RESUMO
Children are more susceptible to Giardia lamblia infection. Cells and hormones contained in human colostrum have an immunoprotective action against giardiasis, but the effects of advanced maternal age on these components are poorly understood. This study analyzed the colostrum of older women to determine melatonin and cortisol levels besides the participation of these hormones on the functional activity of phagocytes against G. lamblia. Colostrum samples were collected from younger (18 to 35 years old) and older (over 36 years old) lactating women. Colostrum samples were subjected to melatonin and cortisol determination, immunophenotyping, quantification of superoxide release, and assessment of phagocytic rate and microbicidal activity of phagocytes treated with hormones and in the presence of G. lamblia. Colostrum from mothers of advanced age contained higher melatonin and cortisol levels and a lower rate of cells expressing CD14+ and CD15+. In the colostru of these older mothers, melatonin increased superoxide release by phagocytes. In both groups, superoxide release by phagocytes treated with cortisol was higher in the presence of G. lamblia. In colostrum from mothers of advanced age, mononuclear (MN) phagocytes treated with melatonin showed higher phagocytosis of G. lamblia and higher microbicidal index. In younger mothers, MN and polymorphonuclear (PMN) colostrum phagocytes exhibited higher rates of G. lamblia elimination when treated with both melatonin and cortisol. In older mothers, cortisol and melatonin regulation for the functional activity of colostrum phagocytes against G. lamblia may represent an additional defense mechanism, relevant for the protection and treatment of parasitic infections in breastfed children.
Assuntos
Envelhecimento/imunologia , Colostro/imunologia , Giardia lamblia/imunologia , Giardíase/imunologia , Hidrocortisona/farmacologia , Melatonina/farmacologia , Neutrófilos/imunologia , Fagocitose/imunologia , Adolescente , Adulto , Animais , Criança , Estudos Transversais , Feminino , Giardíase/parasitologia , Giardíase/prevenção & controle , Humanos , Hidrocortisona/análise , Lactação/fisiologia , Antígenos CD15/biossíntese , Receptores de Lipopolissacarídeos/biossíntese , Idade Materna , Melatonina/análise , Fagócitos/imunologia , Gravidez , Superóxidos/metabolismo , Adulto JovemRESUMO
OBJECTIVES: To evaluate the anti-inflammatory potential of Pterodon polygalaeflorus hexane extract (HE) and its fractions on macrophage migration in vitro and in vivo. METHODS: Hexane extract from P. polygalaeflorus fruits was fractionated and yielded four fractions. RAW 264.7 cells were treated with samples to evaluate cell viability (MTT assay), cell migration (wound healing and transwell assays), CD14 expression (flow cytometry), iNOS and cytokine mRNA expression (RT-qPCR), NO (Griess reaction) and cytokine (ELISA) production. In vivo migration was evaluated on the thioglycollate-induced peritonitis model. Qualitative analysis was performed by GC-MS. KEY FINDINGS: All fractions inhibited the NO production by LPS-stimulated RAW 264.7 cells. Fr3 and Fr4 presented the lowest IC50 values. The expressions of iNOS and IL-1ß, TNF-α and IL-10 cytokines were inhibited by Fr3 and Fr4, whereas the CD14 expression was only inhibited by Fr3. All the samples inhibited RAW 264.7 migration in the wound healing and transwell assays. Fr3 and Fr4 reduced the migration of Mac-1+ Gr-1- cells to the peritoneum and presented in their compositions: 6α-hydroxy-7ß-acetoxyvouacapan-17ß-oate, methyl 6α,7ß-dihydroxyvouacapan-17ß-oate, methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate, geranylgeraniol and 14,15-epoxy-geranylgeraniol. CONCLUSIONS: The anti-inflammatory effects of Fr3 and Fr4 involve inhibition of cell migration, iNOS expression and NO production, cytokine expression (mRNA and proteins) and CD14 expression (Fr3).
Assuntos
Anti-Inflamatórios/farmacologia , Movimento Celular/efeitos dos fármacos , Citocinas/biossíntese , Diterpenos/farmacologia , Fabaceae/química , Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Frutas/química , Receptores de Lipopolissacarídeos/biossíntese , Macrófagos/citologia , Camundongos , Óxido Nítrico Sintase Tipo II/biossíntese , Extratos Vegetais/químicaRESUMO
The nuclear receptor PPARγ acts as a key modulator of lipid metabolism, inflammation and pathogenesis in BCG-infected macrophages. However, the molecular mechanisms involved in PPARγ expression and functions during infection are not completely understood. Here, we investigate signaling pathways triggered by TLR2, the involvement of co-receptors and lipid rafts in the mechanism of PPARγ expression, lipid body formation and cytokine synthesis in macrophages during BCG infection. BCG induces NF-κB activation and increased PPARγ expression in a TLR2-dependent manner. Furthermore, BCG-triggered increase of lipid body biogenesis was inhibited by the PPARγ antagonist GW9662, but not by the NF-κB inhibitor JSH-23. In contrast, KC/CXCL1 production was largely dependent on NF-κB but not on PPARγ. BCG infection induced increased expression of CD36 in macrophages in vitro. Moreover, CD36 co-immunoprecipitates with TLR2 in BCG-infected macrophages, suggesting its interaction with TLR2 in BCG signaling. Pretreatment with CD36 neutralizing antibodies significantly inhibited PPARγ expression, lipid body formation and PGE2 production induced by BCG. Involvement of CD36 in lipid body formation was further confirmed by decreased BCG-induced lipid body formation in CD36 deficient macrophages. Similarly, CD14 and CD11b/CD18 blockage also inhibited BCG-induced lipid body formation, whereas TNF-α synthesis was not affected. Disruption of rafts recapitulates the latter result, inhibiting lipid body formation, but not TNF-α synthesis in BCG-infected macrophages. In conclusion, our results suggest that CD36-TLR2 cooperation and signaling compartmentalization within rafts, divert host response signaling through PPARγ-dependent and NF-κB-independent pathways, leading to increased macrophage lipid accumulation and down-modulation of macrophage response.
Assuntos
Quimiocina CXCL1/biossíntese , Metabolismo dos Lipídeos , Mycobacterium bovis , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Tuberculose , Fator de Necrose Tumoral alfa/biossíntese , Anilidas/farmacologia , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Antígenos CD18/biossíntese , Antígenos CD18/genética , Antígenos CD36/biossíntese , Antígenos CD36/genética , Quimiocina CXCL1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/biossíntese , PPAR gama/genética , Fenilenodiaminas/farmacologia , Receptor 2 Toll-Like/genética , Tuberculose/metabolismo , Tuberculose/patologia , Tuberculose/veterinária , Fator de Necrose Tumoral alfa/genéticaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple functional alterations affecting immune cells, such as B cells, T cells, dendritic cells (DCs) and monocytes. During SLE, the immunogenicity of monocytes and DCs is significantly up-regulated, promoting the activation of self-reactive T cells. Accordingly, it is important to understand the contribution of these cells to the pathogenesis of SLE and the mechanisms responsible for their altered functionality during disease. One of the key enzymes that control monocyte and DC function is haem oxygenase-1 (HO-1), which catalyses the degradation of the haem group into biliverdin, carbon monoxide and free iron. These products possess immunosuppressive and anti-inflammatory capacities. The main goal of this work was to determine HO-1 expression in monocytes and DCs from patients with SLE and healthy controls. Hence, peripheral blood mononuclear cells were obtained from 43 patients with SLE and 30 healthy controls. CD14(+) monocytes and CD4(+) T cells were sorted by FACS and HO-1 expression was measured by RT-PCR. In addition, HO-1 protein expression was determined by FACS. HO-1 levels in monocytes were significantly reduced in patients with SLE compared with healthy controls. These results were confirmed by flow cytometry. No differences were observed in other cell types, such as DCs or CD4(+) T cells, although decreased MHC-II levels were observed in DCs from patients with SLE. In conclusion, we found a significant decrease in HO-1 expression, specifically in monocytes from patients with SLE, suggesting that an imbalance of monocyte function could be partly the result of a decrease in HO-1 expression.
Assuntos
Células Dendríticas/enzimologia , Heme Oxigenase-1/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Monócitos/enzimologia , Adulto , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Feminino , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Monócitos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia , Adulto JovemRESUMO
Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Glândula Pineal/metabolismo , Receptor 4 Toll-Like/metabolismo , Análise de Variância , Animais , Extratos Celulares/química , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunidade Inata/fisiologia , Imuno-Histoquímica , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Masculino , Glândula Pineal/citologia , Glândula Pineal/imunologia , RNA Mensageiro , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Técnicas de Cultura de Tecidos , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/genéticaRESUMO
BACKGROUND: Hookworms survive for several years (5 to 7 years) in the host lumen, inducing a robust but largely ineffective immune response. Among the most striking aspects of the immune response to hookworm (as with many other helminths) is the ablation of parasite-specific T cell proliferative response (hyporesponsiveness). While the role of the adaptive immune response in human helminth infection has been well investigated, the role of the innate immune responses (e.g., dendritic cells and eosinophils) has received less attention and remains to be clearly elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We report on the differentiation/maturation of host dendritic cells in vitro and the eosinophil activation/function associated with human hookworm infection. Mature DCs (mDCs) from Necator americanus (Necator)-infected individuals showed an impaired differentiation process compared to the mDCs of non-infected individuals, as evidenced by the differential expression of CD11c and CD14. These same hookworm-infected individuals also presented significantly down-regulated expression of CD86, CD1a, HLA-ABC, and HLA-DR. The lower expression of co-stimulatory and antigen presentation molecules by hookworm-infected-derived mDCs was further evidenced by their reduced ability to induce cell proliferation. We also showed that this alternative DC differentiation is partially induced by excreted-secreted hookworm products. Conversely, eosinophils from the same individuals showed a highly activated status, with an upregulation of major cell surface markers. Antigen-pulsed eosinophils from N. americanus-infected individuals induced significant cell proliferation of autologous PBMCs, when compared to non-infected individuals. CONCLUSION: Chronic N. americanus infection alters the host's innate immune response, resulting in a possible modulation of the maturation process of DCs, a functional change that may diminish their ability for antigen presentation and thus contribute to the ablation of the parasite-specific T cell proliferative response. Interestingly, a concomitant upregulation of the major cell surface markers of eosinophils was observed in hookworm-infected individuals, indicative of antigen-specific immune responses, especially antigen presentation. We showed that in addition to the postulated role of the eosinophils as effector cells against helminth infection, activated cells may also be recruited to sites of inflammation and contribute to the immune response acting as antigen presenting cells.
Assuntos
Células Dendríticas/imunologia , Eosinófilos/imunologia , Necator americanus/imunologia , Necatoríase/imunologia , Adulto , Animais , Antígenos CD1/biossíntese , Antígeno B7-2/biossíntese , Antígenos CD11/biossíntese , Diferenciação Celular , Doença Crônica , Células Dendríticas/parasitologia , Eosinófilos/parasitologia , Antígenos HLA-DR/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Pessoa de Meia-IdadeRESUMO
Sepsis is defined as systemic inflammation caused by infection. The membrane bound CD14 (mCD14) or the soluble form (sCD14) play a crucial role facing Gram-negative and Gram-positive sepsis since they are pattern recognition receptors of the innate immune response enabling cells to produce inflammatory cytokines against bacterial infections. A -260C>T single nucleotide polymorphism (SNP) was detected in the promoter modulating the CD14 gene expression. We hypothesized that the CD14 expression depends of the genetic inheritance of -260C>T CD14 SNP and it is modulated by sepsis condition. We investigated human CD14 expression on early sepsis diagnosis (in vivo) and after LPS stimulation (in vitro), and determined the -260C>T CD14 SNP. We found that TT homozygotes showed higher mCD14 density (p = 0.0207), but not different sCD14 levels when compared to the CT+CC genotypes. Monocyte mCD14 density and sCD14 serum levels in our sample of early 14 septic patients were significantly higher than normal 30 controls (p<0.0001). Our results suggest that the -260TT CD14 genotype is associated with higher monocyte mCD14, but not sCD14 expression, and that in the first 24 h after sepsis diagnosis, both monocyte mCD14 density and sCD14 levels are elevated, similarly to what is observed in vitro upon challenge with LPS.
Assuntos
Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/genética , Sepse/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Citocinas/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Imunidade Inata/genética , Inflamação , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Sepse/metabolismo , Sepse/mortalidade , Sepse/fisiopatologia , Análise de Sobrevida , Fatores de TempoRESUMO
This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01-5 mug/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.
Assuntos
Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Escherichia coli/química , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Corticosterona/sangue , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/genética , Masculino , NF-kappa B/biossíntese , NF-kappa B/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Testosterona/sangueRESUMO
INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.
Assuntos
Células Dendríticas/citologia , Regulação Neoplásica da Expressão Gênica , Interleucina-4/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-1/biossíntese , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Masculino , Pessoa de Meia-IdadeRESUMO
Protein-energy malnutrition (PEM) modifies resistance to infection, impairing a number of physiological processes, including hematopoiesis. In this study, we examined a few aspects of the inflammatory response to LPS in a model of PEM. We evaluated the cellularity of the blood, bone marrow and spleen, as well as phagocytic, fungicidal and spreading activity, the production in vivo and in vitro of TNF-alpha, IL-1alpha and IL-6, and the expression of CD14 and TLR-4/MD-2 receptors in macrophages. Two-month-old male Swiss mice were submitted to PEM with a low-protein diet containing 4% protein as compared to 20% protein in the control diet. When the experimental group had attained about 20% loss of their original body weight, they were used in the experiments. Malnourished animals presented anemia, leucopenia and severe reduction in bone marrow, spleen and peritoneal cavity cellularity. The production of TNF-alpha, IL-1alpha and IL-6 stimulated in vivo with LPS and the production of IL-6 in bone marrow cells cultured with LPS and the production of TNF-alpha in bone marrow, spleen and peritoneal cells cultured with LPS were significantly lower in malnourished animals. The expression of CD14 and TLR-4/MD-2 receptors was found to be significantly lower in macrophages of malnourished animals. These findings suggest that malnourished animals present a deficient response to LPS. The lower expression of the CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed in the malnourished mice. These data lead us to infer that the nutritional state interferes with the activation of macrophages and with the capacity to mount an immune response.