Early initiation of antibiotic therapy and short-term outcomes in preterm infants: a single-centre retrospective cohort analysis.

BACKGROUND: Sepsis is one of the most important complications in preterm infants. For this reason, many such infants receive antibiotics during their hospital stay. However, early antibiotic therapy has also been associated with adverse outcome. It is yet largely unclear if the time of onset of antibiotic therapy influences the outcome. We here investigated whether the timing of initiation of antibiotic therapy plays a role in the association between antibiotic exposure and short-term outcome. METHODS: Retrospective analysis of data from 1762 very low birthweight infants born in a German neonatal intensive care unit (NICU) between January 2004 and December 2021. RESULTS: Antibiotics were administered to 1214 of the 1762 (68.9%) infants. In 973 (55.2%) of the 1762 of infants, antibiotic therapy was initiated within the first two postnatal days. Only 548 (31.1%) infants did not have any antibiotic prescription during their stay in the NICU. Antibiotic exposure at every timepoint was associated with an increased risk of all short-term outcomes analysed in univariable analyses. In multivariable analyses, initiation of antibiotic therapy within the first two postnatal days and initiation between postnatal days 3 and 6 was independently associated with an increased risk of developing bronchopulmonary dysplasia (BPD) (OR 3.1 and 2.8), while later initiation of antibiotic therapy was not. CONCLUSION: Very early initiation of antibiotic therapy was associated with an increased risk of BPD. Due to the study design, no conclusions on causality can be drawn. If confirmed, our data suggest that an improved identification of infants at low risk of early-onset sepsis is needed to reduce antibiotic exposure.

Expression of immune checkpoint molecules on adult and neonatal T-cells.

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.

Decreased expression of hypoxia-inducible factor 1α (HIF-1α) in cord blood monocytes under anoxia.

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.

Modulatory activity of adenosine on the immune response in cord and adult blood.

BACKGROUND: Neonatal sepsis is a leading cause of neonatal morbidity and mortality, associated with immunosuppression. Myeloid-derived suppressor cells (MDSCs) are cells with immunosuppressive activity, present in high amounts in cord blood. Mechanisms regulating MDSC expansion are incompletely understood. Adenosine is a metabolite with immunoregulatory effects that are elevated in cord blood. METHODS: Impact of adenosine on peripheral and cord blood mononuclear cells (PBMCs and CBMCs) was analysed by quantification of ectonucleotidases and adenosine receptor expression, MDSC induction from PBMCs and CBMCs, their suppressive capacity on T cell proliferation and effector enzyme expression by flow cytometry. RESULTS: Cord blood monocytes mainly expressed CD39, while cord blood T cells expressed CD73. Adenosine-induced MDSCs from PBMCs induced indoleamine-2,3-dioxygenase (IDO) expression and enhanced arginase I expression in monocytes. Concerted action of IDO and ArgI led to effective inhibition of T cell proliferation. In addition, adenosine upregulated inhibitory A3 receptors on monocytes. CONCLUSION: Adenosine acts by inducing MDSCs and upregulating inhibitory A3 receptors, probably as a mode of autoregulation. Thus, adenosine contributes to immunosuppressive status and may be a target for immunomodulation during pre- and postnatal development. IMPACT: Immune effector cells, that is, monocytes, T cells and MDSCs from cord blood express ectonucleotidases CD39 and CD73 and may thus serve as a source for adenosine as an immunomodulatory metabolite. Adenosine mediates its immunomodulatory properties in cord blood by inducing MDSCs, and by modulating the inhibitory adenosine A3 receptor on monocytes. Adenosine upregulates expression of IDO in MDSCs and monocytes potentially contributing to their suppressive activity.

Microbiological analyses of nasally guided catheters after less invasive surfactant administration - a pilot study.

BACKGROUND: Respiratory distress syndrome (RDS) is a frequent complication of premature birth. Treating RDS by continuous positive airway pressure and less invasive surfactant administration (LISA) may reduce bronchopulmonary dysplasia. Surfactant, however, can be inactivated by bacterial infection. Therefore, potential routes of microbe transmission into the airway are of interest. The aim of this study was to evaluate microbiological contamination of catheters used for LISA procedures and its association with postnatal age. METHODS: Catheter tips used for LISA procedures via the nasal route (LISA-n) in infants with RDS were placed into a sterile eSwab container directly after the procedure, cultured and examined for microbiological contamination. RESULTS: Interpretable results could be collected from 20 catheter tips. Four showed positive culture results (20%) with microbes potentially associated with the development of early onset neonatal sepsis. Risk of positive microbe detection increased with postnatal age (< 4 h: 10%; 4-18 h: 20%; > 18 h: 40%). CONCLUSIONS: In this pilot study, the risk of tracheal microbe transmission following the LISA-n procedure increased with postnatal age. Although the clinical relevance of this finding is unclear, earlier surfactant administration might reduce the risk of catheter contamination. TRIAL REGISTRATION NUMBER: Substudy of the registered Trial: feasibility study - Neofact: NCT04086095, www.ClinicalTrials.gov, September 11, 2019.

Cord blood granulocytic myeloid-derived suppressor cells impair monocyte T cell stimulatory capacity and response to bacterial stimulation.

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.

Metalloproteinases TACE and MMP-9 Differentially Regulate Death Factors on Adult and Neonatal Monocytes After Infection with Escherichia coli.

Background: Cleaving ligands and receptors of the tumor necrosis factor (TNF) superfamily can critically regulate the induction of apoptosis. Matrix metalloproteinases (MMPs) such as MMP-9 and tumor necrosis factor-α-converting enzyme (TACE) have been shown to cleave CD95-Ligand (CD95L) and TNF/(TNF receptor-1) TNFR1 which induce phagocytosis induced cell death (PICD) in adult monocytes. This process is reduced in neonatal monocytes. Methods: Here we tested in vitro, whether Escherichia coli infection mounts for activation of MMP-9 and TACE in monocytes and whether this process regulates PICD. Results: The surface expression of TACE was most prominent on infected adult monocytes. In contrast, surface presentation of MMP-9 was highest on infected neonatal monocytes. Selective blocking of MMP-9 decreased CD95L secretion, while inhibition of TACE left CD95L secretion unaltered. Blocking of MMP-9 increased surface CD95L (memCD95L) expression on infected neonatal monocytes to levels comparable to infected adult monocytes. Moreover, MMP-9 inhibition raised PICD of infected neonatal monocytes to levels observed for infected adult monocytes. In contrast, TACE inhibition decreased PICD in infected monocytes. Addition of extracellular TNF effectively induced memCD95L presentation and PICD of adult monocytes and less of neonatal monocytes. Conclusion: MMP-9 activity is crucial for downregulating cell-contact dependent PICD in E. coli infected neonatal monocytes. By this mechanism, MMP-9 could contribute to reducing sustained inflammation in neonates.

HIF-1α-Deficiency in Myeloid Cells Leads to a Disturbed Accumulation of Myeloid Derived Suppressor Cells (MDSC) During Pregnancy and to an Increased Abortion Rate in Mice.

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.

Granulocytic Myeloid-Derived Suppressor Cells (GR-MDSC) in Breast Milk (BM); GR-MDSC Accumulate in Human BM and Modulate T-Cell and Monocyte Function.

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.

Reduced internalization of TNF-ɑ/TNFR1 down-regulates caspase dependent phagocytosis induced cell death (PICD) in neonatal monocytes.

Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with Escherichia coli-GFP (E. coli-GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with E. coli). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with E. coli was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis (all p < 0.05 vs. PBMO). Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with E.coli and is reduced in CBMO.

Outcome of primary repair in extremely and very low-birth-weight infants with esophageal atresia/distal tracheoesophageal fistula.

PURPOSE: The optimal surgical management of extremely (ELBW) and very low-birth-weight (VLBW) neonates with esophageal atresia and distal tracheoesophageal fistula (EA/TEF) (Gross type C) is still debated. The aim of this study was to evaluate the surgical outcome of primary repair in these patients and compare it to ≥1500g neonates. METHODS: Medical records of neonates with repaired EA from 2002 to 2016 were reviewed. RESULTS: 4 ELBW, 7 VLBW, and 24 ≥1500g infants had type C EA/TEF and underwent primary repair. Anastomotic leakage occurred in 0% ELBW, 0% VLBW and 8.3% ≥1500g patients and anastomotic stricture in 25% ELBW, 28.5% VLBW and 37.5% ≥1500g patients. 50% ELBW, 14.2% VLBW and 20.8% ≥1500g patients underwent secondary fundoplication. One patient of the VLBW group and one patient of the ≥1500g group died postoperatively of causes not related to EA/TEF. CONCLUSIONS: In extremely and very low-birth-weight neonates with type C EA/TEF surgical outcome after primary repair is comparable to the outcome in ≥1500g neonates. Primary repair can be performed in most of these patients and staged repair can be restricted to unstable patients. LEVEL OF EVIDENCE: Treatment study level III.

Neonatal myeloid derived suppressor cells show reduced apoptosis and immunosuppressive activity upon infection with Escherichia coli.

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.

NOD2 Loss-of-Function Mutations and Risks of Necrotizing Enterocolitis or Focal Intestinal Perforation in Very Low-birth-weight Infants.

BACKGROUND: NOD2 loss-of-function mutations, that is, R702W [rs2066844], G908R [rs2066845], and Leu1007fsinsC [rs5743293], have been linked to inflammatory bowel diseases. It is yet unknown whether these variants are also associated with necrotizing enterocolitis (NEC) or focal intestinal perforation (FIP) in infants of very low birth weight (VLBW). METHODS: To test this hypothesis, we genotyped 9082 VLBW infants with European ancestry enrolled in a prospective, population-based cohort study of the German Neonatal Network. We assessed the effect of the NOD2 gene variants on the risk for major morbidities of the gastrointestinal tract, that is, NEC/FIP requiring surgery in multivariable logistic regression analyses. RESULTS: In the whole cohort of VLBW infants, carriers of ≥ 2 NOD2 variant alleles had an increased risk for NEC requiring surgery (odds ratio [OR], 3.57; 95% confidence interval [CI], 1.27-10.04; P = 0.03) and NEC or FIP requiring surgery (OR, 3.81; 95% CI, 1.70-8.51; P = 0.004) as compared with wild-type genotypes. In a multivariable logistic regression analysis including gestational age, birth weight, gender, multiple birth, and inborn delivery, the association between ≥ 2 NOD2 variant alleles and NEC surgery (OR, 4.14; 95% CI, 1.41-12.12; P = 0.009), FIP surgery (OR, 3.50; 95% CI, 1.02-12.04; P = 0.047), and NEC or FIP surgery (OR, 4.10; 95% CI, 1.74-9.73; P = 0.001) proved to be independent. We also performed a regression analysis in the subgroup of infants with available information on Lactobacillus acidophilus/Bifidobacterium infantis probiotic supplementation (n = 3638). Although probiotics had a protective effect on NEC and NEC or FIP requiring surgery, the NOD2 variants had no significant impact in this subgroup. CONCLUSIONS: VLBW infants carrying ≥ 2 NOD2 genetic risk factors of inflammatory bowel disease in adults have an increased risk for severe gastrointestinal complications, such as NEC requiring surgery. Therefore, infants might benefit from NOD2 genotyping followed by supplementation with probiotics. Replication studies are needed along with genome-wide arrays to allow risk-adapted prevention and therapeutic strategies.

Monocyte-induced development of Th17 cells and the release of S100 proteins are involved in the pathogenesis of graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. However, the pathophysiology of GvHD remains poorly understood. In this study, we analyzed the induction of Th17 cells by monocytes of patients with GvHD in vitro, demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I-IV and chronic GvHD induce significantly increased levels of Th17 cells compared with patients without GvHD. S100 proteins are known to act as innate amplifier of inflammation. We therefore investigated the presence of S100 proteins in the stool, serum, and bowel tissue of patients with GvHD and the influence of S100 proteins on the induction of Th17 cells. Elevated levels of S100 proteins could be detected in patients with acute GvHD, demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, stimulation of monocytes with S100 proteins was found to promote Th17 development, emphasizing the role of S100 proteins in Th17-triggered inflammation. Altogether, our results indicate that induction of Th17 cells by activated monocytes and the stimulatory effects of proinflammatory S100 proteins might play a relevant role in the pathogenesis of acute GvHD. Regarding our data, S100 proteins might be novel markers for the diagnosis and follow-up of GvHD.

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.

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.

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.

Bacterial reprogramming of PBMCs impairs monocyte phagocytosis and modulates adaptive T cell responses.

Septic diseases are characterized by an initial systemic, proinflammatory phase, followed by a period of anti-inflammation. In the context of the latter, monocytes have been described to display altered functions, including reduced TNF secretion and T cell-stimulating capacities in response to recall antigens. This hyporesponsiveness is supposed to be detrimental for coping with secondary infections. We here characterize bacterially reprogrammed PBMC-derived monocytes with special focus on their phagocytic activity. Hence, we have implemented a surrogate model of the early, postinflammatory period by exposing PBMCs to Escherichia coli on d0 and rechallenging them with bacteria on d2. This induced the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naïve T cell stimulation. The compromising effects on phagocytosis required the presence of bacteria and were not mimicked by TLR4 ligation or exposure to isolated cytokines alone. Moreover, the impairments were specific for the engulfment of bacteria and were coupled to a selective down-regulation of FcγR and SR expression. Intriguingly, this monocytic phenotype contributed to the stimulation of a T(H)17-polarized adaptive immune response in the context of secondary infection. Our findings extend the current knowledge of monocytic reprogramming and identify the phagocytic capacity of monocytes as a putative sepsis biomarker.

Monocytes derived from humanized neonatal NOD/SCID/IL2Rγ(null) mice are phenotypically immature and exhibit functional impairments.

Trials of immune-modulating drugs in septic patients have mostly failed to demonstrate clinical efficacy. Thus, we sought to generate a surrogate model of myelomonocytic lineage differentiation that would potentially allow sepsis induction and preclinical testing of anti-inflammatory drugs. Comparing transplantation of cord blood-derived stem cells in neonatal NOD/SCID/IL2Rγ(null) (neonatal huNSG) mice with transplantation of adult peripheral mobilized stem cells into adult NSG (adult huNSG) recipients, we demonstrate that myelomonocytic lineage differentiation in neonatal huNSG mice is retarded and monocytes are phenotypically immature with respect to HLA-DR expression and the emergence of CD80(+)CD86(+) monocytes. Functionally, neonatal huNSG mice were less sensitive toward interferon-γ-induced upregulation of CD86 and exhibited a reduced T-cell stimulating capacity when compared with adult huNSG mice, whereas the phagocytic activity and the ability for cytokine secretion were mature. However, comparison of these data with data obtained from human neonates indicate that absence of the CD80(+)CD86(+) population and the reduced T-cell stimulating capacity of neonatal huNSG monocytes resemble functional immaturities observed in human neonatal monocytes. Thus, these two mouse models might well serve as 2 independent surrogate models for studying the neonatal myelomonocytic lineage differentiation or for testing the efficacy of immunomodulatory drugs on functionally mature monocytes.