key: cord-0931701-b5ca580l authors: Brazão, Vânia; Santello, Fabricia Helena; Colato, Rafaela Pravato; Duarte, Andressa; Goulart, Amanda; Sampaio, Pedro Alexandre; Nardini, Viviani; Sorgi, Carlos Arterio; Faccioli, Lúcia Helena; do Prado, José Clóvis title: Melatonin down-regulates steroidal hormones, thymocyte apoptosis and inflammatory cytokines in middle-aged T. cruzi infected rats date: 2020-08-06 journal: Biochim Biophys Acta Mol Basis Dis DOI: 10.1016/j.bbadis.2020.165914 sha: 07224bbf85be6014f41c08ef9d41394777284f60 doc_id: 931701 cord_uid: b5ca580l Abstract Chagas disease, triggered by the flagellate protozoan Tryanosoma cruzi (T. cruzi) plays a potentially threat to historically non-endemic areas. Considerable evidence established that the immuno-endocrine balance could deeply influence the experimental T. cruzi progression inside the host’s body. A high-resolution multiple reaction monitoring approach (MRMHR) was used to study the influence of melatonin on adrenal and plasma steroidal hormones profile of T. cruzi infected Wistar rats. Young (5 weeks) and middle-aged (18 months) male Wistar rats received melatonin (5 mg/Kg, orally) during the acute Chagas disease. Corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, progesterone and melatonin concentration were evaluated. Interleukin-1 alpha and β (IL-1α and β), IL-6 and transforming growth factor beta (TGF-β) were also analyzed. Our results revealed an increased production of corticosterone, cortisone, cortisol and aldosterone in middle-aged control animals, thus confirming the aging effects on the steroidal hormone profile. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats. Melatonin treatment reduced the corticosterone, 11-DHC, cortisol, cortisone, aldosterone and progesterone in response to T. cruzi infection. Decreased IL-1 α and β concentrations were also found in melatonin treated middle-aged infected animals. Melatonin treated middle-aged control rats displayed reduced concentrations of TGF-β. Melatonin levels were significantly higher in all middle-aged rats treated animals. Reduced percentages of early and late thymocyte apoptosis was found for young and middle-aged melatonin supplemented rats. Finally, our results show a link between the therapeutic and biological effects of melatonin controlling steroidal hormones pathways as well as inflammatory mediators. Chagas disease is an anthropozoonosis from the Central and South American continent, caused by the protozoan flagellate Trypanosoma cruzi (T. cruzi) and considered a neglected parasitic illness which has spread from its original boundaries to historically non-endemic areas due to widespread migration 1,2 . Around 6-7 million subjects are infected with T. cruzi worldwide. According to the Bulletin of the World Health Organization the increased frequency of T. cruzi infected people in the United States, Europe and the Western Pacific Region is evidenced 2 . Chagas disease has become an important threat being considered an extremely debilitating illness, normally linked to poverty, affecting populations with low visibility and exerts a considerable impact on morbidity and mortality with more than 10 000 deaths annually in Latin America 3 Chagas disease has increased incidence in immunocompromised people and elderly population 4 . It is well established that immune cells share receptors for hormones and cytokines through the action of systemic and local regulatory mechanisms. When one of these systems is disturbed by pathogen invasion, the physiological profile of these interactions changes, triggering the release of proinflammatory cytokines and hormones. The immuno-endocrine balance can deeply influence the experimental T. cruzi progression inside the host's body 5 . Some available data also reveal that a deregulation of the hypothalamicpituitary-adrenal (HPA) axis during the acute T. cruzi infection induces an immunosuppression that is related to endocrine changes involving a circuit with regulatory properties in which cytokines and hormones produced by the HPA axis play essential role 6 . This immuno-endocrine cross-talk begins when HPA axis stimulation promotes the secretion of pituitary adrenocorticotropic hormone (ACTH) and adrenocortical glucocorticoids (GCs), triggered by the release pro-inflammatory cytokines like IL-6 or IL-1β into the circulation 7, 8 . Others data show that glucocorticoids J o u r n a l P r e -p r o o f drive changes in the mitochondrial biogenesis 9 interfering with the stress response. Although active glucocorticoids in the circulation are primarily derived from the adrenal gland as part of the HPA axis, they can also be produced from their inactive substrate 11-dehydrocorticosterone (11-DHC) by the enzyme 11β-HSD1 10 as well in other sites such as thymus, brain and intestinal tract 11 . Additionally, elevated corticosterone levels contributes to the aging process and age-related diseases 12 . Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic 13 signaling substance with vital role in adjusting the circadian rhythmicity 14, 15 and mitochondrial homeostasis 16 . The finding of melatonin synthesis in mitochondria 16 where it likely functions as an effective antioxidant 13, 16, 17 , anti-apoptotic 18 , anti-aging 19 , oncostatic 20 , immunomodulatory [21] [22] [23] , free radical scavenger 24 and progesterone) at tissue and systemic level by using a high-resolution multiple reaction monitoring (MRM HR ) -based mass spectrometry approach. The cytokine repertoire, including IL-1α and IL-1β, IL-6 and TGF-β were also evaluated during the development of acute T. cruzi infection. The molecular standards cortisol, cortisone, corticosterone, progesterone, aldosterone, cortisone-d8, corticosterone-d4 and formic acid were purchased from Male Wistar rats were infected with the Y strain of T. cruzi (1 x 10 5 blood trypomastigotes/animal, intraperitoneally). Melatonin treatment (5 mg/kg/day; orally; cruzi infected melatonin treated (MMI). After a short time animals were humanely anaesthetized with tribromoethanol 2.5% by administration of 0.1mL/ 10 g of body weight and decapitated (at 9 th dpi) for blood, serum and tissue collection for the performance of experimental protocols. Corticosterone and 11DHC from plasma and adrenal were quantified by mass Kyoto, Japan). Data were processed using PeakView and MultiQuant software. Serum samples were used for the detection IL-1β, IL-6, TGF-β and melatonin levels using two-site sandwich ELISA with monoclonal antibody. Standard curves were used for evaluating cytokine concentrations (pg/mL) read at 450nm and quantified by using an automated microplate ELISA reader (BIOTEK SYNERGY H1M). MILLIPLEX assay kit (Cat. # RECYTMAG-65K Millipore) and MAGPIX Multiplexing System (MilliporeSigma) was used for the detection of IL-1α. Graph Pad Prism version 5.0 was used to analyze all data (GraphPad Software, Inc., San Diego, CA, USA; one-way ANOVA with Bonferroni's post test) based on variance difference significance among groups. Significance was assured for p < 0.05 (mean ± standard error of the mean (SEM). Studies in rats have demonstrated that during T. cruzi infection proinflammatory mediators such as IL-1α and IL-6 trigger the stimulation of the HPA axis 6 The next step, the pro-inflammatory cytokines involved in the HPA regulation: IL-1α, IL-1β, IL-6 and TGF-β were evaluated. A significant increase in IL-1α ( Fig. 7A and B) concentrations was observed for infected middle-aged and young rats, when compared with the control ones. As expected, the immunosenescence process was linked to elevated levels of IL-1 β ( Fig. 7C and D) and IL-6 ( Fig. 8A and B) as found for all middle-aged rats as compared to juvenile ones. We further examine if melatonin's actions were related with alterations in the kinetics of cytokine output during the early T. cruzi infection. Our results demonstrated reduced IL-1α and IL-1β production, a key cytokine that has been entangled with chagasic cardiac hypertrophy, for middle-aged J o u r n a l P r e -p r o o f infected melatonin supplemented rats ( Fig. 7B and D) . Melatonin treated middle-aged rats displayed lower levels of IL-6, excepting the young ones ( Fig. 8A and B) . Reduced concentrations of TGF-β were triggered for young infected rats when compared to the untreated ones (Fig. 8D ). Melatonin treated middle-aged control groups displayed reduced concentrations of TGF-β (Fig. 8C) . Since, an important decay in endogenous melatonin synthesis occurs with aging, systemic levels of this hormone were quantified in young and middle-aged groups. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats compared to control animals (Fig 9) . Melatonin levels were significantly higher in all middle-aged rats (control or infected) supplemented with melatonin, as compared to untreated ones. The anti-apoptotic abilities of melatonin were evidenced in our study. Early and late thymocyte apoptosis from all middle-aged melatonin-treated animals were statistically reduced as compared to non-supplemented ones respectively (Fig 10A and B) . However, as shown in Fig. 10 , the percentages of early (A) and late thymocyte apoptosis (B) did not significantly decline among all young melatonin-treated animals. Significantly higher proportions of both early (A) and late apoptotic thymocytes (B) were displayed for all middle-aged animals, as compared to young counterparts ( Fig. 9) triggered by the ageing process. The percentage of viable thymocytes in all young and middle-aged melatonin treated infected rats, was statistically enhanced when compared to untreated counterparts (Fig.10C ). Oppositely, a significant drop in the proportion of viable thymocytes in middle-aged control animals was observed as compared to young groups (Fig. 10 ). A bulk of evidence has shown that altered steroidal hormone production might be related to a number of stress-induced disorders such as major depression, Currently is also known that a prolonged aldosterone treatment (between 4 and 5 weeks) triggers enhanced oxidative stress production and accounts for a proinflammatory phenotype. Although the exact role of melatonin on aldosterone production has remained controversial, we found that melatonin treatment decreased (2017) demonstrated that in lung tissue exposed to lipopolysaccharide, melatonin treatment downregulated the expression of TGF-β1 81 . Our findings constitute the first report of reduced serum IL-1β and IL-6 levels in middle-aged T. cruzi infected rats under melatonin therapy. Melatonin also exerts a negative influence on TGF-β production, with reduced levels for young infected and middle-aged control rats. In summary, for the first time our compelling data clearly showed the agerelated changes in adrenal and plasma steroidal hormones profiling as well cytokine production in Wistar rats confirming the efficiency of melatonin intervention in the regulation and signaling hormonal pathways and inflammatory mediators. and middle-aged melatonin infected (MMI). Results are shown as the means ± SEM of n= 5 to 6 rats. One-way ANOVA followed by Bonferroni's multiple comparison test was used to compare groups (*P < 0.05). Chronic Chagas Heart Disease Management: From Etiology to Cardiomyopathy Treatment Differential susceptibility to acute Trypanosoma cruzi infection in BALB/c and C57BL/6 mice is not associated with a distinct parasite load but cytokine abnormalities Could age and aging change the host response to systemic parasitic infections? A systematic review of preclinical evidence Thymus and aging: morphological, radiological, and functional overview Endogenous glucocorticoids cause thymus atrophy but are protective during acute Trypanosoma cruzi infection Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action Immune-neuro-endocrine interactions: facts and hypotheses Mitochondrial allostatic load puts the "gluc" back in glucocorticoids Will treating diabetes with 11β-HSD1 inhibitors affect the HPA axis? Extra-adrenal glucocorticoids and mineralocorticoids: evidence for local synthesis, regulation, and function Aging, glucocorticoids and developmental programming Melatonin as an antioxidant: under promises but over delivers Melatonin, the circadian multioscillator system and health: the need for detailed analyses of peripheral melatonin signaling Melatonin, hormone of darkness and more: occurrence, control mechanisms, actions and bioactive metabolites Mitochondria: the birth place, battle ground and the site of melatonin metabolism in cells Melatonin: an ancient molecule that makes oxygen metabolically tolerable Immunomodulatory properties and antiapoptotic effects of zinc and melatonin in an experimental model of chronic Chagas disease Melatonin and the theories of aging: a critical appraisal of melatonin's role in antiaging mechanisms Melatonin down-regulates MDM2 gene expression and enhances p53 acetylation in MCF-7 cells Melatonin: clinical relevance Human pineal physiology and functional significance of melatonin The role of melatonin in the cells of the innate immunity: a review Melatonin: a well-documented antioxidant with conditional prooxidant actions Aging, immunity and cancer The effect of age on thymic function Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells Pathogenic conversion of Foxp3+ T cells into TH17 cells in autoimmune arthritis Thymic emigration: conveyor belts or lucky dips? A high plasma concentration of TNF-alpha is associated with dementia in centenarians Marked increase with age of type 1 cytokines within memory and effector/cytotoxic CD8+ T cells in humans: a contribution to understand the relationship between inflammation and immunosenescence Cytokine serum profile in a group of Sicilian nonagenarians Astrocytic metabolic and inflammatory changes as a function of age Inhibition of IL-1, IL-6, and TNF-alpha in immune-mediated inflammatory diseases Isocitrate dehydrogenase 2 deficiency induces endothelial inflammation via p66sh-mediated mitochondrial oxidative stress Possible function of IL-6 and TNF as intraadrenal factors in the regulation of adrenal steroid secretion Neuroendocrine dysfunction in African trypanosomiasis. The role of cytokines Hypogonadism in Rhodesian sleeping sickness: evidence for acute and chronic dysfunction of the hypothalamic-pituitary-gonadal axis Hypothalamus-pituitaryadrenal axis during Trypanosoma cruzi acute infection in mice Pathophysiological role of the cytokine network in the anterior pituitary gland The thymus is a common target organ in infectious diseases Glucocorticoids exacerbate lipopolysaccharide-induced signaling in the frontal cortex and hippocampus in a dosedependent manner Dysregulated Network of Immune, Endocrine and Metabolic Markers is Associated to More Severe Human Chronic Chagas Cardiomyopathy A review of the molecular aspects of melatonin's anti-inflammatory actions: recent insights and new perspectives Effect of a combined treatment with growth hormone and melatonin in the cardiological aging on male SAMP8 mice Beneficial effect of melatonin treatment on inflammation, apoptosis and oxidative stress on pancreas of a senescence accelerated mice model Therapeutic effects of melatonin on heatstroke-induced multiple organ dysfunction syndrome in rats Melatonin attenuates airway inflammation via SIRT1 dependent inhibition of NLRP3 inflammasome and IL-1β in rats with COPD Melatonin suppresses fibrotic responses induced by cigarette smoke via downregulation of TGF-β1 FIGURE LEGENDS FIGURE 1: Corticosterone quantification in adrenal tissue (ng/adrenal gland) and serum (ng/mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue /mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue, (C-D)-Plasma samples, (E)-representative chromatogram retention time and CAD spectra for 11-DHC One-way ANOVA followed by Bonferroni's multiple comparison test were used to compare groups Cortisone quantification in adrenal tissue (ng/adrenal gland) and serum (ng/mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue Cortisol quantification in adrenal tissue (ng/adrenal gland) and serum (ng/mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue Aldosterone quantification in adrenal tissue (ng/adrenal gland) and serum (ng/mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue Progesterone quantification in adrenal tissue (ng/adrenal gland) and serum (ng/mL) samples evaluated by MRM HR method through liquid chromatographic tandem mass spectrometry (HPLC-MS/MS), from young and middle-aged Wistar rats, on the 9 th day post-infection of experimental T. cruzi infection, for the following groups: young control (YC), young melatonin control (YMC), young infected (YI), young melatonin infected (YMI), middle-aged control (MC), middle-aged melatonin control (MMC), middle-aged infected (MI) and middle-aged melatonin infected (MMI). (A-B)-Adrenal tissue %) of early apoptotic cells (annexin V+/PI−), (B) late apoptotic cells (annexin V+/PI+) (%), (C) cell viability of thymocytes (%) We hereby certify that it is an original publication and the manuscript has not been previously submitted or published elsewhere. VB, FS and RC: participated in the conception and design of the study. AD, AG, PS and VN: performed the analysis. CS, LF, JP and VB: helped in drafting of the manuscript. All authors have made substantial contributions and final approval of the conceptions, drafting, and final version. None of the authors has any potential financial conflict of interest related to this manuscript.J o u r n a l P r e -p r o o f