Abstract:
The goal of this study is to build upon a previous validation of assays and reagents to identify T helper cell subsets and elucidate their functions in a group of dolphins from Sarasota Bay, Florida, USA. In addition, we wanted to test several immune functions in dolphins from Barataria Bay, LA and Dauphin Island, AL. In order to identify dolphin Treg cells, we used antibodies to CD4 and FOXP3. In order to determine the functionality of dolphin Treg cells, used porcine reagents to measure TGFß, the major Treg effector cytokine. In order to assess the capacity of dolphin Th cell subsets to respond to a Th1, Th2 or Treg stimulus, we tested the relative expression of effector cytokines upon in vitro stimulation with human recombinant cytokines. To understand the balance of cytokines in wild dolphins, we measured serum concentrations of 12 cytokines including the Th1 cytokines IL-2, IL-12 and IFNγ, the Th2 cytokines IL-4, IL-5 and IL-13, the Treg cytokines IL-10 and TGFß, and the inflammatory cytokines IL-1ß, IL-8, TNFα and GM-CSF, in 34 wild Barataria Bay dolphins and 17 wild Dauphin Island dolphins.
Suggested Citation:
Sylvain De Guise, Milton Levin, Lindsay Jasperse, Guillermo Risatti. 2019. Data from immunoloigcal testing of Barataria Bay, Louisiana, and Dauphin Island, Alabama common bottlenose dolphins (Tursiops truncatus). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-ymp8-x712
Data Parameters and Units:
Specific outcomes depend on the viability of the specificity of the immunoassays developed by the laboratory, and include parameters such as number of regulatory T cells (n), proportion of regulatory T cells (%), CD154/CD69 expression levels (fluorescence intensity and cell counts), cytokine levels (fluorescence intensity or real-time PCR transcript counts).
The detail description of data parameters and common abbreviations used in the dataset can be found in the worksheet "Read.me".
Methods:
Long-term resident bottlenose dolphins from Barataria Bay, LA, United States, and Dauphin Island, AL, United States, were captured, sampled, and released as part of health assessment programs (which included the immunological data presented here). Whole blood was collected in Vacutainer tubes with sodium heparin as part of the routine physical examinations, kept cool and shipped overnight for functional immunological assays. In addition, 1 ml serum was collected and immediately frozen prior to shipping on dry ice for cytokine analysis. Dolphin samples were collected under National Marine Fisheries Service Scientific Research Permit No. 18786, issued to Dr. Teri Rowles. All samples were received and experiments performed following approval from the University of Connecticut IACUC.
Instruments:
CD4 and FOXP3: The fluorescence of approximately 20,000 lymphocytes was read using a BD Biosciences LSRFortessa X-20 Cell Analyzer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) and the FACSDiva software (Becton Dickinson Immunocytometry System, San Jose, CA 95131, USA).
TGFb: TGFß concentrations were measured using the Luminex platform and either porcine reagents (TGFb.pig).
Cytokines: After the incubation and conjugation process, the plates were measured on the Bio-Plex 200 system (Bio-Rad, Hercules, CA 94547, USA), and analyzed using Bio-Rad Manager 5.0. The observed concentration (pg/ml or ng/ml) of each analyte for each sample was calculated using a curve fit generated for each analyte from seven or eight standards (depending on kit). If a sample concentration was extrapolated outside the standard curve and designated as “Value extrapolated beyond standard range” by the software, that sample concentration was accepted as the calculated value. If a sample concentration was reported as “out of range” by the software, that sample concentration was given a 0 pg/ml or 0 ng/ml value or the highest value on the standard curve, depending on whether it was below or above the measurable range. Prior to each use of the Bio-Plex 200 system, instrument calibration and validation procedure using the Bio-Rad Validation and Calibration kit (Bio-Rad, Hercules, CA 94547, USA) was performed to assure the instrument was performing properly, as per manufacturer’s instruction. The instrument passed both calibration and validation tests prior to each use.
Gene expression: Real-time PCR (qRT-PCR) reactions were performed using SYBR green (Thermo Fisher Scientific, Grand Island, NY 14072, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA 94547, USA). All samples were analyzed for housekeeping genes HPRT1 and S-9. Reactions containing water, but no cDNA, were used as negative controls. Product specificity was monitored by analysis of melting curves. Gene expression data were analyzed using the Comparative CT (ddCT) Method. Samples for which the amplification of the housekeeping genes was outside of the expected range were discarded, so as to not misinterpret a change in the expression of a target gene as an inadequate PCR reaction.
Lymphocyte proliferation: Lymphocyte proliferation was evaluated by the incorporation of 5-Bromo-2’-deoxyuridine (BrdU), a thymidine analogue, detected with a monoclonal antibody and colorimetric enzymatic reaction (Cell Proliferation ELISA BrdU (colorimetric), Roche Diagnostics GmbH, Mannheim, Germany) as per manufacturer’s instructions using an ELISA plate reader (Multiskan EX v.1.0) at 450 nm with a reference wavelength of 690 nm. Results were expressed as optical density (OD). Cells from mice (one mouse for each experimental day) were assayed concurrently with dolphin samples for quality control. After field sampling, mouse data were assessed for the presence of outliers using the SPSS software (IBM SPSS Statistics version 21, Armonk, NY 10504, USA). If outliers were detected, it was assumed that normal daily variability for the assay was exceeded, and the corresponding dolphin data for that assay on that day were eliminated from the dataset.
Provenance and Historical References:
Funding: This study was supported by a grant from the Gulf of Mexico Research Initiative (GoMRI) to the Consortium for Advanced Research on Marine Mammal Health Assessment (CARMMHA) under award # SA 18-12 from Ocean Leadership, under a Subaward with the National Marine Mammal Foundation (NMMF).
Acknowledgements: The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-Human CD4 Monoclonal (Sim.4) from Dr. James Hildreth. The monoclonal antibody to cetacean CD4 was graciously provided by Dr. Tracy Romano, Mystic Aquarium. Dolphin samples were collected under National Marine Fisheries Service Scientific Research Permit No. 20455, issued to RSW. This work would not have been possible without the help of countless volunteers who participated in the capture and sampling of dolphins.
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