Anja Leiber

Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T-cell responses

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.

Phagocytosis and postphagocytic reaction of cord blood and adult blood monocyte after infection with green fluorescent protein-labeled Escherichia coli and group B Streptococci.

Neonatal sepsis is characterized by an excessive inflammatory response induced by immune cells (monocytes). We investigated the initial stage of monocyte‐pathogen interaction, i.e. bacterial ingestion and degradation at the single‐cell level, by comparing a new flow cytometric procedure with culture methods. We also examined the hypothesis that, in terms of phagocytosis‐induced cell death (PICD), phenotype, or cytokine production, cord blood monocytes (CBMO) differ from monocytes derived from adults (peripheral blood monocytes, PBMO).

Diminished Phagocytosis-Induced Cell Death (PICD) in Neonatal Monocytes upon Infection with Escherichia coli

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.

Granulocytic Myeloid-Derived Suppressor Cells Accumulate in Human Placenta and Polarize toward a Th2 Phenotype

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.

The CD95/CD95L pathway is involved in phagocytosis-induced cell death of monocytes and may account for sustained inflammation in neonates

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.

Infection-induced bystander-apoptosis of monocytes is TNF-alpha-mediated.

Phagocytosis induced cell death (PICD) is crucial for controlling phagocyte effector cells, such as monocytes, at sites of infection, and essentially contributes to termination of inflammation. Here we tested the hypothesis, that during PICD bystander apoptosis of non-phagocyting monocytes occurs, that apoptosis induction is mediated via tumor necrosis factor-alpha (TNF-α and that TNF-α secretion and -signalling is causal. Monocytes were infected with Escherichia coli (E. coli), expressing green fluorescent protein (GFP), or a pH-sensitive Eos-fluorescent protein (EOS-FP). Monocyte phenotype, phagocytic activity, apoptosis, TNF-receptor (TNFR)-1, -2-expression and TNF-α production were analyzed. Apoptosis occured in phagocyting and non-phagocyting, bystander monocytes. Bacterial transport to the phagolysosome was no prerequisite for apoptosis induction, and desensitized monocytes from PICD, as confirmed by EOS-FP expressing E. coli. Co-cultivation with non-infected carboxyfluorescein-succinimidyl-ester- (CFSE-) labelled monocytes resulted in significant apoptotic cell death of non-infected bystander monocytes. This process required protein de-novo synthesis and still occurred in a diminished way in the absence of cell-cell contact. E. coli induced a robust TNF-α production, leading to TNF-mediated apoptosis in monocytes. Neutralization with an anti-TNF-α antibody reduced monocyte bystander apoptosis significantly. In contrast to TNFR2, the pro-apoptotic TNFR1 was down-regulated on the monocyte surface, internalized 30 min. p.i. and led to apoptosis predominantly in monocytes without phagocyting bacteria by themselves. Our results suggest, that apoptosis of bystander monocytes occurs after infection with E. coli via internalization of TNFR1, and indicate a relevant role for TNF-α. Modifying monocyte apoptosis in sepsis may be a future therapeutic option.

Clearance of Apoptotic Neutrophils Is Diminished in Cord Blood Monocytes and Does Not Lead to Reduced IL-8 Production

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.

Neonatal monocytes express antiapoptotic pattern of Bcl-2 proteins and show diminished apoptosis upon infection with Escherichia coli

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.