RESUMO
Term and especially preterm neonates are much more susceptible to serious bacterial infections than adults. But not only the susceptibility to infection is increased in neonates, but also their risk for developing post-inflammatory diseases such as bronchopulmonary dysplasia (BPD) and periventricular leukomalacia (PVL). This may be due to an impaired ability to terminate inflammation. In the study presented here, we aimed to investigate the proliferative response and the expression of immune-checkpoint molecules (ICM) and activation markers on neonatal T-cells in comparison to adult T-cells with the hypothesis that an increased activation of neonatal T-cells may contribute to the failure of inflammation resolution observed in neonates. We show that neonatal CD4+ and CD8+ T-cells show an increased proliferative capacity and an increased expression of activation markers compared to adult T-cells upon stimulation with OKT3 as well as a decreased expression of ICM, especially PD-L1 on their surface. This decreased expression of PD-L1 by neonatal T-cells was also observed after stimulation with GBS, but not after stimulation with E. coli, the two most important pathogens in neonatal sepsis. Expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 did not differ between adult and neonatal T-cells upon bacterial stimulation. Decreased expression of ICM upon T-cell activation may be a reason for the increased risk of neonates to develop post-inflammatory diseases.
Assuntos
Linfócitos T CD8-Positivos , Proteínas de Checkpoint Imunológico , Adulto , Humanos , Recém-Nascido , Antígeno B7-H1 , Escherichia coli , InflamaçãoRESUMO
BACKGROUND: Infections are a major cause for morbidity and mortality in neonates; however, the underling mechanisms for increased infection susceptibility are incompletely understood. Hypoxia, which is present in inflamed tissues, has been identified as an important activation signal for innate immune cells in adults and is mainly mediated by hypoxia-inducible factor 1α (HIF-1α). Fetal tissue pO2 physiologically is low but rises immediately after birth. METHODS: In this study, the effect of low oxygen partial pressure (pO2) on HIF-1α expression and its targets phagocytosis, reactive oxygen species (ROS) production and vascular endothelial growth factor (VEGF) secretion was compared in vitro between immune cells from adult peripheral blood and cord blood using anoxia, HIF-1α stabilizer desferroxamin (DFO) and E. coli as stimuli. RESULTS: We show that anoxia-induced HIF-1α protein accumulation, phagocytosis, ROS-production and VEGF-expression were greatly diminished in cord blood compared to adult cells. E. coli led to HIF-1α gene expression in adult and cord blood immune cells; however, cord blood cells failed to accumulate HIF-1α protein and VEGF upon E. coli stimulation. CONCLUSIONS: Taken together, our results show a diminished activation of cord blood immune cells by low pO2, which might contribute to impaired reactivity in the context of infection. IMPACT: Neonatal immune cells do not accumulate HIF-1α under low oxygen partial pressure leading to decreased phagocytosis and decreased ROS production. We demonstrate a previously unknown mechanism of reduced activation of neonatal immune cells in the context of an inflammatory response. This could contribute to the increased susceptibility of newborns and preterm infants to infection.
Assuntos
Sangue Fetal , Fator A de Crescimento do Endotélio Vascular , Humanos , Recém-Nascido , Adulto , Fator A de Crescimento do Endotélio Vascular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sangue Fetal/metabolismo , Monócitos/metabolismo , Escherichia coli/metabolismo , Recém-Nascido Prematuro , Hipóxia , Oxigênio , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
BACKGROUND: Sepsis is one of the leading causes of morbidity and mortality in the neonatal period. Compared to adults, neonates are more susceptible to infections, especially to systemic infections with Group B Streptococcus (GBS). Furthermore, neonates show defects in terminating inflammation. The immunological causes for the increased susceptibility to infection and the prolonged inflammatory response are still incompletely understood. METHODS: In the present study, we aimed to investigate the reaction of cord blood mononuclear cells (MNC) to stimulation with GBS in comparison to that of MNC from adult blood with focus on the proliferative response in an in vitro infection model with heat-inactivated GBS. RESULTS: We demonstrate that after stimulation with GBS the proliferation of T cells from adult blood strongly decreased, while the proliferation of cord blood T cells remained unchanged. This effect could be traced back to a transformation of adult monocytes, but not cord blood monocytes, to a suppressive phenotype with increased expression of the co-inhibitory molecule programmed death ligand 1 (PD-L1). CONCLUSIONS: These results point towards an increased inflammatory capacity of neonatal MNC after stimulation with GBS. Targeting the prolonged inflammatory response of neonatal immune cells may be a strategy to prevent complications of neonatal infections. IMPACT: Neonatal sepsis often leads to post-inflammatory complications. Causes for sustained inflammation in neonates are incompletely understood. We show that cord blood T cells exhibited increased proliferative capacity after stimulation with group B streptococci (GBS) in comparison to adult T cells. Adult monocytes but not cord blood monocytes acquired suppressive activity and expressed increased levels of PD-L1 after GBS stimulation. Increased proliferative capacity of neonatal T cells and decreased suppressive activity of neonatal monocytes during GBS infection may contribute to prolonged inflammation and development of post-inflammatory diseases in newborns.
Assuntos
Sepse , Infecções Estreptocócicas , Humanos , Antígeno B7-H1 , Streptococcus agalactiae , Sangue Fetal , MonócitosRESUMO
BACKGROUND: Neonatal sepsis is a leading cause of perinatal morbidity and mortality. In comparison to adults, neonates exhibit a higher susceptibility to infections. Myeloid-derived suppressor cells (MDSCs) are myeloid cells with suppressive activity on other immune cells accumulating during foetal life and controlling inflammation in neonates. Most studies investigating the mechanisms for MDSC-mediated immune suppression have been focused on T-cells. Thus far, little is known about the role of MDSC for monocyte function. METHODS: The impact of human cord blood MDSCs (CB-MDSCs) on monocytes was investigated in an in vitro model. CB-MDSCs were co-cultured with peripheral blood mononuclear cells and monocytes were analysed for expression of surface markers, T cell stimulatory and phagocytic capacity, as well as the production of intracellular cytokines by flow cytometry. RESULTS: CB-MDSCs increased the expression of co-inhibitory molecules and decreased the expression of major histocompatibility complex class II molecules on monocytes, leading to an impaired T-cell stimulatory capacity. Upon bacterial stimulation, expression of phagocytosis receptors, phagocytosis rates and production of tumor necrosis factor-α by monocytes was diminished by CB-MDSCs. CONCLUSION: We show that CB-MDSCs profoundly modulate monocyte functions, thereby indirectly impairing T-cell activation. Further research is needed to figure out if MDSCs could be a therapeutic target for inflammatory diseases in neonates like neonatal sepsis.
Assuntos
Escherichia coli/imunologia , Sangue Fetal/citologia , Granulócitos/imunologia , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Proliferação de Células , Técnicas de Cocultura , Humanos , Recém-NascidoRESUMO
Abortions are the most important reason for unintentional childlessness. During pregnancy, maternal immune cells are in close contact to cells of the semi-allogeneic fetus. Dysregulation of the maternal immune system leading to defective adaptation to pregnancy often plays a role in pathogenesis of abortions. Myeloid-derived suppressor cells (MDSC) are myeloid cells that suppress functions of other immune cells, especially T-cells, thereby negatively affecting diseases such as cancer, sepsis or trauma. They seem, however, also necessary for maintenance of maternal-fetal tolerance. Mechanisms regulating MDSC expansion and function during pregnancy are only incompletely understood. In tumor environment, hypoxia is crucial for MDSC accumulation and activation. Hypoxia is also important for early placenta and embryo development. Effects of hypoxia are mediated through hypoxia-inducible factor 1α (HIF-1α). In the present study we aimed to examine the role of HIF-1α in myeloid cells for MDSC accumulation and MDSC function during pregnancy and for pregnancy outcome. We therefore used a mouse model with targeted deletion of HIF-1α in myeloid cells (myeloid HIF-KO) and analyzed blood, spleens and uteri of pregnant mice at gestational day E 10.5 in comparison to non-pregnant animals and wildtype (WT) animals. Further we analyzed pregnancy success by determining rates of failed implantation and abortion in WT and myeloid HIF-KO animals. We found that myeloid HIF-KO in mice led to an abrogated MDSC accumulation in the pregnant uterus and to impaired suppressive activity of MDSC. While expression of chemokine receptors and integrins on MDSC was not affected by HIF-1α, myeloid HIF-KO led to increased apoptosis rates of MDSC in the uterus. Myeloid-HIF-KO resulted in increased proportions of non-pregnant animals after positive vaginal plug and increased abortion rates, suggesting that activation of HIF-1α dependent pathways in MDSC are important for maintenance of pregnancy.
Assuntos
Aborto Espontâneo/etiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Aborto Induzido , Animais , Apoptose , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Células Mieloides/imunologia , Células Mieloides/metabolismo , GravidezRESUMO
Nosocomial bacterial infections (NBI) and necrotizing enterocolitis (NEC) are among the main reasons for death in preterm infants. Both are often caused by bacteria coming from the infected infant's gut and feeding with breast milk (BM) seems beneficial in their pathogenesis. However, mechanisms causing the protective effect of BM are only incompletely understood. Myeloid-derived suppressor cells (MDSC) are myeloid cells with suppressive activity on other immune cells, recently described to play a role in mediating maternal-fetal tolerance during pregnancy and immune adaptation in newborns. Until now, nothing is known about occurrence and function of MDSC in BM. We analyzed MDSC in BM and peripheral blood of breastfeeding mothers and found that granulocytic MDSC, but not monocytic MDSC, accumulate in BM, exhibit an activated phenotype and increased suppressive activity and modulate TLR-expression on monocytes. Furthermore, we found that the lactotrophic hormones prolactin and oxytocin do not induce MDSC from peripheral blood. This is the first study to describe MDSC with immune-modulatory properties in human BM. Our results point toward a role for MDSC in local immune modulation in the gut possibly protecting infants from NBI and NEC.
Assuntos
Leite Humano/citologia , Leite Humano/imunologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Biomarcadores , Medula Óssea/imunologia , Medula Óssea/metabolismo , Feminino , Granulócitos/imunologia , Granulócitos/metabolismo , Humanos , Imunomodulação , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Prolactina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Susceptibility to infection during the neonatal period and reduced control of inflammation in neonates are attributed to immunosuppression persisting from fetal life. Myeloid-derived suppressor cells (MDSCs) are immature myeloid progenitors with suppressive activity and increased numbers in cord blood. We hypothesized that MDSCs contribute to innate host defence in neonates, paralleled by anti-inflammatory signalling.Phagocytic activity, infection induced apoptosis, expression of B-cell lymphoma (Bcl)-2 family proteins, production of reactive oxygen species (ROS), cytokine production and T-cell suppression of neonatal granulocytic-MDSCs (G-MDSCs) after infection with Escherichia coli (E. coli) were compared to neonatal autologous mature polymorphonuclear leukocytes (PMNs). Phagocytic activity of G-MDSCs upon infection with E. coli was equal to that of mature PMNs, however, apoptosis of G-MDSCs was decreased. G-MDSCs showed enhanced Bcl-2-expression and lower ROS production compared to PMNs. Inhibition of Bcl-2 reduced apoptosis rates of G-MDSCs to that of mature PMNs. Induction of anti-inflammatory transforming growth factor beta (TGF-ß) was enhanced, while pro-inflammatory IL-8 decreased in G-MDSCs compared to PMNs. Infected G-MDSCs strongly suppressed proliferation of T cells. We show a direct role of G-MDSCs for anti-bacterial host defence. Prolonged survival and anti-inflammatory capacity suggest that G-MDSCs are important for immune-regulation after bacterial infection.
Assuntos
Apoptose , Escherichia coli/imunologia , Tolerância Imunológica , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/microbiologia , Antígeno CD11b/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Sangue Fetal/citologia , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Ativação Linfocitária , Células Supressoras Mieloides/fisiologia , Neutrófilos/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismoRESUMO
Infections are a leading cause of perinatal morbidity and mortality. The outstandingly high susceptibility to infections early in life is mainly attributable to the compromised state of the neonatal immune system. One important difference to the adult immune system is a bias towards T helper type 2 (Th2) responses in newborns. However, mechanisms regulating neonatal T-cell responses are incompletely understood. Granulocytic myeloid-derived suppressor cells (GR-MDSC) are myeloid cells with a granulocytic phenotype that suppress various functions of other immune cells and accumulate under physiological conditions during pregnancy in maternal and fetal blood. Although it has been hypothesized that GR-MDSC accumulation during fetal life could be important for the maintenance of maternal-fetal tolerance, the influence of GR-MDSC on the immunological phenotype of neonates is still unclear. Here, we investigated the impact of GR-MDSC isolated from cord blood (CB-MDSC) on the polarization of Th cells. We demonstrate that CB-MDSC inhibit Th1 responses and induced Th2 responses and regulatory T (Treg) cells. Th1 inhibition was cell-contact dependent and occurred independent of other cell types, while Th2 induction was mediated independently of cell contact through expression of ArgI and reactive oxygen species by CB-MDSC and partially needed the presence of monocytes. Treg cell induction by CB-MDSC also occurred cell-contact independently but was partially mediated through inducible nitric oxide synthase. These results point towards a role of MDSC in regulating neonatal immune responses. Targeting MDSC function in neonates could be a therapeutic opportunity to improve neonatal host defence.
Assuntos
Plasticidade Celular , Sangue Fetal/imunologia , Granulócitos/imunologia , Inflamação/prevenção & controle , Células Supressoras Mieloides/imunologia , Células Th2/imunologia , Arginase/imunologia , Arginase/metabolismo , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Sangue Fetal/citologia , Granulócitos/metabolismo , Humanos , Recém-Nascido , Inflamação/imunologia , Inflamação/metabolismo , Células Supressoras Mieloides/metabolismo , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Establishing and maintaining maternal-fetal tolerance is essential for a successful pregnancy; failure of immunological adaptation to pregnancy leads to severe complications such as abortion or preterm delivery. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that suppress T-cell responses, expand during pregnancy and thus may play a role in tolerance induction. Human leucocyte antigen G (HLA-G) is a major histocompatibility complex (MHC) I molecule with immune-modulatory properties, which is expressed during pregnancy. Here, we investigated the impact of HLA-G on MDSCs accumulation and activation in pregnant women. We demonstrate that granulocytic MDSCs (GR-MDSCs) express receptors for HLA-G, namely immunoglobulin-like transcript (ILT) 2 and 4, and that ILT4-expression by GR-MDSCs is regulated during pregnancy. Stimulation with soluble HLA-G (sHLA-G) increased suppressive activity of GR-MDSCs, induced MDSCs from peripheral blood mononuclear cells (PBMCs) and led to phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and induction of indoleamine-2,3-dioxygenase (IDO) in myeloid cells. Effects of sHLA-G on MDSC accumulation were mediated through ILT4. These results suggest an interaction between MDSCs and HLA-G in humans as a potential mechanism for maintaining maternal-fetal tolerance. Modulating MDSC function during pregnancy via HLA-G might provide new opportunities for a therapeutic manipulation of immunological pregnancy complications.
Assuntos
Antígenos HLA-G/metabolismo , Relações Materno-Fetais , Glicoproteínas de Membrana/metabolismo , Células Supressoras Mieloides/imunologia , Receptores Imunológicos/metabolismo , Fator de Transcrição STAT3/metabolismo , Adolescente , Adulto , Células Cultivadas , Feminino , Humanos , Tolerância Imunológica , Imunidade Inata , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Pessoa de Meia-Idade , Gravidez , Ligação Proteica , Transdução de Sinais , Adulto JovemRESUMO
Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter.
Assuntos
Amebíase/complicações , Helmintíase/complicações , Complicações Parasitárias na Gravidez/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Amebíase/genética , Amebíase/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Antígenos de Helmintos/imunologia , Antígenos de Protozoários/imunologia , Feminino , Sangue Fetal , Helmintíase/genética , Helmintíase/imunologia , Humanos , Recém-Nascido , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Gravidez , Complicações Parasitárias na Gravidez/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Transcriptoma/imunologiaRESUMO
Tolerance induction toward the semiallogeneic fetus is crucial to enable a successful pregnancy; its failure is associated with abortion or preterm delivery. Skewing T cell differentiation toward a Th2-dominated phenotype seems to be pivotal in maternal immune adaption, yet underlying mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that mediate T cell suppression and are increased in cord blood of healthy newborns and in peripheral blood of pregnant women. In this study, we demonstrate that granulocytic MDSCs (GR-MDSCs) accumulate in human placenta of healthy pregnancies but are diminished in patients with spontaneous abortions. Placental GR-MDSCs effectively suppressed T cell responses by expression of arginase I and production of reactive oxygen species and were activated at the maternal-fetal interface through interaction with trophoblast cells. Furthermore, GR-MDSCs isolated from placenta polarized CD4(+) T cells toward a Th2 cytokine response. These results highlight a potential role of GR-MDSCs in inducing and maintaining maternal-fetal tolerance and suggest them as a promising target for therapeutic manipulation of pregnancy complications.
Assuntos
Granulócitos/imunologia , Tolerância Imunológica/imunologia , Placenta/imunologia , Gravidez/imunologia , Células Th2/imunologia , Aborto Espontâneo/imunologia , Adolescente , Adulto , Diferenciação Celular/imunologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células Mieloides/imunologia , Fenótipo , Adulto JovemRESUMO
Immune tolerance toward the semiallogeneic fetus plays a crucial role in the maintenance of pregnancy. Myeloid-derived suppressor cells (MDSCs) are innate immune cells characterized by their ability to modulate T-cell responses. Recently, we showed that MDSCs accumulate in cord blood of healthy newborns, yet their role in materno-fetal tolerance remained elusive. In the present study, we demonstrate that MDSCs with a granulocytic phenotype (GR-MDSCs) are highly increased in the peripheral blood of healthy pregnant women during all stages of pregnancy compared with nonpregnant controls, whereas numbers of monocytic MDSCs were unchanged. GR-MDSCs expressed the effector enzymes arginase-I and iNOS, produced high amounts of ROS and efficiently suppressed T-cell proliferation. After parturition, GR-MDSCs decreased within a few days. In combination, our results show that GR-MDSCs expand in normal human pregnancy and may indicate a role for MDSCs in materno-fetal tolerance.
Assuntos
Arginase/imunologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células Mieloides/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Gravidez/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Humanos , Tolerância Imunológica/fisiologia , Pessoa de Meia-Idade , Células Mieloides/citologia , Linfócitos T/citologiaRESUMO
BACKGROUND: Neonates show sustained inflammation after a bacterial infection, which is associated with inflammatory diseases like bronchopulmonary dysplasia or periventricular leucomalacia. Physiologically, inflammation is terminated early after the removal of the invading pathogens by phagocytosis-induced cell death (PICD) of immune effector cells. Earlier results showed reduced PICD in neonatal monocytes. The underlying molecular mechanisms are unknown. We hypothesize that the reduced PICD in neonatal monocytes is regulated through the proteins of the B-cell lymphoma 2 (Bcl-2) protein family. METHODS: mRNA and protein expression of Bcl-2 family proteins in cord blood and adult peripheral blood monocytes infected with Escherichia coli were analyzed by quantitative real-time PCR and flow cytometry and cytochrome c release by fluorescence microscopy. RESULTS: mRNA expression of antiapopototic Bcl-xL was upregulated in cord blood monocytes (CBMO), whereas proapoptotic Bim tended to be higher in peripheral blood monocytes (PBMO). Upon infection, Bax was more strongly expressed in PBMO compared with CBMO. The pro/antiapoptotic balance was skewed toward survival in CBMO and apoptosis in PBMO. Cytochome c release into the cytosol was enhanced in PBMO compared with CBMO. CONCLUSION: Bcl-2 proteins are involved in reduced PICD in neonatal monocytes. These findings are another step toward the understanding of sustained inflammation in neonates.
Assuntos
Apoptose/imunologia , Infecções por Escherichia coli/imunologia , Monócitos/imunologia , Fagocitose/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Adulto , Análise de Variância , Citocromos c/metabolismo , Primers do DNA/genética , Feminino , Citometria de Fluxo , Humanos , Recém-Nascido , Microscopia de Fluorescência , Monócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: The propensity for sustained inflammation after bacterial infection in neonates, resulting in inflammatory sequelae such as bronchopulmonary dysplasia and periventricular leucomalacia, is well known, but its molecular mechanisms remain elusive. Termination of inflammatory reactions physiologically occurs early after removal of bacteria by phagocytosis-induced cell death (PICD) of immune effector cells such as monocytes. PICD from cord blood monocytes (CBMOs) was shown to be reduced as compared with that of peripheral blood monocytes (PBMOs) from adult donors in vitro. METHODS: PBMOs, CBMOs, and Fas (CD95)-deficient (lpr) mouse monocytes were analyzed in an in vitro infection model using green fluorescence protein-labeled Escherichia coli (E. coli-GFP). Phagocytosis and apoptosis were quantified by flow cytometry and CD95L secretion was quantified by enzyme-linked immunosorbent assay. RESULTS: We demonstrate the involvement of the CD95/CD95 ligand pathway (CD95/CD95L) in PICD and provide evidence that diminished CD95L secretion by CBMOs may result in prolonged activation of neonatal immune effector cells. CONCLUSION: These in vitro results offer for the first time a molecular mechanism accounting for sustained inflammation seen in neonates.
Assuntos
Apoptose , Proteína Ligante Fas/metabolismo , Sangue Fetal/imunologia , Inflamação/imunologia , Monócitos/imunologia , Fagocitose , Receptor fas/metabolismo , Adulto , Animais , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Recém-Nascido , Inflamação/sangue , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/microbiologia , Monócitos/patologia , Transdução de Sinais , Receptor fas/deficiência , Receptor fas/genéticaRESUMO
BACKGROUND: Immunoglobulins (IVIG) have been shown to be useful in adults suffering from sepsis. In contrast, prophylactic and curative IVIG trials failed to show beneficial effects in neonates. We tested the hypothesis that IVIG, have different effects on monocytes from cord blood (CBMO) and peripheral blood monocytes from adults (PBMO) with respect to survival, phenotype, and function. METHODS: Mononuclear cells, or purified monocytes, were cultured in 5% human serum, incubated with polyvalent IVIG (1 mg/ml), stimulated with green fluorescent protein (GFP)-labeled Escherichia coli (E. Coli-GFP), Interferon-γ (IFN-γ, 50 U/ml), or the T cell mitogen anti-CD3 monoclonal antibody, αCD3-mAb, (5 µg/ml). Phagocytosis, phenotype, T cell proliferation, and apoptosis were assessed by flow cytometry. RESULTS: IVIG enhanced phagocytosis in PBMO or CBMO when infected directly after isolation, while IVIG had no effect on monocytes cultured 48 h prior to infection. In contrast to PBMO, IVIG inhibited the IFN-γ mediated up-regulation of CD80, CD86, and HLA-DR on CBMO. In the presence of IVIG, stimulation with αCD3 in cord blood enhanced deletion, inhibited blast formation and CD28 up-regulation of T cells (P < 0.05 vs. T cells from adults). IVIG induced monocyte apoptosis, associated with up-regulation of Annexin V and loss of nuclear DNA, which was more pronounced in CBMO. Although phagocytosis induced cell death (PICD) was lower in CBMO (P < 0.05 vs. PBMO), the addition of IVIG enhanced PICD levels of CBMO to the extent of PBMO. CONCLUSIONS: IVIG inhibits co-stimulatory receptors and functions of CBMO and induces apoptosis. These findings may be of clinical relevance for the failure of IVIG benefit in neonatal sepsis.
Assuntos
Sangue Fetal/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , Fatores Imunológicos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/administração & dosagem , Apoptose/efeitos dos fármacos , Complexo CD3/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Interferon gama/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Monócitos/citologia , Monócitos/imunologia , Fagocitose/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are known to reside in specialized niches at the endosteum in the trabecular bone. Osteoblasts are the major cell type of the endosteal niche. It is well established that secreted proteases are involved in cytokine-induced mobilization processes that release stem cell from their niches. However, migratory processes such as the regular trafficking of HSPCs between their niches and the periphery are not fully understood. In the current study we analyzed whether osteoblast-secreted cysteine cathepsins are able to reduce the direct interaction of HSPCs with bone-forming osteoblasts. Isolated human osteoblasts were shown to secrete proteolytically active cysteine cathepsins, such as cathepsins B, K, L, and X. All of these cathepsins were able to digest, although with different efficacy, the chemokine CXCL12, which is known to be important for retaining HSPCs in their niches. Of the 4 identified cathepsins, only cathepsin X was able to reduce binding of HSPCs to osteoblasts. Interestingly, nonactivated pro-cathepsin X and mature cathepsin X did not interfere with HSPC-osteoblast interactions. Only pro-cathepsin X treated with dithiothreitol, which unfolds but does not lead to full maturation of cathepsin X, significantly reduced HSPC adhesion to osteoblasts. These observations argue for a role of the accessible cathepsin X prodomain in diminishing cell binding. Our findings strongly suggest that the cysteine cathepsins B, K, and L constitutively secreted by osteoblasts are part of the fine-tuned regulation of CXCL12 in the bone marrow, whereas pro-cathepsin X with its prodomain can affect HSPC trafficking in the niche.
Assuntos
Catepsinas/química , Quimiocina CXCL12/química , Osteoblastos/química , Precursores de Proteínas/química , Antígenos CD34/química , Catepsina B/química , Catepsina B/farmacologia , Catepsina K/química , Catepsina K/farmacologia , Catepsinas/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular , Microambiente Celular , Meios de Cultivo Condicionados/química , Ditiotreitol/farmacologia , Ativação Enzimática , Ensaios Enzimáticos , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Cultura Primária de Células , Precursores de Proteínas/farmacologia , Proteólise , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TempoRESUMO
Phagocytosis of apoptotic cells, e.g., neutrophils, by monocytes is essential for resolution of inflammation. Delayed removal leads to secondary necrosis, perpetuating inflammation, and tissue destruction. Common histologic features in neonatal chronic inflammatory disorders are an accumulation of apoptotic cells in inflamed tissues. We hypothesized that apoptotic cell removal by monocytes is compromised in newborns. PKH-26 labeled autologous or allogeneic apoptotic neutrophils were fed to monocytes of adult donors (PBMO) and cord blood (CBMO), and phagocytic activity was analyzed by flow cytometry and confocal microscopy. Relative mRNA-expression levels of 21 surface receptors and bridging molecules relevant for apoptotic cell removal were measured, as was postphagocytic IL-8 production upon LPS-stimulation. Compared with PBMO, CBMO exhibited a significantly diminished phagocytotic competence for autologous and allogeneic apoptotic neutrophils. mRNA-expression levels of milk fat globule-EGF factor 8 and T cell immunoglobulin- and mucin-domain-containing molecule, two crucial members of the phagocytic synapse of apoptotic cell removal, were reduced in CBMO. In PBMO, interaction with autologous apoptotic neutrophils reduced LPS-induced IL-8 production whereas it was enhanced in CBMO. Our data suggest a specific defect in CBMO during clearance of apoptotic neutrophils resulting in impaired anti-inflammatory capacity.
Assuntos
Apoptose , Sangue Fetal/citologia , Interleucina-8/metabolismo , Monócitos/citologia , Neutrófilos/citologia , Células Cultivadas , Feminino , Humanos , Recém-Nascido , Leucócitos Mononucleares/citologia , Lipopolissacarídeos/metabolismo , Microscopia Confocal/métodos , Neutrófilos/metabolismo , Fagocitose , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An imbalance in apoptosis or survival of immune cells plays an essential role in the pathophysiology of sepsis. Phagocytosis-induced cell death (PICD) is a common result of the pathogen-host cell interaction mediated by reactive oxygen species (ROS). Neonatal sepsis is frequently characterized by hyperinflammation. Cord blood monocytes (CBMO) are equivalent to monocytes of adults [peripheral blood monocytes (PBMO)], both in terms of phagocytosis and killing of Escherichia coli. We investigated whether CBMO are less sensitive toward PICD compared with PBMO. Monocytes were infected with green fluorescent protein (GFP)-labeled E. coli. Phagocytic activity, cell-count, Annexin V staining, hypoploid DNA content, CD95 and CD95L expression, and caspase-8 and -9 activities were analyzed by flow cytometry, ROS production by chemiluminescence, and CD95L mRNA expression by reverse-transcriptase polymerase chain reaction. With equal phagocytic activity and ROS production, PBMO cell count was decreased by 82 +/- 6% versus 28 +/- 8% for CBMO after infection. Annexin V binding was enhanced fivefold on PBMO; 56 +/- 15% of PBMO showed a hypodiploid DNA content compared with 9 +/- 6% of CBMO. Caspases CD95L and CD95L mRNA were up-regulated in PBMO. Our results indicate that CBMO are less sensitive toward E. coli-mediated PICD than PBMO. Modifying monocyte apoptosis may be a target for future interventions in sepsis.
Assuntos
Apoptose , Infecções por Escherichia coli/patologia , Escherichia coli , Monócitos/patologia , Fagocitose , Adulto , Anexina A5/metabolismo , Antígenos CD/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Células Cultivadas , DNA/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Sangue Fetal/citologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Recém-Nascido , Glicoproteínas de Membrana/metabolismo , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G , Fatores de TempoRESUMO
Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and alphaCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf(R)), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissaminetrade mark-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited alphaCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions.
Assuntos
Macrófagos/efeitos dos fármacos , Fosfatidilcolinas/farmacologia , Surfactantes Pulmonares/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Fagocitose/efeitos dos fármacos , Fenótipo , Surfactantes Pulmonares/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Depuradores/metabolismo , Especificidade por Substrato , Tensoativos/farmacologia , Linfócitos T/citologiaRESUMO
Monocyte-derived macrophage (MPhi) subsets are generated by antagonistic induction pathways. A helper MPhi-type (Mh-MPhi) is induced by interferon gamma (IFN-gamma), whereas a cytotoxic MPhi-type (Mc-MPhi), induced by interleukin-10 (IL-10), is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Compared with MPhi from healthy adults [peripheral blood monocyte-derived macrophages (PBMPhi)], cord blood MPhi (CBMPhi) were found less capable of generating Mh-MPhi. Here we tested the hypothesis that their generation of Mc-MPhi via IL-10 is also impaired. MPhi surface markers were phenotyped. IL-10 protein and mRNA production were detected after stimulation [alphaCD3 monoclonal antibody (mAb)]. CBMPhi or PBMPhi were co-cultured with MPhi-depleted mononuclear cells of adults and CD4-targeting antibodies as models for ADCC were added. In cord blood, we found diminished alphaCD3-induced IL-10 protein and mRNA production (p < 0.05 versus adults). Basal CD16 and HLA-DR expressions on CBMPhi of preterm and full-term neonates were lower (p < 0.05 versus PBMPhi). IL-10 had reduced effects on CD16 up- and HLA-DR down-modulation on CBMPhi (p < 0.05 versus PBMPhi). CD4-directed receptor modulation and deletion were reduced in the presence of CBMPhi (p < 0.05 versus PBMPhi). IL-10 failed to enhance their ADCC capacity, which was in contrast to PBMPhi (p < 0.05). These data suggest that CBMPhi have an impaired cytotoxic capacity via lower sensitivity toward IL-10.