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Our previous studies showed more severe experimental autoimmune encephalomyelitis (EAE) in male compared with female adult rats, and moderating effect of propranolol-induced β-adrenoceptor blockade on EAE in females, the effect associated with transcriptional stimulation of Nrf2/HO-1 axis in spinal cord microglia. This study examined putative sexual dimorphism in propranolol action on EAE severity. Propranolol treatment beginning from the onset of clinical EAE mitigated EAE severity in rats of both sexes, but to a greater extent in males exhibiting higher noradrenaline levels and myeloid cell β 2 -adrenoceptor expression in spinal cord. This correlated with more prominent stimulatory effects of propranolol not only on CX3CL1/CX3CR1/Nrf2/HO-1 cascade, but also on Stat3/Socs3 signaling axis in spinal cord microglia/myeloid cells (mirrored in the decreased Stat3 and the increased Socs3 expression) from male rats compared with their female counterparts. Propranolol diminished the frequency of activated cells among microglia, increased their phagocyting/endocyting capacity, and shifted cytokine secretory profile of microglia/blood-borne myeloid cells towards an anti-inflammatory/neuroprotective phenotype. Additionally, it downregulated the expression of chemokines (CCL2, CCL19/21) driving T-cell/monocyte trafficking into spinal cord. Consequently, in propranolol-treated rats fewer activated CD4+ T cells and IL-17+ T cells, including CD4+IL17+ cells coexpressing IFN-γ/GM-CSF, were recovered from spinal cord of propranolol-treated rats compared with sex-matched saline-injected controls. All the effects of propranolol were more prominent in males. The study as a whole disclosed that sexual dimorphism in multiple molecular mechanisms implicated in EAE development may be responsible for greater severity of EAE in male rats and sexually dimorphic action of substances affecting them. Propranolol moderated EAE severity more effectively in male rats, exhibiting greater spinal cord noradrenaline (NA) levels and myeloid cell β 2 -adrenoceptor (β 2 -AR) expression than females. Propranolol affected CX3CR1/Nrf2/HO-1 and Stat3/Socs3 signaling axes in myeloid cells, favored their anti-inflammatory/neuroprotective phenotype and, consequently, reduced Th cell reactivation and differentiation into highly pathogenic IL-17/IFN-γ/GM-CSF-producing cells., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This review summarizes recent findings related to the role of the sympathetic nervous system (SNS) in pathogenesis of multiple sclerosis (MS) and its commonly used experimental model - experimental autoimmune encephalomyelitis (EAE). They indicate that noradrenaline, the key end-point mediator of the SNS, acting through β-adrenoceptor, has a contributory role in the early stages of MS/EAE development. This stage is characterized by the SNS hyperactivity (increased release of noradrenaline) reflecting the net effect of different factors, such as the disease-associated inflammation, stress, vitamin D hypovitaminosis, Epstein-Barr virus infection and dysbiosis. Thus, the administration of propranolol, a non-selective β-adrenoceptor blocker, readily crossing the blood-brain barrier, to experimental rats before the autoimmune challenge and in the early (preclinical/prodromal) phase of the disease mitigates EAE severity. This phenomenon has been ascribed to the alleviation of neuroinflammation (due to attenuation of primarily microglial activation/proinflammatory functions) and the diminution of the magnitude of the primary CD4+ T-cell autoimmune response (the effect associated with impaired autoantigen uptake by antigen presenting cells and their migration into draining lymph nodes). The former is partly related to breaking of the catecholamine-dependent self-amplifying microglial feed-forward loop and the positive feedback loop between microglia and the SNS, leading to down-regulation of the SNS hyperactivity and its enhancing influence on microglial activation/proinflammatory functions and the magnitude of autoimmune response. The effects of propranolol are shown to be more prominent in male EAE animals, the phenomenon important as males (like men) are likely to develop clinically more severe disease. Thus, these findings could serve as a firm scientific background for formulation of a new sex-specific immune-intervention strategy for the early phases of MS (characterized by the SNS hyperactivity) exploiting anti-(neuro)inflammatory and immunomodulatory properties of propranolol and other relatively cheap and safe adrenergic drugs with similar therapeutic profile., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Considering significance of mechanisms of central tolerance for development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), and suppressive influence of circulating proinflammatory cytokines and alterations in brain-thymus communication, characteristic for the central nervous system (CNS) autoimmune diseases, on thymopoiesis, the study interogated putative strain-based thymus-related specificities relevant for the opposite effects of ageing on susceptibility of Dark Agouti (DA) and Albino Oxford (AO) rats to EAE., Methods: Quantitative and qualitative changes in thymopoiesis including underlying mechanisms were examined using flow cytometry and RT-qPCR quantification of mRNAs for molecules relevant for integrity of stroma and T-cell development, respectively., Results: With ageing, differently from DA rats, in AO rats the surface density of CD90, a negative regulator of selection threshold, on thymocytes undergoing lineage commitment was upregulated (consistent with TGF-β expression downregulation), whereas the generation of natural CD4+CD25+Foxp3+ regulatory T cells (nTregs) was impaired reflecting differences in thymic expression of cytokines supporting their development. Additionally, specifically in old AO rats, in whom EAE development depends on IL-17-producing CD8+ T cells, their thymic differentiation was augmented, reflecting augmented thymic IL-4 expression. In turn, differently from old DA rats developing self-limiting EAE, in age-matched AO rats developing EAE of prolonged duration, EAE development led to impaired generation of nTregs and accumulation of proinflammatory, cytotoxic CD28-CD4+ T cells in the periphery., Discussion: The study indicates that strain differences in age-related changes in the efficacy of central tolerance, in addition to enhanced thymic generation of CD8+ T cells prone to differentiate into IL-17-producing cells, could partly explain the opposite effect of ageing on DA and AO rat susceptibility to EAE induction. Additionally, it suggested that EAE development leading to a less efficient thymic output of CD4+ cells and nTregs in old AO rats than their DA counterparts could contribute to prolonged EAE duration in AO compared with DA rats., Conclusion: The study warns to caution when designing therapeutic interventions to enhance thymic activity in genetically diverse populations, e.g., humans, and interpreting their outcomes. Furthermore, it indicates that CNS autoimmune pathology may additionally worsen thymic involution and age-related immune changes., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

The incidence of multiple sclerosis (MS) and susceptibility of animals to experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, decrease with aging. Generally, autoimmune diseases develop as the ultimate outcome of an imbalance between damaging immune responses against self and regulatory immune responses (keeping the former under control). Thus, in this review the age-related changes possibly underlying this balance were discussed. Specifically, considering the central role of T cells in MS/EAE, the impact of aging on overall functional capacity (reflecting both overall count and individual functional cell properties) of self-reactive conventional T cells (Tcons) and FoxP3+ regulatory T cells (Tregs), as the most potent immunoregulatory/suppressive cells, was analyzed, as well. The analysis encompasses three distinct compartments: thymus (the primary lymphoid organ responsible for the elimination of self-reactive T cells - negative selection and the generation of Tregs, compensating for imperfections of the negative selection), peripheral blood/lymphoid tissues ("afferent" compartment), and brain/spinal cord tissues ("target" compartment). Given that the incidence of MS and susceptibility of animals to EAE are greater in women/females than in age-matched men/males, sex as independent variable was also considered. In conclusion, with aging, sex-specific alterations in the balance of self-reactive Tcons/Tregs are likely to occur not only in the thymus/"afferent" compartment, but also in the "target" compartment, reflecting multifaceted changes in both T-cell types. Their in depth understanding is important not only for envisaging effects of aging, but also for designing interventions to slow-down aging without any adverse effect on incidence of autoimmune diseases., (Copyright © 2021. Published by Elsevier B.V.)

Sympathetic dysfunction is suggested to contribute to development of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) alike. Considering important role of microglia in development/resolution of neuroinflammation, contribution of noradrenaline, the key sympathetic end-point mediator, in modulation of microglial phenotypic and functional properties in rat EAE model was examined. The study showed that noradrenaline acting in neurocrine and autocrine/paracrine way might influence microglia during EAE. Propranolol treatment over the effector phase moderated EAE course. This was associated with the increased proportion of microglia expressing CX3CR1, the key molecule in their immunomodulatory/neuroprotective action, and upregulation of CX3CR1 downstream Nrf2 gene. This correlated with the increased heme oxygenase-1 (HO-1) expression and phagocytic capacity of microglia, and their phenotypic changes mirrored in increased proportion of CD163- and CD83-expressing cells. The enhanced HO-1 expression was linked with the decreased proportion of microglial cells expressing IL-1β and IL-23, and possibly IL-6, followed by increased proportion of IL-10-expressing microglia, and downregulated MCP-1/CCL2 expression. Consistently, spinal cord infiltration with blood-borne myeloid and CD4+ T cells, as well as CD4+ T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17+ cells co-producing IFN-γ and GM-CSF were decreased in propranolol-treated rats compared with saline-injected controls. The in vitro investigations of the effects of noradrenaline on microglia showed that noradrenaline through β-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests β-adrenoceptor-mediated neuroinflammation-promoting role of noradrenaline in EAE via modulation of microglial Nrf2 expression, and thereby forms the basis for further translational pharmacological research to improve multiple sclerosis therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Males exhibit stronger sympathetic nervous system (SNS) activity, but weaker primary CD4+ T-cell (auto)immune responses. To test the role of catecholamines, major end-point SNS mediators, in this dimorphism, influence of propranolol (β-adrenoceptor blocker) on mitogen/neuroantigen-stimulated CD4+ T cells from female and male EAE rat draining lymph node (dLN) cell cultures was examined. Male rat dLNs exhibited higher noradrenaline concentration and frequency of β 2 -adrenoceptor-expressing CD4+ T lymphocytes and antigen presenting cells. Propranolol, irrespective of exogenous noradrenaline presence, more prominently augmented IL-2 production and proliferation of CD4+ lymphocytes in male than female rat dLN cell cultures. In neuroantigen-stimulated dLN cells of both sexes propranolol increased IL-1β and IL-23/p19 expression and IL-17+ CD4+ cell frequency, but enhanced IL-17 production only in male rat CD4+ lymphocytes, thereby abrogating sexual dimorphism in IL-17 concentration observed in propranolol-free cultures. Thus, β-adrenoceptor-mediated signalling may contribute to sex bias in rat IL-17-producing cell secretory capacity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: We examined the effect of β-adrenoceptor (AR) blockade in the preclinical phase of experimental autoimmune encephalomyelitis (EAE), the most commonly used model of multiple sclerosis, on the development of primary CD4+ T-cell responses in draining lymph nodes (dLNs)., Methods: CD11b+ cell migration to dLNs, CD4+ T-cell activation/proliferation, and IL-17+ CD4+ (Th17) cell numbers in dLN and spinal cord (SC) were examined in male and female Dark Agouti rats using flow cytometry analysis., Results: Irrespective of sex, in propranolol-treated (PT) rats, migration of CD11b+ antigen-presenting cells from the site of immunization to dLNs was impaired compared with saline-treated controls and consequently the frequency of all CD11b+ cells in dLNs and activated cells among them, too. This correlated with decreased expression of CCL19/21 transcripts in dLNs. Consistently, the frequency of activated/proliferating cells among dLN CD4+ T cells was reduced in PT rats. Additionally, propranolol reduced the number of Th17 cells in dLNs and SC. Consistently, male and female PT rats exhibited a decreased incidence of EAE and prolonged duration of the asymptomatic disease phase., Conclusion: This study suggests that sympathetic dysregulation is involved in the outbreak of clinical EAE., (© 2019 S. Karger AG, Basel.)

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCRαβ-mediated signaling and activation thresholds, on CD4+CD8+ TCRαβlo/hi thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCRαβhi thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16INK4a accumulation). The higher circulating level of TNF-α in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE., Competing Interests: The authors have declared that no competing interests exist.

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-γ+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCRαβ- 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+TCRαβ- 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.

The study investigated strain specificities in age-related differences in CD8+ T cell- and microglial cell-mediated mechanisms implicated in induction/perpetuation and/or control of neuroinflammation in experimental autoimmune encephalomyelitis (EAE) in Albino Oxford (AO) and Dark Agouti (DA) rats exhibiting age-related changes in the susceptibility to EAE in the opposite direction (increase in relatively resistant AO rats vs decrease in DA rats). In the inductive phase of EAE, the greater number of fully differentiated effector CD8+ T lymphocytes was found in draining lymph nodes (dLNs) from aged rats of both strains than in strain-matched young rats, but this was particularly prominent in AO rats, which exhibited milder EAE of prolonged duration compared with their DA counterparts. Consistently, dLN IFN-γ+ and IL-17+ CD8+ T cell counts were greater in aged AO than in DA rats. Additionally, the magnitudes of myelin basic protein (MBP)-induced rise in the frequency of IFN-γ+ and IL-17+ CD8+ T cells (providing important help to neuroantigen-specific CD4+ T cells in EAE models characterized by clinically mild disease) were greater in dLN cell cultures from aged AO rats. Consistently, the magnitudes of MBP-induced rise in the frequency of both IFN-γ+ and IL-17+ CD8+ T cells were greater in spinal cord mononuclear cell cultures from aged AO rats compared with their DA counterparts. Besides, with aging CD4+CD25+Foxp3+/CD8+CD25+Foxp3+ regulatory T cell ratio changed in spinal cord in the opposite direction. Consequently, in aged AO rats it was shifted towards CD8+CD25+Foxp3+ regulatory T cells (exhibiting lower suppressive capacity) when compared with DA rats. Moreover, the frequency of CX3CR1+ cells among microglia changed with aging and the disease development. In aged rats, in the effector phase of EAE it was lower in AO than in DA rats. This was accompanied by higher frequency of cells expressing IL-1β (whose down-regulation is central for CX3CR1-mediated neuroprotection), but lower that of phagocyting cells among microglia from aged AO compared their DA counterparts. The study indicates the control points linked with strain differences in age-related changes in EAE pathogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To understand strain-specificities of immune system in aged rats and their immunopathological implications, CD4+ T lymphocyte-mediated neuroinflammation in experimental autoimmune encephalomyelitis (EAE) was studied in two strains. Upon immunization for EAE, 22-24-month-old Albino Oxford (AO) rats developed milder neurological deficit of prolonged duration compared with their Dark Agouti (DA) counterparts. Consistently, they exhibited: (i) diminished neuroantigen-specific CD4+ T lymphocyte generation in draining lymph nodes (reflecting lower density of high-affinity IL-2 receptor complex on their surface and higher CD4+FoxP3+CD25+ regulatory cell frequency); (ii) less favorable spinal cord expression of CXCL12 and CCL2, and consequently diminished infiltration of neuroantigen-specific CD4+ T lymphocytes, including highly pathogenic IL-17+IFN-γ+ ones, and inflammatory monocytes into the spinal cord and (iii) subsequently impaired CD4+ T lymphocyte reactivation/survival and differentiation into highly pathogenic IL-17+ cells (reflecting downregulated expression of IL-1β, IL-6 and IL-23/p19). On the other hand, when the neurological deficit reached maximum/plateau, in AO rat spinal cord was found lower CD4+FoxP3+CD25+ cell frequency followed by higher frequency of IL-10-producing CD8+ T cells, which most likely also belong to regulatory T lymphocytes. Thus, the altered relation between regulatory T cell and effector CD4+ T cell subsets was linked with persistence of mild neuroinflammation in AO rat EAE model., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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-γ+, IL-17+IFN-γ-, and IL-17-IFN-γ+ cells accompanied by higher frequency of IL-17-IFN-γ- 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-γ+ Th17 cells in SC) on GM-CSF+IFN-γ+ 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-γ+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1β, 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 (CD45hi 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., Competing Interests: The authors have declared that no competing interests exist.

Considering the crucial pathogenic role of CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and the opposite direction of the sexual dimorphism in the severity of the disease in 22-24-and 3-month-old dark agouti rats, sex differences in CD4+ T-cell-mediated immune response in aged rats immunized for EAE were examined and compared with those in young animals. In the inductive phase of EAE, fewer activated CD4+ lymphocytes were retrieved from draining lymph nodes of male (developing less severe disease) compared with female rats, due, at least partly, to their lesser expansion. The former reflected a greater suppressive capacity of CD4+CD25+Foxp3+ cells. Consequently, CD4+ lymphocyte infiltration into the spinal cord of aged male rats was diminished. At the peak of EAE, the frequency of reactivated cells was lower, whereas that of the regulatory CD4+ cells was higher in male rat spinal cord. Consistently, microglial activation and the expression of proinflammatory/damaging cytokines in male rat spinal cord mononuclear cells were diminished. Additionally, the frequency of the highly pathogenic IL-17+IFN-γ+ T lymphocytes infiltrating their spinal cord was lower. Together, these results point to (i) an age-specificity in CD4+ cell-mediated immune response and (ii) mechanisms underlying the sex differences in this response in aged rats., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freund's 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 Freund's 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aging is associated with the decline in immune response to infectious agents and tumors and increasing risk of autoimmunity, but the incidence of autoimmune diseases does not increase in the elderly. To elucidate the cellular and molecular mechanisms influencing clinical expression of autoimmunity in aged animals, the phenotypic and functional characteristics of mononuclear cells isolated from the spinal cords of 3-month-old (young) and 26-month-old (aged) Dark Agouti rats immunized to induce experimental autoimmune encephalomyelitis (EAE) - the model of multiple sclerosis, the most common autoimmune disease of the central nervous system, were examined. Aged rats were less susceptible to EAE induction, and the neurological and histological picture was milder in those rats which developed the clinically manifested disease. At the peak of the disease, several times fewer mononuclear cells and T lymphocytes were isolated from the spinal cords of aged rats compared with the young ones. The frequency of CD4+ cells among TCRαβ+ lymphocytes, as well as that of reactivated CD134(OX40)+ cells within its CD4+ T-lymphocyte subpopulation, was less in spinal cords of aged compared with young rats. Additionally, CD134 surface density on CD4+ lymphocytes was decreased in the spinal cord of aged rats. The changes in CD134 expression most likely reflected in part age-related intrinsic changes in CD4+ lymphocytes as the expression of this molecule was also impaired on in vitro stimulated naïve CD4+ splenocytes from aged rats compared with young animals. In addition, greater frequency of CD8+ lymphocytes with regulatory phenotypes could also contribute to impaired CD4+ cell reactivation in aged rats. The increased apoptosis of CD4+ cells from aged rats was consistent with their impaired reactivation and it was accompanied by the greater frequency of CD4+CD11b+CD45(int/high) cells, which are supposed to be actively engaged in apoptotic cell phagocytosis and to have immunoregulatory properties. Compared with young rats, following short-term PMA and ionomycin stimulation in vitro, the frequency of IL-17+ and IFN-γ+CD4+ T lymphocytes among the spinal cord mononuclear cells from aged rats and the cytokine expression density on a per lymphocyte basis were reduced. Additionally, the increase in the proportion of autoregulatory IL-17+IL-10+ cells on the account of proinflammatory IL-17+IFN-γ+ cells within IL-17+ lymphocytes suggested their lower pathogenic capacity in aged rats. This most likely reflected alterations in the aged rat spinal cord cytokine milieu, which were mirrored in a diminished expression of IL-1β mRNA followed by an enhanced expression of IL-6 and TGF-β mRNA. Overall, the study points to age-related changes in T lymphocytes and other cells from the spinal cord infiltrate which could contribute to the decreased susceptibility of aged rats to the induction of EAE., (Copyright © 2014 Elsevier Inc. All rights reserved.)