Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype

Jesús Beltrán-García; Rebeca Osca-Verdegal; Daniel Pérez-Cremades; Susana Novella; Carlos Hermenegildo; Federico V Pallardó; José Luis García-Giménez

doi:10.2147/JIR.S363693

Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype

J Inflamm Res. 2022 Jul 25:15:4217-4238. doi: 10.2147/JIR.S363693. eCollection 2022.

Authors

Jesús Beltrán-García^{1

2

3}, Rebeca Osca-Verdegal^{1

3}, Daniel Pérez-Cremades^{2

3}, Susana Novella^{2

3}, Carlos Hermenegildo^{2

3}, Federico V Pallardó^{1

2

3}, José Luis García-Giménez^{1

2

3}

Affiliations

¹ Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain.
² Instituto de Investigación Sanitaria INCLIVA, Valencia, Spain.
³ Departamento de Fisiología, Facultad de Medicina y Odontología, Universitat de València, València, Spain.

Abstract

Introduction: Circulating extracellular histones acquire relevance as cytotoxic mediators in sepsis. Extracellular histones act as damage-associated molecular patterns (DAMPs), which induce oxidative stress and NLRP3 inflammasome activation. Inflammasome mediates pyroptosis, a programmed cell death mechanism that produces inflammation. Despite evidence for inflammasome activation in immune cells during sepsis, it was unknown whether extracellular histones can produce endothelial inflammasomes activation.

Methods: We used human umbilical vein endothelial cells (HUVEC) to explore the activation of pyroptosis, endothelial function and inflammation by extracellular histones. We evaluated pyroptosis by flow cytometry, caspase-1 activity assay, and gene and protein expression analysis by RT-qPCR and Western blot, respectively. The upstream molecular responses involved in pyroptosis activation by extracellular histones were validated by means of using antioxidant glutathione ethyl ester and NLRP3 inflammasome inhibitors. Finally, using mass spectrometry, we measured circulating histones in blood from critically-ill patients and demonstrated that circulating histone levels correlated with the expression of pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity.

Results: We found that extracellular histones mediate the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how the hyperacetylation of extracellular histones or the use of antioxidants decreased pyroptosis. In addition, we showed that pyroptosis is a feasible process occurring in septic shock patients.

Discussion: Circulating histone levels correlated with the expression of pro-inflammatory and pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. We propose to block histone-mediated pyroptosis as a feasible therapeutic strategy in sepsis.

Keywords: NLRP3; endothelium; extracellular histones; histone acetylation; inflammasome; sepsis.

Grants and funding

J.B-G. is supported by a Contratos i-PFIS grant (IFI18/00015) and co-financed by the European Social Fund. R.O-V. is supported by Contratos PFIS grant (FI20/00202) from AES-ISCIII and co-financed by the European Regional Development Fund (ERDF). D.P-C. Thanks Spanish Ministerio de Ciencia e Innovación for the fellowship “Juan de la Cierva - Incorporación” (grant number IJC2019-040237-I). J.L.G-G. thanks INCLIVA and GVA for the starting grants GV/2014/132) and AES2016 (ISCIII) for grant numbers PI16/01036, DTS21/00193 and PI19/00994, co-financed by the European Regional Development Fund (ERDF). S.N. and C.H. thank AES2016 (ISCIII) for grant number PI16/00229 and PI19/01714, co-financed by the European Regional Development Fund (ERDF). J.L.G-G. and FVP thank Grand Challenges Canada and the Spanish Ministry of Science and Innovation, ISCIII through CIBERer (Biomedical Network Research Center for Rare Diseases). We would like to thank INCLIVA’s Biobank ISCIII, FEDER for grant PT13/0010/0004 and PT20/00029.