A human antithrombin isoform dampens inflammatory responses and protects from organ damage during bacterial infection

Abstract

Severe infectious diseases are often characterized by an overwhelming and unbalanced systemic immune response to microbial infections. Human antithrombin (hAT) is a crucial coagulation inhibitor with anti-inflammatory activities. Here we identify three hAT-binding proteins (CD13, CD300f and LRP-1) on human monocytes that are involved in blocking the activity of nuclear factor-κB. We found that the modulating effect is primarily restricted to the less abundant β-isoform (hβAT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of hβAT and analysis of their blood, which was stimulated ex vivo with lipopolysaccharide, showed a decreased inflammatory response. Similar findings were recorded when heterozygotic mice expressing hAT or hβAT were challenged with lipopolysaccharide or infected with Escherichia coli bacteria. Our results finally demonstrate that in a lethal E. coli infection model, survival rates increased when mice were treated with hβAT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Determination of hAT levels in plasma samples from infected patients.
Fig. 2: Analysis of the interaction between hαAT and hβAT and their binding partners.
Fig. 3: Modulation of the inflammatory response by hαAT and hβAT.
Fig. 4: Ex vivo experiments with blood from hβAT-overexpressing individuals.
Fig. 5: Modulation of inflammatory responses in transgenic mice expressing hAT or hβAT.
Fig. 6: hβAT treatment of LPS-challenged or E. coli-infected mice.

Data availability

The data that support the findings of this study are available from the corresponding author on request.

References

  1. 1.

    van der Poll, T., van de Veerdonk, F. L., Scicluna, B. P. & Netea, M. G. The immunopathology of sepsis and potential therapeutic targets. Nat. Rev. Immunol. 17, 407–420 (2017).

    Article  Google Scholar 

  2. 2.

    Chaudhry, H. et al. Role of cytokines as a double-edged sword in sepsis. In Vivo 27, 669–684 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Denstaedt, S. J., Singer, B. H. & Standiford, T. J. Sepsis and nosocomial infection: patient characteristics, mechanisms, and modulation. Front. Immunol. 9, 2446 (2018).

    Article  Google Scholar 

  4. 4.

    Picard, V., Ersdal-Badju, E. & Bock, S. C. Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity. Biochemistry 34, 8433–8440 (1995).

    CAS  Article  Google Scholar 

  5. 5.

    Andersen, O., Flengsrud, R., Norberg, K. & Salte, R. Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human beta-antithrombin. Eur. J. Biochem. 267, 1651–1657 (2000).

    CAS  Article  Google Scholar 

  6. 6.

    Allen, D. H. & Tracy, P. B. Human coagulation factor V is activated to the functional cofactor by elastase and cathepsin G expressed at the monocyte surface. J. Biol. Chem. 270, 1408–1415 (1995).

    CAS  Article  Google Scholar 

  7. 7.

    Ghosh, M., Subramani, J., Rahman, M. M. & Shapiro, L. H. CD13 restricts TLR4 endocytic signal transduction in inflammation. J. Immunol. 194, 4466–4476 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Lee, S. M., Kim, E. J., Suk, K. & Lee, W. H. CD300F blocks both MyD88 and TRIF-mediated TLR signaling through activation of Src homology region 2 domain-containing phosphatase 1. J. Immunol. 186, 6296–6303 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Gaultier, A. et al. Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor. Blood 111, 5316–5325 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Zhang, J., Somani, A. K. & Siminovitch, K. A. Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signalling. Semin. Immunol. 12, 361–378 (2000).

    CAS  Article  Google Scholar 

  11. 11.

    Kim, E. J., Lee, S. M., Suk, K. & Lee, W. H. CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines. Immunology 135, 226–235 (2012).

    CAS  Article  Google Scholar 

  12. 12.

    Strickland, D. K., Muratoglu, S. C. & Antalis, T. M. Serpin-enzyme receptors LDL receptor-related protein 1. Methods Enzym. 499, 17–31 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Toldo, S. et al. Low-density lipoprotein receptor-related protein-1 is a therapeutic target in acute myocardial infarction. JACC Basic Transl. Sci. 2, 561–574 (2017).

    Article  Google Scholar 

  14. 14.

    Janciauskiene, S., Lindgren, S. & Wright, H. T. The C-terminal peptide of alpha-1-antitrypsin increases low density lipoprotein binding in HepG2 cells. Eur. J. Biochem. 254, 460–467 (1998).

    CAS  Article  Google Scholar 

  15. 15.

    Zurhove, K., Nakajima, C., Herz, J., Bock, H. H. & May, P. Gamma-secretase limits the inflammatory response through the processing of LRP1. Sci. Signal. 1, ra15 (2008).

    Article  Google Scholar 

  16. 16.

    Pasi, K. J. et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N. Engl. J. Med. 377, 819–828 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Corral, J., de la Morena-Barrio, M. E. & Vicente, V. The genetics of antithrombin. Thromb. Res. 169, 23–29 (2018).

    CAS  Article  Google Scholar 

  18. 18.

    Luxembourg, B. et al. Molecular basis of antithrombin deficiency. Thromb. Haemost. 105, 635–646 (2011).

    CAS  Article  Google Scholar 

  19. 19.

    Bayston, T. A. et al. Familial overexpression of beta antithrombin caused by an Asn135Thr substitution. Blood 93, 4242–4247 (1999).

    CAS  Article  Google Scholar 

  20. 20.

    Kalle, M. et al. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection. PLoS ONE 9, e102577 (2014).

    Article  Google Scholar 

  21. 21.

    Delano, M. J. & Ward, P. A. The immune system’s role in sepsis progression, resolution, and long-term outcome. Immunol. Rev. 274, 330–353 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Yan, S. B., Helterbrand, J. D., Hartman, D. L., Wright, T. J. & Bernard, G. R. Low levels of protein C are associated with poor outcome in severe sepsis. Chest 120, 915–922 (2001).

    CAS  Article  Google Scholar 

  23. 23.

    Van Den Boogaard, F. E. et al. Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia. J. Thromb. Haemost. 9, 122–132 (2011).

    Article  Google Scholar 

  24. 24.

    Levy, J. H., Sniecinski, R. M., Welsby, I. J. & Levi, M. Antithrombin: anti-inflammatory properties and clinical applications. Thromb. Haemost. 115, 712–728 (2016).

    Article  Google Scholar 

  25. 25.

    Shorr, A. F. et al. Protein C concentrations in severe sepsis: an early directional change in plasma levels predicts outcome. Crit. Care 10, R92 (2006).

    Article  Google Scholar 

  26. 26.

    Gando, S. et al. Tissue factor production not balanced by tissue factor pathway inhibitor in sepsis promotes poor prognosis. Crit. Care Med. 30, 1729–1734 (2002).

    CAS  Article  Google Scholar 

  27. 27.

    Wiedermann, C. J. et al. High-dose antithrombin III in the treatment of severe sepsis in patients with a high risk of death: efficacy and safety. Crit. Care Med. 34, 285–292 (2006).

    CAS  Article  Google Scholar 

  28. 28.

    Poole, D., Bertolini, G. & Garattini, S. Withdrawal of ‘Xigris’ from the market: old and new lessons. J. Epidemiol. Community Health 66, 571–572 (2012).

    Article  Google Scholar 

  29. 29.

    Laterre, P. F. et al. A clinical evaluation committee assessment of recombinant human tissue factor pathway inhibitor (tifacogin) in patients with severe community-acquired pneumonia. Crit. Care 13, R36 (2009).

    Article  Google Scholar 

  30. 30.

    Allingstrup, M., Wetterslev, J., Ravn, F. B., Moller, A. M. & Afshari, A. Antithrombin III for critically ill patients. Cochrane Database Syst. Rev. 2, CD005370 (2016).

    PubMed  Google Scholar 

  31. 31.

    Kerschen, E. J. et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J. Exp. Med. 204, 2439–2448 (2007).

    CAS  Article  Google Scholar 

  32. 32.

    Minnema, M. C. et al. Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli. Blood 95, 1117–1123 (2000).

    CAS  Article  Google Scholar 

  33. 33.

    Rello, J. et al. Towards precision medicine in sepsis: a position paper from the European Society of Clinical Microbiology and Infectious Diseases. Clin. Microbiol. Infect. 24, 1264–1272 (2018).

    CAS  Article  Google Scholar 

  34. 34.

    Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    CAS  Article  Google Scholar 

  35. 35.

    de la Morena-Barrio, M. E. et al. A new method to quantify beta-antithrombin glycoform in plasma reveals increased levels during the acute stroke event. Thromb. Res. 136, 634–641 (2015).

    Article  Google Scholar 

  36. 36.

    Abdillahi, S. M. et al. Collagen VI is upregulated in COPD and serves both as an adhesive target and a bactericidal barrier for Moraxella catarrhalis. J. Innate Immun. 7, 506–517 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 490–505 (2015).

    Google Scholar 

  38. 38.

    Yang, H., Wang, H. & Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat. Protoc. 9, 1956–1968 (2014).

    CAS  Article  Google Scholar 

  39. 39.

    Martin Gonzalez, J. et al. Embryonic stem cell culture conditions support distinct states associated with different developmental stages and potency. Stem Cell Rep. 7, 177–191 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Linder, A., Christensson, B., Herwald, H., Bjorck, L. & Akesson, P. Heparin-binding protein: an early marker of circulatory failure in sepsis. Clin. Infect. Dis. 49, 1044–1050 (2009).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Alfred Österlund Foundation (to P.P. and H.H.), the Crafoord Foundation (grant no. 20180506 to P.P.), the Knut and Alice Wallenberg Foundation (grant no. 2011.0037 to H.H.), the Medical Faculty at Lund University (to H.H.), the Swedish Foundation for Strategic Research (grant no. SB12-0019 to A.E. and H.H.), the Swedish Research Council (grant no. 2013-3078 to A.E. and grant no. 2016-01104 to H.H.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors thank R. Bhongir for performing the microarray assays, M. Baumgarten for her help with the electron microscopy experiments, J. Martin for his support in the generation of mice expressing hAT and hβAT and Lund University Bioimaging Centre.

Author information

Affiliations

Authors

Contributions

P.P., M.R., F.D.H., C.Naudin., E.S., J.W. and G.Kassety. performed the in vitro and in vivo experiments. S.V. generated heat maps. A.A., M.A., C.Novembrino. and I.M. assisted with experiments using blood from hβAT-overexpressing individuals. A.E., I.M.-M., M.E.d.l.M.-B. and J.C. analysed and interpreted the data. C.H.B. designed CRISPR−Cas experiments. A.L. provided patient plasma samples. P.P. and H.H. designed and supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Heiko Herwald.

Ethics declarations

Competing interests

P.P., A.E., G.K. and H.H. have filed a provisional patent on the possibility of using hβAT as an antimicrobial treatment. All other authors have no competing interests.

Additional information

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

Supplementary Information

Supplementary Information

Supplementary Figs. 1−14 and Tables 1−2.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Papareddy, P., Rossnagel, M., Doreen Hollwedel, F. et al. A human antithrombin isoform dampens inflammatory responses and protects from organ damage during bacterial infection. Nat Microbiol 4, 2442–2455 (2019). https://doi.org/10.1038/s41564-019-0559-6

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing