Cadherin-based adhesion is Ca2+-dependent. In vitro, the assembly of intercellular junctions in keratinocytes can be induced by a switch from low to high Ca2+ medium (28, 29). Vice versa, reduction of the extracellular Ca2+ concentration leads to translocation of cadherins from cell-cell borders to the cytosol, followed by disassembly of adherens junctions and desmosomes (28, 30). Since inhibition of Src was protective for adhesion of kerationcytes after treatment with AK23 and PV3-IgG, we investigated the role of Src for desmosomal reassembly. Ca2+ depleted HaCaTs were re-exposed to Ca2+ for 8 h in combination with c-IgG, AK23, and PV3-IgG with or without PP2 and then subjected to a dispase-based dissociation assay. Ca2+ repletion in combination with autoantibodies did not restore cell adhesion indicating that autoantibodies interfered with desmosome reassembly, which likely contributes to loss of cell cohesion when autoantibodies are applied for longer periods such as shown in **Figure 1**. However, PP2 reduced this negative effect of both AK23 and PV3-IgG on reconstitution of cell cohesion (**Figure 2A**).

### The Src Target Cortactin Colocalizes and Interacts With Dsg3

The actin binding protein cortactin was identified as a major substrate of Src (17), and is proposed to be activated by Src-mediated tyrosine phosphorylation (31). Therefore, we were interested if cortactin plays a role in Src-mediated effects on desmosomal adhesion. First, we investigated the correlation of cortactin and Dsg3 in desmosomal assembly by immunofluorescence analysis under Ca2+ switch conditions. Under control conditions, both proteins are located at cell borders and cortactin was phosphorylated on the Src-dependent phosphorylation site Tyr 421. Depletion of Ca2+ resulted in Dsg3 dissociation form the cell borders, accompanied by disruption of cortactin and phospho-cortactin immunostaining. Re-administration of Ca2+ led to restored Dsg3, cortactin, and phospho-cortactin staining at cell-cell borders (**Figure 2B**). Moreover, immunostaining showed that cortactin and Dsg3 partially co-localized at the cell membrane (**Figure 2C**) which was validated by a proximity ligation assay (PLA), also revealing a close association with the cortical actin cytoskeleton (**Figure 2D**, upper images) at the cell periphery

autoantibody fractions were effective to activate Src (n = 3).

(**Figure 2D**, XZ presentation). Next, immunoprecipitation analysis verified that cortactin co-precipitated with Dsg3 in the triton-soluble protein pool but not in the triton-insoluble fraction (**Figure 2E**). This implied that cortactin may play a role in Src-mediated regulation of desmosome assembly. In contrast, phosphorylation of Pkp3 after incubation with AK23 was not detectable (**Supplemental Figure 1**).

### Cortactin Is Important for Src-Mediated Reconstitution of Cell Cohesion in vitro and in a Pemphigus Mouse Model in vivo

To study the role of cortactin in more detail, we isolated primary keratinocytes form newborn cortactin wild type (wt) and cortactin-deficient mice (32). Cortactin-deficient (CTTN−/−) mouse keratinocyte mono-layers showed significantly more fragments in a dispase-based dissociation assay, indicating that cortactin is required for cell cohesion (**Figure 3A**). The knockout of cortactin was verified by Western blot analysis (**Figure 3B**), which also showed no changes of desmosomal proteins such as desmoplakin (DP) and Dsg3 in CTTN−/− cells. Next, we subjected cells to a dissociation assay after application of PP2 in combination with AK23 for 2 h (**Figure 3C**) or 24 h (**Figure 3D**). In wt mouse keratinocytes, cell adhesion was rescued by Src inhibition when AK23 was applied for 2 h or for 24 h. In contrast, in CTTN−/− cells, PP2 blocked AK23-induced loss of adhesion only when AK23 was incubated for 2 h but not for 24 h. This suggested that short-term effects auf AK23 were Src- but not cortactin-dependent, whereas long-term effects at least in part required both Src and cortactin. To examine the importance of cortactin for restoring of cell adhesion, Ca2+-switch assay was performed under the same conditions (**Figure 3E**). Again, dissociation assay experiments showed that Src inhibition by PP2 was not effective to restore cell cohesion in CTTN−/− cells in presence of AK23, indicating that reconstitution of cell adhesion is both Src- and cortactin-dependent.

To verify these findings in vivo, we used the neonatal passive immuno-transfer mouse model as described previously using AK23 (23). Therefore, we injected AK23 alone and in combination with the Src-inhibitor PP2 into the skin of newborn CTTN+/+ and CTTN−/− mice. After incubation for 24 h, defined shear stress was applied. H.E. staining of serial sections showed AK23-induced gross blistering at injection sites of both wt and cortactin-deficient pubs (**Figures 4A–C**). However, in wt animals co-incubation with the Src inhibitor abrogated blister formation almost completely whereas in cortactin-deficient mice, blisters were still observed. These results indicate that AK23 induced skin blistering in vivo is Src- and cortactin-dependent, at least, in mice.

### Inhibition of Src Was Not Protective Against PV-IgG-Induced Blistering in Human Skin ex vivo

Finally, to study the role of Src for skin blistering in intact human skin, we used a human ex vivo skin model as reported previously using PV-IgG (22, 33). After injection and incubation of human skin with PV4-IgG alone or in combination with the Src inhibitor PP2 for 24 h, HE-stained serial sections revealed blister formation after treatment with PV-IgG alone as well as in combination with PP2 (**Figures 5A,B**). Analysis of the ultrastructure showed no differences after PV-IgG incubation in conjunction with vehicle or PP2 treatment (**Figure 5A**, lower panel, **Figures 5C–E**). Under both experimental conditions, the number of desmosomes (**Figure 5C**) and desmosome length (**Figure 5D**) were reduced in comparison to control conditions and inter-desmosomal widening was observed (**Figure 5E**). This data show that PP2 was in the long term not protective against PV-IgG-induced skin blistering in human skin.

### DISCUSSION

The study provides further insights into the role and mechanisms of Src-mediated epidermal blistering in pemphigus. Inhibition of Src was protective against AK23-induced skin blistering in an in vivo mouse model but not against PV-IgG-induced skin blistering in human epidermis. Moreover, the role of Src for loss of cell cohesion appeared to be patient-dependent and to negatively correlate at least in part with autoantibody scores against Dsg1. Src-mediated loss of keratinocyte cohesion engaged mechanisms which were both cortactin-dependent and independent, the former of which were involved in reconstitution of keratinocyte adhesion.

### Role of Src in Pemphigus Autoantibody-Induced Skin Blistering

In accordance with the literature, PV-IgG fractions as well as AK23 led to activation of Src (4, 5, 34, 35). In this context, it was shown that PV-IgG induce Src activation early after application of autoantibodies and thus before other signaling molecules such as p38MAPK or EGFR were activated (5). The first question is by which autoantibodies Src signaling is triggered. It was shown that activation of Src was detectable only when PV-IgG fractions contained antibodies against Dsg3 but not when pemphigus foliaceus (PF) autoantibodies against Dsg1 were applied (4). Since AK23, which is specifically directed against Dsg3, was effective to activate Src it can be concluded that Dsg3 is sufficient to modulate Src signaling (4). Nevertheless, experiments using siRNA-mediated depletion of Dsg1 and Dsg3 suggest that antibodies against other targets may also be capable of activating Src (5).

Consistent with these previous findings, our data confirmed activation of Src after PV-IgG and AK23 treatment for 15 min and up to 2 h. Furthermore, loss of cell adhesion caused by AK23 was completely abolished by PP2 in cell culture in vitro as well as in the neonatal mouse model in vivo, demonstrating that Src plays an important role in loss of cell cohesion caused by autoantibodies directed against Dsg3. This supports previous findings where either broad-spectrum tyrosin kinase inhibitors or PP2 were efficient to inhibit PV-IgG-induced skin blistering in mice (35, 36).

However, inhibition of Src was not effective to consistently protect against PV-IgG-induced loss of adhesion when autoantibody fractions from different donors were applied.

For one fraction, Src inhibition abrogated loss of cell adhesion only when autoantibodies were incubated short-time. Using a second fraction, no protection against loss of cell cohesion was detectable at all (27). Since all autoantibody fractions were equally effective to induce loss of cell cohesion in vitro, this suggests that different mechanisms maybe involved in different patients. This is supported by the observation that PP2 was not effective to inhibit PV-IgG-induced skin blistering in human skin ex vivo. The data presented here do not allow the conclusion that Src is not involved in human skin at all. Nevertheless, the data show that PP2 under the conditions where it is effective to block AK23-mediated blistering in mice and to prevent loss of

keratinocyte cohesion in response to some patients' IgG fractions in vitro, is not consistently protective in intact human skin.

A limitation of the study is that the conditions used in the different experimental models do not necessarily reflect the situation in patients. Regarding the inhibition of Dsg3 with the monoclonal autoantibody AK23 derived from a pempighus mouse model (27), it should be considered that the concentration used was supraphysiologic compared to patients' autoantibody levels. This could lead to an excessive direct inhibition of Dsg3 binding and activation of signaling by Src and p38MAPK (37), both of which may not be typical in PV patients. However, in contrast to the neonatal mouse model used here, AK23 in a

human skin model was not effective to induce blister formation and reduce desmosome numbers indicating that mechanisms triggered by autoantibodies against Dsg1 may be required for blister formation as well (33). Similarly, the autoantibody profiles of the PV-IgG fractions and the abundance of aDsg1 autoantibodies used in this study may at least in part explain the different roles of Src for loss of keratinocyte adhesion. PV3- IgG, the effects of which were consistently ameliorated by Srcinhibition, contained comparable amounts of autoantibodies against Dsg1 and Dsg3, whereas in all other PV-IgG fractions the relative amount of Dsg1 autoantibodies was higher. Especially, PV4-IgG used for the ex vivo human skin model contained excessive levels of autoantibodies against Dsg1. However, for PV2-IgG this explanation falls short because Src inhibition was not protective at all despite comparable levels of antibodies against Dsg1 and Dsg3. Since we have no information about antibodies against other antigens including desmocollin isoforms or others, we cannot rule out that such antibodies may trigger mechanisms others than Src in loss of keratinocyte adhesion.

Nevertheless, the results are in line with the hypothesis that the different clinical phenotypes of pemphigus may at least in part be determined by the mechanisms which are involved in loss of cell cohesion and engaged by antibodies targeting Dsg3 or Dsg1 (4). Moreover, the data indicate that a therapeutic paradigm to modulate Src activity alone unlikely would be effective to treat the majority of patients with epidermal blistering as this usually is associated with antibodies against Dsg1 (1). This may be related to the different mechanisms induced by autoantibodies against Dsg1 in pemphigus pathogenesis.

### Mechanisms by Which Src Regulates Cell Cohesion In Pemphigus

For some of the PV-IgG fractions such as PV1-IgG and PV2- IgG, loss of cell cohesion was clearly progressive from 15 min up to 24 h as studied in dispase assays. For other autoantibody fractions such as PV3-IgG and AK23, this effect was less pronounced which is in line with previous observations (37). This may indicate that at early time-points the underlying mechanisms may be different from later stages of adhesion loss. This is supported by the observation that short time coincubation of AK23 with PP2 in cortactin-deficient cells can abrogate loss of cell cohesion whereas long-term incubation is not affected by Src inhibition. We conclude that short time effects of autoantibodies are in part Src- but not cortactin-dependent whereas cortactin at later stages of cell cohesion loss becomes more relevant.

We observed that autoantibodies indeed interfered with reconstitution of cell adhesion in a manner dependent on Src. Moreover, because phosphorylation of the Src substrate cortactin at cell junctions correlated with junction integrity and cortactin binds to extra-desmosomal Dsg3, we reasoned that cortactin may be involved in desmosome assembly. The interaction of cortactin with desmosomal cadherins recently has been shown for Dsg1 also (38). More detailed studies are required to elucidate how cortactin is important in this context. Here, we observed that short-time effects of autoantibodies cause loss of cell cohesion by mechanisms which do not impair formation of new desmosomes whereas at later stages, impaired reformation of junctions may contribute to adhesion loss. This can be concluded from Ca2+-switch assays in cortactin-deficient monolayers where Src inhibition was no longer protective against autoantibodyinduced adhesion reconstitution. These data are in line with the observation that Dsg3 and Src form a complex together with E-cadherin, which appears to be involved in formation of new desmosomes (12). Since both in intact human skin ex vivo as well as in neonatal mouse skin in vivo autoantibodies were applied for 24 h, it is possible that cell cohesion in neonatal skin is more sensitive to mechanisms impairing formation of new cell contacts.

Since the pathogenesis of pemphigus appears to be complex and to include direct inhibition of Dsg3 binding as well as signaling events (2), the question arises about the role of Src and cortactin for loss of cell cohesion when compared to other mechanisms. Here we show that the short-time effects by which autoantibodies interfere with keratinocyte adhesion are at least in part dependent on Src but are independent of cortactin. Therefore, it is possible that phosphorylation of plakophilin (Pkp) 3 and its dissociation from desmosomes may be involved as has been suggested for experiments with PV-IgG (15). However, for AK23 we did not observe Pkp3 phosphorylation (**Supplemental Figure 1**) indicating that phosphorylation of Pkp3 can be independent of Dsg3. Aside from Src, other signaling molecules likely contribute to loss of cell cohesion in pemphigus. Electron microscopy in ex vivo human skin revealed that ultrastructural alterations such as loss of desmosomes, shortening of desmosomes and inter-desmososomal widening, all of which are observed in patients' lesions as well (39), were independent of Src. In contrast, these effects were shown previously to be mediated by p38MAPK (33). This is in line with the observation that pemphigus autoantibody fractions including autoantibodies targeting Dsg3 activate both Src and p38MAPK (4).

Finally, it is well-established that autoantibody titers correlate with the clinical phenotype of pemphigus (1, 3). For pemphigus autoantibodies from patients with muco-cutaneous PV or with PF, influx of Ca2+ and activation of ERK were also detected

(4). Since Dsg1 was shown to suppress EGFR/ERK signaling by interacting with the ErbB2 binding protein Erbin (40), it is possible that autoantibodies targeting Dsg1 interfere with ERK signaling via this pathway. Taken together, we show that Src contributes to loss of cell adhesion primarily downstream of antibodies against Dsg3 in pemphigus by mechanisms which are both cortactin-dependent and –independent. The notion that actin-binding proteins such as cortactin and adducin become increasingly recognized to control desmosome function and cell behavior (12, 38), opens a new field for research on desmosome regulation.

### AUTHOR CONTRIBUTIONS

DK, VR, EW, DE, MF, and HV-R performed experiments. DK, VR, EW, DE, and MF analyzed data. DK, EW, FV, MS, MH, RE, KR, AS, VS, and JW discussed data and interpreted results. DK and JW designed the study and wrote the manuscript.

### ACKNOWLEDGMENTS

We thank Martina Hitzenbichler and Thomas Korbica for their excellent technical assistance, as well as Jessica Plewa, Michael Becker, and Axel Unverzagt for assistance in preparation of human body donors' skins. This work was supported by DFG grants WA 2474/4-3 and FOR2497 (PEGASUS) JW.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00626/full#supplementary-material

### REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kugelmann, Rötzer, Walter, Egu, Fuchs, Vielmuth, Vargas-Robles, Schnoor, Hertl, Eming, Rottner, Schmidt, Spindler and Waschke. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# ST18 Enhances PV-IgG-Induced Loss of Keratinocyte Cohesion in Parallel to Increased ERK Activation

Mariya Y. Radeva1†, Elias Walter 1†, Ramona Alexandra Stach2 , Amir S. Yazdi 2,3 , Nicolas Schlegel 4 , Ofer Sarig5 , Eli Sprecher 5,6 and Jens Waschke1 \*

1 Department I, Institute of Anatomy and Cell Biology, Ludwig-Maximilians-Universität (LMU) Munich, Munich, Germany, 2 Department of Dermatology, University Medical Center Tübingen, Eberhard Karls University, Tübingen, Germany, 3 Department of Dermatology, RWTH Aachen, Aachen, Germany, 4 Department of General, Visceral, Vascular and Paediatric Surgery, Julius-Maximilians-Universität, Würzburg, Germany, 5 Department of Dermatology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel, 6 Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

#### Edited by:

Takashi Hashimoto, Osaka City University, Japan

#### Reviewed by:

Giovanni Di Zenzo, Institute of Dermatology Immaculate (IRCCS), Italy Akira Ishiko, Toho University, Japan

\*Correspondence:

Jens Waschke<EMAIL_ADDRESS>
> †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 24 January 2019 Accepted: 25 March 2019 Published: 17 April 2019

#### Citation:

Radeva MY, Walter E, Stach RA, Yazdi AS, Schlegel N, Sarig O, Sprecher E and Waschke J (2019) ST18 Enhances PV-IgG-Induced Loss of Keratinocyte Cohesion in Parallel to Increased ERK Activation. Front. Immunol. 10:770. doi: 10.3389/fimmu.2019.00770 Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1α, IL-6, TNFα, and IFN-γ was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1α and IL-6 was enhanced in some experiments but not in others whereas release of TNF-α dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.

Keywords: pemphigus, desmosome, desmoglein, ST18, ERK, cytokines

### INTRODUCTION

Pemphigus vulgaris (PV) is a potentially fatal autoimmune disease of the skin and the mucous membranes. The skin disorder is manifested by mucocutaneous blister formation caused by loss of keratinocytes intercellular cell adhesion, which is primarily promoted by IgG autoantibodies directed against desmosomal adhesion molecules such as desmoglein (Dsg) 1 and Dsg 3 (1). However, autoantibodies targeting desmocollin (Dsc) 3, which are rarely present in cases when antibodies against Dsg3 are missing, were also shown to be pathogenic (2– 4). These desmosomal cadherins are transmembrane proteins forming the adhesive core of desmosomes, a special intercellular junction maintaining the mechanical integrity of tissues and bearing tension primarily if exposed to high levels of mechanical stress such as in the epidermis (5). With their cytoplasmic tail, via Plakoglobin (PG), and Desmoplakin (DP), the cadherins are linked to the intermediate filament skeleton, another structural unit providing mechanical strength to cells (6, 7). Besides their mechanical functions, desmosomes serve as signaling hubs coordinating tissue-specific functions with intercellular adhesion, which is required for instance during wound healing (8).

In response to binding of pemphigus autoantibodies to the keratinocyte surface, Dsg3-mediated homophilic interaction is impaired and desmosomal proteins are depleted from the membrane leading to keratin retraction and Dsg internalization (9–12). These structural alterations are induced by various PV-mediated outside-in intracellular signaling events such as p38 mitogen-activated protein kinase (p38MAPK) (13– 15) and extracellular-signal regulated kinase (ERK) (16, 17). Signaling mechanisms have been shown to be important for loss of cell adhesion (17) and the importance of several signaling mechanisms is highlighted by the protective effects of pharmacological inhibitors in vivo, in vitro, or ex vivo (18–20).

Despite all efforts, the etiology of the disease is still not completely understood. Interestingly studies reporting ethnic susceptibility and familial occurrence of the PV- skin disorder provided evidence for a genetic predisposition to PV (21–25). In this respect, most of the reports provided data for PV associated genes belonging to a human leukocyte antigen (HLA) locus. Few studies, however, described PV genetic association of non-HLA genes (25). Amongst the latter is suppression of tumorigenicity 18 (ST18), the product of which functions as a transcription factor and thereby controls the mRNA levels of numerous proappoptotic and pro-inflammatory genes (26), participating in regulation of processes with potential relevance for loss of cell adhesion in PV (27, 28). Recently, a genetic variant located within the ST18 promoter was reported to cause ST18 up-regulation, associated with PV-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocytes cohesion (29). In line with this, single nucleotide polymorphisms (SNP) identified in the ST18 gene were proposed to predispose to PV in a population-specific manner. SNPs promoted augmentation in ST18 expression was shown to be associated with more severe disease manifestation, indicative for the direct role of ST18 in PV pathogenesis (30, 31).

In the present study we investigated in both HaCaT and normal human epidermal keratinocytes (NHEK) the release of key pro-inflammatory molecules such as IL-1α, IL-6, TNF-α, and IFN-γ upon PV-IgG treatment. Additionally, the effect of ST18 overexpression on cytokine release but also on the modulation of pemphigus-associated ERK signaling was evaluated since both events would render keratinocytes more susceptible to PV-IgGinduced loss of keratinocyte adhesion.

### MATERIALS AND METHODS

### Cell Culture, Isolation of Human Primary Keratinocytes

For all experiments, primary normal human epithelial keratinocytes (NHEK) and HaCaT, a spontaneously immortalized human skin keratinocyte cell line were used. HaCaT cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), (Life Technologies; Carlsbad; CA; USA) supplemented with 10% FCS (Biochrom, Berlin, Germany), 50 U/ml penicillin and 50 U/ml streptomycin, both antibiotics were purchased from AppliChem, Darmstadt, Germany.

NHEK were generated at Universitäts-Hautklinik Tübingen. The procedure was approved by the medical ethical committee of the Eberhard Karls University Tübingen (ethical approval: 547/2011BO2). Briefly, the cells were isolated from juvenile foreskin derived from patients, who have given written informed consent. The skin was divided into epidermis and dermis by using 50 mg/ml Dispase II (Roche; Basel, Switzerland). Epidermal cells were then separated from each other by using a trypsin-EDTA solution (Merck, Darmstadt, Germany) and afterwards cultivated in CnT-07 medium (CELLnTEC Advanced Cell Systems AG, Switzerland) containing 10µg/ml gentamycin and 0.25µg/ml amphotericin B. The cells were cultured in low calcium conditions (0.06 mM Ca2+). 24 h prior treatment, cell differentiation was induced by increasing the Ca2+ concentration to 1.8 mM. All experiments were conducted on NHEK cells between passages 3 to 8.

Both cell lines were grown in a humidified atmosphere containing 5% CO2 at 37◦C. After cells reached confluency the medium was changed and 24 h later cell monolayers were treated accordingly.

### Constructs and Transfection

The human ST18 construct (pCMV6-ST18-Myc/DDK) was generated by inserting the translated region of a human ST18 cDNA into the pCMV6-Entry target vector (Origene Technologies Company, Rockville, MD, USA). The latter was used also for a control transfection.

ST18 overexpression was accomplished by transient transfection with a plasmid DNA into HaCaT cells at 80% confluence using Turbofect transfection reagent (Fermentas, ThermoFisher Scientific). 24 h after transfection, medium was changed and cell monolayers were further exposed to treatment with various IgG fractions.

## RNA Isolation, Complementary DNA (cDNA) Preparation, and PCR Performance

To confirm sufficient transfection efficiency with ST18 construct, PCR was performed with cDNA transcribed from an equal amount of total RNA. The latter was isolated by using RNeasy Plus mini kit (Qiagen, Venlo, Netherlands). cDNA was synthesized through the usage of SuperScript II reverse transcriptase kit (Invitrogen, ThermoFisher Scientific). All preparations were carried out according to manufacturer's instructions.

The transfection efficiency of each experiment requesting ST18 overexpression was tested by PCR using following primer pair:

hST18-164bp-Fw CACTAATCCAGGAGCTCAGTGTTG hST18-164bp-Rev CCTGTCACTGCGGTCTTCTTG

To validate the PCR assay, positive (pCMV-ST18-Myc/DDK vector) and negative (instead of cDNA, H2O was used as a template) controls were used. An empty negative control reflects the lack of contaminations, whereas manifestation of amplicon with correct size in the positive control guarantees accurate PCR conditions.

PCR was carried out as follows: 95◦C for 3 min, followed by 22 cycles at 95◦C for 30 s, 60◦C for 30 s and 72◦C for 30 s, the final extension was at 72◦C for 5 min. Then, the PCR products were visualized by gel electrophoresis.

## Dispase-Based Keratinocytes Dissociation Assay

Keratinocytes were seeded in 24-well plates and grown to confluency. NHEK cells were subsequently differentiated for 24 h by increasing the Ca2+-concentration to 1.8 mM. HaCaT monolayers were transiently transfected either with control or pCMV-ST18-Myc/DDK vector 24 h prior to experimental procedure. Monolayers treated with PBS (Vehicle) or control IgG (c-IgG) for 24 h were considered as controls. Experimental conditions were treated with AK23 or different PV-IgG in a similar fashion.

The loss of cell cohesion was tested by dispase-based dissociation assay. The latter was performed as described elsewhere (32, 33). Briefly, after treatment, cell monolayers were washed with pre-warmed Hank's Balanced Salt Solution (HBSS) and subsequently released from the well-bottom by incubation with 2.4 U/ml Dispase II (Sigma-Aldrich) dissolved in HBSS for 20 min at 37◦C. Next, the protease solution was substituted by HBSS and each condition was treated additionally for 10 min with thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich) at a final concentration of 10µM to better visualize the monolayer sheets. The integrity of a single intact monolayer was compromised by applying controlled shear stress through pipetting the monolayers with an electrical 1 ml pipette. Resulting fragments were counted under a binocular microscope (Leica, Mannheim, Germany) as their number is an inverse measure of intercellular adhesion. Higher number of fragments correspond to stronger loss of intercellular adhesion. A Canon EOS 750D camera was used to record the dissociation experiments. Each experiment was repeated 6 to 10 times.

### Analysis of the Cytokine Secretion

Simultaneous quantitative cytokine measurements were accomplished by high-throughput bead-based multiplex assays. A Milliplex kit (HCYTOMAG-60K, Merck KGaA, Darmstadt, Germany), precustomized for cytokines of interest, i.e., IL-1α, IL-6, IFN-γ, TNF-α, was used. Along with the serial standards, each sample was analyzed in duplicate or triplicate following the manufacturer's instructions. Collection of the data was achieved by using Luminex LX100/200 system (Luminex Corporation, Austin, Texas). Data analysis was performed using MILLIPLEX R Analyst 5.1.Software (Merck KGaA, Darmstadt, Germany). To estimate the cytokine concentration (expressed in pg/ml), standard curves derived from known reference concentrations supplied by the manufacturing company were utilized.

### Immunostaining

Human keratinocytes (HaCaT and NHEK) were grown to confluence on glass coverslips. Twenty four hours prior treatment, the medium was changed and in the case of NHEK cells, the Ca2+ concentration was increased to 1.8 mM in order to induce cell differentiation. After 24 h of incubation at 37◦C with different IgG fractions the culture medium was removed, cell monolayers were washed with PBS and subsequently fixed (with 2% paraformaldehyde (PFA) for 10 min) and permeabilized (with 0,1% Triton-X-100 for 5 min). To prevent unspecific binding the intact monolayers were blocked with a mix of 1% normal goat serum and 3% bovine serum albumin solved in PBS for at least 30 min at room temperature (RT). Consequent overnight incubation at 4◦C with primary rabbit anti-Dsg3 antibody (Biozol Diagnostica Vertrieb GmbH, Germany) was performed. After washing with PBS, the cell monolayers were incubated with Cy3- conjugated secondary goat-anti-rabbit antibody (Dianova, Hamburg, Germany) for an hour at RT. After rinsing the monolayers with PBS and H2O the cells were mounted on a glass slide with N-propyl gallate, an anti-fade reagent, which is used to reduce photobleaching of the fluorescent probes. Images were collected with 63xNA1.4 PL APO objective using a Leica SP5 confocal microscope.

### PV Sera and Purification

Patient blood sera were obtained from the Department of Dermatology and Allergology, Philipps-Universität Marburg, Marburg, Germany. Prior serum collection, all patients gave their written informed consent. For disease determination, clinical and histological studies were carried out and the antibody scores for aDsg1 and 3 were obtained during disease diagnosis. Control serum was donated from healthy volunteers. Before experimental usage, patient sera were purified by column affinity chromatography as described elsewhere (34). Briefly, Protein-A Agarose (ThermoFisher, Waltham, USA) was washed with PBS. Subsequently, beads were incubated with patient sera for 2 h at room temperature on a rotator, which enables IgG binding to the beads. After washing the beads with PBS, IgGs were detached from the Agarose by using sodium citrate (20 mM, pH 2.4) followed up by neutralization with sodium carbonate. Next, a filter unit (Amicon Ultra−4, 100 k; Merck Millipore, Darmstadt, Germany) was used to wash the resulting salts out of the IgG

fractions by centrifugation at 19,000 g for 20 min. As a final step the IgGs were resuspended in PBS. Protein amount of purified IgG fractions was determined by Bicinchoninic acid kit (BCA) (Thermo Fisher Scientific, Schwerte, Germany) and used for further experiments at a dilution of 1:50.

The monoclonal antibody AK23 reacting to Dsg3 was purchased from Biozol (Eching, Germany).

### Triton X-100 Protein Fractionation, Electrophoresis, Western Blotting, and Tested Antibodies

Signaling pathway modulation was examined in either nontransfected or transfected keratinocytes exposed to different IgGs or AK23 for a time period of 30 min. After the incubation was accomplished, cell monolayers were washed with ice cold PBS. As next, extraction buffer (0.5% Triton X-100, 50 mmol/L MES, 25 mmol/L EGTA, 5 mmol/L MgCl2) containing 0.1% of Leupeptin, Pepstatin and Aprotinin as well as 1% PMSF was applied for 15 min, on ice, under moderate shaking. Lysates were collected by scraping. In order to achieve successful fractionation of the cytoskeletal bound insoluble from the soluble fraction, a centrifugation step at 19,000 g for 10 min at 4◦C was performed. Resulting supernatants defined the cytosolic pools. The obtained pellet, representing the cytoskeletal bound fraction, was further washed with Triton extraction buffer, centrifuged for 10 min and consequently re-suspended in SDS-buffer (25 mmol/l HEPES, 2 mmol/l EDTA, 25 mmol/l NaF, and 1% SDS, pH 7,4, complete Protease Inhibitor Cocktail). After sonication, protein concentration of both fractions was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Schwerte, Germany), following the manufacturer's instructions. Equal amounts of samples were mixed with Laemmli buffer and consequently subjected to Western blotting as described elsewhere (17).

Following primary antibodies were incubated overnight at 4◦C in 5% bovine serum albumin (BSA) in Tris-bufferedsaline (TBS) with 0.05% tween (TSB-T): p-ERK (Cell Signaling Technologies, Cambridge, United Kingdom), p44/42 MAPK pAB (Erk1/2) (Cell Signaling Technologies), GAPDH mAB (Santa Cruz), Desmoplakin I/II pAB (H-300) (Santa Cruz). Following peroxidase coupled secondary antibodies were used for 1 h at room temperature in TBS-T: HRP-coupled goat anti-mouse, a HRP-coupled goat anti-rabbit (Dianova, Hamburg, Germany). Afterwards antibodies were visualized by chemiluminescence with ECL reaction (GE Healthcare, Munich, Germany).

### Data Analysis and Statistic

One-way ANOVA followed by Bonferroni correction (GraphPad Software, La Jolla, CA) was used to assess the differences among three or more treatments in dispase-based assay. To determine the cell response to different treatments in cells transfected with EV and ST18- vector either two-way ANOVA followed by Sidak's multiple comparisons test or multiple t-tests corrected by Holm-Sidak's method were applied as indicated. Each data point represents one single experiment. Data are presented as mean ± SEM. P-values less or equal to 0.05 assume significant differences between analyzed groups.

### RESULTS

## Pathogenicity of Pemphigus Autoantibodies on Human Keratinocytes

The pathogenic effect of IgG fractions derived either from an active pemphigus mouse model (AK23, a monoclonal autoantibody targeting Dsg3, (35)) or from pemphigus patients (PV-IgG) was evaluated by dispase-based dissociation assay in cultured immortalized HaCaT and primary normal human epidermal keratinocytes (NHEK) (**Figure 1A**). In contrast to control conditions, in which confluent monolayer exposed to PBS (vehicle) or control IgG (c-IgG) obtained from healthy volunteers remained intact, application of both AK23 and PV-IgG (PV-1 and PV-2) for 24 h induced loss of cell adhesion.

In HaCaT cell monolayers, the effect of pathogenic IgG-fractions on desmosomes was visualized by Dsg3 immunostaining. In all controls (vehicle and c-IgG), linearly organized Dsg3 staining at cell borders was observed. In contrast, 24 h incubation with autoantibodies induced fragmentation of the Dsg3 staining pattern along the borders (**Figure 1B**). Under the same conditions, NHEKs exhibited changes similar to those observed in HaCaT cells (**Supplementary Figure 1**).

### ST18 Overexpression Promotes PV-IgG-Mediated Loss of Keratinocyte Cohesion

Recently, it has been reported that a PV-associated SNP leads to a significant increase in the expression of ST18 gene. As a consequence, in NHEK cells PV-IgG caused loss of keratinocyte adhesion as well as the release of key inflammatory molecules (29). In order to validate the effect of ST18 overexpression in human HaCaT keratinocytes, cells were transiently transfected with either empty vector (EV) or with ST18 expression vector. Consequently, the intact monolayers were exposed to control or pemphigus autoantibodies. Twenty four hours later, dispase-based dissociation assay was performed. Upon ST18 overexpression, the fragmentation of cell monolayers increased significantly in response to PV-IgG and AK23 incubation (**Figures 2A,B**). For each single experiment, sufficient transfection efficiency was confirmed by PCR analysis (**Figure 2C**).

### Effect of Pemphigus Autoantibodies and ST18 Overexpression on Cytokine Release

Besides preserving the structure of the skin by forming strong intercellular junctions and thus functioning as a physical barrier, keratinocytes also mediate inflammation by synthesizing and secreting various cytokines in response to physical or chemical damage, inflammatory signals or UV radiation (36– 38). Therefore, in the present study, the pattern of cytokines expressed by non-transfected or transiently transfected human keratinocytes exposed to either c-IgG or pathogenic IgG was further analyzed.

Twenty four hours after reaching confluency HaCaT and NHEK cells were exposed to pathogenic IgG for another 24 h. Respective controls (PBS or c-IgG treated) were run in parallel. After the incubation was completed, supernatants were collected and simultaneously assessed for multiple cytokines by Luminex assay. Cytokine secretion of IL-1α, IL-6, TNF-α, and IFNγ in both HaCaT and primary NHEK cells was not affected significantly (**Figures 3A,B)**. In case of IFN-γ, independent on the treatment, cytokine release in HaCaT was either not altered, similar as in NHEK cells, or often under the detection limit (**Supplementary Figures 2A,B**). The latter observation was in line with a recent study in PV-patients, where the serum levels of IFN-γ were undetectable (39). Taken together, these data demonstrate that at least in vitro, cytokine release is not strictly required for loss of keratinocyte cohesion.

It was reported that ST18 overexpression in NHEKs results in a PV-IgG-mediated release of key inflammatory cytokines (29). In order to validate the effect of ST18 overexpression in human HaCaT keratinocytes, cells were transiently transfected with either EV or ST18- expression vector. For the recent study, experiments were carried out in HaCaT keratinocytes since the transfection efficiency in NHEK was not consistent. The intact monolayers were consequently exposed to IgG fractions. Supernatants were collected and secretion profiles of IL-1α, IL-6, TNF-α, and IFN-γ were analyzed by Luminex assay. ST18 overexpression induced no significant increase in the secretion of any cytokine analyzed (**Figure 3C**). However, IL-1α and IL-6 levels were augmented in some experiments, but not in others. When compared to c-IgG treatment, the release of TNFα was even reduced significantly upon PV-IgG application in both EV and ST18 transfected monolayers. Secretion of IFN-γ was either undetectable or not affected by ST18 overexpression (**Supplementary Figure 2C**).

### Modulation of ERK Signaling Upon Treatment With PV-IgG Fractions and ST18 Overexpression

Several signaling pathways, including ERK signaling, have been implicated in the pathogenesis of pemphigus (16, 17). In order to evaluate ERK activity upon pemphigus autoantibody treatment, 24 h after differentiation with 1.8 mM Ca2+, confluent NHEK

vector) and negative (H2O) controls were run in parallel.

monolayers were incubated with different control or pathogenic IgGs for 30 min. ERK phosphorylation was investigated by triton fractionation and subsequent western blot analysis. The bands of phosphorylated and total ERK were densitometrically quantified and ERK activity was expressed as a ratio of phosphorylated-ERK to non-phosphorylated total ERK. Analysis revealed that compared to c-IgG, treatment with PV-2 IgG but not with AK23 induced significant activation of ERK (**Figure 4A**).

To evaluate the effect of ST18 overexpression on ERK signaling, 24 h after transfection HaCaT cells were incubated for 30 min with different IgGs (**Figure 4B**). Overexpression of ST18 resulted in significantly increased activity of ERK after c-IgG and AK23 treatment. Prominent but not significant elevation of ERK activity was observed in response to PV-2 application. Thus, we hypothesize that activation of ERK signaling is required to induce significant loss of cell cohesion as a result of ST18 overexpression. In order to test this hypothesis, inhibition of ERK activation by U0126, a widely used MEK inhibitor (40, 41), was performed in dispase assays using cell monolayers transiently transfected with either EV or ST18. Twenty four hours after transfection HaCaT cells were treated with 5µM U0126 for an hour followed by incubation with either c-IgG, PV-IgG, or AK23 for another 2 h. Inhibition of ERK activation completely abolished autoantibody-induced loss of cell adhesion in ST18 transfected monolayers (**Figure 4C**).

Next, to test whether overexpression of ST18 modulates baseline adhesion via ERK, dispase-based dissociation assays were performed in vehicle or U0126-treated HaCaT keratinocytes transiently transfected with either EV or ST18 expression vector. Analysis revealed that neither in EV- nor in ST18- transfected cells adhesion was affected by ERK inhibition (**Figure 4D**). This indicates that an additional factor such as PV-IgG is required to facilitate ST18/ERK-mediated loss of adhesion.

### DISCUSSIONS

Autoantibodies targeting Dsg1 and Dsg3 are pathogenic and cause blister formation by inducing structural desmosomal changes in the skin of PV patients (42). However, the mechanisms underlying disease development and the factors enhancing its manifestation have not yet been fully elucidated. Secondary

collected from cell monolayers, which were either treated with controls (Vehicle, c-IgG), AK23 or PV-IgGs, was evaluated in both HaCaT (A) and NHEK (B) cells. (C) Cytokine release by HaCaT cells transiently transfected with either EV or ST18 expression vectors and exposed to PV-IgG or AK23 was determined. All data are presented as percent of Vehicle (n ≥ 6, two-way ANOVA; \*p ≤ 0.05 vs. respective c-IgG).

factors promoting severity of the disease include non-Dsg antibodies (43) as well as genetic alterations such as recently reported ST18 SNPs (29–31) and may also entail keratinocytederived cytokine release.

Here, we investigate the effect of ST18 overexpression and cytokine secretion on PV-IgG mediated loss of adhesion. We observed that in both HaCaT and NHEK the release of key proinflammatory molecules such as IL-1α, IL-6, TNF-α, and IFN-γ is not strictly required for PV-IgG-induced loss of cell cohesion. However, ST18 overexpression did not only modulate cytokine release in some of the experiments but also altered ERK activity.

Pemphigus, as an autoimmune disease, depends highly on the activity of T-cells, the differentiation of which is regulated by autocrine cytokine signaling and is modulated by paracrine cytokine secretion from other cell type such as keratinocytes (44, 45). In skin, the production and secretion of cytokines has been

dissociation assays (n ≥ 8, two-way ANOVA, \*p < 0.05).

recognized long ago and therefore intensely studied (46). These pro-inflammatory cytokines can also modulate keratinocyte behavior and could therefore be relevant for blister formation in pemphigus (47, 48).

Most studies on PV and cytokine release, however, are conflicting and report patient serum cytokine levels, which are most likely derived from T-cell signaling (49–52). Only few studies include cytokine assessment of blister fluid and only single reports evaluate production and secretion of cytokines in vitro (53). In the latter, polyclonal IgG fractions containing a plethora of antibodies, which could also include non-Dsg antibodies, were used (53). Therefore, in the present study, in addition to patient-derived PV-IgG the ability of a specific aDsg3 antibody, i.e., AK23, to induce cytokine secretion in keratinocytes was investigated. Both, AK23 and PV-IgG did not provoke significant cytokine secretion in HaCaT and NHEK cells (**Figures 3A,B**) but were efficient to impair keratinocyte adhesion. Therefore, secretion of keratinocyte-derived cytokines seems not to be primarily important for induction of loss of cell adhesion. Other antibodies such as mitochondrial antibodies or soluble FAS-ligand might change cytokine secretion profiles and thereby augment the disease as a secondary factor (54, 55).

Studies available on the transcription factor ST18 are limited. However, one of the first reports validated ST18 as a suppressor of tumor growth in breast cancer cell lines (56). Additionally, in fibroblasts, ST18 was reported to promote the expression of pro-inflammatory and pro-apoptotic genes, including cytokines (26). Furthermore, a relationship between a ST18 polymorphism and PV has now been shown to be relevant for Israelian, Egyptian (30) as well as Iranian (31) but not German (30) or Chinese (57) populations. In this respect, it was reported that increased loss of adhesion was associated with cytokine secretion in primary NHEK cells after introduction of ST18 expression vector and incubation with PV-IgG (29). Similarly, in HaCaT cells, we also detected decrease of cell adhesion upon simultaneous ST18 overexpression and pathogenic autoantibody treatment. However, we found elevated cytokine secretion in some but not all experiments following ST18 overexpression independently of IgG treatment. The discrepancy between both studies could possibly be based on the usage of different cell lines since in the study of Vodo et al. (29) NHEK cells were used whereas our experiments were performed with HaCaT cells. Nevertheless, these results indicate that mechanisms independent of cytokine secretion may also contribute to ST18-induced enhancement of adhesion loss.

Activation of several signaling pathways regulates desmosome stability and is involved in pemphigus skin blistering (58, 59). We focused on ERK because it was observed previously that this pathway is activated only when antibodies against Dsg1, which are known to be primarily important for epidermal blistering in pemphigus, were present in autoantibody fractions (1, 16, 17, 60). Similarly to ST18, ERK modulation also was shown to be associated with cytokine signaling (61–65) and could thereby link desmosome destabilization and ST18 expression. Here we show, that expression of ST18 induced significant ERK activation after c-IgG and AK23 treatment, which led to the hypothesis that ST18 promoted increase in disease severity is mediated by ERK activation independent of IgG treatment. In order to validate this hypothesis, ERK activation was inhibited by U0126. The mediator abolished loss of cell cohesion in monolayers transfected with ST18 and incubated with pemphigus antibodies indicating that ST18-mediated loss of adhesion is at least in part induced by ERK signaling pathway modulation.

Mechanisms underlying loss of cell adhesion in response to AK23 or PV-IgG may differ as the latter contain antibodies against Dsg1 and possibly against various other antigens (43). Moreover, it has been shown that in contrast to antibodies targeting Dsg3 such as AK23, which are capable to prevent homophilic interactions between Dsg3 molecules (66), antibodies targeting Dsg1 were not observed to inhibit Dsg1 interactions (34).

We previously reported that ERK was not activated by AK23 or mucosal-dominant PV-IgG both of which are targeting Dsg3, but rather by IgG fractions containing aDsg1 antibodies (17). Here we confirmed these results as we detected increased ERK phosphorylation after incubation with mucocutaneous PV-IgG but not AK23 in NHEK cells. However, the underlying ERKdependent mechanisms relevant for pemphigus skin blistering are not yet completely understood. Interestingly, upon ST18 overexpression in HaCaT monolayers, ERK was activated under AK23 as well as c-IgG treatment, suggesting that ST18 may generally modulate ERK signaling. Nevertheless, further analysis revealed that baseline adhesion in ST18 transfected cells was unaffected by ERK inhibition (**Figure 4D**) indicating that PV-IgG is required as an additional factor to facilitate ERK-mediated loss of adhesion in response to ST18 overexpression.

Taken together, our study adds new insight on the mechanisms by which ST18 may contribute to pemphigus pathogenesis and indicates that altered signaling mechanisms regulating desmosomal adhesion may render keratinocytes more susceptible to autoantibody-induced loss of cell adhesion.

### ETHICS STATEMENT

Human sera were collected and used in accordance with recommendations of the ethics committee of the Philipps-Universität Marburg, Marburg, Germany. Name of the indicated project: Phänotypische und funktionelle Analyse von Immunzellen des peripheren Bluts beim Pemphigus vulgaris.

### AUTHOR CONTRIBUTIONS

MR and EW performed the experiments, participated in study design, interpreted results, and wrote the manuscript. RS isolated the primary NHEK cells. JW designed the experiments, interpreted results, revised the manuscript and provided the funding. AY, NS, OS, and ES revised the manuscript and actively participate in the scientific discussions.

### FUNDING

The study was supported by DFG FOR 2497 to JW and AY.

### ACKNOWLEDGMENTS

The authors thank Martina Hitzenbichler (LMU München) and Mariola Dragan (JMU, Würzburg) for their excellent technical assistance. Additionally, we are very thankful to Prof. Dr. med. Michael Hertl and Prof. Dr. med Rüdiger Eming for providing us with a patients' sera.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00770/full#supplementary-material

Supplementary Figure 1 | NHEK monolayers treated with c-IgG, AK23 and PV-IgG were immunostained for Dsg3. Representative immunofluorescence images from n ≥ 4. Bar scale is 20µm.

### REFERENCES

Supplementary Figure 2 | Release of IFN-γ is unaffected by pemphigus autoantibody application or ST18 overexpression. Confluent keratinocyte monolayers were exposed to PV-IgG or AK23 for 24 h. Respective controls were run in parallel. IFN-γ concentration was assessed in HaCaT (A) and NHEK (B) cells. (C) HaCaT cells transiently transfected with control (EV) or ST18 expression vectors were treated as indicated. 24 h later, the release of IFN-γ was evaluated. Secretion of IFN-γ is presented as raw data (pg/ml), as each data point represents the mean of a single experiment. A dotted red line depicts the detection limit of the cytokine assay; n ≥ 6.

Pemphigus disease among Iranian population. Exp Dermatol. (2018) 7:1395– 8. doi: 10.1111/exd.13778

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Radeva, Walter, Stach, Yazdi, Schlegel, Sarig, Sprecher and Waschke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Current Clinical Trials in Pemphigus and Pemphigoid

Kentaro Izumi 1,2, Katja Bieber 2 and Ralf J. Ludwig2 \*

1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan, 2 Lübeck Institute of Experimental Dermatology, Center for Research on Inflammation of the Skin, University of Lübeck, Lübeck, Germany

Autoimmune bullous dermatoses (AIBDs) are a group of rare chronic inflammatory skin diseases, which clinically manifest as blisters and erosions of the skin and/or mucosa. Immunologically, AIBDs are characterized and caused by autoantibodies targeting adhesion molecules in the skin and mucosa. According to the histological location of the blistering, AIBDs are classified into the following two main subtypes: pemphigus (intraepidermal blistering) and pemphigoid (subepidermal blistering). Most AIBDs were potentially life-threatening diseases before the advent of immunosuppressive drugs, especially systemic steroid therapies, which suppress pathogenic immunological activity. Although there have been recent advancements in the understanding of the pathogenesis of AIBDs, glucocorticosteroids and/or adjuvant immunosuppressive drugs are still needed to control disease activity. However, the long-term use of systemic immunosuppression is associated with major adverse events, including death. Based on the growing understanding of AIBD pathogenesis, novel treatment targets have emerged, some of which are currently being evaluated in clinical trials. Within this article, we review the current clinical trials involving pemphigus and pemphigoid and discuss the rationale that lead to these trials. Overall, we aim to foster insights into translational research in AIBDs to improve patient care.

#### Keywords: autoimmune blistering skin disease, pemphigus, pemphigoid, clinical trial, treatment

## INTRODUCTION

Autoimmune bullous dermatoses (AIBDs) are a heterogeneous group of skin diseases that are characterized and caused by autoantibodies targeting adhesion molecules in the skin and/or mucous membranes. Depending on the targeted adhesion molecules and the location of the blistering, AIBDs are classified into the following two major types: pemphigus diseases with autoantibodies targeting desmosomal proteins (1) and pemphigoid diseases with autoantibodies targeting the structural proteins of dermal-epidermal junction (2). In pemphigus diseases, including pemphigus vulgaris (PV) and pemphigus foliaceus (PF), autoantibody binding leads to the disruption of epidermal adhesion, resulting in the clinical finding of flaccid blisters and the histological finding of intraepidermal blistering (1). In pemphigoid diseases, the linear deposition of autoantibodies along the dermal-epidermal junction causes subepidermal blistering, resulting in tense blisters. Based on the target molecules of the autoantibodies and the clinical manifestations, pemphigoid diseases are classified as bullous pemphigoid (BP), mucous membrane pemphigoid (MMP), epidermolysis bullosa acquisita (EBA), anti-laminin-γ1/p200 pemphigoid (p200), pemphigoid gestationis (PG), lichen planus pemphigoides (LPP), linear IgA bullous dermatosis (LAD), and dermatitis herpetiformis (DH), which is associated with gluten-sensitive enteropathy and characteristic granular IgA deposits in the upper dermis (3–5).

#### Edited by:

Takashi Hashimoto, Osaka City University, Japan

#### Reviewed by:

Raghavendra Rao, Manipal Academy of Higher Education, India Pilar Iranzo, Hospital Clínic de Barcelona, Spain Cassian Sitaru, Freiburg University Medical Center, Germany Jun Yamagami, Keio University Hospital, Japan Branka Marinovic, University Hospital Centre Zagreb, Croatia

> \*Correspondence: Ralf J. Ludwig<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology

> Received: 29 January 2019 Accepted: 16 April 2019 Published: 03 May 2019

#### Citation:

Izumi K, Bieber K and Ludwig RJ (2019) Current Clinical Trials in Pemphigus and Pemphigoid. Front. Immunol. 10:978. doi: 10.3389/fimmu.2019.00978

**62**

Although the advent of systemic steroid therapy significantly improved the prognosis of AIBDs, these groups of diseases are still potentially life-threatening, now mainly due to adverse events resulting from corticosteroid treatment (6). Due to the chronicity of AIBDs, the prolonged administration of systemic steroids is often needed to induce and maintain clinical remission, leading to various adverse effects such as cytopenia, diabetes mellitus, osteoporosis, hypertension, gastrointestinal ulcers, and infections due to immunosuppression (7). Furthermore, severe infection induced by an immunocompromised state is one of the most important causes of death during AIBD treatment (8, 9). Thus, the development of alternative treatment modalities that have fewer adverse events is urgently needed for the treatment of AIBD patients.

Based on the growing understanding of AIBD pathogenesis (1, 10, 11), novel therapeutic targets and/or treatment modalities have been identified (12–15). Some of those new treatments are currently being evaluated in clinical trials. To foster translational AIBD research, in this article, we summarize the current clinical trials involving pemphigus and pemphigoid diseases. For this purpose, we searched clinicaltrials.gov1 and the EU clinical trials register2 through December 2018 and selected clinical trials on pemphigus or pemphigoid disease with the status "recruiting (the study is currently recruiting participants)," "active, not recruiting (the study is ongoing, and participants are receiving an intervention or being examined, but not currently being recruited or enrolled)," and "completed (the study has ended normally, and participants are no longer being examined or treated)," and an initiation in 2013 or later (**Tables 1**, **2**, **Figure 1**). To provide a more comprehensive overview, we list the trials before this time frame in **Supplementary Tables 1, 2**. In addition, we included one clinical trial that is registered with the Australia and New Zealand Clinical Trials Registry, which was recently presented at the 5th International Pemphigus and Pemphigoid Foundation Scientific Conference in Orlando (16).

### PEMPHIGUS

### Anti-CD20 in Pemphigus (Rituximab or Ofatumumab)

Rituximab is a human chimeric IgG1 monoclonal antibody targeting CD20, which is a cell surface marker expressed by B cells (17). Rituximab exerts its treatment effects via the depletion of B-cells following its binding to CD20. Regarding the treatment of pemphigus with rituximab, case reports have suggested the efficacy of rituximab as a second- or third-line therapy (18– 20). There are two main rituximab regimens for pemphigus; one regimen, which is based on the lymphoma protocol, is composed of a total of 4 doses of 375 mg/m2 weekly infusions (21), while the other regimen, which is based on the rheumatoid arthritis protocol, consists of a total of two doses of 1,000 mg (or 500 mg) biweekly intravenous infusions (22). Prior to evaluating the efficacy of a single cycle regimen of rituximab in refractory pemphigus cases, a multicenter, single arm, phase 2/3 clinical trial was conducted in France (23). At 3 months after rituximab treatment, 18 (86%) of 21 cases achieved complete remission (CR). Furthermore, 18 (86%) of 21 cases maintained CR after a median follow-up of 34 months. In addition, 8 of these 18 cases did not receive systemic corticosteroid therapy. To evaluate the safety and efficacy of rituximab in a controlled clinical trial, a prospective, parallel-group, open-label, randomized, phase 3 clinical trial of rituximab as a first-line treatment for moderate to severe cases of pemphigus (**NCT00784589**) was initiated (**Table 1**). Patients with PV or PF were randomized to receive either oral prednisolone at 1.0 to 1.5 mg/kg/day that was tapered over 12–18 months or oral prednisolone at 0.5–1.0 mg/kg/day that was tapered over 3–6 months plus rituximab 1 g on days 0 and 14 and 0.5 g at months 12 and 18. The primary endpoint was the proportion of patients who achieved CR off-therapy at month 24. The results of this clinical trial were recently reported (24). Regarding the primary endpoint, 34% of patients in the prednisolone arm achieved CR at month 24. In the rituximab plus prednisolone arm, CR was reached by 89% of the patients; the difference was significant. Furthermore, fewer grade 3–4 adverse events were observed in the rituximab plus prednisone group than in the prednisone alone group (27 events in 16 out of 46 patients; mean 0.59 [SD 1.15] vs. 53 events in 29 out of 44 patients; mean 1.20 [1.25]). Overall, this trial demonstrated that the first-line treatment of pemphigus with rituximab and lower doses of prednisolone is more effective and safer compared to high-dose prednisolone treatment. Based on the results of this clinical trial, the Food and Drug Administration approved the expansion of health insurance coverage of rituximab for pemphigus vulgaris in the United States.

In addition to rituximab, ofatumumab, a fully human anti-CD20 monoclonal antibody, has been demonstrated to be safe and effective for the treatment of autoimmune disorders other than pemphigus or pemphigoid (25). Preclinical studies suggested that ofatumumab shows a high affinity for CD20 and activates complement-dependent cytotoxicity (26). A double-blind, randomized, placebocontrolled, phase 3 clinical trial evaluating the efficacy of ofatumumab in pemphigus was completed in January 2018 (**NCT01920477**). This clinical trial enrolled moderate

1https://clinicaltrials.gov/

2https://www.clinicaltrialsregister.eu/

**Abbreviations:** AIBDs, autoimmune bullous dermatoses; PV, pemphigus vulgaris; PF, pemphigus foliaceus; BP, bullous pemphigoid; EBA, epidermolysis acquisita; p200, anti-laminin-γ1/p200 pemphigoid; PG, pemphigoid gestationis; LPP, lichen planus pemphigoides; LAD, linear IgA bullous dermatosis; DH, dermatitis herpetiformis; RA, rheumatoid arthritis; MS, multiple sclerosis; CR, complete remission; MMF, mycophenolate mofetil; BAFF, B cell activating factor of the tumor necrosis factor family; BAFF-R, B cell activating factor of the tumor necrosis factor family receptor; BCR, B cell receptor; PDAI, Pemphigus Disease Area Index; FcRn, neonatal Fc receptor; IVIg, intravenous immunoglobulin; BTK, Bruton's tyrosine kinase; Tregs, regulatory T cells; GVHD, graft vs. host disease; Dsg, desmoglein; PRP, Platelet-rich plasma; CAR, chimeric antigen receptor; Dsg3-CAART, Dsg3 chimeric autoantibody receptor T cell; IL, interleukin; EAE, experimental autoimmune encephalomyelitis; DAS, disease activity score; NLRP3, nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing 3; BPDAI, bullous pemphigoid disease area index; ECP, eosinophil cationic protein; SYK, spleen tyrosine kinase; FcγR, Fc gamma receptor; LTB4, leukotriene B4.

TABLE 1 | Current clinical trials in pemphigus.

PV, pemphigus vulgaris; PF, pemphigus foliaceus; BAFF-R, B cell activating factor of the tumor necrosis factor family receptor; FcRn, neonatal Fc receptor; BTK, Bruton's tyrosine kinase; MMF, mycophenolate mofetil; IMPDH, inosine 5′ -monophosphate dehydrogenase; Tregs, regulatory T cells; PRP, Platelet rich plasma.

No clinical trials are being performed for any other pemphigoid diseases. FcRn, neonatal Fc receptor.

to severe PV patients with a history of at least failure of tapered steroid therapy. Participants received a stable dose of prednisone/prednisolone from a minimum of 20 mg/day up to a maximum of 120 mg/day or 1.5 mg/kg/day for 2 weeks prior to randomization. Thirty-five patients with PV were randomized to receive either ofatumumab or a placebo. Ofatumumab at 40 mg was subcutaneously injected at week 0 and week 4. From week 8, subjects were subcutaneously administered ofatumumab 20 mg every 4 weeks through week 56. The primary endpoints were the time to sustained CR on minimal steroid therapy (prednisone/prednisolone dose to <10 mg/day) and the duration of CR on minimal steroid therapy. The results of this study have thus far not been reported.

Similar to these two trials, another double-blind, randomized, phase 3 clinical trial of adjuvant rituximab vs. mycophenolate mofetil (MMF) as a therapy for pemphigus is ongoing (**NCT02383589**). In that study, 135 PV patients who are receiving standard systemic steroid treatment (oral prednisone 60–120 mg/day or equivalent) will randomly receive either (i) MMF and a rituximab-matched placebo or (ii) an MMFmatched placebo and rituximab. MMF is orally administered twice daily from day 1 to week 56. The initial dose of MMF is 500 mg, and the dose will be titrated to achieve a goal of 1,000 mg. Rituximab is administered at a dose of 1,000 mg intravenously on days 1, 15, 168, and 182. The primary endpoint is the proportion of patients who achieve sustained CR.

### VAY736 in Pemphigus

B cell activating factor of the tumor necrosis factor family (BAFF) is a crucial cytokine for regulating B cell development in mice and humans (27, 28). BAFF functions by binding to the BAFF receptor (BAFF-R), B cell maturation antigen, and tumor necrosis factor receptor superfamily member (29). Although at physiological concentrations BAFF cannot rescue B cell apoptosis due to a strong B cell death signal, which is transduced via the B cell receptor (BCR) stimulated by autoantigens, at higher concentrations, BAFF causes the survival of autoreactive B cells, which contributes to the pathogenesis of autoimmune diseases (27, 30, 31). For example, elevated BAFF serum levels have been detected in various autoimmune diseases such as RA, systemic lupus erythematosus, Sjögren's syndrome, and systemic sclerosis (32–34). Taken together, BAFF is a likely therapeutic target for the treatment of autoimmune diseases. Regarding clinical translation, the anti-BAFF antibody belimumab was licensed for the treatment of systemic lupus erythematosus in 2011 (35). However, clinical trials of belimumab have not been conducted in pemphigus thus far. Alternatively, with regard to the inhibition of BAFF, its receptor can be blocked to achieve similar results. BAFF-R signaling drives B cell differentiation, proliferation and survival (36). VAY736 is a novel, defucosylated, human IgG1 monoclonal antibody targeting BAFF-R, providing both enhanced antibody-dependent cellular cytotoxicity-mediated depletion of B cells and the blockade of BAFF. To investigate the safety, tolerability and efficacy of VAY736 in PV, a randomized, placebo-controlled, double-blind,

FIGURE 1 | Schematic representation of the pathophysiology and new therapeutic targets of pemphigus and pemphigoid diseases. The pathophysiology of pemphigus and pemphigoid diseases consists of the following three phases: (1) CD4+T cells promote autoreactive B cell activation, proliferation, and differentiation to plasma cells that produce pathogenic autoantibodies. (2) Circulating pathogenic antibodies are transferred to the dermal epidermal junction or intracellular space of the epidermis. Neonatal Fc receptor (FcRn) plays a role in prolonging the half-life of IgG antibodies during this phase. (3) After the binding of pathogenic autoantibodies to target molecules, pro-inflammatory cells such as granulocytes and macrophages are recruited to the immune complex in lesional skin by chemokines (e.g., eotaxin). Then, granulocytes elicit reactive oxygen species (ROS), elastases, and proteases, resulting in tissue damage such as blisters and/or erythema, which are clinical symptoms in pemphigoid diseases but not in pemphigus diseases. Cytokines [e.g., interleukin (IL)-1beta and IL-18] and Th17 polarization are thought to enhance local inflammation. During the antibody production phase, rituximab and ofatumumab deplete autoreactive B cells to prevent their differentiation to plasma cells. PolyTregs act on CD4+ T cells, and VAY736 and PRN1008 act on autoreactive B cells, resulting in less activation of autoreactive B cells. In the transcytosis phase, intravenous immunoglobulin (IVIg), SYNT001, and ARGX-113 saturate FcRn, contributing to the shortened half-life of pathogenic autoantibodies. In the effector phase, ixekizumab restores Th17 polarization and suppresses inflammatory augmentation. AC-203 modulates cytokines such as IL-1beta and IL-18, contributing to decreased inflammation. The inhibition of eotaxin with bertilimumab ameliorates the recruitment of eosinophils to local inflammation sites in pemphigoid disease, especially bullous pemphigoid. Platelet-rich plasma (PRP) is thought to promote wound healing in erosions.

phase 2 clinical trial is currently ongoing (**NCT01930175**). In this trial, 16 mild-moderate PV patients will randomly receive either intravenous VAY736 or a placebo once. The primary endpoint is the efficacy of single cycle VAY736 administration in reducing Pemphigus Disease Area Index (PDAI) scores at week 12 compared to at the baseline. No results of this trial are currently available.

### Anti-neonatal Fc Receptor (FcRn) in Pemphigus (SYNT001, ARGX-113)

FcRn plays an essential role in regulating host circulating IgG levels (37). FcRn protects IgG from intracellular digestion, leading to the prolongation of its half-life. This "IgG recycling system" is essential for host defense. However, it also maintains the concentration of circulating pathogenic IgG in various autoimmune diseases, including AIBD. Specifically, FcRndeficient mice do not (or to a lesser extent) develop experimental AIBD after the injection of AIBD-inducing antibodies (10). Interestingly, saturation of the FcRn by the administration of high-dose human IgG (IVIg) reduces the pathogenic effects in antibody-transfer AIBD models. Thus, FcRn inhibition is one possible mode of action of IVIg therapy in AIBD (38, 39). Based on these observations, anti-FcRn targeting treatments have been developed. Currently, the following 2 clinical trials targeting FcRn are being conducted in pemphigus: SYNT001 (**NCT03075904**) and ARGX-113 (**NCT03334058**). SYNT001 is a humanized, deimmunized IgG4 monoclonal antibody that blocks the binding of FcRn to the Fc portion of IgG (40). ARGX-113 is a human IgG1-derived Fc-modified fragment with increased affinity for FcRn that reduces the circulating IgG concentration (41). In addition, both SYNT001 and ARGX-113 do not alter serum levels of albumin. As both trials are ongoing, no results have been published so far. Regarding **NCT03075904**, 16 PV or PF patients will be sequentially assigned to receive three different doses of intravenous SYNT001 weekly for either 5 or 14 weeks, and the primary endpoint is the count and percentage of adverse events (time frame; days 0–112 or days 0–175). At the 2018 pre-International Investigative Dermatology meeting in Orlando, the first results were reported for SYNT001. The infusion of SYNT001 in human subjects resulted in a rapid lowering of the circulating levels of IgG (mean total IgG reduction of 56% by day 30) with good safety and tolerability. Furthermore, 5 of the 7 subjects showed a reduction in disease activity by day 42 (42). Regarding **NCT03334058**, 12 newly diagnosed or relapsed PV patients will receive ARGX-113 intravenously. The primary endpoints are safety and tolerability up to 17 weeks.

### PRN1008 in Pemphigus

B cell receptor signaling is a key player in B cell development and function. Bruton's tyrosine kinase (BTK) belongs to the Tec family of nonreceptor tyrosine kinases, and it is a vital component of B cell receptor signaling (43). BTK is predominantly expressed by B-lymphocytes from the pre-B cell stage to the mature B cell stage (44, 45). Based on the crucial role of BTK in B cell function, it has been identified as a potential target for the treatment of autoimmune disorders. Several studies have shown that ibrutinib, which is one of the BTK inhibitors under development, binds to BTK with high affinity, leading to the inhibition of B cell receptor signaling and resulting in the reduction of B cell activation involved in autoimmunity (46). In pemphigus treatment, a previous case report showed that ibrutinib improved clinical cutaneous lesions of paraneoplastic pemphigus complicated with chronic lymphocytic leukemia (47). However, there have been no case reports or series investigating the effects of this BTK inhibitor in PNP, PV and/or PF. PRN1008 is another BTK inhibitor that was evaluated in a phase 1 clinical trial that enrolled 80 healthy volunteers (Australian New Zealand Clinical Trials Registry No. **ACTRN12614000359639**). In that study, PRN1008 was considered safe. Regarding the pharmacokinetics and pharmacodynamics, BTK occupancy of more than 90% was observed within 4 h after dosing in both the single and multiple dose regimens and was closely correlated with the maximum plasma concentration (48). Based on the promising results from the phase 1 trial, an open-label, singlearmed, phase 2 clinical trial of PRN1008 for PV treatment has been conducted (**NCT02704429**). In this trial, 27 pemphigus patients (including PV and PF) received PRN1008 orally for 12 weeks with a 12-week follow-up period. The primary efficacy endpoint was the initial control of disease activity during the first 4 weeks of therapy, during which new lesions cease to form and existing lesions begin to heal, without the need for prednisoneequivalent corticosteroid doses >0.5 mg/kg/day. More than 50% of patients have achieved control of disease activity within 4 weeks of starting PRN1008 thus far3 . Furthermore, Principia has recently initiated a global, randomized, double-blind, placebocontrolled, pivotal phase 3 clinical trial (**NCT03762265**) in approximately 120 PV or PF patients to evaluate PRN1008 vs. a placebo. The primary endpoint is the proportion of participants who are in CR from week ≤ 29 to 37 with a prednisone dose of ≤5 mg/day.

### Polyclonal Regulatory T Cells (PolyTregs) in Pemphigus

Among the T cell subtypes, regulatory T cells (Tregs) play an important role in regulating the immune system and preventing autoimmune disease development. Based on findings in animal models, including AIBD (49, 50), it is hypothesized that naturally occurring Tregs may be utilized for the treatment of autoimmune diseases and potentially replace the use of chronic immunosuppressive therapies that are associated with multiple adverse effects. A clinical trial of Treg adoptive therapy was started by treating graft vs. host diseases (GVHD) with expanded allogeneic Tregs (51). There has been a small study demonstrating the safe administration of autologous Tregs with decreased disease activity in patients with insulin-dependent diabetes (52). Subsequently, Brunstein et al. reported that HLA-matched umbilical cord blood-derived Tregs decreased the incidence of GVHD after double umbilical cord blood transplantation (53), indicating the potential efficacy and safety profile of the passive transfer of autologous Tregs in humans. The application of Tregs for lupus, cancer and organ transplantation

3https://ir.principiabio.com/news-releases/news-release-details/principiabiopharma-reports-positive-prn1008-phase-2-top-line

has been addressed (54). The suppressive effects of Tregs have been reported by studies using an active PV mouse model (55). A recent study indicated that Treg induction via the anti-CD28 antibody reduces pathogenic IgG directing desmoglein (Dsg) 3 in the HLA-DRB1∗ 04:02- transgenic PV mouse model (56). Although Tregs are expected to improve PV symptoms, there are no case reports or case series of autologous Treg injections in patients with PV to date. To evaluate the effects of Tregs on the manifestation of PV, a nonrandomized, open-label, phase 1 clinical trial is ongoing (**NCT03239470**). In this trial, 12 PV or PF patients will receive one infusion of autologous expanded Tregs (CD4+CD127lo/negCD25+) at one of the following doses: either 2.5 x 108 poly Tregs or 10 × 108 poly Tregs. The primary endpoint is the number of significant grade 3 or higher adverse events by week 52. The results from this study have thus far not been reported.

### Comparison of Injections of Steroids to Autologous Platelet-Rich Plasma (PRP) in Oral Erosions in PV

Oral erosive lesions are a major hallmark of PV. These oral lesions cause severe pain, resulting in problems with eating and drinking (57). To improve oral lesions in PV, adjuvant topical or intralesional steroids are used, and based on evidence from case reports, the treatment is effective (58, 59). To date, however, no controlled clinical trial of the treatment of oral lesions in PV has been performed. PRP, which is concentrated plasma derived from autologous whole blood, is believed to promote wound healing (60). In a case report series, El-Komy et al. reported that six of seven PV patients showed improvement of their oral PDAI scores after PRP intralesional injection (61). To evaluate the effects of PRP in PV, an open-label, doseescalation, multicenter phase 1 trial using autologous PRP has been conducted in adults with active PV (**NCT02828163**). This clinical trial was designed to compare the effects of PRP to those of intralesional steroid injection. Eleven PV patients received a 10 mg/mL triamcinolone injection on one side of the oral mucosa and a 1-mL PRP injection on the other side every 2 weeks for 3 months. The primary endpoint was the improvement of oral PV lesions in 3 months. Nine out of 11 participants completed the protocol, and 7 (78%) of those 9 patients showed improvement in oral PDAI and/or pain scores at the PRP injection sites. Although PRP resulted in clinical improvement, as in the previous study, there were no significant differences between PRP and intralesional steroid injections (62). Thus, autologous PRP might be used for the treatment of resistant oral erosions in pemphigus patients when intralesional steroid injection is contraindicated.

### Future Potential Clinical Trials in Pemphigus

B cell depletion therapy using an anti-CD20 monoclonal antibody leads to short-term remission in the majority of pemphigus patients; however, we often observe relapsing disease after treatment. Although disease remission is related to the depletion of circulating Dsg3-specific B cells, the expansion of the same pathogenic B cell clone is observed during relapsing disease (63). Therefore, to maintain complete remission in PV, targeted removal of anti-Dsg3 memory B cells is essential. Recently, chimeric antigen receptor (CAR) technology was developed and resulted in novel treatments that led to the prolonged remission of refractory B cell leukemia and lymphoma. Adapted from that strategy, Dsg3 chimeric autoantibody receptor T cell (Dsg3- CAART) therapy has been reported to result in serological and histological improvements in experimental pemphigus mice without detectable off-target toxicity (15). A phase 1 clinical trial of Dsg3-CAART in PV patients is planned to investigate its safety and therapeutic potential.

## PEMPHIGOID

### Ixekizumab in BP

Th17 cells were first identified by their production of interleukin (IL)-17 (64, 65). A previous study indicated that the transfer of IL-17-producing Th17 cells into healthy SJL/J mice induced experimental autoimmune encephalomyelitis an animal model of MS (66). The prominent role of the Th17 polarization of T cells, as well as that of IL-17, in inflammation and autoimmunity was recognized after this observation. In addition to EAE, the Th17-IL-17 axis is also important in the pathogenesis of RA (67–69). Notably, neutralizing anti-IL-17 antibodies significantly decreased joint inflammation, cartilage destruction, and bone erosion in a collagen-immunized arthritis mouse model. Several recent studies suggest that Th17 cells and their cytokines also play roles in the pathogenesis of AIBD, including BP (70). More specifically, Le Jan et al. reported that IL-17A expression was significantly higher in both serum and lesional skin in BP patients than in normal healthy individuals (71). Furthermore, Chakievska et al. reported that IL-17A -/- mice were protected from the development of experimental BP induced by the transfer of anti-COL17 IgG (72). Taken together, these results indicate that IL-17A is a potential target for the treatment of BP. Ixekizumab is a recombinant fully humanized monoclonal antibody targeting IL-17 and is used for the treatment of psoriasis (73). However, there are neither case reports nor clinical studies reporting the effect of ixekizumab or other IL-17-targeting compounds on BP. To determine the efficacy of ixekizumab for the treatment of BP, an open-label, single-group phase 2 clinical trial is currently ongoing (**NCT03099538**). In this trial, twelve BP patients will receive subcutaneous injections of 160 mg ixekizumab on day 0, and then they will receive 80 mg of ixekizumab every 2 weeks until week 12. The primary endpoint is the median time from the start of treatment to the cessation of blister formation during the 12 weeks of therapy. There is no available report of the results of this trial to date.

### NPB-01 in BP Unresponsive to Corticosteroids

With regard to pemphigus, the efficacy and safety of IVIg in refractory BP patients has been suggested by case reports and case report series (74–76). Based on these observations, several guidelines and consensus statements recommend the use of IVIg for refractory BP (77–79). Recently, Sasaoka Izumi et al. Current Clinical Trials in Pemphigus and Pemphigoid

et al. reported that IVIg reduced serum IL-6 concentrations, circulating pathogenic IgG levels, and skin inflammation disease activity in an active BP mouse model (80). Kamaguchi et al. reported that an anti-idiotypic antibody in IVIg significantly reduced the BP180 depletion of cultured keratinocytes stimulated with BP pathogenic IgG (81). To evaluate the effectiveness of IVIg in BP, a multicenter, randomized, double-blind, placebocontrolled, phase 3 clinical trial was conducted (**NCT01408550**). In this trial, 56 BP patients were parallelly assigned to receive either IVIg or a placebo. The IVIg group received an intravenous drip infusion of human IgG at 400 mg/kg/day for 5 consecutive days. The primary endpoint was the disease activity score (DAS) of skin lesions on day 15. The results of that study have recently been published (82). The DAS on day 15 was evaluated as the primary endpoint. Although the DAS on day 15 in the IVIg group (19.8 ± 22.2) was 12.5 points lower than that in the placebo group (32.3 ± 31.5), the difference between the groups was not significant (p = 0.089). However, a post hoc analysis of covariance using the DAS on day 1 as a covariate showed a significant difference between the IVIg and placebo groups (p = 0.041). Regarding the safety of IVIg in BP, the incidence of adverse drug reactions was 37.9% (n = 11/29) in the IVIg group vs. 18.5% (n = 5/27) in the placebo group. No significant difference in the incidence of adverse drug reactions was observed between the IVIg and placebo groups (p = 0.143). No patients in either group experienced any severe adverse drug reactions. Therefore, this report suggested that IVIg is a beneficial treatment modality for refractory BP cases treated with a moderate dose of systemic steroids.

### Topical AC-203 in BP

The nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing 3 (NLRP3) inflammasome regulates the activation of caspase-1 and the release of pro-inflammatory cytokines such as IL-1β and IL-18 in macrophages (83–87). It is an essential innate immune sensor that is activated in response to various damage-associated molecular patterns (83, 84). Previous reports have suggested that dysregulation of the inflammasome attenuates several chronic diseases, including autoimmune diseases such as EAE (88) and systemic lupus erythematosus (89, 90). In addition, polymorphisms in both NLRP3 and caspase-1 recruitment domain-8 genes led to increased IL-1β production and are related to disease susceptibility and severity in RA (91). A recent study demonstrated that the expression levels of NLRP3 and inflammasome components are significantly higher in peripheral blood mononuclear cells from BP patients than in those from healthy donors (92). Furthermore, higher NLRP3 levels were positively correlated with BP disease activity. Although the precise mechanism by which NLRP3 contributes to pathogenesis in BP is still unclear, pharmacological modulation of the inflammasome pathway could be a novel therapeutic strategy in BP. However, in a mouse model of inflammatory EBA, caspase-1/11-deficient mice developed clinical disease manifestations identical to those of the wild-type controls after the injection of autoantibodies against type VII collagen (93).

To evaluate the therapeutic efficacy of caspase-1 inhibition in BP, AC-203, which is a topical ointment formulation of a modulator of the inflammasome and IL-1β pathways, was developed. To investigate the effects of AC-203 on BP, a randomized, open-label phase 2 clinical trial is currently conducted (**NCT03286582**). Forty BP patients received either topical AC-203 or clobetasol 0.05% topical ointment twice a day (time frame is 10 weeks). The primary endpoint is the incidence of adverse effects during the treatment period. No results of this trial are currently available.

### Bertilimumab in BP

While the presence of eosinophils in the dermal infiltrate is one of the histological hallmarks of BP, their contribution to the pathogenesis of BP remains to be determined (94–97). Recent data, however, support the notion that eosinophils promote subepidermal blistering in BP (98). In line with the presence of eosinophils in the dermal infiltrate in BP, elevated expression levels of IL-5, eotaxin, and eosinophil colony-stimulating factor were detected in BP blister fluids (99). Furthermore, matrix metallopeptidase 9 is secreted by eosinophils in BP lesional skin (100), eosinophil cationic protein (ECP) is detected at the dermal-epidermal junction of lesional skin, and serum concentrations of ECP correlate with BP disease activity (101, 102). Based on these findings, eotaxin, which is released upon eosinophil activation (103), has emerged as a therapeutic target for the treatment of BP.

Bertilimumab is a fully human monoclonal antibody targeting eotaxin-1 (104). Therefore, the administration of bertilimumab is expected to impair eosinophil infiltration into the skin in BP. This hypothesis has been recently tested in an open-label, single arm, phase 2 clinical trial (**NCT02226146**). Eleven moderate to severe BP patients received 10 mg/kg of bertilimumab intravenously on days 0, 14, and 28 with approximately 13 weeks of followup. The primary endpoint was safety, including the incidence of adverse effects. The results of the study were presented at the 2018 American Academy of Dermatology annual meeting. Of the 11 subjects enrolled, 2 patients withdrew consent and 9 received bertilimumab. Bertilimumab was well tolerated in all 9 subjects, and no drug-associated serious adverse events were reported4 Preliminary analyses indicated that the subjects had an 81% decline in their Bullous Pemphigoid Disease Area Index (BPDAI) scores. Based on these results, bertilimumab has been granted fast track designation for the treatment of BP5 .

### Rituximab in BP

Rituximab has a well-documented efficacy in treating pemphigus (24). However, controlled clinical trials in pemphigoid diseases, including BP, are missing. Several case reports and case report series reported good outcomes of rituximab treatment for BP. Although the efficiency of rituximab for the treatment of BP varied among the studies, the overall CR rates were

4https://www.immunepharma.com/what-we-do/bertilimumab

5https://ir.immunepharma.com/press-releases/detail/168/bertilimumab-grantedfast-track-designation-for-the

60–70% and the relapse rates were approximately 20% in BP patients receiving rituximab therapy (105, 106). To evaluate the efficacy and safety of rituximab for the treatment of BP, an open-label, prospective, phase 3 clinical trial was conducted (**NCT00525616**). Eighteen BP patients received two intravenous injections of 1,000 mg of rituximab at 15-day intervals. The primary endpoint was clinical and biological disease control for up to 2 years. To date, no results of this trial are available.

### Future Potential Clinical Trials in Pemphigoid

Spleen tyrosine kinase (SYK) is a nonreceptor cytoplasmic enzyme that is mainly expressed in hematopoietic cells and plays a pivotal role in regulating cellular responses to extracellular pathogens or antigen-immunoglobulin complexes (107). Because SYK acts downstream of activating Fc gamma receptor (FcγR), it has been considered a candidate drug target for antibodyinduced diseases, such as pemphigoid diseases. In fact, a previous study has shown that SYK inhibition led to less activation of neutrophils stimulated with immune complexes and prevented the development of skin lesions in a preclinical EBA mouse model (108). In lesional skin in pemphigoid diseases, both eicosanoid leukotriene B4 (LTB4) and complement factor C5, the precursor of anaphylatoxin C5a, are found; however, their significance in the pathogenesis of pemphigoid diseases still needs to be elucidated. A previous preclinical mouse model of pemphigoid disease indicated a critical role of LTB4 in the recruitment of polymorph nuclear cells to the dermal epidermal junction (109). It is suggested that LTB4 may closely interact with C5a in the regulation of skin inflammation. Thus, inhibiting these two factors individually or in parallel might be effective for the treatment of pemphigoid diseases. These newly identified therapeutic targets should be addressed in future clinical trials.

### REFERENCES

### CONCLUSIONS

The clinical trials discussed here, which include several trials investigating novel therapeutic targets, demonstrate that translational research in pemphigus and pemphigoid is a fastgrowing field. We thus expect that several novel treatments will be shortly available for the treatment of pemphigus and pemphigoid patients. Given the high, and thus far unmet, medical need in this field (110), this is highly encouraging and will hopefully improve the quality of life of the affected patients. In addition to the compounds and targets described here, several new targets have been recently identified in preclinical model systems, such as PDE4 or PI3Kδ inhibitors (111, 112). Hence, the preclinical pipeline is well developed and will contribute to the growing number of clinical trials in pemphigus and pemphigoid.

### AUTHOR CONTRIBUTIONS

KI and RL wrote the manuscript. KB prepared the figure. All authors read, commented on, and approved the final version of the manuscript.

### FUNDING

This work was supported by research grants from the Deutsche Forschungsgemeinschaft: Clinical Research Unit Pemphigoid Diseases (KFO 303), the Excellence Cluster Precision Medicine in Chronic Inflammation (EXC 2167-390884018) and the Uehara Memorial Foundation.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00978/full#supplementary-material

therapy of autoimmune disease. Science. (2016) 353:179–84. doi: 10.1126/science.aaf6756

based rheumatic diseases. Arthritis Rheum. (2001) 44:1313–9. doi: 10.1002/1529-0131(200106)44:6<1313::AID-ART223>3.0.CO;2-S

**Conflict of Interest Statement:** During the last 3 years, RL has received research funding from Almirall, True North Therapeutics, UCB Pharma, ArgenX, TxCell, Topadur, Incyte and Admirx and fees for consulting or speaking from ArgenX, Immunogenetics, Novartis and Lilly.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Izumi, Bieber and Ludwig. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Preferential Reactivity of Dipeptidyl Peptidase-IV Inhibitor-Associated Bullous Pemphigoid Autoantibodies to the Processed Extracellular Domains of BP180

#### Yosuke Mai, Wataru Nishie\*, Kentaro Izumi and Hiroshi Shimizu

*Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan*

#### Edited by:

*Takashi Hashimoto, Osaka City University, Japan*

#### Reviewed by: *Cassian Sitaru,*

*Freiburg University Medical Center, Germany Giovanni Di Zenzo, Institute of Dermatology Immaculate (IRCCS), Italy*

> \*Correspondence: *Wataru Nishie<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology*

> Received: *30 January 2019* Accepted: *14 May 2019* Published: *29 May 2019*

#### Citation:

*Mai Y, Nishie W, Izumi K and Shimizu H (2019) Preferential Reactivity of Dipeptidyl Peptidase-IV Inhibitor-Associated Bullous Pemphigoid Autoantibodies to the Processed Extracellular Domains of BP180. Front. Immunol. 10:1224. doi: 10.3389/fimmu.2019.01224* Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies target the hemidesmosomal components BP180 and/or BP230 in basal keratinocytes. In BP, 80 to 90% of autoantibodies target the juxtamembranous extracellular non-collagenous 16th A (NC16A) domain of BP180. Recently, the administration of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used as antihyperglycemic drugs, has been recognized to be a causative factor for BP. DPP4i-associated BP (DPP4i-BP) autoantibodies tend to target epitopes on non-NC16A regions of BP180, and the pathomechanism for the development of the unique autoantibodies remains unknown. To address the characteristics of DPP4i-BP autoantibodies in detail, we performed epitope analysis of 18 DPP4i-BP autoantibodies targeting the non-NC16A domains of BP180 using various domain-specific as well as plasmin-digested polypeptides derived from recombinant BP180. Firstly, Western blotting showed that only one DPP4i-BP serum reacted with the epitopes on the intracellular domain of BP180, and no sera reacted with the C-terminal domain of the molecule. In addition, only 2 DPP4i-BP sera reacted with BP230 as determined by enzyme-linked immunosorbent assay. Thus, DPP4i-BP autoantibodies were found to mainly target the non-NC16A mid-portion of the extracellular domain of BP. Interestingly, Western blotting using plasmin-digested BP180 as a substrate revealed that all of the DPP4i-BP sera reacted more intensively with the 97-kDa processed extracellular domain of BP180, which is known as the LABD97 autoantigen, than full-length BP180 did. All of the DPP4i-BP autoantibodies targeting the LABD97 autoantigen were IgG1, and IgG4 was observed to react with the molecule in only 7 cases (38.9%). In summary, the present study suggests that IgG1-class autoantibodies targeting epitopes on the processed extracellular domain of BP180, i.e., LABD97, are the major autoantibodies in DPP4i-BP.

Keywords: bullous pemphigoid, dipeptidyl peptidase-IV inhibitor, dipeptidyl peptidase-IV inhibitor-associated bullous pemphigoid, autoantibodies, IgG subclass, BP180, BP230, autoimmune disease

## INTRODUCTION

Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies target the hemidesmosomal components BP180 and/or BP230 in basal keratinocytes (1). In BP, almost 90% of autoantibodies target the juxtamembranous extracellular non-collagenous 16th A (NC16A) domain of BP180 (2). Recently, the administration of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used as antihyperglycemic drugs, is recognized to be a causative factor for BP (3). In 2016, a strong relationship between the onset of BP under DPP4i exposure was reported from the France Pharmacovigilance Database (4). Thereafter, retrospective case-control studies from Finland, (5) Switzerland, (6) France, (6) Israel (7), and Japan (the Japanese Adverse Drug Event Report Database) (8) also confirmed that the administration of DPP4i, especially vildagliptin, was associated with increased risk of BP development. In addition, based on the National Healthcare Insurance Agency of France, the observed frequencies of DPP4i intake in the BP population are higher than those in the general population (9). Although the increased risk of BP development after the use of DPP4i has been intensively studied, the mechanisms whereby DPP4i administration causes BP development remains to be elucidated.

We and other groups have recently reported that Japanese patients with DPP4i-associated BP (DPP4i-BP) may show unique clinical and immunological characteristics (10–14). Clinically, DPP4i-BP patients tend to show a less severe erythematous phenotype than typical non-DPP4i BP patients show. In terms of the Bullous Pemphigoid Disease Area Index (BPDAI) (15) a commonly used disease severity score for BP, patients with typical BP do not differ significantly from patients with DPP4i-BP in terms of erosion/blister scores, whereas patients with DPP4i-BP have significantly less severe erythema/urticaria than patients with typical BP have. Immunologically, DPP4i-BP autoantibodies preferentially target the mid-portion of the extracellular region of BP180 without reactivity toward the NC16A domain (10). Interestingly anti-BP180 NC16A autoantibodies may be produced during the clinical course of DPP4i-BP as a result of epitope spreading (13, 14). After the production of anti-BP180 NC16A autoantibodies, clinical manifestations of DPP4i-BP may resemble those of typical non-DPP4i-BP (13, 14). Thus, anti-BP180 NC16A autoantibodies may be observed also in DPP4i-BP.

In this study, we collected 18 cases of "pure" DPP4i-BP in which autoantibodies did not target the NC16A domain of BP180. As a result, it has been revealed that DPP4i-BP autoantibodies are IgG1-class autoantibodies which target epitopes on the non-NC16A processed extracellular domains of BP180.

## MATERIALS AND METHODS

### Patient Characteristics

BP was diagnosed based on clinical, histopathological, and immunological findings (1). The clinical and immunological characteristics of the DPP4i-BP cases in this study are listed in **Table 1** and summarized in **Table 2**. Direct immunofluorescence study showed positive results for IgG and/or C3 deposition at the basement membrane zone in all cases. The mean age was 78.8 and ranged from 57 to 93. The female/male ratio was 6:12. The mean index value of full-length BP180 ELISA was 108.2 and ranged from 60.9 to 171.7 (cutoff < 4.64). None of our cases was positive for BP180 NC16A by chemiluminescent enzyme immunoassay. BPDAI scores for blister/erosion, urticaria/erythema, and total mucosa are summarized in **Table 1**. Vildagliptin was the most common DPP4i (38.9%), followed by teneligliptin (33.3%), and Sitagliptin (27.8%) (**Table 3**).

### Preparation of Recombinant Proteins

Full-length human BP180 recombinant protein (Met1 – Pro1497) and polypeptides corresponding to the intracellular domain (Met1 to Trp467) and the C-terminus region (Leu1281 – Pro1497) of BP180 were produced using the Flp-In 293 system (Invitrogen, CA) as previously reported (10). Processed BP180 extracellular fragments of 120-kDa and 97-kDa forms, which are known as LAD-1 and LABD97, respectively, were generated by limited plasmin digestion of the full-length recombinant BP180 protein (10). Schematics of the recombinant proteins and the plasmindigested proteins are given in **Figure 1A**. Mixture substrate samples of full-length BP180, LAD-1, and LABD97 were used for Western blotting, of which even doses were confirmed by Coomassie Blue staining (**Figure 1B**).

### Immunofluorescence Study

For indirect immunofluorescence study using 1 M NaCl-split skin, normal human skin was incubated in 1 M NaCl solution for 48 h at 4◦C. Thereafter, the skin was mounted and snap-frozen in OCT compound (Thermo Fisher Scientific, MA), and 5-µm cryosections were prepared. The sections were then incubated with patients' sera (dilution 1:10–20) for 40 min at 37◦C. After washing with PBS 3 times, the sections were incubated with FITC-conjugated antibodies to human IgG (dilution 1:100) (Dako Cytomation, Denmark) for 30 min at 37◦C.

### Western Blotting

Protein samples were separated by SDS-PAGE electrophoresis using 7 or 10% SDS-polyacrylamide gel. The gels were transferred to nitrocellulose membranes (Bio Rad, CA). The membranes were blocked for 30 min at room temperature with 2% skimmed milk in TBS. After incubation with 1:200 diluted patient serum with 2% skimmed milk in TBS for 1 h at room temperature, horseradish peroxidase-conjugated secondary anti-human IgG (1:1,000) (Dako Cytomation, Denmark), IgG1 (1:500) (Thermo Fisher Scientific, MA), or IgG4 (1:500) (Thermo Fisher Scientific, MA) antibodies in the same buffer were reacted at room temperature for 1 h. Signals were visualized with Clarity Western ECL Substrate (Bio Rad, CA). Each protein band was quantified using Fiji (16). Relative intensities were calculated for each band

**Abbreviations:** BP, bullous pemphigoid; NC16A, the 16th non-collagenous domain; DPP4i, dipeptidyl peptidase-IV inhibitor(s); DPP4i-BP, dipeptidyl peptidase-IV inhibitor-associated bullous pemphigoid; ELISA, enzyme-linked immunosorbent assay; BPDAI, bullous pemphigoid disease area index.

#### TABLE 1 | List of DPP4i-BP cases.

*Full-length BP180 ELISA: Index values, cutoff*<*4.64. BP230ELISA: Index values, cutoff* < *9.0. NA, means not available.*

based on the intensity of the 180-kDa bands. Case No.11 was excluded since the intensity of the 180-kDa band was faint.

### Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA using full-length BP180 recombinant proteins was performed as previously described, with a minor modification (10). Briefly, 96-well plates (Thermo Fisher Scientific, MA) were coated with 1 µg/well of the recombinant proteins in 50 mM carbonate buffer pH 9.5 and then blocked with Blocking Reagent for ELISA (Roche, Swiss) for 2 h at room temperature. Patient sera were diluted to 1:100 and incubated for 1 h at room temperature. After washing, the plates were incubated with 1:500 diluted horseradish peroxidase-conjugated anti-human IgG antibody for 1 h at room temperature. Subsequently, the plates were reacted with a substrate solution, 3, 3′ , 5, 5′ tetramthylbenzidine dihydrochloride

single solution (Thermo Fisher Scientific, MA). After color development, the reaction was stopped with the addition of 0.12 N hydrochloric acid, and absorbance was measured at 450 nm by a microplate reader with the correlation wavelength set at 620 nm (Tecan Austria GmbH, Austria). To detect anti-BP230 autoantibodies, BP230 ELISA (MESACUP BP230 ELISA kit, MBL, Japan) was performed according to the manufacturer's instructions.

### Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, USA). Data were analyzed using two-way analysis of variance, followed by Tukey's multiple comparison test. p-values are indicated as ∗ 0.01 < p < 0.05, ∗∗0.001 < p < 0.01, and ∗∗∗∗p < 0.0001.

### RESULTS

### Preferential Reactivity of DPP4i-BP Autoantibodies to the Processed Extracellular Domain of BP180

Firstly, we performed Western blotting using the recombinant intracellular and C-terminal proteins of BP180 (**Figures 1C,D**). Only one case had autoantibodies targeting the intracellular region of BP180, and no cases had autoantibodies targeting both fragments (**Figures 1C,D**). In addition, autoantibodies targeting BP230 were only detected in two cases (11.1%) by BP230 ELISA (cutoff < 9.0) (**Tables 1**, **2**). We also confirmed that there were no IgG autoantibodies targeting the dermal side of the artificial blisters in 1 M NaCl-split skin indirect immunofluorescence (**Supplemental Figure 1**).

To identify the epitopes of IgG autoantibodies in DPP4i-BP, we performed Western blotting using mixture samples of fulllength BP180 and plasmin-digested 120-kDa (LAD-1) and 97 kDa (LABD97) fragments (**Figure 1E**). IgG autoantibodies of all cases except for case No.11 reacted to the full-length BP180 (**Figure 1E**), which is mostly consistent with the result of fulllength BP180 ELISA (**Table 1**). Interestingly, IgG autoantibodies showed more intense reactivity to LABD97 than to LAD-1 or full-length BP180 (**Figures 1E,F**). In contrast, the autoantibodies in typical non-DPP4i BP reacted almost equally to each band (**Figures 1E,F**). Since none of the cases reacted with the NC16A domain of BP180, all of the DPP4i-BP autoantibodies tested in this study showed preferential reactivities toward the processed extracellular domain of BP180.

### IgG1 Autoantibodies to LABD97 Are Major Autoantibodies in DPP4I-BP

It is known that both IgG1 and IgG4 are the predominant autoantibodies in BP (17). Therefore, we evaluated IgG1 and IgG4 autoantibody reactivities to the full-length BP180 and the plasmin-digested LAD-1 and LABD97 fragments of BP180. Although all cases had IgG1 autoantibodies targeting each form of BP180, it should be noted that significant reactivities to LABD97 were observed (**Figure 2**). In contrast, 7 of 18 cases (38.9%) had IgG4 autoantibodies targeting

various forms of BP180 (**Figure 2**). These findings suggest that IgG1 autoantibodies to LABD97 are major autoantibodies in DPP4i-BP.

### DISCUSSION

In this study, we analyzed autoantibodies of 18 DPP4i-BP cases without reactivity to the NC16A domain of BP180. Consistent with our previous study using small numbers of DPP4i-BP cases, (10) all of the autoantibodies showed positive reactivity to the processed extracellular domain of BP180. Interestingly, all of the DPP4i-BP autoantibodies tested in this study showed preferential reactivity to LABD97. A similar characteristic of autoantibodies can be found in another autoimmune blistering skin disease, linear IgA bullous dermatosis. In linear IgA bullous dermatosis, autoantibodies preferentially react with neoepitopes that selectively develop on LAD-1 and LABD97, and they do not react with full-length BP180 (18, 19). BP180 can be processed by several proteases under physiological or pathological conditions, Franzke et al. (20), Hofmann et al. (21) and the N-terminal deletion resulting from cleavage within the NC16A domain of BP180 results in the production of LAD-1 (20). In addition, further C-terminal deletion produces LABD97 (21). This processing of BP180 may induce conformational changes on the molecule, which also may induce new epitopes distinct from those on the native full-length form of BP180 (22–25). Several studies have shown that antibodies targeting the 15th collagenous domain or its neighboring regions in BP180 preferentially react with LAD-1 and LABD97 (22, 23). In addition, our previous study revealed that the C-terminus processing of BP180 induces neoepitopes on the 15th collagenous domain of BP180 (24). Thus, not only does cleavage within the NC16A domain cause neoepitopes to develop on the 15th collagenous domain of BP180, but so does C-terminal processing (25). Although fine epitope mapping of DPP4i-BP autoantibodies will be necessary in future studies, the preferential reactivity of DPP4i-BP autoantibodies to LABD97 may suggest the possibility that putative epitopes may exit on the 15th collagenous domain or in its neighboring regions.

The present study also revealed that all of the DPP4i-BP autoantibodies which preferentially targeted the LABD97 fragment of BP180 were IgG1. In contrast, IgG4-subclass autoantibodies were only observed in 38.9% of our cases. These results indicate that IgG1 autoantibodies targeting epitopes on LABD97 are the main autoantibodies in DPP4i-BP. This finding was unexpected, because it is well-known that IgG1 BP autoantibodies are able to activate the complement pathway associated with the development of inflammation, (26) whereas DPP4i-BP tends to show a non-inflammatory phenotype associated with scant urticarial erythema and fewer infiltrating eosinophils than those of non-DPP4i BP, despite activating the complement at dermal-epidermal junctions (10–14). Thus, the predominance of IgG1 autoantibodies in DPP4i-BP stands in contrast to the previous concept of BP pathogenesis (26). Although further studies are necessary to resolve this issue, the present study indicates that complement activation is insufficient to induce inflammation in DPP4i-BP.

The pathogenicity of IgG4 is controversial. An experimental mouse model showed that IgG4 plays a protective role against the development of BP (27). In contrast, it is experimentally shown that IgG4 BP autoantibodies have the capacity to induce leucocyte-dependent dermal-epidermal separation (28). In addition, BP cases with only IgG4 autoantibodies have been reported (29). As this IgG4-type BP shows a noninflammatory phenotype, IgG4 autoantibodies are assumed to play complement-independent roles in BP development, such as a role in BP180 depletion in keratinocytes, the Fcdependent activation of neutrophils, or leucocyte-dependent dermal-epidermal separation (26, 28, 30). In our subclass analysis of DPP4i-BP, IgG4 autoantibodies preferentially reacted to LABD97, similar to IgG1 autoantibodies. However, the positive rates for IgG4 autoantibodies to LABD97 and full-length BP180 were lower than those of IgG1 autoantibodies, especially to full-length BP180. This discrepancy may suggest that the development of IgG1 autoantibodies might precede IgG4 autoantibody development in DPP4i-BP, although other possibilities cannot be ruled out.

### CONCLUDING REMARKS

In conclusion, major epitopes of DPP4i-BP were located in the mid-portion of the extracellular domain, and these epitopes can be presented on LABD97. All such autoantibodies were in the IgG1 class. The unique immunological characteristics of DPP4i-BP autoantibodies may help us understand the pathological mechanism behind the development of the disease.

### REFERENCES

### ETHICS STATEMENT

This report on a single patient complies with the Declaration of Helsinki. The patient gave written informed consent for publication. This study was approved by the institutional review board of Hokkaido University (institutional review board number: 016-0061).

### AUTHOR CONTRIBUTIONS

YM, WN, and KI drafted the paper and collected the clinical data. HS supervised the writing of the manuscript.

### FUNDING

This work was supported in part by Grant-in-Aid for Scientific Research (B) (#24390274 to WN), and Challenging Exploratory Research (#15K15409 to WN).

### ACKNOWLEDGMENTS

The authors thank the patient and express their appreciation to Nami Ikeshita for her technical assistance.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01224/full#supplementary-material

Supplemental Figure 1 | Indirect Immunofluorescence Study Using 1 M NaCl-split Skin. NC, Negative control using healthy individual sera.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Mai, Nishie, Izumi and Shimizu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dermatitis Herpetiformis: Novel Perspectives

Emiliano Antiga† , Roberto Maglie\* † , Lavinia Quintarelli, Alice Verdelli, Diletta Bonciani, Veronica Bonciolini and Marzia Caproni\*

*Section of Dermatology, Department of Health Sciences, University of Florence, Florence, Italy*

#### Edited by:

*Takashi Hashimoto, Osaka City University, Japan*

#### Reviewed by:

*Russell P. Hall, Duke University, United States Chika Ohata, Kurume University School of Medicine, Japan*

#### \*Correspondence:

*Roberto Maglie<EMAIL_ADDRESS>Marzia Caproni<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology*

> Received: *01 March 2019* Accepted: *21 May 2019* Published: *11 June 2019*

#### Citation:

*Antiga E, Maglie R, Quintarelli L, Verdelli A, Bonciani D, Bonciolini V and Caproni M (2019) Dermatitis Herpetiformis: Novel Perspectives. Front. Immunol. 10:1290. doi: 10.3389/fimmu.2019.01290* Dermatitis herpetiformis (DH) is an inflammatory disease of the skin, considered the specific cutaneous manifestation of celiac disease (CD). Both DH and CD occur in gluten-sensitive individuals, share the same Human Leukocyte Antigen (HLA) haplotypes (DQ2 and DQ8), and improve following the administration of a gluten-free diet. Moreover, almost all DH patients show typical CD alterations at the small bowel biopsy, ranging from villous atrophy to augmented presence of intraepithelial lymphocytes, as well as the generation of circulating autoantibodies against tissue transglutaminase (tTG). Clinically, DH presents with polymorphic lesions, including papules, vesicles, and small blisters, symmetrically distributed in typical anatomical sites including the extensor aspects of the limbs, the elbows, the sacral regions, and the buttocks. Intense pruritus is almost the rule. However, many atypical presentations of DH have also been reported. Moreover, recent evidence suggested that DH is changing. Firstly, some studies reported a reduced incidence of DH, probably due to early recognition of CD, so that there is not enough time for DH to develop. Moreover, data from Japanese literature highlighted the absence of intestinal involvement as well as of the typical serological markers of CD (i.e., anti-tTG antibodies) in Japanese patients with DH. Similar cases may also occur in Caucasian patients, complicating DH diagnosis. The latter relies on the combination of clinical, histopathologic, and immunopathologic findings. Detecting granular IgA deposits at the dermal-epidermal junction by direct immunofluorescence (DIF) from perilesional skin represents the most specific diagnostic tool. Further, assessing serum titers of autoantibodies against epidermal transglutaminase (eTG), the supposed autoantigen of DH, may also serve as a clue for the diagnosis. However, a study from our group has recently demonstrated that granular IgA deposits may also occur in celiac patients with non-DH inflammatory skin diseases, raising questions about the effective role of eTG IgA autoantibodies in DH and suggesting the need of revising diagnostic criteria, conceivably emphasizing clinical aspects of the disease along with DIF. DH usually responds to the gluten-free diet. Topical clobetasol ointment or dapsone may be also applied to favor rapid disease control. Our review will focus on novel pathogenic insights, controversies, and management aspects of DH.

Keywords: dermatitis herpetiformis, epidermal transglutaminase, coeliac disease, non-coeliac gluten sensitivity, direct immunofluorescence

## INTRODUCTION

About 135 years ago, the American dermatologist Luis Duhring reported for the first time an itching and multiform skin eruption with erythema, papules, bullae, pustules, and above all, grouped vesicles localized at typical areas (1). At that time, he had seen 15 cases of such a disease, and he decided to name it "dermatitis herpetiformis," due to its clinical similarity to herpes virus infection (1). After the identification of dermatitis herpetiformis, several years passed before the second important step in its early story until 1950, when dapsone was accidentally found to be effective for the treatment of the disease (2). Then, 50 years ago, Van der Meer described the typical IgA deposits at the papillary tips, which represent the immunological hallmark of DH as well as the clue for the diagnosis of the disease (3).

However, it was only in the 60s and, above all, in the 70s that DH was clearly related to gluten intolerance, allowing to consider the disease as the specific cutaneous manifestation of celiac disease (CD). In fact, patients with DH were shown to have in most cases intestinal changes similar to those found in CD (4) and to share the same genetic background of CD (5), and gluten-free diet (GFD) was demonstrated to improve skin symptoms (6), although more slowly than the gastrointestinal ones.

Since then, several issues about the pathogenesis and the management of DH have been pointed out (7). Among them, the identification of reliable serologic markers of the disease (8) and the discovery of epidermal transglutaminase (eTG) as the autoantigen of DH were some of the most remarkable steps (9).

DH can be defined as a disease presenting with: (i) symmetrical polymorphic lesions involving typical areas such as the extensor aspects of the limbs and sacral region, (ii) a predominant neutrophilic infiltrate at the dermal papillae at histopathology, (iii) granular IgA deposits along the dermalepidermal junction, (iv) an invariable association with CD, and (v) a response to a lifelong GFD (7).

However, despite the growing knowledge about the disease, several issues have to be clarified yet, and some of the cornerstones of DH need further discussion. For example, the specificity of IgA deposits in the perilesional skin of patients with DH could be questioned and the diagnostic algorithm should be revised (10), taking also into account the identification of non-coeliac gluten sensitivity (NCGS) as a new entity among the spectrum of gluten-related disorders (11); moreover, the pathogenic role of IgA antieTG antibodies needs further demonstration, since they may also occur in patients without DH (12). Finally, even the therapeutic role of GFD is discussed, since some authors suggest that it can be interrupted in the (rare) case of DH remission (13).

The current review will try to address or at least to focus on these and other issues that are still under debate in the context of DH. The picture that hopefully will come out would be that of a lively disease that represents both a pathogenic model of autoimmunity and a paradigmatic example of how the skin can be the mirror of an internal condition such as CD.

### THE EPIDEMIOLOGY OF DERMATITIS HERPETIFORMIS: A (RARE) DISEASE OF THE WESTERN COUNTRIES

DH is a rare disease that occurs prevalently in Caucasian individuals. In Europe and USA, the prevalence of DH ranges from 11.2 to 75.3 per 100.000, with the highest reported in Finland; whereas, the incidence ranges from 0.4 to 2.6 per 100.000 people per year (14–16). DH is extremely rare among African and Asian populations. Reasons explaining the low prevalence of DH among such populations include the absence of DH predisposing human leukocyte haplotypes (HLA) DQ2 and DQ8, which are always found in Caucasian DH patients, and the low wheat consumption in these geographic areas (17). However, in the Asians, including Chinese and Japanese populations, the association between DH and CD seems to be weaker than in Western countries, despite DH presents with similar clinical and immunopathological features. The extreme rarity of CD in these populations may have led, indeed, to overlook the diagnosis of DH and possibly underestimate the prevalence of the disease (17, 18).

During the last decades, the overall incidence of DH is significantly decreased, although the incidence of CD is increasing (19–22). One plausible explanation for the opposite trends in the epidemiology of CD and DH could be the increased awareness of CD among physicians and patients and the wide prescription of CD screening tests even in patients without typical gastrointestinal manifestations, leading to identify early patients with latent or potential CD.

DH can occur at any age, but typically occurs during adulthood, and mostly between the third and the fourth decade of life (14). Interestingly, in one Finnish study including 477 patients diagnosed with DH over a 40 year period, the patients' age at DH diagnosis was shown to significantly increase over time. This is presumably related to a parallel decrease of the annual consumption of wheat during the same period, leading to a lower lifetime gluten load (16).

DH has been also reported to occur in pediatric patients, but the exact incidence of DH during childhood is unknown. In one study by our group in 2013 including 159 DH patients, about 36% were diagnosed below the age of 20 (23). Other authors have suggested a possible underestimation of pediatric DH due to clinical overlapping features with atopic dermatitis, which still accounts for the most prevalent dermatologic disease among children (24). Unlike CD, DH seems to occur most commonly in man, although the male/female ratio has reportedly ranging from 2:1 to 1:1 according to different studies (16, 19, 24). Interestingly, a Finnish study found that in females there is a longer diagnostic delay compared to male (25).

### Associated Diseases

Like CD, DH has been found to be associated with several autoimmune disorders, including type I diabetes mellitus, autoimmune thyroid diseases, and connective tissue diseases, such as Sjögren syndrome (24). Of interest is also the reported association between DH and bullous pemphigoid (BP), a subepidermal autoimmune blistering disease characterized by autoimmunity against the hemidesmosomal antigens BP180 and BP230 (26, 27). Accordingly, in a retrospective case-control study, diagnosis of DH was found to increase of 22 folds the risk of BP, compared to only a 2-fold higher risk of developing BP amongst coeliac patients. The mean time between the diagnosis of DH and BP development was about 3 years (27).

Since virtually all patients with DH have CD, DH patients also carry an increased risk of non-Hodgkin lymphomas and gastrointestinal malignancies (28). However, unlike CD, mortality of DH patients is not increased (29–31). Accordingly, in a large population-based study including 476 patients with DH, a reduced mortality for all the causes of death compared to the general population and a significantly reduced mortality related to cerebrovascular diseases were shown. Mortality due to non-Hodgkin lymphomas was increased only during the first 5 years following the diagnosis but not thereafter. DH patients were also found to have less hypercholesterolemia, and there were fewer smoker compared to the control population (32). Ali and Lear speculated that smoking might have a protective effect against DH, since it suppresses natural killer (NK) lymphocytes and reduces intestinal IgA secretion (33–36). Finally, an association between DH and higher social class have been reported, and may contribute to the observed reduced mortality reflecting the "many facets of better living" of a high socioeconomic status (33).

### Clinical Manifestations

The polymorphism and the symmetrical distribution of the lesions represent the major clinical hallmarks of DH (37, 38).

The disease usually presents with grouped erythematous papules and urticarial plaques with overwhelming vesicles. The latter may then coalesce into small tense blisters with serohemorrhagic content, which are characterized by a centrifugal growth pattern. Erosions, excoriations, and crusts are likely to occur because of blisters rupture and because of the scratching secondary to the associated pruritus (15, 38, 39). Lesions eventually heal leaving post-inflammatory hypo- and hyperpigmentation (**Figures 1**, **2**).

Typically, DH lesions symmetrically localized at the extensor surfaces of upper and lower limbs, mostly at the elbows and knees, buttocks, and the sacral region; the abdomen, upper back, shoulders, nuchal area, and the scalp may also be involved, whereas the face and groin are rarely affected.

Likewise, mucosal involvement has sporadically been reported. Oral manifestations of DH mainly consist of erosions affecting both the oral mucosa and the tongue; associated symptoms include pain and burning sensation (15, 40). However, it is not clear whether oral involvement has to be considered as a specific manifestation of DH or, rather, a sign of the underlying CD; accordingly, oral aphthosis, erosions, and/or ulcerations are also frequently found in CD (40–44).