Catecholaminergic signalling through thymic nerve fibres, thymocytes and stromal cells is dependent on both circulating and locally synthesized glucocorticoids

Glucocorticoids have been shown to modulate the expression of noradrenaline metabolizing enzymes and beta(2)- and alpha(1B)-adrenoceptors in a tissue- and cell- specific manner. In the thymus, apart from extensive sympathetic innervation, a regulatory network has been identified that encompasses catecholamine-containing non-lymphoid and lymphoid cells. We examined a putative role of adrenal- and thymus-derived glucocorticoids in modulation of rat thymic noradrenaline levels and adrenoceptor expression. Seven days postadrenalectomy, the thymic levels of mRNAs encoding tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase-A and, consequently, noradrenaline were decreased. Catecholamine content was diminished in autofluorescent nerve fibres (judging by the intensity of fluorescence) and thymocytes (considering HPLC measurements of noradrenaline and the frequency of tyrosine hydroxylase-positive cells), while it remained unaltered in non-lymphoid autofluorescent cells. In addition, adrenalectomy diminished the thymocyte expression of beta(2)- and alpha(1B)-adrenoceptors at both mRNA and protein levels. Administration of ketoconazole (an inhibitor of glucocorticoid synthesis/action; 25 mg kg(-1) day(-1), s.c.) to glucocorticoid-deprived rats increased the thymic levels of tyrosine hydroxylase, dopamine beta-hydroxylase and, consequently, noradrenaline. The increased intensity of the autofluorescent cell fluorescence in ketoconazole-treated rats indicated an increase in their catecholamine content, and suggested differential glucocorticoid-mediated regulation of catecholamines in thymic lymphoid and non-lymphoid cells. In addition, ketoconazole increased the thymocyte expression of alpha(1B)-adrenoceptors. Thus, this study indicates that in the thymus, as in some other tissues, glucocorticoids not only act in concert with cateholamines, but they may modulate catecholamine action by tuning thymic catecholamine metabolism and adrenoceptor expression in a cell-specific manner. Additionally, the study indicates a role of thymus-derived glucocorticoids in this modulation

GM-CSF-Producing Th Cells in Rats Sensitive and Resistant to Experimental Autoimmune Encephalomyelitis

Given that granulocyte macrophage colony-stimulating factor (GM-CSF) is identified as the key factor to endow auto-reactive Th cells with the potential to induce neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models, the frequency and phenotype of GM-CSF-producing (GM-CSF+) Th cells in draining lymph nodes (dLNs) and spinal cord (SC) of Albino Oxford (AO) and Dark Agouti (DA) rats immunized for EAE were examined. The generation of neuroantigen-specific GM-CSF+ Th lymphocytes was impaired in dLNs of AO rats (relatively resistant to EAE induction) compared with their DA counterparts (susceptible to EAE) reflecting impaired CD4+ lymphocyte proliferation and less supportive of GM-CSF+ Th cell differentiation dLN cytokine microenvironment. Immunophenotyping of GM-CSF+ Th cells showed their phenotypic heterogeneity in both strains and revealed lower frequency of IL-17+ IFN-gamma+, IL-17+ IFN-gamma-, and IL-17-IFN-gamma+ cells accompanied by higher frequency of IL-17-IFN-gamma- cells among them in AO than in DA rats. Compared with DA, in AO rats was also found (i) slightly lower surface density of CCR2 (drives accumulation of highly pathogenic GM-CSF+ IFN-gamma+ Th17 cells in SC) on GM-CSF+ IFN-gamma+ Th17 lymphocytes from dLNs, and (ii) diminished CCL2 mRNA expression in SC tissue, suggesting their impaired migration into the SC. Moreover, dLN and SC cytokine environments in AO rats were shown to be less supportive of GM-CSF+ IFN-gamma+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1 beta, IL-6 and IL-23/p19). In accordance with the (i) lower frequency of GM-CSF+ Th cells in dLNs and SC of AO rats and their lower GM-CSF production, and (ii) impaired CCL2 expression in the SC tissue, the proportion of proinflammatory monocytes among peripheral blood cells and their progeny (CD45(hi) cells) among the SC CD11b+ cells were reduced in AO compared with DA rats. Collectively, the results indicate that the strain specificities in efficacy of several mechanisms controlling (auto) reactive CD4+ lymphocyte expansion/differentiation into the cells with pathogenic phenotype and migration of the latter to the SC contribute to AO rat resistance to EAE

Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action

In the present study, upon showing sexual dimorphism in dimethyl fumarate (DMF) efficacy to moderate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats, cellular and molecular substrate of this dimorphism was explored. In rats of both sexes, DMF administration from the day of immunization attenuated EAE severity, but this effect was more prominent in males leading to loss of the sexual dimorphism observed in vehicle-administered controls. Consistently, in male rats, DMF was more efficient in diminishing the number of CD4+ T lymphocytes infiltrating spinal cord (SC) and their reactivation, the number of IL-17+ T lymphocytes and particularly cellularity of their highly pathogenic IFN-gamma+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCR alpha beta- microglia/proinflammatory monocyte progeny, substantiated in a more prominent increase in the frequency of anti-inflammatory phygocyting CD163+ cells and the cells expressing high surface levels of immunoregulatory CD83 molecule (associated with apoptotic cells phagocytosis and implicated in downregulation of CD4+ T lymphocyte reactivation) among CD11b+CD45+TCR alpha beta- cells in male rat SC. These changes were associated with greater increase in the nuclear factor (erythroid-derived 2)-like 2 expression in male rats administered with DMF. In accordance with the previous findings, DMF diminished reactive nitrogen and oxygen species generation and consistently, SC level of advanced oxidation protein products, to the greater extent in male rats. Overall, our study indicates sex-specificity in the sensitivity of DMF cellular and molecular targets and encourages sex-based clinical research to define significance of sex for action of therapeutic agents moderating autoimmune neuroinflammation-/oxidative stress-related nervous tissue damage

Aging diminishes the resistance of AO rats to EAE: putative role of enhanced generation of GM-CSF Expressing CD4+T cells in aged rats

Background: Aging influences immune response and susceptibility to EAE in a strain specific manner. The study was designed to examine influence of aging on EAE induction in Albino Oxford (AO) rats. Results: Differently from 3-month-old (young) rats, which were resistant to EAE induction, the majority of aged (24-26-month-old) rats developed mild chronic form of EAE. On 16th day post-immunization, when in aged rats the neurological deficit reached plateau, more mononuclear cells, including CD4+ T lymphocytes was retrieved from spinal cord of aged than young rats. The frequencies of IL-17+ and GM-CSF+ cells within spinal cord infiltrating CD4+ lymphocytes were greater in aged rats. To their increased frequency contributed the expansion of GM-CSF + IL-17 + IFN-gamma+ cells, which are highly pathogenic in mice. The expression of the cytokines (IL-1 beta and IL-23/p19) driving GM-CSF + IL-17 + IFN-gamma + cell differentiation in mice was also augmented in aged rat spinal cord mononuclear cells. Additionally, in aged rat spinal cord the expansion of GM-CSF + IL-17-IFN-gamma- CD4+ T lymphocytes was found. Consistently, the expression of mRNAs for IL-3, the cytokine exhibiting the same expression pattern as GM-CSF, and IL-7, the cytokine driving differentiation of GM-CSF + IL-17-IFN-gamma- CD4 + lymphocytes in mice, was upregulated in aged rat spinal cord mononuclear cells, and the tissue, respectively. This was in accordance with the enhanced generation of the brain antigen-specific GM-CSF+ CD4+ lymphocytes in aged rat draining lymph nodes, as suggested by (i) the higher frequency of GM-CSF+ cells (reflecting the expansion of IL-17-IFN-gamma- cells) within their CD4+ lymphocytes and (ii) the upregulated GM-CSF and IL-3 mRNA expression in fresh CD4+ lymphocytes and MBP-stimulated draining lymph node cells and IL-7 mRNA in lymph node tissue from aged rats. In agreement with the upregulated GM-CSF expression in aged rats, strikingly more CD11b + CD45(int) (activated microglia) and CD45(hi) (mainly proinflammatory dendritic cells and macrophages) cells was retrieved from aged than young rat spinal cord. Besides, expression of mRNA for SOCS1, a negative regulator of proinflammatory cytokine expression in innate immunity cells, was downregulated in aged rat spinal cord mononuclear cells. Conclusions: The study revealed that aging may overcome genetic resistance to EAE, and indicated the cellular and molecular mechanisms contributing to this phenomenon in AO rats

Sex differences in the pathogenesis of experimental autoimmune encephalomyelitis in the rat

Multipla skleroza (MS) se, kao i većina drugih autoimunskih bolesti, češće javlja kod žena nego kod muškaraca. Polni dimorfizam je zapažen i u kliničkom ispoljavanju MS-e. Kod muškaraca se bolest javlja kasnije, motorni ispadi su teži i primarno progresivni tok češći nego kod žena. Eksperimentalni autoimunski encefalomijelitis (EAE) je animalni model MS-e, koji se kod osetljivih oglednih životinja izaziva različitim indukcionim protokolima. Podaci vezani za polni dimorfizam u kliničkoj prezentaciji EAE-a su relativno oskudni, a postojeći inkonzistentni, pre svega zbog genetskih razlika između korišćenih životinjskih vrsta i sojeva, ali i varijacija u indukcionim protokolima. Hronobiološko starenje organizma uključuje i promene u imunskom sistemu koje karakteriše značajan porast autoimunskih fenomena. Uprkos tome, incidenca mnogih autoimunskih bolesti, uključujući i MS-u, se smanjuje kod starih. Podaci o osetljivosti starih oglednih životinja na indukciju EAE-a su heterogeni, u zavisnosti od vrste i soja oglednih životinja, kao i od modela EAE-a. Mehanizmi koji stoje u osnovi manje incidence autoimunskih bolesti kod starih jedinki nisu u potpunosti razjašnjeni. Još su manje poznati mehanizmi polnog dimorfizma u razvoju ovih bolesti kod starih životinja, a posebno u čemu se oni razlikuju u odnosu na mehanizme odgovorne za ovaj fenomen kod mladih životinja. Ciljevi ove doktorske disertacije su bili da se: 1) ispitaju polne razlike u kliničkim parametrima indukovane autoimunske neuroinflamacije, kao važne patogenetske komponente MS-e, na modelu aktivnog EAE-a, kod mladih adultnih (uzrast 3 meseca) i starih (uzrast 22-26 meseci) pacova Dark Agouti soja i 2) identifikuju ćelijski i molekularni mehanizmi odgovorni za uočene polne razlike. Dobijeni rezultati su pokazali postojanje polnog dimorfizma u incidenci i težini EAE-a i kod mladih i kod starih pacova. Incidenca EAE-a je kod pacova oba uzrasta bila manja kod mužjaka nego kod ženki, s tim što su mladi mužjaci, za razliku od starih, imali teži neurološki deficit u odnosu na ženke odgovarajućeg uzrasta...Multiple sclerosis (MS) is one of the most common organ-specific autoimmune diseases of the central nervous system. As in other autoimmune diseases, the prevalence of MS is higher in women than in men. The clinical manifestations of MS are also sexually dimorphic. Men exhibit later onset of the disease, more severe motor symptoms and primary progressive course more often than women. Experimental autoimmune encephalomyelitis (EAE) is an animal model induced in susceptible strains of animals. Data on sexual dimorphism in the clinical presentation of EAE are limited and inconsistent, reflecting, most likely, the differences in the genetic background of the experimental animals and the induction protocols. Chronobiological ageing of the organism is accompanied by ageing of the immune system. Immunosenescence is characterized by an increase in autoimmune phenomena. However, despite this phenomenon, the incidence of many autoimmune diseases, including MS, declines with ageing. Data on the influence of aging on the incidence and severity EAE are inconsistent. Additionally, data on sex differences in the clinical presentation of EAE in aged animals are extremely limited. The aim of the study was to 1) investigate sex differences in the incidence and severity of autoimmune neuroinflammation, an important pathogenetic component of MS, on an active EAE model in 3-month-old (young adult) and 22- 26-month-old (aged) Dark Agouti rats and 2) identify the cellular and molecular mechanisms behind the observed sex differences. Irrespective of age, the incidence of EAE was lower in male than in age-matched female rats. However, contrary to aged male rats, young male rats, which developed clinically manifested disease, exhibited more severe motor deficit than the age-matched female rats. Irrespective of age, at the peak of EAE, the greater mean maximal score was associated with: (i) greater number of overall and reactivated CD4+ T cells isolated from spinal cord (SC); (ii) upregulated expression of mRNA for CD4+ T helper (Th)17 polarizing cytokines (IL-6, IL-1β, IL-23/subunit p19) in SC mononuclear cells and, consequently, greater percentage of Th17 cells among the T-lymphocytes and (iii) greater activation of myeloid cells (according to the mean fluorescence intensity of CD45 and CD11b molecules on the surface of these cells), accompanied by upregulated expression mRNA for TNF-α and iNOS in SC mononuclear cells..

Sex differences in the pathogenesis of experimental autoimmune encephalomyelitis in the rat

Multipla skleroza (MS) se, kao i većina drugih autoimunskih bolesti, češće javlja kod žena nego kod muškaraca. Polni dimorfizam je zapažen i u kliničkom ispoljavanju MS-e. Kod muškaraca se bolest javlja kasnije, motorni ispadi su teži i primarno progresivni tok češći nego kod žena. Eksperimentalni autoimunski encefalomijelitis (EAE) je animalni model MS-e, koji se kod osetljivih oglednih životinja izaziva različitim indukcionim protokolima. Podaci vezani za polni dimorfizam u kliničkoj prezentaciji EAE-a su relativno oskudni, a postojeći inkonzistentni, pre svega zbog genetskih razlika između korišćenih životinjskih vrsta i sojeva, ali i varijacija u indukcionim protokolima. Hronobiološko starenje organizma uključuje i promene u imunskom sistemu koje karakteriše značajan porast autoimunskih fenomena. Uprkos tome, incidenca mnogih autoimunskih bolesti, uključujući i MS-u, se smanjuje kod starih. Podaci o osetljivosti starih oglednih životinja na indukciju EAE-a su heterogeni, u zavisnosti od vrste i soja oglednih životinja, kao i od modela EAE-a. Mehanizmi koji stoje u osnovi manje incidence autoimunskih bolesti kod starih jedinki nisu u potpunosti razjašnjeni. Još su manje poznati mehanizmi polnog dimorfizma u razvoju ovih bolesti kod starih životinja, a posebno u čemu se oni razlikuju u odnosu na mehanizme odgovorne za ovaj fenomen kod mladih životinja. Ciljevi ove doktorske disertacije su bili da se: 1) ispitaju polne razlike u kliničkim parametrima indukovane autoimunske neuroinflamacije, kao važne patogenetske komponente MS-e, na modelu aktivnog EAE-a, kod mladih adultnih (uzrast 3 meseca) i starih (uzrast 22-26 meseci) pacova Dark Agouti soja i 2) identifikuju ćelijski i molekularni mehanizmi odgovorni za uočene polne razlike. Dobijeni rezultati su pokazali postojanje polnog dimorfizma u incidenci i težini EAE-a i kod mladih i kod starih pacova. Incidenca EAE-a je kod pacova oba uzrasta bila manja kod mužjaka nego kod ženki, s tim što su mladi mužjaci, za razliku od starih, imali teži neurološki deficit u odnosu na ženke odgovarajućeg uzrasta...Multiple sclerosis (MS) is one of the most common organ-specific autoimmune diseases of the central nervous system. As in other autoimmune diseases, the prevalence of MS is higher in women than in men. The clinical manifestations of MS are also sexually dimorphic. Men exhibit later onset of the disease, more severe motor symptoms and primary progressive course more often than women. Experimental autoimmune encephalomyelitis (EAE) is an animal model induced in susceptible strains of animals. Data on sexual dimorphism in the clinical presentation of EAE are limited and inconsistent, reflecting, most likely, the differences in the genetic background of the experimental animals and the induction protocols. Chronobiological ageing of the organism is accompanied by ageing of the immune system. Immunosenescence is characterized by an increase in autoimmune phenomena. However, despite this phenomenon, the incidence of many autoimmune diseases, including MS, declines with ageing. Data on the influence of aging on the incidence and severity EAE are inconsistent. Additionally, data on sex differences in the clinical presentation of EAE in aged animals are extremely limited. The aim of the study was to 1) investigate sex differences in the incidence and severity of autoimmune neuroinflammation, an important pathogenetic component of MS, on an active EAE model in 3-month-old (young adult) and 22- 26-month-old (aged) Dark Agouti rats and 2) identify the cellular and molecular mechanisms behind the observed sex differences. Irrespective of age, the incidence of EAE was lower in male than in age-matched female rats. However, contrary to aged male rats, young male rats, which developed clinically manifested disease, exhibited more severe motor deficit than the age-matched female rats. Irrespective of age, at the peak of EAE, the greater mean maximal score was associated with: (i) greater number of overall and reactivated CD4+ T cells isolated from spinal cord (SC); (ii) upregulated expression of mRNA for CD4+ T helper (Th)17 polarizing cytokines (IL-6, IL-1β, IL-23/subunit p19) in SC mononuclear cells and, consequently, greater percentage of Th17 cells among the T-lymphocytes and (iii) greater activation of myeloid cells (according to the mean fluorescence intensity of CD45 and CD11b molecules on the surface of these cells), accompanied by upregulated expression mRNA for TNF-α and iNOS in SC mononuclear cells..

Supplementary Material for: Androgens Contribute to Age-Associated Changes in Peripheral T-Cell Homeostasis Acting in a Thymus-Independent Way

<b><i>Objective:</i></b> Considering a causal role of androgens in thymic involution, age-related remodeling of peripheral T-cell compartments in the absence of testicular hormones was evaluated. <b><i>Methods:</i></b> Rats were orchidectomized (ORX) at the age of 1 month, and T-peripheral blood lymphocytes (PBLs) and splenocytes from young (75-day-old) and aged (24-month-old) rats were examined for differentiation/activation and immunoregulatory marker expression. <b><i>Results:</i></b> In ORX rats, following the initial rise, the counts of CD4+ and CD8+ PBLs diminished with aging. This reflected the decline in thymic export as shown by recent thymic emigrant (RTE) enumeration. Orchidectomy increased the count of both of the major T-splenocyte subsets in young rats, and they (differently from controls) remained stable with aging. The CD4+:CD8+ T-splenocyte ratio in ORX rats shifted towards CD4+ cells compared to age-matched controls. Although in the major T-cell subsets in the blood and spleen from aged ORX rats the numbers of RTEs were comparable to the corresponding values in age-matched controls, the numbers of mature naïve and memory/activated cells substantially differed. Compared with age-matched controls, in aged ORX rats the numbers of CD4+ mature naïve PBLs and splenocytes were reduced, whereas those of CD4+ memory/activated cells (predictive of early mortality) were increased. Additionally, in spleens from aged ORX rats, despite unaltered thymic export, CD4+CD25+FoxP3+ and natural killer T cell counts were greater than in age-matched controls. <b><i>Conclusion:</i></b> (i) Age-related decline in thymopoietic efficacy is not dependent on androgen presence, and (ii) androgens are involved in the maintenance of peripheral T-cell (particularly CD4+ cell) homeostasis during aging

Sex Difference in Oxidative Stress Parameters in Spinal Cord of Rats with Experimental Autoimmune Encephalomyelitis: Relation to Neurological Deficit

The study examined (a) whether there is sex difference in spinal cord and plasma oxidative stress profiles in Dark Agouti rats immunised for experimental autoimmune encephalomyelitis (EAE), the principal experimental model of multiple sclerosis, and (b) whether there is correlation between the oxidative stress in spinal cord and neurological deficit. Regardless of rat sex, with the disease development xanthine oxidase (XO) activity and inducible nitric oxide synthase (iNOS) mRNA expression increased in spinal cord, whereas glutathione levels decreased. This was accompanied by the rise in spinal cord malondialdehyde level. On the other hand, with EAE development superoxide dismutase (SOD) activity decreased, while O-2 (-) concentration increased only in spinal cord of male rats. Consequently, SOD activity was lower, whereas O-2 (-) concentration was higher in spinal cord of male rats with clinically manifested EAE. XO activity and iNOS mRNA expression were also elevated in their spinal cord. Consistently, in the effector phase of EAE the concentration of advanced oxidation protein product (AOPP) was higher in spinal cord of male rats, which exhibit more severe neurological deficit than their female counterparts. In as much as data obtained in the experimental models could be translated to humans, the findings may be relevant for designing sex-specific antioxidant therapeutic strategies. Furthermore, the study indicated that the increased pro-oxidant-antioxidant balance in plasma may be an early indicator of EAE development. Moreover, it showed that plasma AOPP level may indicate not only actual activity of the disease, but also serve to predict severity of its course

Androgens Contribute to Age-Associated Changes in Peripheral T-Cell Homeostasis Acting in a Thymus-Independent Way

Objective: Considering a causal role of androgens in thymic involution, age-related remodeling of peripheral T-cell compartments in the absence of testicular hormones was evaluated. Methods: Rats were orchidectomized (ORX) at the age of 1 month, and T-peripheral blood lymphocytes (PBLs) and splenocytes from young (75-day-old) and aged (24-month-old) rats were examined for differentiation/activation and immunoregulatory marker expression. Results: In ORX rats, following the initial rise, the counts of CD4+ and CD8+ PBLs diminished with aging. This reflected the decline in thymic export as shown by recent thymic emigrant (RTE) enumeration. Orchidectomy increased the count of both of the major T-splenocyte subsets in young rats, and they (differently from controls) remained stable with aging. The CD4+:CD8+ T-splenocyte ratio in ORX rats shifted towards CD4+ cells compared to age-matched controls. Although in the major T-cell subsets in the blood and spleen from aged ORX rats the numbers of RTEs were comparable to the corresponding values in age-matched controls, the numbers of mature naive and memory/activated cells substantially differed. Compared with age-matched controls, in aged ORX rats the numbers of CD4+ mature naive PBLs and splenocytes were reduced, whereas those of CD4+ memory/activated cells (predictive of early mortality) were increased. Additionally, in spleens from aged ORX rats, despite unaltered thymic export, CD4+CD25+FoxP3+ and natural killer T cell counts were greater than in age-matched controls. Conclusion: (i) Age-related decline in thymopoietic efficacy is not dependent on androgen presence, and (ii) androgens are involved in the maintenance of peripheral T-cell (particularly CD4+ cell) homeostasis during aging. (C) 2014 S. Karger AG, Base