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Characterizing the proteome of bullous pemphigoid blister fluid utilizing tandem mass tag labeling coupled with LC–MS/MS

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Abstract

Bullous pemphigoid is an autoimmune blistering disease caused by autoantibodies against components of the cutaneous basement membrane zone. Autoantibodies lead to complement-dependent and -independent inflammation and blistering. Blister fluid is a valuable biologic resource, as it provides insight into both systemic and local microenvironment responses. Here, we utilized liquid chromatography with tandem mass spectrometry to characterize the bullous pemphigoid blister fluid proteome. We then depleted exosomes to better understand the exosomal versus non-exosomal proteome. We identified 339 proteins in the blister fluid of bullous pemphigoid patients. Gene ontology demonstrated enrichment of several key biologic processes including innate immune response, neutrophil degranulation, platelet degranulation, and complement activation. Exosome depletion resulted in a significant decrease in normalized reporter intensities of 192 proteins, consistent with our observation of a large number of exosomal proteins found in the blister fluid. We then compared the bullous pemphigoid blister fluid proteome to prior proteomic datasets in suction blister fluid, snake bites, and thermal burns, identifying 76 proteins unique to bullous pemphigoid. These include major basic protein, eosinophil peroxidase, galectin-10, and the immunoglobulin epsilon heavy constant region, consistent with tissue eosinophilia. We lastly validated several previously reported blister fluid exosomal components. Blister fluid in bullous pemphigoid contains a mixture of numerous biologic processes. While many of these processes are shared with blistering from alternative causes, we have identified several notable features unique to bullous pemphigoid.

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Availability of data and materials

All databases are available in the supplementary materials.

Code availability

Not Applicable. No new software created. Data analysis code available on request.

References

  1. Amber KT, Murrell DF, Schmidt E, Joly P, Borradori L (2018) Autoimmune subepidermal bullous diseases of the skin and mucosae: clinical features, diagnosis, and management. Clin Rev Allergy Immunol 54(1):26–51. https://doi.org/10.1007/s12016-017-8633-4

    Article  PubMed  Google Scholar 

  2. Kowalski EH, Kneibner D, Kridin K, Amber KT (2019) Serum and blister fluid levels of cytokines and chemokines in pemphigus and bullous pemphigoid. Autoimmun Rev 18(5):526–534. https://doi.org/10.1016/j.autrev.2019.03.009

    Article  CAS  PubMed  Google Scholar 

  3. Muller AC, Breitwieser FP, Fischer H, Schuster C, Brandt O, Colinge J et al (2012) A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling. J Proteome Res 11(7):3715–3727. https://doi.org/10.1021/pr3002035

    Article  CAS  PubMed  Google Scholar 

  4. Khan AQ, Akhtar S, Prabhu KS, Zarif L, Khan R, Alam M et al (2020) Exosomes: emerging diagnostic and therapeutic targets in cutaneous diseases. Int J Mol Sci. https://doi.org/10.3390/ijms21239264

    Article  PubMed  PubMed Central  Google Scholar 

  5. Wang WM, Wu C, Jin HZ (2019) Exosomes in chronic inflammatory skin diseases and skin tumors. Exp Dermatol 28(3):213–218. https://doi.org/10.1111/exd.13857

    Article  CAS  PubMed  Google Scholar 

  6. Fang H, Shao S, Jiang M, Dang E, Shen S, Zhang J et al (2018) Proinflammatory role of blister fluid-derived exosomes in bullous pemphigoid. J Pathol 245(1):114–125. https://doi.org/10.1002/path.5061

    Article  CAS  PubMed  Google Scholar 

  7. Sethi S, Chourasia D, Parhar IS (2015) Approaches for targeted proteomics and its potential applications in neuroscience. J Biosci 40(3):607–627. https://doi.org/10.1007/s12038-015-9537-1

    Article  CAS  PubMed  Google Scholar 

  8. Zhao Y, Wang J, Chen J, Zhang X, Guo M, Yu G (2020) A literature review of gene function prediction by modeling gene ontology. Front Genet 11:400. https://doi.org/10.3389/fgene.2020.00400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Franzen O, Gan LM, Bjorkegren JLM (2019) PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database. https://doi.org/10.1093/database/baz046

    Article  PubMed  PubMed Central  Google Scholar 

  10. Zang T, Broszczak DA, Cuttle L, Broadbent JA, Tanzer C, Parker TJ (2016) Mass spectrometry based data of the blister fluid proteome of paediatric burn patients. Data Brief 8:1099–1110. https://doi.org/10.1016/j.dib.2016.07.033

    Article  PubMed  PubMed Central  Google Scholar 

  11. Macedo JKA, Joseph JK, Menon J, Escalante T, Rucavado A, Gutierrez JM et al (2019) Proteomic analysis of human blister fluids following envenomation by three snake species in india: differential markers for venom mechanisms of action. Toxins. https://doi.org/10.3390/toxins11050246

    Article  PubMed  PubMed Central  Google Scholar 

  12. Dimson OG, Giudice GJ, Fu CL, Van den Bergh F, Warren SJ, Janson MM et al (2003) Identification of a potential effector function for IgE autoantibodies in the organ-specific autoimmune disease bullous pemphigoid. J Invest Dermatol 120(5):784–788. https://doi.org/10.1046/j.1523-1747.2003.12146.x

    Article  CAS  PubMed  Google Scholar 

  13. Schmidt E, Bröcker EB, Zillikens D (1995) High levels of soluble CD23 in blister fluid of patients with bullous pemphigoid. Arch Dermatol 131(8):966–967

    Article  CAS  Google Scholar 

  14. Zhou S, Wakelin SH, Allen J, Wojnarowska F (1998) Blister fluid for the diagnosis of subepidermal immunobullous diseases: a comparative study of basement membrane zone autoantibodies detected in blister fluid and serum. Br J Dermatol 139(1):27–32. https://doi.org/10.1046/j.1365-2133.1998.02311.x

    Article  CAS  PubMed  Google Scholar 

  15. Margaroli C, Bradley B, Thompson C, Brown MR, Giacalone VD, Bhatt L et al (2020) Distinct compartmentalization of immune cells and mediators characterizes bullous pemphigoid disease. Exp Dermatol 29(12):1191–1198. https://doi.org/10.1111/exd.14209

    Article  CAS  PubMed  Google Scholar 

  16. Patel PM, Jones VA, Kridin K, Amber KT (2020) The role of Dipeptidyl Peptidase-4 in cutaneous disease. Exp Dermatol. https://doi.org/10.1111/exd.14228

    Article  PubMed  Google Scholar 

  17. Cugno M, Borghi A, Garcovich S, Marzano AV (2019) Coagulation and Skin Autoimmunity. Front Immunol 10:1407. https://doi.org/10.3389/fimmu.2019.01407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ishihara J, Ishihara A, Starke RD, Peghaire CR, Smith KE, McKinnon TAJ et al (2019) The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing. Blood 133(24):2559–2569. https://doi.org/10.1182/blood.2019000510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tedeschi A, Marzano AV, Lorini M, Balice Y, Cugno M (2015) Eosinophil cationic protein levels parallel coagulation activation in the blister fluid of patients with bullous pemphigoid. J Eur Acad Dermatol Venereol 29(4):813–817. https://doi.org/10.1111/jdv.12464

    Article  CAS  PubMed  Google Scholar 

  20. Amber KT, Valdebran M, Kridin K, Grando SA (2018) The role of eosinophils in bullous pemphigoid: a developing model of eosinophil pathogenicity in mucocutaneous disease. Front Med 5:201. https://doi.org/10.3389/fmed.2018.00201

    Article  Google Scholar 

  21. Jones VA, Patel PM, Amber KT (2020) Eosinophils in Bullous Pemphigoid. Panminerva Med. https://doi.org/10.23736/s0031-0808.20.03997-x

    Article  PubMed  Google Scholar 

  22. Amber KT, Chernyavsky A, Agnoletti AF, Cozzani E, Grando SA (2018) Mechanisms of pathogenic effects of eosinophil cationic protein and eosinophil-derived neurotoxin on human keratinocytes. Exp Dermatol 27(12):1322–1327. https://doi.org/10.1111/exd.13782

    Article  CAS  PubMed  Google Scholar 

  23. Amber KT, Agnoletti A, Chernyavsky A, Grando S (2017) 615 Eosinophil major basic protein has a concentration-dependent cytotoxic effect on cultured keratinocytes. J Invest Dermatology 137(5):S106. https://doi.org/10.1016/j.jid.2017.02.637

    Article  Google Scholar 

  24. de Graauw E, Sitaru C, Horn M, Borradori L, Yousefi S (2017) Evidence for a role of eosinophils in blister formation in bullous pemphigoid. Allergy 72(7):1105–1113. https://doi.org/10.1111/all.13131

    Article  CAS  PubMed  Google Scholar 

  25. Acharya KR, Ackerman SJ (2014) Eosinophil granule proteins: form and function. J Biol Chem 289(25):17406–17415. https://doi.org/10.1074/jbc.R113.546218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hashimoto T, Kursewicz CD, Fayne RA, Nanda S, Shah SM, Nattkemper L et al (2020) Pathophysiologic mechanisms of itch in bullous pemphigoid. J Am Acad Dermatol 83(1):53–62. https://doi.org/10.1016/j.jaad.2019.07.060

    Article  CAS  PubMed  Google Scholar 

  27. Rodriguez-Alcazar JF, Ataide MA, Engels G, Schmitt-Mabmunyo C, Garbi N, Kastenmuller W et al (2019) Charcot–Leyden crystals activate the NLRP3 inflammasome and cause IL-1beta Inflammation in human macrophages. J Immunol 202(2):550–558. https://doi.org/10.4049/jimmunol.1800107

    Article  CAS  PubMed  Google Scholar 

  28. Fang H, Shao S, Cao T, Lei J, Dang E, Zhang J et al (2016) Increased expression of NLRP3 inflammasome components and interleukin-18 in patients with bullous pemphigoid. J Dermatol Sci 83(2):116–123. https://doi.org/10.1016/j.jdermsci.2016.04.009

    Article  CAS  PubMed  Google Scholar 

  29. Le Jan S, Muller C, Plee J, Durlach A, Bernard P, Antonicelli F (2019) IL-23/IL-17 axis activates IL-1beta-associated inflammasome in macrophages and generates an auto-inflammatory response in a subgroup of patients with bullous pemphigoid. Front Immunol 10:1972. https://doi.org/10.3389/fimmu.2019.01972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fukuchi M, Miyabe Y, Furutani C, Saga T, Moritoki Y, Yamada T et al (2020) How to detect eosinophil ETosis (EETosis) and extracellular traps. Allergol Int. https://doi.org/10.1016/j.alit.2020.10.002

    Article  PubMed  PubMed Central  Google Scholar 

  31. Simon D, Hoesli S, Roth N, Staedler S, Yousefi S, Simon HU (2011) Eosinophil extracellular DNA traps in skin diseases. J Allergy Clin Immunol 127(1):194–199. https://doi.org/10.1016/j.jaci.2010.11.002

    Article  CAS  PubMed  Google Scholar 

  32. de Graauw E, Sitaru C, Horn M, Borradori L, Yousefi S, Simon HU et al (2017) Evidence for a role of eosinophils in blister formation in bullous pemphigoid. Allergy 72(7):1105–1113. https://doi.org/10.1111/all.13131

    Article  CAS  PubMed  Google Scholar 

  33. Tukaj S, Grüner D, Zillikens D, Kasperkiewicz M (2014) Hsp90 blockade modulates bullous pemphigoid IgG-induced IL-8 production by keratinocytes. Cell Stress Chaperones 19(6):887–894. https://doi.org/10.1007/s12192-014-0513-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tukaj S, Kleszczynski K, Vafia K, Groth S, Meyersburg D, Trzonkowski P et al (2013) Aberrant expression and secretion of heat shock protein 90 in patients with bullous pemphigoid. PLoS ONE 8(7):e70496. https://doi.org/10.1371/journal.pone.0070496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tukaj S, Zillikens D, Kasperkiewicz M (2015) Heat shock protein 90: a pathophysiological factor and novel treatment target in autoimmune bullous skin diseases. Exp Dermatol 24(8):567–571. https://doi.org/10.1111/exd.12760

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Creative Proteomics (Shirley, NY) for assistance with LC–MS/MS. Lei Bao received financial support from the Albert H. and Mary Jane Slepyan Endowed Fellowship. Farzan Soliamni and Michael Hertl were funded by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG); FOR 2497.

Funding

Lei Bao received financial support from the Albert H. and Mary Jane Slepyan Endowed Fellowship. Farzan Soliamni and Michael Hertl were funded by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG); FOR 2497.

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Authors and Affiliations

  1. Department of Dermatology, Venereology and Allergology, Charitèe–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin and Berlin Institute of Health, Berlin, Germany

    Farzan Solimani

  2. Department of Dermatology and Allergology, Philipps University, Marburg, Germany

    Farzan Solimani, Dario Didona & Michael Hertl

  3. Department of Dermatology, University of Illinois at Chicago, Chicago, IL, USA

    Jing Li, Lei Bao & Payal M. Patel

  4. Department of Health Sciences and Department of Experimental Medicine, University of Genoa, Genoa, Italy

    Giulia Gasparini

  5. Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany

    Khalaf Kridin

  6. Department of Health Sciences (DISSAL), Section of Dermatology, University of Genoa, San Martino Policlinic Hospital IRCCS, Genoa, Italy

    Giulia Gasparini & Emanuele Cozzani

  7. Division of Dermatology, Rush University Medical Center, 707 S Wood St. Suite 220, Chicago, IL, USA

    Kyle T. Amber

  8. Department of Internal Medicine, Rush University Medical Center, Chicago, IL, USA

    Kyle T. Amber

  9. Department of Otorhinolaryngology, Rush University Medical Center, Chicago, IL, 60612, USA

    Kyle T. Amber

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  1. Farzan Solimani
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Contributions

FS, DD, JL, LB and GG helped perform experiments. KK assisted with statistical analysis. EC, MH, and PP provided critical review of the manuscript. KA performed statistical analysis and wrote the manuscript. All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to Kyle T. Amber.

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Conflict of interest

The authors declare no potential conflicts of interest.

Ethical approval

This study was approved by the Ethics Committee at the University of Illinois at Chicago, Philipps University, and University of Genoa.

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Written consent was obtained from all patients in accordance with the Helsinki declaration.

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All authors reviewed and approved of the final manuscript for publication.

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Supplementary Information

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403_2021_2253_MOESM1_ESM.pdf

Supplementary file1 (PDF 148 KB) sFig. 1: Peptide length distribution, protein mass distribution and distribution of protein sequence cover from identified peptides identified by LC–MS/MS of whole blister fluid and exosome-depleted blister fluid.

Supplementary file2 (PDF 308 KB) sFig. 2: Principal component analysis of BP blister fluid samples.

403_2021_2253_MOESM3_ESM.xlsx

Supplementary file3 (XLSX 3016 KB) sFig. 3: Transmission electron microscopy of exosome-depleted blister fluid (A) confirms absence of characteristic cup shaped vesicles precipitated with ultracentrifugation (B).

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Solimani, F., Didona, D., Li, J. et al. Characterizing the proteome of bullous pemphigoid blister fluid utilizing tandem mass tag labeling coupled with LC–MS/MS. Arch Dermatol Res 314, 921–928 (2022). https://doi.org/10.1007/s00403-021-02253-8

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  • DOI: https://doi.org/10.1007/s00403-021-02253-8

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