A unique protein profile of peripheral neutrophils from COPD patients does not reflect cytokine-induced protein profiles of neutrophils in vitro

Contains fulltext : 96603.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Inflammation, both local and systemic, is a hallmark of chronic obstructive pulmonary disease (COPD). Inflammatory mediators such as TNFalpha and GM-CSF are secreted by lung epithelium, alveolar macrophages and other inflammatory cells and are thought to be important contributors in the pathogenesis of COPD. Indeed, neutrophils are activated by these cytokines and these cells are one of the major inflammatory cell types recruited to the pulmonary compartment of COPD patients. Furthermore, these inflammatory mediators are found in the peripheral blood of COPD patients and, therefore, we hypothesized that TNFalpha/GM-CSF-induced protein profiles can be found in peripheral neutrophils of COPD patients. METHODS: Using fluorescence 2-dimensional difference gel electrophoresis we investigated differentially regulated proteins in peripheral neutrophils from COPD patients and healthy age-matched control subjects. Furthermore, protein profiles from COPD patients were compared with those of neutrophils of healthy age-matched controls that were stimulated with TNFalpha and/or GM-CSF in vitro. Protein gels were compared using DeCyder 7.0 software. RESULTS: We identified 7 significantly regulated protein spots between peripheral neutrophils from COPD patients and age-matched healthy control subjects. Stimulation of peripheral neutrophils with TNFalpha, GM-CSF or TNFalpha + GM-CSF in vitro resulted in 13, 20 and 22 regulated protein spots, respectively. However, these cytokine-induced protein differences did not correspond with the protein differences found in neutrophils from COPD patients. CONCLUSION: These results show that neutrophils from COPD patients have a unique protein profile compared to neutrophils from healthy age-matched controls. Furthermore, the neutrophil profiles of COPD patients do not reflect putative dominant signals induced by TNFalpha, GM-CSF or their combination. Our results indicate that systemic neutrophil responses in COPD patients are caused by a unique but subtle interplay between multiple inflammatory signals

Effect of bovine milk fat-based infant formulae on microbiota, metabolites and stool parameters in healthy term infants in a randomized, crossover, placebo-controlled trial

Background: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants’ gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. Methods: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. Results: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p &lt; 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. Conclusions: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. Trial registration: Netherlands Trial Registry Identifier: NL7815 19/06/2019.</p

Management of Cow's Milk Allergy from an Immunological Perspective : What Are the Options?

The immunological mechanism underlying Immunoglobuline E (IgE)-mediated cow's milk allergy has been subject to investigations for many years. Identification of the key immune cells (mast cells, B cells) and molecules (IgE) in the allergic process has led to the understanding that avoidance of IgE-crosslinking epitopes is effective in the reduction of allergic symptoms but it cannot be envisioned as a treatment. For the treatment and prevention of IgE-mediated cow's milk allergy, it is thought that the induction of a sustained state of immunological tolerance is needed. In this review, we will discuss various approaches aimed at achieving immunological tolerance and their success. Furthermore, we will speculate on the involved immunological mechanism

In vitro induction of trained innate immunity by bIgG and whey protein extracts

Bovine immunoglobulin G (bIgG) was previously shown to enhance innate immune responses to toll-like receptor (TLR) stimulation, via induction of trained immunity. In this study, we investigated whether minimally processed dairy streams with high levels of whey proteins as potential infant nutrition ingredients could also induce trained immunity, and to what extent this can be explained by the presence of bIgG. The minimally processed whey ingredients serum protein concentrate (SPC) and whey protein concentrate (WPC) were tested for their ability to induce trained immunity in human peripheral blood monocytes. Both ingredients induced trained immunity as evidenced by an increased production of TNF-α and, to a lesser extent, of IL-6 upon stimulation with TLR ligands. This was comparable to isolated bovine immunoglobulin G (bIgG) that served as positive control. Depletion of bIgG from both whey protein-containing ingredients did not significantly inhibit the induction of trained immunity, suggesting that the streams contain other components in addition to bIgG that are able to induce trained immunity. These results indicate that minimally processed whey ingredients may contribute to protection against infections through enhancing innate immune responsiveness to pathogens</p

Differences in potency of CXC chemokine ligand 8-, CC chemokine ligand 11-, and C5a-induced modulation of integrin function on human eosinophils

The hypothesis was tested that different chemoattractants have different effects on the activity of integrins expressed by the human eosinophil. Three chemoattractants, CXCL8 (IL-8), CCL11 (eotaxin-1), and C5a were tested with respect to their ability to induce migration and the transition of eosinophils from a rolling interaction to a firm arrest on activated endothelial cells under flow conditions. CCL11 and C5a induced a firm arrest of eosinophils rolling on an endothelial surface, whereas CXCL8 induced only a transient arrest of the cells. The CXCL8- and CCL11-induced arrest was inhibited by simultaneously blocking alpha4 integrins (HP2/1) and beta2 integrins (IB4). In contrast, the C5a-induced arrest was only inhibited by 30% under these conditions. The potency differences of C5a>CCL11>CXCL8 to induce firm adhesion under flow condition was also observed in migration assays and for the activation of the small GTPase Rap-1, which is an important signaling molecule in the inside-out regulation of integrins. Interestingly, only C5a was able to induce the high activation epitope of alphaMbeta2 integrin recognized by MoAb CBRM1/5. The C5a-induced appearance of this epitope and Rap activation was controlled by phospholipase C (PLC), as was shown with the PLC inhibitor U73122. These data show that different chemoattractants are able to induce distinct activation states of integrins on eosinophils and that optimal chemotaxis is associated with the high activation epitope of the alphaMbeta2 integrin. Furthermore, PLC plays an important role in the inside-out signaling and, thus, the activation status of integrins on eosinophil

Immunomodulating protein aggregates in soy and whey hydrolysates and their resistance to digestion in an in vitro infant gastrointestinal model:New insights in the mechanism of immunomodulatory hydrolysates

Hydrolysates, which are used in hypoallergenic infant formulas, have been found to possess immune modulating effects. For an optimal utilization of hydrolysates, the working mechanisms and responsible proteins underlying the effects should be elucidated. In this study, the immunomodulating activity of whey and soy hydrolysates was studied by quantifying TLR activation and assessing cytokine production in hydrolysate stimulated dendritic cells. The responsible protein fraction was identified and characterized by gel electrophoresis. The immune effects under gastrointestinal conditions were studied by digesting the hydrolysates in an in vitro infant digestion model, after which the digests were analyzed. In both soy and whey hydrolysates, TLR activation and cytokine production in dendritic cells were induced by a fraction containing protein aggregates larger than 1000 kDa, which were formed by electrostatic interactions and disulfide bonds. Only soy aggregates remained intact during duodenal digestion, and maintained the TLR activating capacity. Soy and whey protein aggregates larger than 1000 kDa possess immunomodulatory properties, but only soy aggregates remain under intestinal digestion conditions. This knowledge is important for a better understanding of the effects of hydrolysates

Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation

Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. PMCs in the circulation might be proatherogenic, and prevention of their formation is a possible therapeutic goa