Immune-pineal-ocular axis in amphibians: unveiling a novel connection?

Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in the ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3h over 24h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10h) or at night (22h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retina light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group.

Immune and endocrine alterations at the early stage of inflammatory assemblage in toads after stimulation with heat-killed bacteria (Aeromonas hydrophila).

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 µl of saline or heat-killed bacteria: groups A1 (3 × 107 cells), A2 (3 × 108 cells), and A3 (3 × 109 cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-6, and IL-10. We found higher expression of IL-6, IL-1ß, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila.

Contrasting effects of transdermal and implant corticosterone treatments in the American bullfrog wound healing.

Glucocorticoid (GC) release is triggered by adverse stimuli that activate the hypothalamus-pituitary-adrenal/interrenal axis. Glucocorticoids may enhance or suppress immune functions depending on the level of elevation. In this study, we investigated the effects of transient and chronic increase of corticosterone (CORT) on the wound healing of the American bullfrog. Frogs were submitted to a daily transdermal hormonal application that acutely elevated CORT plasma levels, or vehicle as a control. Other frogs were surgically implanted with a silastic tube filled with CORT that resulted in chronic elevation of CORT plasma levels or received empty implants as a control. A dermal biopsy was performed to create a wound and was photographed every 3 days. Individuals treated with transdermal CORT started healing faster than their control 32 days after the biopsy. Frogs that received CORT implants tended to heal slower than control subjects. Plasma bacterial killing ability was not affected by treatment, which reinforces the constitutive nature of this innate immune trait. By the end of the experiment, frogs from the acute CORT treatment had smaller wounds compared with those receiving the CORT-filled implants, highlighting the differential effects of acute (immunoenhancing) and chronic (immunosuppressive) elevation of CORT plasma levels. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Liver vs. spleen: Time course of organ-dependent immune gene expression in an LPS-stimulated toad (Rhinella diptycha).

The inflammatory response comprises highly orchestrated events that are conserved amongst vertebrate groups. Hepatic and splenic cytokines are major mediators of the systemic inflammatory processes. However, the liver is still neglected as an immune organ in amphibians. This study reports organ-dependent gene expression using an anuran model. We tracked mRNA levels of immune proteins [C1s (subcomponent S of the complement protein 1), IFN-γ, IL-1ß, IL-6, and IL-10] at four time-points (1 h, 3 h, 6 h, and 18 h post-injection) in spleens and livers of intraperitoneal LPS-challenged (2 mg/kg) adult male toads (Rhinella diptycha) using independent samples. We found acute C1s up-regulation in the liver 1 h post-injection, with no treatment effect in the spleen. The LPS injection did not show any effect in splenic IFN-γ gene expression while eliciting only a marginal effect in the hepatic tissue. IL-1ß was up-regulated in both organs, with the liver initially displaying early expression (1 h and 3 h) and the spleen taking over late expression (18 h). Both organs exhibited similar patterns for IL-6, with early up-regulation (1 h and 3 h) and late peak (18 h). Although IL-10 was early detected and up-regulated only in the liver, both organs showed up-regulation in 6 h and 18 h post-injection. Our results show an exclusive hepatic prominence in complement protein expression during the acute-phase response. Furthermore, hepatic pro-inflammatory cytokine expression was more pronounced in earliest time-points, while the spleen offers a slower and more consistent response overall. Our data provide an organ-integrative outlook into the initial hours of the inflammation in amphibians, confirming the liver's pivotal role as a regulator in the acute-phase of the inflammatory response in amphibians.

Time Course of Splenic Cytokine mRNA and Hormones during a Lipopolysaccharide-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 cytokine (IL-1ß, 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-1ß and IL-6 were up-regulated after 1 h, but IL-1ß 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-1ß was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1ß 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.

Immune and endocrine responses of Cururu toads (Rhinella icterica) in their natural habitat after LPS stimulation.

Glucocorticoids and melatonin display immunomodulatory functions, with both immune-stimulatory and suppressor effects, depending on the context. While their immune properties are well-explored in mammals, there are still few studies on this immune-endocrine interaction in an inflammatory context in amphibians, all of them under captivity conditions, which can constitute a stressor for these animals. Evaluating how amphibians react to an immune challenge in the field would reveal relevant information regarding how immune-physiological parameters are modulated in natural conditions. This study aimed to investigate the effects of lipopolysaccharide (LPS) injection in male toads (Rhinella icterica) recently captured in their natural habitat in the Atlantic Forest at two different times of the day. We evaluated: splenic cytokines mRNA (interleukin [IL]-1ß, IL-6, IL-10, interferon-γ) and complement system protein (C1s), plasma bacterial killing ability (BKA), plasma corticosterone (CORT), melatonin (MEL), and testosterone (T) levels, and neutrophil to lymphocyte ratio (NLR), two hours post-injections. LPS-injection increased NLR, the gene expression of IL-1ß, and less evidently CORT levels independently of the time of the day. These results evidence LPS-induced inflammation, similarly observed in toads in captivity. Saline and LPS-injected toads showed a positive correlation between IL-1ß and IL-6, both cytokines with pro-inflammatory effects. Also, CORT was negatively associated with T, suggesting inhibition of the hypothalamus-pituitary-gonadal axis in the LPS-stimulated group. Our results are associated with the first stage of the inflammatory assemblage. Studies evaluating further steps of this process might lead to a better understanding of the immune-endocrine relations in amphibians.

Day vs. night variation in the LPS effects on toad's immunity and endocrine mediators.

The immune-endocrine interactions following an immune challenge have been demonstrated in amphibians. When considering immune challenges, the immune-endocrine implications can vary with the injection time (day or night), a pattern not explored in amphibians. We investigated the immune response following a lipopolysaccharide - LPS injection, measured as plasma bacterial killing ability - BKA, phagocytosis of blood cells - PP, and neutrophil to lymphocyte ratio - NLR, splenic proinflammatory cytokines mRNA (IL-1ß and IL-6), and also endocrine mediators (corticosterone - CORT and melatonin - MEL plasma levels) in Rhinella icterica adult male toads injected at day (10 am) or night (10 pm). LPS induced increases in CORT, NLR, PP, and IL-1ß mRNA compared with amphibian phosphate-buffer saline-injected individuals. For plasma CORT, the response was more pronounced during the night. While for the PP and IL-1ß mRNA, the effect was more evident during the day. For NLR, the increase happened at both times, day and night, in the LPS-injected toads. Meanwhile, no changes were observed in BKA, IL-6 mRNA, and MEL levels. Overall, our results demonstrated an LPS-induced inflammatory response in R. icterica toads, characterized by higher PP, NLR, and IL-1ß mRNA, followed by activation of the hypothalamic-pituitary-interrenal axis (higher CORT levels). The time in which the toads received the LPS injection affected the endocrine and immune mediators. The higher CORT and lower inflammatory response at night suggested a potential functional interaction between CORT and immune reactivity associated with the differences in night vs. day in R. icterica toads. These results highlight the relevance of investigating different injection times and mechanistic pathways to understand LPS-induced immunomodulation in anurans.

Stress Response, Immunity, and Organ Mass in Toads (Rhinella diptycha) Living in Metal-Contaminated Areas.

Mining is one of the main activities that drive the economy of Brazil. Mining activity is associated with risk of contamination of environment and local fauna by metals. Amphibians have a life cycle that requires a transition between aquatic and terrestrial environments, increasing their vulnerability to metal contamination in the water and substrate. Metals are ubiquitous, with high bioaccumulative and biomagnifying potential, and may lead to immune and endocrine disruption. In this study, we analyzed two different components of the innate immune response, bacterial killing ability (BKA) and phytohemagglutinin edema (PHA), and two stress biomarkers, corticosterone plasma levels (CORT) and the neutrophil to lymphocyte ratio (N:L), of toads (Rhinella diptycha) living in places contaminated by metals. Blood samples were collected pre- and post-restraint (1h), followed by an immune challenge with PHA and tissue collection (liver, spleen, and kidneys). Toads liver metal bioaccumulation did not correlate with the immune response or stress biomarkers. Post-restraint, animals had increased CORT and reduced BKA, independently of the collection site, and these variables were not correlated with liver metal bioaccumulation. Interestingly, toads with the larger spleen (immune organ) showed increased N:L post-restraint and greater edema after the PHA challenge. Our results indicate that toads living in metal-contaminated environments responded to acute stressor, activating the hypothalamic-pituitary-interrenal axis and the immune response. Keep tracking the physiological variables of these animals and the presence of metals in the environment and tissues should provide valuable health status indicators for the population, which is vital for proposing amphibian conservation strategies in these areas.

Immune and hormonal modulation in the postprandial period of bullfrogs (Lithobates catesbeianus).

Mammals show immune up-regulation and increased plasma and local (gastrointestinal tract) concentrations of some immunoregulatory hormones, such as corticosterone and melatonin, after feeding. However, little is known about the endocrine and immune modulation in the postprandial period of ectothermic animals. This study investigated the effects of feeding on endocrine and immune responses in the bullfrog (Lithobates catesbeianus). Frogs were fasted for 10 days and divided into two groups: fasted and fed with fish feed (5% of body mass). Blood and gastrointestinal tract tissues (stomach and intestine) were collected at 6, 24, 48, 96 and 168 h to measure neutrophil/lymphocyte ratio, plasma bacterial killing ability, phagocytosis of blood leukocytes, plasma corticosterone and melatonin, and stomach and intestine melatonin. Feeding increased plasma corticosterone at 24 h and decreased it at 168 h, and increased neutrophil/lymphocyte ratio at 6, 24 and 96 h. We also observed decreased bacterial killing ability 48 h after feeding. Stomach melatonin increased after 17 days of fasting. We show that feeding activates the hypothalamic-pituitary-interrenal axis and promotes transient immunosuppression, without stimulating an inflammatory response. Increased corticosterone may mobilize energy to support digestive processes and melatonin may protect the stomach during fasting. We conclude that feeding modulates secretion of immunoregulatory hormones, initially increasing plasma corticosterone levels, followed by a decrease at the end of meal digestion, and causes systemic immune cell redistribution, increasing neutrophil/lymphocyte ratio for almost the entire period of meal digestion in bullfrogs. Also, fasting modulates secretion of melatonin in the stomach.

Systemic hormonal and immune regulation induced by intraperitoneal LPS injection in bullfrogs (Lithobates catesbeianus).

Host's defense against external challenges activates an inflammatory response regulated by a set of chemical signals, including hormones. These immunomodulatory hormones, such as corticosterone, testosterone, and melatonin, trigger the systemic immune responses responsible for inflammatory assembly and resolution. This study aimed to investigate the effects of an immune challenge on endocrine and innate immune responses in the bullfrog (Lithobates catesbeianus). Adult males were intraperitoneally injected with lipopolysaccharide (LPS; 2 mg/kg) or saline, and blood samples were collected 6 and 24 h after injection for measurement of neutrophil/lymphocyte ratio, blood leukocyte phagocytosis, plasma bacterial killing ability, and plasma levels of corticosterone, melatonin, and testosterone. Our results showed LPS-induced increased neutrophil/lymphocyte ratio and leukocyte phagocytosis, and decreased melatonin and testosterone plasma levels, which were more pronounced 24 h after injection. Overall, we conclude that LPS intraperitoneal injection can activate the innate immune response and modulate the hormonal profile of the bullfrogs, with effects more pronounced 24 h than 6 h after treatment.

Thermal sensitivity of Bullfrog's immune response kept at different temperatures.

Climate change and emerging infectious diseases are often described as the main factors associated with the worldwide amphibian population decline. In this context, rising temperatures due to global warming might act as a chronic stressor for many amphibians, leading to immunosuppression. This study aimed to characterize the thermal sensitivity of the Bullfrog's (Lithobates catesbeianus) immune response and the effect of acclimation at different temperatures on it. Plasma bacterial killing ability (BKA) and phagocytosis activity of blood leukocytes were measured at different incubation temperatures (5-40°C) in individuals kept at 28°C and 34°C. First, all individuals were held under 28°C and sampled on the 16th day. Subsequently, one group was kept at 28°, and the other one was transferred to 34°C. Both groups were sampled at 83 and 106 days of maintenance. Plasma corticosterone (CORT) and testosterone (T) were assessed to evidence thermal stress and possible endocrine correlates of immune changes over time. The incubation temperature affected BKA both on animals kept at 28°C and 34°C, with maximum values at lower temperatures (5-20°C). Phagocytosis activity was constant over the range of assay temperatures. Immune and endocrine variables decreased over time in both thermal regimes, but frogs maintained at 34°C showed lower T and immunosuppression, evidencing stress response. Therefore, exposure to high temperatures might decrease immune function in bullfrogs due to chronic stress response and by exposition to temperatures of lower performance according to the thermal sensitivity curve, which might increase vulnerability to diseases in this anuran species.