Ageing Affects Thymopoiesis and Experimental Autoimmune Encephalomyelitis Development in a Strain-Dependent Manner.

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-ß 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.

Adrenoceptors as potential target for add-on immunomodulatory therapy in multiple sclerosis.

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 ß-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 ß-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.

Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development.

The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of "inflammescent" cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy.

ß-Adrenoceptor Blockade Moderates Neuroinflammation in Male and Female EAE Rats and Abrogates Sexual Dimorphisms in the Major Neuroinflammatory Pathways by Being More Efficient in Males.

Our previous studies showed more severe experimental autoimmune encephalomyelitis (EAE) in male compared with female adult rats, and moderating effect of propranolol-induced ß-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 ß2-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 ß2-adrenoceptor (ß2-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.

B-cell response to seasonal influenza vaccine in mice is amenable to pharmacological modulation through ß-adrenoceptor.

AIMS: Given that deprivation of noradrenaline acting on lymphocytes through ß-adrenoceptor influences antibody response, the effects of propranolol treatment beginning two days before immunization with quadrivalent inactivated influenza vaccine (QIV) on IgG response and underlying cellular molecular mechanism in mice were investigated. MAIN METHODS: Twenty-one days post-immunization the total QIV antigen-specific IgG titer and IgG subclass titers in sera were determined using ELISA. Additionally, the total counts of germinal centre (GC) B cells, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in draining lymph nodes (dLNs) and spleens, in vitro proliferation of interacting B cells and Th cells and IL-21 synthesis in Th cells in response to QIV antigens and/or mitogen were attested using flow cytometry analysis. In QIV antigen-stimulated dLN cell and splenocyte cultures were also measured concentrations of INF-γ and IL-4, cytokines upregulating IgG2a and IgG1 synthesis, respectively. KEY FINDINGS: Propranolol decreased the total QIV antigen-specific IgG titer. This correlated with lower GC B cell count and the shift in Tfr/Tfh cell and Tfr/GC B cell ratio towards Tfr in propranolol-treated mice compared with controls. Consistently, QIV antigen-stimulated proliferation of B cells and Th cells from propranolol-treated mice in vitro was impaired. This correlated with the lower frequency of QIV antigen-specific IL-21-producing cells among Th cells. Additionally, in propranolol-treated mice, in accordance with the changes in INF-γ/IL-4 ratio in dLN cell/splenocyte cultures, serum IgG2a/IgG1 ratio was shifted towards IgG1 reflecting decreased IgG2a response. SIGNIFICANCE: The study indicates that chronic propranolol treatment may impair response to QIV.

Sex-specific remodeling of T-cell compartment with aging: Implications for rat susceptibility to central nervous system autoimmune diseases.

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.

Infrared radiation from cage bedding moderates rat inflammatory and autoimmune responses in collagen-induced arthritis.

The development of collagen type II (CII)-induced arthritis (CIA), a model of rheumatoid arthritis, in rats housed in cages with bedding composed of Celliant fibres containing ceramic particles, which absorb body heat and re-emit the energy back to the body in the form of infrared radiation (+IRF rats), and those housed in cages with standard wooden shaving bedding (-IRF control rats) was examined. The appearance of the first signs of CIA was postponed, while the disease was milder (judging by the arthritic score, paw volume, and burrowing behaviour) in +IRF compared with -IRF rats. This correlated with a lower magnitude of serum anti-CII IgG antibody levels in +IRF rats, and lower production level of IL-17, the Th17 signature cytokine, in cultures of their paws. This could be partly ascribed to impaired migration of antigen-loaded CD11b + dendritic cells and their positioning within lymph nodes in +IRF rats reflecting diminished lymph node expression of CCL19 /CCL21. Additionally, as confirmed in rats with carrageenan-induced paw inflammation (CIPI), the infrared radiation from Celliant fibres, independently from immunomodulatory effects, exerted anti-inflammatory effects (judging by a shift in pro-inflammatory mediator to anti-inflammatory/immunoregulatory mediator ratio towards the latter in paw cultures) and ameliorated burrowing behaviour in CIA rats.

CD8+ T Cell-Mediated Mechanisms Contribute to the Progression of Neurocognitive Impairment in Both Multiple Sclerosis and Alzheimer's Disease?

Neurocognitive impairment (NCI) is one of the most relevant clinical manifestations of multiple sclerosis (MS). The profile of NCI and the structural and functional changes in the brain structures relevant for cognition in MS share some similarities to those in Alzheimer's disease (AD), the most common cause of neurocognitive disorders. Additionally, despite clear etiopathological differences between MS and AD, an accumulation of effector/memory CD8+ T cells and CD8+ tissue-resident memory T (Trm) cells in cognitively relevant brain structures of MS/AD patients, and higher frequency of effector/memory CD8+ T cells re-expressing CD45RA (TEMRA) with high capacity to secrete cytotoxic molecules and proinflammatory cytokines in their blood, were found. Thus, an active pathogenetic role of CD8+ T cells in the progression of MS and AD may be assumed. In this mini-review, findings supporting the putative role of CD8+ T cells in the pathogenesis of MS and AD are displayed, and putative mechanisms underlying their pathogenetic action are discussed. A special effort was made to identify the gaps in the current knowledge about the role of CD8+ T cells in the development of NCI to "catalyze" translational research leading to new feasible therapeutic interventions.

Sex as a confounding factor in the effects of ageing on rat lymph node t cell compartment.

The study examined the influence of sex on the alterations occurring with ageing in rat lymph node (LN) T cell compartment. In female and male rats the decrease in LN T cell counts was followed by a shift in CD4+/CD8+ T cell ratio towards CD8+ T cells, which was more prominent in males than in females. With ageing, in both major LN T cell subpopulations naïve (recent thymic emigrants and mature naïve cells) to memory/activated T cell ratio shifted to the side of memory/activated cells in female, and particularly in male rats. The frequency of regulatory CD25+Foxp3+ cells increased among LN CD4+/CD8+ T cells with ageing, reflecting, at least partly, an enhanced conversion of effector T cells into regulatory cells. This was also more prominent in male rats. The more prounounced increase in LN oxidative damage and the expression levels of proinflammatory cytokines in male rats with ageing, most likely contributed to the greater frequency of proinflammatory, replicatively senescent CD28- cells expressing CD11b (innate cell marker), among T cells of old male rats compared with age-matched females. The increase in LN oxidation/proinflammatory state with ageing was also consistent with the accumulation of exhausted PD-1high cells among T lymphocytes, particularly prominent among CD8+ T cells from male rats. Finally, by calculating a summary score for the key ageing-relevant parameters (an ageing index), a faster development of the deleterious changes in the T cell compartment occurring with ageing was confirmed in male rat LNs. Additionally, the study pointed to indices of LN T cell compartment ageing which correlate with those in peripheral blood.

Sex-Based Differences in Monocytic Lineage Cells Contribute to More Severe Collagen-Induced Arthritis in Female Rats Compared with Male Rats.

Monocytes' plasticity has an important role in the development of rheumatoid arthritis (RA), an autoimmune disease exhibiting greater prevalence in women. Contribution of this phenomenon to sex bias in RA severity was investigated in rat collagen-induced arthritis (CIA) model of RA. The greater severity of CIA in females (exhibiting signs of bone resorption) was accompanied by the higher blood level of advanced oxidation protein products and a more pro-oxidant profile. Consistently, in females, the greater density of giant multinuclear cells (monocytes/macrophages and osteoclasts) in inflamed joint tissue was found. This correlated with the higher frequencies of CCR2- and CX3CR1- expressing cells (precursors of inflammatory monocytes/macrophages and osteoclasts) among CD11b+ splenocytes. This in conjunction with the enhanced migratory capacity of CD11b+ monocytic cells in females compared with males could be linked with the higher frequencies of CCR2+CX3CR1-CD43lowCD11b+ and CCR2-CX3CR1+CD43hiCD11b+ cells (corresponding to "classical" and "non-classical" monocytes, respectively) and the greater density of CD68+ cells (monocytes/macrophages and osteoclast precursors/osteoclasts) in blood and inflamed paws from female rats, respectively. Consistently, the higher levels of GM-CSF, TNF-α and IL-6, IL-1ß (driving Th17 cell differentiation), and IL-17 followed by the lower level of IL-10 were measured in inflamed paw cultures from female compared with male rats. To the greater IL-17 production (associated with enhanced monocyte immigration and differentiation into osteoclasts) most likely contributed augmented Th17 cell generation in the lymph nodes draining arthritic joints from female compared with male rats. Overall, the study suggests the sex-specific contribution of monocytic lineage cells to CIA, and possibly RA development.

Strain specificities in influence of ageing on germinal centre reaction to inactivated influenza virus antigens in mice: Sex-based differences.

Considering variability in vaccine responsiveness across human populations, in respect to magnitude and quality, and importance of vaccines in the elderly, the influence of recipient genetic background on the kinetics of age-related changes in the serum IgG antibody responses to seasonal trivalent inactivated split-virus influenza bulk (TIV) was studied in BALB/c and C57BL/6 mice showing quantitative and qualitative differences in this responses in young adult ages. With ageing the total serum IgG response to influenza viruses declined, in a strain-specific manner, so the strain disparity observed in young adult mice (the greater magnitude of IgG response in BALB/c mice) disappeared in aged mice. However, the sexual dimorphisms in this response (more prominent in females of both strains) remained in aged ones. The strain-specific differences in age-related decline in the magnitude of IgG response to TIV correlated with the number of germinal centre (GC) B splenocytes. The age-related decline in GC B cell number was consistent with the decrease in the proliferation of B cells and CD4+ cells in splenocyte cultures upon restimulation with TIV. Additionally, the age-related decrease in the magnitude of IgG response correlated with the increase in follicular T regulatory (fTreg)/follicular T helper (fTh) and fTreg/GC B splenocyte ratios (reflecting decrease in fTh and GC B numbers without changes in fTreg number), and the frequency of CD4+ splenocytes producing IL-21, a key factor in balancing the B cell and fTreg cell activity. With ageing the avidity of virus influenza-specific antibody increased in females of both strains. Moreover, ageing affected IgG2a/IgG1 and IgG2c/IgG1 ratios (reflecting Th1/Th2 balance) in male BALB/c mice and female C57BL/6 mice, respectively. Consequently, differently from young mice exhibiting the similar ratios in male and female mice, in aged female mice of both strains IgG2a(c)/IgG1 ratios were shifted towards a less effective IgG1 response (stimulated by IL-4 cytokines) compared with males. The age-related alterations in IgG subclass profiles in both strains correlated with those in IFN-γ/IL-4 production level ratio in splenocyte cultures restimulated with TIV. These findings stimulate further research to formulate sex-specific strategies to improve efficacy of influenza vaccine in the elderly.

Sex differences in Tfh cell help to B cells contribute to sexual dimorphism in severity of rat collagen-induced arthritis.

The study examined germinal centre (GC) reaction in lymph nodes draining inflamed joints and adjacent tissues (dLNs) in male and female Dark Agouti rat collagen type II (CII)-induced arthritis (CIA) model of rheumatoid arthritis. Female rats exhibiting the greater susceptibility to CIA mounted stronger serum CII-specific IgG response than their male counterparts. This correlated with the higher frequency of GC B cells in female compared with male dLNs. Consistently, the frequency of activated/proliferating Ki-67+ cells among dLN B cells was higher in females than in males. This correlated with the shift in dLN T follicular regulatory (Tfr)/T follicular helper (Tfh) cell ratio towards Tfh cells in females, and greater densities of CD40L and CD40 on their dLN T and B cells, respectively. The higher Tfh cell frequency in females was consistent with the greater dLN expression of mRNA for IL-21/27, the key cytokines involved in Tfh cell generation and their help to B cells. Additionally, in CII-stimulated female rat dLN cell cultures IFN-γ/IL-4 production ratio was shifted towards IFN-γ. Consistently, the serum IgG2a(b)/IgG1 CII-specific antibody ratio was shifted towards an IgG2a(b) response in females. Thus, targeting T-/B-cell interactions should be considered in putative further sex-based translational pharmacology research.

Propranolol diminished severity of rat EAE by enhancing immunoregulatory/protective properties of spinal cord microglia.

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-1ß 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 ß-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests ß-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.

Age and sex determine CD4+ T cell stimulatory and polarizing capacity of rat splenic dendritic cells.

The study investigated influence of sex and age on splenic myeloid dendritic cells (DCs) from Dark Agouti rats. Freshly isolated DCs from young males exhibited less mature phenotype and greater endocytic capacity compared with those from age-matched females. Upon LPS stimulation in vitro they were less potent in stimulating allogeneic CD4+ cells in mixed leukocyte reaction (MLR), due to lower expression of MHC II, and greater NO and IL-10 production. In accordance with higher TGF-ß production, young male rat DCs were less potent in stimulating IL-17 production in MLR than those from young females. Irrespective of sex, endocytic capacity and responsiveness of DCs to LPS stimulation in culture, judging by their allostimulatory capacity in MLR decreased with age, reflecting decline in MHC II surface density followed by their greater NO production; the effects more prominent in females. Additionally, compared with LPS-stimulated DCs from young rats, those from sex-matched aged rats were more potent in stimulating IL-10 production in MLR, whereas capacity of DCs from aged female and male rats to stimulate IL-17 production remained unaltered and decreased, respectively. This reflected age-related shift in IL-6/TGF-ß production level ratio in LPS-stimulated DC cultures towards TGF-ß, and sex-specific age-related remodeling CD4+ cell cytokine pathways. Additionally, compared with LPS-stimulated DCs from young rats, those cells from sex-matched aged rats were less potent in stimulating IFN-γ production in MLR, the effect particularly prominent in MLRs encompassing male rat DCs. The study showed that stimulatory and polarizing capacity of DCs depends on rat sex and age.

Noradrenaline modulates CD4+ T cell priming in rat experimental autoimmune encephalomyelitis: a role for the α1-adrenoceptor.

Pharmacological blockade of α1-adrenoceptor is shown to influence development of experimental autoimmune encephalomyelitis (EAE), an IL-17-producing CD4+TCR+ (Th17) cell-mediated disease mimicking multiple sclerosis. Considering significance of CD4+ cell priming for the clinical outcome of EAE, the study examined α1-adrenoceptor-mediated influence of catecholamines, particularly those derived from draining lymph node (dLN) cells (as catecholamine supply from nerve fibers decreases with the initiation of autoimmune diseases) for CD4+ cell priming. The results confirmed diminishing effect of immunization on nerve fiber-derived noradrenaline supply and showed that antigen presenting and CD4+ cells synthesize catecholamines, while antigen presenting cells and only CD4+CD25+Foxp3+ regulatory T cells (Tregs) express α1-adrenoceptor. The analysis of influence of α1-adrenoceptor antagonist prazosin on the myelin basic protein (MBP)-stimulated CD4+ lymphocytes in dLN cell culture showed their diminished proliferation in the presence of prazosin. This was consistent with prazosin enhancing effect on Treg frequency and their Foxp3 expression in these cultures. The latter was associated with upregulation of TGF-ß expression. Additionally, prazosin decreased antigen presenting cell activation and affected their cytokine profile by diminishing the frequency of cells that produce Th17 polarizing cytokines (IL-1ß and IL-23) and increasing that of IL-10-producing cells. Consistently, the frequency of all IL-17A+ cells and those co-expressing GM-CSF within CD4+ lymphocytes was decreased in prazosin-supplemented MBP-stimulated dLN cell cultures. Collectively, the results indicated that dLN cell-derived catecholamines may influence EAE development by modulating interactions between distinct subtypes of CD4+ T cells and antigen presenting cells through α1-adrenoceptor and consequently CD4+ T cell priming.

Sexual dimorphism in rat thymic involution: a correlation with thymic oxidative status and inflammation.

The study investigated mechanisms underlying sex differences in thymic involution in Dark Agouti rats. Adverse effects of aging on thymus were more pronounced in males than in females. Thymi from old males exhibited more prominent: (i) fibro-adipose degeneration which correlated with greater intensity of thymic oxidative stress and enhanced thymic TGF-ß and IL-6 expression and (ii) decline in thymopoiesis, as suggested by the number of the most mature CD4+CD8-/CD4-CD8+ single positive (SP) TCRαßhigh thymocytes. The greater accumulation of adipose tissue in old male thymus was linked with greater age-related increase in thymic expression of PPARγ and STAT3, a transcription factor regulating the expression of PPARγ downstream genes, in male than in female rats. In aged thymi of both sexes the early CD4-CD8- double negative (DN) stage of thymocyte development was affected, so relative accumulation of the least mature CD45RC+CD2- cells followed by decreased frequency of their DN and CD4+CD8+ double positive (DP) TCRαß- descendants was observed. Additionally, in old males, because of the increased thymic expression of Nur77, a nuclear receptor involved in negative selection, and decreased CD90 (a negative regulator of thymocyte selection threshold) MFI on DP TCRαßint thymocytes, less efficient positive/more efficient negative selection was found. Moreover, in male rats, thymocyte post-selection differentiation/maturation was skewed towards CD4-CD8+ SP TCRαßhigh cells compared with age-matched females, reflecting, at least partly, greater IL-15 expression in their thymi. The study indicated mechanisms underlying sex-based differences in age-related thymic changes and consequently necessity of sex-specific approaches in designing strategies to rejuvenate thymus.

Influence of aging on germinal centre reaction and antibody response to inactivated influenza virus antigens in mice: sex-based differences.

The study examined sex-specificities in age-related changes in BALB/c mice IgG antibody responses to immunisation with trivalent inactivated split-virus influenza bulk. Aging diminished the total serum IgG antibody responses to H1N1 and H3N2 and B influenza virus antigens in mice of both sexes, but they remained greater in aged females. This sex difference in aged mice correlated with the greater post-immunisation increase in the frequency of spleen germinal centre (GC) B cells and more favourable T follicular regulatory (Tfr)/GC B cell ratio, as Tfr cells are suggested to control antibody production through suppression of glycolysis. The greater post-immunisation GC B cell response in aged females compared with males correlated with the greater proliferation of B cells and CD4+ cells in splenocyte cultures from aged females restimulated with inactivated split-virus influenza from the bulk. To support the greater post-immunisation increase in the frequency GC B cell in aged females was more favourable Tfr/T follicular helper (Tfh) cell ratio. Additionally, compared with aged males, in age-matched females the greater avidity of serum IgG antibodies was found. However, in aged females IgG2a/IgG1 antibody ratio, reflecting spleen Th1/Th2 cytokine balance, was shifted towards IgG1 when compared with age-matched male mice. This shift was ascribed to a more prominent decline in the titres of functionally important IgG2a antibodies in females with aging. The study suggest that biological sex should be considered as a variable in designing strategies to manipulate with immune outcome of immunisation in aged animals, and possibly, at very long distance, humans.

Propranolol Impairs Primary Immune Responses in Rat Experimental Autoimmune Encephalomyelitis.

OBJECTIVE: We examined the effect of ß-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.

Noradrenaline through ß-adrenoceptor contributes to sexual dimorphism in primary CD4+ T-cell response in DA rat EAE model?

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 (ß-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 ß2-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-1ß 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, ß-adrenoceptor-mediated signalling may contribute to sex bias in rat IL-17-producing cell secretory capacity.