Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome

  • P. Galozzi | paola.galozzi@unipd.it Rheumatology Unit, Department of Medicine - DIMED, University of Padova, Italy.
  • O. Negm School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom; Medical Microbiology and Immunology Departement, Faculty of Medicine, Mansoura University, Egypt.
  • E. Greco Rheumatology Unit, Department of Medicine - DIMED, University of Padova, Italy.
  • N. Alkhattabi School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom.
  • A. Gava Rheumatology Unit, Department of Medicine - DIMED, University of Padova, Italy.
  • P. Sfriso Rheumatology Unit, Department of Medicine - DIMED, University of Padova, Italy.
  • L. Fairclough School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom.
  • I. Todd School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom.
  • P. Tighe School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom.
  • L. Punzi Rheumatology Unit, Department of Medicine - DIMED, University of Padova, Italy.

Abstract

The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.

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Author Biographies

P. Galozzi, Rheumatology Unit, Department of Medicine - DIMED, University of Padova
O. Negm, School of Life Sciences, Department of Immunology, University of Nottingham, Nottingham, United Kingdom; Medical Microbiology and Immunology Departement, Faculty of Medicine, Mansoura University
Published
2015-03-31
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Articles
Keywords:
Pro-inflammatory cytokines, Blau syndrome, Autoinflammatory disease, Interleukin-1β.
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How to Cite
Galozzi, P., Negm, O., Greco, E., Alkhattabi, N., Gava, A., Sfriso, P., Fairclough, L., Todd, I., Tighe, P., & Punzi, L. (2015). Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome. Reumatismo, 66(4), 277-284. https://doi.org/10.4081/reumatismo.2014.772