Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47, on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamab- mediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy control lymph nodes. CRISPR-mediated CD47 overexpression affected tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. A combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro-generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor-targeting immunotherapies such as tafasitamab and suggest that there is value in exploring the combination in the clinic.

Generalized pustular psoriasis is a severe psoriatic subtype characterized by epidermal neutrophil infiltration. Although variants in IL36RN and MPO have been shown to affect immune cells, a systematic analysis of neutrophils and PBMC subsets and their differential gene expression dependent on MPO genotypes was not performed yet. We assessed the transcriptomes of MPO-deficient patients using single-cell RNA sequencing of PBMCs and RNA sequencing of neutrophils in a stable disease state. Cell-type annotation by multimodal reference mapping of single-cell RNA-sequencing data was verified by flow cytometry of surface and intracellular markers; the proportions of CD4+ cytotoxic T lymphocytes and other CD4+ effector cells were increased in generalized pustular psoriasis, whereas the frequencies of na??ve CD4+ T cells were significantly lower. The expression of FGFBP2 marking CD4+ cytotoxic T lymphocytes and CD8+ effector memory T cells was elevated in patients with generalized pustular psoriasis with disease-contributing variants compared with that in noncarriers (P = 0.0015). In neutrophils, differentially expressed genes were significantly enriched in genes of the classical complement activation pathway. Future studies assessing affected cell types and pathways will show their contribution to generalized pustular psoriasis???s pathogenesis and indicate whether findings can be transferred to the acute epidermal situation and whether depletion or inactivation of CD4+ cytotoxic T lymphocytes may be a reasonable therapeutic approach.

Psoriasis is a common inflammatory skin disorder with a strong impact on patients' QOL. The most common psoriasis form, psoriasis vulgaris (PsV), is characterized by demarcated, erythematous, raised plaques along with silvery scales. Up to 30% of patients with PsV develop an inflammatory joint disease named psoriatic arthritis (PsA) (Mease et al., 2013Mease P.J. Gladman D.D. Papp K.A. Khraishi M.M. Thaçi D. Behrens F. et al.Prevalence of rheumatologist-diagnosed psoriatic arthritis in patients with psoriasis in European/North American dermatology clinics.J Am Acad Dermatol. 2013; 69: 729-735Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar; Reich et al., 2009Reich K. Krüger K. Mössner R. Augustin M. Epidemiology and clinical pattern of psoriatic arthritis in Germany: a prospective interdisciplinary epidemiological study of 1511 patients with plaque-type psoriasis.Br J Dermatol. 2009; 160: 1040-1047Crossref PubMed Scopus (273) Google Scholar). PsV and PsA are genetically complex diseases with >65 susceptibility loci identified in one or both psoriatic subtypes (Tsoi et al., 2017Tsoi L.C. Stuart P.E. Tian C. Gudjonsson J.E. Das S. Zawistowski M. et al.Large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants.Nat Commun. 2017; 8: 15382Crossref PubMed Scopus (125) Google Scholar). In contrast to PsV and PsA, pustular psoriatic subtypes—palmoplantar pustular psoriasis or palmoplantar pustulosis (PPP) and generalized pustular psoriasis (GPP)—are rarer. PPP is characterized by localized epidermal neutrophil pustules, whereas more generalized pustules in severe multisystemic inflammation are typical for GPP. The genetic etiology of PPP is unsolved; discrepant results related to association with IL36RN (encoding the IL-36 receptor antagonist) variants were reported in British patients (Twelves et al., 2019Twelves S. Mostafa A. Dand N. Burri E. Farkas K. Wilson R. et al.Clinical and genetic differences between pustular psoriasis subtypes.J Allergy Clin Immunol. 2019; 143: 1021-1026Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) versus in German and Estonian patients (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (43) Google Scholar). Conversely, IL36RN has been acknowledged as a major gene for GPP (Marrakchi et al., 2011Marrakchi S. Guigue P. Renshaw B.R. Puel A. Pei X.Y. Fraitag S. et al.Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.N Engl J Med. 2011; 365: 620-628Crossref PubMed Scopus (623) Google Scholar), although further factors contribute to the pathogenesis of GPP, and oligogenic inheritance is discussed (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (43) Google Scholar). Patients diagnosed with syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis exhibit different combinations of inflammatory skin and bone diseases considerably overlapping with common psoriatic subtypes and pustular psoriasis; the genetic basis of syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis is largely unknown. Recently, we and others identified MPO as an additional major susceptibility gene in GPP (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar; Vergnano et al., 2020Vergnano M. Mockenhaupt M. Benzian-Olsson N. Paulmann M. Grys K. Mahil S.K. et al.Loss-of-function myeloperoxidase mutations are associated with increased neutrophil counts and pustular skin disease.Am J Hum Genet. 2020; 107: 539-543Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The deficiency of neutrophilic enzyme MPO impairs phagocytosis of dying neutrophils and increases the activity of IL-36 activating proteases, thereby driving inflammation in pustules (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). In addition, patients with GPP with MPO variants are more likely to have PPP, tongue manifestations, and a positive family history of psoriasis than those without MPO variants (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar); an increased serum amount of MPO has been detected in patients with PsV and PsA, suggesting a potentially protective role of MPO variants in common psoriatic manifestations (Cretu et al., 2018Cretu D. Gao L. Liang K. Soosaipillai A. Diamandis E.P. Chandran V. Differentiating psoriatic arthritis from psoriasis without psoriatic arthritis using novel serum biomarkers.Arthritis Care Res (Hoboken). 2018; 70: 454-461Crossref PubMed Scopus (26) Google Scholar). These findings prompted us to investigate the role of MPO variants in other psoriatic subtypes. This study was approved by the ethical committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg (Erlangen, Germany) and the ethical committees of the further involved universities. Written informed consent was obtained from each affected and control individual before enrolment. We analyzed the segregation of MPO, IL36RN, and CARD14 variants in available family members of seven patients with GPP and one patient with acute generalized exanthematous pustulosis, previously identified to carry MPO variants (Figure 1). On the basis of self-reported diagnoses, 14 of those family members were affected by PsV, 2 were affected by inflammatory joint disease, and 1 was affected by PPP. Overall, 6 of the 12 analyzed family members with PsV (50%) and 8 of the 13 analyzed healthy family members (62%) carried a heterozygous MPO variant. When the families were analyzed on the basis of associated enzyme activity of MPO variants, from complete to partial MPO deficiency in the homozygous state (Supplementary Table S1), the proportion of patients with PsV with MPO variants associated with complete deficiency was slightly higher (6 of 10, 60%) but was similar to the one in healthy family members (7 of 11, 64%). In two families with IL36RN variants in addition to the ones in MPO (pedigrees VI and VII, Figure 1), double heterozygous individuals were healthy, whereas the two individuals who were heterozygous or compound heterozygous only for IL36RN presented with PsV (family VI) or PsV and PPP (pedigree VII). Because these findings were based on a limited number of individuals and we could neither establish nor exclude a role of MPO variants in susceptibility to PsV, we investigated MPO variants in larger study groups of patients with psoriasis. We genotyped 3,663 individuals (1,105 with PsV; 1,313 with PsA; 271 with PPP; 52 with syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis; and 922 control individuals) for seven of the most common functionally relevant MPO variants (Supplementary Table S1) (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) using global screening array and single SNP genotyping assays (Supplementary Materials and Methods including Supplementary Figure S2, Supplementary Tables S7 and S8). We extensively validated the genotyping quality with several approaches, including repeated genotyping with different assays and Sanger sequencing in individuals with MPO variants and randomly selected genotypes (Supplementary Materials and Methods). We determined the cumulative frequency of all variants in patients with psoriatic subtypes and in control individuals as described before (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) (Table 1). Whereas the frequencies of all MPO variants were similar between two control groups and comparable in PsV; PsA; and syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis, we observed evidence for marginal significant association of PPP with MPO variants (P = 0.015). Analyses at the single variant level pointed to a prominent contribution of variant c.995C>T/p.(Ala332Val), associated with partial MPO deficiency, in PPP (P = 0.008; Supplementary Table S1).Table 1Allele Numbers and Frequencies of Rare MPO Variants in 1,105 Patients with PsV, 1,313 Patients with PsA, 271 Patients with PPP, 52 Patients with SAPHO Syndrome, 922 German Control Indivs., and 64,591 NFE Indivs. of gnomADVariant at Nucleotide and Protein LevelPsoriasis SubtypesControl Indivs.PsV, n (%)PsA, n (%)PPP, n (%)SAPHO Syndrome, n (%)Control Indivs., n (%)NFE Indivs., n (%)c.752T>C/p.(Met251Thr)27 (1.22)38 (1.45)8 (1.48)2 (1.92)25 (1.36)1,735 (1.34)c.995C>T/p.(Ala332Val)33 (1.49)38 (1.45)19 (3.51)1 (0.96)34 (1.84)2,314 (1.79)c.1555_1568del/ p.(Met519Profs∗21)6 (0.27)6 (0.23)2 (0.37)1 (0.96)2 (0.11)0 (.15)c.1642C>T/p.(Arg548Trp)0 (0)0 (0)0 (0)0 (0)1 (0.05)67 (0.05)c.1705C>T/p.(Arg569Trp)6 (0.27)6 (0.23)3 (0.55)1 (0.96)3 (0.16)370 (0.29)c.1768C>T/p.(Arg590Cys)0 (0)0 (0)0 (0.00)0 (0.00)0 (0)9 (0.01)c.2031-2A>C15 (0.68)15 (0.57)4 (0.74)1 (0.96)11 (0.6)936 (0.72)Σ mutant alleles87 (3.94)103 (3.92)36 (6.64)6 (5.77)Σ 5,696 (4.35)Σ WT alleles2,123 (96.06)2,523 (96.08)506 (93.36)98 (94.23)Σ 125,330 (95.65)P-valuen.s. (0.399)n.s. (0.333)0.015n.s. (0.464)——Abbreviations: indivs., individuals; n, number of patients; NFE, non-Finnish European; no., number; n.s., not significant; PPP, palmoplantar pustulosis; PsA, psoriatic arthritis; PsV, psoriasis vulgaris; SAPHO, syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis; WT, wild type.Σ indicates sum.Cumulative frequency distributions of missense or splice variants in patient and control groups were compared using Fisher's exact test with P-values not adjusted for the no. of tests (four psoriatic subtypes).Source from Karczewski et al., 2020Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. et al.The mutational constraint spectrum quantified from variation in 141,456 humans [published correction appears in Nature 2021;590:E53].Nature. 2020; 581: 434-443Crossref PubMed Scopus (1605) Google Scholar. Open table in a new tab Abbreviations: indivs., individuals; n, number of patients; NFE, non-Finnish European; no., number; n.s., not significant; PPP, palmoplantar pustulosis; PsA, psoriatic arthritis; PsV, psoriasis vulgaris; SAPHO, syndrome of synovitis, acne, pustulosis, hyperostosis, osteitis; WT, wild type. Σ indicates sum. Cumulative frequency distributions of missense or splice variants in patient and control groups were compared using Fisher's exact test with P-values not adjusted for the no. of tests (four psoriatic subtypes). Source from Karczewski et al., 2020Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. et al.The mutational constraint spectrum quantified from variation in 141,456 humans [published correction appears in Nature 2021;590:E53].Nature. 2020; 581: 434-443Crossref PubMed Scopus (1605) Google Scholar. At the genotype level when considering the combination of two MPO variants, we observed mainly combinations resulting in partial MPO deficiency and estimated loss in enzyme activity of 65‒90% in patients (Supplementary Table S2) (Haskamp et al., 2020Haskamp S. Bruns H. Hahn M. Hoffmann M. Gregor A. Löhr S. et al.Myeloperoxidase modulates inflammation in generalized pustular psoriasis and additional rare pustular skin diseases.Am J Hum Genet. 2020; 107: 527-538Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar; Marchetti et al., 2004Marchetti C. Patriarca P. Solero G.P. Baralle F.E. Romano M. Genetic characterization of myeloperoxidase deficiency in Italy.Hum Mutat. 2004; 23: 496-505Crossref PubMed Scopus (23) Google Scholar; Nauseef et al., 1994Nauseef W.M. Brigham S. Cogley M. Hereditary myeloperoxidase deficiency due to a missense mutation of arginine 569 to tryptophan.J Biol Chem. 1994; 269: 1212-1216Abstract Full Text PDF PubMed Google Scholar). Although the percentage of patients with psoriasis with two variants was increased 1.5-fold compared with that of control individuals owing to higher percentages in PsV and PPP (0.54% and 0.74%, respectively, vs. 0.27%), we did not observe a significant difference in the overall numbers of MPO variants (Supplementary Table S3). Similar results were found when combining MPO genotype data with IL36RN variants (Supplementary Table S4). By investigating the relationships of MPO variants with the age of onset (Supplementary Figure S1), the severity of disease (Supplementary Table S5), and positive family history (Supplementary Table S6), we obtained negative findings, with a tendency to a negative correlation regarding the severity of disease (P = 0.09). Our study found a marginally significant association of MPO variants with intermediate effect size with PPP, although the P-value indicating association did not withstand correction for multiple testing most probably owing to the size of our patient cohort. We found no association with both PsV and PsA. This might be due to the rarity of these alleles, limiting the power of our study. Previous GWASs in psoriasis did not describe MPO as a susceptibility locus (Tsoi et al., 2017Tsoi L.C. Stuart P.E. Tian C. Gudjonsson J.E. Das S. Zawistowski M. et al.Large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants.Nat Commun. 2017; 8: 15382Crossref PubMed Scopus (125) Google Scholar). Because our analyses focused on German patients, we cannot exclude a potential role in patients of other ethnic backgrounds. Nevertheless, individuals carrying two MPO variants may be more susceptible to psoriasis, because the proportion of patients with psoriasis with two variants (PPP and PsV) was nominally higher than that in controls. These findings require independent replication but point to a situation where other genetic factors largely increase susceptibility to the four psoriasis subtypes. Overall, our findings differentiate the genetic etiology of GPP from those of the other psoriasis subtypes. Relevant data generated and/or analyzed are available in this manuscript and its supplementary file. Stefan Haskamp: http://orcid.org/0000-0003-3760-9074 Joseph Simon Horowitz: http://orcid.org/0000-0002-4456-6984 Vinzenz Oji: http://orcid.org/0000-0003-1380-4828 Sandra Philipp: http://orcid.org/0000-0002-9421-5461 Michael Sticherling: http://orcid.org/0000-0001-9396-3938 Knut Schäkel: http://orcid.org/0000-0001-6344-7799 Sarah Schuhmann: http://orcid.org/0000-0002-6775-3547 Jörg C. Prinz: http://orcid.org/0000-0002-8857-5495 Harald Burkhardt: http://orcid.org/0000-0002-6261-3131 Frank Behrens: http://orcid.org/0000-0001-8750-7186 Beate Böhm: http://orcid.org/0000-0001-5823-1184 Michaela Köhm: http://orcid.org/0000-0002-3579-5574 Jürgen Rech: http://orcid.org/0000-0002-2569-2029 David Simon: http://orcid.org/0000-0001-8310-7820 Georg Schett: http://orcid.org/0000-0001-8740-9615 Kirsten Morrison: http://orcid.org/0000-0002-5308-0249 Sascha Gerdes: http://orcid.org/0000-0002-6667-7757 Gunter Assmann: http://orcid.org/0000-0002-3216-9340 Ali Nimeh: http://orcid.org/0000-0003-0912-4758 Volker Schuster: http://orcid.org/0000-0001-5735-1838 Arnd Jacobi: http://orcid.org/0000-0002-0684-3426 Ansgar Weyergraf: http://orcid.org/0000-0001-5221-2492 André Reis: http://orcid.org/0000-0002-6301-6363 Steffen Uebe: http://orcid.org/0000-0002-4819-7637 Dagmar Wilsmann-Theis: http://orcid.org/0000-0002-5594-7192 Rotraut Mößner: http://orcid.org/0000-0003-2476-7647 Ulrike Hüffmeier: http://orcid.org/0000-0001-6448-4671 JSH is employed in the psychiatric department of Klinik am Europakanal, Erlangen, Germany. MS is involved in clinical studies on anti‒IL-36 antibody in generalized pustular psoriasis and palmoplantar pustulosis by Boehringer Mannheim. The remaining authors state no conflict of interest. We are grateful to the patients and controls who participated in this study. We thank Anne Gerschütz, Valentina Rabinovich, and Iris Kerker for excellent technical assistance. We thank Peter Morrison, PhD (Department of Dermatology, Førde Sentralsjukehus, Førde, Norway) for proofreading this manuscript. This project was funded by grants to UH from the Deutsche Forschungsgemeinschaft (CRC1181, project A05; HU 2163/1-1), by a grant to UH from the Dr. Pfleger-Stiftung, and by grants to AR and UH from the Federal Ministry of Education and Research (Metarthros 01EC1407A) and a grant to UH from the Interdisciplinary Centre for Clinical Research Erlangen (Project A85). Conceptualization: UH; Investigation: JSH, UH; Formal Analysis: SH, JSH, SU, UH; Resources: VO, SP, MS, KS, SS, JCP, HB, FB, BB, MK, JR, DS, GS, KM, SG, GA, AN, VS, AJ, AW, DWT, RM, UH; Writing - Original Draft Preparation: SH, UH; Writing - Review and Editing: SH, JSH, VO, SP, MS, KS, SS, JCP, HB, FB, BB, MK, JR, DS, GS, KM, SG, GA, AN, VS, AJ, AW, AR, SU, DWT, RM, UH Download .pdf (.62 MB) Help with pdf files Supplementary File

Generalized pustular psoriasis (GPP) represents a rare pustular disease manifesting as a multisystemic inflammation in a chronic or episodic course, sometimes as life-threatening incidents. Biallelic IL36RN mutations are known to be disease-causing or -contributing in 21–41% of patients with GPP (Hussain et al., 2015Hussain S. Berki D.M. Choon S.E. Burden A.D. Allen M.H. Arostegui J.I. et al.IL36RN mutations define a severe autoinflammatory phenotype of generalized pustular psoriasis.J Allergy Clin Immunol. 2015; 135: 1067-1070.e9Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Marrakchi et al., 2011Marrakchi S. Guigue P. Renshaw B.R. Puel A. Pei X.Y. Fraitag S. et al.Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.N Engl J Med. 2011; 365: 620-628Crossref PubMed Scopus (668) Google Scholar, Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar, Onoufriadis et al., 2011Onoufriadis A. Simpson M.A. Pink A.E. Di Meglio P. Smith C.H. Pullabhatla V. et al.Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis.Am J Hum Genet. 2011; 89: 432-437Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, Sugiura et al., 2013Sugiura K. Takemoto A. Yamaguchi M. Takahashi H. Shoda Y. Mitsuma T. et al.The majority of generalized pustular psoriasis without psoriasis vulgaris is caused by deficiency of interleukin-36 receptor antagonist.J Invest Dermatol. 2013; 133: 2514-2521Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). IL-36 binds to the IL-36 receptor complex, thereby triggering the activation of mitogen-activated protein kinase and NF-κB, resulting in the production of proinflammatory cytokines. The IL-36 receptor antagonist competitively binds to the IL-36 receptor, inhibiting signal transduction (Hahn et al., 2017Hahn M. Frey S. Hueber A.J. The novel interleukin-1 cytokine family members in inflammatory diseases.Curr Opin Rheumatol. 2017; 29: 208-213Crossref PubMed Scopus (39) Google Scholar). IL36RN mutations lead to reduced antagonism of the IL-36 receptor, with an imbalance in the IL-36 pathway supportive of proinflammatory IL-36α, -β, and -γ cytokines (Marrakchi et al., 2011Marrakchi S. Guigue P. Renshaw B.R. Puel A. Pei X.Y. Fraitag S. et al.Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.N Engl J Med. 2011; 365: 620-628Crossref PubMed Scopus (668) Google Scholar). Additional disease-associated variants in CARD14 and/or AP1S3 were identified in 15% of patients carrying IL36RN mutations, suggesting an oligogenic rather than monogenic inheritance (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar). Because variants in known susceptibility genes cannot be identified in 64% of patients with GPP (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (31) Google Scholar), we aimed to detect more disease-relevant variants by performing whole exome sequencing in 25 patients with GPP. Our studies were approved by the ethical committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg and the University of Göttingen. Written informed consent was obtained from each patient and healthy volunteer before enrollment. Whole exome sequencing did not reveal any new candidate gene with autosomal-recessive inheritance in ≥2 patients. As effects of truncating variants can be predicted more reliably than effects of nonsynonymous variants, we selected genes with truncating variants in ≥2 patients. By analyzing gene function and expression, we prioritized SERPINA3 as the most relevant gene. We identified the same heterozygous deletion c.966delT/p.Tyr322Ter in two patients with GPP and confirmed it by Sanger sequencing (Supplementary Figure S1a and b). This rare variant was significantly associated with GPP (Pc = 2.65E-04, Table 1). Haplotype analyses indicated that the mutation localizes to the same common haplotype, leaving open whether the variant arose once or recurrently. Sanger sequencing of a further 54 patients with GPP, acrodermatitis continua suppurativa Hallopeau, or acute generalized exanthematous pustulosis revealed a rare heterozygous missense variant c.1046C>A/p.Ala349Asp (Supplementary Table S1).Table 1Number of Absolute Alleles and Frequencies of Rare Indel Variants in SERPINA3 in Patients with Pustular Psoriasis and Control Individuals and Results of Comparisons of Allele Frequency DistributionsVariant at nucleotide/ protein levelPosition in hg19 (dbSNP-ID)79 patients with GPP/ACH/AGEP398 patients with GPP/ACH/AGEP/PPP64,583 Non-Finnish European individuals (gnomAD)mutant – no. of alleles (%)wildtype – no. of alleles (%)mutant – no. of alleles (%)wildtype – no. of alleles (%)mutant – no. of alleles (%)wildtype – no. of alleles (%)c.966delT/p.Tyr322Terchr14:95,088,726 (rs771543687)2 (1.27)156 (98.73)2 (0.25)794 (99.75)8 (0.01)129,166 (99.99)Pc2.65E-040.0064OR (95% CI)207.52 (21.24–1011.41)40.65 (4.20–204.93)319 patients with PPP398 patients with GPP/ACH/AGEP/PPP64,523 Non-Finnish European individuals (gnomAD)c.221_223delTCT/p.Phe75delchr14:95,080,999-95,081,001 (rs748990999)1 (0.16)637 (99.84)1 (0.13)795 (99.87)14 (0.01)129,046 (99.99)Pc0.2840.352OR (95% CI)14.47 (0.34–95.39)11.59 (0.27–76.48)Abbreviations: ACH, acrodermatitis continua suppurativa Hallopeau; AGEP, acute generalized exanthematous pustulosis; GPP, generalized pustular psoriasis; OR, odds ratio; PPP, palmoplantar pustular psoriasis.Patients tested included 79 patients with GPP/ACH/AEP or 319 PPP patients or combined study groups. Control individuals were non-Finnish Europeans from gnomAD (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Google Scholar). Comparisons were made using Fisher's exact test. Open table in a new tab Abbreviations: ACH, acrodermatitis continua suppurativa Hallopeau; AGEP, acute generalized exanthematous pustulosis; GPP, generalized pustular psoriasis; OR, odds ratio; PPP, palmoplantar pustular psoriasis. Patients tested included 79 patients with GPP/ACH/AEP or 319 PPP patients or combined study groups. Control individuals were non-Finnish Europeans from gnomAD (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Google Scholar). Comparisons were made using Fisher's exact test. SERPINA3 encodes serine protease inhibitor A3 (serpin A3), which specifically inhibits several proteases (Cooperman et al., 1993Cooperman B.S. Stavridi E. Nickbarg E. Rescorla E. Schechter N.M. Rubin H. Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex.J Biol Chem. 1993; 268: 23616-23625Abstract Full Text PDF PubMed Google Scholar, Djie et al., 1997Djie M.Z. Stone S.R. Le Bonniec B.F. Intrinsic specificity of the reactive site loop of alpha 1-antitrypsin, alpha 1-antichymotrypsin, antithrombin III, and protease nexin I.J Biol Chem. 1997; 272: 16268-16273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The key functional site of serpin A3 is the reactive center loop, mediating binding of the target protease. After cleavage of the reactive center loop by the protease, a covalent serpin-protease complex is formed, leading to irreversible protease inactivation. Theoretically, the entire reactive center loop is missing in c.966delT/p.Tyr322Ter (Figure 1a), indicating a functionally inactive protein, whereas our in vitro experiments indicate nonsense-mediated mRNA decay and therefore haploinsufficiency of SERPINA3. The effect of c.1046C>A/p.Ala349Asp was predicted to be uncritical because of the residue's orientation to the solvent. Our quantitative expression analyses revealed the most abundant expression of SERPINA3 in the liver and lower expression in skin, dermal fibroblasts, HaCaT cells, trachea, and lung (Supplementary Figure S2a). In controls, seral serpin A3 was in the range of 0.05–0.23 mg/ml, the level in the two frameshift variant carriers at the range's low end (Figure 1b), matching the finding of a haploinsufficient gene. In male controls, we observed evidence for some correlation of serpin A3 with age (Figure 1c), confirming previous results (Herman et al., 2009Herman W.A. Seńko A. Korczowska I. Lacka K. Assessment of selected serum inflammatory markers of acute phase response and their correlations with adrenal androgens and metabolic syndrome in a population of men over the age of 40.Pol Arch Med Wewn. 2009; 119: 704-711PubMed Google Scholar). More importantly, the finding underlines the relevance of very low levels of serpin A3 in our 75-year-old male patient with GPP. We observed highly reduced mRNA expression after transfection of the mutant vector compared with the wildtype (Figure 1d), indicating nonsense-mediated mRNA decay. In the supernatants of cells transfected with the mutant, we could not detect any protein (Figure 1e). Immunohistochemistry revealed a confined expression of serpin A3 in the upper epidermis in patients with GPP carrying the frameshift variant, more pronounced at the edge of pustules (Figure 1f–h). As serpin A3 belongs to the acute phase proteins (Aronsen et al., 1972Aronsen K.F. Ekelund G. Kindmark C.O. Laurell C.B. Sequential changes of plasma proteins after surgical trauma.Scand J Clin Lab Invest Suppl. 1972; 124: 127-136Crossref PubMed Scopus (265) Google Scholar), we stimulated SERPINA3's expression in several cell lines. In HaCaT cells, we observed an earlier stimulation, whereas induction was highest in dermal fibroblasts (Supplementary Figure S2b–e). Sequencing analysis of SERPINA3 by Sanger in 319 patients with palmoplantar pustular psoriasis revealed two rare heterozygous coding variants. The effect of c.8G>A/p.Arg3Lys was predicted to be negligible, whereas the other variant's (in-frame-deletion c.221_223delTCT/p.Phe75del) effect was a significant conformational rearrangement of the adjacent residues of the shutter region (Supplementary Figure S3, Supplementary Results). c.221_223delTCT/p.Phe75del was not significantly associated with palmoplantar pustular psoriasis (Table 1). A targeted analysis of the frameshift variant in 2,223 patients with psoriasis vulgaris and psoriatic arthritis did not identify a single carrier. Serpin A3's interaction with cathepsin G is the strongest among the proteases (Beatty et al., 1980Beatty K. Bieth J. Travis J. Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin.J Biol Chem. 1980; 225: 3931-3934Abstract Full Text PDF Google Scholar). In vitro analyses indicated that neutrophilic serine proteases including cathepsin G can escalate inflammation by cleavage leading to an increased activation of less active IL-36β by a factor >500 (Henry et al., 2016Henry C.M. Sullivan G.P. Clancy D.M. Afonina I.S. Kulms D. Martin S.J. Neutrophil-derived proteases escalate inflammation through activation of IL-36 family cytokines.Cell Rep. 2016; 14: 708-722Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) (Supplementary Figure S4a). In an acute GPP episode with infiltration of neutrophil granulocytes in the upper epidermis, there is a close proximity of neutrophils to serpin A3. The known inhibitory effect of serpin A3 on cathepsin G suggests a regulatory interaction of both proteins in normal skin and an imbalance in the case of patients with GPP carrying SERPINA3 mutations (Supplementary Figure S4b). SERPINA3 has been identified to be expressed at higher levels in psoriasis vulgaris skin than normal skin (Li et al., 2014Li B. Tsoi L.C. Swindell W.R. Gudjonsson J.E. Tejasvi T. Johnston A. et al.Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms.J Invest Dermatol. 2014; 134: 1828-1838Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Based on our own experiences (D'Erme et al., 2015D'Erme A.M. Wilsmann-Theis D. Wagenpfeil J. Hölzel M. Ferring-Schmitt S. Sternberg S. et al.IL-36gamma (IL-1F9) is a biomarker for psoriasis skin lesions.J Invest Dermatol. 2015; 135: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), this differentiates psoriasis vulgaris from further chronic skin diseases such as atopic dermatitis and lichen planus without upregulated SERPINA3. A more recent comparison of the transcriptome in GPP skin with psoriasis vulgaris skin indicated that serpin A3 was upregulated in both psoriatic subtypes (Johnston et al., 2017Johnston A. Xing X. Wolterink L. Barnes D.H. Yin Z. Reingold L. et al.IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis.J Allergy Clin Immunol. 2017; 140: 109-120Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Although serum amount was in the lowest normal range in both carriers of c.966delT/p.Tyr322Ter, serpin A3 was determined in affected, but not normal, epidermis. This disparity might be explained by different disease phases, an acute GPP episode with upregulation of acute phase proteins during skin biopsy in contrast to the healthy interval of blood sampling. Lack of differences in serum levels of IL-36α and IL-36β between patients and controls (Supplementary Figure S5) might also reflect the healthy interval or lack of sufficiently sensitive ELISAs. In conclusion, we detected a loss-of-function variant in SERPINA3 in two independent patients, and in combination with our experimental data, we propose SERPINA3 as a new candidate gene for GPP. The small fraction of patients carrying mutations in SERPINA3 fits the known heterogeneity in GPP, and the inherited variant by a healthy parent in one case would be in accordance with the previously suggested oligogenic inheritance pattern. Genetic studies of independent patient groups are necessary to replicate our findings, and further experimental studies will shed light on SERPINA3's role in GPP and other pustular psoriasis subtypes. Whole exome sequencing was performed to detect new candidate genes in GPP. Because of privacy regulations, the whole datasets are not publicly available, but authors can be contacted for specific issues. Silke Frey: http://orcid.org/0000-0001-9411-8357 Heinrich Sticht: http://orcid.org/0000-0001-5644-045X Dagmar Wilsmann-Theis: http://orcid.org/0000-0002-5594-7192 Anne Gerschütz: http://orcid.org/0000-0002-3202-562X Katharina Wolf: http://orcid.org/0000-0001-7931-6056 Sabine Löhr: http://orcid.org/0000-0001-7720-0762 Stefan Haskamp: http://orcid.org/0000-0003-3760-9074 Benjamin Frey: http://orcid.org/0000-0001-6743-3351 Madelaine Hahn: http://orcid.org/0000-0001-7028-0456 Arif B. Ekici: http://orcid.org/0000-0001-6099-7066 Steffen Uebe: http://orcid.org/0000-0002-4819-7637 Christian Thiel: http://orcid.org/0000-0003-3817-7277 André Reis: http://orcid.org/0000-0002-6301-6363 Harald Burkhardt: http://orcid.org/0000-0002-6261-3131 Frank Behrens: http://orcid.org/0000-0001-8750-7186 Michaela Köhm: http://orcid.org/0000-0002-3579-5574 Jürgen Rech: http://orcid.org/0000-0002-2569-2029 Georg Schett: http://orcid.org/0000-0001-8740-9615 Gunter Assmann: http://orcid.org/0000-0002-3216-9340 Külli Kingo: http://orcid.org/0000-0001-6301-9612 Sulev Kõks: http://orcid.org/0000-0001-6087-6643 Rotraut Mössner: http://orcid.org/0000-0003-2476-7647 Jörg C. Prinz: http://orcid.org/0000-0002-8857-5495 Vinzenz Oji: http://orcid.org/0000-0003-1380-4828 Peter Schulz: http://orcid.org/0000-0002-3168-2660 Luis E. Muñoz: http://orcid.org/0000-0002-5395-804X Andreas E. Kremer: http://orcid.org/0000-0002-9263-948X Jörg Wenzel: http://orcid.org/0000-0002-4744-5993 Ulrike Hüffmeier: http://orcid.org/0000-0001-6448-4671 KK is an investigator for Celgene, Merck, Mitsubishi Pharma, Novartis, Regeneron Pharmaceuticals, Inc., and Sandoz. All other authors state no conflict of interest. The authors are grateful to all patients and healthy volunteers for participation in this project. The authors thank Ingo Ganzleben and Christoph Becker for providing the cell-line BEAS-2B (epithelial of lung bronchus). This project was partly funded by a grant to SF and UH from the DFG (CRC1181, project A05), by a grant to AR and UH from the BMBF (Metarthros 01EC1407A), by grants to UH from the DFG (HU 2163/1-1) and from the Interdisciplinary Centre for Clinical Research (laboratory rotation) of the Clinical Center Erlangen of the Friedrich-Alexander-Universität Erlangen-Nürnberg , and by support of the Innovative Medicine Initiative (IMI RTCure). Formal Analysis: HS, AG, KW, SL, SH, MH, SU; Funding Acquisition: UH; Investigation: SF, AG, KW, SL, MH; Methodology: AG, SH, BF, LEM; Project Administration: AG, UH; Resources: SF, DWT, HB, FB, MK, JR, GS, GA, KK, SK, RM, JP, AK, VO, PS, AEK; Software: HS, ABE, CT, SU; Supervision: SF, UH, AEK, JW; Validation: AG, SL; Visualization: SH, JW, UH; Writing - Original Draft Preparation: UH; Writing - Review and Editing: SF, AR, AEK We sequenced whole exomes of 25 patients with generalized pustular psoriasis (GPP), published within a larger group previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar). Those patients were selected based on a negative carrier status of IL36RN mutations. In addition, we used DNAs of 47 patients with independent GPP, 3 patients with acute generalized exanthematous pustulosis, and 4 patients with acrodermatitis continua suppurativa Hallopeau—also described previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar)—for a targeted SERPINA3 analysis. We also investigated DNAs of 273 German and 46 Estonian patients with palmoplantar pustular psoriasis (PPP) and 1,099 patients with psoriasis vulgaris, 1,124 patients with psoriatic arthritis, and 934 control individuals, described in similar compositions previously (Löhr et al., 2019Löhr S. Ekici A.B. Uebe S. Büttner C. Köhm M. Behrens F. et al.Association analyses of psoriatic arthritis and psoriasis vulgaris with functional NCF1 variants.Rheumatology(Oxford). 2019; 58: 915-917Crossref PubMed Scopus (3) Google Scholar, Mössner et al., 2015Mössner R. Frambach Y. Wilsmann-Theis D. Löhr S. Jacobi A. Weyergraf A. et al.Palmoplantar pustular psoriasis is associated with missense variants in CARD14, but not with loss-of-function mutations in IL36RN in European patients.J Invest Dermatol. 2015; 135: 2538-2541Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The investigations were conducted according to Declaration of Helsinki principles. All DNAs used for whole exome sequencing passed quality control and were enriched using the SureSelect Human All Exons Kits (version 4 [n = 12] or version 5 [n = 13], Agilent, Santa Clara, CA). Sequencing was performed on a SOLiD 5500 XL (n = 19) (Thermo Fisher Scientific, Waltham, MA) or an Illumina HiSeq 2500 (n = 6) (Illumina, San Diego, CA). Reads were aligned with the bwa mem algorithm (Li and Durbin, 2009Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (24737) Google Scholar) using the human genome assembly hg19 as a reference. Single nucleotide variants and small insertions and deletions were called using the algorithms GATK Haplotype Caller, GATK Universal Genotyper, SNVer, ATLAS2, diBayes, freeBayes, LifeScope InDel Caller, and Platypus and annotated with ANNOVAR (Wang et al., 2010Wang K. Li M. Hakonarson H.H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7264) Google Scholar). On average, coverage of the target was 144.1×, and 89.0% of the target sequence was covered ≥20×. We included variants with an allele frequency of ≤0.3% in all European individuals of the Exome Aggregation Consortium (Lek et al., 2016Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. et al.Analysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6277) Google Scholar) and an allele frequency of ≤0.5% in the Exome Variant Server and 1000 Genomes. Selected variants had coverage of ≥20× and were identified ≤2× in a group of 421 independent individuals sequenced in-house for projects analyzing nonimmunological diseases. All sequenced patients with GPP were independent individuals. We prioritized for variants in overlapping genes using different modes of inheritance. As mutations in IL36RN, the first major gene in GPP, have been identified on both alleles, we selected for homozygous or compound heterozygous variants in overlapping genes but were unable to identify a single gene. Therefore, we prioritized for heterozygous truncating variants and identified two truncating variants in six different genes (Supplementary Tables S2 and S3), which were further evaluated using the Integrative Genomics Viewer (Thorvaldsdóttir et al., 2013Thorvaldsdóttir H. Robinson J.T. Mesirov J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.Brief Bioinform. 2013; 14: 178-192Crossref PubMed Scopus (4562) Google Scholar). Function and expression of candidate genes were considered to further select one candidate gene. Variants in NLRP5 and SERPINA3 were confirmed by Sanger sequencing. Parents of one of the two patients were available for targeted testing of the SERPINA3 variant. Supplementary Table S4 gives an overview on rare variants in other genes in the two carriers of the SERPINA3 frameshift variant. Structural analysis of serine protease inhibitor A3 (serpin A3) was performed on the basis of the known crystal structure (PDB:1QMN) (Gooptu et al., 2000Gooptu B. Hazes B. Chang W.S. Dafforn T.R. Carrell R.W. Read R.J. et al.Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease.Proc Natl Acad Sci USA. 2000; 97: 67-72Crossref PubMed Scopus (175) Google Scholar). Variant p.Pro55Leu (p.Pro78Leu according to NM_001085) present in the structure was reverted to the wildtype using Swiss-PDBViewer (Guex and Peitsch, 1997Guex N. Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). ModLoop (Fiser and Sali, 2003Fiser A. Sali A. ModLoop: automated modeling of loops in protein structures.Bioinformatics. 2003; 19: 2500-2501Crossref PubMed Scopus (566) Google Scholar) was applied to model the deletion of phenylalanine and to add missing residues of the reactive center loop (PDB:7API) (Engh et al., 1989Engh R. Löbermann H. Schneider M. Wiegand G. Huber R. Laurell C.B. The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism.Protein Eng. 1989; 2: 407-415Crossref PubMed Scopus (55) Google Scholar). RasMol (Sayle and Milner-White, 1995Sayle R.A. Milner-White E.J. RASMOL: biomolecular graphics for all.Trends Biochem Sci. 1995; 20: 374Abstract Full Text PDF PubMed Scopus (2298) Google Scholar) was used for visualization. We sequenced exons of SERPINA3 in 54 independent patients with rare pustular psoriasis (GPP, acrodermatitis continua suppurativa Hallopeau, and acute generalized exanthematous pustulosis) by Sanger (primer sequences, Supplementary Table S5; reference sequence, NM_001085). Furthermore, we established a multiplex ligation-dependent probe amplification for exons if possible or regions closed to exons (sequences of oligos, Supplementary Table S6) and performed multiplex ligation-dependent probe amplification in all patients with rare pustular psoriasis forms (n = 79). We applied Fisher's exact test to determine allele frequency differences between patients and control individuals as described previously (Mössner et al., 2018Mössner R. Wilsmann-Theis D. Oji V. Gkogkolou P. Löhr S. Schulz P. et al.The genetic basis for most patients with pustular skin disease remains elusive.Br J Dermatol. 2018; 178: 740-748Crossref PubMed Scopus (52) Google Scholar) and to obtain evidence for the association of combination of GPP, acrodermatitis continua suppurativa Hallopeau, and acute generalized exanthematous pustulosis and of pustular psoriasis in general with c.966delT/p.Tyr322Ter and for the association of PPP and of pustular psoriasis in general with c.221_223delTCT/p.Phe75del, correcting the P-value for Bonferroni (n = 4). For differential expression analyses, we used a multitissue panel (Clontech, Mountain View, CA) including RNAs collected from the following human tissues: bone marrow, whole brain, fetal brain, fetal liver, heart, kidney, liver, lung, placenta, prostate, skeletal muscle, spleen, testis, thymus, trachea, uterus, colon, small intestine, spinal cord, and stomach. In addition, we used human mRNA from a lymphoblastoid cell line, two skin biopsies described previously (Hüffmeier et al., 2009Hüffmeier U. Lascorz J. Becker T. Schürmeier-Horst F. Magener A. Ekici A.B. et al.Characterisation of psoriasis susceptibility locus 6 (PSORS6) in patients with early onset psoriasis and evidence for interaction with PSORS1.J Med Genet. 2009; 46: 736-744Crossref PubMed Scopus (24) Google Scholar), a HaCaT cell line (Cell Lines Service, Eppelheim, Germany), two liver cell lines (American Tissue Culture Collection, Manassas, Virginia), CD14+ cells (negative selection with magnetic beads [Stemcell Technologies, Cologne, Germany]), and CD16+ cells (cell separation with a ficoll gradient). Tissues were provided by healthy donors. We performed quantitative PCR of cDNAs for expression of SERPINA3 in different tissues using a predesigned assay (Hs01038298_m1; Thermo Fisher Scientific) including an exon-spanning probe as described previously (Löhr et al., 2019Löhr S. Ekici A.B. Uebe S. Büttner C. Köhm M. Behrens F. et al.Association analyses of psoriatic arthritis and psoriasis vulgaris with functional NCF1 variants.Rheumatology(Oxford). 2019; 58: 915-917Crossref PubMed Scopus (3) Google Scholar, Uebe et al., 2017Uebe S. Ehrlicher M. Ekici A.B. Behrens F. Böhm B. Homuth G. et al.Genome-wide association and targeted analysis of copy number variants with psoriatic arthritis in German patients.BMC Med Genet. 2017; 18: 92Crossref PubMed Scopus (4) Google Scholar). ACTB, B2M, and PKG1 were used as housekeeping genes. The amount of serpin A3 in sera was determined with a commercially available ELISA (KA2128, Abnova, Taipei City, Taiwan). After a test run using three different dilutions of control sera (1:1,000, 1:2,500, and 1:5,000), we selected a dilution of 1:5,000 for measurements in two patients and 13 healthy volunteers (5 males and 8 females). We performed immunohistochemistry of serpin A3 in the skin of patients with GPP carrying the SERPINA3 frameshift mutation and of controls by using antibodies provided by Abcam (Cambridge, United Kingdom). Immunofluorescence of the skin of patients and control probands was performed as described previously (D'Erme et al., 2015D'Erme A.M. Wilsmann-Theis D. Wagenpfeil J. Hölzel M. Ferring-Schmitt S. Sternberg S. et al.IL-36gamma (IL-1F9) is a biomarker for psoriasis skin lesions.J Invest Dermatol. 2015; 135: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). To induce expression of SERPINA3, we used 1 μM dexamethasone (Sigma-Aldrich/Merck, St. Louis, MO); the cytokines tumor necrosis factor-α, IL-6, and IL-1β (ImmunoTools, Friesoythe, Germany); and lipopolysaccharide. We induced expression with dexamethasone only or combinations of dexamethasone with other stimuli in skin-derived cell lines and single stimuli in Hep3B and Hep2G cells. Cells were harvested at 6 hours and 24 hours after incubation with different concentrations of stimuli. Levels of saturation of SERPINA3's induction were shown (Supplementary Figure 2b–e). A plasmid containing the open reading frame of the SERPINA3 gene (Hölzel, Cologne, Germany) was used for transfection of HaCaTs; its sequence was confirmed by Sanger. To introduce the 1 base pair deletion, we performed mutagenesis using QuikChange Site-Directed Kit (Agilent) (primers, Supplementary Table S7). HaCaTs were transfected with lipofectamine 3000 using an empty vector, wildtype plasmid, or 1 base pair–deletion plasmid. Transfection of HaCaTs was performed in three independent experiments in duplicates or triplicates. Cell nuclei were labeled with DAPI (Sigma-Aldrich/Merck), whereas constructs were detectable by orange fluorescent protein. We determined transfection efficiency in HaCaTs using Image J (https://imagej.nih.gov/ij/index.html). Cells stained with DAPI and with or without the vector's orange fluorescent protein were counted in three different images containing >900 cells per image. We ob

Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.