The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.

The transcriptional control of the VEGFA-VEGFR1 (FLT1) axis in alternatively polarized murine and human macrophages.

Introduction: Macrophages significantly contribute to the regulation of vessel formation under physiological and pathological conditions. Although the angiogenesis-regulating role of alternatively polarized macrophages is quite controversial, a growing number of evidence shows that they can participate in the later phases of angiogenesis, including vessel sprouting and remodeling or regression. However, the epigenetic and transcriptional regulatory mechanisms controlling this angiogenesis-modulating program are not fully understood. Results: Here we show that IL-4 can coordinately regulate the VEGFA-VEGFR1 (FLT1) axis via simultaneously inhibiting the proangiogenic Vegfa and inducing the antiangiogenic Flt1 expression in murine bone marrow-derived macrophages, which leads to the attenuated proangiogenic activity of alternatively polarized macrophages. The IL-4-activated STAT6 and IL-4-STAT6 signaling pathway-induced EGR2 transcription factors play a direct role in the transcriptional regulation of the Vegfa-Flt1 axis. We demonstrated that this phenomenon is not restricted to the murine bone marrow-derived macrophages, but can also be observed in different murine tissue-resident macrophages ex vivo and parasites-elicited macrophages in vivo with minor cell type-specific differences. Furthermore, IL-4 exposure can modulate the hypoxic response of genes in both murine and human macrophages leading to a blunted Vegfa/VEGFA and synergistically induced Flt1/FLT1 expression. Discussion: Our findings establish that the IL-4-activated epigenetic and transcriptional program can determine angiogenesis-regulating properties in alternatively polarized macrophages under normoxic and hypoxic conditions.

Ultrafast high-resolution analysis of human milk oligosaccharides by multicapillary gel electrophoresis.

There is recently growing interest towards synthesized human milk oligosaccharides (HMOs) as baby formula additives, and interestingly also as dietary supplements for adults. Currently quite a few manufacturers synthesize HMOs, however, their analysis is challenging, both in resolution and speed. In this paper an ultrafast high-resolution method is introduced for the separation of HMOs by multicapillary gel electrophoresis. Two gel compositions were evaluated with complementary resolving power. One was a conventionally used industrial standard carbohydrate separation matrix, resolving oligosaccharides according to their charge to hydrodynamic volume ratios. The other one was a borate-buffered dextran gel, which utilized the secondary equilibrium of the borate-vicinal diol complexation to enhance resolution. Considering the rapid analysis time and multiplexing (12-channel system), a 96 well sample plate can be analyzed in less than 80 min with the conventional type carbohydrate separation matrix and in less than one hour with the borate-buffered dextran gel. Exploiting the one fluorophore per molecule labeling stoichiometry, the limit of detection (S/N > 3) and limit of quantitation (S/N > 10) were determined as 0.025 and 0.100 mg/mL, respectively, with good linearity. Based on the calibration plot, the quantities of several low concentration HMOs were determined from a human milk sample.

Embryonic exposure to low concentrations of aflatoxin B1 triggers global transcriptomic changes, defective yolk lipid mobilization, abnormal gastrointestinal tract development and inflammation in zebrafish.

Aflatoxin B1-contaminated feeds and foods induce various health problems in domesticated animals and humans, including tumor development and hepatotoxicity. Aflatoxin B1 also has embryotoxic effects in different livestock species and humans. However, it is difficult to distinguish between the indirect, maternally-mediated toxic effects and the direct embryotoxicity of aflatoxin B1 in mammals. In the present study, we investigated the aflatoxin B1-induced direct embryotoxic effects in a zebrafish embryo model system combining toxicological, transcriptomic, immunological, and biochemical approaches. Embryonic exposure to aflatoxin B1 induced significant changes at the transcriptome level resulting in elevated expression of inflammatory gene network and repression of lipid metabolism and gastrointestinal tract development-related gene sets. According to the gene expression changes, massive neutrophil granulocyte influx, elevated nitric oxide production, and yolk lipid accumulation were observed in the abdominal region of aflatoxin B1-exposed larvae. In parallel, aflatoxin B1-induced defective gastrointestinal tract development and reduced L-arginine level were found in our model system. Our results revealed the complex direct embryotoxic effects of aflatoxin B1, including inhibited lipid utilization, defective intestinal development, and inflammation.