Skip to main content

Advertisement

SpringerLink
Log in

Proteomic analysis of peripheral blood polymorphonuclear cells (PBMCs) reveals alteration of neutrophil extracellular trap (NET) components in uncontrolled diabetes

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Neutrophils have been thought to play a major role in inflammation and diabetic complications especially in poor glycemic control patients as demonstrated by their aberrant inflammatory markers. The aim of the present study was to compare neutrophil proteome profiles between diabetic patients with good glycemic control and those with poor glycemic control to see whether there might be any differences that could be related to the cause of complications which are found more commonly in the latter. Using 2-dimensional gel electrophoresis (2-DE) followed by quadrupole time of flight mass spectrometry (Q-TOF MS) and/or tandem mass spectrometry (MS/MS), we identified 35 differentially expressed proteins, some of which were protein components of neutrophil extracellular traps (NETs), in the poor glycemic control group compared to the good glycemic control group. The observed alterations of protein components of NETs included downregulation of myeloperoxidase, azurocidin (CAP37), and S100A9; and upregulation of the glycolytic enzymes transketolase and alpha-enolase. Manganese superoxide dismutase (MnSOD), functioning in cellular response and defense, was also found downregulated in the poor control group. Most of the glycolysis-related proteins were downregulated in the good control group but upregulated in the poor control group, including phosphoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase B chain (LDHB). The findings of this study demonstrate the dysregulation of protein components of NETs in neutrophils in patients with poorly controlled diabetes. More specifically, these findings suggest association between NETs and inflammation in diabetes and provide further insights into the role of neutrophils in the complications of poorly controlled diabetes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

2-DE:

Two-dimensional gel electrophoresis

ACN:

Acetonitrile

AGE:

Advanced glycation end product

CAP37:

Cationic antimicrobial protein of molecular weight 37 kDa

CHAPS:

3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate

DM:

Diabetes mellitus

DNA:

Deoxyribonucleic acid

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HbA1c:

Hemoglobin A1c

HDL:

High-density lipoprotein

IEF:

Isoelectric focusing

IL:

Interleukin

LDH:

l-Lactate dehydrogenase

LDHB:

l-Lactate dehydrogenase B chain

LDL:

Low-density lipoprotein

LPS:

Lipopolysaccharide

MnSOD:

Manganese superoxide dismutase

MPO:

Myeloperoxidase

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

NADPH:

Nicotinamide adenine dinucleotide phosphate

NETs:

Neutrophil extracellular traps

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate-buffered saline

PGK1:

Phosphoglycerate kinase 1

PKC:

Protein kinase C

PMA:

Phorbol 12-myristate 13-acetate

PMN:

Polymorphonuclear leukocyte

Q-TOF MS:

Quadrupole time of flight mass spectrometry

ROS:

Reactive oxygen species

SDS-PAGE:

Sodiumdeodecyl sulfate polyacrylamide gel electrophoresis

T2DM:

Type 2 diabetes mellitus

TFA:

Trifluoroacetic acid

TNF:

Tumor necrosis factor

References

  1. Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA, Hadden D, Turner RC, Holman RR (2000) Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ 321(7258):405–412

    Article  CAS  Google Scholar 

  2. Donath MY, Shoelson SE (2011) Type 2 diabetes as an inflammatory disease. Nat Rev Immunol 11(2):98–107. https://doi.org/10.1038/nri2925

    Article  CAS  PubMed  Google Scholar 

  3. Fernandez-Real JM, Pickup JC (2008) Innate immunity, insulin resistance and type 2 diabetes. Trends Endocrinol Metab 19(1):10–16. https://doi.org/10.1016/j.tem.2007.10.004

    Article  CAS  PubMed  Google Scholar 

  4. Brownlee M (2005) The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54(6):1615–1625

    Article  CAS  Google Scholar 

  5. Fiorentino TV, Prioletta A, Zuo P, Folli F (2013) Hyperglycemia-induced oxidative stress and its role in diabetes mellitus related cardiovascular diseases. Curr Pharm Des 19(32):5695–5703

    Article  CAS  Google Scholar 

  6. Ayilavarapu S, Kantarci A, Fredman G, Turkoglu O, Omori K, Liu H, Iwata T, Yagi M, Hasturk H, Van Dyke TE (2010) Diabetes-induced oxidative stress is mediated by Ca2+-independent phospholipase A2 in neutrophils. J Immunol 184(3):1507–1515. https://doi.org/10.4049/jimmunol.0901219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shurtz-Swirski R, Sela S, Herskovits AT, Shasha SM, Shapiro G, Nasser L, Kristal B (2001) Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients. Diabetes Care 24(1):104–110

    Article  CAS  Google Scholar 

  8. Gkrania-Klotsas E, Ye Z, Cooper AJ, Sharp SJ, Luben R, Biggs ML, Chen LK, Gokulakrishnan K, Hanefeld M, Ingelsson E, Lai WA, Lin SY, Lind L, Lohsoonthorn V, Mohan V, Muscari A, Nilsson G, Ohrvik J, Chao Qiang J, Jenny NS, Tamakoshi K, Temelkova-Kurktschiev T, Wang YY, Yajnik CS, Zoli M, Khaw KT, Forouhi NG, Wareham NJ, Langenberg C (2010) Differential white blood cell count and type 2 diabetes: systematic review and meta-analysis of cross-sectional and prospective studies. PLoS ONE 5(10):e13405. https://doi.org/10.1371/journal.pone.0013405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Marhoffer W, Stein M, Maeser E, Federlin K (1992) Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes. Diabetes Care 15(2):256–260

    Article  CAS  Google Scholar 

  10. Grykiel K, Zozulinska D, Kostrzewa A, Wiktorowicz K, Wierusz-Wysocka B (2001) Evaluation of expression of polymorphonuclear neutrophil surface receptors in patients with type 1 diabetes. Pol Arch Med Wewn 105(5):377–381

    CAS  PubMed  Google Scholar 

  11. van Oostrom AJ, van Wijk JP, Sijmonsma TP, Rabelink TJ, Castro Cabezas M (2004) Increased expression of activation markers on monocytes and neutrophils in type 2 diabetes. Neth J Med 62(9):320–325

    PubMed  Google Scholar 

  12. Hatanaka E, Monteagudo PT, Marrocos MS, Campa A (2006) Neutrophils and monocytes as potentially important sources of proinflammatory cytokines in diabetes. Clin Exp Immunol 146(3):443–447. https://doi.org/10.1111/j.1365-2249.2006.03229.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wiersma JJ, Meuwese MC, Van Miert JN, Kastelein A, Tijssen JG, Piek JJ, Trip MD (2008) Diabetes mellitus type 2 is associated with higher levels of myeloperoxidase. Med Sci Monit 14(8):CR406–CR410

    CAS  PubMed  Google Scholar 

  14. Menegazzo L, Ciciliot S, Poncina N, Mazzucato M, Persano M, Bonora B, Albiero M, Vigili de Kreutzenberg S, Avogaro A, Fadini GP (2015) NETosis is induced by high glucose and associated with type 2 diabetes. Acta Diabetol 52(3):497–503. https://doi.org/10.1007/s00592-014-0676-x

    Article  CAS  PubMed  Google Scholar 

  15. Wong SL, Demers M, Martinod K, Gallant M, Wang Y, Goldfine AB, Kahn CR, Wagner DD (2015) Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med 21(7):815–819. https://doi.org/10.1038/nm.3887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Berezin A (2018) Neutrophil extracellular traps: the core player in vascular complications of diabetes mellitus. Diabetes Metab Syndr. https://doi.org/10.1016/j.dsx.2018.07.010

    Article  PubMed  Google Scholar 

  17. McLeish KR, Merchant ML, Klein JB, Ward RA (2013) Technical note: proteomic approaches to fundamental questions about neutrophil biology. J Leukoc Biol 94(4):683–692. https://doi.org/10.1189/jlb.1112591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lominadze G, Ward RA, Klein JB, McLeish KR (2006) Proteomic analysis of human neutrophils. Methods Mol Biol 332:343–356. https://doi.org/10.1385/1-59745-048-0:343

    Article  CAS  PubMed  Google Scholar 

  19. Tomazella GG, da Silva I, Laure HJ, Rosa JC, Chammas R, Wiker HG, de Souza GA, Greene LJ (2009) Proteomic analysis of total cellular proteins of human neutrophils. Proteome Sci 7:32. https://doi.org/10.1186/1477-5956-7-32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tomazella GG, daSilva I, Thome CH, Greene LJ, Koehler CJ, Thiede B, Wiker HG, de Souza GA (2010) Analysis of detergent-insoluble and whole cell lysate fractions of resting neutrophils using high-resolution mass spectrometry. J Proteome Res 9(4):2030–2036. https://doi.org/10.1021/pr1000253

    Article  CAS  PubMed  Google Scholar 

  21. Loi ALT, Hoonhorst S, van Aalst C, Langereis J, Kamp V, Sluis-Eising S, Ten Hacken N, Lammers JW, Koenderman L (2017) Proteomic profiling of peripheral blood neutrophils identifies two inflammatory phenotypes in stable COPD patients. Respir Res 18(1):100. https://doi.org/10.1186/s12931-017-0586-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ramos-Mozo P, Madrigal-Matute J, Martinez-Pinna R, Blanco-Colio LM, Lopez JA, Camafeita E, Meilhac O, Michel JB, Aparicio C, Vega de Ceniga M, Egido J, Martin-Ventura JL (2011) Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression. Arterioscler Thromb Vasc Biol 31(12):3011–3019. https://doi.org/10.1161/atvbaha.111.237537

    Article  CAS  PubMed  Google Scholar 

  23. Uchida T, Nagai K, Sato T, Iizuka N, Arito M, Takakuwa Y, Nakano H, Ooka S, Kurokawa MS, Suematsu N, Okamoto K, Ozaki S, Kato T (2013) Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis. J Proteom 91:259–269. https://doi.org/10.1016/j.jprot.2013.07.021

    Article  CAS  Google Scholar 

  24. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A (2009) Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog 5(10):e1000639. https://doi.org/10.1371/journal.ppat.1000639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Remijsen Q, Kuijpers TW, Wirawan E, Lippens S, Vandenabeele P, Vanden Berghe T (2011) Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ 18(4):581–588. https://doi.org/10.1038/cdd.2011.1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Delgado-Rizo V, Martinez-Guzman MA, Iniguez-Gutierrez L, Garcia-Orozco A, Alvarado-Navarro A, Fafutis-Morris M (2017) Neutrophil extracellular traps and its implications in inflammation: an overview. Front Immunol 8:81. https://doi.org/10.3389/fimmu.2017.00081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Miyoshi A, Yamada M, Shida H, Nakazawa D, Kusunoki Y, Nakamura A, Miyoshi H, Tomaru U, Atsumi T, Ishizu A (2016) Circulating neutrophil extracellular trap levels in well-controlled type 2 diabetes and pathway involved in their formation induced by high-dose glucose. Pathobiology 83(5):243–251. https://doi.org/10.1159/000444881

    Article  CAS  PubMed  Google Scholar 

  28. Loria V, Dato I, Graziani F, Biasucci LM (2008) Myeloperoxidase: a new biomarker of inflammation in ischemic heart disease and acute coronary syndromes. Mediat Inflamm 2008:135625. https://doi.org/10.1155/2008/135625

    Article  CAS  Google Scholar 

  29. Umsa-Ard W, Thongboonkerd V, Soongsathitanon J (2015) Activated status and altered functions of neutrophils in poorly controlled diabetes. JAFES 30(1):9–17. https://doi.org/10.15605/jafes.030.01.02

    Article  Google Scholar 

  30. Heinzelmann M, Mercer-Jones MA, Flodgaard H, Miller FN (1998) Heparin-binding protein (CAP37) is internalized in monocytes and increases LPS-induced monocyte activation. J Immunol 160(11):5530–5536

    CAS  PubMed  Google Scholar 

  31. Lee TD, Gonzalez ML, Kumar P, Grammas P, Pereira HA (2003) CAP37, a neutrophil-derived inflammatory mediator, augments leukocyte adhesion to endothelial monolayers. Microvasc Res 66(1):38–48

    Article  CAS  Google Scholar 

  32. Pereira HA, Moore P, Grammas P (1996) CAP37, a neutrophil granule-derived protein stimulates protein kinase C activity in endothelial cells. J Leukoc Biol 60(3):415–422

    Article  CAS  Google Scholar 

  33. Lee TD, Gonzalez ML, Kumar P, Chary-Reddy S, Grammas P, Pereira HA (2002) CAP37, a novel inflammatory mediator: its expression in endothelial cells and localization to atherosclerotic lesions. Am J Pathol 160(3):841–848. https://doi.org/10.1016/s0002-9440(10)64907-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Madsen-Bouterse SA, Zhong Q, Mohammad G, Ho YS, Kowluru RA (2010) Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase. Free Radic Res 44(3):313–321. https://doi.org/10.3109/10715760903494168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chen H, Yu M, Li M, Zhao R, Zhu Q, Zhou W, Lu M, Lu Y, Zheng T, Jiang J, Zhao W, Xiang K, Jia W, Liu L (2012) Polymorphic variations in manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPX1), and catalase (CAT) contribute to elevated plasma triglyceride levels in Chinese patients with type 2 diabetes or diabetic cardiovascular disease. Mol Cell Biochem 363(1–2):85–91. https://doi.org/10.1007/s11010-011-1160-3

    Article  CAS  PubMed  Google Scholar 

  36. Mollsten A, Marklund SL, Wessman M, Svensson M, Forsblom C, Parkkonen M, Brismar K, Groop PH, Dahlquist G (2007) A functional polymorphism in the manganese superoxide dismutase gene and diabetic nephropathy. Diabetes 56(1):265–269. https://doi.org/10.2337/db06-0698

    Article  CAS  PubMed  Google Scholar 

  37. Raj V, Ojha S, Howarth FC, Belur PD, Subramanya SB (2018) Therapeutic potential of benfotiamine and its molecular targets. Eur Rev Med Pharmacol Sci 22(10):3261–3273

    CAS  PubMed  Google Scholar 

  38. Hammes HP, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J, Bierhaus A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, Brownlee M (2003) Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med 9(3):294–299. https://doi.org/10.1038/nm834

    Article  CAS  PubMed  Google Scholar 

  39. Du Y, Kowluru A, Kern TS (2010) PP2A contributes to endothelial death in high glucose: inhibition by benfotiamine. Am J Physiol Regul Integr Comp Physiol 299(6):R1610–1617. https://doi.org/10.1152/ajpregu.00676.2009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Diaz-Ramos A, Roig-Borrellas A, Garcia-Melero A, Lopez-Alemany R (2012) Alpha-enolase, a multifunctional protein: its role on pathophysiological situations. J Biomed Biotechnol 2012:156795. https://doi.org/10.1155/2012/156795

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ji H, Wang J, Guo J, Li Y, Lian S, Guo W, Yang H, Kong F, Zhen L, Guo L, Liu Y (2016) Progress in the biological function of alpha-enolase. Anim Nutr 2(1):12–17. https://doi.org/10.1016/j.aninu.2016.02.005

    Article  PubMed  PubMed Central  Google Scholar 

  42. Lu N, Zhang Y, Li H, Gao Z (2010) Oxidative and nitrative modifications of alpha-enolase in cardiac proteins from diabetic rats. Free Radic Biol Med 48(7):873–881. https://doi.org/10.1016/j.freeradbiomed.2010.01.010

    Article  CAS  PubMed  Google Scholar 

  43. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA 97(22):12222–12226. https://doi.org/10.1073/pnas.97.22.12222

    Article  CAS  PubMed  Google Scholar 

  44. Du X, Matsumura T, Edelstein D, Rossetti L, Zsengeller Z, Szabo C, Brownlee M (2003) Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells. J Clin Investig 112(7):1049–1057. https://doi.org/10.1172/jci18127

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge Kittisak Sintiprungrat for technical assistance. This study was supported by a Mahidol University Research Grant (Grant No. IRN60W0004) and a grant from the Thailand Research Fund (Grant No. IRG5980006). Umsa-ard W. was partially supported by a Siriraj Graduate Thesis Scholarship.

Author information

Authors and Affiliations

  1. Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand

    J. Soongsathitanon

  2. Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    W. Umsa-Ard

  3. Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    V. Thongboonkerd

Authors
  1. J. Soongsathitanon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. Umsa-Ard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V. Thongboonkerd
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JS, WU, and VT conceived the study; JS and VT designed the experiments; WU performed the experiments; WU and VT analyzed the data; and, JS wrote the manuscript. All authors approved the final manuscript.

Corresponding author

Correspondence to J. Soongsathitanon.

Ethics declarations

Conflict of interest

All authors declare no personal or professional conflicts of interest, and no financial support from the companies that produce and/or distribute the drugs, devices, or materials described in this report.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research ethics committee (Siriraj Institutional Review Board COA No. Si 012/2008) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all participants.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 395 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Soongsathitanon, J., Umsa-Ard, W. & Thongboonkerd, V. Proteomic analysis of peripheral blood polymorphonuclear cells (PBMCs) reveals alteration of neutrophil extracellular trap (NET) components in uncontrolled diabetes. Mol Cell Biochem 461, 1–14 (2019). https://doi.org/10.1007/s11010-019-03583-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-019-03583-y

Keywords

Search

Navigation