Neonatal Fc Receptor Inhibition Reduces Bullous Pemphigoid Anti–Basement Membrane Zone IgG Deposition and Blistering in 3-Dimensional Human Skin Equivalents

Bullous pemphigoid (BP) is an autoimmune blistering disease whereby patients develop autoantibodies against the basement membrane zone (BMZ) antigens BP180 and/or BP230. These autoantibodies exert numerous pathogenic Fc receptor (FcR)-dependent and FcR-independent effects (Cole et al., 2022Cole C. Vinay K. Borradori L. Amber K.T. Insights Into the Pathogenesis of Bullous Pemphigoid: The Role of Complement-Independent Mechanisms.Front Immunol. 2022; 13912876Crossref Scopus (15) Google Scholar), and can be readily visualized for diagnosis by direct immunofluorescence (DIF) and indirect immunofluorescence. The neonatal Fc receptor (FcRn) mediates recycling of IgG, extending the half-life of IgG. FcRn is expressed on numerous cell types, including keratinocytes (Cauza et al., 2005Cauza K. Hinterhuber G. Dingelmaier-Hovorka R. Brugger K. Klosner G. Horvat R. et al.Expression of FcRn, the MHC class I-related receptor for IgG, in human keratinocytes.J Invest Dermatol. 2005; 124: 132-139Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). FcRn inhibition has been shown to be efficacious using in vivo models of pemphigoid by reducing circulating antibodies (Pigors et al., 2023Pigors M. Patzelt S. Reichhelm N. Dworschak J. Khil'chenko S. Emtenani S. et al.Bullous pemphigoid induced by IgG targeting type XVII collagen non-NC16A/NC15A extracellular domains is driven by Fc gamma receptor- and complement-mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade.J Pathol. 2023; PubMed Google Scholar) as well as in vivo or using in vitro models of keratinocytes in pemphigus (Chen et al., 2015Chen Y. Chernyavsky A. Webber R.J. Grando S.A. Wang P.H. Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris.J Biol Chem. 2015; 290: 23826-23837Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Li et al., 2005Li N. Zhao M. Hilario-Vargas J. Prisayanh P. Warren S. Diaz L.A. et al.Complete FcRn dependence for intravenous Ig therapy in autoimmune skin blistering diseases.J Clin Invest. 2005; 115: 3440-3450Crossref PubMed Scopus (222) Google Scholar, Zakrzewicz et al., 2022Zakrzewicz A. Würth C. Beckert B. Feldhoff S. Vanderheyden K. Foss S. et al.Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?.Cells. 2022; 11Crossref Scopus (11) Google Scholar). The contribution of keratinocyte FcRn to BP pathogenesis is unclear. As the persistence of autoantibodies along the BMZ would presumably sustain inflammation, we questioned whether therapeutic FcRn inhibition could exert a potentially acute therapeutic benefit, accelerating local depletion of deposited anti-BMZ antibodies. To test this, we affinity purified IgG from patients with BP or healthy controls. All donors provided written, informed consent. Enrollment criteria and additional methods are described in the Supplementary Methods. We generated 3D human skin equivalents (HSE), pretreating them with efgartigimod, a recombinant Fc mutant with high affinity for FcRn (Ulrichts et al., 2018Ulrichts P. Guglietta A. Dreier T. van Bragt T. Hanssens V. Hofman E. et al.Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans.J Clin Invest. 2018; 128: 4372-4386Crossref PubMed Scopus (171) Google Scholar), or wild type recombinant Fc (FcWT). 3D HSE were then subsequently treated with BP-IgG, and IgG deposition and blistering measured by DIF and H&E respectively. Pretreatment with efgartigimod resulted in a significant decrease in histologic blistering (Fig 1a, b). Notably, the efgartigimod treated group demonstrated re-attachment by day 7. In contrast, FcWT treated 3D HSE demonstrated stabilization in blistering by day 3 without significant re-attachment. Efgartigimod treated 3D HSE demonstrated a significant decrease in IgG deposition along the BMZ by day 3 (Fig 1 c, d). Interestingly, by day 7, fluorescent intensity of FcWT treated 3D HSE returned to levels consistent with that of the efgartigimod group, demonstrating that IgG presumably becomes depleted in the culture conditions between day 3 and 7. We next questioned whether these findings were due to accelerating metabolism of local IgG via FcRn blockade, or whether BP180/BP-IgG complexes relied on FcRn for internalization. Primary human keratinocytes were treated with BP-IgG to induce characteristic internalization of BP180 as previously described (Hiroyasu et al., 2013Hiroyasu S. Ozawa T. Kobayashi H. Ishii M. Aoyama Y. Kitajima Y. et al.Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway.Am J Pathol. 2013; 182: 828-840Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) (Fig 2a). We next pre-treated cultures with efgartigimod or FcWT prior to the addition of BP-IgG. The percentage of cells demonstrating BP180 internalization at 2 hours was quantified, demonstrating no significant change in BP180 internalization (Fig 2b, c). To further validate this, we added AlexaFluor-488 conjugated BP-IgG and AlexaFluor-568 conjugated dextran to visualize internalization kinetically. Again, efgartigimod did not appear to alter the internalization kinetics, indicating that blockade of FcRn does not abrogate BP-IgG mediated BP180 macropinocytosis (Fig 2d, e). Our data provides critical insight into the role of FcRn inhibition in clearing local deposition of anti-BMZ IgG in pemphigoid. FcRn inhibition causes a decrease in circulating IgG levels, resulting in therapeutic improvement in experimental settings. However, in patients, IgG deposition has already occurred upon onset of symptoms. Finding an efficacious treatment to expedite depletion of antibodies already deposited in the skin remains an important acute therapeutic goal. Our data demonstrate that FcRn inhibition expedites clearance of deposited pathogenic IgG at the BMZ, as well as decreases immune cell-independent blistering. In therapeutic application of FcRn inhibition in vivo, the effect of clearance of already deposited IgG may be counteracted by the rate of autoantibody production by plasma cells, however a systemic blockade of IgG recycling would decrease autoantibodies arriving at the target site. BP-IgG exerts several complement independent, and potentially pathologic effects (Cole et al., 2022Cole C. Vinay K. Borradori L. Amber K.T. Insights Into the Pathogenesis of Bullous Pemphigoid: The Role of Complement-Independent Mechanisms.Front Immunol. 2022; 13912876Crossref Scopus (15) Google Scholar). We confirm this functionally using 3D HSE. In contrast to prior cryosection models of blistering, 3D HSE offer the benefit of a viable epithelium. Thus, the direct contribution of autoantibodies on keratinocytes can be further studied (Bao et al., 2022Bao L. Perez White B.E. Li J. Patel P.M. Amber K.T. Gene expression profiling of laminin α3-blocked keratinocytes reveals an immune-independent mechanism of blistering.Exp Dermatol. 2022; 31: 615-621Crossref PubMed Scopus (5) Google Scholar). Several limitations must be considered. To preserve basal keratinocyte morphology, we utilized serum free culture conditions. A potential-downside of this is the inability to perform serum starvation, thus resulting in asynchronous macropinocytosis. This was evident in our experiments as we noted significant variability between replicate experiments, with macropinocytosis being observed in anywhere from 20% to 80% of BP-IgG treated cells at 2 hours depending on the culture length. Despite this, macropinocytosis was comparable between efgartigimod and FcWT treated keratinocytes in each experiment. We also observed a faster onset of macropinocytosis as early as 30 minutes in contrast to that seen by Hiroyasu et al (Hiroyasu et al., 2013Hiroyasu S. Ozawa T. Kobayashi H. Ishii M. Aoyama Y. Kitajima Y. et al.Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway.Am J Pathol. 2013; 182: 828-840Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Lastly, deciphering the kinetics of anti-BMZ deposition and blistering must be taken with some caution. Presumably, anti-BMZ antibodies saturate the cutaneous BMZ. As blistering and intrinsic protease release occurs, autoantigens are lost and then renewed, eventually depleting the free autoreactive IgG. Likewise, decreased blistering presumably extends the half-life of free IgG in the media, due to lack of proteolytic activity resulting in destruction, as evidenced by a decrease in fluorescent signal in areas of blistering (Fig. 1b). We, however, did not assess supernatant IgG concentrations given the need for media changes, and thus could not assess local levels after prolonged courses. There is also likely a delay between early blistering and loss of IgG deposition, as indicated by our finding of blistering occurring before significant loss of fluorescent signal. It is also unclear whether IgG deposits along the BMZ originate from soluble IgG in the media or are caused by keratinocyte FcRn mediated transcytosis. As IgG is found in interstitial fluid and blister fluid, both mechanisms are likely physiologic and would be improved through FcRn inhibition. This mechanistic data provides potential clues for translation to therapy. Acute depletion of deposited autoreactive IgG would potentially indicate a mechanism for acute clinical improvement. This is supported by clinical trial data of IVIg where even a single course resulted in improvement in disease severity despite unchanged circulating anti-BP180 antibody levels (Amagai et al., 2017Amagai M. Ikeda S. Hashimoto T. Mizuashi M. Fujisawa A. Ihn H. et al.A randomized double-blind trial of intravenous immunoglobulin for bullous pemphigoid.J Dermatol Sci. 2017; 85: 77-84Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In summary, we demonstrate that FcRn inhibition can expedite the clearance of BMZ-bound autoantibodies in vitro, independently of anti-BP180/BP180 complex internalization. This corresponds with an observed decrease in histologic blistering. Further studies with keratinocyte specific Fcgrt knockouts would provide additional insight into the contributory role of keratinocyte FcRn on pemphigoid. No large datasets were generated or analyzed. KV, PV, MS, and MB are employees of argenx and may hold stock or stock options. KTA has a collaborative research agreement with argenx. argenx provided reagent and collaborative discussion regarding study design, but the research was performed and analyzed independently of argenx. During the peer review process argenx was offered an opportunity to review the manuscript. Authors retained full editorial control over the content. Conceptualization – KTA, KV, PV, MS, MB; Data curation – LB, BPW, RC, JL; Formal Analysis RC, KTA; Investigation - LB, BPW, RCC, J; Methodology – KTA, Project administration – KTA, Resources – KV, PV, MS, MB; Supervision – KTA, Validation – BPW, RCC, KTA; Visualization – KTA, Writing – original draft – KTA, writing – review & Editing, LB, BPW, RCC, JL KV, PV, MS, MB. This work was supported by argenx. KTA is supported in part by the Office of Research Infrastructure Programs of the National Institute of Health (R21OD030057) The study was approved by the Rush University Institutional Review Board (IRB#20121406). The study was performed in accordance with the Declaration of Helsinki. All donors provided written informed consent. After obtaining informed consent, blood was drawn from patients with a confirmed diagnosis of BP. BP diagnosis was defined based on current international guidelines1, using the following criteria. 1) clinical suspicion for BP, 2) Positive direct immunofluorescence demonstrating deposition of IgG along the cutaneous BMZ, 3) A positive serologic test detecting BP180 and/or BP230 antibodies, and/or indirect immunofluorescence demonstrating deposition along the roof of salt-split skin. Patient serology is summarized below.Tabled 1PatientMucous Membrane InvolvementDPP4 ExposureBP180 ELISABP230 ELISAIIF titer on human SSSPatient 1NoYes18151:160Patient 2NoNo78621:20,480Patient 3NoNo9341:2,560Patient 4NoNo631011:20,480 Open table in a new tab DPP4 – Dipeptidyl peptidase inhibitor, IIF – indirect immunofluorescence, SSS salt split skin Affinity purification was performed on pooled serum samples utilizing the Nab Protein G spin Column (Thermo Fischer Scientific, Waltham, MA) to minimize variability in the ratio of BP180/BP230 antibodies. Purified IgG was further washed using 3kDa Amicon Ultra-15 centrifugal filter units with a 3kDa filter in PBS (MilliporeSigma, Burlington, MA), followed by azide removal (Nanopartz, Loveland, CO). For 3D HSE experiments, multiple single donor sera were purchased from Innovative Research. For 2D experiments, each biologic replicate utilized pooled IgG from different vendors to increase diversity of normal control (MP Biochemicals, Irvine, CA; Cell Sciences, Newburyport, MA; Sigma-Aldrich, St. Louis, MO). Affinity purified BP-IgG was conjugated to AlexaFluor-488 using the Lightning-Link Conjugation Kit (Abcam, Cambridge, MA). 3D HSEs were initiated and propagated as previously described2 using primary neonatal human keratinocytes. A minimum of three independent cell lines derived from three donors were used for all 3D HSEs. Cultures were treated with efgartigimod (Lonza, UK) or wild type Fc (Evitria, Switzerland) were added to after 7 days of 3D HSE growth at a concentration of 25ug/mL based on prior reported dosing3 (Defined as day 0). After 60 minutes of preincubation, 2mg/mL of BP-IgG or control IgG was added. Cultures were harvested on day 1, 3, and 7. Media changes occurred on day 3 and 5. Primary adult human keratinocytes (PHKs) (Thermo Fisher Scientific) were cultured in EpiLife Medium with Human Keratinocyte Growth Supplement in a CO2 incubator and grown on chamber slides. Culture media changes occurred every 48 h. Cells were pretreated with 25ug/mL of efgartigimod or FcWT followed 30 minutes later by 4mg/mL of BP-IgG. In a second set of experiments, cells were pretreated with efgartigimod or FcWT as previously described, followed by 4mg/mL of AF488-conjugated BP-IgG and 0.1mg/ml of 70kDa AF568-conjugated dextran (Thermo Fisher Scientific). AF-conjugated IgG has previously been shown to functionally bind to FcRn4. To detect anti-BMZ antibodies, 3D HSE were placed in OCT, followed by sectioning. Sections were stained with 1:100 AlexaFluo488 conjugated anti-human IgG (Thermo Fisher Scientific). For 2D cultures, Chamber slides were at 36, 60, 90, or 120 minutes. For imaging BP180 internalization, cells were fixed, permeabilized, then stained with anti-COL17 antibody (Clone SR46-05, Thermo Fisher Scientific). For dextran imaging, cells permeabilization was skipped. All samples were imaged on an Evos FL II . Formalin fixed paraffin embedded portions of 3D HSE were sectioned at 4 μm, stained with hematoxylin and eosin per standard protocol, and imaged using an Zeiss Axioplan 2 light microscope with high resolution AxioCamHR camera. Images were analyzed in ImageJ (National Institute of Health, Bethesda, MD) by an investigator blinded to the experimental conditions. For BP180 and dextran internalization, the morphology of prototypical BP-IgG treated cells demonstrating internalization versus Ctrl-IgG cells were used to define the two populations for performing a count of randomized slide sections. For quantifying the percentage of histologic blistering, length of detached tissue at the BMZ was divided by the total length of tissue and multiplied by 100. For direct immunofluorescence, Ctrl-IgG treated sections were used to establish autofluorescent intensity gates on a scale of 0-255. Mean pixel intensity of intact BMZ exceeding an intensity of 90 (the autofluorescence of control tissue) was measured for each section, and mean RFU was considered as mean RFU minus 90. Data was analyzed using Prism 9 software (Graphpad, La Jolla, CA). Pairwise measures were made by Mann-Whitney U test. All tests were 2 tailed with significance defined as P < 0.05. 1. Borradori L, Van Beek N, Feliciani C, et al. Updated S2 K guidelines for the management of bullous pemphigoid initiated by the European Academy of Dermatology and Venereology (EADV). J Eur Acad Dermatol Venereol. 2022;36(10):1689-1704. 2. Arnette C, Koetsier JL, Hoover P, Getsios S, Green KJ. In Vitro Model of the Epidermis: Connecting Protein Function to 3D Structure. Methods Enzymol. 2016;569:287-308. 3. Zakrzewicz A, Würth C, Beckert B, et al. Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus? Cells. 2022;11(6). 4. Tzaban S, Massol RH, Yen E, et al. The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity. J Cell Biol. 2009;185(4):673-684.