ltb4 kge006b  (R&D Systems)

Journal: Advanced Science

Article Title: Boron Nitride Nanomaterials Trigger Immunomodulatory Effects in Human Broncho‐Epithelial Cells by Modulating Eicosanoid Lipid Signaling

doi: 10.1002/advs.202516401

Figure Lengend Snippet: Global lipid profiling of healthy and asthmatic HBE cultures after exposure to h ‐BN and BNNTs indicates effects on the production of eicosanoid lipid intermediates. a) Summary of cellular eicosanoid signaling involving enzymes to target cell membrane lipids (PLA 2 , phospholipase A2) and to further process resulting arachidonic acids intracellularly to produce prostaglandins (using COX1/2) or leukotrienes (5‐lipooxygenase, also known as Alox‐5). b) Scatter plot of the first two components of the supervised sparse Partial Least Squares ‐ Discriminant Analysis (sPLS‐DA) model of global lipidomic data in healthy and asthmatic samples with a 95% confidence interval. c) Hierarchical clustering showing the top 100 most significantly altered lipid expression values based on One‐way ANOVA, and the additional Heatmap on the right side further shows the annotation of lipids within the highlighted cluster with FDR values < 0.05. d) Immunoblot indicating expression of Alox‐5 (an enzyme that produces leukotriene B4) in healthy HBE cultures. β‐actin was used as a reference for loading uniformity. Refer to Data file (Supporting Information) for full immunoblots. e) Densitometry analysis of the protein blot showing Alox‐5 expression with respect to β‐actin (loading control). f) diff‐HL60 cell migration after 24 h of exposure to conditioned medium from healthy (H) and asthmatic (As) HBE cell cultures in the presence or absence of leukotriene B4 receptor inhibitor ( LY293111 , 25 n m ). Lipopolysaccharide (LPS, 100 ng mL −1 ) and fetal bovine serum (10%) were used as positive controls for chemotaxis‐mediated cell migration. The results in panels (e,f) are presented as mean + SD ( n = 3). The statistical significance was calculated using One‐Way ANOVA analysis with Tukey`s post hoc test. * p = 0.06 and *** p < 0.001 ‐ statistical significance with respect to negative control. ### p < 0.001 ‐ statistical significance between treatment groups (with and without inhibitor LY293111 ).

Article Snippet: Leukotriene content in BAL fluid was determined by using LTB4 ELISA (R&D Systems, #KGE006B) and performed by following the manufacturer's instructions.

Techniques: Membrane, Expressing, Western Blot, Control, Migration, Chemotaxis Assay, Negative Control

Journal: Advanced Science

Article Title: Boron Nitride Nanomaterials Trigger Immunomodulatory Effects in Human Broncho‐Epithelial Cells by Modulating Eicosanoid Lipid Signaling

doi: 10.1002/advs.202516401

Figure Lengend Snippet: Single‐cell immune profiling of primary human PBMCs using mass cytometry (CyToF) after exposure (24 h) to conditioned media (CM) from h‐ BN or BNNT‐treated healthy (H) and asthmatic (As) HBE cell cultures. a) Schematic representation of leukotriene–BLT1 lipid chemo‐attractant signaling and immune regulation in lungs: the extracellular release of leukotriene from lung epithelial cells could lead to the recruitment (by chemo‐attraction) and functional stimulation of BLT‐1 expressing immune cells from peripheral blood that subsequently sensitize airway tissues by releasing cytokine‐chemokine molecules and modulate disease progression. b) CyTOF results are represented in a T‐distributed stochastic neighbor embedding (tSNE) map with 12 clusters of cells identified in PBMCs with the most notable changes observed in NK cells, cytotoxic (CD8+) memory T cells, and naïve T cells (CD4+ and CD8+). c,d) Using the generalized linear model diffcyt‐DA‐EdgeR, we extracted the clusters that were significantly different between the conditions. Bar plots show the relative abundance of these clusters and that incubation of PBMCs with CM‐BNNT from asthmatic cultures triggered an increase in the number of NK cells and cytotoxic memory T cells (c), however, decreased the number of helper and cytotoxic naïve T cells (d). e) Heatmap showing median expression profiles of cell state‐specific markers across control and treatment groups for each donor using diffcyt‐DS‐limma ( n = 3).

Article Snippet: Leukotriene content in BAL fluid was determined by using LTB4 ELISA (R&D Systems, #KGE006B) and performed by following the manufacturer's instructions.

Techniques: Single Cell, Mass Cytometry, Functional Assay, Expressing, Biomarker Discovery, Incubation, Control

Journal: Advanced Science

Article Title: Boron Nitride Nanomaterials Trigger Immunomodulatory Effects in Human Broncho‐Epithelial Cells by Modulating Eicosanoid Lipid Signaling

doi: 10.1002/advs.202516401

Figure Lengend Snippet: Leukotriene B4 biosynthesis pathway in mice lungs 28 days after pharyngeal aspiration to h ‐BN and BNNT (1 µg µL −1 ; total administered volume 30 µL). a) Evaluation of mRNA transcripts in lung tissue for the genes involved in lipid biosynthesis and phospholipid remodeling. b) Leukotriene B4 content in mouse BAL fluid. c,d) Alox‐5 protein expression in lung tissues with respect to β‐actin (loading control), (b) protein blots, and (c) corresponding densitometry plot, showing Alox‐5 expression in five individual mice from control and h ‐BN or BNNT exposures. The full protein blot for Alox‐5 and β‐actin can be found in Figure (Supporting Information). e) Evaluation of mRNA transcript levels for the asthma marker genes in lung tissue using RT‐qPCR. The data in (b) and (d) are presented as mean + SD ( n = 5) and statistical significance was calculated by One‐Way ANOVA analysis with Tukey`s post hoc test. * * p < 0.01 . The statistical analysis in (a) and (e) was performed by applying an unpaired t ‐test to compare the fold change between the negative control and individual treatments, n = 5; (*) p < 0.05, (**) p < 0.01.

Article Snippet: Leukotriene content in BAL fluid was determined by using LTB4 ELISA (R&D Systems, #KGE006B) and performed by following the manufacturer's instructions.

Techniques: Expressing, Control, Marker, Quantitative RT-PCR, Negative Control