Abstract:

Bottlenose dolphin peripheral blood mononuclear cells were experimentally exposed to oil in culture medium (high-energy medium accommodated fraction; HEMAF) and cytokine gene transcripts were measured to assess the cells' ability to polarize towards a Th1, Th2 or Treg response.

Suggested Citation:

Sylvain De Guise, Milton Levin, and Lindsay Jasperse. 2020. Immune function of peripheral blood mononuclear cells exposed to Deepwater Horizon oil in laboratory experiments. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-vjnn-yz30

Purpose:

The purpose of this project is to evaluate the direct effects of oil exposure and to investigate the cause and effect relationship (in vitro using dolphin cells) and strengthen the weight of evidence between the changes observed in live dolphins and oil exposure.

Data Parameters and Units:

PCR.ID Laboratory identification number for PCR assay Subject Sample origin from a wild bottlenose dolphin in Sarasota, FL. Treatment Dolphin lymphocytes were treated with cytokines associated with either a Th1, Th2, or Treg stimulus, or unstimulated. Th1 = IL-12 and IFNy (25 ng/ml each). Th2 = IL-4 (25 ng/ml). Th2+IL-2 = IL-4 and IL-2 (25 ng/ml each) Treg = IL-2 and TGFb (10 ng/ml each). Treatment.conc_ng/ml Treatment concentration in ng/ml HEMAF_perc Treatment with High Energy Medium Accommodated Fraction (HEMAF) in vitro oil exposure (0%, 12.5%, 25% or 50% HEMAF in culture medium) HPRT1.63_Ct Gene expression via the Comparative Ct method, with annealing temperature at 63˚C. HPRT1.63_flag A warning that the housekeeping gene expression level is above the expected range, in which case, the data for the gene of interest is rejected. S9.63_Ct Gene expression via the Comparative Ct method, with annealing temperature at 63˚C. S9.63_flag A warning that the housekeeping gene expression level is above the expected range, in which case, the data for the gene of interest is rejected. IFNy_Ct Gene expression via the Comparative Ct method. IL-13_Ct Gene expression via the Comparative Ct method. TGFb_Ct Gene expression via the Comparative Ct method. HPRT1.54_Ct Gene expression via the Comparative Ct method, with annealing temperature at 54˚C. HPRT1.54_flag A warning that the housekeeping gene expression level is above the expected range, in which case, the data for the gene of interest is rejected. S9.54_Ct Gene expression via the Comparative Ct method, with annealing temperature at 54˚C. S9.54_flag A warning that the housekeeping gene expression level is above the expected range, in which case, the data for the gene of interest is rejected. IL-4_Ct Gene expression via the Comparative Ct method. IL-10_Ct Gene expression via the Comparative Ct method.

Methods:

Blood samples were collected from live-captured bottlenose dolphins from Sarasota Bay (unexposed to DWH oil) and shipped to the lab overnight. Peripheral blood mononuclear cells were isolated using density-gradient centrifugation and cultured with IL-12 and IFNy to stimulate a Th1 polarization, IL-4 or IL-4 and IL-2 to stimulate a TH2 polarization, or IL-2 and TGFb to stimulate a Treg polarization. Following RNA isolation, RT-PCR was performed to measure the expression of the genes for the effector respective effector cytokines (IFNy for Th1, iL-4 and IL-13 for Th2, and TGFb and IL-10 for Treg).

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