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Mouse Models of Alopecia Areata: C3H/HeJ Mice Versus the Humanized AA Mouse Model

  • Amos Gilhar
    Correspondence
    Correspondence: Amos Gilhar, Laboratory for Skin Research, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
    Affiliations
    Skin Research Laboratory, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
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  • Rimma Laufer Britva
    Affiliations
    Skin Research Laboratory, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
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  • Aviad Keren
    Affiliations
    Skin Research Laboratory, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
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  • Ralf Paus
    Affiliations
    Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA

    Centre for Dermatology Research, University of Manchester, and NIHR Biomedical Research Centre, Manchester, United Kingdom

    Monasterium Laboratory Skin & Hair Research Solutions GmbH, Muenster, Germany
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      The C3H/HeJ model has long dominated basic alopecia areata (AA) in vivo research and has been used as proof-of-principle that Jak inhibitors are suitable agents for AA management in vivo. However, its histologic features are not typical of human AA, and it is questionable whether it is sufficiently clinically predictive for evaluating the therapeutic effects of candidate AA agents. Instead, the humanized mouse model of AA has been used to functionally demonstrate the role of key immune cells in AA pathogenesis and to discover human-specific pharmacologic targets in AA management. Therefore, we advocate the use of both models in future preclinical AA research.

      Abbreviation:

      AA (alopecia areata)

      Introduction

      The mainstream autoimmunity research community has been slow to recognize and acknowledge that the hair loss disorder, alopecia areata (AA), is one of the most common human autoimmune diseases (
      • Gilhar A.
      • Etzioni A.
      • Paus R.
      Alopecia areata.
      ,
      • McElwee K.J.
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      • Tobin D.J.
      • Ramot Y.
      • Sundberg J.P.
      • Nakamura M.
      • et al.
      What causes alopecia areata?.
      ), exceeded in incidence and prevalence only by type 1 diabetes mellitus and rheumatoid arthritis. The clinical and psychosocial importance of this disease is notoriously underestimated because AA is neither life-threatening nor recognized to be life-shortening or physically crippling. However, patients affected by AA, namely by its maximal variants, alopecia totalis or universalis, often experience AA as a psychologically devastating illness, whose burden of disease is very substantial (
      • Gilhar A.
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      Alopecia areata.
      ,
      • Matzer F.
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      Psychosocial stress and coping in alopecia areata: a questionnaire survey and qualitative study among 45 patients.
      ,
      • Monselise A.
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      Examining the relationship between alopecia areata, androgenetic alopecia, and emotional intelligence.
      ). This finding is aggravated by the fact that there is neither fully satisfactory, universally effective therapy available for the established disease, nor convincing management strategies for the reliable prevention of AA progression (
      • Harries M.J.
      • Sun J.
      • Paus R.
      • King Jr., L.E.
      Management of alopecia areata.
      ,
      • Paus R.
      • Bulfone-Paus S.
      • Bertolini M.
      Hair follicle immune privilege revisited: the key to alopecia areata management.
      ).
      These facts alone call for clinically relevant AA research models, in which the as yet insufficiently understood pathobiology of AA can be systematically and mechanistically dissected and in which new AA treatment and prevention strategies can be explored in vivo at the preclinical level, ideally with good predictive value for the outcomes that can realistically be expected in patients with AA. Mouse models have provided invaluable research tools for dissecting the roles of extrinsic and intrinsic factors and various underlying immune pathologies in various autoimmune skin diseases (
      • Yu X.
      • Huang Q.
      • Petersen F.
      History and milestones of mouse models of autoimmune diseases.
      ,
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      A methodological review of induced animal models of autoimmune diseases.
      ). Lessons that have been learned from mouse models for psoriasis (
      • Jin S.P.
      • Koh S.J.
      • Yu D.A.
      • Kim M.W.
      • Yun H.T.
      • Lee D.H.
      • et al.
      Imiquimod-applied interleukin-10 deficient mice better reflects severe and persistent psoriasis with systemic inflammatory state.
      ,
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      • Baumann C.
      • Auriemma M.
      • Sternemann C.
      • Soeberdt M.
      • et al.
      The tripeptide KdPT ameliorates ongoing psoriasis-like skin inflammation in murine and human skin.
      ,
      • Nakajima K.
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      Mouse models of psoriasis and their relevance.
      ), atopic dermatitis (
      • Lin L.
      • Xie M.
      • Chen X.
      • Yu Y.
      • Liu Y.
      • Lei K.
      • et al.
      Toll-like receptor 4 attenuates a murine model of atopic dermatitis through inhibition of langerin-positive DCs migration.
      ,
      • Nakajima S.
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      • Common J.
      • Kabashima K.
      Insights into atopic dermatitis gained from genetically defined mouse models.
      ,
      • Yamada Y.
      • Ueda Y.
      • Nakamura A.
      • Kanayama S.
      • Tamura R.
      • Hashimoto K.
      • et al.
      Immediate-type allergic and protease mediated reactions are involved in scratching behavior induced by topical application of Dermatophagoides farinae extract in NC/Nga mice.
      ), and vitiligo (
      • Riding R.L.
      • Richmond J.M.
      • Harris J.E.
      Mouse model for human vitiligo.
      ) have greatly advanced our understanding of disease pathogenesis and for exploring novel therapeutic strategies at the preclinical level.
      According to the literature, there are at least 20 models for psoriasis, 19 for atopic dermatitis, and 11 for vitiligo but only three for AA. Because one of the latter, that is, the Dundee experimental balding rat model (
      • McElwee K.J.
      • Spiers E.M.
      • Oliver R.F.
      Partial restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4+ T cells.
      ,
      • McElwee K.J.
      • Pickett P.
      • Oliver R.F.
      Hair follicle autoantibodies in DEBR rat sera.
      ), is no longer available, this has left us with just two in vivo models of AA: the most widely used murine AA model that has long dominated preclinical AA in vivo research, that is, aging C3H/HeJ mice (
      • Dai Z.
      • Xing L.
      • Cerise J.
      • Wang E.H.
      • Jabbari A.
      • de Jong A.
      • et al.
      CXCR3 blockade inhibits T cell migration into the skin and prevents development of alopecia areata.
      ,
      • de Jong A.
      • Jabbari A.
      • Dai Z.
      • Xing L.
      • Lee D.
      • Li M.M.
      • et al.
      High-throughput T cell receptor sequencing identifies clonally expanded CD8+ T cell populations in alopecia areata.
      ,
      • Shin J.M.
      • Choi D.K.
      • Sohn K.C.
      • Koh J.W.
      • Lee Y.H.
      • Seo Y.J.
      • et al.
      Induction of alopecia areata in C3H/HeJ mice using polyinosinic-polycytidylic acid (poly[I:C]) and interferon-gamma.
      ,
      • Sundberg J.P.
      • Boggess D.
      • Montagutelli X.
      • Hogan M.E.
      • King Jr., L.E.
      C3H/HeJ mouse model for alopecia areata.
      ,
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ), in which spontaneously developing AA-like lesions can be studied, and the humanized AA mouse model (
      • Gilhar A.
      • Schrum A.G.
      • Etzioni A.
      • Waldmann H.
      • Paus R.
      Alopecia areata: animal models illuminate autoimmune pathogenesis and novel immunotherapeutic strategies.
      ,
      • Gilhar A.
      • Keren A.
      • Shemer A.
      • d’Ovidio R.
      • Ullmann Y.
      • Paus R.
      Autoimmune disease induction in a healthy human organ: a humanized mouse model of alopecia areata.
      a).
      The C3H/HeJ mouse model has produced many novel results with important implications for human AA by accessing the powerful tools of mouse genetics (
      • de Jong A.
      • Jabbari A.
      • Dai Z.
      • Xing L.
      • Lee D.
      • Li M.M.
      • et al.
      High-throughput T cell receptor sequencing identifies clonally expanded CD8+ T cell populations in alopecia areata.
      ) and has helped to identify candidate autoantigens (
      • Wang E.H.C.
      • Yu M.
      • Breitkopf T.
      • Akhoundsadegh N.
      • Wang X.
      • Shi F.T.
      • et al.
      Identification of autoantigen epitopes in alopecia areata.
      ) and novel treatment strategies (
      • Dai Z.
      • Xing L.
      • Cerise J.
      • Wang E.H.
      • Jabbari A.
      • de Jong A.
      • et al.
      CXCR3 blockade inhibits T cell migration into the skin and prevents development of alopecia areata.
      ,
      • Jalili R.B.
      • Kilani R.T.
      • Li Y.
      • Khosravi-Maharlooie M.
      • Nabai L.
      • Wang E.H.C.
      • et al.
      Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model.
      ,
      • Xing L.
      • Dai Z.
      • Jabbari A.
      • Cerise J.E.
      • Higgins C.A.
      • Gong W.
      • et al.
      Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by Jak inhibition.
      ). It also provided insight into the role that could be played by psychoemotional stressors and associated neuropeptides in AA pathogenesis (
      • Paus R.
      • Arck P.
      Neuroendocrine perspectives in alopecia areata: does stress play a role?.
      ,
      • Peters E.M.
      • Liotiri S.
      • Bodó E.
      • Hagen E.
      • Bíró T.
      • Arck P.C.
      • et al.
      Probing the effects of stress mediators on the human hair follicle: substance P holds central position.
      ,
      • Siebenhaar F.
      • Sharov A.A.
      • Peters E.M.
      • Sharova T.Y.
      • Syska W.
      • Mardaryev A.N.
      • et al.
      Substance P as an immunomodulatory neuropeptide in a mouse model for autoimmune hair loss (alopecia areata).
      ,
      • Zhang X.
      • Yu M.
      • Yu W.
      • Weinberg J.
      • Shapiro J.
      • McElwee K.J.
      Development of alopecia areata is associated with higher central and peripheral hypothalamic-pituitary-adrenal tone in the skin graft induced C3H/HeJ mouse model.
      ) and has confirmed the key role of IFN-γ in AA pathogenesis (
      • Freyschmidt-Paul P.
      • McElwee K.J.
      • Hoffmann R.
      • Sundberg J.P.
      • Vitacolonna M.
      • Kissling S.
      • et al.
      Interferon-gamma-deficient mice are resistant to the development of alopecia areata.
      ;
      • Ito T.
      • Ito N.
      • Bettermann A.
      • Tokura Y.
      • Takigawa M.
      • Paus R.
      Collapse and restoration of MHC class-I-dependent immune privilege: exploiting the human hair follicle as a model.
      ). Advanced variations of this model that accelerate the development of AA by transplanting lesional skin from older mice to young ones (
      • McElwee K.J.
      • Boggess D.
      • King Jr., L.E.
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ) or that transfer draining lymph node-derived cells from affected older to as yet unaffected young C3H/HeJ cells (
      • Wang E.H.C.
      • Khosravi-Maharlooei M.
      • Jalili R.B.
      • Yu R.
      • Ghahary A.
      • Shapiro J.
      • et al.
      Transfer of alopecia areata to C3H/HeJ mice using cultured lymph node-derived cells.
      ) have further facilitated the use of is murine AA model.
      The C3H/HeJ mouse model was also used to identify key immune cell and molecular principles in murine AA and proof-of-principle that Jak inhibitors are suitable agents for AA management in vivo because both IFN-γ and IL-15 signal through the Jak pathway, thus rendering Jak inhibitor therapy a highly promising intervention strategy in AA management (
      • Phan K.
      • Sebaratnam D.F.
      Jak inhibitors for alopecia areata: a systematic review and meta-analysis.
      ,
      • Wang E.H.C.
      • Sallee B.N.
      • Tejeda C.I.
      • Christiano A.M.
      Jak inhibitors for treatment of alopecia areata.
      ,
      • Xing L.
      • Dai Z.
      • Jabbari A.
      • Cerise J.E.
      • Higgins C.A.
      • Gong W.
      • et al.
      Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by Jak inhibition.
      ). However, their potential adverse effects deserve to be more rigorously contemplated and evaluated, especially when Jak inhibitors are systemically administered long-term to children (
      • Gilhar A.
      • Keren A.
      • Paus R.
      Jak inhibitors and alopecia areata.
      ).
      Despite its undisputed usefulness for and major contributions to preclinical AA research, the C3H/HeJ mouse model carries several important disadvantages that are often ignored, yet must be kept in mind (Table 1). These include a major constitutive toll-like receptor signaling defect (
      • Kamath A.T.
      • Sheasby C.E.
      • Tough D.F.
      Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma.
      ,
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ) that is absent in patients with AA and alopecic lesions induced itch-related grooming behavior-induced (
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ,
      • King Jr., L.E.
      • Silva K.A.
      • Kennedy V.E.
      • Sundberg J.P.
      Lack of response to laser comb in spontaneous and graft-induced alopecia areata in C3H/HeJ mice.
      ). A key disease-promoting “danger” signal in AA pathobiology, the NKG2D agonistic ligand, MICA (
      • Ito T.
      • Ito N.
      • Saatoff M.
      • Hashizume H.
      • Fukamizu H.
      • Nickoloff B.J.
      • et al.
      Maintenance of hair follicle immune privilege is linked to prevention of NK cell attack.
      ,
      • Li J.
      • van Vliet C.
      • Rufaut N.W.
      • Jones L.N.
      • Sinclair R.D.
      • Carbone F.R.
      Laser capture microdissection reveals transcriptional abnormalities in alopecia areata before, during, and after active hair loss.
      ,
      • 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.
      ), is strikingly absent in mice (the murine homolog protein has only 27% amino acid identity with human MICA) (
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ). Moreover, the histologic features are not typical for human AA (
      • McElwee K.J.
      • Boggess D.
      • King Jr., L.E.
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ,
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ) because in the murine AA-like phenotype, the inflammatory cell infiltrate extends to the distal follicle between the hair bulb and sebaceous gland, sometimes reaching the bulge, whereas human AA is characterized by a largely peribulbar lymphocytic infiltrate (
      • Sundberg J.P.
      • Cordy W.R.
      • King Jr., L.E.
      Alopecia areata in aging C3H/HeJ mice.
      ,
      • King Jr., L.E.
      • Silva K.A.
      • Kennedy V.E.
      • Sundberg J.P.
      Lack of response to laser comb in spontaneous and graft-induced alopecia areata in C3H/HeJ mice.
      ). In addition, these mice cannot serve as a valid model for evaluating therapeutic effects of selected immunoinhibitory agents of interest in AA, such as Kv1.3 blockers because the K+ channel expression pattern of mouse T cells is different from that of human T cells (
      • Beeton C.
      • Wulff H.
      • Standifer N.E.
      • Azam P.
      • Mullen K.M.
      • Pennington M.W.
      • et al.
      Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases.
      ). It has been argued that, given the major differences between the immune systems of mice and humans (
      • Zschaler J.
      • Schlorke D.
      • Arnhold J.
      Differences in innate immune response between man and mouse.
      ), spontaneous models of autoimmunity that arise in mice do not satisfactorily recapitulate the human condition and thus make the development of new therapeutic strategies that will also work in the human system particularly challenging (
      • Walsh N.C.
      • Kenney L.L.
      • Jangalwe S.
      • Aryee K.E.
      • Greiner D.L.
      • Brehm M.A.
      • et al.
      Humanized mouse models of clinical disease.
      ). Unsurprisingly, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments (
      • Beura L.K.
      • Hamilton S.E.
      • Bi K.
      • Schenkel J.M.
      • Odumade O.A.
      • Casey K.A.
      • et al.
      Normalizing the environment recapitulates adult human immune traits in laboratory mice.
      ).
      Table 1Differences between C3H/HeJ and Humanized AA Mouse Model
      Typical PropertiesC3H/HeJ Mice
      Skin graft-induced hair loss variant (McElwee et al., 1998).
      Humanized AA Mouse Model
      Ease of useBasic surgical skillsAdvanced surgical skills
      CostRelatively lowRelatively high
      ConvenienceGoodDifficult to find donors willing to provide both scalp skin and blood
      Grooming-induced hair lossPresentAbsent
      PredictivenessUnclearGood
      Disadvantages
      • 1.
        The histologic feature is not characteristic to human AA
      • 1.
        Need specific conditions
      • 2.
        Absence of MICA, a recognized pathogenic key NKG2D ligand in human AA
      • 2.
        Lack of genetic background
      • 3.
        Mouse-specific hair follicle immunopathology
      • 3.
        Small area of scalp skin xenotransplants
      AdvantagesCan easily be employed as first stage assay for in vivo candidate drug testing, even though a negative outcome may not be predictive
      • 1.
        The histologic feature is characteristic to human AA
      • 2.
        Mimics human AA more closely than any other animal model, good predictive power
      Abbreviation: AA, alopecia areata.
      a Skin graft-induced hair loss variant (
      • McElwee K.J.
      • Boggess D.
      • King Jr., L.E.
      • Sundberg J.P.
      Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.
      ).
      Furthermore, the major differences between nonconventional T cells populations in humans and mice must be taken into account when using mice as preclinical models of human disease (
      • Zschaler J.
      • Schlorke D.
      • Arnhold J.
      Differences in innate immune response between man and mouse.
      ). For example, there are several distinct subsets of γδ T cells in mice and humans, but mouse and human subsets have notably different TCR use, antigen reactivity, and patterns of tissue homing (
      • Godfrey D.I.
      • Uldrich A.P.
      • McCluskey J.
      • Rossjohn J.
      • Moody D.B.
      The burgeoning family of unconventional T cells.
      ).
      • Buscher K.
      • Ehinger E.
      • Gupta P.
      • Pramod A.B.
      • Wolf D.
      • Tweet G.
      • et al.
      Natural variation of macrophage activation as disease-relevant phenotype predictive of inflammation and cancer survival.
      demonstrated that mouse models failed to account for the natural diversity in human immune responses, and as a result, insights gained in the laboratory may be lost in translation.
      Therefore, it constituted an important advance in the field when lesional human skin from patients with AA was successfully transplanted onto SCID mice (
      • Gilhar A.
      • Landau M.
      • Assy B.
      • Shalaginov R.
      • Serafimovich S.
      • Kalish R.S.
      Mediation of alopecia areata by cooperation between CD4+ and CD8+ T lymphocytes: transfer to human scalp explants on Prkdc(scid) mice.
      ,
      • Gilhar A.
      • Landau M.
      • Assy B.
      • Shalaginov R.
      • Serafimovich S.
      • Kalish R.S.
      Melanocyte-associated T cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc(scid) mice.
      ,
      • Gilhar A.
      • Ullmann Y.
      • Berkutzki T.
      • Assy B.
      • Kalish R.S.
      Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice.
      ). This advance permitted one, for the first time, to study and experimentally manipulate the AA-affected human target organ directly in a preclinical in vivo setting. Moreover, this model confirmed the proposed key role of CD8+ T cells and anagen hair follicle-derived autoantigens in AA (
      • Paus R.
      • Bulfone-Paus S.
      • Bertolini M.
      Hair follicle immune privilege revisited: the key to alopecia areata management.
      ,
      • Paus R.
      • Slominski A.
      • Czarnetzki B.M.
      Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb?.
      ) and the importance of CD4+ T cell help for developing a maximal AA phenotype (
      • Gilhar A.
      • Landau M.
      • Assy B.
      • Ullmann Y.
      • Shalaginov R.
      • Serafimovich S.
      • et al.
      Transfer of alopecia areata in the human scalp graft/Prkdc(scid) (SCID) mouse system is characterized by a TH1 response.
      ,
      • Gilhar A.
      • Landau M.
      • Assy B.
      • Shalaginov R.
      • Serafimovich S.
      • Kalish R.S.
      Mediation of alopecia areata by cooperation between CD4+ and CD8+ T lymphocytes: transfer to human scalp explants on Prkdc(scid) mice.
      ). However, this model has not been adopted by the field because it is too unpractical as it requires the availability of substantial amounts of diseased human scalp skin from patients with AA as well as autologous, intracutaneous T cell populations from lesional skin (
      • Gilhar A.
      • Ullmann Y.
      • Berkutzki T.
      • Assy B.
      • Kalish R.S.
      Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice.
      ).
      Therefore, a more practical “humanized” mouse model of AA with a wider range of applications had been developed. Helpful leads for this came from the previous observation that the intracutaneous injection of PBMCs enriched in cell populations that express NK cell markers into split-thickness transplants of healthy human corporeal skin onto beige SCID mice (which lack T, B, and have a low level of NK cells [
      • Thomsen M.
      • Galvani S.
      • Canivet C.
      • Kamar N.
      • Böhler T.
      Reconstitution of immunodeficient SCID/beige mice with human cells: applications in preclinical studies.
      ] suffices to induce psoriasis [
      • Bracke S.
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      • Guerrero-Aspizua S.
      • Desmet E.
      • Illera N.
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      • et al.
      Targeted silencing of DEFB4 in a bioengineered skin-humanized mouse model for psoriasis: development of siRNA SECosome-based novel therapies.
      ;
      • Guerrero-Aspizua S.
      • García M.
      • Murillas R.
      • Retamosa L.
      • Illera N.
      • Duarte B.
      • et al.
      Development of a bioengineered skin-humanized mouse model for psoriasis: dissecting epidermal-lymphocyte interacting pathways.
      ,
      • Nickoloff B.J.
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      ,
      • Nousbeck J.
      • Ishida-Yamamoto A.
      • Bidder M.
      • Fuchs D.
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      • et al.
      IGFBP7 as a potential therapeutic target in Psoriasis.
      ]).
      In the new model, AA-like hair loss lesions are induced in normal, full-thickness human scalp skin transplanted onto SCID beige mice by injecting autologous PBMCs enriched for NKG2D+/CD56+ cells treated stimulated with IL-2 (
      • Gilhar A.
      • Keren A.
      • Shemer A.
      • d’Ovidio R.
      • Ullmann Y.
      • Paus R.
      Autoimmune disease induction in a healthy human organ: a humanized mouse model of alopecia areata.
      ,
      • Gilhar A.
      • Keren A.
      • Shemer A.
      • Ullmann Y.
      • Paus R.
      Blocking potassium channels (Kv1.3): a new treatment option for alopecia areata?.
      ). Given that PBMCs from the same patient are used who has donated the scalp skin xenotransplants, a graft-versus-host scenario, which would anyway not generate an AA phenotype but permanent, cicatricial alopecia, is avoided (
      • Gilhar A.
      • Schrum A.G.
      • Etzioni A.
      • Waldmann H.
      • Paus R.
      Alopecia areata: animal models illuminate autoimmune pathogenesis and novel immunotherapeutic strategies.
      ).
      Using this model permits one to circumvent many of the disadvantages of the C3H/HeJ AA model, as they do not apply to the humanized one (Table 1). This model should encourage investigators to widely use the humanized AA model, for example, as a second step after initial screening experiments in C3H/HeJ mice, to optimize preclinical AA in vivo research and its predictive power for clinical outcomes. Moreover, this in vivo model should facilitate at least initial progress in the long-overdue challenge to dissect which role human hair follicles and their immune privilege actually play in the establishment, maintenance, and collapse of peripheral autoantigens (
      • Oelert T.
      • Gilhar A.
      • Paus R.
      T-cell “induced-self” MHC class I/peptide complexes may enable “de novo” tolerance induction to neo-antigens occurring outside of the thymus: lessons from the hair follicle.
      ).
      By using the humanized AA mouse model, it has been elucidated that AA pathogenesis in human skin is also affected by unconventional T cell subtypes such as NKT, iNKT10, ILC1, γ/δ-T, and γ/δ-regulatory T cells, whose numbers are significantly increased in AA compared with healthy human skin (
      • Ghraieb A.
      • Keren A.
      • Ginzburg A.
      • Ullmann Y.
      • Schrum A.G.
      • Paus R.
      • et al.
      iNKT cells ameliorate human autoimmunity: lessons from alopecia areata.
      ,
      • Kaufman G.
      • d’Ovidio R.
      • Kaldawy A.
      • Assy B.
      • Ullmann Y.
      • Etzioni A.
      • et al.
      An unexpected twist in alopecia areata pathogenesis: are NK cells protective and CD49b+ T cells pathogenic?.
      ,
      • Laufer Britva R.
      • Keren A.
      • Ullmann Y.
      • Paus R.
      • Gilhar A.
      Possible role of ILC1 in the pathogenesis of alopecia areata (AA).
      ), whose likely role in AA pathobiology had previously escaped murine AA research. Because the experiments demonstrated that nonconventional T cells might play a role in human AA (
      • Ghraieb A.
      • Keren A.
      • Ginzburg A.
      • Ullmann Y.
      • Schrum A.G.
      • Paus R.
      • et al.
      iNKT cells ameliorate human autoimmunity: lessons from alopecia areata.
      ,
      • Laufer Britva R.
      • Keren A.
      • Ullmann Y.
      • Paus R.
      • Gilhar A.
      Possible role of ILC1 in the pathogenesis of alopecia areata (AA).
      ), they suggest that targeting these immunocytes offers new opportunities for innovative therapeutic intervention. The humanized mouse model has been used to discover human-specific pharmacologic targets such as the potassium channel Kv1.3 (
      • Gilhar A.
      • Keren A.
      • Shemer A.
      • d’Ovidio R.
      • Ullmann Y.
      • Paus R.
      Autoimmune disease induction in a healthy human organ: a humanized mouse model of alopecia areata.
      ). In addition, the model demonstrated both preventive and therapeutic effects of α-galactosylceramide, which stimulates IL-10 production by iNKT cells and their expansion (
      • Ghraieb A.
      • Keren A.
      • Ginzburg A.
      • Ullmann Y.
      • Schrum A.G.
      • Paus R.
      • et al.
      iNKT cells ameliorate human autoimmunity: lessons from alopecia areata.
      ), thus introducing a promising a new candidate treatment strategy into translational AA research. Finally, in vivo results in the humanized AA mouse model elegantly recapitulate the reported differential clinical trial results in patients with AA with the Jak inhibitor, tofacitinib versus the phosphodiesterase type 4 inhibitor, apremilast (
      • Liu L.Y.
      • King B.A.
      Tofacitinib for the treatment of severe alopecia areata in adults and adolescents.
      ;
      • Mikhaylov D.
      • Pavel A.
      • Yao C.
      • Kimmel G.
      • Nia J.
      • Hashim P.
      • et al.
      A randomized placebo-controlled single-center pilot study of the safety and efficacy of apremilast in subjects with moderate-to-severe alopecia areata.
      ): just as in patients with AA, poor therapeutic effects were seen in the humanized AA mouse model with apremilast, contrasted by a strong therapeutic effect of tofacitinib (Gilhar et al., unpublished data).

      Conclusion

      In summary, we advocate making it a routine practice in future preclinical AA research to use both the C3H/HeJ (e.g., for screening purposes) and the humanized AA model as perfectly complementary investigation tools and to test new candidate AA therapeutics also in the humanized AA model before entering into clinical trials.

      Conflict of Interest

      RP is the founder & CEO of a CRO (Monasterium Laboratory GmbH; https://www.monasteriumlab.com) that is involved in research on the humanized alopecia areata model described here. The remaining authors state no conflict of interest.

      Author Contributions

      Conceptualization: AG; Supervision: AG; Writing: AG and RP; Data curation: AK and RLB; Visualization: AK and RLB

      Acknowledgments

      This article is published as part of a supplement sponsored by the National Alopecia Areata Foundation .
      Funding for the Summit and publication of this supplement was provided by the National Alopecia Areata Foundation. This Summit was supported (in part) by the National Institute of Arthritis and Musculoskeletal and Skin Diseases under award number R13AR074890 . The opinions or views expressed in this professional supplement are those of the authors and do not necessarily reflect the official views, opinions, or recommendations of the National Institutes of Health or the National Alopecia Areata Foundation.

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