Time Course of Splenic Cytokine mRNA and Hormones during a Lipopolysaccharide-Induced Inflammation in Toads

Synopsis 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 cytokine (IL-1 beta, 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 18 h) after an immune challenge with lipopolysaccharide (LPS) using independent samples. Toads were responsive to LPS, with all hormones and cytokines affected by LPS. IL-1 beta and IL-6 were up-regulated after 1 h, but IL-1 beta decreased right after 3 h, while IL-6 sustained up-regulation throughout all time-points. IL-10 had not been detected until 6 h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18 h, CORT levels were still high, and IL-1 beta was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1 beta 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 hypothalamus-pituitary-interrenal (HPI) axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians.

Automated summary

The study investigates the hormone levels and cytokine expression in amphibians during the inflammatory response. The results show that corticosterone and melatonin levels are affected by LPS in the early stages of immune response. IL-1 beta and IL-6 are up-regulated after 1 hour, while IL-1 beta decreases after 3 hours and IL-6 remains up-regulated throughout. IL-10 is detected after 6 hours of LPS stimulation, accompanied by an increase in corticosterone levels. After 18 hours, corticosterone levels remain high, IL-1 beta is up-regulated again, and IL-6 is up-regulated with a decrease in IL-10. Positive correlations are observed between IL-1 beta and IL-6 in the LPS group.