{"paper_id":"ec2e6f81-34a7-479f-95b4-03849e92eb67","body_text":"Caspase-activation powers anti-Desmoglein 3-induced acantholysis in human epidermis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Caspase-activation powers anti-Desmoglein 3-induced acantholysis in human epidermis Amir Yazdi, Morna Schmidt, Maria Feoktistova, Diana Panayotova-Dimitrova, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7382893/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Cell Death Discovery → Version 1 posted 9 You are reading this latest preprint version Abstract Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease caused by circulating autoantibodies against desmoglein (Dsg) 1 and 3. Whether acantholysis in PV results exclusively from antibody binding to Dsg3, or involves additional factors remains controversial. Given that Fas-Ligand (FasL), an activator of apoptotic caspase-8, is increased in the serum and the skin of patients with PV, we investigated the role of caspases in anti-Dsg3-mediated acantholysis using both ex vivo and in vitro models. Our results demonstrate that anti-Dsg3 antibodies induce acantholysis ex vivo in the absence of caspase activation, primarily through the redistribution of Dsg3 to intracellular compartments. FasL-induced caspase activation leads to a synergistic amplification of anti-Dsg3-mediated loss of cell adhesion by promoting Dsg3 cleavage. This dual mechanism provides new insights into the disease heterogeneity of PV and may also explain the rapid response of PV to high-dose glucocorticosteroids despite the persistence of antibodies. Biological sciences/Cell biology/Cell death/Apoptosis Health sciences/Diseases/Skin diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pemphigus vulgaris (PV) is a chronic, life-threatening blistering disease caused by IgG autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 ( 1 , 2 ), essential for cell-cell adhesion in skin and mucous membranes ( 3 , 4 ). Antibody binding induces acantholysis, leading to blisters and erosions. While direct inhibition of Dsg3 interaction is a major mechanism, antibody-induced acantholysis also involves intracellular signaling pathways (p38 MAPK, protein kinase C, or tyrosine kinase Src) ( 5 – 10 ) and apoptosis as evidenced by chromatin condensation, nuclear fragmentation and TUNEL positivity in keratinocytes ( 11 , 12 ), ( 7 , 11 – 14 ), along with an altered distribution of Dsg1 or Dsg3 on the cell surface ( 12 , 15 ). Despite relatively homogenous distribution of autoantibody binding in the epidermis of the entire body, blisters develop only at distinct sites and often heal despite persistent antibodies, suggesting that active signaling contributes significantly to acantholysis ( 16 , 17 ). One such pathway involves Fas-Fas ligand (FasL) interactions: FasL levels are elevated in the serum of untreated patients ( 11 , 18 ), and FasL-deficient mice display reduced acantholysis ( 19 ). FasL activates the extrinsic apoptotic caspase cascade via Fas, leading to the formation of Death Inducing Signaling Complex (DISC) at the intracellular death domain of Fas, activation of caspase-8 and caspase-3 what ultimately results in apoptosis. Herein, we demonstrate that anti-Dsg3 antibody binding triggers the acantholysis mainly by depleting Dsg3 from the keratinocyte surface. FasL-mediated activation of caspase-8 amplifies this effect by cleaving Dsg3. This study provides compelling evidence that cell death is an event that occurs late after the Dsg3-depletion from the keratinocyte surface, therefore not being related to desmosomal detachment. Results Caspase-activation leads to increased acantholysis and desmosomal vesicle formation To investigate apoptotic features related to PV, we examined human lesional PV skin. Immunohistology revealed strong active caspase-8 staining (exemplary, Fig. 1 A, black arrows), but no TUNEL-positive keratinocytes (Fig. 1 A), unlike toxic epidermal necrolysis (TEN), where both were present (Supplemental Fig. 1A). Thus, caspase activation in PV may occur without apoptosis. We next examined: ( 1 ) how pathogenic anti-Dsg3 antibodies affect the anchoring of Dsg3 on keratinocyte membranes, and ( 2 ) whether FasL-induced caspase activation amplifies these effects. Healthy skin explants were injected with anti-Dsg3 antibodies (AK23), control IgG and/ or FasL into the upper dermis (Fig. 1 B). AK23 is known to induce acantholysis in mice ( 20 ) and loss of cohesion in vitro in keratinocyte monolayers following overnight stimulation ( 21 ). AK23 caused discrete suprabasal acantholysis at 24 h, progressing with time (Fig. 1 C, green arrows). To explore the role of caspase activation, we utilized a sublethal concentration of FasL (Supplemental Fig. 1B). Co-injection of anti-Dsg3 antibodies and FasL led to discrete suprabasal acantholysis within 24 h (Fig. 1 C, black arrows) and severe disruption of basal keratinocytes with blister formation and acantholytic cells by 48 h (Fig. 1 C, black stars), whereas FasL alone did not affect epidermal cohesion (Supplemental Fig. 1C). Caspase-8 was active either in developing lesion sites or blister roofs, mirroring patient findings (Fig. 1 D, red staining; Supplemental Fig. 1D). In summary, these findings indicate that anti-Dsg3 induced acantholysis is enhanced by activation of apoptotic caspases, independent of cell death. Ultrastructural transmission electron microscopy (TEM) analysis of HaCaT monolayers or ex vivo skin models revealed that anti-Dsg3 antibody treatment increased intracellular vesicles both ex vivo (Fig. 1 E) and in vitro (Fig. 1 F and G), an effect significantly amplified by FasL (Fig. 1 F and G), indicating synergy. Conversely, anti-Dsg3 treatment reduced desmosome length in vitro (Supplemental Fig. 2A and B) and ex vivo (Supplemental Fig. 2), but FasL had no additive effect (Supplemental Fig. 2A and B). Both treatments caused only a minor (Supplemental Fig. 2D-F). In summary, anti-Dsg3 monoclonal antibody induces acantholysis, enhanced and accelerated by co-stimulation with FasL, in the presence of caspase-8 activation, but with no signs of cell death. Ultrastructurally, the binding of Dsg3-antibodies induces vesicle formation, which is significantly enhanced by the addition of FasL. This points to distinct mechanisms, controlling the fate of Dsg3 in PV. An altered cellular distribution of endogenous Dsg3 contributes to antibody mediated acantholysis The depletion of desmosomal Dsg3 is a key driver in the loss of cell adhesion ( 22 , 23 ). In our ex vivo skin model, we investigated the effect of anti-Dsg3 antibody on the distribution of Dsg3. Antibody binding was confirmed by IgG staining (Fig. 2 A, green staining). In control treated samples, endogenous Dsg3 was evenly distributed across the cell surface, with occasional cytoplasmic localization (Fig. 2 A red staining, upper row). Treatment with AK23 resulted in a decrease of Dsg3 in the suprabasal layer with concomitant enrichment in the apical region of certain suprabasal keratinocytes (Fig. 2 A, red staining, lower row). Additionally, granular structures were formed in the cytoplasm (Fig. 2 A, inset, white arrows), with most granules containing both Dsg3 and AK23, suggesting their co-translocation. Within 30 min of anti-Dsg3 antibody treatment of epidermal monolayers, endogenous Dsg3 levels in the total cell lysate decreased (Fig. 2 B). This reduction corresponded with a time-dependent decline in Dsg3 levels in the cytosolic fraction (Triton-soluble fraction, TSF), alongside the AK23 heavy chain (Fig. 2 C). However, we observed that both Dsg3 and AK23 accumulated in the membranous/ vesicles fraction (Triton-insoluble fraction, TIF) in a time-dependent manner (Fig. 2 C). These data strongly correspond to our histological observation demonstrating the formation of AK23 and Dsg3 positive granules upon anti-Dsg3 treatment (Fig. 2 A). Importantly, caspase inhibition by pan-caspase inhibitor Z-VAD-fmk (zVAD) did not prevent the reduction of Dsg3 in the lysate (Supplemental Fig. 3A), indicating that this process is caspase-independent. Dsg3 is known to degrade via endosomal and lysosomal pathways ( 24 , 25 ). However, neither the proteasome inhibitor bortezomib (Supplemental Fig. 3B), nor chloroquine (a lysosome inhibitor) (Supplemental Fig. 3C) alone or combined (Supplemental Fig. 3D), prevented the depletion of Dsg3. Altogether, these findings demonstrate that anti-Dsg3 might support the redistribution of endogenous Dsg3 to the membranous fraction or other cell compartments (e.g. lipid rafts ( 26 )). FasL synergistically enhances anti Dsg3-induced loss of cell adhesion via caspase-8 While Dsg3 depletion via sequestration in lipid-containing cellular compartments contributes to anti-Dsg3 induced acantholysis, the protein can also be altered by proteolytic cleavage. It has previously been shown that induction of apoptosis causes Dsg3 cleavage, as observed with staurosporine treatment ( 27 ). We found that FasL stimulation induced time-dependent cleavage into ~ 100 kDa and ~ 75 kDa fragments (Fig. 3 A). In a dispase-based keratinocyte dissociation assay ( 28 ), anti-Dsg3 treatment alone reduced keratinocyte cohesion within 4h (Fig. 3 B), unaffected by the pan-caspase inhibitors zVAD or QVD-OPH (QVD) (Supplemental Fig. 4A) and without inducing caspase-8 activation (Supplemental Fig. 4B) or sensitizing cells to apoptosis (Supplemental Fig. 4C). As FasL is released by lesional keratinocytes in PV ( 19 ), is present in the serum of PV-patients ( 11 ) and enhances antibody-induced acantholysis ex vivo , we investigated its effect on desmosomal detachment. FasL at sublethal levels (Supplemental Fig. 4D) alone had no impact on keratinocyte cohesion, but markedly increased antibody-mediated loss of cell cohesion in a caspase-dependent manner (Fig. 3 B). To specifically assess the role of caspase-8, we utilized HaCaT cells overexpressing the short isoform of cFLIP (cFLIPs OE), which blocks caspase-8 activation (Supplemental Fig. 4E) ( 29 ). Overexpression of cFLIP completely abolished the synergistic effect of AK23 and FasL on the loss of cell cohesion, confirming the critical role of caspase-8 activation in this mechanism (Fig. 3 C). Notably, the cleavage of Dsg3 occurs in both the cytosolic (TSF) and membranous (TIF) fractions and is completely prevented by zVAD (Fig. 3 D). This indicates that two different mechanisms reduce full-length Dsg3 in the cytosolic fraction: anti-Dsg3 treatment promotes protein translocation, while FasL decreases full-length Dsg3 through cleavage. This unravels a synergistic effect of anti-Dsg3 monoclonal antibodies and FasL by promoting loss of cell-adhesion (Fig. 3 E). In conclusion, FasL enhances anti-Dsg3-induced loss of cell cohesion by caspase-8-induced cleavage of Dsg3 in the presence of anti-Dsg3 antibody. Discussion Mechanisms leading to antibody-related acantholysis reamin debated, with surface Dsg3 levels critical fort skin integrity. Reduced Dsg3 levels ( 15 , 22 , 30 , 31 ) are attributed to protein degradation ( 32 ), cleavage ( 33 , 34 ) or translocation (24, 35 ). We show that anti-Dsg3 antibody rapidly internalizes complexes consisting of endogenous Dsg3 and anti-Dsg3 antibodies, depleting Dsg3 from the cell surface and causing loss of keratinocytes cohesion without initial degradation. Internalized Dsg3/antibody complexes may later co-localize with lysosomal markers ( 25 , 32 ). Consistent with these observations, we detected a degradation in suprabasal skin layers coincing with late-stage acantholysis, in agreement with Jolly et al. ( 36 ). However, this late Dsg3 degradation may be modulated by additional signaling pathways activated by other PV-IgG or serum components. We have identified intracellular Dsg3 cleavage as one possible mechanism resulting in increased acantholysis. Previous studies have shown that Dsg3 can translocate into lipid rafts ( 37 ) or intracellular vesicles ( 38 ). TEM revealed that anti-Dsg3 treatment increases intracellular vesicles near desmosomes and shortens desmosoems in vitro and ex vivo , consistent with findings from Egu et al. ( 39 ). Moreover, reduced cell cohesion and increased acantholysis were evident, consistent with other reports ( 22 , 40 ). Simultaneously, we detected an increase of both Dsg3 protein and anti-Dsg3 antibodies in fractions enriched for endosomes and lipid rafts, indicating co-translocation of Dsg3 and anti-Dsg3 antibody. Lipid rafts play a key role in PV pathogenesis, as desmosome disassembly and endocytosis triggered by PV-IgG depend on these membrane microdomains ( 26 , 35 , 41 ). Of note, the active Fas-DISC is known to localize in lipid rafts ( 42 ), and elevated FasL have been detected in PV sera, originating from keratinocyte- or cytotoxic T-cell driven sources ( 11 , 43 , 44 ). Blocking soluble FasL has been shown to reduce blister formation in an ex vivo PV-model ( 45 ). Fas-signaling synergizes with anti-Dsg3 antibodies, promoting endocytosis ( 46 ). Here we demonstrate combined treatment with FasL and anti-Dsg3 antibodies produce a pronounced synergistic, markedly promoting endocytosis and blister formation ex vivo . The relevance of apoptosis on PV-related blister formation remains controversial: while some studies link PV-IgG to caspase-8/-3 activation, resulting in apoptosis ( 12 , 31 , 47 ), others claim acantholysis occurring independently of cell death, considering it a secondary, irrelevant side effect ( 15 , 48 ) or a parallel event ( 49 ). In our study, we demonstrated that binding of anti-Dsg3 antibody alone is insufficient to activate caspases or induce apoptosis. However, the activation of the FasL-mediated apoptotic pathway significantly enhances the effect of anti-Dsg3 antibody both in vitro and ex vivo . Notably, we observed active caspase-8 in lesional skin without cell death, which contrasts with reports of TUNEL-positive keratinocytes ( 11 , 13 , 47 , 50 , 51 ), and suggests mechanistic or temporal heterogeneity. In our patient samples, TUNEL positive cells were absent in blisters, but we detected caspase-8 positive cells. These data, together with the results from our ex vivo model demonstrate that caspase-8 activation precedes or coincides with blister formation. The presence of TUNEL positive cells may reflect cell death occurring at a later stage. Consistently, our ex vivo experiments demonstrated presence of caspase-8 activity only when the skin was treated with both anti-Dsg3 antibody and FasL, while no cell death was detected under the same conditions in vitro . Overall, these results reveal that additional signaling pathways, such as FasL signaling, act synergistically with anti-Dsg3 antibodies to promote acantholysis. In conclusion, anti-Dsg3 antibodies drive acantholysis primarily through caspase-independent Dsg3 internalization. Caspase activation (e.g. caspase-8 via FasL) acts as an enhancer through Dsg3 cleavage prior to cell death (Fig. 4 ). This dual mechanism provides a novel insight in the manifestation of PV at distinct body sites and disease heterogeneity in PV. Materials and methods Antibodies and reagents The following antibodies were used for Western Blot (WB): β-actin (A2103) and LC3 (L8918) (both Sigma-Aldrich, St. Louis, USA), Dsg3 (sc-23912, Santa Cruz, California, USA); NIK (#4994, Cell Signaling, Danvers, USA), cFLIP (NF6) and caspase-8 (C-15) were kindly gifted by P.H. Krammer; caspase-3 (cpp32/19, BD Bioscience, San Jose, USA) and caspase 3 active (cleaved caspase-3, AF 835, R&D, Minneapolis, USA); HRP-conjugated goat anti-rabbit (4030-05) and HRP-conjugated goat anti-mouse IgG1 (1070-05), IgG2a antibody (1080-05) (all Southern Biotechnology Associates, Birmingham, USA). The following antibodies were used for IF or IHC: Polyclonal rabbit anti-human IgG (F0315, Agilent Technologies, Santa Clara, California, USA), Mouse IgG1 Alexa Fluor® 488-conjugated Antibody (IC002G, R&D, Minneapolis, USA), Cleaved caspase-8 (#9496, Cell Signaling, Danvers, USA), anti-Desmoglein 3 antibody (ab183743, Abcam, Cambridge, United Kingdom). Secondary antibodies were purchased by Thermo Fisher Scientific Inc., Waltham, USA: # A-11010 for staining Dsg3 and # A-11001 for staining AK23. DAPI was used for nuclear staining (1198406, AppliChem GmbH, Darmstadt, Hessen). The following stimuli and reagents were used: the IgG human isotype control (# 02-7102, Thermo Fisher Scientific Inc., Waltham, USA), Dulbecco´s Phosphate Buffered Saline (DPBS, P04-36500, PAN-Biotech GmbH, Aidenbach, Germany), pan-caspase inhibitors Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) (4026865, Bachem GmbH, Bubendorf, Switzerland) and Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh) (SML0063, Sigma-Aldrich St. Louis, USA,). For expression of Fc-FasL we used constructs published previously ( 52 ); kindly provided by P. Schneider, Epalinges, Switzerland. One unit of Fc-FasL was determined as a 1:1000 dilution of the stock Fc-FasL supernatant, and one unit/ml of Fc-FasL supernatant was sufficient to kill 50 percent (LD50) of HaCaT cells, seeded at 50% confluence and stimulated overnight. Chloroquine diphosphate salt (C6628, Sigma Aldrich, St. Louis, Missouri, United States). Bortezomib (BTZ) (5.04314, Sigma Aldrich, St. Louis, Missouri, United States). Poly(I:C) (#tlrl-pic, InvivoGen, San Diego, USA). For expression of His-FLAG-TRAIL (HF-TRAIL) we used constructs published previously ( 53 ). IgG-production and -purification Hybridoma cells for the mouse monoclonal anti-Dsg3 AK23 antibody were kindly provided by M. Amagai. The cells were cultured in suspension using RPMI 1640 medium at 37°C in a humidified atmosphere at 5% CO 2 . The culture medium was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate (all from Gibco/Life Technologies, Carlsbad, CA, USA), and 55 µM β-mercaptoethanol (Sigma-Aldrich, Munich, Germany). For antibody production, the cells were grown in a medium with 60% of ISF-I hybridoma medium (Sigma-Aldrich) and 40% culture medium in 1 L volume in EZ flasks (KDBIO, Berstett, France), and grown for 30 days before harvesting by centrifugation ( 20 ). Anti-Dsg3 antibody (AK23) purification was performed following Beckert et al. ( 54 ). Generation of cell lines cFLIP s OE HaCaT cells were generated using a pCFG5-IEGZ retroviral vector, as described previously ( 55 ). The expression of cFLIP s was confirmed by WB. Cell culture The spontaneously immortalized HaCaT human keratinocyte cell line (kindly provided by P. Boukamp, formerly DZFK Heidelberg), HaCaT cFLIP s OE and control cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (P04-04515, PAN-Biotech GmbH, Aidenbach) supplemented with 10% Fetal Bovine Serum (FBS) standard (equivalent to Fetal Calf Serum (FCS)) (P30-3306, PAN-Biotech GmbH, Aidenbach, Germany) at 37°C in 5% CO 2 atmosphere. Conditions for cell stimulation HaCaT cells were grown to confluent monolayers. The cells were used up to passage 48. The following stimulation conditions were used: pre-stimulation with zVAD-fmk (10 µM) or QVD (10 µM) for one hour. Pre-stimulation with BTZ (1µM) and/ or chloroquine (100 µM) was conducted for five hours. 1 Unit of Fc-FasL was determined as a 1:1000 dilution of the stock Fc- FasL supernatant, and 1 Unit/ml of Fc- FasL supernatant was sufficient to kill 50% (LD50) of A375 melanoma cells, as previously described ( 55 ). For Dispase-based keratinocyte dissociation assay (DDA) and electron microscopy, parental or transduced HaCaT cells were pre-stimulated for 1 h with IgG or AK23 (20 µg/mL for parental HaCaT and 30 µg/ml for transduced HaCaT cells), followed by FasL (0.8 U/ml) stimulation for 3 h. For WB, IgG or AK23 (30 µg/mL) and FasL (0.8U/ml). Poly(I:C) (10 µg/ml) and HF-TRAIL (500 ng/ml) were added for indicated time points. Cells were pre-incubated with IgG or AK23 for 4 h, followed by FasL stimulation for 3h. For transmission electron microscopy, HaCaT cells were grown confluent on 8-well chamber slides and were pre-incubated with IgG or AK23 (20 µg/mL) for 1 h, followed by FasL (0.8 U/mL) stimulation for further 3 h. Stimulation was performed using DMEM supplemented with chelated FCS (1.6 mM final concentration of Ca 2+ ) at 37°C in 5% CO 2 atmosphere. Chelated FCS was produced and purified as described previously ( 28 ). Propidium iodide staining A 100% confluent layer of HaCaT cells on 96-well cell culture plates was stimulated for indicated time points as described above. The cells were trypsinized, washed with DPBS and stained with PI (10 µg/mL) for 15 min. BD Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, New Jersey, U.S.) was used for FACS analysis. Dispase-based keratinocyte dissociation assay The method was performed under previously established conditions ( 28 ). Resulting fragments were quantified using ImageJ software (1708195; Bio-Rad Laboratories Inc., Hercules, CA, USA) ( 56 ) or alternatively counted manually. Western Blot analysis Cells were washed with DPBS and lysed as described previously ( 57 ). Sonication was performed to further analyze the Triton-insoluble fraction in the cell pellet ( 58 ). Five µg of the protein were separated on a 4–12% gradient gel (NP0329BOX; Thermo Fisher Scientific Inc., Waltham, MA, USA) with SDS-PAGE technique after heat denaturation of the proteins (95°C, 5 min) and then transferred to membranes (IB24001X3; Thermo Fisher Scientific Inc., Waltham, MA, USA). Membranes were then blocked in TPBS containing 5% milk powder (70166-500G; Sigma Aldrich, St. Louis, Missouri, United States) for 2 h at room temperature (RT) and washed with TPBS. Blots were then incubated with primary antibodies overnight at 4°C, followed by incubation with an appropriated secondary antibody for 1 h at RT. Protein bands were visualized with an Immobilon Forte Western HRP substrate (WBLUF0500, Merck, Darmstadt, Germany). Electron microscopic analysis Stimulated cells were fixed in 3% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (Roth, Karlsruhe, Germany). Samples were post-fixed in 1% OsO 4 (E19100, Science Services, Munich Germany) in 25 mM sucrose buffer (1.07651.1000, Merck, Darmstadt, Germany), dehydrated by ascending ethanol series, and embedded in Epon. Ultrathin sections were cut in horizontal plane. Contrast was enhanced by staining with 0.5% uranyl acetate (E22499-05, Science Services, Munich, Germany) and 1% Sato`s lead citrate. Samples were examined using a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) operating at an acceleration voltage of 100 kV. Analysis was performed by ImageJ. In each independent experiment (n = 2), at least 50 images per condition were captured, each containing at least one desmosome. Desmosomes were measured using ImageJ with the following criteria: desmosomal length was determined as the longest continuous visible segment (50k magnification); interdesmosomal space was measured three times at different points along the desmosome (100k magnification); and the number of vesicles was counted as the total vesicles per microscopic field (50k magnification) containing at least one desmosome. The mean of the measurements from each experiment was used for statistical analysis. Ex vivo skin models The skin was obtained from safety margins after surgeries at the Department of Dermatology, University Hospital RWTH Aachen. Excess skin from excised safety margins was used on the same day. Before further use, the tissue was washed three times for 15 min each in DPBS. The subcutis was dissected, 8 mm pieces of skin were taken by punch biopsy and placed in 6-well culture plates with cell culture inserts (353091, Corning (Corning Inc.), Somerville, USA). 2 ml of medium/well (equal amounts of DMEM + 1% antibiotics/antimycotics (15240096, Thermo Fisher Scientific Inc., Waltham, USA) + 10% FBS and KBM-2 Keratinocyte Growth media (CC31-03, Lonza, Basel Switzerland)) was added. Human IgG (40 µg); AK23 (40µg) and/ or FasL (1:10) were diluted in PBS in a volume of 50 µl/ punch and injected by a needle (0.4 mm diameter) into the upper dermis. The culture medium was changed daily. The samples were frozen in liquid nitrogen. Histology and immunohistochemistry Cryosections or formalin-fixed and paraffin-embedded tissues were used for histopathology (H&E staining) and immunohistochemistry. Image processing was applied identically to all samples and controls. Immunofluorescence Cryosections were used for immunofluorescence. Image processing was applied identically to all samples and controls. TUNEL TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining was performed according to the manufacturer’s instructions (TUNEL-kit C10617, Thermo Fisher Scientific Inc., Waltham, USA). Statistics All data are expressed as the mean ± SEM (standard error of the mean). A two-tailed Student′s t-test for two groups was used to assess the significance of differences. ns = p > 0.05; * p = < 0.05; ** p = < 0.01; *** p = < 0.001. Declarations Ethics approval The study was approved by the Independent Ethics Committee RWTH Aachen University (Aachen, Germany) according to the Declaration of Helsinki Principles (EK 318/21). The written, informed consent was given prior to participation. Data Availability Statement Original data are available from the corresponding author upon a reasonable request. Acknowledgments We thank Petra Boukamp for providing the HaCaT cell line; Manuela Busch, Katharina Fietkau, Manuela Jansen, Linda Lopopolo and Yvonne Marquardt for their technical support and Hiltrud Königs-Werner for preparing the samples for electron microscopy. We also thank Peter H. Krammer for the caspase-8- and cFLIP- antibodies; Pascal Schneider for providing the Fc-FasL construct- The graphical abstract and Fig. 1B were created with BioRender.com. Author Contribution The study was concepted by MFS, MAF and ASY. The funding was acquired by JW, RT, JMB, ASY. MFS and MAF established the methodology, collected the data, performed the formal analysis, validated and visualized the data. The investigation was supported by DPD, EMB, MR and JW. RT provided further resources. ASY supervised the project. The first draft of the manuscript was written by MFS and MAF and all authors commented on previous versions of the manuscript. MR assisted with editing the manuscript and contributed the study design. All authors reviewed and approved the final version of the manuscript. Funding This project was supported by the German Research Foundation (DFG) to the Unit FOR 2497 PEGASUS (TP 5 to JW, TI 291/10-2 to RT, BA1803/9-2 to JMB and YA-182/4-2 to ASY). MFS was funded by scholarships from the state of North Rhine-Westphalia (FF-med) and the University RWTH Aachen (Kurzzeitstipendium), an intramural fellowship of the RWTH Aachen. DPD is supported by the DFG (DI 2440/3-1); PB is supported by the DFG (Project IDs 322900939, 432698239 & 445703531), European Research Council (ERC Consolidator Grant No 101001791), and the Federal Ministry of Education and Research (BMBF, STOP-FSGS-01GM2202C). Conflict of Interest The authors have declared that no conflict of interest exists. References Amagai M, Klaus-Kovtun V, Stanley JR. Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell. 1991;67(5):869-77. Hashimoto T, Amagai M, Garrod DR, Nishikawa T. Immunofluorescence and immunoblot studies on the reactivity of pemphigus vulgaris and pemphigus foliaceus sera with desmoglein 3 and desmoglein 1. Epithelial Cell Biol. 1995;4(2):63-9. Amagai M. Autoantibodies against cell adhesion molecules in pemphigus. J Dermatol. 1994;21(11):833-7. Schmidt E, Kasperkiewicz M, Joly P. Pemphigus. Lancet. 2019;394(10201):882-94. Osada K, Seishima M, Kitajima Y. Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes. J Invest Dermatol. 1997;108(4):482-7. Berkowitz P, Hu P, Liu Z, Diaz LA, Enghild JJ, Chua MP, et al. Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem. 2005;280(25):23778-84. Frusic-Zlotkin M, Raichenberg D, Wang X, David M, Michel B, Milner Y. Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis--possible involvement of the EGF receptor. Autoimmunity. 2006;39(7):563-75. Cirillo N, AlShwaimi E, McCullough M, Prime SS. Pemphigus vulgaris autoimmune globulin induces Src-dependent tyrosine-phosphorylation of plakophilin 3 and its detachment from desmoglein 3. Autoimmunity. 2014;47(2):134-40. Egu DT, Schmitt T, Waschke J. Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin. Front Immunol. 2022;13:884067. Schmitt T, Waschke J. Autoantibody-Specific Signalling in Pemphigus. Front Med (Lausanne). 2021;8:701809. Puviani M, Marconi A, Cozzani E, Pincelli C. Fas ligand in pemphigus sera induces keratinocyte apoptosis through the activation of caspase-8. J Invest Dermatol. 2003;120(1):164-7. Wang X, Bregegere F, Frusic-Zlotkin M, Feinmesser M, Michel B, Milner Y. Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins. Apoptosis. 2004;9(2):131-43. Pelacho B, Natal C, Espana A, Sanchez-Carpintero I, Iraburu MJ, Lopez-Zabalza MJ. Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes. FEBS Lett. 2004;566(1-3):6-10. Gniadecki R, Jemec GB, Thomsen BM, Hansen M. Relationship between keratinocyte adhesion and death: anoikis in acantholytic diseases. Arch Dermatol Res. 1998;290(10):528-32. Schmidt E, Gutberlet J, Siegmund D, Berg D, Wajant H, Waschke J. Apoptosis is not required for acantholysis in pemphigus vulgaris. Am J Physiol Cell Physiol. 2009;296(1):C162-72. Bystryn JC, Rudolph JL. Pemphigus. Lancet. 2005;366(9479):61-73. Grando SA. Pemphigus autoimmunity: hypotheses and realities. Autoimmunity. 2012;45(1):7-35. Moravvej H, Yousefi M, Farrokhi B, Mosaffa N. Soluble Fas in pemphigus vulgaris. Arch Iran Med. 2011;14(3):200-1. Lotti R, Shu E, Petrachi T, Marconi A, Palazzo E, Quadri M, et al. Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus. Front Immunol. 2018;9:370. Tsunoda K, Ota T, Aoki M, Yamada T, Nagai T, Nakagawa T, et al. Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3. J Immunol. 2003;170(4):2170-8. Ishii K, Harada R, Matsuo I, Shirakata Y, Hashimoto K, Amagai M. In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris. J Invest Dermatol. 2005;124(5):939-46. Aoyama Y, Kitajima Y. Pemphigus vulgaris-IgG causes a rapid depletion of desmoglein 3 (Dsg3) from the Triton X-100 soluble pools, leading to the formation of Dsg3-depleted desmosomes in a human squamous carcinoma cell line, DJM-1 cells. J Invest Dermatol. 1999;112(1):67-71. Sato M, Aoyama Y, Kitajima Y. Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy. Lab Invest. 2000;80(10):1583-92. Mao X, Choi EJ, Payne AS. Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies. J Invest Dermatol. 2009;129(4):908-18. Nguyen B, Dusek RL, Beaudry VG, Marinkovich MP, Attardi LD. Loss of the desmosomal protein perp enhances the phenotypic effects of pemphigus vulgaris autoantibodies. J Invest Dermatol. 2009;129(7):1710-8. Lajoie P, Nabi IR. Regulation of raft-dependent endocytosis. J Cell Mol Med. 2007;11(4):644-53. Weiske J, Schoneberg T, Schroder W, Hatzfeld M, Tauber R, Huber O. The fate of desmosomal proteins in apoptotic cells. J Biol Chem. 2001;276(44):41175-81. Schmidt MF, Feoktistova M, Panayotova-Dimitrova D, Eichkorn RA, Yazdi AS. Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris. Vaccines (Basel). 2022;10(2). Kavuri SM, Geserick P, Berg D, Dimitrova DP, Feoktistova M, Siegmund D, et al. Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex. J Biol Chem. 2011;286(19):16631-46. Schmitt T, Hudemann C, Moztarzadeh S, Hertl M, Tikkanen R, Waschke J. Dsg3 epitope-specific signalling in pemphigus. Front Immunol. 2023;14:1163066. Peng X, Wang S, Wu K, Cook C, Li L, Wang Z, et al. Effect of opioid receptor antagonist on mitigating tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-induced apoptolysis in pemphigus pathogenesis. J Autoimmun. 2024;149:103307. Calkins CC, Setzer SV, Jennings JM, Summers S, Tsunoda K, Amagai M, et al. Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies. J Biol Chem. 2006;281(11):7623-34. Cirillo N, Campisi G, Gombos F, Perillo L, Femiano F, Lanza A. Cleavage of desmoglein 3 can explain its depletion from keratinocytes in pemphigus vulgaris. Exp Dermatol. 2008;17(10):858-63. Cirillo N, Dell' Ermo A, Gombos F, Lanza A. The specific proteolysis hypothesis of pemphigus: does the song remain the same? Med Hypotheses. 2008;70(2):333-7. Delva E, Jennings JM, Calkins CC, Kottke MD, Faundez V, Kowalczyk AP. Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism. J Biol Chem. 2008;283(26):18303-13. Jolly PS, Berkowitz P, Bektas M, Lee HE, Chua M, Diaz LA, et al. p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis. J Biol Chem. 2010;285(12):8936-41. Levental I, Levental KR, Heberle FA. Lipid Rafts: Controversies Resolved, Mysteries Remain. Trends Cell Biol. 2020;30(5):341-53. Moldovan NI, Heltianu C, Simionescu N, Simionescu M. Ultrastructural evidence of differential solubility in Triton X-100 of endothelial vesicles and plasma membrane. Exp Cell Res. 1995;219(1):309-13. Egu DT, Kugelmann D, Waschke J. Role of PKC and ERK Signaling in Epidermal Blistering and Desmosome Regulation in Pemphigus. Front Immunol. 2019;10:2883. Yamamoto Y, Aoyama Y, Shu E, Tsunoda K, Amagai M, Kitajima Y. Anti-desmoglein 3 (Dsg3) monoclonal antibodies deplete desmosomes of Dsg3 and differ in their Dsg3-depleting activities related to pathogenicity. J Biol Chem. 2007;282(24):17866-76. Stahley SN, Saito M, Faundez V, Koval M, Mattheyses AL, Kowalczyk AP. Desmosome assembly and disassembly are membrane raft-dependent. PLoS One. 2014;9(1):e87809. George KS, Wu S. Lipid raft: A floating island of death or survival. Toxicol Appl Pharmacol. 2012;259(3):311-9. Pacheco-Tovar MG, Avalos-Diaz E, Vega-Memije E, Bollain-y-Goytia JJ, Lopez-Robles E, Hojyo-Tomoka MT, et al. The final destiny of acantholytic cells in pemphigus is Fas mediated. J Eur Acad Dermatol Venereol. 2009;23(6):697-701. Arnold R, Seifert M, Asadullah K, Volk HD. Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines. J Immunol. 1999;162(12):7140-7. Lotti R, Hundt JE, Ludwig RJ, Bennett B, Amato A, Marconi A, et al. Blocking soluble Fas Ligand ameliorates pemphigus: PC111 efficacy in ex-vivo human pemphigus models. Front Immunol. 2023;14:1193032. Degli Esposti M, Matarrese P, Tinari A, Longo A, Recalchi S, Khosravi-Far R, et al. Changes in membrane lipids drive increased endocytosis following Fas ligation. Apoptosis. 2017;22(5):681-95. Frusic-Zlotkin M, Pergamentz R, Michel B, David M, Mimouni D, Bregegere F, et al. The interaction of pemphigus autoimmunoglobulins with epidermal cells: activation of the fas apoptotic pathway and the use of caspase activity for pathogenicity tests of pemphigus patients. Ann N Y Acad Sci. 2005;1050:371-9. Lee HE, Berkowitz P, Jolly PS, Diaz LA, Chua MP, Rubenstein DS. Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis. J Biol Chem. 2009;284(18):12524-32. Grando SA, Bystryn JC, Chernyavsky AI, Frusic-Zlotkin M, Gniadecki R, Lotti R, et al. Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis. Exp Dermatol. 2009;18(9):764-70. Arredondo J, Chernyavsky AI, Karaouni A, Grando SA. Novel mechanisms of target cell death and survival and of therapeutic action of IVIg in Pemphigus. Am J Pathol. 2005;167(6):1531-44. Sanath AK, Devy AS, Aithal S, Kumar GS, Prasad BG, Pradeep PS. Caspase cascade pathways of apoptosis in oral pemphigus: An immunohistochemical study. J Oral Maxillofac Pathol. 2018;22(1):48-53. Bossen C, Ingold K, Tardivel A, Bodmer JL, Gaide O, Hertig S, et al. Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J Biol Chem. 2006;281(20):13964-71. Diessenbacher P, Hupe M, Sprick MR, Kerstan A, Geserick P, Haas TL, et al. NF-kappaB inhibition reveals differential mechanisms of TNF versus TRAIL-induced apoptosis upstream or at the level of caspase-8 activation independent of cIAP2. J Invest Dermatol. 2008;128(5):1134-47. Beckert B, Panico F, Pollmann R, Eming R, Banning A, Tikkanen R. Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris. Int J Mol Sci. 2019;20(13). Geserick P, Hupe M, Moulin M, Wong WW, Feoktistova M, Kellert B, et al. Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment. J Cell Biol. 2009;187(7):1037-54. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5. Feoktistova M, Makarov R, Yazdi AS, Panayotova-Dimitrova D. RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation. Int J Mol Sci. 2021;22(22). Wirths O. Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords. Bio Protoc. 2017;7(15):e2422. Additional Declarations There is no conflict of interest Supplementary Files SFig1.jpg Supplemental Figure 1 SFig2.jpg Supplemental Figure 2 SFig3.jpg Supplemental Figure 3 SFig4.jpg Supplemental Figure 4 SupplementaryoriginalWesternblots.pdf Supplemental Material - original Western blot data Graphicalabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 29 Sep, 2025 Review # 2 received at journal 24 Sep, 2025 Review # 1 received at journal 11 Sep, 2025 Reviewer # 2 agreed at journal 10 Sep, 2025 Reviewer # 1 agreed at journal 09 Sep, 2025 Reviewers invited by journal 08 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 Editor assigned by journal 31 Aug, 2025 First submitted to journal 31 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7382893\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":512156159,\"identity\":\"d0c0bca9-7c79-455b-8e8d-0e4bf01dd049\",\"order_by\":0,\"name\":\"Amir Yazdi\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYLACHgMbBgkgzQzmHSBOSxrJWhgOk6CFf0by4w9vCs4nzmw//PBzQcUdBr7jDfi1SNxIMzCcY3A7cTZPmrH0jDPPGCTPELLmRoJBMg9QyzyGBDNm3rbDDAY3EvDrkL+R/uEwj8G5xHn8z78x8/4Darn/AL8Wgxs5hs08BgcSZ0vkAG1pANlCwF2GZ94UM84xSDaeOeNNsTTPscM8kmcIOEzuePrmD2/+2MnOOJ++8TNPzWE5vuMHCFgjgGYmDwH1QMBPyMxRMApGwSgYBQANoUktqEMRewAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"University Hospital RWTH Aachen\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Amir\",\"middleName\":\"\",\"lastName\":\"Yazdi\",\"suffix\":\"\"},{\"id\":512156160,\"identity\":\"56740889-94d4-4f28-aae4-b825237a695e\",\"order_by\":1,\"name\":\"Morna Schmidt\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital RWTH Aachen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Morna\",\"middleName\":\"\",\"lastName\":\"Schmidt\",\"suffix\":\"\"},{\"id\":512156161,\"identity\":\"3bc8122c-424d-45ff-aca6-d10d47130f88\",\"order_by\":2,\"name\":\"Maria Feoktistova\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Maria\",\"middleName\":\"\",\"lastName\":\"Feoktistova\",\"suffix\":\"\"},{\"id\":512156162,\"identity\":\"bf179ef1-6652-4319-9258-d51aecb7c0e8\",\"order_by\":3,\"name\":\"Diana Panayotova-Dimitrova\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital RWTH Aachen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Diana\",\"middleName\":\"\",\"lastName\":\"Panayotova-Dimitrova\",\"suffix\":\"\"},{\"id\":512156163,\"identity\":\"2a68805b-ed33-43b7-986b-0a59daf7460c\",\"order_by\":4,\"name\":\"Eva Buhl\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0003-0627-9228\",\"institution\":\"Universitätsklinikum Aachen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Eva\",\"middleName\":\"\",\"lastName\":\"Buhl\",\"suffix\":\"\"},{\"id\":512156164,\"identity\":\"34a8ac79-dba2-453d-bcae-6f8e4a4c5efa\",\"order_by\":5,\"name\":\"Peter Boor\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-9921-4284\",\"institution\":\"RWTH Aachen University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Peter\",\"middleName\":\"\",\"lastName\":\"Boor\",\"suffix\":\"\"},{\"id\":512156165,\"identity\":\"d8ab72e0-8c7a-4c10-95e2-594c8a51c591\",\"order_by\":6,\"name\":\"Jens Waschke\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Munchen Uni\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jens\",\"middleName\":\"\",\"lastName\":\"Waschke\",\"suffix\":\"\"},{\"id\":512156166,\"identity\":\"4c012047-5902-4fcd-b568-a49f03697058\",\"order_by\":7,\"name\":\"Ritva Tikkanen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ritva\",\"middleName\":\"\",\"lastName\":\"Tikkanen\",\"suffix\":\"\"},{\"id\":512156167,\"identity\":\"dfe9171f-e043-4258-9be4-c36374dcb92b\",\"order_by\":8,\"name\":\"Jens Malte Baron\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital RWTH Aachen\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jens\",\"middleName\":\"Malte\",\"lastName\":\"Baron\",\"suffix\":\"\"},{\"id\":512156168,\"identity\":\"5afeaba8-33e6-4a1a-8304-60e4c540c8ac\",\"order_by\":9,\"name\":\"Martin Röcken\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Martin\",\"middleName\":\"\",\"lastName\":\"Röcken\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-08-15 16:30:45\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7382893/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7382893/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41420-026-02963-w\",\"type\":\"published\",\"date\":\"2026-02-19T05:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":91505586,\"identity\":\"61cb5448-91ca-47a1-b1e3-97fc8d9c929a\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:18:11\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1053146,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCaspase-activation enhances anti-Dsg3-induced acantholysis through desmosomal vesicle formation. A\\u003c/strong\\u003e Representative images of patient skin sections stained with H\\u0026amp;E, TUNEL, and IHC for cleaved caspase-8. Black arrows indicate keratinocytes positive for cleaved caspase-8. The dotted line marks the basal membrane. Scale bar: 100 µm. \\u003cstrong\\u003eB\\u003c/strong\\u003e Schematic illustration of the \\u003cem\\u003eex vivo\\u003c/em\\u003e skin model. Stimuli are injected intradermally into 8 mm punch biopsies of healthy skin, which are then maintained in organ culture medium prior to analysis. \\u003cstrong\\u003eC \\u003c/strong\\u003eRepresentative H\\u0026amp;E staining of \\u003cem\\u003eex vivo\\u003c/em\\u003eskin punches following stimulation for indicated time points. Arrows highlight areas of early acantholysis (green: 24 h; black: 48 h), black stars indicate acantholytic keratinocytes. Scale bar: 50µm. \\u003cstrong\\u003eC\\u003c/strong\\u003e Representative staining of stimulated skin punches for indicated time points: cleaved caspase-8 (red) and AK23 (green), DAPI (blue) used to visualize the nucleus. The dotted line marks the basal membrane. Scale bar: 50 µm. \\u003cstrong\\u003eC\\u003c/strong\\u003e Quantification of vesicles adjacent to desmosomes in transmission electron microscopy (TEM) images of stimulated \\u003cem\\u003eex vivo\\u003c/em\\u003e skin models. \\u003cstrong\\u003eF \\u003c/strong\\u003eRepresentative TEM pictures of HaCaT monolayers preincubated with AK23 or IgG for 1 h, then stimulated with FasL for further 3 h. White arrows indicate intracytoplasmic vesicles in proximity to desmosomes. Scale bar: 200 nm. \\u003cstrong\\u003eG \\u003c/strong\\u003eQuantification of vesicles adjacent to desmosomes in TEM images.Each dot represents an individual measurement; the horizontal line indicates the mean \\u003cstrong\\u003eE and G\\u003c/strong\\u003e. *p \\u0026lt; 0.05, **p \\u0026lt; 0.01; comparisons without asterisks are not statistically significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/27bcbc8ec181e5c700a7c874.jpg\"},{\"id\":91506923,\"identity\":\"ba2981fe-6fae-46a0-990d-aa378baf000f\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:26:11\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":382953,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAK23 disrupts the endogenous distribution of Dsg3 in a time-dependent manner.\\u003c/strong\\u003e \\u003cstrong\\u003eA \\u003c/strong\\u003eImmunofluorescence staining of skin punches stimulated with AK23 or control IgG for 48 h, showing IgG (green) and Dsg3 (red) localization. Arrows indicate intracellular granules. The dotted line marks the basal membrane. Scale bar: 50 µm. \\u003cstrong\\u003eB-C\\u003c/strong\\u003e WB analysis of Dsg3 in HaCaT keratinocyte monolayers under different treatment conditions: \\u003cstrong\\u003eB\\u003c/strong\\u003eTime-course of Dsg3 expression following AK23 treatment; \\u003cstrong\\u003eC\\u003c/strong\\u003e Comparison of Dsg3 presence in Triton soluble (TSF) and Triton insoluble fraction (TIF) of HaCaT monolayers treated with AK23 for the indicated time points. Caspase-8 and calveolin confirm the purity of the respective TIF and TSF. Equal protein amounts were analyzed in all WB experiments.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/44194f669536c5cfa1c24977.jpg\"},{\"id\":91505599,\"identity\":\"e1ded3af-75d3-4bb3-bccf-604c45ded417\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:18:11\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":570970,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFasL synergistically enhances AK23-induced loss of cell adhesion via a caspase-8-dependent mechanism. A\\u003c/strong\\u003e WB analysis of HaCaT monolayers stimulated with FasL. \\u003cstrong\\u003eB\\u003c/strong\\u003e HaCaT monolayers were\\u003cstrong\\u003e \\u003c/strong\\u003epre-stimulated with zVAD for 1 h, then stimulated with AK23 or control IgG for further 1 h, followed by FasL for 3h, before conducting a dispase-based keratinocytes dissociation assay (DDA) (n=6). \\u003cstrong\\u003eC\\u003c/strong\\u003eHaCaT cFLIPs OE or control monolayers were pre-stimulated with AK23 or IgG, followed by FasL stimulation, and analyzed by DDA (n=4). Error bars (\\u003cstrong\\u003eA/B\\u003c/strong\\u003e) represent the SEM, ns: p\\u0026gt;.05; ∗p\\u0026lt;.05, ∗∗p\\u0026lt;.01, ∗∗∗p \\u0026lt;.001). \\u003cstrong\\u003eD-E\\u003c/strong\\u003e WB analysis of HaCaT monolayers pre-stimulated with zVAD 1 h, followed by FasL incubation for 3 h C or pre-stimulated with AK23 or IgG for 1 h, followed by FasL stimulation for 3 h C. Equal protein amounts were analyzed in all WB experiments. fr: fragment; h.c.: heavy chain; TIF: triton insoluble fraction; TSF: Triton soluble fraction.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/ef6ed5853ffddaa8568ca453.jpg\"},{\"id\":91509882,\"identity\":\"3a180275-1d1c-48a4-bd8d-9e30368b89a0\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:42:11\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":218047,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe role of caspase-dependent signaling in anti-Desmoglein 3-induced acantholysis.\\u003c/strong\\u003e \\u003cstrong\\u003eA\\u003c/strong\\u003eAnti-Dsg3 antibodies (such as AK23) binding to Dsg3 within the desmosomes triggers the translocation of Dsg3 to lipid rafts and its internalization, ultimately leading to acantholysis. \\u003cstrong\\u003eB\\u003c/strong\\u003eFasL interacts with the Fas receptor, initiating Death-Inducing Signaling Complex (DISC) formation and caspase activation. Active caspases cleave Dsg3, further enhancing antibody-induced acantholysis, independently of cell death. Dsg3, Desmoglein 3; FasL, Fas ligand.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/28bc901662b7e96284cf4700.jpg\"},{\"id\":103637648,\"identity\":\"86dacd19-1e3e-43c6-8ad2-4bf1103fd912\",\"added_by\":\"auto\",\"created_at\":\"2026-02-28 08:11:05\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3187317,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/0ad2ef8e-2b2a-4d98-a1d1-a6bf019bc2e5.pdf\"},{\"id\":91505587,\"identity\":\"ddd239f2-c242-45e0-a115-a15e30b5bd3c\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:18:11\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":379910,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplemental Figure 1\",\"description\":\"\",\"filename\":\"SFig1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/7ba4349a9441cb3e51a882a3.jpg\"},{\"id\":91506926,\"identity\":\"92d2b17a-d9af-4515-a934-5cc347ed67f1\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:26:11\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":686746,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSupplemental Figure 2\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SFig2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/2b9297d122eada97a3484dac.jpg\"},{\"id\":91507450,\"identity\":\"a064f33e-64df-495d-9f52-4e144226085f\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:34:11\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":276473,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSupplemental Figure 3\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SFig3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/04a37f062d8899550522903b.jpg\"},{\"id\":91507451,\"identity\":\"0a485d1f-3adb-4897-b805-5cb15311d950\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:34:11\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":384346,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSupplemental Figure 4\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SFig4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/6ec66be022435d69a1e13524.jpg\"},{\"id\":91505595,\"identity\":\"219e1278-0939-4b1b-96f0-9bae99245270\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:18:11\",\"extension\":\"pdf\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":648606,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSupplemental Material - original Western blot data\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SupplementaryoriginalWesternblots.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/910082a215e5a2e40abe739d.pdf\"},{\"id\":91506930,\"identity\":\"4a1339f1-d383-4e9b-b83f-18a2d3d8c881\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 08:26:11\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":165335,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Graphicalabstract.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7382893/v1/ce7098dbfa5c7b981e3e775f.jpg\"}],\"financialInterests\":\"There is no conflict of interest\",\"formattedTitle\":\"Caspase-activation powers anti-Desmoglein 3-induced acantholysis in human epidermis\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePemphigus vulgaris (PV) is a chronic, life-threatening blistering disease caused by IgG autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e), essential for cell-cell adhesion in skin and mucous membranes (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). Antibody binding induces acantholysis, leading to blisters and erosions. While direct inhibition of Dsg3 interaction is a major mechanism, antibody-induced acantholysis also involves intracellular signaling pathways (p38 MAPK, protein kinase C, or tyrosine kinase Src) (\\u003cspan additionalcitationids=\\\"CR6 CR7 CR8 CR9\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e) and apoptosis as evidenced by chromatin condensation, nuclear fragmentation and TUNEL positivity in keratinocytes (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e), (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR12 CR13\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e), along with an altered distribution of Dsg1 or Dsg3 on the cell surface (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eDespite relatively homogenous distribution of autoantibody binding in the epidermis of the entire body, blisters develop only at distinct sites and often heal despite persistent antibodies, suggesting that active signaling contributes significantly to acantholysis (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). One such pathway involves Fas-Fas ligand (FasL) interactions: FasL levels are elevated in the serum of untreated patients (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e), and FasL-deficient mice display reduced acantholysis (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). FasL activates the extrinsic apoptotic caspase cascade via Fas, leading to the formation of Death Inducing Signaling Complex (DISC) at the intracellular death domain of Fas, activation of caspase-8 and caspase-3 what ultimately results in apoptosis.\\u003c/p\\u003e\\u003cp\\u003eHerein, we demonstrate that anti-Dsg3 antibody binding triggers the acantholysis mainly by depleting Dsg3 from the keratinocyte surface. FasL-mediated activation of caspase-8 amplifies this effect by cleaving Dsg3. This study provides compelling evidence that cell death is an event that occurs late after the Dsg3-depletion from the keratinocyte surface, therefore not being related to desmosomal detachment.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCaspase-activation leads to increased acantholysis and desmosomal vesicle formation\\u003c/h2\\u003e\\u003cp\\u003eTo investigate apoptotic features related to PV, we examined human lesional PV skin. Immunohistology revealed strong active caspase-8 staining (exemplary, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, black arrows), but no TUNEL-positive keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA), unlike toxic epidermal necrolysis (TEN), where both were present (Supplemental Fig.\\u0026nbsp;1A). Thus, caspase activation in PV may occur without apoptosis. We next examined: (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) how pathogenic anti-Dsg3 antibodies affect the anchoring of Dsg3 on keratinocyte membranes, and (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) whether FasL-induced caspase activation amplifies these effects. Healthy skin explants were injected with anti-Dsg3 antibodies (AK23), control IgG and/ or FasL into the upper dermis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). AK23 is known to induce acantholysis in mice (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e) and loss of cohesion \\u003cem\\u003ein vitro\\u003c/em\\u003e in keratinocyte monolayers following overnight stimulation (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). AK23 caused discrete suprabasal acantholysis at 24 h, progressing with time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, green arrows). To explore the role of caspase activation, we utilized a sublethal concentration of FasL (Supplemental Fig.\\u0026nbsp;1B). Co-injection of anti-Dsg3 antibodies and FasL led to discrete suprabasal acantholysis within 24 h (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, black arrows) and severe disruption of basal keratinocytes with blister formation and acantholytic cells by 48 h (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, black stars), whereas FasL alone did not affect epidermal cohesion (Supplemental Fig.\\u0026nbsp;1C). Caspase-8 was active either in developing lesion sites or blister roofs, mirroring patient findings (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD, red staining; Supplemental Fig.\\u0026nbsp;1D). In summary, these findings indicate that anti-Dsg3 induced acantholysis is enhanced by activation of apoptotic caspases, independent of cell death.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eUltrastructural transmission electron microscopy (TEM) analysis of HaCaT monolayers or \\u003cem\\u003eex vivo\\u003c/em\\u003e skin models revealed that anti-Dsg3 antibody treatment increased intracellular vesicles both \\u003cem\\u003eex vivo\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE) and \\u003cem\\u003ein vitro\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF and G), an effect significantly amplified by FasL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF and G), indicating synergy. Conversely, anti-Dsg3 treatment reduced desmosome length \\u003cem\\u003ein vitro\\u003c/em\\u003e (Supplemental Fig.\\u0026nbsp;2A and B) and \\u003cem\\u003eex vivo\\u003c/em\\u003e (Supplemental Fig.\\u0026nbsp;2), but FasL had no additive effect (Supplemental Fig.\\u0026nbsp;2A and B). Both treatments caused only a minor (Supplemental Fig.\\u0026nbsp;2D-F).\\u003c/p\\u003e\\u003cp\\u003eIn summary, anti-Dsg3 monoclonal antibody induces acantholysis, enhanced and accelerated by co-stimulation with FasL, in the presence of caspase-8 activation, but with no signs of cell death. Ultrastructurally, the binding of Dsg3-antibodies induces vesicle formation, which is significantly enhanced by the addition of FasL. This points to distinct mechanisms, controlling the fate of Dsg3 in PV.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eAn altered cellular distribution of endogenous Dsg3 contributes to antibody mediated acantholysis\\u003c/h3\\u003e\\n\\u003cp\\u003eThe depletion of desmosomal Dsg3 is a key driver in the loss of cell adhesion (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). In our \\u003cem\\u003eex vivo\\u003c/em\\u003e skin model, we investigated the effect of anti-Dsg3 antibody on the distribution of Dsg3. Antibody binding was confirmed by IgG staining (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, green staining). In control treated samples, endogenous Dsg3 was evenly distributed across the cell surface, with occasional cytoplasmic localization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA red staining, upper row). Treatment with AK23 resulted in a decrease of Dsg3 in the suprabasal layer with concomitant enrichment in the apical region of certain suprabasal keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, red staining, lower row). Additionally, granular structures were formed in the cytoplasm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, inset, white arrows), with most granules containing both Dsg3 and AK23, suggesting their co-translocation. Within 30 min of anti-Dsg3 antibody treatment of epidermal monolayers, endogenous Dsg3 levels in the total cell lysate decreased (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). This reduction corresponded with a time-dependent decline in Dsg3 levels in the cytosolic fraction (Triton-soluble fraction, TSF), alongside the AK23 heavy chain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). However, we observed that both Dsg3 and AK23 accumulated in the membranous/ vesicles fraction (Triton-insoluble fraction, TIF) in a time-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). These data strongly correspond to our histological observation demonstrating the formation of AK23 and Dsg3 positive granules upon anti-Dsg3 treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Importantly, caspase inhibition by pan-caspase inhibitor Z-VAD-fmk (zVAD) did not prevent the reduction of Dsg3 in the lysate (Supplemental Fig.\\u0026nbsp;3A), indicating that this process is caspase-independent. Dsg3 is known to degrade via endosomal and lysosomal pathways (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). However, neither the proteasome inhibitor bortezomib (Supplemental Fig.\\u0026nbsp;3B), nor chloroquine (a lysosome inhibitor) (Supplemental Fig.\\u0026nbsp;3C) alone or combined (Supplemental Fig.\\u0026nbsp;3D), prevented the depletion of Dsg3. Altogether, these findings demonstrate that anti-Dsg3 might support the redistribution of endogenous Dsg3 to the membranous fraction or other cell compartments (e.g. lipid rafts (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e)).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003eFasL synergistically enhances anti Dsg3-induced loss of cell adhesion via caspase-8\\u003c/h3\\u003e\\n\\u003cp\\u003eWhile Dsg3 depletion via sequestration in lipid-containing cellular compartments contributes to anti-Dsg3 induced acantholysis, the protein can also be altered by proteolytic cleavage. It has previously been shown that induction of apoptosis causes Dsg3 cleavage, as observed with staurosporine treatment (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). We found that FasL stimulation induced time-dependent cleavage into ~\\u0026thinsp;100 kDa and ~\\u0026thinsp;75 kDa fragments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). In a dispase-based keratinocyte dissociation assay (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e), anti-Dsg3 treatment alone reduced keratinocyte cohesion within 4h (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB), unaffected by the pan-caspase inhibitors zVAD or QVD-OPH (QVD) (Supplemental Fig.\\u0026nbsp;4A) and without inducing caspase-8 activation (Supplemental Fig.\\u0026nbsp;4B) or sensitizing cells to apoptosis (Supplemental Fig.\\u0026nbsp;4C). As FasL is released by lesional keratinocytes in PV (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e), is present in the serum of PV-patients (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e) and enhances antibody-induced acantholysis \\u003cem\\u003eex vivo\\u003c/em\\u003e, we investigated its effect on desmosomal detachment. FasL at sublethal levels (Supplemental Fig.\\u0026nbsp;4D) alone had no impact on keratinocyte cohesion, but markedly increased antibody-mediated loss of cell cohesion in a caspase-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). To specifically assess the role of caspase-8, we utilized HaCaT cells overexpressing the short isoform of cFLIP (cFLIPs OE), which blocks caspase-8 activation (Supplemental Fig.\\u0026nbsp;4E) (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e). Overexpression of cFLIP completely abolished the synergistic effect of AK23 and FasL on the loss of cell cohesion, confirming the critical role of caspase-8 activation in this mechanism (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Notably, the cleavage of Dsg3 occurs in both the cytosolic (TSF) and membranous (TIF) fractions and is completely prevented by zVAD (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). This indicates that two different mechanisms reduce full-length Dsg3 in the cytosolic fraction: anti-Dsg3 treatment promotes protein translocation, while FasL decreases full-length Dsg3 through cleavage. This unravels a synergistic effect of anti-Dsg3 monoclonal antibodies and FasL by promoting loss of cell-adhesion (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, FasL enhances anti-Dsg3-induced loss of cell cohesion by caspase-8-induced cleavage of Dsg3 in the presence of anti-Dsg3 antibody.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eMechanisms leading to antibody-related acantholysis reamin debated, with surface Dsg3 levels critical fort skin integrity. Reduced Dsg3 levels (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e) are attributed to protein degradation (\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e), cleavage (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e) or translocation (24, 35 ). We show that anti-Dsg3 antibody rapidly internalizes complexes consisting of endogenous Dsg3 and anti-Dsg3 antibodies, depleting Dsg3 from the cell surface and causing loss of keratinocytes cohesion without initial degradation. Internalized Dsg3/antibody complexes may later co-localize with lysosomal markers (\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). Consistent with these observations, we detected a degradation in suprabasal skin layers coincing with late-stage acantholysis, in agreement with Jolly et al. (\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e). However, this late Dsg3 degradation may be modulated by additional signaling pathways activated by other PV-IgG or serum components. We have identified intracellular Dsg3 cleavage as one possible mechanism resulting in increased acantholysis. Previous studies have shown that Dsg3 can translocate into lipid rafts (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e) or intracellular vesicles (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). TEM revealed that anti-Dsg3 treatment increases intracellular vesicles near desmosomes and shortens desmosoems \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003eex vivo\\u003c/em\\u003e, consistent with findings from Egu et al. (\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). Moreover, reduced cell cohesion and increased acantholysis were evident, consistent with other reports (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e). Simultaneously, we detected an increase of both Dsg3 protein and anti-Dsg3 antibodies in fractions enriched for endosomes and lipid rafts, indicating co-translocation of Dsg3 and anti-Dsg3 antibody. Lipid rafts play a key role in PV pathogenesis, as desmosome disassembly and endocytosis triggered by PV-IgG depend on these membrane microdomains (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). Of note, the active Fas-DISC is known to localize in lipid rafts (\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e), and elevated FasL have been detected in PV sera, originating from keratinocyte- or cytotoxic T-cell driven sources (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e). Blocking soluble FasL has been shown to reduce blister formation in an \\u003cem\\u003eex vivo\\u003c/em\\u003e PV-model (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e). Fas-signaling synergizes with anti-Dsg3 antibodies, promoting endocytosis (\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e). Here we demonstrate combined treatment with FasL and anti-Dsg3 antibodies produce a pronounced synergistic, markedly promoting endocytosis and blister formation \\u003cem\\u003eex vivo\\u003c/em\\u003e.\\u003c/p\\u003e\\u003cp\\u003eThe relevance of apoptosis on PV-related blister formation remains controversial: while some studies link PV-IgG to caspase-8/-3 activation, resulting in apoptosis (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e), others claim acantholysis occurring independently of cell death, considering it a secondary, irrelevant side effect (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e) or a parallel event (\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e). In our study, we demonstrated that binding of anti-Dsg3 antibody alone is insufficient to activate caspases or induce apoptosis. However, the activation of the FasL-mediated apoptotic pathway significantly enhances the effect of anti-Dsg3 antibody both \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003eex vivo\\u003c/em\\u003e. Notably, we observed active caspase-8 in lesional skin without cell death, which contrasts with reports of TUNEL-positive keratinocytes (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e), and suggests mechanistic or temporal heterogeneity. In our patient samples, TUNEL positive cells were absent in blisters, but we detected caspase-8 positive cells. These data, together with the results from our \\u003cem\\u003eex vivo\\u003c/em\\u003e model demonstrate that caspase-8 activation precedes or coincides with blister formation. The presence of TUNEL positive cells may reflect cell death occurring at a later stage. Consistently, our \\u003cem\\u003eex vivo\\u003c/em\\u003e experiments demonstrated presence of caspase-8 activity only when the skin was treated with both anti-Dsg3 antibody and FasL, while no cell death was detected under the same conditions \\u003cem\\u003ein vitro\\u003c/em\\u003e. Overall, these results reveal that additional signaling pathways, such as FasL signaling, act synergistically with anti-Dsg3 antibodies to promote acantholysis.\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, anti-Dsg3 antibodies drive acantholysis primarily through caspase-independent Dsg3 internalization. Caspase activation (e.g. caspase-8 via FasL) acts as an enhancer through Dsg3 cleavage prior to cell death (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). This dual mechanism provides a novel insight in the manifestation of PV at distinct body sites and disease heterogeneity in PV.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eAntibodies and reagents\\u003c/h2\\u003e\\u003cp\\u003eThe following antibodies were used for Western Blot (WB): β-actin (A2103) and LC3 (L8918) (both Sigma-Aldrich, St. Louis, USA), Dsg3 (sc-23912, Santa Cruz, California, USA); NIK (#4994, Cell Signaling, Danvers, USA), cFLIP (NF6) and caspase-8 (C-15) were kindly gifted by P.H. Krammer; caspase-3 (cpp32/19, BD Bioscience, San Jose, USA) and caspase 3 active (cleaved caspase-3, AF 835, R\\u0026amp;D, Minneapolis, USA); HRP-conjugated goat anti-rabbit (4030-05) and HRP-conjugated goat anti-mouse IgG1 (1070-05), IgG2a antibody (1080-05) (all Southern Biotechnology Associates, Birmingham, USA).\\u003c/p\\u003e\\u003cp\\u003eThe following antibodies were used for IF or IHC: Polyclonal rabbit anti-human IgG (F0315, Agilent Technologies, Santa Clara, California, USA), Mouse IgG1 Alexa Fluor\\u0026reg; 488-conjugated Antibody (IC002G, R\\u0026amp;D, Minneapolis, USA), Cleaved caspase-8 (#9496, Cell Signaling, Danvers, USA), anti-Desmoglein 3 antibody (ab183743, Abcam, Cambridge, United Kingdom). Secondary antibodies were purchased by Thermo Fisher Scientific Inc., Waltham, USA: # A-11010 for staining Dsg3 and # A-11001 for staining AK23. DAPI was used for nuclear staining (1198406, AppliChem GmbH, Darmstadt, Hessen).\\u003c/p\\u003e\\u003cp\\u003eThe following stimuli and reagents were used: the IgG human isotype control (# 02-7102, Thermo Fisher Scientific Inc., Waltham, USA), Dulbecco\\u0026acute;s Phosphate Buffered Saline (DPBS, P04-36500, PAN-Biotech GmbH, Aidenbach, Germany), pan-caspase inhibitors Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) (4026865, Bachem GmbH, Bubendorf, Switzerland) and Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh) (SML0063, Sigma-Aldrich St. Louis, USA,). For expression of Fc-FasL we used constructs published previously (\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e); kindly provided by P. Schneider, Epalinges, Switzerland. One unit of Fc-FasL was determined as a 1:1000 dilution of the stock Fc-FasL supernatant, and one unit/ml of Fc-FasL supernatant was sufficient to kill 50 percent (LD50) of HaCaT cells, seeded at 50% confluence and stimulated overnight. Chloroquine diphosphate salt (C6628, Sigma Aldrich, St. Louis, Missouri, United States). Bortezomib (BTZ) (5.04314, Sigma Aldrich, St. Louis, Missouri, United States). Poly(I:C) (#tlrl-pic, InvivoGen, San Diego, USA). For expression of His-FLAG-TRAIL (HF-TRAIL) we used constructs published previously (\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eIgG-production and -purification\\u003c/h3\\u003e\\n\\u003cp\\u003eHybridoma cells for the mouse monoclonal anti-Dsg3 AK23 antibody were kindly provided by M. Amagai. The cells were cultured in suspension using RPMI 1640 medium at 37\\u0026deg;C in a humidified atmosphere at 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. The culture medium was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate (all from Gibco/Life Technologies, Carlsbad, CA, USA), and 55 \\u0026micro;M β-mercaptoethanol (Sigma-Aldrich, Munich, Germany). For antibody production, the cells were grown in a medium with 60% of ISF-I hybridoma medium (Sigma-Aldrich) and 40% culture medium in 1 L volume in EZ flasks (KDBIO, Berstett, France), and grown for 30 days before harvesting by centrifugation (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). Anti-Dsg3 antibody (AK23) purification was performed following Beckert et al. (\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eGeneration of cell lines\\u003c/h3\\u003e\\n\\u003cp\\u003ecFLIP\\u003csub\\u003es\\u003c/sub\\u003e OE HaCaT cells were generated using a pCFG5-IEGZ retroviral vector, as described previously (\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e). The expression of cFLIP\\u003csub\\u003es\\u003c/sub\\u003e was confirmed by WB.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCell culture\\u003c/h2\\u003e\\u003cp\\u003eThe spontaneously immortalized HaCaT human keratinocyte cell line (kindly provided by P. Boukamp, formerly DZFK Heidelberg), HaCaT cFLIP\\u003csub\\u003es\\u003c/sub\\u003e OE and control cell lines were cultured in Dulbecco\\u0026rsquo;s Modified Eagle Medium (DMEM) (P04-04515, PAN-Biotech GmbH, Aidenbach) supplemented with 10% Fetal Bovine Serum (FBS) standard (equivalent to Fetal Calf Serum (FCS)) (P30-3306, PAN-Biotech GmbH, Aidenbach, Germany) at 37\\u0026deg;C in 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eConditions for cell stimulation\\u003c/h2\\u003e\\u003cp\\u003eHaCaT cells were grown to confluent monolayers. The cells were used up to passage 48.\\u003c/p\\u003e\\u003cp\\u003eThe following stimulation conditions were used: pre-stimulation with zVAD-fmk (10 \\u0026micro;M) or QVD (10 \\u0026micro;M) for one hour. Pre-stimulation with BTZ (1\\u0026micro;M) and/ or chloroquine (100 \\u0026micro;M) was conducted for five hours. 1 Unit of Fc-FasL was determined as a 1:1000 dilution of the stock Fc- FasL supernatant, and 1 Unit/ml of Fc- FasL supernatant was sufficient to kill 50% (LD50) of A375 melanoma cells, as previously described (\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eFor Dispase-based keratinocyte dissociation assay (DDA) and electron microscopy, parental or transduced HaCaT cells were pre-stimulated for 1 h with IgG or AK23 (20 \\u0026micro;g/mL for parental HaCaT and 30 \\u0026micro;g/ml for transduced HaCaT cells), followed by FasL (0.8 U/ml) stimulation for 3 h.\\u003c/p\\u003e\\u003cp\\u003eFor WB, IgG or AK23 (30 \\u0026micro;g/mL) and FasL (0.8U/ml). Poly(I:C) (10 \\u0026micro;g/ml) and HF-TRAIL (500 ng/ml) were added for indicated time points. Cells were pre-incubated with IgG or AK23 for 4 h, followed by FasL stimulation for 3h.\\u003c/p\\u003e\\u003cp\\u003eFor transmission electron microscopy, HaCaT cells were grown confluent on 8-well chamber slides and were pre-incubated with IgG or AK23 (20 \\u0026micro;g/mL) for 1 h, followed by FasL (0.8 U/mL) stimulation for further 3 h.\\u003c/p\\u003e\\u003cp\\u003eStimulation was performed using DMEM supplemented with chelated FCS (1.6 mM final concentration of Ca\\u003csup\\u003e2+\\u003c/sup\\u003e) at 37\\u0026deg;C in 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. Chelated FCS was produced and purified as described previously (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePropidium iodide staining\\u003c/h2\\u003e\\u003cp\\u003eA 100% confluent layer of HaCaT cells on 96-well cell culture plates was stimulated for indicated time points as described above. The cells were trypsinized, washed with DPBS and stained with PI (10 \\u0026micro;g/mL) for 15 min. BD Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, New Jersey, U.S.) was used for FACS analysis.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eDispase-based keratinocyte dissociation assay\\u003c/h2\\u003e\\u003cp\\u003eThe method was performed under previously established conditions (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). Resulting fragments were quantified using ImageJ software (1708195; Bio-Rad Laboratories Inc., Hercules, CA, USA) (\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e) or alternatively counted manually.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eWestern Blot analysis\\u003c/h2\\u003e\\u003cp\\u003eCells were washed with DPBS and lysed as described previously (\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e). Sonication was performed to further analyze the Triton-insoluble fraction in the cell pellet (\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e). Five \\u0026micro;g of the protein were separated on a 4\\u0026ndash;12% gradient gel (NP0329BOX; Thermo Fisher Scientific Inc., Waltham, MA, USA) with SDS-PAGE technique after heat denaturation of the proteins (95\\u0026deg;C, 5 min) and then transferred to membranes (IB24001X3; Thermo Fisher Scientific Inc., Waltham, MA, USA). Membranes were then blocked in TPBS containing 5% milk powder (70166-500G; Sigma Aldrich, St. Louis, Missouri, United States) for 2 h at room temperature (RT) and washed with TPBS. Blots were then incubated with primary antibodies overnight at 4\\u0026deg;C, followed by incubation with an appropriated secondary antibody for 1 h at RT. Protein bands were visualized with an Immobilon Forte Western HRP substrate (WBLUF0500, Merck, Darmstadt, Germany).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eElectron microscopic analysis\\u003c/h2\\u003e\\u003cp\\u003eStimulated cells were fixed in 3% glutaraldehyde in 0.1 M Soerensen\\u0026rsquo;s phosphate buffer (Roth, Karlsruhe, Germany). Samples were post-fixed in 1% OsO\\u003csub\\u003e4\\u003c/sub\\u003e (E19100, Science Services, Munich Germany) in 25 mM sucrose buffer (1.07651.1000, Merck, Darmstadt, Germany), dehydrated by ascending ethanol series, and embedded in Epon. Ultrathin sections were cut in horizontal plane. Contrast was enhanced by staining with 0.5% uranyl acetate (E22499-05, Science Services, Munich, Germany) and 1% Sato`s lead citrate. Samples were examined using a Hitachi HT7800 transmission electron microscope (Hitachi, Japan) operating at an acceleration voltage of 100 kV. Analysis was performed by ImageJ. In each independent experiment (n\\u0026thinsp;=\\u0026thinsp;2), at least 50 images per condition were captured, each containing at least one desmosome. Desmosomes were measured using ImageJ with the following criteria: desmosomal length was determined as the longest continuous visible segment (50k magnification); interdesmosomal space was measured three times at different points along the desmosome (100k magnification); and the number of vesicles was counted as the total vesicles per microscopic field (50k magnification) containing at least one desmosome. The mean of the measurements from each experiment was used for statistical analysis.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eEx vivo skin models\\u003c/h2\\u003e\\u003cp\\u003eThe skin was obtained from safety margins after surgeries at the Department of Dermatology, University Hospital RWTH Aachen. Excess skin from excised safety margins was used on the same day. Before further use, the tissue was washed three times for 15 min each in DPBS. The subcutis was dissected, 8 mm pieces of skin were taken by punch biopsy and placed in 6-well culture plates with cell culture inserts (353091, Corning (Corning Inc.), Somerville, USA). 2 ml of medium/well (equal amounts of DMEM\\u0026thinsp;+\\u0026thinsp;1% antibiotics/antimycotics (15240096, Thermo Fisher Scientific Inc., Waltham, USA)\\u0026thinsp;+\\u0026thinsp;10% FBS and KBM-2 Keratinocyte Growth media (CC31-03, Lonza, Basel Switzerland)) was added. Human IgG (40 \\u0026micro;g); AK23 (40\\u0026micro;g) and/ or FasL (1:10) were diluted in PBS in a volume of 50 \\u0026micro;l/ punch and injected by a needle (0.4 mm diameter) into the upper dermis. The culture medium was changed daily. The samples were frozen in liquid nitrogen.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eHistology and immunohistochemistry\\u003c/h2\\u003e\\u003cp\\u003eCryosections or formalin-fixed and paraffin-embedded tissues were used for histopathology (H\\u0026amp;E staining) and immunohistochemistry. Image processing was applied identically to all samples and controls.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eImmunofluorescence\\u003c/h2\\u003e\\u003cp\\u003eCryosections were used for immunofluorescence. Image processing was applied identically to all samples and controls.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eTUNEL\\u003c/h2\\u003e\\u003cp\\u003eTUNEL (TdT-mediated dUTP-biotin nick end labeling) staining was performed according to the manufacturer\\u0026rsquo;s instructions (TUNEL-kit C10617, Thermo Fisher Scientific Inc., Waltham, USA).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStatistics\\u003c/h2\\u003e\\u003cp\\u003eAll data are expressed as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SEM (standard error of the mean). A two-tailed Student\\u0026prime;s t-test for two groups was used to assess the significance of differences. ns\\u0026thinsp;=\\u0026thinsp;p \\u0026gt;\\u0026thinsp;0.05; * p\\u0026thinsp;=\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; ** p\\u0026thinsp;=\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; *** p\\u0026thinsp;=\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cem\\u003eEthics approval\\u0026nbsp;\\u003c/em\\u003eThe study was approved by the Independent Ethics Committee RWTH Aachen University (Aachen, Germany) according to the Declaration of Helsinki Principles (EK 318/21). The written, informed consent was given prior to participation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eData Availability Statement\\u003c/em\\u003e Original data are available from the corresponding author upon a reasonable request.\\u003cem\\u003e\\u003cbr\\u003e\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u0026nbsp;\\u003c/strong\\u003eWe thank Petra Boukamp for providing the HaCaT cell line; Manuela Busch, Katharina Fietkau, Manuela Jansen, Linda Lopopolo and Yvonne Marquardt for their technical support and Hiltrud K\\u0026ouml;nigs-Werner for preparing the samples for electron microscopy. We also thank Peter H. Krammer for the caspase-8- and cFLIP- antibodies; Pascal Schneider for providing the Fc-FasL construct- The graphical abstract and Fig. 1B were created with BioRender.com.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contribution\\u003c/strong\\u003e\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eThe study was concepted by MFS, MAF and ASY. The funding was acquired by JW, RT, JMB, ASY. MFS and MAF established the methodology, collected the data, performed the formal analysis, validated and visualized the data. The investigation was supported by DPD, EMB, MR and JW. RT provided further resources. ASY supervised the project. The first draft of the manuscript was written by MFS and MAF and all authors commented on previous versions of the manuscript. MR assisted with editing the manuscript and contributed the study design. All authors reviewed and approved the final version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003eThis project was supported by the German Research Foundation (DFG) to the Unit FOR 2497 PEGASUS (TP 5 to JW, TI 291/10-2 to RT, BA1803/9-2 to JMB and YA-182/4-2 to ASY). MFS was\\u0026nbsp;funded by scholarships from the state of North Rhine-Westphalia (FF-med) and the University RWTH Aachen (Kurzzeitstipendium), an intramural fellowship of the RWTH Aachen. DPD is supported by the DFG (DI 2440/3-1); PB is supported by the DFG (Project IDs 322900939, 432698239 \\u0026amp; 445703531), European Research Council (ERC Consolidator Grant No 101001791), and the Federal Ministry of Education and Research (BMBF, STOP-FSGS-01GM2202C).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eConflict of Interest\\u0026nbsp;\\u003c/em\\u003eThe authors have declared that no conflict of interest exists.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAmagai M, Klaus-Kovtun V, Stanley JR. Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell. 1991;67(5):869-77.\\u003c/li\\u003e\\n\\u003cli\\u003eHashimoto T, Amagai M, Garrod DR, Nishikawa T. Immunofluorescence and immunoblot studies on the reactivity of pemphigus vulgaris and pemphigus foliaceus sera with desmoglein 3 and desmoglein 1. Epithelial Cell Biol. 1995;4(2):63-9.\\u003c/li\\u003e\\n\\u003cli\\u003eAmagai M. Autoantibodies against cell adhesion molecules in pemphigus. J Dermatol. 1994;21(11):833-7.\\u003c/li\\u003e\\n\\u003cli\\u003eSchmidt E, Kasperkiewicz M, Joly P. Pemphigus. Lancet. 2019;394(10201):882-94.\\u003c/li\\u003e\\n\\u003cli\\u003eOsada K, Seishima M, Kitajima Y. Pemphigus IgG activates and translocates protein kinase C from the cytosol to the particulate/cytoskeleton fractions in human keratinocytes. J Invest Dermatol. 1997;108(4):482-7.\\u003c/li\\u003e\\n\\u003cli\\u003eBerkowitz P, Hu P, Liu Z, Diaz LA, Enghild JJ, Chua MP, et al. Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem. 2005;280(25):23778-84.\\u003c/li\\u003e\\n\\u003cli\\u003eFrusic-Zlotkin M, Raichenberg D, Wang X, David M, Michel B, Milner Y. Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis--possible involvement of the EGF receptor. Autoimmunity. 2006;39(7):563-75.\\u003c/li\\u003e\\n\\u003cli\\u003eCirillo N, AlShwaimi E, McCullough M, Prime SS. Pemphigus vulgaris autoimmune globulin induces Src-dependent tyrosine-phosphorylation of plakophilin 3 and its detachment from desmoglein 3. Autoimmunity. 2014;47(2):134-40.\\u003c/li\\u003e\\n\\u003cli\\u003eEgu DT, Schmitt T, Waschke J. Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin. Front Immunol. 2022;13:884067.\\u003c/li\\u003e\\n\\u003cli\\u003eSchmitt T, Waschke J. Autoantibody-Specific Signalling in Pemphigus. Front Med (Lausanne). 2021;8:701809.\\u003c/li\\u003e\\n\\u003cli\\u003ePuviani M, Marconi A, Cozzani E, Pincelli C. Fas ligand in pemphigus sera induces keratinocyte apoptosis through the activation of caspase-8. J Invest Dermatol. 2003;120(1):164-7.\\u003c/li\\u003e\\n\\u003cli\\u003eWang X, Bregegere F, Frusic-Zlotkin M, Feinmesser M, Michel B, Milner Y. Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins. Apoptosis. 2004;9(2):131-43.\\u003c/li\\u003e\\n\\u003cli\\u003ePelacho B, Natal C, Espana A, Sanchez-Carpintero I, Iraburu MJ, Lopez-Zabalza MJ. Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes. FEBS Lett. 2004;566(1-3):6-10.\\u003c/li\\u003e\\n\\u003cli\\u003eGniadecki R, Jemec GB, Thomsen BM, Hansen M. Relationship between keratinocyte adhesion and death: anoikis in acantholytic diseases. Arch Dermatol Res. 1998;290(10):528-32.\\u003c/li\\u003e\\n\\u003cli\\u003eSchmidt E, Gutberlet J, Siegmund D, Berg D, Wajant H, Waschke J. Apoptosis is not required for acantholysis in pemphigus vulgaris. Am J Physiol Cell Physiol. 2009;296(1):C162-72.\\u003c/li\\u003e\\n\\u003cli\\u003eBystryn JC, Rudolph JL. Pemphigus. Lancet. 2005;366(9479):61-73.\\u003c/li\\u003e\\n\\u003cli\\u003eGrando SA. Pemphigus autoimmunity: hypotheses and realities. Autoimmunity. 2012;45(1):7-35.\\u003c/li\\u003e\\n\\u003cli\\u003eMoravvej H, Yousefi M, Farrokhi B, Mosaffa N. Soluble Fas in pemphigus vulgaris. Arch Iran Med. 2011;14(3):200-1.\\u003c/li\\u003e\\n\\u003cli\\u003eLotti R, Shu E, Petrachi T, Marconi A, Palazzo E, Quadri M, et al. Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus. Front Immunol. 2018;9:370.\\u003c/li\\u003e\\n\\u003cli\\u003eTsunoda K, Ota T, Aoki M, Yamada T, Nagai T, Nakagawa T, et al. Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3. J Immunol. 2003;170(4):2170-8.\\u003c/li\\u003e\\n\\u003cli\\u003eIshii K, Harada R, Matsuo I, Shirakata Y, Hashimoto K, Amagai M. In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris. J Invest Dermatol. 2005;124(5):939-46.\\u003c/li\\u003e\\n\\u003cli\\u003eAoyama Y, Kitajima Y. Pemphigus vulgaris-IgG causes a rapid depletion of desmoglein 3 (Dsg3) from the Triton X-100 soluble pools, leading to the formation of Dsg3-depleted desmosomes in a human squamous carcinoma cell line, DJM-1 cells. J Invest Dermatol. 1999;112(1):67-71.\\u003c/li\\u003e\\n\\u003cli\\u003eSato M, Aoyama Y, Kitajima Y. Assembly pathway of desmoglein 3 to desmosomes and its perturbation by pemphigus vulgaris-IgG in cultured keratinocytes, as revealed by time-lapsed labeling immunoelectron microscopy. Lab Invest. 2000;80(10):1583-92.\\u003c/li\\u003e\\n\\u003cli\\u003eMao X, Choi EJ, Payne AS. Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies. J Invest Dermatol. 2009;129(4):908-18.\\u003c/li\\u003e\\n\\u003cli\\u003eNguyen B, Dusek RL, Beaudry VG, Marinkovich MP, Attardi LD. Loss of the desmosomal protein perp enhances the phenotypic effects of pemphigus vulgaris autoantibodies. J Invest Dermatol. 2009;129(7):1710-8.\\u003c/li\\u003e\\n\\u003cli\\u003eLajoie P, Nabi IR. Regulation of raft-dependent endocytosis. J Cell Mol Med. 2007;11(4):644-53.\\u003c/li\\u003e\\n\\u003cli\\u003eWeiske J, Schoneberg T, Schroder W, Hatzfeld M, Tauber R, Huber O. The fate of desmosomal proteins in apoptotic cells. J Biol Chem. 2001;276(44):41175-81.\\u003c/li\\u003e\\n\\u003cli\\u003eSchmidt MF, Feoktistova M, Panayotova-Dimitrova D, Eichkorn RA, Yazdi AS. Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris. Vaccines (Basel). 2022;10(2).\\u003c/li\\u003e\\n\\u003cli\\u003eKavuri SM, Geserick P, Berg D, Dimitrova DP, Feoktistova M, Siegmund D, et al. Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex. J Biol Chem. 2011;286(19):16631-46.\\u003c/li\\u003e\\n\\u003cli\\u003eSchmitt T, Hudemann C, Moztarzadeh S, Hertl M, Tikkanen R, Waschke J. Dsg3 epitope-specific signalling in pemphigus. Front Immunol. 2023;14:1163066.\\u003c/li\\u003e\\n\\u003cli\\u003ePeng X, Wang S, Wu K, Cook C, Li L, Wang Z, et al. Effect of opioid receptor antagonist on mitigating tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-induced apoptolysis in pemphigus pathogenesis. J Autoimmun. 2024;149:103307.\\u003c/li\\u003e\\n\\u003cli\\u003eCalkins CC, Setzer SV, Jennings JM, Summers S, Tsunoda K, Amagai M, et al. Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies. J Biol Chem. 2006;281(11):7623-34.\\u003c/li\\u003e\\n\\u003cli\\u003eCirillo N, Campisi G, Gombos F, Perillo L, Femiano F, Lanza A. Cleavage of desmoglein 3 can explain its depletion from keratinocytes in pemphigus vulgaris. Exp Dermatol. 2008;17(10):858-63.\\u003c/li\\u003e\\n\\u003cli\\u003eCirillo N, Dell\\u0026apos; Ermo A, Gombos F, Lanza A. The specific proteolysis hypothesis of pemphigus: does the song remain the same? Med Hypotheses. 2008;70(2):333-7.\\u003c/li\\u003e\\n\\u003cli\\u003eDelva E, Jennings JM, Calkins CC, Kottke MD, Faundez V, Kowalczyk AP. Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism. J Biol Chem. 2008;283(26):18303-13.\\u003c/li\\u003e\\n\\u003cli\\u003eJolly PS, Berkowitz P, Bektas M, Lee HE, Chua M, Diaz LA, et al. p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis. J Biol Chem. 2010;285(12):8936-41.\\u003c/li\\u003e\\n\\u003cli\\u003eLevental I, Levental KR, Heberle FA. Lipid Rafts: Controversies Resolved, Mysteries Remain. Trends Cell Biol. 2020;30(5):341-53.\\u003c/li\\u003e\\n\\u003cli\\u003eMoldovan NI, Heltianu C, Simionescu N, Simionescu M. Ultrastructural evidence of differential solubility in Triton X-100 of endothelial vesicles and plasma membrane. Exp Cell Res. 1995;219(1):309-13.\\u003c/li\\u003e\\n\\u003cli\\u003eEgu DT, Kugelmann D, Waschke J. Role of PKC and ERK Signaling in Epidermal Blistering and Desmosome Regulation in Pemphigus. Front Immunol. 2019;10:2883.\\u003c/li\\u003e\\n\\u003cli\\u003eYamamoto Y, Aoyama Y, Shu E, Tsunoda K, Amagai M, Kitajima Y. Anti-desmoglein 3 (Dsg3) monoclonal antibodies deplete desmosomes of Dsg3 and differ in their Dsg3-depleting activities related to pathogenicity. J Biol Chem. 2007;282(24):17866-76.\\u003c/li\\u003e\\n\\u003cli\\u003eStahley SN, Saito M, Faundez V, Koval M, Mattheyses AL, Kowalczyk AP. Desmosome assembly and disassembly are membrane raft-dependent. PLoS One. 2014;9(1):e87809.\\u003c/li\\u003e\\n\\u003cli\\u003eGeorge KS, Wu S. Lipid raft: A floating island of death or survival. Toxicol Appl Pharmacol. 2012;259(3):311-9.\\u003c/li\\u003e\\n\\u003cli\\u003ePacheco-Tovar MG, Avalos-Diaz E, Vega-Memije E, Bollain-y-Goytia JJ, Lopez-Robles E, Hojyo-Tomoka MT, et al. The final destiny of acantholytic cells in pemphigus is Fas mediated. J Eur Acad Dermatol Venereol. 2009;23(6):697-701.\\u003c/li\\u003e\\n\\u003cli\\u003eArnold R, Seifert M, Asadullah K, Volk HD. Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines. J Immunol. 1999;162(12):7140-7.\\u003c/li\\u003e\\n\\u003cli\\u003eLotti R, Hundt JE, Ludwig RJ, Bennett B, Amato A, Marconi A, et al. Blocking soluble Fas Ligand ameliorates pemphigus: PC111 efficacy in ex-vivo human pemphigus models. Front Immunol. 2023;14:1193032.\\u003c/li\\u003e\\n\\u003cli\\u003eDegli Esposti M, Matarrese P, Tinari A, Longo A, Recalchi S, Khosravi-Far R, et al. Changes in membrane lipids drive increased endocytosis following Fas ligation. Apoptosis. 2017;22(5):681-95.\\u003c/li\\u003e\\n\\u003cli\\u003eFrusic-Zlotkin M, Pergamentz R, Michel B, David M, Mimouni D, Bregegere F, et al. The interaction of pemphigus autoimmunoglobulins with epidermal cells: activation of the fas apoptotic pathway and the use of caspase activity for pathogenicity tests of pemphigus patients. Ann N Y Acad Sci. 2005;1050:371-9.\\u003c/li\\u003e\\n\\u003cli\\u003eLee HE, Berkowitz P, Jolly PS, Diaz LA, Chua MP, Rubenstein DS. Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis. J Biol Chem. 2009;284(18):12524-32.\\u003c/li\\u003e\\n\\u003cli\\u003eGrando SA, Bystryn JC, Chernyavsky AI, Frusic-Zlotkin M, Gniadecki R, Lotti R, et al. Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis. Exp Dermatol. 2009;18(9):764-70.\\u003c/li\\u003e\\n\\u003cli\\u003eArredondo J, Chernyavsky AI, Karaouni A, Grando SA. Novel mechanisms of target cell death and survival and of therapeutic action of IVIg in Pemphigus. Am J Pathol. 2005;167(6):1531-44.\\u003c/li\\u003e\\n\\u003cli\\u003eSanath AK, Devy AS, Aithal S, Kumar GS, Prasad BG, Pradeep PS. Caspase cascade pathways of apoptosis in oral pemphigus: An immunohistochemical study. J Oral Maxillofac Pathol. 2018;22(1):48-53.\\u003c/li\\u003e\\n\\u003cli\\u003eBossen C, Ingold K, Tardivel A, Bodmer JL, Gaide O, Hertig S, et al. Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J Biol Chem. 2006;281(20):13964-71.\\u003c/li\\u003e\\n\\u003cli\\u003eDiessenbacher P, Hupe M, Sprick MR, Kerstan A, Geserick P, Haas TL, et al. NF-kappaB inhibition reveals differential mechanisms of TNF versus TRAIL-induced apoptosis upstream or at the level of caspase-8 activation independent of cIAP2. J Invest Dermatol. 2008;128(5):1134-47.\\u003c/li\\u003e\\n\\u003cli\\u003eBeckert B, Panico F, Pollmann R, Eming R, Banning A, Tikkanen R. Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris. Int J Mol Sci. 2019;20(13).\\u003c/li\\u003e\\n\\u003cli\\u003eGeserick P, Hupe M, Moulin M, Wong WW, Feoktistova M, Kellert B, et al. Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment. J Cell Biol. 2009;187(7):1037-54.\\u003c/li\\u003e\\n\\u003cli\\u003eSchneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5.\\u003c/li\\u003e\\n\\u003cli\\u003eFeoktistova M, Makarov R, Yazdi AS, Panayotova-Dimitrova D. RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation. Int J Mol Sci. 2021;22(22).\\u003c/li\\u003e\\n\\u003cli\\u003eWirths O. Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords. Bio Protoc. 2017;7(15):e2422.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-discovery\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddiscovery\",\"sideBox\":\"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)\",\"snPcode\":\"41420\",\"submissionUrl\":\"https://mts-cddiscovery.nature.com/\",\"title\":\"Cell Death Discovery\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7382893/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7382893/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003ePemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease caused by circulating autoantibodies against desmoglein (Dsg) 1 and 3. Whether acantholysis in PV results exclusively from antibody binding to Dsg3, or involves additional factors remains controversial. Given that Fas-Ligand (FasL), an activator of apoptotic caspase-8, is increased in the serum and the skin of patients with PV, we investigated the role of caspases in anti-Dsg3-mediated acantholysis using both \\u003cem\\u003eex vivo\\u003c/em\\u003e and \\u003cem\\u003ein vitro\\u003c/em\\u003e models. Our results demonstrate that anti-Dsg3 antibodies induce acantholysis \\u003cem\\u003eex vivo\\u003c/em\\u003e in the absence of caspase activation, primarily through the redistribution of Dsg3 to intracellular compartments. FasL-induced caspase activation leads to a synergistic amplification of anti-Dsg3-mediated loss of cell adhesion by promoting Dsg3 cleavage. This dual mechanism provides new insights into the disease heterogeneity of PV and may also explain the rapid response of PV to high-dose glucocorticosteroids despite the persistence of antibodies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Caspase-activation powers anti-Desmoglein 3-induced acantholysis in human epidermis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-17 08:18:06\",\"doi\":\"10.21203/rs.3.rs-7382893/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"revise\",\"date\":\"2025-09-29T08:42:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2025-09-24T08:57:37+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"editorInvitedReview\",\"content\":\"This content is not available.\",\"date\":\"2025-09-11T07:14:15+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2025-09-10T14:18:40+00:00\",\"index\":2,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewerAgreed\",\"content\":\"This content is not available.\",\"date\":\"2025-09-09T05:21:32+00:00\",\"index\":1,\"fulltext\":\"This content is not available.\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-09-08T22:57:39+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-09-02T07:11:01+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-08-31T19:04:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cell Death Discovery\",\"date\":\"2025-08-31T19:04:30+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cell-death-discovery\",\"isNatureJournal\":false,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"cddiscovery\",\"sideBox\":\"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)\",\"snPcode\":\"41420\",\"submissionUrl\":\"https://mts-cddiscovery.nature.com/\",\"title\":\"Cell Death Discovery\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"41ccd0df-710c-4ce3-b8a7-9942a5d0074e\",\"owner\":[],\"postedDate\":\"September 17th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":54397726,\"name\":\"Biological sciences/Cell biology/Cell death/Apoptosis\"},{\"id\":54397727,\"name\":\"Health sciences/Diseases/Skin diseases\"}],\"tags\":[],\"updatedAt\":\"2026-02-28T08:10:43+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7382893\",\"link\":\"https://doi.org/10.1038/s41420-026-02963-w\",\"journal\":{\"identity\":\"cell-death-discovery\",\"isVorOnly\":false,\"title\":\"Cell Death Discovery\"},\"publishedOn\":\"2026-02-19 05:00:00\",\"publishedOnDateReadable\":\"February 19th, 2026\"},\"versionCreatedAt\":\"2025-09-17 08:18:06\",\"video\":\"\",\"vorDoi\":\"10.1038/s41420-026-02963-w\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41420-026-02963-w\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7382893\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7382893\",\"identity\":\"rs-7382893\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}