Development and Application of Specific Cytokine Assays in Tissue Samples from a Bottlenose Dolphin with Hyperinsulinemia

Abstract

Chronic inflammation has been associated with insulin resistance and type 2 diabetes (T2D) in humans. Postmortem hepatic and splenic tissue from a 46-year-old geriatric male bottlenose dolphin (Tursiops truncatus) with insulin resistance (chronic hyperinsulinemia with hyperglycemia), chronic inflammation (white blood cell count greater than 12,000 cells/μL), and mild fatty liver disease was evaluated for elevated pro-inflammatory mediators. Cytokine mRNA expression in postmortem hepatic and splenic tissue, as determined by real-time PCR, included an array of cytokines: TGF-β, TNF-α, IFN-γ, IL-2, IL-4, IL-10, IL-12p40, IL-13, and IL-18. Values from this dolphin were compared to a younger reference dolphin with no known chronic metabolic perturbations or inflammation. Levels of TGF-β, TNF-α, and IL-4 were higher in the case dolphin’s liver compared to that of the reference dolphin. In the case dolphin’s spleen, IL-10 and IFN-γ mRNA was upregulated while IL-4 was less than the reference dolphin. IL-18 and IL-13 were upregulated in both tissues. Fluorescent immunohistochemistry (IHC) utilized the following antibodies: anti-porcine IL-6, anti-bovine IFN-γ, IL-4, and IL-10, anti-human TGF-β, anti-ovine IL-1β, and anti-dolphin IL-8. Fluorescent IHC in spleen from the case dolphin revealed staining of IL-4, IL-6, IL-8, and TGF-β throughout the tissue. IL-10 and IFN-γ were seen to predominate in areas surrounding the follicles of splenic tissue. This is the first characterization of cytokine levels in dolphin hepatic and splenic tissue. While there are limitations to a case study, this report of inflammatory biomarkers in tissues of a dolphin with insulin resistance and fatty liver disease are similar to those observed in human patients.

T Helper Cell Subsets and Their Functions in Common Bottlenose Dolphins (Tursiops truncatus)

“…Cytokines are important mediators of the immune response, and several studies have explored the use of cytokines to relate to health status of marine mammals in different manners. Amplification of mRNA for cytokines using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to quantify cytokine expression in marine mammals, with or without stimulation of PBMCs, has been used in several studies (18, 46–53). While useful to measure responsiveness to a signal, the quantification of mRNA does not necessarily represent the circulating bioactive protein, which requires important steps, in which translation and secretion need to take place.…”

“…While powerful at associating cytokine production with specific cell types using simultaneous extra-cellular labeling, this approach is tedious and time consuming, and allows the labeling for a relatively low number of cytokines at a time, in this case only two cytokines. Similarly, immunohistochemistry has been used to detect cytokines in cetacean tissue section (53, 55, 56). While informative on the distribution of the cell types secreting cytokines in different tissues, this method is limited to the use of tissues from dead animals, or biopsies in live animals, which would be a rather invasive procedure.…”

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus)

“…We and others have used cross-reactive antibodies specific for human, mouse, porcine, and bovine on cetacean samples [ 5 , 13 , 14 ]. However, cross-species reactivity of available Ab to cytokines or chemokines cannot be universally expected for all cytokines.…”

“…Increased production of a variety of pro-inflammatory cytokines is the hallmark for many diseases. Using real-time qPCR and fluorescent IHC, Eberle et al showed that a dolphin with hyperinsulinemia, hyperglycemia, chronic inflammation, and fatty liver disease displayed a similar pro-inflammatory state as humans with type 2 diabetes and hyperinsulinemia [ 5 ]. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) are two cytokines specifically useful to measure the inflammatory state of an animal [ 6 , 7 ].…”

Dolphins and Diabetes: Applying One Health for Breakthrough Discoveries

“…Dr. Neeley et al’s discovery of disrupted associations between adiponectin and glucagon in dolphins with high insulin and iron overload, paired with high sequence similarity of dolphin and human adiponectin, continue to support increasing high-weight adiponectin as a potential therapeutic target for people and other animals with these diseases ( 16 ). Dr. Kirsten Eberle et al use their newly developed, dolphin-specific immunoassays to characterize inflammation present in a 46-year old dolphin with metabolic syndrome and iron overload compared to a dolphin without these diseases ( 17 ). Parallels in chronic inflammation among people and dolphins with metabolic syndrome are reported, enhancing the emerging story of chronic inflammation and metabolic perturbations.…”