RESUMO
Decidual immunological mediators modulate placental formation, decidualization and fetal development. However, the effect of maternal hyperthyroidism on decidual immunology needs further research. The aim of this study was to evaluate the population of uterine natural killer cells (uNKs) and the expression of immunological mediators in the decidua of female rats throughout pregnancy. Wistar rats were used and hyperthyroidism was induced by daily administration of L-thyroxine (T4) throughout pregnancy. The population of uNK cells in decidua was evaluated by immunostaining Lectin DBA, as well as the expression of interferon γ (INFγ), macrophage migration inhibitory factor (MIF), interleukin 15 (IL-15) and inducible nitric oxide synthase (iNOS) at 7, 10, 12, 14 and 19 days of gestation (DG). Maternal hyperthyroidism reduced the DBA+ uNK cell population in the decidua at 7 (P < 0.05) and 10 (P < 0.01) DGs compared to that in the control group, while it increased in the basal decidua (P < 0.05) and metrial gland (P < 0.0001) at the 12th DG. Hyperthyroidism also increased immunostaining of IL-15 (P < 0.0001), INFγ (P < 0.05), and MIF (P < 0.05) in the 7th DG, and increased immunostaining of IL-15 (P < 0.0001) and MIF (P < 0.01) in the 10th DG. However, excess thyroxine reduced IL-15 expression in the metrial gland and/or basal decidua in the 12th (P < 0.05), 14th (P < 0.01), and 19th (P < 0.001) DGs, as was also observed for INFγ in the basal decidua (P<0.001) and metrial gland (P < 0.0001) in the 12th DG. Regarding iNOS, an antiinflammatory cytokine, lower expression was observed in the basal decidua of hyperthyroid animals at 7 and 12 DGs (P < 0.05), whereas an increase occurred in the 10th DG (P < 0.05). These data demonstrate that maternal hyperthyroidism in female rats, particularly between 7 and 10 DGs, reduces the population of DBA+ uNKs in the decidua and increases the expression of inflammatory cytokines, suggesting a more proinflammatory environment in early pregnancy caused by this gestational disease.
Assuntos
Hipertireoidismo , Placenta , Ratos , Gravidez , Feminino , Animais , Placenta/metabolismo , Decídua/metabolismo , Interleucina-15/metabolismo , Interleucina-15/farmacologia , Ratos Wistar , Células Matadoras Naturais/metabolismo , Hipertireoidismo/metabolismoRESUMO
Triple Negative Breast Cancer (TNBC) treatment is still challenging, and immunotherapy is a potential approach in this tumor subtype. Cetuximab is an IgG1 monoclonal antibody (mAb) directed against Epidermic Growth Factor Receptor (EGFR), a protein overexpressed in a subgroup of TNBC patients and associated with poor prognosis. Previously, we demonstrated in vitro that Cetuximab triggers Ab-dependent cell cytotoxicity against TNBC cells. In this study, using co-cultures including TNBC cells, and NK and Dendritic Cells (DCs) from healthy donors, we studied the effect of Cetuximab-activated NK cells on DC function. Given that we already demonstrated that TNBC has an immunosuppressive effect on NK cells, we also tested Cetuximab combination with IL-15. We determined that Cetuximab opsonization of TNBC cells increased IFN-γ and TNF-α production by NK cells co-cultured with DCs. Moreover, we showed that NK cells activated by TNBC cells opsonized with Cetuximab promoted tumor material uptake and maturation of DCs, as well as their ability to produce IL-12. Furthermore, the stimulation with IL-15 increased the activation of NK cells and the maturation of DCs. These results suggest that IL-15 may enhance the efficacy of Cetuximab in the treatment of TNBC by promoting activation of both NK cells and DCs.
Assuntos
Cetuximab/farmacologia , Interleucina-15/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Fagocitose/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
How human macrophages can control the intracellular infection with Leishmania is not completely understood. IL-15 and IL-32 are cytokines produced by monocytes/macrophages that can induce antimicrobial mechanisms. Here, we evaluated the effects of recombinant human IL-15 (rhIL-15) on primary human macrophage infection and response to L. braziliensis. Priming with rhIL-15 reduced the phagocytosis of L. braziliensis and increased the killing of the parasites in monocyte-derived macrophages from healthy donors. rhIL-15 induced TNFα and IL-32 in uninfected cells. After infection, the high levels of rhIL-15-induced TNFα and IL-32 were maintained. In addition, there was an increase of NO and an inhibition of the parasite-induced IL-10 production. Inhibition of NO reversed the leishmanicidal effects of rhIL-15. Although rhIL-15 did not increase L. braziliensis-induced reactive oxygen intermediates (ROS) production, inhibition of ROS reversed the control of infection induced by rhIL-15. Treatment of the cells with rhIL-32γ increased microbicidal capacity of macrophages in the presence of high levels of vitamin D (25D3), but not in low concentrations of this vitamin. rhIL-15 together with rhIL-32 lead to the highest control of the L. braziliensis infection in high concentrations of vitamin D. In this condition, NO and ROS mediated rhIL-32γ effects on microbicidal activity. The data showed that priming of human macrophages with rhIL-15 or rhIL-32γ results in the control of L. braziliensis infection through induction of NO and ROS. In addition, rhIL-32γ appears to synergize with rhIL-15 for the control of L. braziliensis infection in a vitamin D-dependent manner.
Assuntos
Antiparasitários/metabolismo , Interleucina-15/metabolismo , Interleucinas/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Vitamina D/metabolismo , Antiparasitários/farmacologia , Interleucina-15/farmacologia , Interleucinas/farmacologia , Leishmania braziliensis/fisiologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Vitamina D/farmacologiaRESUMO
The prevention and treatment of type-2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), which are disorders with high incidence rates, is of primary importance. In this study, we analyzed the effect of 1,25-(OH)2D3 and lipopolysaccharide (LPS) in combination with interleukin (IL)-15 on the inflammatory immune response and expression of vitamin D receptor (VDR) in mononuclear cells of T2DM and DN uremia (DNU) patients. The human acute monocytic leukemia cell line THP-1 was treated with peripheral blood serum isolated from 30 healthy controls and T2DM and DNU patients each, cultured in the presence or absence of 1,25-(OH)2D3, and subsequently treated with LPS and IL-15. The VDR mRNA and protein expression in THP-1 cells was detected by real-time polymerase chain reaction and western blot (and immunofluorescence assay), respectively, and IL-6 and IL-10 concentrations in the culture supernatant were detected by enzyme-linked immunosorbent assay. LPS treatment induced a significant decrease in VDR mRNA expression in T2DM and DNU serum-treated THP-1 cells compared to the control cells (P < 0.05). The VDR protein expression in DNU serum-treated THP-1 cells was also significantly down-regulated (P < 0.05). LPS treatment induced IL-6 secretion in serum-treated THP-1 cells (P < 0.05), while 1,25-(OH)2D3 treatment inhibited IL-6 secretion to some extent. These findings suggested that LPS down-regulates the expression of VDR in mononuclear cells of T2DM and DNU patients and induces an imbalance in the pro-inflammatory and anti-inflammatory cytokine response, while 1,25-(OH)2D3 partially reversed the effect of LPS and protected patients with T2DM and DNU.
Assuntos
Calcitriol/farmacologia , Diabetes Mellitus Tipo 2/imunologia , Nefropatias Diabéticas/imunologia , Monócitos/efeitos dos fármacos , Receptores de Calcitriol/genética , Uremia/imunologia , Estudos de Casos e Controles , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Soros Imunes/farmacologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-15/antagonistas & inibidores , Interleucina-15/farmacologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Masculino , Monócitos/citologia , Monócitos/imunologia , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/antagonistas & inibidores , Receptores de Calcitriol/imunologia , Uremia/sangue , Uremia/patologiaAssuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sobrevivência de Enxerto/efeitos dos fármacos , Interleucina-15/farmacologia , Movimento Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacosRESUMO
Paracoccidiodomycosis is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), which is endemic in Latin America. The host innate immune response against the fungus has been well characterized and several studies have shown the important role played by phagocytic cells. Our laboratory has studied the relationship between human neutrophils (PMNs)/Pb, focusing the effector mechanisms of these cells against the fungus. However, in last years, studies have shown that in addition to their phagocytic and killer functions, PMNs can modulate and instruct the immune response, since these cells have been shown to produce and release several cytokines. Thus, we evaluated whether PMNs stimulated with Pb can modulate the immune response to a Th1 phenotype through the production of IFN-γ, as well as the role of "pattern-recognition receptors" (PRRs) such as TLR2, TLR4 and Dectin-1 in this production. Furthermore, we asked whether activation of the cells with the cytokines IL-12, IL-15 and IL-18 could result in increased levels of this cytokine. Peripheral blood PMNs obtained from 20 healthy donors were nonactivated or activated with IL-12, IL-15 or IL-18 in different concentrations and challenged with strain 18 Pb (Pb18) for 2 h, 4 h, 12 h, 24 h and 48 h and evaluated for IFN-γ production, by ELISA. In other experiments, PMNs were treated with monoclonal antibodies anti-TLR2, TLR4 and Dectin-1, challenged with Pb and evaluated for IFN-γ production. We found that Pb induces human PMNs to produce IFN-γ, probably by binding to TLR4 and Dectin-1 receptors expressed by these cells. Moreover, IFN-γ levels were significantly increased when cells were activated with each of the tested cytokines or a combination of two of them, being the association IL-12 plus IL-15 the most effective. The results support our hypothesis that during infection by Pb, human PMNs modulate the adaptive immune response to a Th1 response pattern, via IFN-γ production.
Assuntos
Interferon gama/biossíntese , Interleucina-12/farmacologia , Interleucina-15/farmacologia , Neutrófilos/imunologia , Paracoccidioides/imunologia , Anticorpos Monoclonais/imunologia , Humanos , Interferon gama/imunologia , Interleucina-18/farmacologia , Lectinas Tipo C/biossíntese , Lectinas Tipo C/imunologia , Ativação de Neutrófilo/imunologia , Paracoccidioidomicose/imunologia , Células Th1/imunologia , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologiaRESUMO
Diabetes mellitus type 2 (DM2), obesity, and the metabolic syndrome are considered that a low-grade chronic inflammatory condition. Although synthesis of different pro- and anti-inflammatory cytokines has been implicated in this process, this low-grade inflammatory condition is characterized especially by an increase in Tumor Necrosis Factor alpha (TNF-α), a cytokine that has been associated to the development of insulin resistance and muscle wasting in the diabetic patients. In consequence, mechanisms implicated in attenuating the deleterious effects of TNF-α are activated. One of these mechanisms may involve interleukin 15 (IL-15), which has proven effect on intermediate metabolisms, regulating blood glucose and lipid levels, and diminishing proteolysis in striated muscle. This suggests that the induction of endogenous IL-15 production by a simple event as physical activity may aid in decreasing or even inhibiting the negative effects of TNF-α in patient with DM2 or obesity, in which a low grade chronic inflammatory condition is present.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Interleucina-15/metabolismo , Atividade Motora/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/terapia , Terapia por Exercício/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Humanos , Resistência à Insulina/fisiologia , Interleucina-15/farmacologia , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Adulto , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cetuximab , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Cytokines play important roles in regulating immune responses. Interleukin-2 (IL-2) has usually been used as an adjuvant to enhance antitumour immune responses. However, its crucial role in activation-induced cell death, inhibition of homeostatic proliferation of CD8+ memory T cells and its notable biological side effects impair its prospect of application. IL-15 has several similar functions to IL-2 and shows potential advantages over IL-2, and is being investigated to enhance antitumour dendritic cell (DC) vaccine strategies in our ongoing studies. OBJECTIVE: In this preliminary study, we evaluated the ability of IL-15, compared with IL-2, to act as an adjuvant to enhance T-cell responses activated by DCs in vitro. MATERIALS AND METHODS: Bone marrow-derived DCs (BMDCs) were pulsed with tumour antigens and used to stimulate lymphocyte responses in the presence of IL-15 or IL-2. The activated T lymphocytes were examined by flow cytometric analysis, and interferon-γ (IFN-γ) enzyme-linked immunospot and cytotoxicity assays. RESULTS: IL-15 was observed to activate lymphocytes with comparable phenotype characteristics of activated/memory CD8+ lymphocytes, compared with IL-2. Both in primary and secondary stimulation with DCs, when using IL-15 as an adjuvant, activated lymphocytes showed higher proportions of IFN-γ-secreting subsets. In secondary stimulation with BMDCs in the presence of IL-15, the activated lymphocytes showed a stronger cytotoxicity to antigen-specific tumour target cells. CONCLUSIONS: Our study suggested that IL-15 might be a prospective adjuvant for a DC vaccine strategy against cancers. The further observation that IL-15 acts as an adjuvant for an antitumour DC vaccine strategy is worth investigating.
Assuntos
Células Dendríticas/imunologia , Interleucina-15/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Células da Medula Óssea/imunologia , Separação Celular , Células Cultivadas , Citotoxicidade Imunológica , Células Dendríticas/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-15/farmacologia , Interleucina-2/imunologia , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacosRESUMO
Triple negative breast cancer (TNBC) patients are not likely to benefit from anti-estrogen or anti-HER2 therapy and this phenotype is associated with a more aggressive clinical course and worse clinical outcome. Taking into account the limited treatment possibilities in TNBC, the aim of the present work was to study a potential therapy based on Cetuximab-mediated immune activity by natural killer (NK) cells. We performed in vitro studies on human breast cancer (BC) cell lines, IIB-BR-G, and the in vivo metastatic variant IIB-BR-G MT. The immunohistochemical analysis showed a TNBC phenotype with high but different levels of EGFR expression on each cell line, measured by flow cytometry. DNA sequencing showed that both cell lines have a mutated K-RAS status, 38 G > A at codon 13. Consequently, Cetuximab did not inhibit cellular proliferation or induce apoptosis. We investigated if Cetuximab could trigger immune mechanisms, and we determined that both cell lines treated with 1 µg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity (ADCC), mediated by peripheral blood mononuclear cells (PBMC). At 50:1 effector:target ratio, lytic activity was 34 ± 2% against IIB-BR-G and 27 ± 6% against IIB-BR-G MT cells. PBMC pretreatment with IL-2 allowed reaching 65 ± 3% of Cetuximab-mediated ADCC against IIB-BR-G and 63 ± 6.5% against IIB-BR-G MT. Furthermore, IL-15 pretreatment increased the ADCC up to 71 ± 3% in IIB-BR-G and 79 ± 3.5% in IIB-BR-G MT. We suggest that NK cells are the effectors present in PBMC since they were able to induce ADCC at lower effector:target ratios. Besides, IL-2- and mainly IL-15-induced upregulation of NK activating receptors CD16 and NKG2D and enhanced IFN-γ production. EGFR-expressing TNBC could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could advantageously replace IL-2 in most of its immunologic activities, stimulating the ability to produce IFN-γ, and paralleling the up-regulation of activating receptors.