Abstract

Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability.¹ The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect the animals of high trophic level via food chain, such as cetacean and human.² Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in modulation of the immune system, and can be classified into Th1 (IL-12, IFN-γ, and IL-18) and Th2 (IL-4, IL-5, and IL-13).^3,4,5 Th1/Th2 balance can be evaluated by the ratio of their polarizing cytokines (i.e., IFN-γ/IL-4), and animals with imbalanced Th1/Th2 response (Th1/Th2 polarization) may become more susceptive to certain kinds of infections.^4,6,7 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 qRT-PCR. Blood samples of 6 common bottlenose dolphins (Tursiops truncatus) in Farglory Ocean Park. cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses of cPBMCs to C-AgNP20 by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The selected cytokines included Interleukin (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 samples were determined and used to normalize the mRNA expression levels of target genes. There was a significant increase in the ratio of late apoptotic/necrotic cells of cPBMCs with or without Con A 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. 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. IFN-γ/IL-4 was significantly decreased at 24 h of culture with 1 μg/ml C-AgNP₂₀ treatments, and IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 μg/ml C-AgNP₂₀ treatments. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 μg/ml C-AgNP₂₀ after 24 h of culture. The present study demonstrated that the sublethal dose of C-AgNP₂₀ on cPBMCs (≤1 μg/ml), and the inhibitory effect of C-AgNP₂₀ on the mRNA expression levels of cytokines of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased mRNA expression level of TNF-α. Th2 response is associated with enhanced immunity against to parasite but with decreased immunity to intracellular organisms.^4,6 TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens.⁸ In summary, our data indicated that C-AgNP₂₀ suppress the cell-mediated immune response, and thus may inhibit the immunity against intracellular organisms, such as Toxoplasma gondii, Brucella spp., and morbillivirus, in cetaceans.^9,10

* Presenting author
+ Student presenter

Acknowledgements

We thank all the personnel of Farglory Ocean Park for blood sample collection and storage, the dolphins in Farglory Ocean Park for donating their blood, Dr. Bang-Yeh Liou for blood sample transportation, and Ministry of Science & Technology, Taiwan for financial support (Grant MOST 106-2313-B-002-054-).

Literature Cited

1.  Massarsky A, Trudeau VL, Moon TW. 2014. Predicting the environmental impact of nanosilver. Environ Toxicol Pharmacol. 38:861–873.

2.  Walters CR, Pool EJ, Somerset VS. 2014. Ecotoxicity of silver nanomaterials in the aquatic environment: a review of literature and gaps in nano-toxicological research. J Environ Sci Health A Tox Hazard Subst Environ Eng. 49:1588–1601.

3.  Kidd P. 2003. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev. 8:223–246.

4.  Owen JA, Punt J, Stranford SA, Jones PP, Kuby J. 2013. Kuby Immunology. New York, NY: W.H. Freeman.

5.  Tizard IR. 2013a. Cell signaling: cytokines and their receptors. In: Tizard IR, ed. Veterinary Immunology. 9th ed. St. Louis, MO: Saunders; 75–83.

6.  Tizard IR. 2013b. Helper T cells and their response to antigen. In: Tizard IR, ed. Veterinary Immunology. 9th ed. St. Louis, MO: Saunders; 137–149.

7.  Raphael I, Nalawade S, Eagar TN, Forsthuber TG. 2015. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 74:5–17.

8.  Korner H, McMorran B, Schluter D, Fromm P. 2010. The role of TNF in parasitic diseases: still more questions than answers. Int J Parasitol. 40:879–888.

9.  Dubey JP, Fair PA, Sundar N, Velmurugan G, Kwok OC, McFee WE, Majumdar D, Su C. 2008. Isolation of Toxoplasma gondii from bottlenose dolphins (Tursiops truncatus). J Parasitol. 94:821–823.

10.  Mazzariol S, Centelleghe C, Di Provvido A, Di Renzo L, Cardeti G, Cersini A, Fichi G, Petrella A, Di Francesco CE, Mignone W, Casalone C, Di Guardo G. 2017. Dolphin morbillivirus associated with a mass stranding of sperm whales, Italy. Emerg Infect Dis. 23:144–146.