SummaryBioactive components of human milk, such as human lactoferrin (hLF), play an essential role in gut microbiome homeostasis and protection against neonatal inflammatory diseases. Neonatal intestinal macrophages display a proinflammatory profile that might contribute to inflammatory mucosal injury. Therefore, the aim of the study was to investigate the immunomodulatory effects of hLF on differentiation and activation of monocyte‐derived macrophages (moMϕ). Monocytes isolated from umbilical cord blood of term neonates and peripheral blood of healthy adults were differentiated in the absence or presence of hLF, and differentiation, apoptosis and phagocytosis were evaluated. Cytokine production, Toll‐like receptor (TLR) signalling and activation marker expression were investigated upon activation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge. We demonstrate that hLF‐differentiated moMϕ exhibit decreased TLR‐4 expression, TLR signalling, proinflammatory cytokine secretion and intracellular tumour necrosis factor (TNF)‐α production. Investigation of differentiation markers, morphology and induction of apoptosis showed no alteration in lactoferrin‐differentiated moMϕ. Taken together, hLF promote anergic/anti‐inflammatory effects by TLR expression and pathway interference, resulting in a diminished proinflammatory moMϕ phenotype. The anergic/anti‐inflammatory properties of hLF might contribute to the prevention of harmful TLR‐mediated inflammatory disorders in the developing gut of premature infants.

Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding—and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.

BackgroundEndothelial cells (ECs) exert immunological functions such as production of proinflammatory cytokines/chemokines as well as facilitation of extravasation of immune cells into infected tissue. Limited data are available on the functionality of ECs from extremely preterm neonates during infection. Accordingly, the aim of our study was to investigate the immune response of premature ECs after proinflammatory stimulation.MethodsCell adhesion receptors' expression and function, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) signaling, and chemokine production were analyzed in umbilical cord ECs from extremely preterm and term neonates after proinflammatory stimulation.ResultsP-selectin and E-selectin surface expression as well as NFκB signaling were lower after lipopolysaccharide (LPS) stimulation in premature ECs. Preterm ECs exhibited lower, but significant, cell-adhesive functions after LPS stimulation compared with term ECs. CCL2/CXCL8 chemokine secretion was significantly upregulated after proinflammatory stimulation in both groups. CXCL10 production was significantly increased in term but not in preterm ECs upon stimulation with tumor necrosis factor compared with unstimulated ECs.ConclusionExtremely premature ECs showed partly reduced expression levels and function of cell adhesion molecules. Both NFκB signaling and chemokine/cytokine production were reduced in premature ECs. The diminished endothelial proinflammatory immune response might result in impaired infection control of preterm newborns rendering them prone to severe infection.

Plasmacytoid dendritic cells (pDCs) are key players in the antiviral immune response and type III IFNs such as IL‐29 appear to play a pivotal role in pDC function. Pronounced susceptibility to viral infections in neonates is partly resulting from diminished antiviral immune mechanisms. Accordingly, the aim of the present study was to investigate the impact of IL‐29 in the altered immune response of neonatal pDCs. PBMCs of adult and term newborns were stimulated with CpG‐ODN2216 in the presence or absence of IL‐29 and assessed for IFN‐α production, downstream‐signaling, and activation marker expression. A significantly lower IL‐29 production after TLR9‐specific stimulation was demonstrated in neonatal pDCs. IL‐29 enhanced the IFN‐α production of pDCs in adults compared to newborns. Newborn pDCs displayed a significantly lower surface expression of IL‐10 and IL‐28Rα receptor resulting in diminished STAT1 and IRF7 activation. Interestingly, concomitant stimulation with CpG‐ODN2216/IL‐29 had no impact on the expression of surface activation and maturation markers of pDCs in neither population. The diminished antiviral immune response of neonatal pDCs is associated with reduced production and cellular responses toward IL‐29. Potential therapeutic agents enhancing the IL‐29 response in neonatal pDCs possibly augment viral protection in newborns.