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Warp Speed Ahead! Technology-Driven Breakthroughs in Skin Immunity and Inflammatory Disease

  • Piotr Konieczny
    Affiliations
    Department of Pathology, Department of Medicine, and Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA
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  • Shruti Naik
    Correspondence
    Correspondence: Shruti Naik, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA.
    Affiliations
    Department of Pathology, Department of Medicine, and Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA
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Open ArchivePublished:June 10, 2020DOI:https://doi.org/10.1016/j.jid.2020.05.084
      The skin’s physical barrier is reinforced by an arsenal of immune cells that actively patrol the tissue and respond swiftly to penetrating microbes, noxious agents, and injurious stimuli. When unchecked, these same immune cells drive diseases such as psoriasis, atopic dermatitis, and alopecia. Rapidly advancing microscopy, animal modeling, and genomic and computational technologies have illuminated the complexity of the cutaneous immune cells and their functions in maintaining skin health and driving diseases. Here, we discuss the recent technology-driven breakthroughs that have transformed our understanding of skin immunity and highlight burgeoning areas that hold great promise for future discoveries.

      Abbreviations:

      3D (three-dimensional), CyTOF (mass cytometry), DC (dendritic cell), DETC (dendritic epidermal T cell), ECM (extracellular matrix), ILC (innate lymphoid cell), LC (Langerhans cell), scRNAseq (single-cell RNA sequencing), TRM (resident memory T cell)

      Introduction

      Owing to its exteriority, the skin has captivated the human imagination since ancient Roman and Egyptian civilizations. Yet, modern-day experimental dermatology and immunology did not take root until the late 19th and 20th centuries, respectively. In parallel, experimental dermatologists and immunologists studied contact hypersensitivity, graft rejection and histocompatibility, and adjuvant responses (
      • Chase M.W.
      Immunology and experimental dermatology.
      ). These foundational works revealed the immune underpinnings of skin diseases while still viewing the skin as an epithelial barrier that recruited immune allies only under duress.
      Advancing technologies illuminated the myriad of immune cells that reside in and continually patrol the skin, shifting the view that the skin is simply an inert barrier (
      • Kobayashi T.
      • Naik S.
      • Nagao K.
      Choreographing immunity in the skin epithelial barrier.
      ). Inspired by the 2019 Montagna Biology of Skin Symposium, we discuss the remarkable discoveries in skin immunity that have resulted from imaging, tissue processing, and genomic techniques (Figure 1). We also highlight the importance of these tools in understanding immune dysfunction in inflammatory skin diseases. Finally, we explore emerging technologies and their potential for further expanding the knowledge of skin immunity.
      Figure thumbnail gr1
      Figure 1Timeline of the technological advances, their implementation, and seminal discoveries in skin immunity. The top row summarizes seminal discoveries and implemented technologies, and the bottom row presents innovations and their precise year of development. ATAC-Seq, assay for transposase-accessible chromatin using sequencing; CyTOF, mass cytometry; RNA-Seq, RNA sequencing; SALT, skin-associated lymphoid tissue.

      Seeing is believing

      In 1868, Paul Langerhans, enabled by rudimentary light microscopy, uncovered cells with dendrites in the epidermis (
      • Langerhans P.
      Üeber die Nerven der menschlichen Haut.
      ), which he concluded were epidermal nerves. His discovery of Langerhans cells (LCs) was the first known observation of immune cells in normal skin. In 1949, Andrews and Andrews distinguished lymphocytes in normal epidermis using light microscopy (
      • Andrew W.
      • Andrew N.V.
      Lymphocytes in the normal epidermis of the rat and of man.
      ). A few decades later,
      • Streilein J.W.
      Lymphocyte traffic, T-cell malignancies and the skin.
      built upon these and other works to propose that the skin had a dedicated immune component, which he termed skin-associated lymphoid tissue.
      Since then, sophisticated imaging techniques, most notably fluorescence microscopy, have been widely used to illuminate the immune microanatomy of the skin (
      • Kabashima K.
      • Honda T.
      • Ginhoux F.
      • Egawa G.
      The immunological anatomy of the skin.
      ). Fluorescence microscopy enabled the simultaneous visualization of multiple cell types and their expressed factors at a higher resolution than simple light microscopy (
      • Sanderson M.J.
      • Smith I.
      • Parker I.
      • Bootman M.D.
      Fluorescence microscopy.
      ). Initially used to detect epidermal-resident dendritic epidermal T cells (DETCs) and LCs (
      • Havran W.L.
      • Allison J.P.
      Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors.
      ,
      • Kissenpfennig A.
      • Henri S.
      • Dubois B.
      • Laplace-Builhé C.
      • Perrin P.
      • Romani N.
      • et al.
      Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.
      ,
      • Steiner G.
      • Koning F.
      • Elbe A.
      • Tschachler E.
      • Yokoyama W.M.
      • Shevach E.M.
      • et al.
      Characterization of T cell receptors on resident murine dendritic epidermal T cells.
      ), microscopic analyses have revealed the tightly controlled spatial distribution of the myriad of immune cells in the skin (
      • Kabashima K.
      • Honda T.
      • Ginhoux F.
      • Egawa G.
      The immunological anatomy of the skin.
      ). LCs and intraepithelial lymphocytes (DETCs [
      • Havran W.L.
      • Allison J.P.
      Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors.
      ] CD8+ resident memory T cells [TRM] [
      • Schenkel J.M.
      • Masopust D.
      Tissue-resident memory T cells.
      ]) and innate lymphoid cells (ILCs) (
      • Kobayashi T.
      • Voisin B.
      • Kim D.Y.
      • Kennedy E.A.
      • Jo J.H.
      • Shih H.Y.
      • et al.
      Homeostatic control of sebaceous glands by innate lymphoid cells regulates commensal bacteria equilibrium.
      ) are capable of traversing the basement membrane reside in the epidermis. The upper dermis houses several dendritic cells (DCs) subsets (
      • Tamoutounour S.
      • Guilliams M.
      • Montanana Sanchis F.
      • Liu H.
      • Terhorst D.
      • Malosse C.
      • et al.
      Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.
      ), γδ T cells (
      • Gray E.E.
      • Suzuki K.
      • Cyster J.G.
      Cutting edge: identification of a motile IL-17-producing gammadelta T cell population in the dermis.
      ), CD4 T helper (
      • Adachi T.
      • Kobayashi T.
      • Sugihara E.
      • Yamada T.
      • Ikuta K.
      • Pittaluga S.
      • et al.
      Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma.
      ), and regulatory T cells (
      • Ali N.
      • Zirak B.
      • Rodriguez R.S.
      • Pauli M.L.
      • Truong H.A.
      • Lai K.
      • et al.
      Regulatory T cells in skin facilitate epithelial stem cell differentiation.
      ) and ILCs (
      • Kobayashi T.
      • Voisin B.
      • Kim D.Y.
      • Kennedy E.A.
      • Jo J.H.
      • Shih H.Y.
      • et al.
      Homeostatic control of sebaceous glands by innate lymphoid cells regulates commensal bacteria equilibrium.
      ). These cells are enriched around hair follicles (
      • Adachi T.
      • Kobayashi T.
      • Sugihara E.
      • Yamada T.
      • Ikuta K.
      • Pittaluga S.
      • et al.
      Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma.
      ), highlighting that this region is a key immunological hub in the skin. The lower dermis houses various macrophage subsets in close apposition to vasculature, nerves, and adipocytes (
      • Silva H.M.
      • Báfica A.
      • Rodrigues-Luiz G.F.
      • Chi J.
      • Santos P.D.A.
      • Reis B.S.
      • et al.
      Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges.
      ). In addition to immune localization, dynamic imaging has divulged immune surveillance function in the normal skin. Images of LCs extending their dendrites through the epidermis captured their homeostatic uptake of external antigens (
      • Ouchi T.
      • Kubo A.
      • Yokouchi M.
      • Adachi T.
      • Kobayashi T.
      • Kitashima D.Y.
      • et al.
      Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome.
      ). LCs and other DCs migrate to the lymph nodes to induce T effector and regulatory cells and/or provide homeostatic signals to maintain these populations in the normal skin (
      • Naik S.
      • Bouladoux N.
      • Linehan J.L.
      • Han S.J.
      • Harrison O.J.
      • Wilhelm C.
      • et al.
      Commensal-dendritic-cell interaction specifies a unique protective skin immune signature.
      ,
      • Seneschal J.
      • Clark R.A.
      • Gehad A.
      • Baecher-Allan C.M.
      • Kupper T.S.
      Human epidermal Langerhans cells maintain immune homeostasis in skin by activating skin resident regulatory T cells.
      ).
      Pioneered in 1990, multiphoton microscopy allowed for deeper tissue penetration and opened the door to intravital imaging (
      • Denk W.
      • Strickler J.H.
      • Webb W.W.
      Two-photon laser scanning fluorescence microscopy.
      ). Coupled with the generation of fluorescence reporter animals, multiphoton imaging was used to view live image immune cells (
      • Kabashima K.
      • Egawa G.
      Intravital multiphoton imaging of cutaneous immune responses.
      ). This enabled the three-dimensional (3D) reconstruction of immune niches and revealed the interaction of leukocytes with the skin’s structural components (
      • Kabashima K.
      • Egawa G.
      Intravital multiphoton imaging of cutaneous immune responses.
      ). Intravital imaging is also a powerful tool to visualize the induction and propagation of inflammatory responses (
      • Obeidy P.
      • Tong P.L.
      • Weninger W.
      Research techniques made simple: two-photon intravital imaging of the skin.
      ). A key feature of inflammatory responses in the skin is a compromised barrier, especially in the case of infectious agents or tissue injury. Live imaging identified neutrophils as first responders infiltrating within hours of epidermal breach (
      • Obeidy P.
      • Tong P.L.
      • Weninger W.
      Research techniques made simple: two-photon intravital imaging of the skin.
      ,
      • Peters N.C.
      • Egen J.G.
      • Secundino N.
      • Debrabant A.
      • Kimblin N.
      • Kamhawi S.
      • et al.
      In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.
      ) and the kinetics of DC migration to the lymph nodes under stress, illustrating that functionally specialized DC subsets migrate with specific kinetics to induce adaptive responses (
      • Tamoutounour S.
      • Guilliams M.
      • Montanana Sanchis F.
      • Liu H.
      • Terhorst D.
      • Malosse C.
      • et al.
      Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.
      ).
      Quantitative imaging combined with pathways-specific modulation of cell–cell interactions, cell–extracellular matrix (ECM) interactions, or motility has unearthed therapeutic targets in inflammation (
      • Matheu M.P.
      • Beeton C.
      • Garcia A.
      • Chi V.
      • Rangaraju S.
      • Safrina O.
      • et al.
      Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.
      ,
      • Overstreet M.G.
      • Gaylo A.
      • Angermann B.R.
      • Hughson A.
      • Hyun Y.M.
      • Lambert K.
      • et al.
      Inflammation-induced interstitial migration of effector CD4+ T cells is dependent on integrin αV.
      ). For instance, perivascular lymphocytes and DCs form clusters in an IL-1R–dependent manner to drive contact dermatitis (
      • Natsuaki Y.
      • Egawa G.
      • Nakamizo S.
      • Ono S.
      • Hanakawa S.
      • Okada T.
      • et al.
      Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin.
      ). Imaging studies provided insight into how innate and adaptive immune cells control skin tumors. To this end, a role for CD8+ TRM and innate immune cells in restraining melanoma and epithelial neoplasms has been identified (
      • Caulin C.
      • Nguyen T.
      • Lang G.A.
      • Goepfert T.M.
      • Brinkley B.R.
      • Cai W.W.
      • et al.
      An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations.
      ,
      • Park S.L.
      • Buzzai A.
      • Rautela J.
      • Hor J.L.
      • Hochheiser K.
      • Effern M.
      • et al.
      Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.
      ). Thus, imaging techniques have provided invaluable insights into the location, migration, interactions, and functions of immune cells in skin health and disease.

      Cytometry and genomic technologies widen the lens

      Perhaps the most underappreciated and widely used methodology in skin immunology is the ability to efficiently extract viable cells from the skin while preserving the expression of surface proteins for phenotypic analysis. This was first accomplished by employing a serine protease, trypsin, to digest ECM and severe cell–cell interactions to obtain DETCs and LCs (
      • Havran W.L.
      • Allison J.P.
      Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors.
      ,
      • Steiner G.
      • Koning F.
      • Elbe A.
      • Tschachler E.
      • Yokoyama W.M.
      • Shevach E.M.
      • et al.
      Characterization of T cell receptors on resident murine dendritic epidermal T cells.
      ). Since then, sophisticated enzymes with minimal nonspecific activity have become available and are used to prepare cell suspensions for a number of downstream analysis platforms (
      • Botting R.A.
      • Bertram K.M.
      • Baharlou H.
      • Sandgren K.J.
      • Fletcher J.
      • Rhodes J.W.
      • et al.
      Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.
      ,
      • Clark R.A.
      • Chong B.F.
      • Mirchandani N.
      • Yamanaka K.
      • Murphy G.F.
      • Dowgiert R.K.
      • et al.
      A novel method for the isolation of skin resident T cells from normal and diseased human skin.
      ).
      Flow cytometry has been the cornerstone of immunology for many decades and is ubiquitously used to analyze cells from healthy and diseased skin (
      • Adan A.
      • Alizada G.
      • Kiraz Y.
      • Baran Y.
      • Nalbant A.
      Flow cytometry: basic principles and applications.
      ). Antibodies raised to specific protein moieties (surface markers, cytokines, transcription factors, and signaling components) are coupled with fluorescent indicators and have empowered researchers to examine multiple cellular parameters simultaneously and quantitatively. In 1969, Herzenberg published a new technique to obtain highly purified cell populations, called FACS (
      • Hulett H.R.
      • Bonner W.A.
      • Barrett J.
      • Herzenberg L.A.
      Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence.
      ). Cells purified directly from the skin with FACS have been used for functional in vitro studies (
      • Seneschal J.
      • Clark R.A.
      • Gehad A.
      • Baecher-Allan C.M.
      • Kupper T.S.
      Human epidermal Langerhans cells maintain immune homeostasis in skin by activating skin resident regulatory T cells.
      ), in vivo cell transfer experiments (
      • Schenkel J.M.
      • Masopust D.
      Tissue-resident memory T cells.
      ), and downstream tissue and cell-specific genomic analysis (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ).
      However, fluorescent indicators have restricted analysis to the visible-light and UV spectrum and limited the number of parameters that could be measured simultaneously. In 2009,
      • Bandura D.R.
      • Baranov V.I.
      • Ornatsky O.I.
      • Antonov A.
      • Kinach R.
      • Lou X.
      • et al.
      Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.
      overcame these limitations by developing mass cytometry (CyTOF). CyTOF blends flow cytometry with mass spectrometry using metal-conjugated antibodies to dramatically increase the number of analytes from as few as 10,000 cells, enabling efficient analysis of small patient samples (
      • Bandura D.R.
      • Baranov V.I.
      • Ornatsky O.I.
      • Antonov A.
      • Kinach R.
      • Lou X.
      • et al.
      Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.
      ,
      • Yao Y.
      • Liu R.
      • Shin M.S.
      • Trentalange M.
      • Allore H.
      • Nassar A.
      • et al.
      CyTOF supports efficient detection of immune cell subsets from small samples.
      ). Multiparametric CyTOF analysis of normal skin and in inflammatory disease revealed a remarkable intraindividual heterogeneity in homeostatic DC populations and highly polarizing impact of inflammatory diseases on immune subsets (
      • Alcántara-Hernández M.
      • Leylek R.
      • Wagar L.E.
      • Engleman E.G.
      • Keler T.
      • Marinkovich M.P.
      • et al.
      High-dimensional phenotypic mapping of human dendritic cells reveals interindividual variation and tissue specialization.
      ,
      • Farrera C.
      • Melchiotti R.
      • Petrov N.
      • Weng Teng K.W.
      • Wong M.T.
      • Loh C.Y.
      • et al.
      T-cell phenotyping uncovers systemic features of atopic dermatitis and psoriasis.
      ).
      Genome-based analysis has radically transformed our understanding of skin immunity. Spurred by the human genome project (
      • Collins F.S.
      • Green E.D.
      • Guttmacher A.E.
      • Guyer M.S.
      US National Human Genome Research Institute
      A vision for the future of genomics research.
      ), the ability to sequence and compile whole-human genomes uncovered genetic susceptibility loci underlying a number of complex inflammatory skin diseases (
      • Paternoster L.
      • Standl M.
      • Chen C.M.
      • Ramasamy A.
      • Bønnelykke K.
      • Duijts L.
      • et al.
      Meta-analysis of genome-wide association studies identifies three new risk loci for atopic dermatitis.
      ,
      • Tsoi L.C.
      • Stuart P.E.
      • Tian C.
      • Gudjonsson J.E.
      • Das S.
      • Zawistowski M.
      • et al.
      Large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants.
      ). These studies provided key insights into the molecular and cellular drivers of complex multifactorial diseases. For instance, GWAS of Alopecia areata were instrumental in identifying the key innate and adaptive immune drivers of hair follicle destruction (
      • Petukhova L.
      • Duvic M.
      • Hordinsky M.
      • Norris D.
      • Price V.
      • Shimomura Y.
      • et al.
      Genome-wide association study in alopecia areata implicates both innate and adaptive immunity.
      ). Similarly, the IL-23 and NF-κB immune pathways were linked to psoriasis with GWAS (
      • Nair R.P.
      • Duffin K.C.
      • Helms C.
      • Ding J.
      • Stuart P.E.
      • Goldgar D.
      • et al.
      Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways.
      ).
      Microarray technology and, more recently, RNA sequencing has provided a global picture of gene expression from skin tissue and purified immune cells (
      • Li B.
      • Tsoi L.C.
      • Swindell W.R.
      • Gudjonsson J.E.
      • Tejasvi T.
      • Johnston A.
      • et al.
      Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms.
      ,
      • Nirschl C.J.
      • Suárez-Fariñas M.
      • Izar B.
      • Prakadan S.
      • Dannenfelser R.
      • Tirosh I.
      • et al.
      IFNγ-dependent tissue-immune homeostasis is co-opted in the tumor microenvironment.
      ). Transcriptional analysis has also been instrumentational in revealing the unique, universal, and synergistic cellular programs induced by inflammatory cytokines (
      • Mehta N.N.
      • Teague H.L.
      • Swindell W.R.
      • Baumer Y.
      • Ward N.L.
      • Xing X.
      • et al.
      IFN-γ and TNF-α synergism may provide a link between psoriasis and inflammatory atherogenesis.
      ,
      • Swindell W.R.
      • Beamer M.A.
      • Sarkar M.K.
      • Loftus S.
      • Fullmer J.
      • Xing X.
      • et al.
      RNA-seq analysis of IL-1b and IL-36 responses in epidermal keratinocytes identifies a shared MyD88-dependent gene signature.
      ). Mechanistic studies using cell culture systems and animal models have defined the causal contributory factors identified by GWAS and transcriptional studies (
      • Billi A.C.
      • Ludwig J.E.
      • Fritz Y.
      • Rozic R.
      • Swindell W.R.
      • Tsoi L.C.
      • et al.
      KLK6 expression in skin induces PAR1-mediated psoriasiform dermatitis and inflammatory joint disease.
      ,
      • Hawkes J.E.
      • Gudjonsson J.E.
      • Ward N.L.
      The snowballing literature on imiquimod-induced skin inflammation in mice: a critical appraisal.
      ) paving the way for the development of targeted therapeutics.
      The power of evaluating the gene expression of a single cell was harnessed by next-generation sequencing platforms to evaluate transcriptomes at cellular resolution (
      • Tang F.
      • Barbacioru C.
      • Wang Y.
      • Nordman E.
      • Lee C.
      • Xu N.
      • et al.
      mRNA-Seq whole-transcriptome analysis of a single cell.
      ). There has since been an explosion in the use of single-cell RNA sequencing (scRNAseq) by skin immunologists to study cellular heterogeneity (
      • Shook B.A.
      • Wasko R.R.
      • Rivera-Gonzalez G.C.
      • Salazar-Gatzimas E.
      • López-Giráldez F.
      • Dash B.C.
      • et al.
      Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.
      ), identify rare cell populations (
      • Kobayashi T.
      • Voisin B.
      • Kim D.Y.
      • Kennedy E.A.
      • Jo J.H.
      • Shih H.Y.
      • et al.
      Homeostatic control of sebaceous glands by innate lymphoid cells regulates commensal bacteria equilibrium.
      ), and map the developmental (
      • Popescu D.M.
      • Botting R.A.
      • Stephenson E.
      • Green K.
      • Webb S.
      • Jardine L.
      • et al.
      Decoding human fetal liver haematopoiesis.
      ) and functional trajectories of distinct cell lineages (
      • Tan L.
      • Sandrock I.
      • Odak I.
      • Aizenbud Y.
      • Wilharm A.
      • Barros-Martins J.
      • et al.
      Single-cell transcriptomics identifies the adaptation of Scart1+ Vγ6+ T cells to skin residency as activated effector cells.
      ). Comparing immune cells in psoriasis, atopic dermatitis, vitiligo, and bullous skin disease (
      • Cheng J.B.
      • Sedgewick A.J.
      • Finnegan A.I.
      • Harirchian P.
      • Lee J.
      • Kwon S.
      • et al.
      Transcriptional programming of normal and inflamed human epidermis at single-cell resolution.
      ; TK Hughes, unpublished data, 2019) has revealed heterogeneity not only in immune cells but also in the functionally responsive stromal cells that they engage. Although scRNAseq has been instrumental in mapping the cellular ecology of cutaneous immunity, just as the genomic techniques that came before, functional follow-up studies will be essential to determine causality and meaningful cellular interactions. Perhaps the most exciting application of scRNAseq is its use in rapid diagnosis, especially in diseases that lack a clear mechanism.
      • Kim D.
      • Kobayashi T.
      • Voisin B.
      • Jo J.H.
      • Sakamoto K.
      • Jin S.P.
      • et al.
      Targeted therapy guided by single-cell transcriptomic analysis in drug-induced hypersensitivity syndrome: a case report.
      recently used scRNAseq to effectively diagnose and treat a patient with drug-induced hypersensitivity syndrome, a disease with elusive pathophysiology.

      Emerging technology and future promise

      Many emerging technologies are melding methods to evaluate multiple modalities in the same sample. For instance, cellular indexing of transcriptomes and epitopes by sequencing (
      • Stoeckius M.
      • Hafemeister C.
      • Stephenson W.
      • Houck-Loomis B.
      • Chattopadhyay P.K.
      • Swerdlow H.
      • et al.
      Simultaneous epitope and transcriptome measurement in single cells.
      ) combines antibody-based protein detection with scRNAseq, allowing for simultaneous evaluation of gene transcript and its protein product within a single cell. Similarly, concurrent assessment of epigenetics state, including chromatin accessibility, DNA and histone modifications and 3D chromatin structure, and the transcriptional landscape of a single cell, may provide a more nuanced understanding of regulatory genomic elements that underlie distinct cell states (
      • Jia G.
      • Preussner J.
      • Chen X.
      • Guenther S.
      • Yuan X.
      • Yekelchyk M.
      • et al.
      Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement.
      ). One of the most exciting techniques on the horizon is spatial transcriptomics (
      • Moncada R.
      • Barkley D.
      • Wagner F.
      • Chiodin M.
      • Devlin J.C.
      • Baron M.
      • et al.
      Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas.
      ), a method that provides gene expression coupled with spatial distribution in tissue. Spatial transcriptomics will be particularly useful to evaluate microanatomical heterogeneity in disease, for instance, the tumor-stromal interface or the edge and bed of a nonhealing wound. Widely implementing these technologies will undoubtedly require tremendous computational power and the use of machine learning. An added challenge posed by these techniques is the integration of large datasets and dissemination for downstream functional validation. Nevertheless, these advances present a tantalizing toolbox with which cutaneous biologists can compose rich portraits of skin immune health and disease.

      Data availability statement

      No datasets were generated or analyzed during this study.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank our friends and colleagues whose works have inspired this perspective, especially the speakers of the 2019 Montagna Biology of Skin Symposium. We apologize for not being able to include all relevant papers published in skin immunology, which are comprehensively reviewed by
      • Kobayashi T.
      • Naik S.
      • Nagao K.
      Choreographing immunity in the skin epithelial barrier.
      , due to space constraints. Our figure was made using Biorender (www.biorender.com). This work was supported by the National Institute of Allergy and Infectious Diseases (1K22AI135099-01. SN).

      Author Contributions

      Conceptualization: SN; Funding Acquisition: SN; Visualization: SN, PK; Writing - Original Draft Preparation: SN, PK, Writing - Review and Editing: SN, PK

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