Immunosenescence: participation of T lymphocytes and myeloid-derived suppressor cells in aging-related immune response changes / Imunosenescência: participação de linfócitos T e células mieloides supressoras nas alterações da resposta imune relacionadas ao envelhecimento

ABSTRACT Healthy aging is partly related to appropriate function of the immune system. As already reported, some changes in this system are observed, including reduced number and repertoire of T cells due to thymic involution, accumulation of memory T cells by chronic infections, homeostatic proliferation compensating for the number of naïve T cells, decreased proliferation of T cells against a stimulus, telomere shortening, replicative senescence of the T cells, and inflammaging, besides the accumulation of myeloid-derived suppressor cells. The purpose of this article is to clarify each of these changes, aiming to minimize limitations of immunosenescence. If such associations can be established, these cells may be used as early and less invasive markers of aging-related diseases, as well as to indicate interventions, evaluate the efficacy of interventions and be a tool to achieve longevity with quality of life.

RESUMO O envelhecimento saudável está relacionado, pelo menos em parte, com a função adequada do sistema imunológico. Isso porque já foi relatado que, com o envelhecimento, algumas mudanças desse sistema são observadas, como a diminuição da percentagem e do repertório de células T pela involução tímica, acúmulo de células T de memória por infecções crônicas, compensação do número de células T naïve por proliferação homeostática, diminuição da capacidade de proliferação das células T frente a um estímulo, encurtamento dos telômeros, senescência replicativa das células T, e inflammaging, além do acúmulo de células mieloides supressoras. Este artigo visa esclarecer cada uma das mudanças, mencionadas, com o intuito de buscar meios de minimizar as limitações da imunosenescência. Caso seja possível estabelecer tais relações, essas células podem ser utilizadas como marcadores precoces e pouco invasivos de doenças relacionadas ao envelhecimento, além da possibilidade de serem utilizadas para indicar intervenções, avaliar a eficácia das intervenções e como ferramenta para alcance da longevidade com qualidade de vida.

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. .

Myeloid-derived suppressor cells and associated events in urethane-induced lung cancer

OBJECTIVES: Myeloid-derived suppressor cells contribute to the immunosuppressive microenvironment during tumor development and limit the efficacy of cancer immunotherapy. Identifying myeloid-derived suppressor cells and associated factors is the first step in creating strategies to reverse the suppressive effects of these cells on the immune system. METHODS: To induce lung cancer, we administered 2 doses of urethane to BALB/c mice and observed these animals for 120 days. After this period, we evaluated the percentage of myeloid-derived suppressor cells in the blood, lung and bone marrow. The expression of alpha-smooth muscle actin, transforming growth factor-β, Toll-like receptor 2, Toll-like receptor 4, and interleukin-6 was also determined in the lung tissue. RESULTS: Myeloid-derived suppressor cells were increased in all evaluated tissues after lung cancer development in association with increased Toll-like receptor 4 expression and decreased interleukin-6 expression in the lung. We observed alpha-smooth muscle actin and transforming growth factor-β expression in lung nodules. CONCLUSIONS: We believe that the early diagnosis of cancer through determining the blood levels of myeloid-derived suppressor cells followed by the depletion of these cells should be further investigated as a possible approach for cancer treatment. .

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.

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

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 percent ± 8.1 percent and male: 38.8 percent ± 10.5 percent; p-value = 0.023). Also, the 80 to 84-year-old group of men had a higher percentage of CD8+ (female: 20.8 percent ± 8.2 percent, and male: 27.2 percent ± 8.2 percent; 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.

Changes in lymphocyte phenotype and increased skin allograft survival after FTY720+FK506 therapy / Modificação do fenótipo linfocitário e aumento da sobrevida do enxerto de pele após a terapia com FTY720 + FK506

The development of new drugs to be associated with calcineurin inhibitors and promote additional immunosuppression with fewer side effects is the goal in transplantation. FTY720 is a new synthetic compound which presents immunomodulatory properties which are not fully understood. It has been reported that the main mechanism of action of FTY720 is to reduce the peripheral lymphocyte count by redirecting these cells toward secondary lymphoid organs. Skin allograft transplantation in a fully mismatched strain combination was used to investigate the potential of FTY720 alone or in combination with a calcineurin inhibitor - FK506 - in preventing rejection. The number and phenotype of immune system cells was also evaluated. FTY720 alone or in combination with FK506 provided significant skin allograft survival. FTY720+FK506 therapy was associated with decreases of total lymphocyte numbers in spleen and blood, and increases in apoptosis levels in splenocytes. In FTY720 isolated treatment, a significant decrease in the CD4 expression and significantly lower expressions of MHC II and ICAM-1 molecules were observed in spleen lymphocytes. Despite of allograft survival being the same in both FTY720 and FTY720+FK506 treated groups, the association of drugs was associated with the absence of macroscopic skin necrosis for a longer period than the other treatments (FTY720, FK506) and histology showed less cell infiltration. Our results suggest that a decrease of effector T cells due to elevated levels of apoptosis and impairment in the appearance of antigens were events associated with FTY720+FK506 administration.

O objetivo na área dos transplantes é o desenvolvimento de novas drogas que possam ser associadas a inibidores da calcineurina para evitar o processo de rejeição e causar menos efeitos colaterais. FTY720 é um novo composto sintético que apresenta propriedades imunomoduladoras não completamente elucidadas. Foi relatado que o principal mecanismo de ação do FTY720 é a redução do número de linfócitos periféricos através do redirecionamento dessas células para órgãos linfóides secundários. O alotransplante de pele entre linhagens de camundongos completamente incompatíveis quanto ao MHC foi usado para investigar o potencial de FTY720 isolado ou em combinação com um inibidor da calcineurina - FK506 - na prevenção da rejeição. Também foram avaliados o número e fenótipo das células do sistema imune. A administração de FTY720 como monoterapia ou FTY720+FK506 associou-se a uma diminuição do número total de linfócitos no baço e no sangue e aumento dos níveis de apoptose nos esplenócitos. No grupo tratado somente com FTY720, foi observada uma diminuição mais importante da expressão de CD4 e expressão significativamente menor de moléculas de MHC II e ICAM-1. Apesar de a sobrevida do aloenxerto ter sido igual para os grupos tratados com FTY720 ou FTY720 +FK506, a associação das drogas promoveu ausência de necrose macroscópica da pele por um período maior do que os outros tratamentos (FTY720, FK506) e os achados histológicos mostraram menor infiltrado celular. Nossos resultados sugerem que uma diminuição do número de células T efetoras devido a elevados níveis de apoptose e o prejuízo da apresentação de antígenos foram os eventos associados à administração de FTY720+FK506.

Ischemic and reperfusion syndrome of hind limbs: functional and histological renal changes in rats

Ischemic and reperfusion injury of the extremities may result in a systemic, severe and complex metabolic syndrome, manifested by acute renal failure, myoglobinuria, metabolic acidosis, hipercalemia and free radicals releasing. We investigated the functional and histologic renal changes after ischemia and reperfusion of the hind limb skeletal muscles. Rats were submitted to the ligature of the infrarenal aorta for 6 and 12 h. The animals were then randomized into four groups of 10 rats: Group I, 6 h of ischemia and 24 h of reperfusion; Group II, 12 h of ischemia and 24 h of reperfusion; Group III, 6 h of ischemia and 10 days of reperfusion; and Group IV, Group sham with no ischemia or reperfusion. Blood samples at the end of the experiment and urine volume in the first 24 h of reperfusion in group I and II and in the last day in group III for functional analysis were collected. The following renal functional parameters were studied: creatinine plasmatic level, creatinine depuration and sodium urinary/creatinine urinary ratio. The kidneys were removed and a histological tubulo-interstitial lesional index was evaluated for each animal. We found higher plasma creatinine levels and morphologic changes in groups submitted to ischemia and reperfusion. Ten days after reperfusion, the histologic changes persisted despite the recovery of renal function.

Avaliaçäo da imunidade periférica, pela técnica do linfonodo poplieteal após a injeçäo intratímica de antígenos do doador / Evaluation of peripherical immunity trough popliteal linfonode technic after intrathymic injection of donor antigens

A injeçäo intratímica (IT) é uma técnica eficiente nainduçäo de tolerância imunológica em modelos experimentais, nos quais se pode alcançar uma imunossupressäoespecífica sem comprometer o restante do sistema imune. Utilizou-se o modelo da técnica do linfonodo popliteal (TLP) para avaliar a resposta imune periférica aos aloantígenos previamente apresentados ao receptor pela IT. Camundongos entre 6 e 8 semanas, BALB/c (H2)foram usados como recptores. Esplenócitos foram isolados de camundongos DBA/2(H2), C57BL/6(H2) e C3H/He(H2), todos com a mesma faixa etária. A IT foi realizada7 dias do estímulo na pata (10 células). Os linfonodos foram retirados e pesados 7 dias após a TLP. Estímulo com células alogênicas aumentaram 3 vezes o peso do linfonodo quando comparado a células isogênicas (5,7+- 1,5 vs 2,6+-0,8p<0,001). IT com C57BL/6 reduziu as células C3H/He näo reduziram o peso do linfonodo subseqüente ao estímulo com C57BL/6 (8,9 +-3,4vc 5,7 +-1,7, p0,01. especificamente as células C3H/He näo reduziram o peso do linfonodo subseqüente ao estímulo com C57BL/6 (8,9 +-3,4 vs 5,7 +-1,5, p=0,08). A IT capaz de reduzir especificamente a resposta imune periférica aos aloantígenos verificada pela TLP

Injeção intratímica de antígenos do doador em modelos experimentais de imunização e transplante de órgãos / Effects of donor antigens intratymic injection in animal models of transplatation and immunization

O uso crônico de drogas imunossupressoras em transplante está associado a complicaçoes severas como infecçoes oportunistas, neoplasias, e toxicidade, além de nao controlar totalmente o processo de rejeiçao. A meta em transplante é a ausência de resposta imune específica ao doador sem comprometer o restante do sistema imune do receptor. Uma possível estratégia é o tratamento do receptor com injeçao intratímica de antígenos do doador associada ao uso de imunossupressao no período peritransplante. Esta estratégia, denominada tolerância intratímica, tem sido utilizada com sucesso em modelos experimentais de transplante de órgaos. Utilizamos a injeçao intratímica de 2xl07 esplenócitos do doador na tentativa de aumentar a sobrevida dos aloenxertos cardíaco e renal, e diminuir a resposta proiiferativa no ensaio do linfonodo popliteal. No protocolo de transplante cardíaco heterotópico abdominal em ratos, utilizamos como doador Munich Wistar (RT- 1a.) e receptor Wistar (outbred). A mesma combinaçao foi utilizada para o transplante renal ortotópico. No modelo de transplante cardíaco a injeçao intratímica foi feita concomitante ou quatro dias antes do transplante, sendo que no transplante renal o procedimento foi realizado apenas concomitante ao transplante. No protocolo de transplante cardíaco heterotópico abdominal em camundongos, C57BL/6 foram doadores e BALB/c receptores, sendo estes animais completamente dispares quanto aos antígenos principais e secundários de histocompatibilidade. A injeçao intratímica foi realizada concomitante ou sete dias antes do transplante. O agente imunossupressor associado foi o anticorpo monocional anti-CD3. No modelo do linfonodo popliteal, as combinaçoes de camundongos foram as mesmas daquelas utilizadas para o transplante cardíaco. O seceptor foi estimulado na pata direita traseira com 107 esplenócitos do doador e após sete dias, o linfono do popliteal drennte foi retirado e pesado. A injeçao intratímica de antígenos do doador foi realizada sete dias antes do estímulo na pata. Nos modelos de transplante em ratos foi possível aumentar significativamente a sobrevida do aloenxerto apenas com o uso da injeçao intratímica concomitante ou previamente ao transplante. O mesmo ocorreu no ensaio de linfonodo popliteal, onde a injeçao intratímica isolada diminuiu a resposta proliferativa específica aos antígenos do doador. Contudo, a injeçao intratímica de antígenos do doador nao...(au)