Valquiria Bueno

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes.

Autoimmune uveitis: the associated proinflammatory molecules and the search for immunoregulation.

Uveitis is an intraocular inflammatory disease causing a significant visual impairment. The disease can be idiopathic, associated with infectious and systemic disorders or arisen from an unknown cause. Over the last 20years the model of EAU in mice has contributed significantly for the establishment of parameters for diagnostic evaluations and therapies for posterior uveitis in human. Many studies using recently discovered molecules which present proinflammatory and anti-inflammatory effects have been described. Moreover, new approaches of research provided by the increasing body of knowledge on components of immune responses such as cytokines, T-cell subpopulations and their associated functions have contributed for the further understanding of uveitis and possible treatment.

Ageing and myeloid-derived suppressor cells: possible involvement in immunosenescence and age-related disease

Infections, cancer and autoimmune diseases occur more frequently in the elderly, and although many factors contribute to this, the age-related remodelling of the immune system, termed immunosenescence, plays a major role. Over the last two decades, studies have evaluated the effect of ageing on both the adaptive and innate arms of the immune system and demonstrated compromised function in several cells including lymphocytes (naïve, effector and memory), regulatory T and B cells, monocytes, neutrophils and NK cells. In addition, a well-documented feature of ageing is the increase in systemic inflammatory status (inflammageing), with raised serum levels of IL6, TNFα and CRP as well as reduced IL10. Recently, myeloid-derived suppressor cells have been the focus of many reports as these cells show immunosuppressive properties and are present in higher frequency during infections, cancer and autoimmunity. Importantly, there have been publications showing increased numbers of myeloid-derived suppressor cells in aged mice and humans. In this review, we discuss the current literature on myeloid-derived suppressor cells, their possible role in altered immune function in the elderly, and whether it may be possible to manipulate these cells to alleviate age-related immune dysfunction.

TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720.

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR+FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR.

FTY720 and lung tumor development.

FTY720 has been shown to prevent cancer development in experimental models but there is no report whether this beneficial effect is associated with the time point of the drug administration. Lung adenoma was induced in mice by urethane injection followed by different periods of FTY720 administration in order to evaluate lung tumor development. BALB/c mice received urethane intraperitoneally in two doses of 1.5 g/kg and were submitted to five daily doses of FTY720 (1 mg/kg/day) starting just after urethane injection (G2 n=5), 4 weeks after urethane injection (G3 n=10), 8 weeks after urethane injection (G4 n=10) and no FTY720 administration (G1 n=5). Twenty-four weeks after urethane administration mice were evaluated for the number of leukocyte in blood, lymphocytes in spleen, and lungs were evaluated for changes in histology, PCNA and VEGF expression. Lung nodules were present in higher numbers both in non treated (G1; 0.0-7.0) and FTY720 treated 8 weeks after urethane injection (G4; 0.0-6.0). G4 Group also presented the highest number of papillary nodules. G1 and G4 groups presented the lower number of splenocytes and neutrophils. In early time FTY720 treated mice (G2) we observed a slight decrease in PCNA staining and also the lower percentage of VEGF intense staining. Therefore, our data suggest that the benefits of FTY720 treatment are time-dependent and when administered in early periods after lung tumor induction this drug could impair cancer development.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system.

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.

Evaluation of lymphocyte levels in a random sample of 218 elderly individuals from São Paulo city

Background Age-associated changes in the immune system cause decreased protection after vaccination and increased rates of infections and tumor development. Methods Lymphocyte percentages were compared by gender and age to establish differences between subtypes. Three mL blood samples were obtained from 218 randomly selected individuals (60-101 years old) who live in São Paulo city. Blood was lysed with Tris phosphate buffer and stained for 30 minutes with monoclonal antibodies (CD3PerCP, CD4FITC, CD8Pe, CD19Pe) for analysis by flow cytometry. Statistical analysis was by ANOVA. Results The percentage of CD4+ T cells (p-value = 0.005) and the CD4/CD8 ratio (p-value = 0.010) were lower in men, whereas the percentage of CD8+ T cells was lower (p-value = 0.002) in women; the percentage of B cells (CD19+ ) was similar between groups. Individuals grouped by gender and age range and compared showed a drop in CD4+ cells in 75 to 79-year-old men (female: 46.1% ± 8.1% and male: 38.8% ± 10.5%; p-value = 0.023). Also, the 80 to 84-year-old group of men had a higher percentage of CD8+ (female: 20.8% ± 8.2%, and male: 27.2% ± 8.2%; p-value = 0.032). Low percentages of B cells were detected in men in the 75 to 79-year-old (p-value = 0.003), 85 to 89-year-old (p-value = 0.020) and older than 90 year old (p-value = 0.002) age ranges. Conclusion Elderly men present with more changes in lymphocyte subsets compared to elderly women. These findings could demonstrate impairment in the immune response since the lower CD4+ in men would provide less help to B cells (also lower in men) in terms of antibody production. In addition, the increase in CD8+ cells in this group could represent chronic inflammation observed during the aging process.

Identification of myeloid-derived suppressor cells and T regulatory cells in lung microenvironment after Urethane-induced lung tumor

Tumor growth occurs by the imbalance between cells with effector function and cells with suppressor/regulatory functions. To investigate this scenario we administered the chemical carcinogen Urethane in BALB/c mice and followed these animals during 120 days to observe lung tumor development. In another set of experiments the same protocol was performed with the harvest of spleen, lung and blood at 20 and 30 days after Urethane injection. The lung was used for histology, spleen cells were evaluated for IFN-γ production, and serum nitrite was measured as an indirect form of nitric oxide (NO) evaluation. The spleen and lung-infiltrating cells were evaluated by flow cytometry for CD11b+Gr-1+ myeloid suppressor cells and CD4+FoxP3+ T regulatory cells. Urethane led to lung nodules development after 120 days and the time point evaluation showed that splenocytes stimulated ex vivo expressed higher levels of IFN-γ 20 days after the chemical injection. Also, the level of nitric oxide in serum was higher after 20 days of Urethane injection. There was no statistical difference in spleen cells percentages for CD11b+Gr-1+ and CD4+Foxp3+ in all groups. However, lung-infiltrating cells presented early (20 days) a higher expression of CD11b+Gr-1+ suggesting suppression at this site. In conclusion, it was possible to observe two distinct events at the very early time point after Urethane injection. In periphery there was an increase at the effector immune response (as depicted by IFN-γ-producing cells) and in tumor development site there was an increase at the suppressor cell (CD11b+Gr-1+) phenotype. Suppressor/regulatory cells are targets for cancer therapy.

Skin Allograft Survival and Analysis of Renal Parameters After FTY720 + Tacrolimus Treatment in Mice

Calcineurin inhibitors such as cyclosporine (CsA) and tacrolimus (FK506) show similar efficacy to prevent rejection within the first year after organ transplantation. However, their use is limited by side effects, such as kidney damage, hypertension, new-onset diabetes, and hyperlipidemia. The consensus opinion suggests that compared with CsA, FK506 has fewer negative effects on blood pressure, serum lipids, and renal function. Nevertheless, FK506 use is associated with a higher incidence of posttransplantation diabetes mellitus. FTY720 is a new compound that has shown beneficial effects in animal models of rejection in transplantation, ischemia/reperfusion injury, autoimmune diseases, and tumor development. Our aim was to investigate whether FTY720 + tacrolimus association could provide additional immunosuppression without causing renal toxicity. FTY720 as a monotherapy or in association with FK506 was administered to C57BL/6 mice for 21 days to prevent skin graft rejection and to evaluate renal function and structure. Increased skin allograft survival in the FTY720 + FK506 group was associated with decreased cell numbers in the spleen, blood, and axillary lymph nodes. Changes in major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1) expressions in splenocytes were also found in this group. The major effects already described for FK506 (diabetes) or FTY720 (lymphopenia) were observed after 21 days administration even when the drugs were associated. FTY720 associated with FK506 caused fewer changes in kidney structure, and blood glucose levels were lower than in FK506 monotherapy.

Evaluation of renal function and immune system cells in elderly individuals from São Paulo City

OBJECTIVES: Both renal function and immune system function decline with age. Although controversial, a significant number of studies have shown that the decline in kidney function is associated with the worsening of the immune system. These findings are reinforced by the increased susceptibility to infections and deficient immunization coverage after vaccination both in patients with chronic renal disease and in elderly individuals. Our objective was to evaluate a non-institutionalized elderly population from São Paulo City and correlate the estimated glomerular filtration rate with the percentage of lymphocytes in circulation. METHODS: A random population of 237 individuals (107 men and 130 women), ranging in age from 60 to 101 years, who were enrolled in the Health, Well-Being and Aging Study was evaluated for renal function (Modification on Diet in Renal Disease formula) and lymphocyte percentage (flow cytometry). RESULTS: Aging was associated with a decrease in the estimated glomerular filtration rate in both male and female individuals. We did not identify a significant correlation between the estimated glomerular filtration rate and either the percentage of CD4, CD8, and B cells or CD4/CD8 ratio. The median percentage of CD8+ T cells was significantly lower in individuals with an estimated glomerular filtration rate ≥60 mL/min/1.73 m2. CONCLUSIONS: In this study, no statistical correlation was found between the estimated glomerular filtration rate and either the lymphocyte phenotype (CD4+, CD8+, and CD19+ cells) or the CD4/CD8 ratio in blood.