IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients
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1
Universidad de Antioquia, Grupo de Inmunodeficiencias Primarias Facultad de Medicina , Colombia
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2
Universidad de Antioquia, Grupo de Investigación en Microbiología Básica y Aplicada (MICROBA) Escuela de Microbiología, Colombia
Neutrophil Extracellular Traps (NETs) are complex variable structures comprised mainly of chromatin and proteins from intracytoplasmic granules that are released by Polymorphonuclear Neutrophils (PMNs) as a defense mechanism that immobilize microorganisms to facilitate their destruction. The process by which these structures are released is known as NETosis and molecular events involved in the formation of NETs are not completely understood. However, reactive oxygen species (ROS) produced by the NADPH oxidase system in phagocytic cells are essential to the decondensation of chromatin and subsequently the formation of NETs.
Chronic Granulomatous Disease (CGD, OMIM # 306400) is a primary immunodeficiency characterized by a profound failure to produce ROS due to mutations in genes that code for different proteins associated with the NADPH oxidase system in phagocytic cells. It has been previously shown that CGD patients do not release NETs and it has been suggested that this mechanism might be partly responsible for their increased susceptibility to infections. The use of recombinant human Interferon gamma (rhIFN) has been shown to reduce this susceptibility in some patients by partly increasing NADPH oxidase activity and consequently the microbicidal activity of PMNs. However, the molecular mechanism by which IFNγ promotes most of its microbicidal activity in PMNs patients remains unclear. Therefore, we aimed to determine whether IFNγ affects the production of NETs in PMNs from healthy individuals and CGD patients.
To evaluate production of ROS and NETs, human PMNs were isolated from healthy individuals and patients with X-CGD from peripheral blood using density gradient separation and cells were placed at 400.000 per tube for flow cytometry and 250.000 per 24-well cell culture plate containing therein round glass cover slip for microscopy and fluorometry. The cells were then primed 100 UI/ml of rhIFNγ for 2 hours at 37oC, and subsequently stimulated with a 50 nM solution of PMA for 15 min at 37°C and ROS production was measured by flow cytometry using the dihydrorhodamine oxidation test (DHR). In addition, for visualization of NETs, PMNs were primed or not with rhIFNγ 100 UI/ml and stimulated with PMA 50 nM or Ionomycin μg/ml was documented with the Hoeschst 33342 stain solution for DNA and rabbit anti-human elastase antibodies, using in a fluorescent microscope (Axio Vert.A1 – Carl Zeiss) and a fluorometer (Spectra Max Gemini – Molecular devices) for quantification of DNA release. Nets production was analyzed by Wilcoxon test with p<0.05.
Initially PMNs from controls and patients were stimulated with rhIFNγ 100 UI/ml alone, and no measurable production of ROS by DHR testing was observed. However, stimulation of PMNs from both groups with PMA 50 nM resulted in high production of ROS as expected. Furthermore, when PMNs were primed with rhIFNγ and subsequently stimulated with PMA 50 nM we observed an increase in the induction of ROS when compared with PMNs stimulated with PMA only. Next, we used fluorescence microscopy with DNA and anti-elastase antibodies to visualize NETs under different stimulation conditions in PMNs from controls and patients. When PMNs from both groups were left unstimulated they retained the classic morphology of multilobed nuclei as well as when PMNs from controls were stimulated with PMA there was significant production of NETs, in contrast to PMNs from X-CGD patients, in whom the production of NETS was low. However, when PMNs from patients were primed with 100 IU/ml rhIFNy for the same time and subsequently activated with PMA 50 nM there was a small increase in production of NETs when compared to controls. Stimulation of PMNs from both groups with 5 μg/ml of ionomycin resulted in high production of NETs, this was evident in fluorescence microscopy and NETs quantification by fluorometry. Priming whit rhIFNγ and subsequent stimulation with ionomycin doesn’t shown difference in comparison with cells that only were stimulated with ionomycin.
Here we demonstrate that production of ROS and NETs in PMNs is induced normally in healthy individuals by stimulation with PMA but not by rhIFNg alone, however priming with rhIFNγ followed by PMA stimulation increased their production. Interestingly, we found that PMNs from CDG patients released NETS when stimulated with PMA and ionomicyn stimuli, in contrast to what has been previously published. Therefore, we suggest that rhIFNcould be potentiating the microbicidal activity of PMNs from by promoting the production of NETs. The high production of NETs when the cells are stimulated whit ionomicyn its related whit an independent ROS NETs release.
Acknowledgements
This work is supported by COLCIENCIAS grant (number 111554531412)
Keywords:
neutrophil extracellular traps,
NETs,
chronic granulomatous disease,
CGD,
Neutrophils
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Immunodeficiencies
Citation:
Vélez
GJ,
Rocha
YC,
Arias
AA and
López
JA
(2015). IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00122
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Received:
28 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. Juan A López, Universidad de Antioquia, Grupo de Inmunodeficiencias Primarias Facultad de Medicina, Medellín, Antioquia, Colombia, alvaro.lopez@udea.edu.co