Nevena Arsenović-Ranin

Quercetin Ameliorates Experimental Autoimmune Myocarditis in Rats

PURPOSE Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and post-myocarditis dilated cardiomyopathy. The pathogenesis of EAM has not been elucidated, but there is accumulating evidence that cytokines secreted from monocytes/macrophages and T cells play a crucial role in the induction and progression of disease. Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, which scavenge oxygen radicals and have anti-inflammatory activities. Having in mind in vivo beneficial effects of flavonoid quercetin in different animal models of immunoinflammatory diseases such as experimental autoimmune encephalomyelitis and adjuvant arthritis, on the one side, and its in vitro suppressive effect on production of tumor necrosis factor-alpha (TNF-α) on the other side, we investigated the effects of quercetin on EAM in rats. METHODS Myocarditis was induced in Dark Agouti (DA) rats by injection of porcine cardiac myosin and quercetin at doses of 10 or 20 mg/kg was orally administered from days 0 to 21 after induction of disease. The severity of myocarditis was evaluated by determination of heart weight/body weight ratio (Hw/Bw) and histopathological examination of hearts. The levels of cytokines (TNF-α, IL-12, IL-17 and IL-10) in serum and lymph node cells (LNC) culture supernatants were measured by ELISA. RESULTS The rats treated with 20 mg/kg of quercetin had significantly decreased incidence of EAM, Hw/Bw, macroscopic and microscopic scores of hearts. Further, in EAM rats treated with quercetin levels of TNF-α and IL-17 were significantly lower, while the level of IL-10 was significantly higher both in serum and culture supernatants of LNC stimulated with concanavalin A compared with vehicle-treated animals. CONCLUSIONS The present study suggests that quercetin ameliorates EAM, at least in part, by interfering production of proinflammatory (TNF-α and IL-17) and/or anti-inflammatory (IL-10) cytokines.

Age-associated changes in CD90 expression on thymocytes and in TCR-dependent stages of thymocyte maturation in male rats.

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRalphabeta and (ii) Thy-1, which is supposed to be a regulator of TCRalphabeta signalling, and thereby the thymocyte selection thresholds. Since an essential role for TCRalphabeta signalling in the development of CD4+25+T(reg)-cells was suggested, the frequency of these cells was also quantified. We demonstrated that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive. Furthermore, we revealed that TCRalphabeta-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression of Thy-1 on TCRalphabeta(low) thymocytes entering selection processes. The diminished frequency of the post-selection CD4+8+ double positive (DP) cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational transition from the DP TCRalphabeta(high) to the SP TCRalphabeta(high) developmental stage, which is followed by an increase in pre-migration proliferation of the mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4+25+T(reg)-cells in aged rats, thus providing a possible explanation for the increased frequency of autoimmune diseases in ageing.

Characterization of thymocyte phenotypic alterations induced by long-lasting β-adrenoceptor blockade in vivo and its effects on thymocyte proliferation and apoptosis

Adult male Wistar rats were subjected to propranolol (P, 0.40 mg/100 g/day) or saline (S) administration (controls) over 14 days. The expression of major differentiation molecules on thymocytes and Thy-1 (CD90) molecules, which are shown to adjust thymocyte sensitivity to TCRαβ signaling, was studied. In addition, the sensitivity of thymocytes to induction of apoptosis and concanavalin A (Con A) signaling was estimated. The thymocytes from P-treated (PT) rats exhibited an increased sensitivity to induction of apoptosis, as well as to Con A stimulation. Furthermore, P treatment produced changes in the distribution of thymocyte subsets suggesting that more cells passed positive selection and further differentiated into mature CD4+ or CD8+ single positive (SP) TCRαβhigh cells. These changes may, at least partly, be related to the markedly increased density of Thy-1 surface expression on TCRαβlow thymocytes from these rats. The increased frequency of cells expressing the CD4+25+ phenotype, which has been shown to be characteristic for regulatory cells in the thymus, may also indicate alterations in thymocyte selection following P treatment. Inasmuch as positive and negative selections play an important role in continuously reshaping the T-cell repertoire and maintaining tolerance, the hereby presented study suggests that pharmacological manipulations with β-AR signaling, or chemically evoked alterations in catecholamine release, may interfere with the regulation of thymocyte selection, and consequently with the immune response. (Mol Cell Biochem xxx: 1–13, 2005)

Role of ovarian hormones in age-associated thymic involution revisited

A commonly held view that ovarian hormones are causally involved in age-associated thymic involution has been recently challenged. In particular, their relevance in the progression of thymic involution has been disputed. To reassess this issue 10-month-old rats with well advanced thymic involutive changes were ovariectomized (Ovx), and after 1 month thymic cellularity, thymocyte development and levels of recent thymic emigrants (RTEs) were examined in peripheral blood and spleen. In addition, the distribution of major conventional and regulatory T-cell subsets was analyzed in the same peripheral lymphocyte compartments. Ovariectomy increased thymic weight and cellularity above the levels in both 10-month-old and age-matched controls indicating that ovarian hormone ablation not only prevented further progression of thymic involution, but also reversed it. The increased thymic cellularity was accompanied by altered thymocyte differentiation/maturation culminating in increased thymic output of naïve T cells as indicated by elevated levels of both CD4+ and CD8+ RTEs in peripheral blood and spleen. The changes in T-cell development produced: (i) a disproportional increase in cellularity across thymocyte subsets, so that relative proportions of cells at all maturational stages preceding the CD4+CD8+ T cell receptor (TCR)alphabeta(low) stage were reduced; the relative numbers of CD4+CD8+ TCRalphabeta(low) cells entering positive selection and their immediate CD4+CD8+ TCRalphabeta(high) descendents were increased, while those of the most mature CD4+CD8- and CD4-CD8+ TCRalphabeta(high) cells remained unaltered; (ii) enhanced cell proliferation across all thymocyte subsets and (iii) reduced apoptosis of cells within the CD4+CD8+ thymocyte subset. The augmented thymic output of naïve T cells in Ovx rats most likely reflected an early disinhibition of thymocyte development followed by increased positive/reduced negative selection, at least partly, due to raised thymocyte surface Thy-1 expression. The greater number of CD4+CD25+Foxp3+ cells in both thymus and peripheral blood suggested augmented thymic production of these cells. In addition, an increased CD4+/CD8+ cell ratio was found in the spleen of Ovx rats. Thus, ovarian hormone ablation led not only to increased diversity of the T-cell repertoire, but also to a new balance among distinct T-cell subsets in the periphery.

Age-associated plasticity of α1-adrenoceptor-mediated tuning of T-cell development

Alpha(1)-adrenoceptors (α(1)-ARs) are involved in neuro-thymic and thymic intercellular communications, and consequently modulation of T-cell development. Ageing is associated with a number of changes in noradrenergic neuro-effector transmission, and possibly intercellular noradrenaline (NA)-mediated communication resulting in altered responses of target cells to NA. Thus, in old animals an altered NA modulation of thymopoiesis via α(1)-ARs may be expected. To test this hypothesis, in old and young adult Wistar rats we examined: 1) thymic NA levels, density of noradrenergic innervation and NA synthesizing cells, as well as α(1)-AR expression, and 2) then the effects of 14-day-long treatment with the α(1)-AR blocker, urapidil, on thymocyte development. Overall, the first part of study suggested augmented NA signalling to thymic cells via α(1)-ARs due to increased NA availability and α(1)-AR thymocyte surface density in old rats. The second part of study supported this assumption. Namely, although in rats of both ages urapidil affected the same thymocyte developmental steps ultimately leading to changes in the relative number of the most mature single positive TCRαβ(high) thymocytes, its effects were generally more prominent in old animals. Following urapidil treatment, the percentages of CD4+CD8- cells, including those showing a regulatory CD4+CD25+RT6.1- phenotype, were increased, while CD4-CD8+ cells decreased. In old rats, an augmented thymic escape of immature CD4+CD8+ cells was also registered. In rats of both ages the thymic changes were accompanied by alterations in the proportions of major cell populations in the T-lymphocyte compartment of both peripheral blood and spleen, leading to an increase in the CD4+/CD8+ T-cell ratio. These alterations were also more pronounced in old rats. Moreover, in old rats following urapidil treatment the proportion of TCRαβ+cells in the periphery was slightly greater reflecting, most likely, partly enhanced thymic production of regulatory CD161+TCRαβ+cells. Thus, the study indirectly suggests an age-associated increase in the basal α(1)-AR-mediated inhibitory influence of NA on thymopoiesis.

Beneficial Effects of Dimethyl Fumarate on Experimental Autoimmune Myocarditis

BACKGROUND Fumaric acid esters (FAE) have been proven to be effective for the systemic treatment of psoriasis and multiple sclerosis, Th1 cell-mediated chronic inflammatory diseases, but their effect on autoimmune myocarditis has not yet been addressed. We investigated the effect of dimethyl fumarate (DMF) on myosin-induced experimental autoimmune myocarditis (EAM). METHODS Dark Agouti (DA) rats immunized with porcine cardiac myosin were orally treated with 5 and 15 mg/kg body weight (bw) DMF either from days 0-10 (early treatment groups) or from days 10-21 (late treatment groups) after induction of EAM. All rats were sacrificed on day 21 after immunization and hearts were evaluated macroscopically and microscopically. Levels of TNF-alpha and IL-10 in serum and lymph node cells culture supernatants were detected by ELISA. RESULTS Both early and late treatment with 15 mg/kg body weight (bw) DMF markedly reduced the severity of myocarditis by comparing the incidence, heart weight/bw ratio, macroscopic and microscopic scores, and number of OX-6+ cells in the myocardium. Further, levels of tumor necrosis factor-alpha (TNF-alpha) in serum and culture supernatants of lymph node cells stimulated with ConA or myosin were significantly lower in DMF-treated EAM animals compared with vehicle-treated EAM rats. There was no significant difference in serum levels of interleukin-10 between DMF- and vehicle-treated EAM rats. CONCLUSIONS These results show for the first time that DMF ameliorates experimental autoimmune myocarditis and may be acted, at least in part, by interfering with the production of TNF-alpha.

Neonatal testosterone imprinting affects thymus development and leads to phenotypic rejuvenation and masculinization of the peripheral blood T-cell compartment in adult female rats

Exposure of female rodents to testosterone in the critical neonatal period produces defeminization/masculinization of the hypothalamo-pituitary-gonadal (HPG) axis, i.e. neonatal androgenization and postpones axis maturation. To address the hypothesis that HPG axis signaling is involved in the programming of thymic maturation/involution and sexual differentiation we studied the impact of neonatal androgenization on thymic cellularity, development of effector and regulatory T cells, and phenotypic characteristics of peripheral blood T lymphocytes in adult rats. A single injection of testosterone on postnatal day 2 postponed thymic maturation/involution as revealed by organ hypercellularity, increased cellularity of the most mature (CD4+CD8- and CD4-CD8+) TCRalphabeta(high) thymocyte and both recent thymic emigrant (RTE) subsets and caused phenotypic defeminization/masculinization of thymic (decreased CD4+CD8-TCRalphabeta(high)/CD4-CD8+TCRalphabeta(high) cell ratio) and peripheral blood T-cell compartments (decreased CD4+RTE/CD8+RTE and CD4+/CD8+ cell ratio). In addition, neonatal androgenization increased the relative and absolute numbers of both CD4+CD25+Foxp3+ and natural killer (NK) regulatory T cells in peripheral blood. These findings, in conjunction with thymocyte overexpression of Thy-1 that is assumed to reduce negative selection affecting self-reactive cell generation, suggest a new relationship between self-reactive and regulatory T cells. In conclusion, our study provides additional evidence for a role of HPG signals (i.e. sex steroids and gonadotropins) in programming the kinetics of thymic maturation/involution and in establishing immunological sexual dimorphism.

Male rats develop more severe experimental autoimmune encephalomyelitis than female rats: Sexual dimorphism and diergism at the spinal cord level

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freunds adjuvant (CFA) exhibited lower incidence of the disease, but the maximal neurological deficit was greater in the animals that developed the disease. Consistently, at the peak of the disease greater number of reactivated CD4+CD134+CD45RC- T lymphocytes was retrieved from male rat spinal cord. Their microglia/macrophages were more activated and produced greater amount of prototypic proinflammatory cytokines in vitro. Additionally, oppositely to the expression of mRNAs for IL-12/p35, IL-10 and IL-27/p28, the expression of mRNA for IL-23/p19 was upregulated in male rat spinal cord mononuclear cells. Consequently, the IL-17+:IFN-γ+ cell ratio within T lymphocytes from their spinal cord was skewed towards IL-17+ cells. Within this subpopulation, the IL-17+IFN-γ+:IL-17+IL-10+ cell ratio was shifted towards IL-17+IFN-γ+ cells, which have prominent tissue damaging capacity. This was associated with an upregulated expression of mRNAs for IL-1β and IL-6, but downregulated TGF-β mRNA expression in male rat spinal cord mononuclear cells. The enhanced GM-CSF mRNA expression in these cells supported the greater pathogenicity of IL-17+ T lymphocytes infiltrating male spinal cord. In the inductive phase of the disease, contrary to the draining lymph node, in the spinal cord the frequency of CD134+ cells among CD4+ T lymphocytes and the frequency of IL-17+ cells among T lymphocytes were greater in male than in female rats. This most likely reflected an enhanced transmigration of mononuclear cells into the spinal cord (judging by the lesser spinal cord CXCL12 mRNA expression), the greater frequency of activated microglia/macrophages and the increased expression of mRNAs for Th17 polarizing cytokines in male rat spinal cord mononuclear cells. Collectively, the results showed cellular and molecular mechanisms underlying the target organ specific sexual dimorphism in the T lymphocyte-dependent immune/inflammatory response, and suggested a substantial role for the target organ in shaping the sexually dimorphic clinical outcome of EAE.

Age-related changes in spleen of Dark Agouti rats immunized for experimental autoimmune encephalomyelitis.

The study was undertaken considering age-related changes in susceptibility to experimental autoimmune encephalomyelitis (EAE) and a putative role of spleen in pathogenesis of this disease. The phenotypic and functional characteristics of T splenocytes were examined in young (3-month-old), middle-aged (8-month-old) and aged (26-month-old) Dark Agouti rats immunized for EAE with rat spinal cord in complete Freunds adjuvant. The rat susceptibility to EAE induction, as well as the number of activated CD4+CD134+ lymphocytes retrieved from their spinal cords progressively decreased with aging. To the contrary, in rats immunized for EAE the number of activated CD4+ splenocytes, i.e., CD4+CD134+, CD4+CD25+FoxP3- and CD4+CD40L+ cells, progressively increased with aging. This was associated with age-related increase in (i) CD4+ splenocyte surface expression of CD44, the molecule suggested to be involved in limiting emigration of encephalitogenic CD4+ cells from spleen into blood and (ii) frequency of regulatory T cells, including CD4+CD25+FoxP3+ cells, which are also shown to control encephalitogenic cell migration from spleen into the central nervous system. In favor of expansion of T-regulatory cell pool in aged rats was the greater concentration of IL-10 in unstimulated, Concanavalin A (ConA)- and myelin basic protein (MBP)-stimulated splenocyte cultures from aged rats compared with the corresponding cultures from young ones. Consistent with the age-related increase in the expression of CD44, which is shown to favor Th1 effector cell survival by interfering with CD95-mediated signaling, the frequency of apoptotic cells among CD4+ splenocytes, despite the greater frequency of CD95+ cells, was diminished in splenocyte cultures from aged compared with young rats. In addition, in control, as well as in ConA- and MBP-stimulated splenocyte cultures from aged rats, despite of impaired CD4+ cell proliferation, IFN-γ concentrations were greater than in corresponding cultures from young rats. This most likely reflected increased abundance of IFN-γ-producing cells in splenocyte cultures from aged compared with young rats. The diminished CD4+ cell proliferation in response to ConA and MBP in splenocyte cultures from aged compared with young rats could be, at least partly, associated with an enhanced splenic expression of iNOS mRNA in aged rats. Thus, the study suggests that age-associated changes leading to entrapping of activated CD4+ cells in the spleen could contribute to the restriction in development of EAE in aged rats.

Role of ovarian hormones in T-cell homeostasis: from the thymus to the periphery.

The study explored the putative role of ovarian hormones in the peripubertal remodelling of peripheral T-cell compartment. Ovariectomy at age of 1 month enhanced the peripubertal rise in CD4+ and CD8+ cell numbers in peripheral blood (PB) and spleen from 2-month-old rats. This reflected maintenance of thymopoietic efficiency at the prepubertal level (judging by numbers of the most mature CD4+ and CD8+ thymocytes and recent thymic emigrants) and alterations in T-cell survival/proliferation in the periphery. Compared with age-matched controls, the frequency of apoptotic cells among CD8+ peripheral blood lymphocytes (PBLs) and CD4+ and CD8+ splenocytes was diminished in ovariectomized (Ox) rats, at least partly, due to lower CD95 surface density. The diminished frequency of the apoptotic T splenocytes could also be associated with the rise in the amount of splenic IL-7 mRNA. Additionally, the latter finding was consistent with the augmented proliferation of CD4+ and CD8+ splenocytes. However, the enhanced proliferation of these cells could also be linked to the rise in IL-2 receptor surface density. This increase was related to the enhanced splenic TNF-α mRNA expression. Additionally, ovariectomy led to the phenotypic alterations in the major PBL and splenic T-cell subsets by diminishing/preventing the peripubertal changes in the frequency of cells at distinct stages of post-thymic differentiation/maturation (recent thymic emigrants, mature naïve and memory cells), and by decreasing the frequency of NKT cells within peripheral CD8+ subsets. In addition to numerical and phenotypic changes in T-cell compartment (due to the lack of ovarian hormone action at both the thymic and peripheral level), Ox rats exhibited a much larger delayed-type hypersensitivity (DTH) response compared with age-matched controls. This suggested the augmented T-cell-mediated immune response in Ox rats compared with aged-matched controls.