DISCUSSION
We present the most comprehensive intra-patient transcriptomic comparison of tape-strips and biopsies in moderate-to-severe AD to date. We previously developed a framework for using tape-strips to measure mRNA and protein in AD in both pediatric and adult populations, including for monitoring skin activity with topical or systemic treatment.29,30,69,70 Here, we provided insights into the common AD phenotype detected by both tape-strips and biopsies as well as potential differences between these approaches in detecting specific disease features. Past reports comparing tape-strips and biopsies in AD are limited either by a small panel of markers,16,35,36 separation of epidermal and dermal layers in biopsies,18,37,71 or focus on non-lesional skin.37,71 An intra-patient, RNA-seq profiling of active AD lesions comparing tape-strips to full-thickness biopsies, thus capturing the full spectrum of immune, barrier, and pruritus-related molecular abnormalities is not available.
Our study showed that both tape-strips and biopsies captured key molecular immune abnormalities of AD, including T-cell and DC activation, innate immunity, Th2/Th1/Th17/Th22, and attenuated negative regulation to varying degrees. However, tape-strips and biopsies also showed some distinct biomarker profiles, with some notable biomarkers differentially expressed in only one of the two techniques. Overall tape-strips outperformed biopsies for most T-helper axes, with greater differentiation from controls for Th2 (IL-4R, IL-13, IL-31, CCL17, TNFRSF4/OX40), Th17 (CCL20, CXCL1, CXCL2), innate immunity (IL-1B, IL-8, TNF), some Th1-related chemokines (CXCL10, CCL3), and negative regulator markers (IL-34, IL-37). IL-34, in particular, was shown to distinguish AD from healthy skin with almost perfect accuracy in tape-strips,30 a property that is likely unique to tape-strips given the >35x higher fold-change between AD and controls in tape-strips compared to biopsies. These findings suggest that chemokines that promote Th2/Th17 differentiation and their defining cytokines are found in high concentration in the upper epidermis, whereas their expression may be diluted in larger biopsy samples. Tape-strips also better captured DC-related products, including myeloid DCs (ITGAM/CD11B, ITGAX/CD11C), LCs, and inflammatory DCs (CD1A, FCER1A, CD207/langerin), mainly localized to the epidermis,48,72,73 which are major sources of chemokines. Inflammatory DCs correlate with clinical AD severity and drive Th2 skewing,74,75 representing a potential pathogenic cell type in AD with better detection by tape-strips.
In contrast, biopsies demonstrated higher fold-changes than tape-strips for hyperplasia related cytokines and chemokines, including the Th22 cytokine IL-22 and the S100As that are Th17 and Th22 co-regulated,76 suggesting that biopsies may be more ideal for evaluating hyperplasia markers. IL-22 is primarily expressed in the dermis during inflammatory states,77,78 while S100As are produced by suprabasal keratinocytes that are likely below the depth of tape-stripping.9 Both promote keratinocyte proliferation and epidermal hyperplasia.77 IFN-γ, the main Th1 cytokine, and CCL26, perhaps the best biomarker of therapeutic response to dupilumab, are additional AD biomarkers primarily localized to the dermis78,79 and accordingly showed dysregulation only in biopsies. Markers of an inflammatory dermal fibroblast population that may orchestrate lymphocyte migration to secondary lymphoid organs and Th2 immunity, including COL6A5, COL6A6, TNC, and CCL19,9 were also predictably solely up-regulated in biopsies. Of note, many markers that are more prominently detected by tape-strips (IL-4R, IL-13, IL-31, TNFRSF4/OX40) or biopsies (IL-22) are therapeutic targets for AD that are either FDA-approved or under investigation,80-88 reinforcing that although both techniques can detect molecular changes in AD in response to therapy,80,82,85,86 the type of therapeutic target may dictate the appropriate sampling technique.
Pruritus, the cardinal clinical feature of AD significantly impacting patients’ quality of life,89-91 is orchestrated by complex cross-talk between immune cells, neurons, and keratinocytes.51,56,63,65,88,92-105 One of the advantages of tape-strips over biopsies was the ability to better detect changes in the neuroimmune pathways mediating pruritus. For example, the Th2 cytokine IL-31, perhaps the key driver of pruritus in AD,56,88,92-95 was uniquely up-regulated in tape-strips, with similar trends in OSM and OSMR, which are part of the same itch pathway and directly interact with IL-31.56STAT3, which mediates IL-31-induced neuronal overgrowth contributing to skin sensitivity to minimal stimuli,93 was also dysregulated only in tape-strips. Several transient receptor channel components (TRPV2, TRPV3, TRPM3) showed more pronounced dysregulation in tape-strips, likely owing to their expression by sensory neurons, as well as keratinocytes and/or immune cells.106,107Among these, TRPV3 is most abundant on keratinocytes,106 and has been shown to be increased in lesional AD,62 and promote a pro-inflammatory cascade via the NF-κB pathway.96 In contrast, TRPV6, which is the only down-regulated TRP gene, with more negative fold-changes in tape-strips than biopsies, is a pre-requisite for keratinocyte entry into differentiation.100 As an example of the complex intercellular interactions that drive itch, IL-31-induced up-regulation of TRPV3 was proposed to induce keratinocyte release of SERPINE1, which then directly activates PLAUR/u-PAR in sensory neurons to perpetuate a cycle of itch and neuroinflammation via the innate immune system.63,108 Accordingly, SERPINE1 and PLAUR/u-PAR were also exclusively dysregulated in tape-strips. Overall, given the large contribution of pruritus to the burden of AD,89-91 the ability to measure the itch signature of AD before and after treatment through minimally invasive approaches, such as tape-strips, which in fact outperformed biopsies, is critical.
In addition to immune/neuroimmune dysregulation, tape-strips and biopsies also preferentially captured distinct aspects of epidermal barrier impairment. Although markers of terminal differentiation109-111 (FLG2, SCEL, PSORS1C2) were decreased in both techniques, at least two-fold greater changes between healthy and lesional AD were seen in tape-strips. In contrast, biopsies showed more pronounced up-regulation of markers of epidermal hyperplasia (KRT6A, KRT16, MKI67), which are primarily expressed in the basal and immediately suprabasal layers.112, Tight junction components, which are variably distributed throughout the epidermis,78 were largely attenuated in AD in both techniques, with some differences. For example, down-regulation of CLDN1, which is expressed at all suprabasal layers and has been described as an AD susceptibility gene with inverse correlations to Th2,113 was only detected in biopsies. In contrast, the gap junction components (GJB3, GJB5), which are primarily expressed in the granular layer,114 were only significantly decreased in tape-strips. Lipid biosynthesis/metabolism abnormalities, which are partly driven by Th2 in AD,115 were observed in both tape-strips and biopsies in line with prior reports,7,86,115-119 but also showed differences between the two techniques. For example, genes involved in the biosynthesis of ceramides, the main lipid constituent of the stratum corneum (DGS2, SPTLC3),120 were only decreased in tape-strips, while products related to phospholipid synthesis (AGPAT3, LPIN1), which are more common in the lower epidermis,120 were only down-regulated in biopsies. The inclusion of lipids from the sebocytes of sebaceous glands in biopsies but not tape strips, which also contribute to barrier function,121 may further contribute to differences in lipid-related genes between the two techniques. FAR2, which regulates synthesis of wax esters exclusively produced in sebaceous glands,122 and FADS2, which regulates production of sapienate, the most abundant fatty acid in sebum,123were down-regulated only in biopsies. Overall, tape-strips and biopsies each preferentially capture gene alterations that complement each other to constitute the defective barrier phenotype characteristic of AD.
Our study has some limitations. Non-lesional biopsies were not available for this cohort, preventing a parallel intra-patient comparison of non-lesional tissues analyzed by both approaches. Nevertheless, prior studies demonstrated that tape-strips capture the non-lesional AD phenotype.29,37,69,71,124 Additionally, our analyses were performed on a primarily adult Caucasian population. Future analysis should also include other age groups and ethnic backgrounds.
In summary, this is the first global, intra-patient, full-thickness molecular profiling of lesional skin from moderate-to-severe AD that compares sampling by tape-strip versus biopsies. Although both capture the main immune and barrier abnormalities of AD, tape-strips may be the preferred technique for Th2/Th17, innate immunity, DC, pruritus, and terminal differentiation related markers. Biopsies may be more appropriate for evaluating genes below the granular layer, including markers of epidermal hyperplasia, dermal cytokines (IFN-γ, IL-22), and inflammatory dermal populations like fibroblasts. The choice of appropriate sampling technique should be determined considering which specific immune or barrier features are of greatest importance to the investigation. Still, this study highlights that tape-strips are not only adequate but can even outperform biopsies in detecting certain AD features, including several biomarkers that are therapeutic targets. Thus, tape-stripping provides a minimally invasive alternative or supplement to biopsies in clinical trials and longitudinal studies with the potential to minimize biopsy-related complications and bolster patient participation in adult and pediatric AD studies.