Transforming growth factor receptor III (Betaglycan) regulates the generation of pathogenic Th17 cells in EAE.

The transforming growth factor receptor III (TßRIII) is commonly recognized as a co-receptor that promotes the binding of TGFß family ligands to type I and type II receptors. Within the immune system, TßRIII regulates T cell development in the thymus and is differentially expressed through activation; however, its function in mature T cells is unclear. To begin addressing this question, we developed a conditional knock-out mouse with restricted TßRIII deletion in mature T cells, necessary because genomic deletion of TßRIII results in perinatal mortality. We determined that TßRIII null mice developed more severe autoimmune central nervous neuroinflammatory disease after immunization with myelin oligodendrocyte peptide (MOG35-55) than wild-type littermates. The increase in disease severity in TßRIII null mice was associated with expanded numbers of CNS infiltrating IFNγ+ CD4+ T cells and cells that co-express both IFNγ and IL-17 (IFNγ+/IL-17+), but not IL-17 alone expressing CD4 T cells compared to Tgfbr3fl/fl wild-type controls. This led us to speculate that TßRIII may be involved in regulating conversion of encephalitogenic Th17 to Th1. To directly address this, we generated encephalitogenic Th17 and Th1 cells from wild type and TßRIII null mice for passive transfer of EAE into naïve mice. Remarkably, Th17 encephalitogenic T cells from TßRIII null induced EAE of much greater severity and earlier in onset than those from wild-type mice. The severity of EAE induced by encephalitogenic wild-type and Tgfbr3fl/fl.dLcKCre Th1 cells were similar. Moreover, in vitro restimulation of in vivo primed Tgfbr3fl/fl.dLcKCre T cells, under Th17 but not Th1 polarizing conditions, resulted in a significant increase of IFNγ+ T cells. Altogether, our data indicate that TßRIII is a coreceptor that functions as a key checkpoint in controlling the pathogenicity of autoreactive T cells in neuroinflammation probably through regulating plasticity of Th17 T cells into pathogenic Th1 cells. Importantly, this is the first demonstration that TßRIII has an intrinsic role in T cells.

Obesity modulates the immune macroenvironment associated with breast cancer development.

Growing evidence demonstrates a strong correlation between obesity and an increased risk of breast cancer, although the mechanisms involved have not been completely elucidated. Some reports have described a crosstalk between adipocytes, cancer cells, and immune cells within the tumor microenvironment, however, it is currently unknown whether obesity can promote tumor growth by inducing systemic alterations of the immune cell homeostasis in peripheral lymphoid organs and adipose tissue. Here, we used the E0771 breast cancer cell line in a mouse model of diet-induced obesity to analyze the immune subpopulations present in the tumors, visceral adipose tissue (VAT), and spleen of lean and obese mice. Our results showed a significant reduction in the frequency of infiltrating CD8+ T cells and a decreased M1/M2 macrophage ratio, indicative of the compromised anti-tumoral immune response reported in obesity. Despite not finding differences in the percentage or numbers of intratumoral Tregs, phenotypic analysis showed that they were enriched in CD39+, PD-1+ and CCR8+ cells, compared to the draining lymph nodes, confirming the highly immunosuppressive profile of infiltrating Tregs reported in established tumors. Analysis of peripheral T lymphocytes showed that tumor development in obese mice was associated to a significant increase in the percentage of peripheral Tregs, which supports the systemic immunosuppressive effect caused by the tumor. Interestingly, evaluation of immune subpopulations in the VAT showed that the characteristic increase in the M1/M2 macrophage ratio reported in obesity, was completely reversed in tumor-bearing mice, resembling the M2-polarized profile found in the microenvironment of the growing tumor. Importantly, VAT Tregs, which are commonly decreased in obese mice, were significantly increased in the presence of breast tumors and displayed significantly higher levels of Foxp3, indicating a regulatory feedback mechanism triggered by tumor growth. Altogether, our results identify a complex reciprocal relationship between adipocytes, immune cells, and the tumor, which may modulate the immune macroenvironment that promotes breast cancer development in obesity.

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells during Staphylococcus aureus infection.

Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1ß (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell.

3-Chloro-1,3,5-pregnatriene derivatives with glucocorticoid activity.

Novel synthetic glucocorticoid analogues were tested for receptor binding and glucocorticoid activity. They were of unusual structure, insofar as they had a 3-chloro rather than a 3-oxo function. 3-Chloro analogues of fluorinated glucocorticoids formed extremely stable complexes with the rat liver glucocorticoid receptor. 3-Chloro derivative of fluocinolone acetonide also had in vivo glucocorticoid activity. It induced tyrosine aminotransferase in the liver and repressed thymidine kinase in the thymus very effectively. It is concluded that 3-chloro analogues may retain glucocorticoid activity as well as the ability to bind to the glucocorticoid receptor protein.

Molybdate effect on the glucocorticoid receptor in cell-free systems and intact lymphocytes.

The effect of sodium molybdate on the stability and activation of the glucocorticoid receptor from chick and rat thymus were investigated. Molybdate, at a concentration range of 1-10 mM, blocked denaturation of the cytosol receptor by elevated (25 and 37 degrees C) temperatures. This effect could be observed only with the aggregated (low-salt) form of the receptor. Molybdate also inhibited transformation of the receptor-hormone complex to the DNA-binding state which occurs either with incubation at 25 degrees C or with salt treatment. The inhibitory effect of molybdate could be observed only on the non-activated receptor; nuclear-and DNA-binding of the activated receptor was not significantly changed by molybdate. Both effects were concentration-dependent. Molybdate had no effect on the activation of the partially purified glucocorticoid receptor. Molybdate effect was also examined using intact lymphocytes. Sodium molybdate had no effect either on the steroid binding of whole cells or on the nuclear transfer of the hormone-receptor complex. While the mechanism of molybdate action remains unknown the results of experiments on purified receptor suggest that molybdate does not act directly on the receptor molecule; rather through some cytosol factor(s). However, these effects could only be seen in cell-free experiments, and not during the conditions of the living cell.

Pyridoxal-5-phosphate affects glucocorticoid receptors in intact lymphocytes similarly as in cell-free systems.

Pyridoxal-5-phosphate markedly decreased the heat-stability of the unbound thymus cytosol receptor as well as that of the receptor-[3H]-triamcinolone acetonide complex in cell-free systems. Treatment with pyridoxal phosphate also decreased DNA-binding of the complex, being present either before or after heat- and salt-activation. 5 mM pyridoxal phosphate was required for 50% inhibition of DNA-binding. Pyridoxine, at similar concentrations, had no inhibitory effect. Pre-incubation of intact thymocytes with 10 mM pyridoxine caused also a marked decrease in the binding of [3H]-triamcinolone acetonide by the cells. Treating the cells with liposomes containing 1-100 mM pyridoxal phosphate caused an increase in intracellular pyridoxal phosphate concentration by about 0.1-10 microM and a decrease in [3H]-triamcinolone acetonide binding of the cells by about 50%. The results suggest that 1. pyridoxal phosphate acts not only on the activated but also on the unbound and non-activated forms of glucocorticoid receptor in cell-free systems; 2. pyridoxal phosphate has a similar effect in the intracellular millieu and thus, 3. pyridoxal phosphate might act as a physiologic regulator of the steroid hormone action.

Physicochemical characterization of the cytosol glucocorticoid receptors in various lymphoid tissues.

Cytosol glucocorticoid receptors of five different lymphoid tissues of chicken and rats were characterized and compared by physicochemical parameters. Binding specificity of receptors was found to be similar, except that cytosol receptors of chick tissues appeared slightly more specific for dexamethasone than those from rats. Association rate constants of the dexamethasone-receptor complex were higher by about one order of magnitude in the case of chicken thymus and bursa than in other cytosols. Dissociation of dexamethasone-receptor complex occurred in one step in rat tissues, whereas in case of chicken tissues two phases of dissociation were discernible. Differences between Ka and the ratio of the association and dissociation rate constants are explained by non-equilibrium conditions. The stability of receptors from the two species differed significantly, the chick receptor being more stable. Hormone-receptor complex from each tissue could be separated into two fractions by DE-52 chromatography. The peak eluted at the lower KCl concentration in the case of chick corresponded to the fast dissociating hormone-receptor complex. Kinetic analyses proved to be just as useful as ion-exchange chromatography for the characterization of, and distinguishing between, glucocorticoid receptors.

Thymidine kinase activity in murine lymphoid organs following glucocorticoid and heparin administration.

Thymidine kinase (TK) activity was studied in thymus and spleen of mice after glucocorticoid and heparin administration. Glucocorticoids (cortisone and hydrocortisone) injected intraperitoneally caused a prolonged 80--90% decrease in TK activity of both lymphoid organs. Heparin per se administered in depot-form subcutaneously did not alter the enzyme activity in the lymphoid organs significantly. By contrast, heparin injected 6 hours before glucocorticoid treatment inhibited the decreasing effect of cortisone but not that of hydrocortisone on TK activity in the thymus and fully inhibited the effect of both hormones on the enzyme activity of spleen. In addition the combined use of heparin and cortisone increased the splenic TK activity above the control value on the 2nd day after treatment. The possible mode of action of heparin is discussed.

Effects of steroids on Tetrahymena.

Phagocytosis of Tetrahymena is inhibited by prednisolone-sodium-succinate and deoxy-corticosterone-glucoside, and stimulated by dexamethasone and prednisolone. Dexamethasone and estradiol enter the cells and are localized first in food vacuoles, and later on in the cytosol. They were never found in the nucleus. The demonstration by biochemical methods of a specific glucocorticoid binding protein failed in all three subcellular fractions examined.

Analysis of thymidine kinase activity and glucocorticoid-binding capacity in the thymuses of healthy and tumor-bearing chickens.

Thymidine kinase activity in the lymphoid organs of healthy and tumor-bearing chickens was studied after hydrocortisone or dexamethasone treatment. Glucocorticoid hormones (10 mg/100 g body wt) administered twice in 48 hours resulted in an 80% decrease in thymidine kinase activity in the thymuses of healthy chickens, whereas birds with transplantable MC 29 chicken hepatoma failed to respond to the hormones. In the bursae of Fabricius of healthy or tumor-bearing chickens, thymidine kinase activity did not change considerably after administration of hydrocortisone or dexamethasone. The steroid-binding capacity in cellfree systems prepared from thymuses of tumor-bearing birds was significantly decreased. Thus the difference in steroid-binding capacity between thymuses of healthy and tumor-bearing chickens correlated well with, and could account for, the failure of hydrocortisone and dexamethasone to reduce the thymidine kinase level in tumor-bearing birds.

Tyrosine aminotransferase induction in normal and tumor-bearing chickens.

Tyrosine aminotransferase (TAT) induction by glucagon and dexamethasone in the liver of tumor-bearing chickens was studied and compared with induction in healthy animals. The transplantable tumor was caused by inoculation of cells from a cell line induced by MC29 avian leukosis virus. TAT was hardly detectable in tumor tissue of control and dexamethasone-treated chickens, but it was induced by glucagon to levels which were significant although very low when compared to those in host liver or the liver of non-tumor-bearing controls after glucagon treatment. Dexamethasone failed to induce TAT in host liver at 8 A.M. while it significantly indiced TAT in the normal liver at the same time of the day. Similar failure of TAT induction was not detectable when glucagon was used instead of dexamethasone. Furthermore, it was found that diurnal variations in basal and dexamethasone or glucagon-induced TAT levels are considerably mitigated in host liver as compared to those observed in the liver of healthy animals. The possible reasons for these findings are discussed.