Abstract Fragment crystallizable gamma receptor (FcγR)-induced signaling is a crucial process that determines the cellular response to immune complexes (IC) in autoimmune diseases. In several diseases including pemphigoid diseases (PD), such as epidermolysis bullosa acquisita (EBA), or rheumatoid arthritis (RA), neutrophils are prominently involved as effector cells, while others such as immune thrombocytopenia (ITP) are independent of neutrophils. At the same time, most diseases are commonly treated by broad-range immunosuppression accompanied by severe risk for adverse effects. Signal transduction inhibitors (STIs) have been successfully applied in cancer therapy. However, their use in autoimmune diseases is an emerging, but so far understudied potential treatment avenue. Therefore, we screened a target-selective compound library consisting of 155 STIs in a neutrophil-based assay and conducted a multiplex kinase activity profiling with IC-stimulated neutrophils. Thus, we found novel potential therapeutic targets that were validated both in vitro in functional neutrophil assays and in vivo in murine models of EBA Here, we demonstrate that both systemic and topical treatment with several individual STIs is effective in a prophylactic approach in these models. Furthermore, therapeutic treatment with the BTK inhibitor ibrutinib in the immunization-induced EBA model reduced disease severity by approximately 85 % and showed efficacy in additional experimental models of EBA, arthritis, and ITP. Together, the present study contributes to the elucidation of FcγR-dependent signaling in neutrophils and identifies multiple novel promising treatment options including inhibition of PLC, PDK-1, PKC, p38, DNA-PK, KSP, c-Met, TBK-1 and BTK for IC-mediated autoimmune diseases.

Neutrophils are key effector cells in antibody-mediated autoimmune diseases, contributing to inflammation via the release of reactive oxygen species (ROS). Extracellular signal-regulated kinase 5 (ERK5), a member of the MAPK family, is expressed in neutrophils but its role in autoimmune disease pathogenesis remains unclear. We investigated the functional relevance of ERK5 in antibody-mediated autoimmune diseases by comparing neutrophil-dependent (epidermolysis bullosa acquisita, EBA; serum transfer arthritis, STA) and neutrophil-independent (immune thrombocytopenia, ITP) murine models using the small-molecule ERK5 inhibitor XMD8-92. In vitro , pharmacological ERK5 inhibition specifically reduced neutrophil ROS release and CD62L shedding without affecting adhesion, chemotaxis, or CD18 expression. No major effects on viability were observed. In vivo , ERK5 inhibition with XMD8-92 significantly ameliorated antibody transfer-induced EBA, supporting a critical role of neutrophil-derived ROS in disease pathogenesis. In STA and ITP, ERK5 inhibition did not affect clinical outcomes. Together, these findings highlight ERK5 as a regulator of neutrophil ROS release and a potential therapeutic target in ROS-driven autoimmune diseases such as EBA. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, EXC 2167, GRK 2633, CRC1526 State of Schleswig-Holstein

BackgroundAutoimmune pancreatitis (AIP) is a multifactorial disease caused by both genetic and environmental factors. Previous studies have implicated Bach2, a key regulator of adaptive immunity, in the pathogenesis of this disease. However, direct experimental evidence is lacking. Here, we used C57BL/6N mice with a targeted deletion of Bach2 (Bach2 knockout mice) to study their susceptibility to AIP under homeostatic conditions and in response to two AIP triggers: a high-fat diet (HFD) and polyinosinic:polycytidylic acid (poly I:C).MethodsIn this multicenter preclinical study, Bach2 wild type and knockout mice were maintained under homeostatic conditions, challenged with a HFD for 3 months, or treated with poly I:C for 6 weeks. The pancreata were examined histopathologically. Additionally, RNA sequencing and PamGene multiplex kinase activation measurements were performed. To assess the effects of the HFD, the livers were evaluated for the presence of fatty liver disease.ResultsConsistent with the results of previous studies, Bach2 knockout mice showed reduced growth and developed eosinophilic crystalline pneumonia, necessitating humane euthanasia at the age of 18 weeks. At 8 and 18 weeks of age, pancreatic infiltrates with lymphocytes typical of AIP were frequently detected in Bach2 knockout mice but not in wild type animals without additional manipulations. RNA sequencing analyses and kinase activity assays revealed the activation of processes linked to adaptive immunity in the pancreatic tissues of Bach2 knockout mice. Wild type mice treated with poly I:C showed lymphocytic infiltrates similar to those of untreated knockout mice, whereas HFD did not induce AIP. In Bach2 knockout mice, HFD and poly I:C did not further enhance the disease. As expected, HFD-fed wild type mice developed fatty liver disease. Strikingly, the livers of Bach2 knockout mice were almost free of fat and histological changes, such as hepatocyte ballooning and degeneration. The data obtained from the two project sites were highly consistent, indicating strong intersite reproducibility.ConclusionBach2-deficient C57BL/6N mice were prone to spontaneous AIP development. This could be due to disturbed immune homeostasis with dysregulated activation of adaptive immune system cells. The protective effect of Bach2 deficiency against the development of fatty liver disease warrants further investigation.

Cyclin-dependent kinases are involved in basic cellular processes like regulation of cell-cycle progression and transcription. However, recent data also indicate a specific role in terminally differentiated neutrophils by promoting reactive oxygen species (ROS) release, degranulation, neutrophil extracellular trap (NET) formation, or apoptosis. Thus, we investigated the effect of pharmacological CDK inhibition on IC-activated neutrophil functions. Inhibitors targeting CDK1, 2, 4/6, 7, 9, 11, and 12 showed effects on different neutrophil functions (surface activation marker expression, ROS release, adhesion, apoptosis) in vitro . To analyse the inhibitors in a more translational approach, we proceeded with the systemic application of the effective inhibitors in a murine antibody transfer-induced local EBA model. In this predominantly neutrophil-driven model, we observed a reduction of disease severity upon treatment with CDK7, 9, or 11 inhibitors. Inhibiting these CDKs with topical THZ2, MC180295, or OTS964 also improved the clinical phenotype. Therefore, the most efficient inhibitor, MC180295, was validated in two other IC-mediated models of autoimmune disease: Serum-transfer arthritis, which also, but not exclusively depends on neutrophils and ITP, which is considered neutrophil-independent. Here, an effect in STA was observed, but not in ITP. Hence, we show the therapeutic potential of CDK7, 11, and especially 9 inhibition in IC-driven neutrophil-mediated diseases such as RA and EBA. ### Competing Interest Statement The authors have declared no competing interest. Cluster of Excellence “Precision Medicine in Chronic Inflammation”, EXC 2167 Research Training Group “Defining and Targeting Autoimmune Pre-Disease”, GRK 2633 Collaborative Research Center “Pathomechanisms of Antibody-mediated Autoimmunity”, CRC 1526

Pemphigus is a group of autoimmune blistering diseases characterized by the presence of autoantibodies against desmogleins, which are critical components of desmosomes. These autoantibodies disrupt the adhesive function of desmosomes, leading to loss of cell-cell adhesion in the epidermis, which manifests clinically as blistering and erosions of the skin and mucous membranes. In this study, we explored the potential use of midostaurin, a multikinase inhibitor commonly employed in the treatment of FLT3-altered cancers, as a therapeutic option for pemphigus. The results demonstrated that midostaurin effectively rescued loss of adhesion and keratin retraction induced by both pemphigus vulgaris and pemphigus foliaceus IgG in cultured keratinocytes. In addition, midostaurin prevented pemphigus vulgaris IgG-mediated relocalization of desmoglein 3 within the cell membrane as well as loss of desmoglein 3 adhesion on single-molecule level as revealed by atomic force microscopy. In ex vivo human skin, midostaurin treatment successfully prevented pemphigus vulgaris IgG-induced blister formation. Ultrastructural analyses revealed that midostaurin restored the integrity of desmosomes. These findings indicate that midostaurin can counteract the pathogenic effects of pemphigus autoantibodies, suggesting its potential as a therapeutic agent for pemphigus.

Myelofibrosis (MF) in 50% of cases is driven by an activating JAK2 mutation, mostly V617F. Ruxolinitib is approved for the treatment of MF. Responses to ruxolitinib in MF are of limited duration. Unexpectedly, treatment of JAK2-V617F expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at activation loop Tyr1007/Tyr1008. The significance of ruxolitinib-induced JAK2 hyperphosphorylation is not well understood. We found that a ruxolitinib-resistant JAK2 variant (V617F + L983F) and a kinase dead mutant (JAK2-V617F + K882R) did not show paradoxical hyperphosphorylation after ruxolitinib treatment indicating that it is an intrinsic mechanism. Antibodies against pTyr1007/1008 failed to immunoprecipitate native JAK2-V617F in the presence of ruxolitinib, although JAK2-V617F was hyperphosphorylated at these sites, suggesting that in the presence of ruxolitinib the JAK2 activation loop is buried within the kinase domain. This stabilization of the activation loop conformation resulted in the protection of pTyr1007/1008 sites from phosphatases. Mutation of Arg975 and Lys999 to Ala reduced the phosphorylation at both Tyr1007/Tyr1008 residues, and notably, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation. Importantly, hyperphosphorylated JAK2 after ruxolitinib dissociation displayed excess rebound activation of STAT5 target gene PIM kinase. Our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolitinib. Subject categories: JAK2-V617F, Ruxolitinib, JAK2 hyperphosphorylation, Phosphatases action, PIM kinases

Pemphigus is an autoimmune blistering disorder caused by autoantibodies against desmogleins (Dsg) 1 and 3, proteins essential for cell adhesion. Diagnosis relies on detecting tissue-bound autoantibodies. These antibodies disrupt cell adhesion, leading to blistering in skin or mucous membranes. While all IgG subclasses (IgG1, IgG2, IgG3, IgG4) are involved, IgG4 largely drives the response due to its unique Fab-arm exchange, making it monovalent and bispecific, unlike other IgGs that enhance crosslinking. This study isolated IgG subclasses from two pemphigus patients, examining their effects on keratinocytes (NHEK) at 0, 5, and 30 minutes. Multiplex kinome analyses showed that all IgG subclasses increased p38 MAPK signaling, linked to Dsg3 internalization, and activated kinases like PKC, CaMK4, and IKK-epsilon. MAP3K8 and mTOR/FRAP activities also rose under both IgG4 and IgG1-3 treatments, but only IgG4 uniquely increased CDK4, 6, 9, and 11. Confocal microscopy confirmed both IgG4 and IgG1-3 caused Dsg3 internalization. However, IgG4-treated keratinocytes showed no clustering, confirming subclass separation. This study highlights IgG subclass-specific kinase activation in pemphigus, especially the role of p38 MAPK and CDKs in IgG4-mediated Dsg3 internalization. Further research should explore subclass-specific signaling to identify kinase-targeted therapies for pemphigus. Basic Autoimmunity (BA)

Neutrophils are key effector cells in antibody-mediated autoimmune diseases, contributing to inflammation via the release of reactive oxygen species (ROS). Extracellular signal-regulated kinase 5 (ERK5), a member of the MAPK family, is expressed in neutrophils but its role in autoimmune disease pathogenesis remains unclear. We investigated the functional relevance of ERK5 in antibody-mediated autoimmune diseases by comparing neutrophil-dependent (epidermolysis bullosa acquisita, EBA; serum transfer arthritis, STA) and neutrophil-independent (immune thrombocytopenia, ITP) murine models using the small-molecule ERK5 inhibitor XMD8-92. In vitro , pharmacological ERK5 inhibition specifically reduced neutrophil ROS release and CD62L shedding without affecting adhesion, chemotaxis, or CD18 expression. No major effects on viability were observed. In vivo , ERK5 inhibition with XMD8-92 significantly ameliorated antibody transfer-induced EBA, supporting a critical role of neutrophil-derived ROS in disease pathogenesis. In STA and ITP, ERK5 inhibition did not affect clinical outcomes. Together, these findings highlight ERK5 as a regulator of neutrophil ROS release and a potential therapeutic target in ROS-driven autoimmune diseases such as EBA.

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3, which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. In this study, we provide evidence that EGFR inhibition by erlotinib ameliorates pemphigus vulgaris IgG-induced acantholysis in intact human epidermis. Pemphigus vulgaris IgG caused phosphorylation of EGFR (Y845) and Rous sarcoma-related kinase in human epidermis. In line with this, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and Rous sarcoma-related kinase family kinase signaling in response to pemphigus vulgaris IgG but not to pemphigus foliaceus autoantibodies. Erlotinib inhibited pemphigus vulgaris IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes, as well as ultrastructural alterations of desmosome size, plaque symmetry, and keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single-molecule DSG3-binding frequency and strength and delayed DSG3 fluorescence recovery, supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy owing to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease, of which the incidence has increased in recent years. BP is characterized by circulating IgG and IgE autoantibodies against the hemidesmosomal proteins BP180 and BP230. Although autoantibodies trigger inflammatory cascades that lead to blister formation, effector cells and cell-mediated autoimmunity must also be considered as important factors in the pathogenesis of BP. The aim of this review is to outline the current knowledge on the role of eosinophils, basophils, and neutrophils in BP.

Class I phosphoinositide 3-kinases (PI3K) have been implemented in pathogenesis of experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disease caused by type VII collagen (COL7) autoantibodies. Mechanistically, inhibition of specific PI3K isoforms, namely PI3Kβ or PI3Kδ, impaired immune complex (IC)-induced neutrophil activation, a key prerequisite for EBA pathogenesis. Data unrelated to EBA showed that neutrophil activation is also modulated by PI3Kα and γ, but their impact on the EBA has, so far, remained elusive. To address this and to identify potential therapeutic targets, we evaluated the impact of a panel of PI3K isoform-selective inhibitors (PI3Ki) on neutrophil function in vitro, and in pre-clinical EBA mouse models. We document that distinctive, and EBA pathogenesis-related activation-induced neutrophil in vitro functions depend on distinctive PI3K isoforms. When mice were treated with the different PI3Ki, selective blockade of PI3Kα (alpelisib), PI3Kγ (AS-604850), or PI3Kβ (TGX-221) impaired clinical disease manifestation. When applied topically, only TGX-221 impaired induction of experimental EBA. Ultimately, multiplex kinase activity profiling in the presence of disease-modifying PI3Ki identified unique signatures of different PI3K isoform-selective inhibitors on the kinome of IC-activated human neutrophils. Collectively, we here identify topical PI3Kβ inhibition as a potential therapeutic target for the treatment of EBA.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

TO THE EDITOR Although the molecular controls that drive murine hair follicle (HF) initiation, morphogenesis, cycling, and wave pattern formation are becoming increasingly well defined (Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: 132-142Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar; Geyfman and Andersen, 2010Geyfman M. Andersen B. Clock genes, hair growth and aging.Aging. 2010; 2: 122-128Crossref PubMed Scopus (44) Google Scholar; Plikus et al., 2011Plikus M.V. Baker R.E. Chen C.C. et al.Self-organizing and stochastic behaviors during the regeneration of hair stem cells.Science. 2011; 332: 586-589Crossref PubMed Scopus (152) Google Scholar; Plikus, 2012Plikus M.V. New activators and inhibitors in the hair cycle clock: targeting stem cells’ state of competence.J Invest Dermatol. 2012; 132: 1321-1324Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Supplementary Reference S13 online), much less is known about the regulation of human HF cycling (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (960) Google Scholar; Supplementary Reference S1 online). Although several growth factors, cytokines, and peptides are recognized to prolong or inhibit active hair growth (anagen) in organ-cultured human scalp HFs and their transformation into the regression stage (catagen) in vitro (e.g., Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: 132-142Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar; Supplementary References S4, S6, and S14 online), the molecular determinants of the endogenous human “hair cycle clock” remain to be deciphered (Supplementary Reference S1 online). Download .pdf (.38 MB) Help with pdf files Supplementary Figures It is well established that both WNT/β-catenin and EDA-A1/EDAR/NF-κB signaling are critical signaling pathways that mutually interact to drive murine primary HF development (Fuchs, 2007Fuchs E. Scratching the surface of skin development.Nature. 2007; 445: 834-842Crossref PubMed Scopus (644) Google Scholar; Zhang et al., 2008Zhang Y. Andl T. Yang S.H. et al.Activation of beta-catenin signaling programs embryonic epidermis to hair follicle fate.Development. 2008; 135: 2161-2172Crossref PubMed Scopus (147) Google Scholar, Zhang et al., 2009Zhang Y. Tomann P. Andl T. et al.Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction.Dev Cell. 2009; 17: 49-61Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar; Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: 132-142Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar; Lefebvre et al., 2012Lefebvre S. Fliniaux I. Schneider P. et al.Identification of ectodysplasin target genes reveals the involvement of chemokines in hair development.J Invest Dermatol. 2012; 132: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Given that HF morphogenesis and anagen induction share multiple characteristics (morphology, signaling) (Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: 132-142Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar), we hypothesized that the transcription factor NF-κB also regulates adult human hair growth. The putative involvement of EDA-A1/EDAR/NF-κB signaling in human hair growth is supported by the severe hair growth abnormalities associated with defects in the EDA-A1/EDAR/NF-κB pathway that lead to hypohydrotic ectodermal dysplasia (Monreal et al., 1999Monreal A.W. Ferguson B.M. Headon D.J. et al.Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia.Nature. 1999; 22: 366-369Google Scholar; Supplementary Reference S19 online). Hypohydrotic ectodermal dysplasia patients display severely impaired hair growth, suggesting that murine primary guard hairs and human scalp hair may be analogous. Moreover, key hair growth regulators such as tumor necrosis factor-α and IL-1β signal via NF-κB (Ghosh and Hayden, 2012Ghosh S. Hayden M.S. Celebrating 25 years of NF-κB research.Immunol Rev. 2012; 246: 5-13Crossref PubMed Scopus (168) Google Scholar; Pasparakis, 2012Pasparakis M. Role of NF-κB in epithelial biology.Immunol Rev. 2012; 246: 346-358Crossref PubMed Scopus (123) Google Scholar) and selected human HF keratin genes are NF-κB responsive (Supplementary References S5 and S10 online). Finally, NF-κB signaling has also been implicated in the formation of HF-derived tumors such as cylindroma, trichoepithelioma, and spiradenoma (Supplementary References S11 and S22 online). A previous study has suggested that there may be NF-κB activity in human HFs (Supplementary Reference S21 online); however, signal specificity, exact localization, and functional significance of NF-κB activity in the human hair cycle all remain unknown. In the current pilot study, we analyzed the possible role for NF-κB in the human HF anagen–catagen transition, as this represents the clinically most relevant HF transformation (shortening of anagen and premature catagen induction underlies most hair loss disorders seen in clinical practice) (Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (960) Google Scholar; Schneider et al., 2009Schneider M.R. Schmidt-Ullrich R. Paus R. The hair follicle as a dynamic miniorgan.Curr Biol. 2009; 19: 132-142Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). First, intact human anagen HFs were examined for the presence of the active nuclear form of NF-κB p65 by using anti-phosho-p65 and anti-p65 antibodies (see Supplementary Material online and figures legends for details; Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3526) Google Scholar; Ghosh and Hayden, 2012Ghosh S. Hayden M.S. Celebrating 25 years of NF-κB research.Immunol Rev. 2012; 246: 5-13Crossref PubMed Scopus (168) Google Scholar). Specific nuclear phospho-p65 immunoreactivity (IR) was observed in the proximal hair bulb epithelium in female scalp skin, most prominently in hair matrix keratinocytes of anagen HFs (Figure 1a, c, and g and Supplementary Figure S2b, c, f, g online). In addition, some proliferating (Ki-67+) hair matrix keratinocytes were positive for phospho-p65 (Figure 1h). Interestingly, phospho-p65 IR was also observed in the bulge, the site of HF epithelial stem cells, albeit at a significantly lower level than in the anagen matrix (Figure 1b, e, and g). In catagen HFs and in the secondary hair germ epithelium of catagen HFs, phospho-p65 IR was clearly reduced compared with anagen HFs (Figure 1d–g). As in unstimulated cultured human dermal papilla fibroblasts in situ dermal paiplla fibroblasts in situ, human HF, human HF mesenchyme did not show substantial phospho-p65 IR (Figure 1a–f and h) (Hill et al., 2012Hill R.P. Haycock J.W. Jahoda C.A. Human hair follicle dermal cells and skin fibroblasts show differential activation of NF-κB in response to pro-inflammatory challenge.Exp Dermatol. 2012; 21: 158-160Crossref PubMed Scopus (8) Google Scholar). In line with this, mRNA transcripts of the canonical NF-κB inhibitor IκBα were detected predominantly in the HF matrix, in the lower inner root sheath, and to a lesser degree in the precortical region, but not in the HF mesenchyme (Figure 1i and j). In the absence of classical canonical NF-κB signals such as TNF family members, IκBα is required to sequester NF-κB subunits p50 and p65 in the cytoplasm (Supplementary Figure S1 online; Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3526) Google Scholar; Hinz et al., 2012Hinz M. Arslan S.Ç. Scheidereit C. It takes two to tango: IκBs, the multifunctional partners of NF-κB.Immunol Rev. 2012; 246: 59-76Crossref PubMed Scopus (120) Google Scholar). Note that IκBα is a bona fide target gene of canonical NF-κB signaling, which results in a negative feedback loop (Supplementary Reference S8 Online). These results reveal that human anagen scalp HFs show prominent NF-κB activity in the rapidly proliferating hair matrix epithelium, indicating that NF-κB activity may be required for anagen maintenance. Furthermore, the expression pattern of previously identified NF-κB target genes, WNT10a, WNT10b, DKK4, SHH (Zhang et al., 2009Zhang Y. Tomann P. Andl T. et al.Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction.Dev Cell. 2009; 17: 49-61Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar; Supplementary Reference S17 online), and several key genes implicated in the control of the anagen–catagen transformation (Supplementary Figure S2a online) were also analyzed. Indeed, WNT10a, WNT10b, DKK4, and SHH mRNA expression colocalized with IκBα expression in the hair matrix and lower inner root sheath (Figure 1k–r). Moreover, human EDAR mRNA was observed at the same sites of NF-κB activity (Figure 1s and t). This suggests that in human HFs, similarly to murine HFs (Zhang et al., 2009Zhang Y. Tomann P. Andl T. et al.Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction.Dev Cell. 2009; 17: 49-61Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), NF-κB may be regulated by EDA-A1/EDAR signaling and may be required to fine-tune canonical WNT signaling in the main proliferation compartment. As small interfering RNA (siRNA) silencing of IKK (IκB kinase) complex component IKKβ was not successful, we modulated NF-κB activity by adding two classical pharmacological NF-κB inhibitors, BAY 11-7082 (BAY) and NEMO (NBD) (Supplementary Reference S12 online) to organ-cultured human scalp HFs and analyzed their influence on the anagen–catagen transition. Of great importance is that the BAY concentration tested was inversely related to the phospho-p65 IR, showing successful inhibition of NF-κB activity. This inhibition promoted apoptosis-driven catagen development in human anagen HFs, when compared with untreated HFs (Figure 2a and b). This observation was supported by a significant increase in apoptotic hair matrix keratinocytes and reduced hair shaft production in BAY-treated HFs (Figure 2c and d). (Although the other tested NF-κB inhibitor, NEMO, also slightly increased the percentage of catagen HFs compared with vehicle controls, excessive toxicity of NEMO prevented the generation of biologically meaningful data (data not shown).) Here, we have analyzed NF-κB activity (respectively anti-phospho-p65 IR) present in human HFs at different stages of the hair cycle. Since investigating selective activation of NF-κB is not possible in human HFs with the currently available tools, it still remains unknown which signal activates NF-κB in mature human HFs. Taken together, the current pilot study suggests a functional importance of NF-κB in maintaining the anagen phase of human HFs. Therefore, targeting intrafollicular NF-κB activity deserves exploration as a potential new strategy to therapeutically promote or inhibit human hair growth in vivo. This study was supported by a grant from Deutsche Forschungsgemeinschaft (DFG) to RP and RS-U (Pa 345/13-1 and SCHM 855/4-1). We thank Nadine Dörwald, Claudia Kremling, and Inge Krahn for excellent technical assistance, and Stephan Tiede and Erika Lisztes for scientific advice and support. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

β1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While β1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, β1 integrin-mediated signaling in ePC biology was explored by β1 integrin siRNA silencing, specific β1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. β1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in β1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded β1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The β1 integrin-inhibitory antibody reduced β1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the β1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require β1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to β1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

Cutaneous nestin+ cells are of substantial interest in regenerative medicine. However, the location of nestin+ cells in situ remains controversial. We therefore sought to determine their location in female human scalp skin, using stringently controlled immunohistochemical techniques, Western blot analysis, and in situ hybridization and complementing those techniques with relative and quantitative reverse transcriptase-PCR of enzymatically digested or laser-capture microdissected human hair follicle (HF) compartments. We show here that the immunoreactivity (IR) patterns obtained with anti-nestin antibodies are highly dependent on the tissue-fixation and immunohistochemical methods used. NESTIN mRNA could not be detected within HF-associated epithelial cells in situ or in RNA extracts of the microdissected HF epithelium. Instead, NESTIN transcripts were found only in intramesenchymal skin compartments. Individual cells showing both, specific nestin IR and NESTIN mRNA were detectable in the connective-tissue sheaths of human HFs, sebaceous and sweat glands. Moreover, stimulation of organ-cultured human scalp skin with the adipokine leptin increased the number of nestin+ cells in these intramesenchymal skin locations, whereas no specific nestin IR could be induced by leptin within the HF epithelium, including the bulge. Therefore, nestin expression at the gene and protein levels in human scalp skin is restricted to the periappendage mesenchyme and can be stimulated by leptin.