Time Course of Splenic Cytokine mRNA and Hormones During an LPS-Induced Inflammation in Toads.

Inflammation comprises alterations in glucocorticoids (in amphibians, corticosterone - CORT) and melatonin (MEL) levels, two hormones with immunomodulatory effects on cytokine production in several vertebrates. Cytokines mediate inflammation progress differently depending on their function. While some are secreted during the acute phase of the immune response, others prevail during the resolution phase. Major efforts have been made to understand the interaction of endocrine mediators and cytokine production in endotherms, but little is known for ectotherms so far. Characterizing the stages of inflammation and their interplay with endocrine mediators is crucial for an assertive and integrative approach to amphibian physiology and ecoimmunology. Herein, we investigated CORT and MEL plasma levels as well as splenic cytokines (IL-1β, IL-6, and IL-10) mRNA levels during the progression of the inflammatory response in toads (Rhinella diptycha) in four time-points (1, 3, 6, and 18h) after an immune challenge with lipopolysaccharide (LPS) using independent samples. Toads were responsive to LPS, with all hormones and cytokines affected by LPS. IL-1β and IL-6 were up-regulated after 1h, but IL-1β decreased right after 3h, while IL-6 sustained up-regulation throughout all time points. IL-10 had not been detected until 6h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18h, CORT levels were still high, and IL-1β was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1β with IL-6 for LPS and saline groups. LPS-treated individuals showed an overall decrease in MEL plasma levels compared to saline counterparts. Our results showcase the early endocrine and molecular events of the amphibian immune response. We also report activation of the HPI axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians.