Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.
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1
Universidad Autónoma de Nayarit. Secretaria de Investigación y Posgrado. Laboratorio de Inmunotoxicologia, Mexico
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2
Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS). Departamento de Inmunologia, Mexico
Introduction:
The essential function of the immune system in all vertebrates is their defense against foreign elements. Teleosts fishes are the first group of organisms that present innate and adaptive immune mechanisms, similar to the ones of mammals, hence the study of these organisms is of great relevance due to the information they give about the evolution of the immune system of vertebrates. Innate immunity mechanisms are crucial, since apart of acting as first defense line, they also regulate importantly the mechanisms of adaptive immunity, the latter mediated by T and B lymphocytes, essential cells in the protection against recurrent infections. The physiology of the immune system cells is extremely complex and is regulated by the cellular microenvironment. Lymphocytes physiology is regulated by different signal transduction pathways and essential elements such as intracellular Ca+2, mechanisms that influence in proliferative and anti-ploriferative processes, such as cellular differentiation, apoptosis and senescence.
Lymphocytes functional parameters have been widely studied in mammals; nevertheless, in lower vertebrates, such as fishes, data are scarce. In this study, Nile tilapia (Oreochromis niloticus), a freshwater teleost fish, with economic importance and widely distributed in almost all subtropical countries of the world, consequently it can be a potential model for research in biomedical and environmental areas such as immunology, immunotoxicology and ecoimmunology.
Objetive:
The aim of this study was to identify spleen cells populations and to evaluate different functional parameters in lymphocytes (intracellular Ca+2, cellular proliferation, mitochondrial membrane potential) and anti-proliferative processes (apoptosis and senescence).
Material and Methods:
Nile tilapia (273 ± 43 g and 20 ± 3 cm) obtained from a local farm were used for this study, which were kept in tanks of 400 L at 28 ± 2°C and constant aeration, under optimal conditions for acclimation in the laboratory (pH 8.0, dissolved oxygen 6.0 mg/L, and oxygen saturation 85%) for four weeks. After acclimation time, fishes were sacrificed on a bath of ice, the spleen was extracted and mechanically disaggregated. The splenocytes were separated by density gradient, using Histopaque-1077, at 2,500 rpm/20 min. After, the cells were quantified by microscopy, using a hemocytometer, adjusting cell suspension at 1x106 cell/mL.
Evaluation of cellular proliferation was determined by the method MTT (3-(4,5-dimethyl-2-thyazole) 2,5- diphenyl-2H- tetrazolium bromide]), in cultivated cells in medium RPM-1640 during 72 h in atmosphere of CO2, using different mitogens concentrations as phorbol 12-myristate 13-acetate (PMA) and ionomycin. Identification of cell populations was made through the use of graphics of forward and side scatter in the Flow cytometer BD AcuriTMC6. Determination of intracellular Ca2+ was made by the use of a fluorescent probe fluo-4 NW and as stimulus PMA and ionomycin were used. Apoptosis quantification was made through annexin V-FITC, using carbobenzoxy-Leu-Leu-leucinal (MG-132) and etoposide as pro-apoptotic stimuli. On the other hand, the mitochondrial membrane potential () was determined by using an assay kit MitoProbe™ DiOC2 (3), using carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) and MG-132 as stimuli. Finally, cellular senescence determination was made through measure of the β-galactosidase activity, using as substrate the 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG), doxorrubicin was used as stimulus. Each functional parameter was contrasted with the corresponding controls. The different stimulus were added 24 h (at 28 ± 2 ° C and 5% of CO2) before analysis of each test.
Results:
Obtained results indicate that lymphocytes of Nile Tilapia presented a proliferation index of 1.22 ± 0.13, 1.37 ± 0.48 and 2.06 ± 0.18 with the combination of mitogens at concentrations of 1.5 µg ionomycin + 40, 60 or 80 ng PMA respectively; and a proliferation index of 1.61 ± 0.21, 1.40 ± 0.50 and 1.72 ± 0.03 with the combination of mitogens at concentrations of 2 µg ionomycin + 40, 60 or 80 ng PMA.
In cellular suspension, previously submitted to density gradient, two cell populations were able to be identified by flow cytometry (using parameters of FSW and SSC), corresponding to lymphocytes and monocytes.
Intracellular calcium parameters, mitochondrial membrane potential, apoptosis and senescence were determined in the lymphocytes population previously identified. Regarding intracellular Ca+2, different concentrations of ionomycin (2, 3 and 5 µg) and PMA (2 µg) were used as stimulus, achieving to obtain a response of 48.8, 56.5 y 53.42 % with the combination of mitogens at concentrations of 2 µg ionomicyn + 2 µg PMA, 3 µg ionomicyn + 2 µg PMA and 5 µg ionomicyn + 2 µg PMA respectively. On the other hand, the mitochondrial membrane potential () was significantly altered by MG-132 (76.9%), in comparison with the CCCP, which caused a damage of 5.4%. Apoptotic quantification was determined using MG-132 (2µM) and etoposide (40µg) as pro-apoptotic inducer; the MG-132 caused apoptosis at 38.5% while the etoposide in 49.9% of the population. Regarding cellular senescence, exposure to 1, 4 and 8 µM doxorubicin provoked an increase of this parameter of 5.5, 30 and 76.8%, respectively.
Conclusion:
The results described above show the response of lymphocytes of fishes to different inducers. This cells presented better proliferation index (2.06 ± 0.18) with the combination of mitogens at a concentration of 1.5 µg/mL of ionomicyn and 80 ng/mL of PMA. Also, these results showed a greater flow of calcium (56.5 %) with the combination of 3 µg ionomicyn and 2 µg PMA. On the other hand, the MG-132 can be used as good indicator for loss mitochondrial membrane potential, while the etoposide as inducer of apoptotic cells. Moreover a concentration of 8 µM doxorubicin is sufficient to generate more than 50% of senescent cells.
Hence, these parameters can be used as cellular biomarkers, and thus the functionality of the immune system of fishes can be evaluated to possible antigenic and/or xenobiotics present in aquatic ecosystems.
Acknowledgements
This work was funded by a grant from the financial resources of SEP-CONACyT-Mexico for Basic Research (Project no. 2012-179508). The first two authors are students of Postgraduate in Biological and Agricultural Sciences (Posgrado en Ciencias Biológico-Agropecuarias CBAP) of the State University of Nayarit (Universidad Autonoma de Nayarit, Mexico).
Keywords:
Nile tilapia,
fish immunology,
Lymphocyte Activation,
Flow Cytometry,
Intracellular signaling
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:
Oral Presentation
Topic:
Veterinary and Comparative Immunology
Citation:
Diaz-Resendiz
KJ,
Toledo Ibarra
GA,
Fonseca-Torres
NX,
Ortiz-Lazareno
PC and
Girón-Pérez
M
(2015). Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry..
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.2015.05.00328
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Received:
29 Apr 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
Dr. Manuel Iván Girón-Pérez, Universidad Autónoma de Nayarit. Secretaria de Investigación y Posgrado. Laboratorio de Inmunotoxicologia, TEPIC, NAYARIT, 63000, Mexico, ivan_giron@hotmail.com