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.

Sexual dimorphism in Th17/Treg axis in lymph nodes draining inflamed joints in rats with collagen-induced arthritis.

Collagen type II-induced arthritis (CIA) in Dark Agouti rats, a model of rheumatoid arthritis (RA), reproduces sexual dimorphism in the incidence and severity of the human disease. Th17 cells are central in the induction/propagation of autoimmune inflammation in CIA and RA. To assess mechanisms underlying this dimorphism in CIA rats, in lymph nodes draining inflamed joints and adjacent tissues (dLNs) from CIA rats of both sexes Th17/CD25+Foxp3+CD4+ T-regulatory cell (Treg) ratio, Th17 cell redifferentiation in functionally distinct subsets and Treg transdifferentiation into IL-17-producing cells (exTregs) were examined. In female rats (developing more severe CIA than their male counterparts) the higher frequency of all Th17 cells (reflecting partly their greater proliferation), followed by the higher frequency of highly pathogenic IFN-γ/GM-CSF-co-producing cells, but lower frequency of less pathogenic/immunoregulatory IL-10-producing cells among them was found. Additionally, compared with male rats, in female rats the lower frequency of Tregs was observed. Moreover, Tregs from female rats exhibited diminished proliferative and suppressive capacity (judging by PD-1 expression) and enhanced conversion into IL-17-producing cells. Given that TGF-ß concentration was comparable in collagen-type II-stimulated dLN cell cultures from female and male rats, the shift in Th17/Treg ratio followed by augmented Th17 cell redifferentiation into IFN-γ/GM-CSF-co-producing cells and Treg transdifferentiation into IL-17-producing cells in female rats was associated with increased concentration of IL-6 in female rat dLN cell cultures, and the higher frequency of IL-1ß- and IL-23-producing cells among their dLN cells. The lower frequency of IL-10-producing B cells, presumably B regulatory cells (Bregs) could also contribute to the shift in Th17/Treg ratio in female rat compared with male rat dLNs. Consistently, the lower expression of IL-35 (the cytokine promoting Treg expansion directly and indirectly, by favoring Breg expansion and conversion into IL-10/IL-35-producing cells) in female rat dLN cells was detected. Thus, the study identified putative cellular and molecular substrates of the sexual dimorphism in the immunopathogenesis and clinical outcome of CIA and suggested mechanisms to be targeted in females to improve control of Th17 response, and consequently clinical outcome of CIA, and possibly RA.

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.

Collagen-induced arthritis in Dark Agouti rats as a model for study of immunological sexual dimorphisms in the human disease.

Collagen-induced arthritis (CIA) is a frequently used animal model of rheumatoid arthritis, human autoimmune disease that exhibits clear sex bias in incidence and clinical course. Female Dark Agouti rats immunized for CIA showed also greater incidence and higher arthritic score than their male counterparts. The study investigated sex differences in mechanisms controlling the primary immune responses in draining lymph nodes (dLNs), as a factor contributing to this dimorphism. The higher frequencies of CD4 + CD25 + Foxp3- cells, presumably activated effector T (Teff) cells, and IL-17+, IFN-γ + and IL-17 + IFN-γ + T cells were found in female compared with male rat dLNs. However, the frequency of CD4 + CD25 + Foxp3+ T regulatory cells (Treg) did not differ between sexes. Thus, CD4+ Teff cells/Treg ratio, and IL-17+ T cells/Treg and IFN-γ + T cells/Treg ratios were higher in female than in male rats, and among them was found lower frequency of PD-1+ cells. This suggested less efficient control of (auto)immune Th1/Th17 cell responses in female rat dLNs. On the contrary, the frequency of IL-4+ T cells was lower in female than in male rat dLNs. Consistently, the ratio of serum levels of collagen-specific IgG2a (IFN-γ-dependent, with an important pathogenic role in CIA) and IgG1 (IL-4-dependent) was shifted towards IgG2a in female compared with male rats. As a whole, the study suggests that sexual dimorphism in the control of T cell activation/polarization could contribute to sex bias in the susceptibility to CIA. Moreover, the study advises the use of animals of both sexes in the preclinical testing of new drugs for rheumatoid arthritis.

Sex bias in mouse humoral immune response to influenza vaccine depends on the vaccine type.

The study explored influence of biological sex on development of humoral immune response to seasonal trivalent whole inactivated virus (WIV) and split virus (SV) influenza vaccines in outbred Swiss mouse model. To this end, mice of both sexes were immunized with WIV (WIV mice) and SV vaccines (SV mice) and examined for specific antibody response. Irrespective of sex, total IgG and neutralizing antibody responses to distinct virus strains were weaker in SV than in WIV mice. In WIV mice of both sexes, irrespective of strain specificity, IgG isotype response was dominated by IgG2a antibodies, while in SV mice nearly equal representation of IgG2a and IgG1 antibodies was found. The analyses of sex differences showed higher titers of H1N1-specific and both H1N1- and H3N2-specific total IgG and neutralizing antibodies in female WIV and SV mice, respectively. Additionally, sexual dimorphism in IgG subclass profile depended on vaccine type. Specifically, compared with males, in females WIV shifted IgG2a/IgG1 antibody ratio towards IgG2a isotype on the account of weaker IgG1 response, whereas in SV mice, irrespective of virus strain, IgG2a and IgG1 isotypes were equally represented in both sexes. These findings indicate the vaccine type-dependent sex bias in antibody response to inactivated influenza vaccines.

Sex and age as determinants of rat T-cell phenotypic characteristics: influence of peripubertal gonadectomy.

The study examined the influence of age, sex and peripubertal gonadectomy on a set of T-cell phenotypic parameters. Rats of both sexes were gonadectomised at the age of 1 month and peripheral blood and spleen T lymphocytes from non-gonadectomised and gonadectomised 3- and 11-month-old rats were examined for the expression of differentiation/activation (CD90/CD45RC) and immunoregulatory markers. Peripheral blood T lymphocytes from non-gonadectomised rats showed age-dependent sexual dimorphisms in (1) total count (lower in female than male 11-month-old rats); (2) CD4+:CD8 + cell ratio (higher in female than male rats of both ages); (3) the proportion of recent thymic emigrants in CD8 + T cells (lower in female than male 3-month-old rats) and (4) the proportions of mature naïve and memory/activated cells (irrespective of age, the proportion of naïve cells was higher, whereas that of memory/activated cells was lower in females). Gonadectomy influenced magnitudes or direction of these sex differences. Additionally, sex differences in peripheral blood T-lymphocyte parameters did not fully correspond to those observed in T-splenocyte parameters, suggesting the compartment-specific regulation of the major T-cell subpopulations' and their subsets' composition. Furthermore, there was no sexual dimorphism in the proportion of either CD25 + Foxp3 + cells among CD4 + or CD161+ (NKT) cells within CD8 + T lymphocytes. However, there was gonadal hormone-independent age-associated sexual dimorphism in the proportion of CD161 + cells (NKT cells) in CD8 + T splenocytes. Overall, the study revealed age-dependent variations in sexual dimorphisms in T-cell parameters relevant for immune response efficacy and showed that they are T-cell compartment-specific and partly gonadal hormone-related.

Sex as a determinant of age-related changes in rat spinal cord inflammation-oxidation state.

To close the gap in our knowledge of sex influence on age-related changes in inflammation-oxidation state in spinal cord (SC) relevant to inflammation/oxidative-stress associated neuropathologies, 2-3 month-old (young) and 18-20 month-old (old) rats, exhibiting increased level of IL-6, a commonly used marker of inflamm-aging, were examined for inflammatory/redox status, and the underlying regulatory networks' molecules expression. With age, rat SC microglia became sensitized ("primed"), while SC tissue shifted towards mild inflammatory state, with increased levels of proinflammatory IL-1ß (key marker of microglial systemic inflammation-induced neurotoxicity), which was more prominent in males. This, most likely, reflected age- and sex-related impairment in the expression of CX3CR1, the receptor for fractalkine (CX3CL1), the soluble factor which regulates microglial activation and diminishes production of IL-1ß (central for fractalkine neuroprotection). Considering that (i) age-related changes in SC IL-1ß expression were not followed by complementary changes in SC IL-6 expression, and (ii) the reversal in the direction of the sex bias in circulating IL-6 level and SC IL-1ß expression, it seems obvious that there are tissue-specific differences in the proinflammatory cytokine profile. Additionally, old male rat SC exhibited greater oxidative damage than female, reflecting, most likely, their lower capacity to maintain the pro-oxidant-antioxidant balance. In conclusion, these findings, apart from highlighting the significance of sex for age-associated changes in SC inflammation-oxidation, may be relevant for understating sex differences in human inflammation/oxidative-stress related SC diseases, and consequently, for optimizing their prevention/therapy.

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 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-1ß and IL-6, but downregulated TGF-ß 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.

Androgens contribute to age-associated changes in peripheral T-cell homeostasis acting in a thymus-independent way.

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

NMDA Receptor Antagonist Memantine Ameliorates Experimental Autoimmune Encephalomyelitis in Aged Rats.

Aging is closely related to the main aspects of multiple sclerosis (MS). The average age of the MS population is increasing and the number of elderly MS patients is expected to increase. In addition to neurons, N-methyl-D-aspartate receptors (NMDARs) are also expressed on non-neuronal cells, such as immune cells. The aim of this study was to investigate the role of NMDARs in experimental autoimmune encephalomyelitis (EAE) in young and aged rats. Memantine, a non-competitive NMDAR antagonist, was administered to young and aged Dark Agouti rats from day 7 after immunization. Antagonizing NMDARs had a more favourable effect on clinical disease, reactivation, and apoptosis of CD4+ T cells in the target organ of aged EAE rats. The expression of the fractalkine receptor CX3CR1 was increased in memantine-treated rats, but to a greater extent in aged rats. Additionally, memantine increased Nrf2 and Nrf2-regulated enzymes' mRNA expression in brain tissue. The concentrations of superoxide anion radicals, malondialdehyde, and advanced oxidation protein products in brain tissue were consistent with previous results. Overall, our results suggest that NMDARs play a more important role in the pathogenesis of EAE in aged than in young rats.

Aging affects AO rat splenic conventional dendritic cell subset composition, cytokine synthesis and T-helper polarizing capacity.

It is well-established that almost all cellular components of innate and adaptive immunity undergo age-related remodelling. The findings on age-related changes in both human and mouse dendritic cells (DCs) are conflicting, whereas there are no data on the influence of aging on rat DCs. In an attempt to fill this gap, freshly isolated splenic DCs expressing CD103 (αOX-62 integrin), a DC specific marker recognized by MRC OX62 monoclonal antibody, from 3- (young) and 26-month-old (aged) Albino Oxford rats were examined for subset composition, expression of activation/differentiation markers (CD80, CD86 and CD40 and MHC II molecules) and endocytic capacity using flow cytometric analysis (FCA). In addition, splenic OX62+ DCs cultured in the presence or absence of LPS were analysed for the activation marker and TNF-α, IL-6, IL-12, IL-23, TGF-ß1, IL-10 expression using FCA, RT-PCR and ELISA, respectively. Moreover, the allostimulatory capacity of OX62+ DCs and IFN-γ, IL-4 and IL-17 production by CD4+ T cells in mixed leukocyte reaction was quantified using FCA and ELISA, respectively. It was found that aging: i) shifts the CD4+:CD4- subset ratio in the OX62+ DCs population towards the CD4- subset and ii) influences DCs maturation (judging by activation marker expression and efficiency of endocytosis) by affecting the expression of intrinsic (TNF-α and IL-10) and extrinsic maturation regulators. Furthermore, in LPS-matured OX62+ DCs from aged rats expression of TNF-α, IL-12, IL-23 and IL-6 was increased, whereas that of IL-10 was diminished compared with the corresponding cells from young rats. Moreover, in MLR, OX62+ DCs from aged rats exhibited enhanced Th1/Th17 driving force and diminished allostimulatory capacity compared with those from young rats.

The Role of Advanced Glycation End Products on Dyslipidemia.

Disorders of lipoprotein metabolism and glucose homeostasis are common consequences of insulin resistance and usually co-segregate in patients with metabolic syndrome and type 2 diabetes mellitus (DM). Insulin-resistant subjects are characterized by atherogenic dyslipidemia, a specific lipid pattern which includes hypertriglyceridemia, reduced high-density lipoprotein cholesterol level, and increased proportion of small, dense low-density lipoprotein (LDL). Chronic hyperglycemia favors the processes of non-enzymatic glycation, leading to the increased production of advanced glycation end products (AGEs). Apart from direct harmful effects, AGEs are also potent inducers of oxidative stress and inflammation. In addition, increased AGEs' production may induce further qualitative modifications of small, dense LDL particles, converting them to glycated LDLs. These particles are even more atherogenic and may confer an increased cardiovascular risk. In this narrative review, we summarize the available evidence of the pathophysiological role and clinical importance of circulating AGEs and glycated LDLs in patients with dyslipidemia, particularly those with DM and related complications. In addition, we discuss recent advances and the issues that should be improved regarding laboratory assessment of AGEs and glycated LDLs, as well as the possibilities for their therapeutic modulation.

In Vitro Antibiofilm Effect of N-Acetyl-L-cysteine/Dry Propolis Extract Combination on Bacterial Pathogens Isolated from Upper Respiratory Tract Infections.

Bacterial biofilms play an important role in the pathogenesis of chronic upper respiratory tract infections. In addition to conventional antimicrobial therapy, N-acetyl-L-cysteine (NAC) and propolis are dietary supplements that are often recommended as supportive therapy for upper respiratory tract infections. However, no data on the beneficial effect of their combination against bacterial biofilms can be found in the scientific literature. Therefore, the aim of our study was to investigate the in vitro effect of N-acetyl-L-cysteine (NAC) and dry propolis extract in fixed combinations (NAC/dry propolis extract fixed combination) on biofilm formation by bacterial species isolated from patients with chronic rhinosinusitis, chronic otitis media, and chronic adenoiditis. The prospective study included 48 adults with chronic rhinosinusitis, 29 adults with chronic otitis media, and 33 children with chronic adenoiditis. Bacteria were isolated from tissue samples obtained intraoperatively and identified using the MALDI-TOF Vitek MS System. The antimicrobial activity, synergism, and antibiofilm effect of NAC/dry propolis extract fixed combination were studied in vitro. A total of 116 different strains were isolated from the tissue samples, with staphylococci being the most frequently isolated in all patients (57.8%). MICs of the NAC/dry propolis extract fixed combination ranged from 1.25/0.125 to 20/2 mg NAC/mg propolis. A synergistic effect (FICI ≤ 0.5) was observed in 51.7% of strains. The majority of isolates from patients with chronic otitis media were moderate biofilm producers and in chronic adenoiditis they were weak biofilm producers, while the same number of isolates in patients with chronic rhinosinusitis were weak and moderate biofilm producers. Subinhibitory concentrations of the NAC/propolis combination ranging from 0.625-0.156 mg/mL to 10-2.5 mg/mL of NAC combined with 0.062-0.016 mg/mL to 1-0.25 mg/mL of propolis inhibited biofilm formation in all bacterial strains. Suprainhibitory concentrations ranging from 2.5-10 mg/mL to 40-160 mg/mL of NAC in combination with 0.25-1 mg/mL to 4-16 mg/mL of propolis completely eradicated the biofilm. In conclusion, the fixed combination of NAC and dry propolis extract has a synergistic effect on all stages of biofilm formation and eradication of the formed biofilm in bacteria isolated from upper respiratory tract infections.

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.

Quercetin ameliorates experimental autoimmune myocarditis in rats.

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

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.

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.

Hashimoto Thyroiditis and Dyslipidemia in Childhood: A Review.

Hashimoto autoimmune thyroiditis (AIT) is the most common cause of acquired hypothyroidism in the pediatric population. Development of AIT is mediated mainly by cellular immune response directed toward thyroid autoantigens, leading to inflammation and impaired function of thyroid gland. Both thyroid dysfunction and inflammation affect the metabolism of plasma lipoproteins. The alterations in lipid profile worsen with the advancement of hypothyroidism, ranging from discrete changes in euthyroid AIT patients, to atherogenic dyslipidemia in the overt hypothyroidism. In this review, characteristics of dyslipidemia in pediatric AIT patients, and the consequences in respect to the risk for cardiovascular disease (CVD) development are discussed. Additionally, benefit of L-thyroxine treatment on serum lipid profile in pediatric AIT patients is addressed. Finally, potential usefulness of novel lipid biomarkers, such as proprotein convertase subtilisin/kexin type 9 (PCSK9), non-cholesterol sterols, low-density lipoprotein particle size and number, and high-density lipoprotein structure and functionality in AIT patients is also covered. Further longitudinal studies are needed in order to elucidate the long-term cardiovascular outcomes of dyslipidemia in pediatric patients with Hashimoto AIT.

Beneficial effects of dimethyl fumarate on experimental autoimmune myocarditis.

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