Alopecia Areata - Not always a 'problem to be fixed'.

Alopecia areata (AA) is a common autoimmune skin disease that results in the loss of hair on the scalp and elsewhere on the body and affects over 146 million people worldwide at some point in their lives. Founded in 1981, the National Alopecia Areata Foundation is a nonprofit organization that supports research to find a cure or acceptable treatment for AA, supports those with the disease, and educates the public about AA. The National Alopecia Areata Foundation conducts research summits every 2 years to review progress and create new directions in its funded and promoted research. The Foundation brings together scientists from all disciplines to get a broad and varied perspective. These AA research summits are part of the Foundation's main strategic initiative, the AA Treatment Development Program, to enhance the understanding of AA and accelerate progress toward a viable treatment.

Editor's evaluation: Involvement of ILC1-like innate lymphocytes in human autoimmunity, lessons from alopecia areata

Decision letter: Involvement of ILC1-like innate lymphocytes in human autoimmunity, lessons from alopecia areata

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Here, we have explored the involvement of innate lymphoid cells-type 1 (ILC1) in the pathogenesis of alopecia areata (AA), because we found them to be significantly increased around lesional and non-lesional HFs of AA patients. To further explore these unexpected findings, we first co-cultured autologous circulating ILC1-like cells (ILC1lc) with healthy, but stressed, organ-cultured human scalp hair follicles (HFs). ILClc induced all hallmarks of AA ex vivo: they significantly promoted premature, apoptosis-driven HF regression (catagen), HF cytotoxicity/dystrophy, and most important for AA pathogenesis, the collapse of the HFs physiological immune privilege. NKG2D-blocking or IFNγ-neutralizing antibodies antagonized this. In vivo, intradermal injection of autologous activated, NKG2D+/IFNγ-secreting ILC1lc into healthy human scalp skin xenotransplanted onto SCID/beige mice sufficed to rapidly induce characteristic AA lesions. This provides the first evidence that ILC1lc, which are positive for the ILC1 phenotype and negative for the classical NK markers, suffice to induce AA in previously healthy human HFs ex vivo and in vivo, and further questions the conventional wisdom that AA is always an autoantigen-dependent, CD8 +T cell-driven autoimmune disease. Editor's evaluation This manuscript provides fundamental data that implicate ILC1-like cells in alopeia areata. The data are solid in the use of cultured human hair follicles co-cultured with ILC-1-like cells and demonstration that alopecia phenotypes emerge. The authors also provide compelling evidence that injection of ILC1-like cells induces alopecia in a mouse model grafted with human hair follicle-containing skin. This work will be of interest to immunologists, skin biologists, and scientists interested in autoimmune disorders. https://doi.org/10.7554/eLife.80768.sa0 Decision letter eLife's review process Introduction Alopecia areata (AA) is both the most common inflammatory hair loss disorder and one of the most common human autoimmune diseases and exerts a major negative impact on quality of life (Gilhar et al., 2012; Gilhar et al., 2019a; Korta et al., 2018; Pratt et al., 2017). Despite major recent advances in AA therapy, a causal therapy does not yet exist, and disease relapse after therapy discontinuation is the rule, not the exception in long-standing AA (Meah et al., 2020; Gilhar et al., 2019a). Thus, the currently available, purely symptomatic AA therapy, including JAK inhibitors (Gilhar et al., 2019b), remains unsatisfactory. Since the exact pathobiology of AA and its clinical variants remains to be fully characterized, the – likely diverse – disease-initiating factors that ultimately result in the characteristic AA hair loss pattern shared by all AA variants, require more comprehensive dissection for optimal, personalized therapeutic targeting (Bertolini et al., 2020; Paus et al., 2018). Specifically, there is increasing awareness that a classical, autoantigen- and CD8 +T cell-dependent autoimmune variant of AA (AAA) and a possibly autoantigen-independent non-autoimmune variant (NAIAA) may have to be distinguished from each other (Gilhar et al., 2019a; Bertolini et al., 2020; Paus et al., 2018; Paus, 2020). This is in line with the long-standing, but often under-appreciated clinical recognition that AA shows a wide spectrum of phenotypes and sub-forms (Gilhar et al., 2012; Ikeda, 1965; Meah et al., 2021; King et al., 2022). One reason why the currently available AA therapy is not entirely satisfactory may be related to as yet insufficient therapeutic targeting of innate immunocytes in the immunopathogenesis of human AA, namely in NAIAA, even though these are now recognized as major players in AA pathobiology (Ghraieb et al., 2018; Ito et al., 2008; Li et al., 2016; Uchida et al., 2020; Uchida et al., 2021). Previously, we had demonstrated that AA lesions are associated with a massive increase in the number of perifollicular NKG2D+NK cells (Gilhar et al., 2013a), which recognize the activating NKG2D ligand MICA, a ‘danger’ signal that is greatly overexpressed by the epithelium of lesional AA HFs (Ito et al., 2008; Li et al., 2016; Connell and Jabbari, 2022). Subsequent work has confirmed the key role of NKG2D and its activating ligands in human and murine AA (Xing et al., 2014; Petukhova et al., 2010). In fact, AA lesions can be induced experimentally in healthy human scalp skin in vivo by the transfer of interleukin 2 (IL-2)-activated NKG2D+ cells (Gilhar et al., 2013a), most of which had NK cell characteristics, with only a small minority of CD8 +T cells being present, that is the best-recognized pathogenic lymphocyte population in AA (Gilhar et al., 2012; Gilhar et al., 2013a; Pratt et al., 2017; Bertolini et al., 2020; de Jong et al., 2018). Moreover, pro-inflammatory mast cells (Bertolini et al., 2014) and (likely autoantigen-non-specific) γδ T-cells are also increased around/in lesional human AA HFs (Uchida et al., 2020). Finally, these ‘intermediate immunity’ protagonists suffice to induce the hallmarks of AA ex vivo (Uchida et al., 2021). Taken together, this questions whether pathogenic, autoreactive CD8 +T cells are the only drivers of disease, and that all cases of AA, represent a genuine, autoantigen-dependent autoimmune disease (Bertolini et al., 2020; Paus et al., 2018) in the strictly defined sense of this term (Rose and Bona, 1993). In our ongoing exploration of the role of innate/transitional immunity in the pathobiology of AA (Paus, 2020; Uchida et al., 2020; Uchida et al., 2021; Gilhar et al., 2019a; Bertolini et al., 2014), we, therefore, have asked in the current study whether innate lymphoid cells type 1 (ILC1 cells) (Zhou et al., 2020; Nabekura and Shibuya, 2021a; Colonna, 2018) can initiate human AA lesions. We were interested in these immunocytes since human ILC1 cells secrete large amounts of interferon-γ (IFN-γ) (Ebbo et al., 2017), the crucial AA pathogenesis-promoting cytokine (Gilhar et al., 2012; Gilhar et al., 2019a; Paus et al., 2018), and this notably independent of classical autoantigen-specific CD8 +T cell activities. These ‘unconventional’ T-cells are placed in strategic tissue locations (Collins et al., 2017; Jiao et al., 2016; Kim et al., 2021) and represent an important link between innate and adaptive immunity (Vivier et al., 2018). While ILC1s play an essential role in human inflammatory bowel disease (IBD) (Ebbo et al., 2017; Luo et al., 2022; Clottu et al., 2021), their role in the pathophysiology of autoimmune hepatitis and rheumatoid arthritis requires further investigation (Ebbo et al., 2017; Fang et al., 2020; Yang et al., 2015), and their role in human autoimmune diseases overall remains insufficiently understood. We hypothesized that AA might offer a good model disease for interrogating this role. ILC1 cells are classified as a component of type 1 immunity (Shannon et al., 2021), express NKG2D, recognize conserved phosphoantigens (Nabekura and Shibuya, 2021a), and contribute to immunity against tumor cells, for example through NKG2D activation (Dadi et al., 2016). The activating receptor NKG2D and its ligands (MICA, ULBP3) play an important role in innate (NK, ILC1), ‘translational’ (γδ T-cells) and CD8 T-cell-mediated immune responses to tumors and in several autoimmune diseases (Frazao et al., 2019; Babic and Romagnani, 2018). Given that ILC1 cells produce TH1-type cytokines (such as IFN-γ) and share several phenotypic markers with NK cells, namely NKG2D (Spits et al., 2016), it is challenging to distinguish NKs and ILC1 cells (Tulic et al., 2019; Zhang et al., 2018; Seillet et al., 2021; Conlon et al., 2021). In fact, how to reliably discriminate between NK cells and ILC1s and unraveling the shared and distinct functions of these cell populations remains an important open quest (Seillet et al., 2021; Lopes et al., 2023; Cheng et al., 2023; Taggenbrock and van Gisbergen, 2023). For example, Eomeshi T-betlo liver-resident NK cells have been described in humans and mice (Park et al., 2019; Harmon et al., 2016), while ILC1s from human tonsil and blood was also found to be Eomes+ (Cella et al., 2019). Therefore, the distinction between NK cells and ILC1s remains provisional – which is exactly why we have cautiously labeled the latter as ‘ILC1lc.’. The transcriptional and functional identity of ILC1 cells in humans is still a matter of debate, given that in contrast to other ILC subsets ILC1 cells seem to lack robust markers that enable their unequivocal identification and isolation (Bennstein et al., 2020). However, although integrin α1 (CD49a) is upregulated on activated NK cells (Albini et al., 2021; Zheng et al., 2016), CD49a and integrin α2 (CD49b) are used as two mutually exclusive markers for distinguishing between NK and ILC1 cells, with NK cells being defined as CD49b+CD49a- and ILC1 as CD49b-CD49a+ (Gao et al., 2017; Vienne et al., 2021; Flommersfeld et al., 2021; Krzywinska et al., 2022) In the current study, we have accepted and employed this consensus. Also, in contrast to ILC1 and ILC1lc, classical NK cells demonstrate high T-bet and Eomes expression (T-bethi /Eomeshi) (Verma et al., 2020). Therefore, for the purpose of this study, we define ILC1lc as CD49a+CD49b- (Verma et al., 2020) and as lin-/CD127+/CD117-/CRTH2-phenotype, which are typical to classical ILC1 cells (Bennstein et al., 2020; Krabbendam et al., 2021), and also as T-betlo/ Eomeshi (Bennstein et al., 2020) (in contrast to classical T-bethi /Eomeslo ILC1 cells Verma et al., 2020). Specifically, we have asked whether (a) their number is increased in lesional AA skin, (b) they can damage human HFs ex vivo in a manner that mimics the AA phenotype, and finally (c) whether ILC1lc alone suffice to induce AA in previously healthy human scalp skin in vivo. To address these questions, we first analyzed the abundance, distribution, and phenotype of ILC1lc in human AA skin lesions compared to healthy human control skin. We then co-cultured autologous ILC1lc with freshly organ-cultured scalp HFs from the same patient, that is under conditions where the epithelium of these HFs transiently undergo an acute stress response and overexpresses MICA (Uchida et al., 2021), to check whether these innate lymphocytes exert any HFs cytotoxicity and/or impact on the physiological immune privilege (IP) of HFs (Bertolini et al., 2020; Paus et al., 2005; Ito et al., 2004; Peters et al., 2007; Bertolini et al., 2016). Finally, we injected autologous ILC1lc intradermally into healthy human scalp skin xenotransplants from the same human volunteers on SCID/beige mice to probe whether this suffices to induce classical AA hair loss lesions in vivo. Taken together, our data show that ILC1lc is increased in AA lesions and suffice to induce an AA phenotype in healthy human HFs ex vivo and in vivo. This provides the first functional evidence of a key role of ILC1lc innate lymphocytes in a model human autoimmune disease (Colonna, 2018; Seillet et al., 2021; Conlon et al., 2021; Flommersfeld et al., 2021; Daussy et al., 2014; Park et al., 2019) - but also questions whether AA always a classical autoimmune disease is and underscore the role of innate immune cells in AA pathobiology. Results Peri- and intrafollicular infiltrates of ILC1lc are seen in both lesional and non-lesional AA skin First, we investigated whether healthy and AA-affected human skin differs in their content and/or distribution of ILC1lc, using a comprehensive set of triple-immunofluorescence (IF) staining best suited to identify these immunocytes (Seillet et al., 2021; Bennstein et al., 2020; Gao et al., 2017). This revealed the presence of only extremely few ILC1lc in healthy control skin with all three staining settings employed (Eomes+, CD49a+, NKG2D+ [Figure 1A and Figure 1—figure supplement 1A], Eomes+, c-KIT-, CD49a+ [Figure 1B and Figure 1—figure supplement 1A], or NKp44+, CD103+, T-bet- cells [Figure 1C and D and Figure 1—figure supplement 1A; Kim, 2015; Fuchs et al., 2013; Salimi and Ogg, 2014]). These cells appeared to be preferentially scattered along the papillary dermis of healthy scalp skin biopsies and around the HFs (Figure 1C). This is reminiscent of the few Vδ1+T cells detectable in healthy human skin that also have a preferential perifollicular location and may ‘police’ the skin for molecular indications of tissue stress, namely of HFs (Uchida et al., 2020; Uchida et al., 2021). Figure 1 with 1 supplement see all Download asset Open asset Immunofluorescence microscopy analyses of ILC1lc and CD8+/NKG2D+ cells in alopecia areata (AA) scalp skin. (A) ILC1lc (EOMES+, CD49a+, and NKG2D+) around HF in normal scalp skin, intrafollicular and perifollicular ILC1lc infiltrates in lesional and in non-lesional AA scalp patient. (B) EOMES+, c-KIT-,CD49a+, and (C) NKp44+, CD103+, T-bet- ILC1lc. For each panel, yellow staining indicates double staining A-EOMES+, NKG2D+; B- EOMES+, CD49a+; C- NKp44+,CD103+ (D) Quantitative immunohistomorphometry (qIHM) shows an increased number of ILC1lc in AA patients as compared to normal volunteers and increased number of the cells in lesional versus non-lesional areas of the patients. There is a significant increased perifollicular than intrafollicular ILC1lc in the lesional and non lesional areas. (E) CD8+/NKG2D+ cells around HF in AA scalp patient and absence of these cells in normal scalp skin of normal scalp skin. (F) There is an increased number of CD8+/NKG2D+ cells in HFs of AA patients compared to normal scalp skin and a significant lower number of ILC1lc versus CD8+/NKG2D+ cells in AA scalp skin. N=6 biopsies /AA patients and six biopsies /healthy donors from six independent donors, three areas were evaluated per section, and three sections per biopsy. Following Shapiro-Wilk test, Student’s t-test: *p<0.05, **p<0.01 or Mann Whitney U test: #p<0.05. Scale bars, 50 µm. CTS- connective tissue sheath, DP - dermal papilla, HM - hair matrix, White arrow- c-KIT stained melanocyte. Figure 1—source data 1 Quantitative data for immunofluorescence microscopy analyses of ILC1lc and CD8+/NKG2D+ cells in AA scalp skin. https://cdn.elifesciences.org/articles/80768/elife-80768-fig1-data1-v1.xlsx Download elife-80768-fig1-data1-v1.xlsx Instead, intra and peri-follicular infiltrates of ILC1lc were frequently present in lesional AA HFs (Figure 1A, B, C and D and Figure 1—figure supplement 1A), typically in conjunction with a dominant infiltrate of CD8+/NKG2D+ cells around the hair bulb (p<0.05) (Figure 1E and F). Importantly, the number of ILC1lc was already significantly increased in/around non-lesional AA HFs compared to healthy scalp skin (p<0.01) (Figure 1A, B, C and D and Figure 1—figure supplement 1A). This may indicate that ILC1lc may actually have arrived around the HFs before the CD8 cells and may have contributed to attracting the CD8 cells into the perifollicular space. This strongly suggested that ILC1lc are not mere bystanders attracted only secondarily to the HFs by CD8 T-cells, similar to, but more pronounced than we have recently observed regarding perifollicular Vδ1+T cells in non-lesional AA skin (Uchida et al., 2020). This invited the hypothesis that ILC1lc is actively involved in transforming healthy human scalp HFs into lesional AA HFs. T-betlo/Eomeshi ILC1lc can be expanded from human peripheral blood mononuclear cells (PBMCs) in vitro To functionally probe this hypothesis, we isolated, purified, and characterized human peripheral blood-derived ILC1lc as the most suitable cell source for the planned HF-immunocyte co-culture studies. The scarcity of ILC1lc in healthy human skin, compared to their relative abundance in peripheral blood (Colonna, 2018; Artis and Spits, 2015) necessitated to isolate autologous ILC1lc from the latter source rather than from skin (Teunissen et al., 2014). To facilitate ILC1lc isolation, PBMCs of healthy volunteers were first cultured with high-dose IL-2 (100 U/mL) in the presence of IL-18 (1 µg/1 ml), IL-33 (1.5 µg/5 ml), and IL-12 (1.5 µg/5 ml), since these cytokines induce ILC1lc expansion (Salimi and Ogg, 2014; Silver et al., 2016; Orimo et al., 2020; Ohne et al., 2016). When ILC1lc were sorted by FACS Aria and characterized by FACS analysis on day seven of culture, low T-bet, and high Eomes expression were observed (Figure 2A), in contrast to classical T-bethi and Eomeslo ILC1 cells (Jiao et al., 2016; Vivier et al., 2018; Zhang et al., 2018). In addition, the ILC1lc expressed and shared the following markers with classical ILC1 cells: LIN- CD3/CD1a/D14/CD19/CD34/CD123/CD11c /BDCH2/FcεR1α/TCRαβ/TCRγδ/CD56, CD127+, CD161+, c-KIT-, and CRTH2- (Zook and Kee, 2016; Bernink et al., 2017; Simoni and Newell, 2017; Figure 2A). Figure 2 with 2 supplements see all Download asset Open asset Circulating ILC1lc expanded and characterized by FACS analysis. (A) PBMCs activated by IL-18, IL-33 and IL-12 were sorted by FACS Aria and characterized by FACS analysis. ILC1lc markers were identified by the expression of CD127+, CD161+, c-KIT-, and CRTH2-, high levels of integrin α1 (CD49a) expression, combined with the absence of integrin α2 (CD49b) and transcription factors Eomeshi and T-betlo (B) unstimulated PBMCs (C) isotype controls. N=10 blood donors, 1.5 × 106 cells/blood donor, analysis was performed in triplicates from each of the blood donors. Following Shapiro-Wilk test, Student’s t-test, p<0.05. Figure 2—source data 1 Quantitative data for circulating ILC1lc expanded and characterized by FACS analysis. https://cdn.elifesciences.org/articles/80768/elife-80768-fig2-data1-v1.xlsx Download elife-80768-fig2-data1-v1.xlsx This immune phenotype suggests that the immune cells used in our study are best classified as ILC1lc (Nabekura and Shibuya, 2021a), and documents that all experiments reported below were indeed performed with autologous ILC1lc rather than with NK cell subpopulations. Indeed, the FACS analysis (Figure 2—figure supplement 1A,B and C) revealed that ILC1lc demonstrates the ILC1 phenotype (CD200R, CD127, CXCR6) (Lopes et al., 2023; Curio and Belz, 2022) but not of the classical NK cell lineage (IRF8, Perforin, NKp80, CD16) (Sagebiel et al., 2019; Brownlie et al., 2021; Krämer et al., 2023), thus further serving as an evidence that EOMES +ILCs represent distinct ILC1 lineage-defining markers. In contrast to NK cells, ILC1lc also expressed the expected high levels of integrin α1 (CD49a), combined with the absence of integrin α2 (CD49b) (Jiao et al., 2016; Figure 2A). All these characteristic markers of ILClc were absent in the control unstimulated PBMCs (Figure 2B and C). This immune phenotype suggests that the immune cells used in our study are best classified as ILC1lc (Nabekura and Shibuya, 2021a), and documents that all experiments reported below were indeed performed with autologous ILC1lc rather than with NK cell subpopulations. Note that we had previously shown that NKG2D+/CD56 +NK cells suffice to induce AA lesions in human skin in vivo (Gilhar et al., 2013a; Laufer Britva et al., 2020) while iNKT cells are AA-protective in the humanized AA mouse model (Ghraieb et al., 2018). Subsequently, these ILC1lc were either used for HF co-culture assays or injected into healthy human scalp skin xenotransplants on SCID/beige mice (Gilhar et al., 2013a; Ito et al., 2005b). As controls, we also isolated ILC2 and ILC3 cells, which failed to induce AA phenotype in a sharp contrast to the ILC1lc (see Materials and methods). In order to exclude the possibility that contamination from the ILC1lc and thus to the observed we a set of FACS data on sorted ILC1lc. Given that ILC1 cells are negative while ILC3 cells are positive et al., 2022; et al., 2021), the data demonstrate that the contamination hypothesis is (Figure 2—figure supplement ILC1lc induces HF cytotoxicity ex vivo we functionally the of ILC1lc with HFs that were investigated as a model human in which the of a healthy human tissue with autologous populations can be ex vivo in the absence of any immune or (Uchida et al., 2021). For organ-cultured human scalp HFs et al., 2015) were co-cultured for six with peripheral purified, ILC1lc, or with autologous human CD8 cells ILC3 cells, or PBMCs activated with Importantly, only scalp HFs in the of the hair were used as et al., that had been freshly placed into HF for since these HFs are in contrast to that is after isolation, or that had already several of to the conditions of (Uchida et al., 2021; et al., These day 1 HFs and while the expression of that is and (Uchida et al., 2021), a transiently but HF immune privilege (Bertolini et al., 2020; Ito et al., The expression of associated with tissue stress, that is the intrafollicular (Ito et al., and the NKG2D ligand is also in day 1 organ-cultured HFs compared to freshly HFs or after day of 1 HFs also show of HF by increased into the and express recognized for their in AA that is and (Uchida et al., 2021; Ito et al., 2020). Thus, day 1 HFs are suited for interrogating human with a transiently but healthy human that overexpresses the ‘danger’ under ex vivo conditions (Uchida et al., 2021; et al., First, we the of ILC1lc on healthy human scalp HF ex vivo by the HF of into the This not only significantly induced by ILC1lc than by co-culture with all three negative control cell populations or but also even HF cytotoxicity levels than induced by CD8+/NKG2D+ cells namely after three of co-culture (Figure These HF cytotoxicity were fully by characteristic of HF following co-culture with while CD8+/NKG2D+ cells induced similar these were not seen after co-culture with (Figure and C). The of significant HF by ILC1lc ex vivo was further by the presence of and intrafollicular et al., 2007; et al., 2005; Figure and and by and increased of hair (Figure and were also seen in the but not in HFs co-cultured with Thus, autologous ILC1lc alone suffice to induce HF cytotoxicity ex vivo co-cultured with transiently but healthy human scalp HFs. Figure Download asset Open asset of ILC1lc on normal human scalp HF ex vivo. These cell populations were placed into with HFs and of these cell populations on normal human scalp HF ex vivo were by the of from the HFs. cytotoxicity of ILC1lc co-cultured with HFs compared to as as to and and from three independent donors analyzed in three independent HF Following Shapiro-Wilk and Figure data 1 Quantitative data for of CD8+/NKG2D+ and ILC1lc on normal human scalp HF ex vivo. Download Figure Download asset Open asset follicles and in normal human scalp HF ex vivo co-cultured with ILC1lc and CD8+/NKG2D+ staining revealed and cells, dermal papilla, and the of cells, from three independent donors. revealed and location of only in HFs co-cultured with ILC1lc, but not in HFs cultured with from three independent donors. Following Shapiro-Wilk t-test: *p<0.05, HFs co-cultured with ILC1lc or CD8+/NKG2D+ cells a significantly and increased wide N=6 from two independent donors, three areas were evaluated per Following Shapiro-Wilk test, Student’s t-test: *p<0.05, in the hair bulb compared to HFs cultured with Scale bars, 50 µm. DP - dermal papilla, HM - hair Figure data 1 Quantitative data for HFs and in normal human scalp HF ex vivo co-cultured with ILC1lc and CD8+/NKG2D+ Download ILC1lc induces HF immune privilege collapse ex vivo NKG2D Given that AA the collapse of HF immune privilege (Gilhar et al., 2012; Bertolini et al., we also investigated the impact of ILC1lc on key markers. Indeed, the co-culture of HFs with ILC1lc as by and overexpressed and along with of the MICA and which with and NKG2D (Uchida et al., 2021; et al., 2022) as compared to HFs with or with ILC3 cells (Figure B, and F). Figure Download asset Open asset of alopecia areata (AA) hair follicles (HFs). (A) MICA, (B) (C) (D) and (E) expression by HFs which had been co-cultured with either ILC1lc or CD8+/NKG2D+ cells but not in the control which had been co-cultured with either ILC1lc or in the HFs. (F) The immune HF immune privilege and in ILC1lc and but were present in ILC1lc and control from three independent donors, three areas were evaluated per Following Shapiro-Wilk t-test, Scale µm. - Figure data 1 Quantitative data for characteristic of AA HFs. Download (qIHM) also that expression of the and (Gilhar et al., 2012; Bertolini et al., 2020; Paus et al., 2018; Ito et al., in the epithelium of HFs co-cultured with autologous ILC1lc or CD8+/NKG2D+ cells (Figure and while these were still expressed in negative control HFs (Figure and Importantly, antibodies HFs collapse and the in the ILC1lc (Figure and This demonstrates that autologous ILC1lc induces human collapse ex vivo – the first that the of collapse by ILC1lc has been in an human ILC1lc are activated by HFs and induce we how autologous ILC1lc on human HF given that of apoptosis-driven HF regression is one of the hallmarks of AA (Gilhar et al., 2012; Bertolini et al., 2020; et al., This that ILC1lc significantly the of into HFs ex vivo et al., compared to all three negative or – thus the of the AA phenotype collapse and ex vivo (Gilhar et al., 2012; Bertolini et al., 2020; et al., Figure as we had previously shown for cells (Uchida et al., 2021). As expected (Gilhar et al., 2012; Pratt et al., 2017;

Mechanism leading to an abrupt hair loss in diffuse alopecia areata (AA) remains unclear. To explore the characteristics of diffuse AA and possible factors involved in its pathogenesis. Clinical and laboratory data of 17 diffuse AA patients and 37 patchy AA patients were analyzed retrospectively. Serum IgE level was evaluated in all diffuse and patchy AA patients, as well as 27 healthy subjects without hair loss to serve as normal control. Univariate analysis was performed using Fisher's exact test and Wilcoxon rank-sum test. Associations between inflammatory cell infiltration and laboratory values were analyzed using Spearman rank correlation test. The mean age of patients with diffuse AA was 27 years with a mean disease duration of 1.77 months. All of them presented in spring or summer with an acute onset of diffuse hair loss preceded by higher incidence of scalp pruritus. Although no statistically significant difference on the incidence of atopic disease among three groups has been found, serum IgE level in diffuse AA was higher than that in healthy controls, but was comparable to that in patchy AA group. Histopathology of lesional scalp biopsies showed more intense infiltration comprising of mononuclear cells, eosinophils, CD3 + , and CD8 + T cells around hair bulbs in diffuse AA group than in patchy AA group. Moreover, IgE level in diffuse AA patients positively correlated with intensity of infiltration by mononuclear cells, eosinophils, and CD8 + T cells. Hypersensitivity may be involved in pathogenesis of diffuse AA. The acute onset of diffuse AA may be related to intense local inflammatory infiltration of hair loss region and an increase in serum IgE level.

Concordance rate of alopecia areata in identical twins supports both genetic and environmental factors

Meaningful patient input to understand disease experience and patient expectations for improvement with treatment is essential for the selection and development of outcome measures for alopecia areata (AA) clinical trials. This study explored the physical signs and symptoms of AA through 30 semistructured interviews with adult (n= 25) and adolescent (n= 5) patients experienced with severe or very severe AA. Scalp hair loss was overwhelmingly the most important sign and symptom of AA. Nearly all patients (90%) considered scalp hair loss in their top three most bothersome physical signs and symptoms of AA, with 77% (n= 23) naming scalp hair loss as the most bothersome symptom. Other identified signs and symptoms in the top three most bothersome included eyebrow, eyelash, nose, body, and facial hair loss, as well as eye irritation and nail damage and/or appearance. Eyebrow (16%, n= 4), eyelash (4%, n= 1), nasal (4%, n= 1), and body (4%, n= 1) hair loss were identified by seven adult patients as the most bothersome signs and symptoms of AA. Conceptual saturation confirmed that a comprehensive understanding of this patient population's physical AA-related signs and symptoms was obtained. These findings indicate that the primary objective for new AA treatments for this patient population should be meaningful improvement in scalp hair growth to address the most troubling unmet need.

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Alopecia areata and myasthenia gravis presenting as paraneoplastic phenomena of breast cancer

Gut instinct: Using tofacitinib to treat alopecia areata in the context of comorbid inflammatory bowel disease

Introduction/Background Studies have shown that patients with vs without alopecia areata (AA) have a higher risk of developing atopic dermatitis (AD). However, the real-world prevalence and incidence of moderate-to-severe AD in patients with AA are not well characterized. Objectives To assess the real-world prevalence and incidence of comorbid AD among patients with AA using data from a US administrative claims database. Methods This retrospective analysis evaluated claims from the Merative Marketscan Commercial Claims and Encounters database. Eligible patients were aged ≥12 years at index date (first AA diagnosis), received an AA diagnosis (≥1 inpatient encounter or ≥2 outpatient encounters/claims based on International Classification of Diseases, Clinical Modification, 10th Revision [ICD-10-CM]: L63.x or 9th Revision [ICD-9-CM]: 704.01) between 01/01/2017–07/31/2023, and had continuous plan enrollment for 5 years prior to index. AA disease severity was evaluated during the 6-month follow-up period post-index date. Patients were considered to have moderate-to-severe AA if they were diagnosed with alopecia universalis (ICD-10-CM: L63.1) or alopecia totalis (ICD-10-CM: L63.0) or if they received prescriptions for any systemic immunomodulators, oral corticosteroids, nonsteroidal systemic agents, or phototherapy within 6 months post-index date. Very severe AA (subset of moderate-to-severe AA) was defined by a diagnosis of alopecia universalis (ICD-10-CM: L63.1) or alopecia totalis (ICD-10-CM: L63.0). AD was defined as ≥1 inpatient or ≥2 outpatient claims with diagnosis codes for AD (ICD-10-CM L20.x) or other AD-related conditions (ICD-9-CM 691.8). Moderate-to-severe AD was defined per the AD criteria plus claims indicating 1 of the following: ≥1 dispensing of dupilumab; ≥2 dispensing of high-potency topical corticosteroids or systemic immunosuppressants; or ≥3 dispensing of medium potency topical corticosteroids, topical tacrolimus, phototherapy, or oral/parenteral corticosteroids. AD prevalence was assessed during the 5 years pre-index period; prevalence was also assessed over a longer duration and included those with ≥1 AD diagnosis from 01/01/2007–index. AD incidence was assessed from the 7th month after the AA index date through the time of AD diagnosis or end of continuous enrollment. Relative risk for developing AD comorbidity among patients with AA was evaluated using Cox proportional hazards model, controlling for age, sex, obesity, Charlson Comorbidity Index (CCI), and geographic region. Results Of 429,903 patients identified with AA in the database, 10,863 met eligibility criteria and 9507 completed ≥6 months of follow-up post-index date (mild AA, n=7087; moderate-to-severe AA, n=2420; very severe AA, n=491). The 1-year period prevalence of AA in the US was 0.16% in 2022. Among the 10,863 eligible patients, 63.0% were female, the mean (SD) age was 40.4 (15.1) years, and the mean (SD) CCI was 0.7 (1.2). Among all eligible patients with AA (n=10,863), the prevalence (within 5 years pre-index date) was 3.1% for any AD and 2.3% for moderate-to-severe AD. Among patients with moderate-to-severe AA (n=2420), the prevalence was 4.8% for any AD and 4.2% for moderate-to-severe AD. The prevalence of any AD any time prior to index among all eligible patients with AA and moderate-to-severe AA was 11.9% and 14.6%, respectively. AD incidence at follow-up was 4 per 1000 person-years in patients with mild AA, 7 per 1000 person-years for moderate-to-severe AA, and 6 per 1000 person-years for very severe AA. Mean (SD) time to AD diagnosis after AA index date was 2.1 (1.7) years for patients with mild AA, 2.0 (1.7) years for moderate-to-severe AA, and 2.0 (1.7) years for very severe AA. Risk of developing AD was greater in patients with moderate-to-severe vs mild AA (adjusted hazard ratio 1.63 [95% CI 1.01, 2.64]). Conclusions Patients with moderate-to-severe vs mild AA had higher prevalence and incidence of AD comorbidity and higher prevalence of moderate-to-severe AD.

The existing literature on alopecia areata (AA) clearly demonstrates patients' concerns related to physical symptoms, emotional well-being, mental health, social functioning, and other dimensions of daily functioning. Although questionnaires such as the Skindex-16 and the Dermatology Life Quality Index have been used, these questionnaires were validated for skin conditions other than AA as a chronic condition. The goals of this study are to develop a measure of quality of life, symptoms, and their impact for patients with AA called the Alopecia Areata Symptom Impact Scale (AASIS) and to provide psychometric evidence for its use. We used data from 1,400 patients from the National Alopecia Areata Registry together with clinical experts' reviews and quantitative approaches. The preliminary version of the AASIS with 13 items was administered to about 210 patients with AA. Results indicated that the AASIS measures three underlying constructs related to AA. These dimensions were impact of AA, hair loss, and physical skin symptoms. The internal consistency reliabilities of these subscales are 0.93, 0.86, and 0.81, respectively. Cognitive debriefing results showed that patients find the AASIS items easy to understand, clear, and concise. Preliminary evidence suggests that the AASIS is a reliable and valid measure of the symptoms and their impact in patients with AA.

Alopecia Areata Is Associated with Atopic Diathesis: Results from a Population-Based Study of 51,561 Patients

Alopecia areata after CoronaVac vaccination.

Successful treatment of corticosteroid-resistant ophiasis-type alopecia areata (AA) with platelet-rich plasma (PRP)