Thymic Stromal Lymphopoietin, Skin barrier dysfunction, and the atopic march

Abstract

Objective:

Atopic dermatitis often precedes the development of other atopic diseases, and the atopic march describes this temporal relationship in the natural history of these diseases. Although the pathophysiological mechanisms that underlie this relationship are poorly understood, epidemiological and genetic data have suggested that the skin might be an important route of sensitization to allergens

Data Sources:

Review of recent studies on the role of skin barrier defects in systemic allergen sensitization.

Study Selections:

Recent publications on the relationship between skin barrier defects and expression of epithelial

Results:

Animal models have begun to elucidate how skin barrier defects can lead to systemic allergen sensitization. Emerging data now suggest that epithelial cell-derived cytokines such as thymic stromal lymphopoietin (TSLP) drive the progression from atopic dermatitis to asthma and food allergy. Skin barrier defects can lead to induction of epithelial cell-derived cytokines, which in turn leads to the initiation and maintenance of allergic inflammation and the atopic march.

Conclusion:

Development of new biologic drug targeting type 2 cytokines provide novel therapeutic interventions for atopic dermatitis.

1. Introduction

The concept of “atopy” is characterized by exaggerated immune responses to allergic stimuli and high IgE that can lead to clinical disease at a variety of anatomic sites. In general, clinical symptoms of atopy are not present at birth; however, atopic individuals are predisposed to the development of allergies¹. For example, the “allergic triad” – atopic dermatitis (eczema), allergic rhinitis, and allergic asthma – frequently presents in a single individual^{2, 3, 4, 5, 6}. Other allergic diseases, such as food allergies, eosinophilic esophagitis (EoE), and allergic conjunctivitis are also common in atopic patients^{7, 8, 9, 10}.

The natural history of atopic diseases tends to follow a characteristic sequence of events: the first manifestation of atopy is frequently atopic dermatitis, followed later by food allergy, allergic rhinitis, and allergic asthma. The term “atopic march” (or “allergic march”) describes this developmental progression of atopic diseases^{3, 4, 5, 6}. The atopic march often begins early in infancy with the development of atopic dermatitis, which peaks in prevalence within the first two years of life^{11, 12}. Of the children who develop atopic dermatitis, many, if not most, develop the disease within the first six months of life^{13, 14}. There is also a link between atopic dermatitis and the development of food allergies, especially to peanut^{15, 16, 17}. Although asthma has substantial heterogeneity that likely represents distinct pathophysiological mechanisms, individuals with chronic asthma often present before the age of five^{18, 19}.

Children with atopic dermatitis are more likely to develop other atopic diseases than those without atopic dermatitis^{17, 20, 21}. Furthermore, the severity and chronicity of atopic dermatitis also correlate with increased incidence of atopic diseases; whereas around 20% of children with mild atopic dermatitis develop asthma, over 60% with severe atopic dermatitis develop asthma^{20, 21, 22, 23}.

Although the mechanisms that underlie the temporal relationship of diseases in the atopic march are still poorly understood, epidemiological studies have provided a framework for experimental models to examine how atopic dermatitis and skin barrier dysfunction can lead to disease at other anatomic sites. This review will focus on the factors that affect the development of atopic diseases and the atopic march, with a focus on the role of skin barrier dysfunction and the cytokine thymic stromal lymphopoetin (TSLP). target for the prevention of the atopic march and treatment of atopic diseases.

The genetics of atopic diseases and the atopic march

While changes in environmental exposures provide some potential as targeted means of decreasing the prevalence of atopic disease, there is a strong influence of genetics on the development of allergies. Based primarily on twin studies of asthma and atopic dermatitis, the heritability of atopic diseases has been estimated to be around 60 to 75%^{24, 25, 26}.

Genetic studies have provided evidence of the importance of epithelial barrier defects in the pathophysiology of atopic dermatitis and other atopic diseases. In particular, mutations in genes encoding three important components of the epithelial barrier of the skin – filaggrin, serine peptidase inhibitor Kazal-type 5 (SPINK5), and corneodesmosin – are associated with atopic dermatitis or atopic dermatitis-like syndromes as well as atopic diseases at other sites.

Filaggrin is expressed as the precursor protein profilaggrin that is subsequently cleaved into filaggrin, which then aggregates and organizes keratin filaments within the skin epithelium. Mutations in the gene encoding filaggrin (FLG) can give rise to ichthyosis vulgaris and lead to an increased susceptibility to atopic dermatitis^{27, 28}. Filaggrin loss-of-function mutations have been shown to be associated with increased risk of food allergy and eosinophilic esophagitis^{29, 30}, but only in individuals with atopic dermatitis³¹. Some studies have also reported increased risk of asthma or increased asthma severity with these filaggrin mutations, but it is unclear whether this effect on asthma incidence or severity was dependent on having coincident atopic dermatitis^{27, 32, 33}.

Another example of a monogenic atopic disease is Netherton syndrome, a severe, autosomal recessive disorder caused by mutations in the SPINK5 gene, which encodes serine protease inhibitor Kazal-type 5 (also referred to as lympho-epithelial Kazal-type-related inhibitor [LEKTI])^{34, 35}. At the neutral pH of the deep stratum corneum, LEKTI binds and inhibits the proteases kallikrein related peptidase 5 (KLK5) and KLK7; however, in the acidic conditions of the upper stratum corneum, LEKTI is inhibited, which allows KLK5 and KLK7 to function and promote skin peeling³⁶. In Netherton syndrome, LEKTI deficiency results in KLK5 and KLK7 proteolytic activity in the deeper levels of the skin, resulting in the development of a severe atopic dermatitis-like syndrome and a specific hair shaft defect (trichorrexis invaginata or 'bamboo hair')³⁷. Netherton syndrome patients also have other atopic manifestations including hay fever, food allergies, high serum IgE levels, and hypereosinophilia^{38, 39}. Importantly, these patients display extremely high levels of circulating TSLP, and elevated TSLP expression in the skin, consistent with a role for TSLP in the allergic manifestations of this mutation⁴⁰.

Autosomal recessive mutations in the corneodesmosin gene (CDSN) result in an inflammatory subtype of generalized skin peeling syndrome (type B), a disorder that overlaps clinically with Netherton’s syndrome^{41, 42}. Mutations associated with generalized skin peeling syndrome type B typically result in a complete loss of corneodesmosin, a secreted glycoprotein component of corneodesmosomes that maintain cell-cell adhesion in the outer layers of the skin. Loss of corneodesmosin in this syndrome results in generalized skin peeling (exfoliation). Although skin peeling syndrome type B is quite rare, atopic manifestations, including food allergies and asthma, do seem to be major features of this syndrome⁴³.

As discussed earlier, mutations in the filaggrin gene are strongly associated with risk for atopic dermatitis but have also been associated with risk for food allergies and asthma. Additional support for a central role of the epithelium in the pathogenesis of allergic diseases is provided by variants of genes encoding epithelial cell-derived cytokines and their receptors that confer increased risk of allergic disease. Single nucleotide polymorphisms (SNP) at the loci for thymic stromal lymphopoietin (TSLP) or its receptor have been implicated in risk for asthma, atopic dermatitis, and eosinophilic esophagitis^{44, 45, 46, 47}, while SNPs in the loci for IL-33 or its receptor are associated with risk for asthma and atopic dermatitis^{48, 49, 50}. These data are consistent with an important role for epithelial cytokines in the initiation and progression of atopic disease.

Although some genetic susceptibility loci seem specific to certain atopic diseases, the numerous loci associated with both atopic dermatitis and asthma suggest shared underlying pathways. Of note, the genetics of atopic dermatitis has highlighted the importance of the skin barrier. The increased susceptibility to allergic diseases at multiple anatomic sites seen in individuals with fillaggrin, SPINK5, and CDSN mutations also suggests a pathophysiological or mechanistic link between skin barrier defects and increased risk of atopic disease at other sites.

Skin sensitization and the atopic march

Although it is difficult to establish directly the frequency to which sensitization occurs through the skin, the observation that atopic dermatitis tends to precede atopic disease at other sites has led to the proposal that the skin may be an important site in the initiation of the atopic march. In fact, even in the absence of atopic dermatitis, children with skin barrier defects are still at a higher risk for asthma than healthy children, suggesting that the skin may serve as a site for sensitization to allergens even when allergic skin inflammation is absent²¹.

Several studies have been able to link skin allergen exposure to increased risk of food allergies to those allergens. A case-control study in Japan showed that use of a wheat-containing facial soap was positively correlated with development of food allergy to wheat⁵¹. In the Avon Longitudinal Study of Parents and Children, skin sensitization was also linked to food allergy by demonstrating that application of peanut oil to inflamed skin was positively associated with the development of peanut food allergies⁵². In this study, maternal consumption of peanuts during pregnancy was not associated with the development of food allergies in the child, and peanut-specific IgE was not detectable in cord blood, suggesting that sensitization to food antigens did not occur in utero. Furthermore, levels of peanut allergens found in breast milk were also not associated with sensitization.

Additional insights into where allergic sensitization takes place have come from the analysis of allergen-specific T cells that become “imprinted” and express specific patterns of homing molecules based on where they are activated and differentiate. In peanut-allergic patients, peanut allergen Ara h 1-specific T cells expressing a memory phenotype also expressed CCR4, a Th2-associated cell trafficking marker^{53, 54, 55}. In another study, memory T cells from peanut-allergic subjects that expressed the skin-homing marker cutaneous lymphocyte antigen (CLA) showed increased proliferation compared to those that expressed α4β7 integrin, a gastrointestinal-homing marker⁵⁶. Taken together these data suggest that in peanut allergy, allergic sensitization may occur through the skin.

In addition to allergen exposure, sensitization usually requires the presence of other factors that may function as adjuvants: exogenous adjuvants, bacteria colonization of lesional skin, allergens with intrinsic protease activity, or skin barrier damage or defects⁵⁷. Most, if not all, of these factors elicit the production of cytokines, notably thymic stromal lymphopoietin (TSLP) and interleukin-33 (IL-33), from the epithelium.

TSLP and the atopic march

Thymic stromal lymphopoietin (TSLP) is a member of the 4-helix bundle cytokine family, and a distant paralog of IL-7⁵⁸. As the name suggests, TSLP was first identified in the supernatant of a mouse thymic stromal cell line as an activity capable of supporting immature B cell proliferation and development^{59, 60, 61}. A humanTSLP homolog was subsequently identified in humans using in silico methods^{62, 63}. Similarly, several groups isolated a TSLP-binding protein in both humans and mice (referred to as TSLP receptor (TSLPR) in mice and cytokine receptor-like factor 2 (CRLF2) in humans), which bound TSLP with low affinity^{64, 65, 66, 67}. Sequence analysis found that TSLPR was most closely related to the common gamma chain (γc; ⁶⁴). It is now known that the functional, high affinity, TSLPR complex is a heterodimer of TSLPR and interleukin 7 receptor alpha (IL-7Rα; Fig. 1)^{64, 65}. Cross-species homology for both the cytokine and its receptor is relatively low (~40% for each), although functionally they appear to be quite similar. Thus, the role of this cytokine axis is conserved between man and mouse despite of a loss of sequence identity.

Figure 1. Schematic of TSLP Receptor Complex.

High affinity signaling TSLP receptor complex consists of the TSLPR and IL-7Rα chains. The tyrosine kinases Jak1 and Jak2 bind IL-7Rα and TSLPR, respectively, and activate the transcriptional regulator Stat5.

A primary cellular target for TSLP are dendritic cells (DCs), which upregulate OX40L, CD80, and CD86 in response to TSLP, and TSLP-treated DCs can drive IL-4, IL-5, and IL-13 production from naïve CD4+ T cells upon co-culture^{68, 69, 70, 71}. In addition to its effects on Th2 cell polarization through antigen presenting cells, TSLP can also act directly on CD4+ T cells, CD8+ T cells, and Treg cells^{69, 72, 73, 74}. TSLP can also promote Th2 cytokine responses through its actions on mast cells, innate lymphoid cells (ILCs), epithelial cells, macrophages, and basophils^{75, 76, 77, 78}. Finally, TSLP was found to play an important role in basophil biology, where in vitro, TSLP could induce basophil maturation from bone marrow precursors in an IL-3 independent manner; furthermore, TSLP-elicited basophils in vivo were phenotypically distinct from IL-3-elicited basophils⁷⁹.

TSLP is expressed at basal levels at mucosal surfaces (e.g., gut and lung), as well as in the skin^{62, 80, 81, 82}. Its expression can be further enhanced through exposure to viral, bacterial, or parasitic pathogens as well as TLR agonists^{78, 83, 84}. A link between TSLP expression and atopic disease first came from work from Soumelis et al. who showed dramatically elevated expression in the lesional skin of individual with atopic dermatitis⁸⁵. Following that finding, TSLP expression was found in the airways of asthmatics and the nasal passages of individuals with allergic rhinitits^{86, 87, 88}. TSLP levels in asthmatic airways correlated with Th2-attracting chemokine expression and disease severity⁸⁶. In eosinophilic esophagitis, a gain-of-function polymorphism in TSLP is associated with disease in pediatric subjects^{44, 45}, and TSLP expression was higher in esophageal biopsy samples from children with active EoE than in biopsy samples from control subjects or subjects with inactive EoE⁸⁹.

Mouse model systems have been important to understand the role of TSLP in atopic disease. For example, skin-specific overexpression of TSLP under a keratinocyte-specific promoter resulted in a spontaneous atopic dermatitis-like phenotype⁹⁰. In models of skin sensitization, TSLP is induced following tape stripping or topical application of MC903 (a low calcemic analogue of vitamin D3 that induces TSLP expression)^{91, 92}. Skin sensitization in these models drives local skin inflammation that resembles atopic dermatitis. After sensitization with MC903, antigen challenge in the lung aggravated airway inflammation^{93, 94}, whereas antigen challenge orally drove esophageal inflammation that resembled eosinophilic esophagitis⁸⁹. We have developed a model of the atopic march in which skin sensitization is induced through intradermal injection of TSLP in the presence of antigen^{95, 96}. Following skin sensitization, intranasal antigen challenge promoted airway inflammation⁹⁵, and oral antigen challenge drove allergic diarrheal disease⁹⁶. Dendritic cell-intrinsic TSLP signaling was required to during sensitization, demonstrating the critical role of TSLP in initiating antigen-specific type 2 responses.

As mentioned above, TSLP has been shown to be important for the development of atopic disease in humans. For example, genetic studies in patients with AD have demonstrated that genetic variants of TSLP are associated with both disease severity and persistence^{97, 98}. The TSLP gene variant rs1898671 has been demonstrated to be significantly associated with less persistent AD in white children, which is further enhanced by two filaggrin protein loss-of-function mutations^{99, 100}. TSLP has been shown to be highly expressed in both acute and chronic lesions of AD compared with non-diseased patients and non-lesional skin of patients with AD⁸⁵ and is correlated with measures of severity and epidermal barrier function¹⁰¹. A small proof-of-concept clinical trial was conducted in patients with moderate-severe AD with tezepelumab, a human anti-TSLP antibody¹⁰². Treatment with tezepelumab resulted in a numerical, but not statistically-significant, improvement in eczema severity scores¹⁰².

An important symptom of AD is chronic pruritis, which is mediated by primary afferent somatosensory neurons that innervate the skin and are activated by endogenous pruritogens to drive itching¹⁰³. TSLP may evoke itching indirectly by activating immune cells that secrete inflammatory mediators and cytokines (e.g., IL-4, IL-13, IL-31) which stimulate sensory neurons¹⁰⁴. TSLP has also been shown to act on a subset of TRPA1-positive sensory neurons to trigger pruritis directly¹⁰⁵. These data suggest that TSLP may contribute to AD early in the course of the disease by causing itching, scratching and breakdown of the skin barrier.

TSLP has also been found to be involved in asthma in humans. Lung epithelium, like skin, is chronically exposed to a variety of environmental substances and forms a critical barrier to the external environment. Many of these environmental and pathogenic insults to the airway epithelium result in the expression of TSLP (Fig. 2). These include respiratory viruses, allergens, TLR agonists and diesel particulate matter^{84, 106, 107, 108, 109, 110}. Genetics studies have also found a link between polymorphisms in the TSLP gene and asthma susceptibility, with the TSLP SNP rs1837253 associated with childhood-onset asthma in 2 genome wide association studies^{111, 112}.

Figure 2. A model of barrier disruption and skin sensitization.

Allergens, infections, and tissue damage can all stimulate release of TSLP (as well as IL-25 and IL-33) from the epithelium. These epithelial cell-deried cytokines license DCs to drive type 2 responses but also act on a variety of cell types, including basophils, eosinophils, mast cells and ILCs to initiate and maintain allergic inflammation.

Several recent studies have examined the therapeutic potential of TSLP inhibition in patients with asthma. The first, a bronchial allergen challenge study in patients with mild allergic asthma, found that after three months of treatment with tezepelumab significant reductions of blood and sputum eosinophils and exhaled nitric oxide, indicating that TSLP plays a key upstream role in regulating the release of type-2 cytokines IL-5 and IL-4/IL-13¹¹³. Tezepelumab also significantly reduced both early- and late-phase allergen-induced bronchoconstriction, suggesting that both immediate (airway mast cell release) and late events (Th2 cytokine release, eosinophil recruitment and activation) were inhibited. In a subsequent placebo-controlled 52 week trial in moderate-severe uncontrolled asthma, tezepelumab treatment was associated with up to a 71% reduction in the annualized asthma exacerbation rate, with significant reductions observed in patients with type 2 (blood eosinophils > 250 cells/mcl) as well as non-type 2 asthma¹¹⁴. These results in the type 2 subgroup suggest that inhibition of TSLP alone has robust effects on asthma exacerbations, largely caused by viral infections, even in the absence of IL-33 and/or IL-25 antagonism¹¹⁵. The unexpected efficacy of tezepelumab in non-type 2 asthma is not well-understood at present, and data from a current phase 3 study will further elucidate the role TSLP plays as a key player in asthma exacerbations across a range of asthma subtypes.

Conclusion

The development of atopic dermatitis early in life predisposes children to subsequent development of food allergy, allergic rhinitis, and asthma – a phenomenon known as the “atopic march.” Data from epidemiologic studies and animal models suggest that skin barrier defects that allow increased exposure and sensitization to allergens may be important factors in the march from allergic skin inflammation to disease at other sites. It is increasingly apparent that the epithelium at barrier sites is not only a protective lining but is also an important source of cytokines such as TSLP, IL-25, and IL-33 that may initiate and drive type 2 inflammation at these sites. Further studies are needed to clarify the specific roles of these cytokines in the atopic march. While some level of redundancy exists, animal models have demonstrated that these cytokines do play distinct roles in allergic inflammation as shown by the effects of TSLP blockade in asthma. The requirements for these epithelial cytokines in both the initiation and maintenance of inflammation make them attractive targets for therapy.

Key Messages.

• Skin barrier defects are associated with susceptibility to atopic dermatitis and subsequent food allergies

• Skin barrier defects can lead to allergen sensitization

• The epithelial cell-derived cytokine TSLP acts as an “alarmin” that is released by the barrier epithelium

• TSLP acts to promote type 2 inflammatory responses

• TSLP blockade can be an important therapeutic intervention in atopic disease.