Jasmina Djikić

Age-associated changes in rat immune system: Lessons learned from experimental autoimmune encephalomyelitis

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.

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.

Effects of catecholamines on thymocyte apoptosis and proliferation depend on thymocyte microenvironment.

The present study, through quantification of tyrosine hydroxylase (TH) expression and catecholamine (CA) content in the presence and in the absence of α-methyl-p-tyrosine (AMPT), a TH inhibitor, in adult thymic organ (ATOC) and thymocyte culture, demonstrated that thymic cells produce CAs. In addition, in ATOC an increase in β2-adrenoceptor (AR) mRNA expression and β2-AR thymocyte surface density was registered. Furthermore, AMPT (10(-4)M), as propranolol (10(-4)M), augmented thymocyte apoptosis and diminished thymocyte proliferation in ATOC. Propranolol exerted these effects acting on CD3(high) thymocytes. However, in thymocyte cultures, propranolol (10(-6)M) acting on the same thymocyte subset exerted the opposing effect on thymocyte apoptosis and ConA-stimulated proliferation. This suggested that, depending on thymocyte microenvironment, differential effects can be induced through the same type of AR. Additionally, arterenol (10(-8) to 10(-6)M), similar to propranolol, diminished apoptosis, but increased ConA-stimulated thymocyte proliferation in thymocyte culture. However, differently from propranolol, arterenol affected manly CD3- thymocyte subset, which harbors majority of α1-AR+thymocytes. Additionally, arterenol showed a dose-dependent decrease in efficiency of thymocyte apoptosis and proliferation modulation with the rise in its concentration. Considering greater affinity of arterenol for α1-ARs than for β2-ARs, the previous findings could be attributable to increased engagement of β2-ARs with the rise of arterenol concentration. Consistently, in the presence of propranolol (10(-6)M), a β-AR blocker, the arterenol (10(-8)M) effects on thymocytes were augmented. In conclusion, thymic endogenous CAs, acting through distinct AR types and, possible, the same AR type (but in different cell microenvironment) may exert the opposing effects on thymocyte apoptosis/proliferation.

17β-Estradiol influences in vitro response of aged rat splenic conventional dendritic cells to TLR4 and TLR7/8 agonists in an agonist specific manner

This study was undertaken considering that, despite the broad use of the unopposed estrogen replacement therapy in elderly women, data on estrogen influence on the functional capacity of dendritic cells (DCs), and consequently immune response are limited. We examined the influence of 17β-estradiol on phenotype, cytokine secretory profile, and allostimulatory and polarizing capacity of splenic (OX62+) conventional DCs from 26-month-old (aged) Albino Oxford rats matured in vitro in the presence of LPS, a TLR4 agonist, and R848, a TLR7/8 agonist. In the presence of 17β-estradiol, DCs from aged rats exhibited an impaired ability to mature upon stimulation with LPS, as shown by the lower surface density of MHC II and costimulatory CD80 and CD86 molecules. 17β-Estradiol alone enhanced CD40 expression in OX62+ DCs without affecting the expression of other costimulatory molecules, thereby confirming that the expression of this molecule is regulated independently from the regulation of other costimulatory molecules. However, although R848 upregulated the expression of MHC II and CD80 and CD40 costimulatory molecules on DCs, 17β-estradiol diminished the effect of this TLR agonist only on MHC II expression. In conjunction, the previous findings suggest that LPS and R848 elicit changes in the expression of costimulatory molecules via triggering differential intracellular signaling pathways. Furthermore, 17β-estradiol diminished the stimulatory influence of both LPS- and R848-matured OX62+ DCs on allogeneic CD4+ T lymphocyte proliferation in a mixed lymphocyte reaction (MLR). Moreover, as shown in MLR, the exposure to 17β-estradiol during LPS- and R848-induced maturation diminished Th1- and enhanced Th17-driving capacity and reduced Th1-driving capacity of OX62+ DCs, respectively. This suggests that LPS and R848 affect not only the surface phenotype, but also functional characteristics of OX62+ DCs triggering distinct intracellular signaling pathways. Collectively, the findings indicate that estrogen directly acting on OX62+ DCs, may affect CD4+ lymphocyte-dependent immune response in aged female rats.

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: a putative role for progesterone deficiency.

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCRαβ+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCRαβ+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, naïve cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-β mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation.

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 β2‐ and α1B‐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 β‐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 β2‐ and α1B‐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 β‐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 α1B‐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.

Ovarian hormone level alterations during rat post-reproductive life-span influence CD8 + T-cell homeostasis

The study examined the putative role of ovarian hormones in shaping of rat peripheral T-cell compartment during post-reproductive period. In 20-month-old rats ovariectomized (Ox) at the very end of reproductive period, thymic output, cellularity and composition of major TCRαβ + peripheral blood lymphocyte and splenocyte subsets were analyzed. Ovariectomy led to the enlargement of CD8 + peripheral blood lymphocyte and splenocyte subpopulations. This reflected: (i) a more efficient thymic generation of CD8 + cells as indicated by increased number of CD4+CD8 + double positive and the most mature CD4-CD8+TCRαβhigh thymocytes and CD8 + recent thymic emigrants (RTEs) in peripheral blood, but not in the spleen of Ox rats, and (ii) the expansion of CD8 + memory/activated peripheral blood lymphocytes and splenocytes. The latter was consistent with a greater frequency of proliferating cells among freshly isolated memory/activated CD8 + peripheral blood lymphocytes and splenocytes and increased proliferative response of CD8 + splenocytes to stimulation with plate-bound anti-CD3 antibody. The former could be related to the rise in splenic IL-7 and IL-15 mRNA expression. Although ovariectomy affected the overall number of CD4 + T cells in none of the examined compartments, it increased CD4+FoxP3 + peripheral blood lymphocyte and splenocyte counts by enhancing their generation in periphery. Collectively, the results suggest that ovariectomy-induced long-lasting disturbances in ovarian hormone levels (mirrored in diminished progesterone serum level in 20-month-old rats) affects both thymic CD8 + cell generation and peripheral homeostasis and leads to the expansion of CD4+FoxP3 + cells in the periphery, thereby enhancing autoreactive cell control on account of immune system efficacy to combat infections and tumors.

Strain specificities in age-related changes in mechanisms promoting and controlling rat spinal cord damage in experimental autoimmune encephalomyelitis

Abstract 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‐&ggr;+ 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‐&ggr;+ 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‐&ggr;+ 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‐1&bgr; (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. HighlightsAging affects rat susceptibility to EAE in a strain‐specific way.Effector CD8+ T cell number in EAE rat draining lymph nodes depends on strain and age.Spinal cord (SC) microglial CX3CR1 expression in EAE rats depends on strain and age.Aging changes CD4+/CD8+ Treg cell ratio in EAE rat SC in a strain‐specific way.The frequency of infiltrating SC CD8+ T cells in EAE rats depends on strain and age.