Allelic and copy-number variations of FcγRs affect granulocyte function and susceptibility for autoimmune blistering diseases

Journal of Autoimmunity 61 (2015) 36e44

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Allelic and copy-number variations of FcgRs affect granulocyte function and susceptibility for autoimmune blistering diseases Andreas Recke a, b, *, Gestur Vidarsson c, Ralf J. Ludwig a, b, Miriam Freitag b, € ller b, Reinhard Vonthein d, e, Julia Schellenberger a, Ozan Haase a, Steffen Mo € rg f, Almut Nebel g, Friederike Flachsbart g, Stefan Schreiber g, Siegfried Go €ser j, Sandrine Benoit k, Miklo s Sa rdy l, Rüdiger Eming m, Wolfgang Lieb h, i, Regine Gla m a, b d € nig , Enno Schmidt a, b, Saleh Ibrahim b, **, , Inke R. Ko Michael Hertl , Detlef Zillikens the German AIBD Genetic Study Group1 a

Department of Dermatology, Allergology and Venereology, University of Lübeck, Lübeck, Germany Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany Department of Experimental Hematology, Sanquin Research Institute, Amsterdam, The Netherlands d €t zu Lübeck, Universita €tsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany Institut für Medizinische Biometrie und Statistik, Universita e €t zu Lübeck, Lübeck, Germany Zentrum für Klinische Studien, Universita f Department of Transfusion Medicine, University of Lübeck, Lübeck, Germany g Institute of Clinical Molecular Biology, Christian-Albrechts-University, Kiel, Germany h Institute of Epidemiology, Christian-Albrechts-University, Kiel, Germany i popgen Biobank, Christian-Albrechts-University, Kiel, Germany j Department of Dermatology, Allergology and Venereology, Christian-Albrechts-University, Kiel, Germany k Department of Dermatology, Allergology and Venereology, University Hospital Würzburg, Würzburg, Germany l Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany m Department of Dermatology and Allergology, Philipp University of Marburg, Marburg, Germany b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2015 Received in revised form 5 May 2015 Accepted 6 May 2015 Available online 29 May 2015

Low-affinity Fcg receptors (FcgR) bridge innate and adaptive immune responses. In many autoimmune diseases, these receptors act as key mediators of the pathogenic effects of autoantibodies. Genes encoding FcgR exhibit frequent variations in sequence and gene copy number that influence their functional properties. FcgR variations also affect the susceptibility to systemic autoimmunity, e.g. systemic lupus erythematosus and rheumatoid arthritis. This raises the question whether FcgR variations are also associated with organ-specific autoimmunity, particularly autoantibody-mediated diseases, such as subepidermal autoimmune blistering diseases (AIBD). A multitude of evidence suggests a pathogenic role of neutrophil granulocyte interaction with autoantibodies via FcgR. In a two-stage study, we analyzed whether the FcgR genotype affects neutrophil function and mRNA expression, and consequently, bullous pemphigoid (BP) disease risk. We compared this to findings in pemphigus vulgaris/ foliaceus (PV/PF), two Fc-independent AIBDs. Our results indicate that both allele and copy number variation of FcgR genes affect FcgR mRNA expression and reactive oxygen species (ROS) release by granulocytes. Susceptibility of BP was associated with FcgR genotypes that led to a decreased ROS release by neutrophils, indicating an unexpected protective role for these cells. BP and PV/PF differed substantially regarding the FcgR genotype association patterns, pointing towards different disease etiologies. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Autoantibodies Fcg receptors Joint copy number and allelic variation Functional genetics Autoimmune blistering dermatoses Neutrophils Reactive oxygen species

* Corresponding author. Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany. Tel.: þ49 451 500 2530; fax: þ49 451 500 5162. ** Corresponding author. Tel.: þ49 451 500 5250; fax: þ49 451 500 5162. E-mail addresses: [email protected] (A. Recke), [email protected] (S. Ibrahim). 1 A complete list of the members of and the German AIBD Genetic Study Group appears in the “Appendix.” http://dx.doi.org/10.1016/j.jaut.2015.05.004 0896-8411/© 2015 Elsevier Ltd. All rights reserved.

1. Introduction A central interface between humoral and cellular immune system is provided by Fcg receptors (FcgR), residing on the surface of effector cells of the innate immune system. IgG molecules bound to

A. Recke et al. / Journal of Autoimmunity 61 (2015) 36e44

antigens are able to cross-link FcgR, thus triggering phagocytosis, release of reactive oxygen species and other cytotoxic agents, and release of signaling molecules like chemokines and cytokines [1e3]. In many autoimmune diseases, these receptors act as key mediators of the pathogenic effects of autoantibodies [2,4e8]. In humans, this family is composed of the activating FcgRI, FcgRIIa, FcgRIIc, FcgRIIIa and FcgRIIIb, and the inhibitory FcgRIIb [9,10]. Activating signals are transduced via immunoreceptor tyrosinebased activation motifs (ITAMs) located in the intracellular domain of FcgRIIa and FcgRIIc, or in the g-chain of FcgRIIIa. The inhibitory FcgRIIb carries corresponding immunoreceptor tyrosinebased inhibition motifs (ITIMs). FcgRIIIb and FcgRIIc are almost unique for the human species, otherwise found in cow and chimpanzee. The first, FcgRIIIb, is a truncated receptor lacking a transmembrane and a cytoplasmic domain. FcgRIIIb resides in lipid rafts on the cell membrane, where it is attached by a glycosylphosphatidylinositol-anchor [11]. Physiologically, it is only expressed by granulocytes, and it may potentially amplify granulocyte activation through interaction with immune complexes that also colligate FcgRIIa, bringing the ITAM docking motifs into the lipid raft signaling platform [12e15]. FcgRIIc, is probably the product of an unequal crossover between the FcgRIIa and FcgRIIb genes [16]; the genetic sequences of its cytoplasmic tail, including the ITAM, is identical to that of FcgRIIa, while that of its extracellular region, together with the gene promoter, is identical to that of FcgRIIb.

37

A major obstacle for discrimination and differential analysis of FcgRIIs is the high degree of homology between functionally different receptors e at protein level [17], but also at mRNA and genomic levels. Genes encoding FcgR exhibit frequent variations in sequence and gene copy number, which can be determined simultaneously by a multiplex ligation-dependent probe amplification (MLPA) (Fig. 1A) [18e21]. The FcgRIIa.H131R (rs1801274), and the FcgRIIIa.F158V (rs396911) polymorphisms influence the affinity to the different IgG subclasses [3,22]. For FcgRIIb, a change of Ile232 to Thr (rs1050501, FCGR2B.I232T) leads to an impaired inhibitory function of this receptor [23]. The NA1 (HNA1a) and NA2 (HNA1b) variants of FcgRIIIb (rs200688856) have different capacities to induce phagocytosis [24,25]. The FcgRIIIb.SH (HNA1c) variant is the result of a point mutation (rs5030738) of the FCGR3B.NA2 allele [26]. FCGR2C and FCGR2B promoters share the rs3219018 (FCGR2B.G-386C and FCGR2C.G-386C) and the rs34701572 (FCGR2B.A-120T and FCGR2C.A-120T) SNPs, which affect the binding of the transcription factors GATA4 and YY1 [27,28]. The FCGR2C gene normally contains an inactivating inframe stop codon (FCGR2C.Stop allele). However, an open-reading frame (FCGR2C.ORF) variant (rs183547105) exists that is translated into a functionally competent receptor that mediates signal activation [18]. Recently, two novel rare alleles of the FCGR2C gene have been described, which are erroneously recognized as FCGR2C.Stop and FCGR2C.ORF alleles, respectively, by the commercially available MLPA method [29].

Fig. 1. Genomic organization of the low-affinity Fcg receptor gene locus. A, consensus map of the low-affinity FcgR gene region at chromosomal position 1q23.3, comprising the genes for FcgRIIa (FCGR2A), FcgRIIIa (FCGR3A), FcgRIIc (FCGR2C), FcgRIIIb (FCGR3B) and FcgRIIb (FCGR2B). The FcgR gene alleles that were identified and quantified by MLPA are indicated [18]. The FCGR2B and FCGR2C promoter sequences are identical, except for the frequency of SNP variants at nucleotide positions 386 and 120. The genes for the heat shock proteins 6 (HSPA6) and 7 (HSPA7) were included in the MLPA analysis for verification purposes. Modified after Recke et al. [47]. B, the duplication and deletion patterns and estimated frequencies (#) of haplotypes, as determined by the hidden Markov model inference using R package “CNAV” (Appendix A2).

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A. Recke et al. / Journal of Autoimmunity 61 (2015) 36e44

FcgR variations also affect the susceptibility to systemic autoimmunity, e.g. systemic lupus erythematosus and rheumatoid arthritis [18,20,23,30,31]. This raises the question whether FcgR variation could also be associated with organ-specific autoimmunity. AIBDs are prototypic organ-specific autoimmune diseases, characterized by autoantibodies against structural proteins of the skin. In subepidermal AIBD, such as BP and epidermolysis bullosa acquisita (EBA), autoantibodies directed against structural components of the dermaleepidermal junction cause activation of the complement system and of neutrophil granulocytes and other cells of the innate immune system [3,5,25,32]. This leads to the release of proteolytic enzymes and ROS and finally to blister formation [32e37]. The activation of neutrophil granulocytes by skin-bound autoantibodies requires low-affinity FcgR [5,6,38]. In case of PV/ PF, autoantibodies cause blistering independently of the Fc portion by binding to desmosomal proteins in the epidermis. This blocks desmosome formation by steric hindrance, but also by altering intracellular signaling pathways leading to desmoglein depletion and keratin retraction [39e41]. We hypothesized that genetic variations leading to increased activity of key pathogenetic players would also lead to an increased risk for the development of a subepidermal blistering disease. In a multi-faceted study (overview in Fig. S1) [57e59], we first analyzed the effect of the FcgR genotype on the expression of FcgR mRNA and the induction of ROS release by neutrophil granulocytes after stimulation with immobilized autoimmune complexes. Secondly, we used a caseecontrol study to estimate the effect of the patient FcgR genotype on the risk to develop BP. For comparison, a similar analysis was performed on PV/PF, which helped us to interpret the specific role of FcgR in both disease types. 2. Materials and methods 2.1. Ethics statement All studies with human materials (Fig. S1) followed the ethical principles established by the Declaration of Helsinki and were approved by the local ethics committee (AZ 10-026 and AZ 08-156). All human participants gave written informed consent. Samples and demographic data of patients and controls were collected in adherence to ethics and German privacy protection regulations. 2.2. Description of human materials Functional studies were performed with neutrophil granulocytes that were freshly isolated from healthy blood donors collected over an 8-month period. The cases and younger controls used for the bullous pemphigoid (BP) and pemphigus vulgaris (PV) or pemphigus foliaceus (PF) association studies were provided by the popgen biological materials collection [42]. Control individuals aged 92 years and older are part of the German longevity sample collection and were recruited as described previously [43]. All patients included in this study were examined at one of the contributing dermatological centers. All BP patients fulfilled the following criteria (inclusion criteria): clinical presentation with prurigo-type lesions, eczema or tense blisters on inflamed skin (BP), IgG deposition at the dermaleepidermal junction by direct and indirect immunofluorescence (IF) microscopy and detection of circulating BP180/NC16A autoantibodies. The inclusion criteria for PV and PF patients were as follows: vulnerable blisters and erosions on non-inflamed skin, IgG deposition at the intercellular junctions between epidermal keratinocytes by direct and indirect IF and detection of circulating

desmoglein 3 (PV) autoantibodies.

or

desmoglein

1

(PF)

or

both

(PV)

2.3. Quantification of ROS released by the in vitro model of blistering dermatoses Neutrophil granulocytes were isolated from freshly collected whole blood by using PolymorphPrep™ (Axis-Shield GmbH, Heidelberg, Germany) according to the manufacturer's recommendations. The detection of luminol-enhanced chemiluminescence from neutrophil granulocytes stimulated with recombinant immunecomplexes was performed as described [38,44]. Briefly, white microwell plates (Greiner BioOne, Frickenhausen, Germany) were coated with a recombinant fragment of human type VII collagen (hCOLE-F) [44] and blocked with bovine serum albumin. Recombinant IgG1, IgG2, IgG3 and IgG4 subclass autoantibodies directed against human type VII collagen [45] were diluted at optimized concentrations and incubated with the plates. Neutrophil granulocytes were subsequently suspended at a density of 1  106/ml in modified dye-free RPMI1640 medium (Lonza, Geneva, Switzerland) containing 0.5% fetal calf serum and 50 mg/ml luminol. Formylmethionine-leucine-proline (FMLP) was added at a final concentration of 1 mM as a positive control. Luminol-enhanced chemiluminescence kinetics were recorded in a VICTOR™ 3 reader (PerkinElmer Inc., Waltham, MA, USA) over a period of 90 min. The area under the curve values from all stimulations was divided by the negative control values for each donor. 2.4. Quantification of Fcg receptor mRNA expression Total RNA was concurrently prepared from neutrophil granulocytes isolated from each blood donor using an RNeasy preparation kit (Qiagen, Hilden, Germany). The total RNA was reverse transcribed with a First-strand cDNA synthesis kit (Fermentas, Schwerte, Germany). Pre-designed TaqMan assays purchased from Applied Biosystems (Foster City, CA, USA) were used to detect mRNA from FcgRIIa (Hs01013401_g1), FcgRIIb (Hs00269610_m1), FcgRIIIa (Hs02388314_m1) and FcgRIIIa/ FcgRIIIb (Hs00275547_m1), as well as the b2 microglobulin (B2M) controls. The primers used to detect FCGR2C were designed according to W.B. Breen's et al. [18]: sense 50 -atc att gtg gct gtg gtc act gg-30 , antisense 50 -ctt tct gat ggc aat cat ttg acg-30 and FAMconjugated internal 50 -gcc aat tcc act gat cct gt-30 . The specificity of the both pre-designed and customized TaqMan assays is depicted in Fig. S2. The TaqMan reactions were performed according to the manufacturer's recommendations, with 10 ng cDNA, PCR MasterMix, assay specific primers and the B2M internal housekeeping reference gene. The external GAPDH housekeeping reference gene was used with the FCGR2C assay, together with Maxima Probe/ROX qPCR 2x Mastermix (Fermentas, Schwerte, Germany). After an initial denaturation step of 95  C for 3 min, 50 cycles of 95  C for 15 s followed by 60  C for 1 min were performed in a realplex2 Mastercycler (Eppendorf, Germany) and analyzed with realplex software. 2.5. Multiplex ligation-dependent amplification (MLPA) Genomic DNA from all healthy blood donors was concurrently isolated from whole blood using a genomic DNA extraction kit (Qiagen, Hilden, Germany). The samples provided by popgen were also highly pure genomic DNA. The P110 and P111 kits (MRCHolland, Amsterdam, The Netherlands) were used according to the manufacturer's instructions for the MLPA of the Fcg receptors. Briefly, 50 ng of genomic DNA was hybridized with twin-probes overnight, followed by a ligation reaction. The ligated probes

A. Recke et al. / Journal of Autoimmunity 61 (2015) 36e44

39

were amplified by PCR, and the size and quantity of the resulting PCR fragments was analyzed by a Beckman CEQ-880 capillary sequencer (Beckman Coulter, Krefeld, Germany). The fragment analysis was performed as recommended by the manufacturer. As readout, MLPA provides the allele counts and gene copy numbers for all analyzed genes of an individual genome (Fig. 1A). MLPA data was further processed as described in Appendix A1. Because of inevitable uncertainty in genotype determination, we used a weighted pseudo-individual approach as described [46,47].

The overall genotype distribution of the locus in the cohort of German controls (Table S1) resembled the data reported for a Dutch cohort by W.B. Breen's et al. [18]. Hidden Markov modeling was used to understand the linkage between multiplications and deletions of different FcgR gene regions (Fig. 1B), as described in Appendix A2 and Recke et al. [47]. Notably, gains or losses in copy numbers of FCGR3A were less frequent than for FCGR3B and FCGR2C; no evidence of copy number variation was observed for FCGR2A and FCGR2B.

2.6. Statistical analyses and graphical presentation

3.2. Gene copy number and allelic variation both influence FcgR mRNA expression

All statistical analyses and data graphics were generated in R version 3.03 “warm puppy” (R project and Comprehensive R Archive Network (CRAN), URL: http://www.r-project.org/), including packages “ENmisc” (Erich Neuwirth miscellaneous, URL: http://cran.r-project.org/web/packages/ENmisc/index.html), “INLA” (Integrated Nested Laplace Approximation (INLA) for R, URL: http://www.r-INLA.org/), “CNAV” [47] (Copy Number and Allelic Variation, URL: https://r-forge.r-project.org/projects/CNAV/) and “wtJonckheere” (Jonckheere Terpstra testing on weighted data, URL: https://r-forge.r-project.org/projects/wtJonckheere/). In case of Bayesian inference, the 95% highest posterior density (HPD) intervals for covariate effects were calculated from the marginal posterior distributions of covariates. For the effect size estimators, HPD intervals were also used for Bayesian hypothesis testing that was analogous to traditional frequentist statistics [48]. The (Bayesian) p value was defined to be the threshold a value, at which an effect size of 0 (point zero) is contained in the (1  a) HPD credibility interval of the marginal posterior distribution of the respective covariate. Bayesian p values < 0.1 are considered as indicative, whereas p values < 0.05 are considered decisive. When frequentist techniques were used, e.g. JonckheereeTerpstra testing, all p values below 0.05 were considered significant. In case of multiple testing of hypotheses, the BonferroniHolm procedure was applied. 3. Results 3.1. FcgRIIIa, IIc and IIIb show very frequent changes in gene copy numbers To investigate the influence of the low-affinity FcgR genotype on cellular function and disease risk, we determined joint copynumber and allelic variation by MLPA in 387 healthy control subjects, 173 patients with BP and 76 patients with PV/PF (Table 1 and Fig. S1). The reliability of MLPA results was verified by TaqMan CNV assays (Supplementary methods and Fig. S3).

Table 1 Characterization of age and sex distribution in the disease and control cohorts. Age (years)

Female 0e20 20e40 40e60 60e80 80e110 N N a b

BPa

Controls Male

Female

PV/PFb Male

Female

Male

1 32 24 99 27

1 53 69 24 57

1 4 17 48 36

0 1 8 39 19

7 23 20 20 1

0 8 10 7 0

183

204

106

67

51

25

387

173

Bullous pemphigoid. Pemphigus vulgaris and pemphigus foliaceus.

76

182 from the 387 healthy control subjects were recruited for cell-based assays and determination of mRNA expression levels (Fig. S1). FcgR mRNA expression levels were successfully determined by TaqMan assays in 105 cases for FcgRIIa, 103 cases for FcgRIIb, 89 cases for FcgRIIc and 106 cases for FcgRIIIb. In accordance with previous studies showing that neutrophil granulocytes do not express FcgRIIIa [49], no detectable levels of FcgRIIIa mRNA were found. The gene polymorphisms exhibiting the most striking effects on mRNA expression are presented in Fig. 2. In-depth analyses of the data set using a Bayesian linear mixed model regression (involving INLA) are shown in Figs. S4eS7. While we observed no differences in mRNA expression among the FCGR2A.H131R (rs1801274) polymorphisms (Figs. 2A and S4), the G-386C and A-120T polymorphism found in both the FCGR2C and FCGR2B promoters (Figs. 2BeC and S5eS6) significantly affected the expression of both receptors, confirming previous reports about these promoter polymorphisms [28]. Interestingly, FcgRIIb expression was also increased by the presence of an ORF variant of FCGR2C (Figs. 2D and S6). However, no reverse influence of the FCGR2B gene on FCGR2C expression could be described. The mRNA expression of FCGR3B increased linearly with overall gene copy number (Figs. 2E and S7), but was also different between the FCGR3B.NA1, FCGR3B.NA2, and FCGR3B.SH alleles, with the latter two variants of FcgRIIIb being expressed upto twofold stronger than the first (Figs. 2F and S7). 3.3. Age, the FCGR2C.ORF/Stop polymorphism and FcgRIIIb gene copy number significantly affect ROS release by neutrophil granulocytes Next, we determined the association of FcgR genotypes with ROS release by neutrophil granulocytes after stimulation with immobilized autoimmune complexes in an in vitro model of subepidermal AIBDs [38,45]. In this model, neutrophil granulocytes isolated from healthy donors were incubated in a highly standardized procedure with autoimmune complexes containing recombinant autoantigen and recombinant human autoantibodies of each of the four recombinant V gene-matched IgG subclass antibodies against collagen VII [45]. We analyzed the AUC values of chemiluminescence kinetics from the four autoantibody variants measured for each donor by principal component analysis (PCA). We evaluated the impact of the FcgR genotype with INLA (see Appendices A3, A4 and A6), using a linear mixed effects model on the first PCA component that represented 90% of the variance in the data set (Fig. 3 and Table S2). We found that the intensity of ROS release decisively increased with each copy of FCGR3B by 20.1% (p ¼ 0.0297). Of similar importance was the balance between FCGR2C alleles: each FCGR2C.ORF allele copy decreased ROS release by 11.7%, while each Stop allele copy increased it by the same amount (p ¼ 0.00621). We dichotomized age as a covariate for presentation of the analysis, because data exploration revealed a quite narrow interval at the age

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Fig. 2. Impact of Fcg receptor genotype on mRNA expression. The key findings regarding the influence of genotype on FcgR mRNA expression in human neutrophil granulocytes (comparison models described in Figs. S4eS7). Box and whisker plots generated with the function “wtd.boxplot” of the R package “ENmisc” showing the normalized mRNA expression levels of the different FcgR genotypes. A dependency between genotypes and mRNA expression was assessed using a two-sided JonckheereeTerpstra test (R package “wtJonckheere”). A, FcgRIIa mRNA expression is not influenced by the FCGR2A.H131R polymorphism (p ¼ 0.306, N ¼ 105). B, FcgRIIc mRNA expression increases with every C allele of the FCGR2C.G-386C promoter polymorphism (p ¼ 6.57$109, N ¼ 89). C, FcgRIIb mRNA expression is increased in the presence of the C variant of the FCGR2B.G-386C promoter polymorphism (p ¼ 4.19$1012, N ¼ 103). D, the FcgRIIb mRNA expression is increased in the presence of the FCGR2C.ORF variant (p ¼ 4.85$1010, N ¼ 103). E, FcgRIIIb mRNA expression increases linearly with each gene copy of FCGR3B (p ¼ 2.4$108, N ¼ 106). F, FcgRIIIb mRNA expression and is a function of FCGR3B.NA1 vs. NA2/SH allele balance; i.e., the number of NA1 allele copies minus the combined copy numbers of NA2 and SH alleles (p ¼ 6.12$1010, N ¼ 106).

of 40 where the ROS release dropped decisively by about 26.2% (p ¼ 0.000126, Fig. 3 and Table S2). In the individual analysis of the four IgG subclasses, we found that the FCGR2A.H131R polymorphism showed a differential impact on ROS release (Fig. S8). This was depending on the IgG subclass, which was partially expected due to the different affinities of FcgRIIa alleles for IgG subclasses [3].

Only two genetic variations showed a decisive impact on the risk for BP (Fig. 3 and Table S3): FCGR2C copy number and FCGR3B copy number. Each gene copy of FCGR3B leads to a 2.1-fold decreased risk for BP (hazard ratio, HR ¼ 0.48, p ¼ 0.00186). In contrast, each FCGR2C gene copy results in a 1.89-fold increased disease risk (p ¼ 0.00158). The risk association pattern of covariate effects resembled the inverted pattern of ROS release.

3.4. Susceptibility to bullous pemphigoid is associated with gains of FcgRIIc and losses of FcgRIIIb gene copy numbers

3.5. Differently from bullous pemphigoid, susceptibility to pemphigus vulgaris/foliaceus is associated with the FCGR2C.ORF/ Stop and FcgRIIb promoter polymorphisms

To investigate the role of functional FcgR gene variants with the pathogenesis of BP, we performed a disease association study using a Cox proportional hazards model. In total, 173 patients with BP were compared with the 387 healthy German control individuals, including old (age > 90 years) and very old (age > 100 years) subjects to match age distributions (Table 1). For optimal comparability between the functional study and the disease association study, genotypes were included as covariates in the same way in this model as in the functional study (Fig. 3, Appendices A3eA4 and A6eA7).

To validate [50] the results of the FcgR genotype on BP disease risk, we evaluated its association with disease risk for PV/PF (Fig. 3 and Table S4). Unlike BP, the pathogenicity of autoantibodies in PV/ PF is independent of FcgR signaling [39,51,52]. In agreement with this, no association was found between disease risk and activating FcgR genotypes expressed on myeloid cells. Instead, an association between disease risk and the FCGR2C.ORF/Stop polymorphism (p ¼ 0.000563) and the FCGR2B.G-386C (p ¼ 0.00243) promoter polymorphism was identified. The FCGR2B.A-120T promoter

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41

Fig. 3. Impact of the Fcg receptor genotype on neutrophil function and the association with bullous pemphigoid or pemphigus vulgaris or foliaceus. Diamond plots representing the posterior distributions of the effect sizes of FcgR genotype covariates on ROS release by neutrophil granulocytes isolated from healthy blood donors (ROS release) in comparison with the risk association of developing BP (BP risk) or PV/PF (PV/PF risk). ROS release from 118 blood donors is represented by the first component of the principal components analysis of the area under curve values from four separately measured luminol-enhanced chemiluminescence kinetics after neutrophil granulocyte stimulation with autoimmunecomplexes from IgG1, IgG2, IgG3 and IgG4 subclass recombinant autoantibodies (Fig. S8). The distribution of covariate effects on ROS release was estimated by a Gaussian linear mixed model (Appendices A3, A4 and A6). The effect sizes (in %) were calculated relative to the mean value of the first principal component of all blood donors. For the risk association analysis, cohorts of 387 healthy controls, 173 cases of BP and 76 cases of PV/PF were analyzed using a Cox proportional hazards model (Appendices A3, A4 and A7). Hazard ratios were given for all FcgR genotype covariates. The diamond shapes indicate the posterior mean effect size (vertical line) and the 95% highest posterior density (HPD) credibility intervals estimated for the posterior distribution of each covariate, calculated by the Integrated Nested Laplace Approximation (INLA from R package “INLA”). For the risk association, age and sex were not included in this figure, as they were both used for stratification of the model. The color of the diamond shapes indicates the degree of significance: black for Bayesian p < 0.05, gray for 0.05 < Bayesian p < 0.1, white for Bayesian p > 0.1. More detailed information is provided in Fig. S8 and Tables S2eS4.

polymorphism that appeared to be highly linked to the FCGR2B.G386C polymorphism was not included in the all-covariate regression model for reasons of mathematical stability. 4. Discussion In autoantibody-mediated diseases like subepidermal AIBD, FcgR have a prominent role in the tissue-damage due to the activation of effector cells of the innate immune system, like neutrophil granulocytes [5,6,38]. We therefore hypothesized that polymorphisms that lead to a stronger activation of effector cells could influence the susceptibility to subepidermal AIBD. To verify this, we evaluated the influence of FcgR variation on ROS release by neutrophil granulocytes after stimulation with autoimmune complexes (functional phenotype), on expression levels of FcgR mRNA (intermediate phenotype) and association with BP and PV/PF (increased risk phenotype). For genotyping, we used an MLPA method which allowed simultaneous determination of allelic and copy number variation of the low-affinity FcgR gene region [18]. The genotypes distribution we found in our German control cohorts closely resembled genotyping data from a Dutch population [19]. We did not observe any variability in gene copy numbers of FCGR2A and FCGR2B. Both receptor genes are flanking a region of high structural variability, in which the genes for FCGR3A, FCGR2C and FCGR3B are located. Interestingly, FCGR2C and FCGR3B seem to form a block where gains and losses of gene copies occur together, while FCGR3A is more independent and more restricted in copy number changes. This indicates that changes in gene copy numbers of FcgRIIIa, and even more of FcgRIIa and FcgRIIb, have critical or even fatal effects

on the organism. In contrast to that, there seems to be a relaxed negative selection pressure for copy number changes in case of the FCGR2C gene. This corresponds to the finding that the FCGR2C gene is normally a non-expressed pseudogene, with the stop codon variant found in 85.4% of gene copies. In contrast, the gene for FcgRIIIb reveals a copy number variability similar to that of FcgRIIc, although this has consequences for cellular responses and for disease risk of BP, as discussed later. One might speculate that these consequences are not fatal, and that FcgRIIIb might be more important for fine-tuning cellular responses. As intermediate phenotype, the mRNA levels of FcgRIIa, FcgRIIb, FcgRIIc and FcgRIIIb were quantified by TaqMan. The mRNA expression of FCGR2A and FCGR2B were both independent of the FCGR2A.H131R and the FCGR2B.I232T polymorphisms, respectively. In contrast, the promoter polymorphisms in FCGR2B and FCGR2C showed the same effects that have been predicted experimentally by Su et al. [27,28]. Additionally, we found an increased expression of FcgRIIb mRNA in the presence of an ORF allele of FCGR2C. It may be possible that the expression of FCGR2B is disturbed in case of the FCGR2C.ORF variant, because both receptors share nearly identical promoter sequences which should respond equally to the same transcription factors. This idea is further supported by the observation that B cells express, besides FcgRIIb, a functionally active FcgRIIc in case of the FCGR2C.ORF variant [53]. Furthermore, the gene copy number of FcgRIIIb has a direct dose-dependent effect on the level of FcgRIIIb mRNA expression; and the mRNA expression level differs between the FcgRIIIb.NA1 (HNA1a) and FcgRIIIb.NA2/FcgRIIIb.SH (HNA1b/c) alleles. Thus, there seems no secondary controlling mechanism to compensate the effect of genetic variability of this receptor on

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expression of its mRNA, possibly associated with differences in the promoter or unknown regulatory elements in this locus. To determine the impact of the FcgR variation on granulocyte effector functions, we used an in vitro assay to measure ROS release after stimulation with immobilized immune complexes. Here, we found that ROS release was increased dependent on the number of FCGR3B gene copies. This correlated well with the dose-dependent increase of mRNA levels for this receptor. Allelic variation of FcgRIIIb did not alter the cellular response, despite different expression levels. This might be explained by a compensating effect due to a stronger responsivity of the FcgRIIIb.NA1 variant over FcgRIIIb.NA2 and FcgRIIIb.SH [25]. In contrast, presence of the FCGR2C.ORF allele led to a paradoxical reduction in ROS release, although this allele is known be expressed as a functionally active receptor on the cell surface [18,53]. A possible explanation for this might be that the presence of FCGR2C.ORF induces the expression of the inhibitory FcgRIIb, as observed on mRNA level. IgG subclass specific differences were only detected due to the FcgRIIa.H131R variation, matching the differences between the affinities of the FcgRIIa.131H and FcgRIIa.131R variant to the different IgG subclasses [3]. In the second stage of this study, we investigated the association of FcgR gene allelic and copy number polymorphisms on disease association with AIBD. Our study demonstrated a negative association of BP with high FCGR3B and low FCGR2C copy numbers. The involvement of FcgRIIIb generally indicates that granulocytes, especially neutrophil granulocytes, are responsible for differences in the risk of BP, as the FcgRIIIb is expressed exclusively by this cell type [12e15]. Although neutrophil granulocytes are responsible for autoantibody-mediated pathogenicity, high expression of FcgRIIIb might protect against events that would finally lead to, or promote, the development of BP. Surprisingly, the copy-number variation of the FcgRIIIb gene that was associated with increased ROS release by neutrophil granulocytes was also associated with decreased risk for developing BP. This is in contrast to findings in mouse models of subepidermal blistering diseases, which suggest that ROS release is a prerequisite for disease manifestation. Our data indicated that ROS release from neutrophil granulocytes after immobilized immune complex stimulation drops after the age of 40. This offers an explanation for both findings. A higher FCGR3B copy number might compensate for physiologically low levels of ROS at middle/old age, thereby attenuating the susceptibility to autoimmunity. These findings suggest that a hypothesis of Holmdahl and colleagues, that increased ROS release protects against organ-specific autoimmunity, could be true in case of BP [54]. Two possible mechanisms might be proposed for this association: (1) the decrease of ROS at old age associated with low FCGR3B copy numbers and low expression of FcgRIIIb might cause an inability to sufficiently control an infectious agent, leading to an inappropriate overstimulation of the immune system and the development of pathogenic autoantibodies; (2) the function of neutrophils as B helper cells [55] could be impaired at old age and low copy number of FCGR3B, leading to uncontrolled development of autoantibodies. Our results did not confirm the observation of an earlier study that suggested an association between BP disease risk and a polymorphism leading to a valine substitution for phenylalanine at position 158 in the FcgRIIIa (rs396911) [55]. However, the mentioned study used an inappropriate genotyping method that ignored the copy number variation of this gene, unknown at the time of publication. In contrast to this finding, FCGR3B genotypes do not influence the risk of PV/PF: it was increased in the presence of the FCGR2C.ORF allele and decreased in the presence of a promoter polymorphism that leads to enhanced expression of the FcgRIIb.

Because the FcgRIIb is involved in peripheral tolerance on B cells, which can be counterbalanced by functional FcgRIIc expression [53], it is possible that the risk of PV/PF increases due to changed threshold for activation and proliferation of autoreactive B cells. This association pattern is completely the opposite from what we observe for BP, indicating a different type of pathogenic mechanism; probably because expression and function of FcgR on myeloid cells are involved in the development of autoimmunity in BP. The involvement of peripheral tolerance in PV/PF risk could correlate with a possible pre-disease status preceding the clinical manifestation of this disease. Such a pre-disease status has been observed in endemic pemphigus foliaceous (Fogo selvagem), where non-pathogenic autoantibodies against Dsg1 are observed in the individuals of the affected population [56]. An impaired peripheral tolerance might increase the probability of a transition between pre-disease to clinically manifest pemphigus. 5. Conclusions In this study, we dissected the impact of allelic and copynumber variation in the complex gene region of low-affinity FcgR on the response of granulocytes, and compared this to the susceptibility to develop organ-specific autoimmune diseases. Our findings indicate that a reduced granulocyte responsiveness might contribute to the development of BP, but not of PV/PF; here, a disturbed peripheral tolerance in B cells might increase the disease risk. Most of the functional knowledge about the role of FcgRs in autoimmune diseases applies for the later stages of pathogenesis. This study provides functional insight into the contribution of FcgRs to processes and conditions that eventually lead to clinically relevant autoimmunity. Contributions A.R., I.R.K and S.I. designed research; A.R., G.V., M.F. and J.S. performed experiments; A.R., G.V., M.F., R.J.L, R.V., I.R.R. and S.I. analyzed the data and made the figures and tables; A.R., G.V., R.J.L, S.M., R.V., O.H. A.N. F.F., W.L., R.G., S.B., M.S., R.E., D.Z., I.R.K., E.S. and S.I. wrote the manuscript; A.R., M.F., O.H., S.G., A.N., F.F., S.S., W.L., R.G., S.B., M.L., R.E., M.H., D.Z. and E.S. and The German AIBD Genetic Study Group collected patient materials. Conflict-of-interest disclosure The authors declare no competing financial interests. Acknowledgments We thank everybody in the Department of Transfusion Medicine (University of Lübeck, Lübeck, Germany) involved in the collection of blood samples and all of the volunteers who donated blood for the experiments performed in this study. This work received infrastructural support from the Deutsche Forschungsgemeinschaft Cluster of Excellence Inflammation at Interfaces (Cluster 306/2) and from Research Training Group Grant 1727/1 (TP2) (to A.R.). Appendix. The German AIBD Genetic Study group members (in alphabetical order) Sandrine Benoit, Department of Dermatology, Venereology and Allergology, University Hospital of Würzburg, Würzburg, Germany. €schlein, Department of Dermatology, University of Georg Da Greifswald, Greifswald, Germany. Rüdiger Eming, Department of Dermatology, Venereology and Allergology, Phillip University of

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€ser, Department of Marburg, Marburg, Germany. Regine Gla Dermatology, Venereology and Allergology, Christian Albrecht University, Kiel, Germany. Mattias Goebeler, Department of Dermatology, Venereology and Allergology, University Hospital of Würzburg, Würzburg, Germany. Steven Goetze, Department of Dermatology, Unviersity Hospital Jena, Jena, Germany. Claudia Günther, Department of Dermatology, University Hospital of Dresden, Dresden, Germany. Eva Hadaschik, Department of Dermatology, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany. Bernhard Homey, Department of Dermatology, Heinrich Heine University, Düsseldorf, Germany. Nicolas Hunzelmann, Department of Dermatology, University of Cologne, Cologne, Germany. Andreas Kreuter, Department of Dermatology, Venereology and Allergological, University of Bochum, Bochum, Germany. Manfred Kunz, Department of Dermatology, Venereology and Allergology, University of Leipzig, Leipzig, Germany. Undine Lippert, Department of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany. Wiebke Ludwig-Peitsch, Department of Dermatology, University Medical Center Mannheim, Heidelberg University, Mannheim, Germany. €hler, Department of Dermatology, Saarland University Claudia Pfo sSa rdy, Department of Hospital, Homburg/Saar, Germany. Miklo Dermatology, Ludwig Maximilian University Munich, Munich, Germany. Michael Sticherling, Department of Dermatology, University of Erlangen-Nuremberg, Erlangen, Germany. Margitta Worm, Department of Dermatology, Venereology and Allergology, , Charite -Medical University Berlin, Berlin, Allergy Center Charite Germany. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jaut.2015.05.004. References [1] F. Nimmerjahn, J.V. Ravetch, Fcgamma receptors: old friends and new family members, Immunity 24 (2006) 19e28, http://dx.doi.org/10.1016/ j.immuni.2005.11.010. [2] S. Mihai, F. Nimmerjahn, The role of Fc receptors and complement in autoimmunity, Autoimmun. Rev. 12 (2013) 657e660, http://dx.doi.org/10.1016/ j.autrev.2012.10.008. [3] P. Bruhns, B. Iannascoli, P. England, D.A. Mancardi, N. Fernandez, S. Jorieux, et al., Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses, Blood 113 (2009) 3716e3725, http://dx.doi.org/10.1182/blood-2008-09-179754. [4] P.M. Hogarth, Fc receptors are major mediators of antibody based inflammation in autoimmunity, Curr. Opin. Immunol. 14 (2002) 798e802. [5] M. Zhao, M.E. Trimbeger, N. Li, L.A. Diaz, S.D. Shapiro, Z. Liu, Role of FcRs in animal model of autoimmune bullous pemphigoid, J. Immunol. 177 (2006) 3398e3405. [6] M. Kasperkiewicz, F. Nimmerjahn, S. Wende, M. Hirose, H. Iwata, M.F. Jonkman, et al., Genetic identification and functional validation of FcgRIV as key molecule in autoantibody-induced tissue injury, J. Pathol. 228 (2012) 8e19, http://dx.doi.org/10.1002/path.4023. [7] E. Schmidt, D. Zillikens, Pemphigoid diseases, Lancet 381 (2013) 320e332, http://dx.doi.org/10.1016/S0140-6736(12)61140-4. [8] T. Takai, Roles of Fc receptors in autoimmunity, Nat. Rev. Immunol. 2 (2002) 580e592, http://dx.doi.org/10.1038/nri856. [9] F. Nimmerjahn, J.V. Ravetch, Fcgamma receptors as regulators of immune responses, Nat. Rev. Immunol. 8 (2008) 34e47, http://dx.doi.org/10.1038/ nri2206. [10] P. Bruhns, Properties of mouse and human IgG receptors and their contribution to disease models, Blood 119 (2012) 5640e5649, http://dx.doi.org/ 10.1182/blood-2012-01-380121. [11] A. David, R. Fridlich, I. Aviram, The presence of membrane proteinase 3 in neutrophil lipid rafts and its colocalization with FcgammaRIIIb and cytochrome b558, Exp. Cell Res. 308 (2005) 156e165, http://dx.doi.org/10.1016/ j.yexcr.2005.03.034. [12] G. Vidarsson, J.G. van de Winkel, Fc receptor and complement receptor-mediated phagocytosis in host defence, Curr. Opin. Infect. Dis. 11 (1998) 271e278. [13] T.W. Huizinga, C.E. van der Schoot, C. Jost, R. Klaassen, M. Kleijer, A.E. von dem Borne, et al., The PI-linked receptor FcRIII is released on stimulation of neutrophils, Nature 333 (1988) 667e669, http://dx.doi.org/10.1038/333667a0.

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