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.

Selectivity of polyclonal repertoire of anti-microbial IgA and its subclasses in saliva and serum in humans.

Increased interest in microbiota calls for the thorough analysis of antibody reactivity to different microorganisms. As salivary IgA represents the first line of defence against microorganisms contacting mucosal surfaces, we explored the binding and specificity of salivary IgA by testing the binding of purified, FITC-labelled salivary IgA to different microorganisms in flow cytometry and conclude that this kind of analysis enables the differentiation of species/strains with high IgA binding capacity, which should be corroborated on a larger sample size. Further we compare, with in-house ELISA, the binding of polyclonal salivary IgA with the binding of polyclonal serum IgA from the same individuals to whole microbial cells and to purified microbial components. High correlations were obtained in total salivary IgA binding to Lactobacillus rhamnosus and Escherichia coli, very distant bacterial species, as well as to isolated bacterial components (r = .70-.97). The binding of total salivary IgA resembled the binding of both salivary IgA1 and IgA2, with IgA2 predominating. For serum polyclonal IgA repertoire, substantially higher specificity was obtained. Serum IgA binding to E. coli correlated best with serum IgA binding to lipopolysaccharide (r = .86), and serum IgA against L. rhamnosus correlated best with the anti-peptidoglycan IgA levels (r = .88). We have also detected that total serum IgA response is governed by either IgA1 or IgA2 response, depending on the nature of the antigen/s. We conclude that steady state salivary IgA repertoire, unlike serum IgA repertoire, consists of polyreactive antibodies with innate specificity, questioning its capacity to select resident microbiota.

Lactobacillus rhamnosus Affects Rat Peritoneal Cavity Cell Response to Stimulation with Gut Microbiota: Focus on the Host Innate Immunity.

Gut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peritoneal cavity cell response to in vitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon in vitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163- and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory priming by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release in vitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host.

The involvement of estrogen receptors α and ß in the in vitro effects of 17ß-estradiol on secretory profile of peritoneal macrophages from naturally menopausal female and middle-aged male rats.

The systemic and extra- gonadal levels of 17ß-estradiol (E2) change during aging, and affect the expression of estrogen receptors (ERs) in the immune cells of both females and males. The age-related cessation of ovarian function in females, as well as the tissue-specific expression of enzyme aromatase (estrogen synthase which significantly rises with the advancing age) in both males and females, both determine the concentration of E2 to which immune cells may be exposed. The present study was set up to investigate the direct influence of E2 in vitro on the secretory profile of peritoneal macrophages from young and naturally menopausal female rats, and from young and middle-aged male rats. The involvement of receptor(s) responsible for mediating the effects of E2 in vitro was examined by use of antagonists specific for ERα or ERß. Whereas in macrophages from young female rats E2 treatment diminished interleukin (IL)-1ß secretion, it increased it in young males, and the middle-aged females. The in vitro E2 treatment increased tumor necrosis factor (TNF)-α release by macrophages from young rats of both sexes, while it increased macrophage IL-6 release independently of both sex and age. At the same time, E2 decreased hydrogen peroxide (H2O2) production in macrophages from females, and increased it in male rats of both ages, whereas it diminished nitric oxide (NO) release in all experimental groups. Inspite of the sex- and age-specific effects of E2 on macrophage urea release, E2 did not affect the NO/urea ratio in macrophages from female rats, and diminished it in macrophages from both young and middle-aged male rats. Independently of the sex and age, E2 stimulated the release of inflammatory cytokines predominantly via macrophage ERα, and inhibited the IL-1ß release in young females via ERß. In contrast, E2 increased macrophage H2O2 and urea production by activating ERß, but diminished their release via ERα. Our study may contribute to better understanding of the complex role(s) that E2 may play in innate immunity during aging, and that are dependent of sex.

Rat strain differences in peritoneal immune cell response to selected gut microbiota: A crossroad between tolerance and autoimmunity?

AIMS: Some gut commensals can be protective, whereas others are implicated as necessary for development of inflammatory/autoimmune diseases. Peritoneal immune cells may play an important role in promoting autoimmunity in response to gut microbiota. This study investigated the phenotype and the function of peritoneal immune cells in the autoimmunity-resistant Albino Oxford (AO), and the autoimmunity-prone Dark Agouti (DA) rat strains upon stimulation with their own colonic E. coli or Enterococcus. MAIN METHODS: Rats were intraperitoneally injected with their own E. coli or Enterococcus. Peritoneal cells isolated two days later were tested for nitric oxide (NO) and cytokine production, and for arginase and myeloperoxidase (MPO) activity. The phenotype of cells was determined using flow cytometry. KEY FINDINGS: While the Enterococcus injection did not affect the composition of peritoneal cells in AO rats, the E. coli treatment increased the percentages of activated CD11bintHIS48hi neutrophils, and decreased the proportion of resident (CD11bhiHIS48int/low, CD163 + CD86+) and anti-inflammatory CD68 + CD206+ macrophages. E. coli increased the production of NO and urea, but preserved their ratio in cells from AO rats. Conversely, both E. coli and Enterococcus diminished the proportion of resident and anti-inflammatory macrophages, increased the proportion of activated neutrophils, and induced inflammatory polarization of peritoneal cells in DA rats. However, injection of E. coli maintained the ratio of typical CD11bintHIS48int neutrophils in DA rats, which correlated with the sustained MPO activity. SIGNIFICANCE: The rat strain differences in peritoneal cell response to own commensal microbiota may contribute to differential susceptibility to inflammatory/autoimmune diseases.

Sex Differences in Macrophage Functions in Middle-Aged Rats: Relevance of Estradiol Level and Macrophage Estrogen Receptor Expression.

The aim of this study was to examine the influence of sex on age-related changes in phenotype and functional capacity of rat macrophages. The potential role of estradiol as a contributing factor to a sex difference in macrophage function with age was also examined. Thioglycollate-elicited peritoneal macrophages derived from the young (2 months old) and the naturally senescent intact middle-aged (16 months old) male and female rats were tested for cytokine secretion and antimicrobial activity (NO and H2O2 production and myeloperoxidase activity). Serum concentration of estradiol and the expression of estrogen receptor (ER)α and ERß on freshly isolated peritoneal macrophages were also examined. Decreased secretion of IL-1ß and IL-6 by macrophages from middle-aged compared to the young females was accompanied with the lesser density of macrophage ERα expression and the lower systemic level of estradiol, whereas the opposite was true for middle-aged male rats. Macrophages in the middle-aged females, even with the diminished circulating estradiol levels, produce increased amount of IL-6, and comparable amounts of IL-1ß, TNF-α, and NO to that measured in macrophages from the middle-aged males. Age-related changes in macrophage phenotype and the antimicrobial activity were independent of macrophage ERα/ERß expression and estradiol level in both male and female rats. Although our study suggests that the sex difference in the level of circulating estradiol may to some extent contribute to sex difference in macrophage function of middle-aged rats, it also points to more complex hormonal regulation of peritoneal macrophage activity in females.

Strain-dependent response to stimulation in middle-aged rat macrophages: A quest after a useful indicator of healthy aging.

Rats of Albino Oxford (AO) strain in our animal facility exhibit a longer average healthy life span than rats of Dark Agouit (DA) strain. Since chronic activation of macrophages contributes to chronic low level inflammation common in older age, elucidation of the changes in middle-aged rats could be useful in prevention of unbalanced inflammatory response in advanced age. We have analysed the phenotype of unelicited and thioglycollate-elicited peritoneal macrophages from young and middle-aged DA and AO rats and tested functions of these cells following stimulation with lipopolysaccharide (LPS) in vitro. Unelicited cells from middle-aged DA rats produced higher amounts of proinflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO), but have a diminished response to LPS stimulation then cells from young rats, in spite of increased frequency of TLR4- and CD14-expressing mature macrophages. Injection of thioglycollate robustly increased overall cytokine production in young rats' macrophages, while diminishing their response to LPS stimulation. In middle-aged DA rats injection of thioglycollate diminished IL-6 production, but increased it in response to LPS stimulation. Quite the contrary to DA rats, the macrophages from middle-aged AO rats have released diminished levels of TNF-α and NO, whereas urea production was strongly increased, when compared to the macrophages from young rats. Although the thioglycollate injection has increased the proportion of CD86+MHCII+ mature macrophages in young rats, and percentages of activated TLR4+ macrophages in both age groups of AO rats, it has not affected the cytokine production in young rats' macrophages, and the TNF-α production in middle-aged rats' macrophages. Moreover, the injection of thioglycollate has robustly increased the production of urea in macrophages derived from both age groups of AO rats. Although middle-aged rats of both strains were healthy during experiment, differences between the inflammatory responses of peritoneal macrophages of middle-aged rats of these strains might be one of the contributing factors defining their health in their advanced age. Development of strategies for the prevention of undesirable inflammatory changes in the elderly would benefit from the prospective study of the middle-aged.

Aging affects the responsiveness of rat peritoneal macrophages to GM-CSF and IL-4.

Macrophages undergo significant functional alterations during aging. The aim of the present study was to investigate changes of rat macrophage functions and response to M1/M2 polarization signals with age. Therefore, resident and thioglycollate-elicited peritoneal macrophages from young (3-month-old) and aged (18-19-month-old) rats were tested for phagocytic capacity and ability to secrete inflammatory mediators following in vitro stimulation with LPS and GM-CSF, and IL-4, prototypic stimulators for classically (M1) and alternatively activated (M2) macrophages, respectively. Aging increased the frequency of monocyte-derived (CCR7+ CD68+) and the most mature (CD163+ CD68+) macrophages within resident and thioglycollate-elicited peritoneal macrophages, respectively. The ability to phagocyte zymosan of none of these two cell subsets was affected by either LPS and GM-CSF or IL-4. The upregulated production of IL-1ß, IL-6 and IL-10 and downregulated that of TGF-ß was observed in response to LPS in resident and thioglycollate-elicited macrophages from rats of both ages. GM-CSF elevated production of IL-1ß and IL-6 in resident macrophages from aged rats and in thioglycollate-elicited macrophages from young rats. Unexpectedly, IL-4 augmented production of proinflammatory mediators, IL-1ß and IL-6, in resident macrophages from aged rats. In both resident and thioglycollate-elicited macrophages aging decreased NO/urea ratio, whereas LPS but not GM-SCF, shifted this ratio toward NO in the macrophages from animals of both ages. Conversely, IL-4 reduced NO/urea ratio in resident and thioglycollate-elicited macrophages from young rats only. In conclusion, our study showed that aging diminished GM-CSF-triggered polarization of elicited macrophages and caused paradoxical IL-4-driven polarization of resident macrophages toward proinflammatory M1 phenotype. This age-related deregulation of macrophage inflammatory mediator secretion and phagocytosis in response to M1/M2 activators may lead to the deficient control of infectious and/or inflammatory diseases in advanced age.

Unopposed Estrogen Supplementation/Progesterone Deficiency in Post-Reproductive Age Affects the Secretory Profile of Resident Macrophages in a Tissue-Specific Manner in the Rat.

PROBLEM: The influence of unopposed estrogen replacement/isolated progesterone deficiency on macrophage production of pro-inflammatory/anti-inflammatory mediators in the post-reproductive age was studied. METHOD OF STUDY: Considering that in the rats post-ovariectomy the circulating estradiol, but not progesterone level rises to the values in sham-operated controls, 20-month-old rats ovariectomized at the age of 10 months served as an experimental model. Estrogen and progesterone receptor expression, secretion of pro- and anti-inflammatory cytokines, and arginine metabolism end-products were examined in splenic and peritoneal macrophages under basal conditions and following lipopolysaccharide (LPS) stimulation in vitro. RESULTS: Almost all peritoneal and a subset of splenic macrophages expressed the intracellular progesterone receptor. Ovariectomy diminished cytokine production by splenic (IL-1ß) and peritoneal (TNF-α, IL-1ß, IL-10) macrophages and increased the production of IL-10 by splenic and TGF-ß by peritoneal cells under basal conditions. Following LPS stimulation, splenic macrophages from ovariectomized rats produced less TNF-α and more IL-10, whereas peritoneal macrophages produced less IL-1ß and TGF-ß than the corresponding cells from sham-operated rats. Ovariectomy diminished urea production in both subpopulations of LPS-stimulated macrophages. CONCLUSION: Although long-lasting isolated progesterone deficiency in the post-reproductive age differentially affects cytokine production in the macrophages from distinct tissue compartments, in both subpopulations, it impairs the pro-inflammatory/anti-inflammatory cytokine secretory balance.