Lipoprotein(a), an Opsonin, Enhances the Phagocytosis of Nontypeable Haemophilus influenzae by Macrophages

We recently showed that both nontypeable Haemophilus influenzae (NTHi) and its surface plasminogen- (Plg-) binding proteins interact with lipoprotein(a) (Lp(a)) in a lysine-dependent manner. Because Lp(a) can be taken up by macrophages, we postulated that it serves as an opsonin to enhance phagocytosis of NTHi by macrophages. Based on colony-forming unit (CFU) counts, Lp(a) was found to increase U937 macrophage-mediated phagocytosis of NTHi49247 and NTHi49766 by 34% and 43%, respectively, after 120 min. In contrast, Lp(a) did not enhance phagocytosis of Escherichia coli BL21 or E. coli JM109, which were unable to bind to Lp(a). As with U937 macrophages, Lp(a) was capable of increasing phagocytosis of NTHi49247 by peripheral blood mononuclear cell-derived macrophages. Opsonic phagocytosis by Lp(a) was inhibited by the addition of recombinant kringle IV type 10 (rKIV10), a lysine-binding competitor; moreover, Lp(a) did not increase phagocytosis of NTHi by U937 macrophages that were pretreated with a monoclonal antibody against the scavenger receptor CD36. Taken together, our observation suggests that Lp(a) might serve as a lysine-binding opsonin to assist macrophages in rapid recognition and phagocytosis of NTHi.

Lactoferrin, Quercetin, and Hydroxyapatite Act Synergistically against Pseudomonas fluorescens

Pseudomonas fluorescens is an opportunistic, psychotropic pathogen that can live in different environments, such as plant, soil, or water surfaces, and it is associated with food spoilage. Bioactive compounds can be used as antimicrobials and can be added into packaging systems. Quercetin and lactoferrin are the best candidates for the development of a complex of the two molecules absorbed on bio combability structure as hydroxyapatite. The minimum inhibiting concentration (MIC) of single components and of the complex dropped down the single MIC value against Pseudomonas fluorescens. Characterization analysis of the complex was performed by means SEM and zeta-potential analysis. Then, the synergistic activity (Csyn) of single components and the complex was calculated. Finally, the synergistic activity was confirmed, testing in vitro its anti-inflammatory activity on U937 macrophage-like human cell line. In conclusion, the peculiarity of our study consists of optimizing the specific propriety of each component: the affinity of lactoferrin for LPS; that of quercetin for the bacterial membrane. These proprieties make the complex a good candidate in food industry as antimicrobial compounds, and as functional food.

Synthetic chalcone derivatives inhibit cytokine secretion via inhibition of ERK and JNK pathways in human U937 macrophage

Purpose: To investigate the inhibitory effects of a chalcone derivative on lipopolysaccharide (LPS)- induced interleukin (IL)-6 and IL-8 secretions and on LPS-induced mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation in human U937 macrophage-like cell line. Methods: The effects of chalcone derivative on LPS-induced secretion of IL-6 and IL-8 in endothelial cells were determined by enzyme-linked immunosorbent assay while the effects of chalcone on the activation of MAPK and NF-kB pathway were determined by Western blotting. Results: The results showed that 3-(5-methyl-furan-2-yl)-naphthalen-1-yl-propenone (compound 1) significantly inhibited the secretion of LPS-induced IL-6 and IL-8 in U937 macrophages. This compound also demonstrated significant suppression of c-Jun N-terminal kinases (JNK) and extracellular signalregulated kinases (ERK) phosphorylation. However, the compound did not reverse the degradation of inhibitor kappa B alpha (IκBα) and did not inhibit the phosphorylation of NF-κB subunit and P-38 MAPK. Conclusion: Compound 1 inhibits the secretion of cytokines via the inhibition of ERK and JNK pathways. These results suggest that chalcone derivative could act as an antiinflammatory agent by altering cytokine secretion and inflammatory pathways.

Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

Eicosanoid content in fetal calf serum accounts for reproducibility challenges in cell culture

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking in. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.