Neonatal Monocyte-derived Macrophages Research Articles

Transcriptomic analysis identifies lactoferrin-induced quiescent circuits in neonatal macrophages.

Upon birth, a hitherto naïve immune system is confronted with a plethora of microbial antigens due to intestinal bacterial colonization. To prevent excessive inflammation and disruption of the epithelial barrier, physiological mechanisms must promote immune-anergy within the neonatal gut. As high concentrations of human lactoferrin (hLF), a transferrin glycoprotein shown to modulate macrophage function, are frequently encountered in colostrum, its direct interaction with intestinal macrophages may satisfy this physiological need. Thus, the primary objective of this study was to investigate transcriptional changes induced by human lactoferrin in neonatal monocyte-derived macrophages. Cord blood-derived monocytes were differentiated with M-CSF in presence or absence of 500 µg/mL hLF for 7 days and afterwards stimulated with 1 ng/mL LPS or left untreated. RNA was then isolated and subjected to microarray analysis. Differentiation of cord blood-derived monocytes in presence of hLF induced a distinct transcriptional program defined by cell cycle arrest in the G2/M phase, induction of IL-4/IL-13-like signaling, altered extracellular matrix interaction, and enhanced propensity for cell-cell interaction. Moreover, near-complete abrogation of transcriptional changes induced by TLR4 engagement with LPS was observed in hLF-treated samples. The global transition towards an M2-like homeostatic phenotype and the acquisition of quiescence elegantly demonstrate the ontogenetical relevance of hLF in attenuating pro-inflammatory signaling within the developing neonatal intestine. The marked anergy towards proinflammatory stimuli such as LPS further underlines the glycoprotein's potential therapeutic relevance.

Dose-dependent immunomodulatory effects of metformin on human neonatal monocyte-derived macrophages

While the association of inflammation with bronchopulmonary dysplasia (BPD) has long been appreciated, M1 proinflammatory macrophage population is emerging as the key element in driving the BPD inflammatory environment. Previous study suggests that low-dose metformin elicits an anti-inflammatory response, possibly through modulating macrophages, to improve disease outcome in a rat BPD model. To investigate this concept further, we examined the dose-dependent immunomodulatory function of metformin directly on human macrophages derived from cord blood (CB) monocytes. We demonstrate that low-dose metformin promotes expansion of M2 anti-inflammatory macrophages, contrasted with high-dose treatment, which exacerbates inflammation by favoring M1 polarization and restricting M2 phenotype. These findings highlight that metformin hold immunomodulatory ability by regulating macrophage polarization in a dose-dependent manner, and only when applied at low dose, exhibiting potential for beneficial anti-inflammatory adjuvant in BPD setting.

Human lactoferrin attenuates the proinflammatory response of neonatal monocyte-derived macrophages.

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.

Enhanced Phagocytosis in Neonatal Monocyte-Derived Macrophages is Associated with Impaired SHP-1 Signaling

Resident macrophages represent a first line of defense through the ingestion of microbial pathogens. Phagocytosis mediated by immunoglobulin-binding Fc receptors is a complex series of events involving tyrosine phosphorylation and dephosphorylation. In the present study we determined that the phagocytic capacity of neonatal monocyte-derived macrophage (MDM) was enhanced in comparison to adult MDM. Cross-linking of surface FcγRIIa receptors enhanced tyrosine phosphorylation of several proteins in both groups, followed by a reduction in tyrosine phosphorylation in adult but not neonatal MDM. Expression of the tyrosine phosphatase SHP-1 was similar in neonatal and adult MDM; however, baseline SHP-1 activity levels were diminished in neonatal MDM. Cross-linking of FcγRIIa receptors induced a greater increase in SHP-1 activity in adult MDM vs. neonatal MDM. The cytoplasmic adapter protein Cbl is a SHP-1 ligand and negatively affects phagocytosis. As determined by co-immunoprecipitation assays, SHP-1 and Cbl did not associate to the same extent in neonatal as compared to adult MDM. Our data suggest that the enhanced phagocytic capacity of neonatal MDM is associated with decreased SHP-1 activity and alteration of downstream signaling pathways.

Decreased CD47 expression during spontaneous apoptosis targets neutrophils for phagocytosis by monocyte-derived macrophages

Background Neutrophils (PMN) are the primary leukocyte responders during acute inflammation. After migrating into the tissues, PMN undergo programmed cell death (apoptosis) and are subsequently removed via phagocytosis by resident macrophages during the resolution phase. Efficient phagocytosis of apoptotic neutrophils is necessary for successful resolution. CD47 plays a critical role in mediating the phagocytic response, although its role in the phagocytosis of apoptotic PMN is incompletely understood. Aims In the present study we tested the hypotheses that CD47 modulates the targeting of apoptotic PMN for phagocytosis, and that this process is altered in neonatal PMN. Study design Adult and neonatal PMN were examined for their expression of CD47. To investigate CD47-mediated functions, apoptotic adult and neonatal PMN were co-cultured with monocyte-derived macrophages (MDM) and the phagocytic index was determined using a flow cytometry-based assay. Results We observed lower basal surface CD47 levels on neonatal vs. adult PMN. In both groups, spontaneous apoptosis led to decreased surface and total cellular CD47 expression. Adult and neonatal MDM ingested apoptotic neonatal target PMN more avidly than apoptotic adult target PMN. Masking of surface CD47 on PMN with a monoclonal antibody enhanced MDM phagocytic activity. Conclusions Our results suggest that age-dependent expression of CD47 on PMN may account for differences in their ingestion by macrophages and in the resolution of inflammation.

Differential Expression of Chemokines and Their Receptors in Adult and Neonatal Macrophages Infected with Human or Avian Influenza Viruses

In 1997, avian influenza virus H5N1 was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H5N1 infection prompted us to compare the chemokine responses between influenza virus–infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H5N1) (H5N1/97), its precursor A/Quail/Hong Kong/G1/97 (H9N2) (H9N2/G1), and human influenza virus A/Hong Kong/54/98 (H1N1) (H1N1/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H5N1/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus–infected adult MDMs. The strong induction of chemokines and their receptors by avian influenza viruses, particularly in adult MDMs, may account for the severity of H5N1 disease

Morphine enhances HIV infection of neonatal macrophages.

Perinatal transmission of HIV accounts for almost all new HIV infections in children. There is an increased risk of perinatal transmission of HIV with maternal illicit substance abuse. Little is known about neonatal immune system alteration and subsequent susceptibility to HIV infection after morphine exposure. We investigated the effects of morphine on HIV infection of neonatal monocyte-derived macrophages (MDM). Morphine significantly enhanced HIV infection of neonatal MDM. Morphine-induced HIV replication in neonatal MDM was completely suppressed by naltrexone, the opioid receptor antagonist. Morphine significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of macrophage inflammatory protein-1beta in neonatal MDM. Thus, morphine, most likely through alteration of beta-chemokines and CCR5 receptor expression, enhances the susceptibility of neonatal MDM to HIV infection, and may have a cofactor role in perinatal HIV transmission and infection.

Cytokine receptor signalling in neonatal macrophages: defective STAT-1 phosphorylation in response to stimulation with IFN-gamma.

We reported earlier that neonatal monocyte-derived macrophages (MDM) could not be fully activated with IFN-gamma, a finding that could not be attributed to lower expression of IFN-gamma receptors on the neonatal cells. In this study we explored elements of IFN-gamma R-mediated signalling in cord monocytes and MDM. Intracellular expression of STAT-1 was analysed by flow cytometry. We have assessed phosphorylation of STAT-1 by using MoAbs that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis. Using MoAbs against the native form of STAT-1 revealed comparable expression of this protein in cord and adult cells (both monocytes and MDM). However, STAT-1 phosphorylation in response to IFN-gamma was significantly decreased in neonatal monocytes (P < 0.05) and MDM (P < 0.01) compared to adult cells (n > 5 for each). These data suggest deficient cytokine-receptor signalling in neonatal mononuclear phagocytes exposed to IFN-gamma. We propose that decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.

Interferon-gamma-induced activation of indoleamine 2,3-dioxygenase in cord blood monocyte-derived macrophages inhibits the growth of group B streptococci.

Neonatal sepsis is most often caused by group B streptococci (GBS) and is a major cause of death in the neonatal period. The response of the immune system in the newborn child has received much attention and is thought to be deficient in a number of ways. The effector response of neonatal monocyte-derived macrophages (MDM) was investigated. Interferon-gamma induced the activation of indoleamine 2,3-dioxygenase in MDM and inhibited the growth of GBS. Both effects were enhanced by the addition of tumor necrosis factor-alpha to the culture conditions. The coincident supplementation of L-tryptophan with the bacteria abrogated the bacterial growth inhibition, thus confirming the causative role of L-tryptophan depletion. Control of the extracellular as well as intracellular L-tryptophan levels may thus be one of the effector mechanisms with which the immune system defends the host against GBS dissemination and disease.

Human neonatal monocyte-derived macrophages and neutrophils exhibit normal nonopsonic and opsonic receptor-mediated phagocytosis and superoxide anion production.

Human neonates are at risk for invasive infections with fungi and gram-negative bacteria, microorganisms which are often susceptible to phagocytosis in either the presence or the absence of opsonins. Although opsonic phagocytosis has been investigated, to date there have been no systematic studies of nonopsonic phagocytosis in neonates. We investigated receptor-mediated phagocytosis by neonatal neutrophils and monocyte-derived macrophages in order to determine if a functional defect exists in any of the classes of nonopsonic or opsonic receptors. Ingestion or binding by mannosyl/fucosyl, Fc and complement receptors and the receptor(s) for unopsonized Pseudomonas aeruginosa were normal in neutrophils and macrophages from the cord blood of 26 healthy full-term neonates as compared to simultaneous normal adult controls. Superoxide anion production by neonatal neutrophils in response to both soluble and particulate stimuli was also normal. We conclude that neonatal nonopsonic and opsonic receptor-mediated phagocytosis are normal.

Phagocytosis-associated functions in neonatal monocyte-derived macrophages.

Using a Teflon culture system we analyzed different aspects of cell maturation and phagocytic activities in neonatal monocyte-derived macrophages. Morphological and cytochemical characteristics as well as the protein composition of neonatal macrophages were identical with those of adult controls; actin binding protein (265,000 Da), myosin (210,000), alpha-actinin (102,000) and actin (42,000) could be identified in cells from either source. All phagocytic functions were shown to be perfectly normal in neonatal macrophages when compared with adult cells: random migration, chemotactic response to zymosan-activated serum and formyl-methionyl-leucyl-phenylalanine, ingestion, and killing of Staphylococcus aureus, phagocytosis-associated chemiluminescence, production of oxygen intermediates (superoxide anion, O2-; hydrogen-peroxide, H2O2). Phorbol myristate acetate-stimulated O2(-)-generation by 1 x 10(5) macrophages was 11.8 +/- 4.7 nmol/h for neonates, and 10.2 +/- 3.9 for controls; production of H2O2 was 7.6 +/- 3.5 nmol/h in neonatal macrophages and 6.4 +/- 2.8 in adult controls. It is unlikely, then, that the increased susceptibility of human neonates to systemic bacterial infections can be related to an abnormality in the essential phagocyte functions of macrophages.