology [7]. Notably, several studies have emphasized the role of innate immune cells in the onset and progression of AD [8, 9]. It has been widely accepted that type 2 innate lymphoid cells (ILCs), a member of the innate immune system, contribute to the initiation of AD. Unlike the paradigmatic Th2‐mediated immune response in the early stage, a mixed Th1/Th2 pattern of inflammation can be observed in the chronic phase, which is characterized by a switch toward a Th1/Th17 environment [10]. This article reviews recent research on the role of innate immune cells in AD, which will help further explain the pathogenesis of AD and give insight for treating AD from the perspective of innate immunity in clinics.

As the largest immune organ in the body, the skin protects against numerous external dangers [11, 12]. Skin immunity can also be divided into adaptive immunity and innate immunity. Innate immune response has two main characteristics. One is that it can distinguish structural components of microbial pathogens, while these specific structural components are absent in the normal host cells, and these main structural components are pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The recognition process is mediated by a variety of proteins present in the host cells such as the PRRs [13, 14]. These PRRs could recognize PAMPs or DAMPs present in microbial pathogens. Then, the relevant cells take their functions. The other characteristic is that the innate immune response does not have the ability to generate immunological memory after the recognition of the pathogen. This is different from adaptive immunity [13]. Besides, innate immune cells could take their function rapidly without the need for cloning or amplification while adaptive immunity does. The cells mainly involved in the innate immunity of the skin are natural killer cells (NK cells), natural killer T cells (NKT cells), dendritic cells (DCs), neutrophils, melanocytes, and keratinocytes. Other innate immune cells, including eosinophils, gamma delta T cells (γδ T cells), ILCs, macrophages, mast cells, and basophils, are also implicated in inflammatory skin diseases such as AD, either in lesional skin or in peripheral blood. Specifically, keratinocytes, macrophages, epidermal and dermal DCs, and mast cells constitute the resident cells (shown in Fig. 1a). Meanwhile, when inflammation happens, mobilized cells such as neutrophils, inflammatory DCs, and eosinophils can be immediately recruited to the skin and produce significant effects (shown in Fig. 1b) [15]. In addition, the epidermis and dermis contain specific immune cells. For example, keratinocytes and epidermal DCs distribute in the epidermis and the latter take residence even before birth [16]. In contrast, mast cells and macrophages are found around the dermal papillary vessel and superficial dermis, respectively, while DCs are found throughout the dermis [17]. Other dermal immune components include basophils and eosinophils [16].

Neutrophil precursors located in the bone marrow, after differentiation and maturation, enter the circulation as neutrophils. During an immune response in the tissue, neutrophils are recruited to detect pathogens and danger signals and to initiate inflammation. Herein, neutrophils are regarded as key innate immune phagocytes in immune defense [18]. An earlier study showed a significant number of neutrophils in the skin of AD lesions [19]. Furthermore, transcriptomic profiling also revealed increased expression of genes associated with neutrophil chemotaxis, such as CXCL8 and GM-CSF [19]. Neutrophils could secrete S100A8 and S100A9, and the latter binds to Toll-like receptor 4 to participate in the pathogenesis of AD [20‒22]. Staphylococcus aureus infection is a common complication of AD. Increased S. aureus may lead to thickening of the epidermis and expansion of immune response cells, leading to the onset and exacerbation of AD [23]. Interestingly, recruited neutrophils failed to inhibit bacterial growth in the skin; as a result, increased S. aureus colonization and persistence were reported. This observation may be caused by the interaction of neutrophil extracellular traps with keratinocytes [24]. Overall, neutrophils may be involved in the pathological process of AD to some extent, but their contribution to AD is much less than that of psoriasis [25].

Eosinophils are part of white blood cells that help destroy foreign substances, such as parasites, and promote inflammation in the body [26]. Eosinophils and their secreted mediators have been implicated in various inflammatory diseases, including AD [27]. The deposition of eosinophils and eosinophil granules has long been established [28]. Compared with healthy controls, the peripheral blood eosinophils count of AD patients is significantly increased [25], which is a typical feature of AD and has a value in differentiating AD from similar diseases [29, 30]. The elevated levels of blood eosinophils also correlate with disease severity [29, 31].

Although the exact role of eosinophils in the pathogenesis of AD is still unclear, Mendelian randomization findings indicated that increased eosinophil and basophil counts may be a potential causal risk factor of AD [32]. Conversely, the depletion of eosinophils improved skin barrier function and alleviated symptoms [33]. In addition, the activity of eosinophils can be regulated and controlled by pathological changes. Previous studies have confirmed that Th2-derived IL-5 induced activation and chemotaxis of eosinophils [29]. Recently, the effects of histamine on eosinophils have attracted attention. Eosinophils from AD patients highly expressed the histamine H4 receptor (H4R), and histamine was crucial in activating human eosinophils [34]. Osiecka et al. [35] linked secretory leukocyte protease inhibitor (SLPI) to the migratory function of eosinophils. Eosinophils are one of the major sources of SLPI, which is stored in the crystalline core of eosinophil granules. SLPI may be involved in the recruitment of eosinophils to tissue either through interaction with the cell surface and potentially cell-surface receptors, and/or via modifying intracellular pathways upon uptake by eosinophils [35]. Subsequently, eosinophils may produce leukotriene (LT) C4/D4/E4 (LTC4, LTD4, LTE4) that amplify the inflammatory cascade, produce reactive oxygen species, and release extracellular eosinophil traps, thus promoting inflammation [36].

ILCs comprise five subsets: NK cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer cells. Group 1 ILCs comprise NK cells and ILC1s; group 2 refers to ILC2s; group 3 includes lymphoid tissue inducer cells and ILC3s [37]. ILCs do not express antigen-specific receptors but respond to infection guided by cytokines and secreted proteins. ILC2s are components of the first line of defense, acting prior to the adaptive immune response [38]. ILC2s and ILC3s are resident cells, while ILC1s constantly traffic and predominantly produce IFN-γ [39].

NK cells are cytotoxic cells of the innate immune systems that limit the spread of infection. They circulate in the blood seeking out infected cells or abnormal cells. NK cells recognize certain molecules on the surface of abnormal cells and release cytotoxic granules to destroy them [40]. Variations in the quantity and quality of NK cells in lesional AD skin and peripheral blood have been reported [41‒44]. Activated NK cells and their chemoattractant (CCL2 and CCL22) were enriched in lesional AD skin [41, 43]. However, total CD56⁺ NK cells and peripheral IFN-γ production were decreased in AD blood samples [42], possibly due to increased NK-cell apoptosis in the blood or increased extravasation in the inflamed skin [42, 44]. The reduced number of NK cells in the blood can also indicate the severity of AD and is of a certain diagnostic value [43‒45]. Histamine may play a role in NK-cell migration into skin lesions. The histamine level was elevated in the skin lesions, and in vitro studies have shown that stimulation of the H4R expressed by NK cells induces chemotaxis [46]. NK cells can function in regulating immune responses in tissues [47]. In skin lesions of AD, NK cells could regulate ILC2s because the loss of NK cells in mice exacerbated pathogenic ILC2 responses [43]. Furthermore, IFN-γ deficiency shifted the Th1/Th2 polarization toward Th2 responses and increased host susceptibility to viral infection [42, 48].

Several changes occur in NK-cell subsets in AD. For example, the proportion of transforming growth factor (TGF)-β-producing regulatory NK (NKreg) subset decreased in peripheral blood mononuclear cells of AD patients, which significantly suppressed Th2 immunity and ILC2 activation in AD mice [49]. In a mouse AD disease model, adoptive transfer of NKreg alleviates the severity of AD [49]. Blood NK cells consist of CD3-CD56^dim and CD3-CD56^bright-cell subsets. High-dimensional mass cytometry technology and RNA-seq analysis revealed a selective loss of a subset of mature CD56^dim NK cells [43]. Additionally, an imbalance in the expression of NK-cell inhibitory and activating receptors as well as a disbalance between resting and activated NK cells have been reported in lesional AD skins [50]. These facts highlight the importance of studying the imbalance of different NK-cell subsets.

ILC2s were originally found to protect the host against intestinal helminth infection by releasing the typical Th2 cytokine IL-13. Later, their function in the production of type 2 inflammatory cytokines, including IL-4, IL-5, and IL-13, was reported, and scholars began to link them to various inflammatory and allergic diseases [51]. Dermal ILC2s can modulate inflammation as they produce IL-13, which affects mast cell activity in normal situations and, when activated, promotes eosinophil influx and mast cell activation [52], with both pro- and anti-inflammatory potential.

The infiltration of ILC2s in AD lesions increased while ILC2s in the blood were rarely detectable [44, 53]. Moreover, several genes encoding Thymic stromal lymphopoietin (TSLP), IL-4, IL-13, and IL-18R are tested to be the susceptibility loci for AD, and all of them are related to the activated behaviors of ILC2s [37, 54, 55]. Experiments in earlier years have already demonstrated the role of ILC2s in MC903 (vitamin D analog calcipotriol)-induced AD-like dermatitis in a mouse model, and deletion of ILCs attenuated the dermatitis [56]. ILC2s are important sources of type 2 cytokines in AD. A subset of ILC2s that express NKp30 can interact with increased B7-H6 [57], a cognate activating ligand, in AD lesions, inducing rapid production of type 2 cytokines, further leading to skin barrier disruption, entry of outer antigens, and induction of effector Th2 cells [58]. Strikingly, though type 2 cytokines serve as a bridge between ILC2s and adaptive immune cells like Th2 cells, ILC2s promote AD independently of adaptive immunity. In a filaggrin-deficient mouse model that tended to AD-like inflammation, the typical clinical manifestations of AD could still be observed under intentional T/B-cell deficiency conditions [59]. Similarly, in another mouse model lacking T/B lymphocytes, AD-like symptoms did not disappear, accompanied by increased ILC2-cell numbers in the skin. These may prove that innate immune response is more important in triggering skin inflammation [60].

In the past, little attention was paid to the role of ILC3s in AD. Although ILC3s are rarely present in human skin, they are significantly increased in eczema [61, 62]. In an AD mouse model built by Kong et al. [62] increases in ILC3s often preceded increases in ILC2s. Moreover, the presence of inflammatory ILC3s roughly correlated with severity. These facts indicate a possible prerequisite role of type 3 innate immune response in AD. In addition, ILC3s can induce IL-33 production by non-immune skin cells, keratinocytes, and fibroblasts, thus promoting type 2 immune responses [63].

Keratinocytes are not considered classical innate immune cells in histology. However, to the extent of infection-related areas, we could classify them into innate immune cells according to the following characteristics: Keratinocytes are the main constituent cells of the epidermis, accounting for more than 80% of the epidermal cells [64]. Besides, keratinocytes form the physical skin barrier and represent the first line of the host defense system by sensing pathogens via PRRs on their cell surfaces. These PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptor-like receptors (NLRs) and C-type lectin receptors [65]. These receptors recognize highly conserved structure components of microorganisms, known as PAMPs and DAMPs, initiating anti-microbial responses and producing various chemokines, anti-microbial peptides and Th2-promoting cytokines, including TSLP, IL-25 and IL-33. These keratinocyte-derived cytokines could regulate effector T-cell differentiation in AD. Meanwhile, keratinocytes participate in antigen presentation by expressing major histocompatibility complex class II antigens, phagocytosis, and crude processing of antigens [66]. In summary, we could classify them into innate immune cells due to their functions in AD. Keratinocytes can act as an initiator and subsequently activate other cells in the pathogenesis of AD. In AD patients, type 2 pro-inflammatory chemokines can be released from keratinocytes, contributing to the infiltration of mast cells, ILC2 cells, eosinophils, monocytes, and DCs [67, 68]. These cells produce type 2 cytokines and chemokines involved in aberrant immunity in AD. Furthermore, several conditions common in AD can stimulate the release process, including epidermal barrier defects, signal transmission, and environmental stimuli such as dysbiosis of S. aureus [68].

DCs are professional antigen-presenting cells (APC) that form a link between innate and adaptive immunity [69]. In particular, human skin has a special type of DCs called Langerhans cells (LCs), which reside in the epidermis and act as the first cellular barrier. DCs in the dermis comprise different subsets of dermal DCs (dDCs) [70]. Under the state of AD, a special DC subset called inflammatory dendritic epidermal cells (IDEC) infiltrates the inflammatory lesional skin and participates in the development of AD [70].

DCs are capable of regulating Th2-cell function and maintaining homeostasis. Both epidermal LCs and dDCs can produce CC chemokine ligands CCL17 and CCL22, which play a critical role in Th2-cell recruitment and immune response maintenance [71]. However, when a disorder in the regulation occurs, the overreaction of Th2 cells happens and leads to allergic diseases such as AD [72]. In patients with AD, both LC and IDEC highly express the FcεRI receptor, a high-affinity receptor for IgE. IgE facilitates allergen uptake and binding and offers a possible explanation for abnormal IgE-mediated hypersensitivity [69]. Unlike LCs and dDCs, IDECs present the allergen to Th1 cells and are likely to associate with the chronicity of eczema by IL-12 and IL-18 production [41, 73].

Skin mast cells are also APCs that involve immune regulation and allergic reactions due to the expression of large numbers of IgE Fc receptors. Mast cells increased in skin lesions in both human and mouse AD models, indicating their participation in AD [74]. Mast cell-deficient mice displayed attenuated skin inflammation, revealing that mast cells are essential in allergen-induced AD-like skin inflammation [75]. In addition, a special receptor, dendritic cell immunoreceptor (DCIR), which mediates allergen binding and uptake, was upregulated, further leading to ROS generation and oxidation of calmodulin kinase II (ox-CaMKII) [75]. Numerous studies also revealed that mast cells are producers of several inflammatory mediators demonstrated to associate with AD, including prostaglandin D2 (PGD2), histamine, IL-31, and IL-33 [76].

γδ T cells are a subset of T cells that perform intrinsic immune functions, and their T-cell receptors (TCRs) are composed of γ and δ chains. Though γδ T cells occupy a tiny percentage of total T cells in the peripheral blood, they are enriched in the skin, especially during acute and chronic skin inflammation [77]. However, blood γδ T cells are decreased due to apoptosis induced by monocytes [48]. Meanwhile, γδ T cells may have the activity of maintaining homeostasis. Recent findings demonstrate that innate neonatal-derived IL-17 producing γδ T (Tγδ17)-cell subset has a crucial role in preventing AD. Spontaneous AD was observed in a Tγδ17 cell-deficient mouse model, indicating the dual homeostatic and inflammatory function of IL-17 in AD [78].

Basophils, similar to mast cells residing in the skin, also express the high-affinity receptor for IgE, FcεRI, and release similar effector molecules such as histamine, 5-hydroxytryptamine, tryptophan-like enzymes, and LTs upon IgE cross-linking. It is noteworthy that basophils are present in the skin lesions among approximately 60% of patients with AD [79], while in peripheral blood, the number of basophils varied [79, 80], indicating distinct endotypes of AD (with or without involvement of basophils). Recent studies report that FcεRIa is upregulated on basophils in inflammation associated with AD [81]. In addition, for AD involving basophils, a retrospective study shows that basophils are associated with epidermal infiltration of helper T cells [82], and might play a role in pruritus (described below).

NKT cells express NK receptors and markers of conventional T cells as well [83], and are usually divided into type I and type II. Type I NKT cells are called semi-invariant NKT cells (iNKT) because they express the typical, semi-invariant TCR, whereas type II NKT cells have a different TCR repertoire. Both type I and type II NKT cells can recognize glycolipid antigens on CD1d molecules.

The significant alterations in the number and function of NKT cells in AD suggest a potential link between NKT cells and AD [84‒86]. Decreased intracellular IFN-γ and increased intracellular IL-4 were observed [84]. Besides, surface immune cell markers, including CXCR4 and CD4/CD8, also undergo quantitative changes in NKT cells [86]. Skin-resident NKT cells uniquely expressed CXCR4 and were enriched in AD skin, which was presumed to be recruited by fibroblast-derived CXCL12 [85]. However, although NKT cells play a critical role in many other allergic diseases, they may not compose a requisite part of allergic skin inflammation [87]. The role of NKT cells in AD requires further investigation.

Macrophages derive from monocytes when they invade the dermis. As a member of APCs, dermal macrophages play a role in wound repair and prevention of microbial invasion. Previous studies have revealed increased infiltration of macrophages in AD skin [88]. Furthermore, reduced phagocytic activity and TLR-2 expression of macrophages were detected in patients with AD, contributing to enhanced susceptibility to skin infections [88]. Recently, increasing evidence has shown how macrophages are involved in the pathogenesis of AD. Transient receptor potential vanilloid 4 (TRPV4) expressed by macrophages exhibited an anti-inflammatory effect, and decreased expression of TRPV4 was detected in AD [89]. Chitinase 3-like 1 (CHI3L1), a recognized player in Th2 inflammation, has been proven to drive AD via M2 macrophage activation [90]. Additionally, histamine stimulated M2 macrophages to express chemokine CCL18, a highly expressed chemokine in lesional skin and serum [91]. In conclusion, the infiltration and activation of macrophages may contribute to the maintenance of inflammation [92].

ILC2s play a pivotal role in the immune network concerning innate immunity. ILC2s can be activated by several innate immune cells. In MC903-induced AD mouse models, basophils regulated ILC2 activation via IL-4 production [93]. Mast cells enhanced IL-5 production by ILC2s via miR103a-3p in extracellular vesicles [94]. Additionally, mast cell-derived mediators such as cysteinyl LTs, LTE4 in particular, can activate ILC2s. Meanwhile, the expression of LT receptors CysLT1 increased on ILC2s [95]. Basophil-generated IL-4 and increased expression of CCL11, IL-5, IL-9, and IL-13 by ILC2s after activation by basophils contributed to the recruitment and accumulation of eosinophils [96, 97].

Keratinocytes form the first line of defense and play a role in the upstream of the network. TSLP expression in keratinocytes significantly increases, directly activating iNKT cells to secrete IL-4 and IL-13 [98]. Besides, TSLP is known as a main regulator of skin ILC2s [98]. Innate immune signaling in keratinocytes, like MyD88 signaling, led to T-cell recruitment and LC emigration [99].

Since the acute phase of AD is dominated by type 2 immune responses, the effect of Th2 cells cannot be ignored. As previously mentioned, TSLP directly interacted with Th2 cells; NK cells promoted Th2 polarization; and DCs participated in Th2-cell recruitment [42, 48, 71]. In the chronic stage, Type 1 inflammation also plays a part. Infiltration of Th1 cells required IL-12 secreted by macrophages, eosinophils, and DCs [100]. In addition, innate immune cells such as ILCs produced Th1 cytokines (IL-2, IFN-γ, etc.) and Th2 cytokines (IL-5, IL-13, etc.) to promote adaptive immunity [101]. Overall, innate immune cells actively participate in both innate and adaptive immunity, constituting a complex network in AD (shown in Fig. 2).

Pruritus, transmitted by non-myelinated C-type free nerve endings in the epidermis and upper dermis [102], is one of the major and diagnostic symptoms of AD. During the induction of pruritus, the interactions between sensory neurons and immune cells require pruritogens to act as intermediaries [103]. It is well known that the primary sources of pruritogens are histamine, neuropeptides, and cytokines (including IL-4, IL-13, and IL-31), and innate cells are the important producers. For example, mast cells, basophils, and eosinophils are the primary sources of pruritogens mentioned above [104]. A basophil-LT axis contributes to the acute itch flare in the chronic course of AD because allergen-stimulated basophils produce enhanced LTC4, which then bind to its receptor CysLTR2 on neurons [81]. Besides, eosinophils in the skin of AD patients mainly locate close to peripheral nerves and express BDNF, one of the neurite growth-stimulating factors, and may induce itch via stimulation of skin nerve branching [102].

Neutrophils play many roles in itch as neutrophils produce and release pruritogens like histamine and LTB4, which then induce itch [105]. Crisaborole (phosphodiesterase 4 inhibitor), an ointment approved to treat mild to moderate AD, inhibits itch by reducing neutrophil infiltration and interaction of neutrophils with sensory neurons in the MC903-treated mouse model [106]. Depletion of neutrophils significantly relieved itch in the mouse model of AD, along with a decrease in several hallmarks of chronic itch inflammatory cytokines [107]. However, above-mentioned evidence of neutrophils inducing itch is mainly based on mouse models. Although the role of neutrophils in itch has been demonstrated in other pruritic skin diseases like psoriasis, the role in AD needs further research.

Keratinocytes are the major source of TSLP, which activates primary afferent neurons through TRPA1 activation [108]. TSLP expression was highly upregulated in AD skin, accounting for pruritus induction [109].

M2 macrophages are essential producers of IL-31, a type 2 cytokine related to itching sensation [110]. The scratching due to the sensation of pruritus will eventually damage the skin barrier and increase the likelihood of skin infections (shown in Fig. 3).

AD can be divided into two types: intrinsic type of AD (IAD) and extrinsic type of AD (EAD) [111]. The difference between these two types includes the concentrations of normal total serum IgE levels and allergen-specific IgE antibodies in the blood. IAD may develop into EAD over time. With the elevation of total serum IgE [112], EAD patients showed high levels of Th2 cytokines (IL-4, IL-5, and IL-13) compared with IAD. Besides, strong CCL18 expression was observed in lesional skin and IDEC-like DCs in EAD patients [113]. These phenomena indicate that there may be some differences in the function of cells between two types of AD, including mast cells, DCs, monocytes, etc.

AD is also considered a biphasic disease, with Th2 predominating in acute disease and a switch to Th1 characterizing chronic disease. In the acute phase of AD, a subset of increased terminal differentiation proteins was found, including S100A7, S100A8, and S100A9 [114]. The last two proteins could be secreted by neutrophils and bind to Toll-like receptor 4 to participate in the pathogenesis of AD [20‒22]. These might indicate that the role of neutrophils in the acute phase of AD is more dominant than that in the chronic phase. Since the acute phase of AD is dominated by type 2 immune responses, the effect of Th2 cells cannot be ignored. TSLP could directly interact with Th2 cells; NK cells promoted Th2 polarization; and DCs participated in Th2-cell recruitment [42, 48, 71]. In the chronic stage, type 1 inflammation also plays a part. Infiltration of Th1 cells required IL-12 secreted by macrophages, eosinophils, and DCs [100]. In addition, innate immune cells such as ILCs produced Th1 cytokines (IL-2, IFN-γ, etc.) and Th2 cytokines (IL-5, IL-13, etc.) to promote adaptive immunity [101].

AD could also be divided into adult-onset AD and child-onset AD. About 13% of American children suffer from AD. While 1 in 4 adults with AD report adult-onset disease, many adults with AD might have child-onset AD [115]. These differences may be due to different stages of growth and immune status.

AD is a chronic inflammatory skin disease closely associated with host immune system dysfunction, manifested by compromised skin barrier. Innate immune cells present variations in quantity and function, contributing to the pathogenesis of AD via various pathways. Additionally, innate immune cells can interact with adaptive immune cells, further complicating the regulatory network. Moreover, some innate immune cells contribute to induce itching. Overall, this review helps understand the roles of innate immune cells in AD and is instructive for treatment.

The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

Yuke Pan, Youyi Wang, Kang Zeng, and Xiaowen Huang reviewed previous related publications. Yuke Pan and Youyi Wang wrote the first draft of the manuscript. Meinian Xu, Meizhen Zhong, Xiaoming Peng, Kang Zeng, and Xiaowen Huang commented on previous versions of the manuscript. Yuke Pan, Youyi Wang, Meinian Xu, Meizhen Zhong, Xiaoming Peng, Kang Zeng, and Xiaowen Huang read and approved the final manuscript.

Yuke Pan and Youyi Wang contributed equally to this work and share first authorship.