Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)
- Published
- Accepted
- Subject Areas
- Toxicology, Veterinary Medicine, Immunology, Ecotoxicology, Environmental Contamination and Remediation
- Keywords
- Cetacean, Silver nanoparticles (AgNPs), Immunotoxicity, Cytokine, Th2 bias, qRT-PCR
- Copyright
- © 2018 Li et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus) PeerJ Preprints 6:e27049v1 https://doi.org/10.7287/peerj.preprints.27049v1
Abstract
Background Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic level, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types, Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/ Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Methods Blood samples were collected from 6 captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-ɑ. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes.
Results The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 μg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 μg/ml C-AgNP20 treatment. At 24 h of culture with 1 μg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 μg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 μg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 μg/ml C-AgNP20 after 24 h of culture.
Discussion The present study demonstrated that the sublethal dose of C-AgNP20 (≤ 1 μg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
The size distribution and zeta pontential of the 100 and 500 μg/ml C-AgNP20 in 2 mM citrate buffer (pH 7.4)
Cytotoxicity of of C-AgNP20 in cPBMCs with or without ConA stimulation
qPCR efficiencies of each primer set (ct values)
Time kinetics of mRNA expression levels of selected cytokines of cPBMCs
The cPBMCs with 0 h incubation were used as control for the calculation of cytokine expression level by △△CT method. In addition, the geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each samples were determined and used to normalize the the expression levels of target genes. The numbers in the sheet were the expression levels.
Effects of C-AgNP20 on mRNA expression levels of selected cytokines of cPBMCs (4 h)
The cPBMCs with 4 h incubation without C-AgNP20 treatment were used as control for the calculation of cytokine expression level by △△CT method. In addition, the geometric means of two HKGs, GAPDH and B2M, of each samples were determined and used to normalize the the expression levels of target genes
Effects of C-AgNP20 on mRNA expression levels of selected cytokines of cPBMCs (24 h)
The cPBMCs with 24 h incubation without C-AgNP20 treatment were used as control for the calculation of cytokine expression level by △△CT method. In addition, the geometric means of two HKGs, GAPDH and B2M, of each samples were determined and used to normalize the the expression levels of target genes.