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

Journal: Cells

Article Title: CD45 and CD148 Are Critically Involved in Neutrophil Recruitment and Function During Inflammatory Arthritis in Mice

doi: 10.3390/cells14151169

Figure Lengend Snippet: CD45 shows overarching modulation of neutrophil recruitment and effector functions compared to CD148. ( A ) Mice were injected intrascrotally with TNF, and rolling velocity, number of adherent and emigrated cells were assessed after 2 h (n = 3–8 mice in each group). ( B ) For analysis of chemokine-induced arrest, the cremaster muscle was prepared, and CXCL1 was administered via a carotid artery catheter (n = 3–5 mice in each group). Binding of the β2-integrin ligands ICAM-1 ( C ) and fibrinogen ( D ) was assessed by flow cytometry after BM-derived neutrophils were stimulated with CXCL1 or LTB4 (n = 3–10 mice in each group). ( E – G ) BM-derived neutrophils were stimulated with CXCL1 or LTB4 and analyzed for expression of indicated surface proteins (n = 4 mice in each group). ( H ) BM-derived neutrophils were plated on uncoated or IC-coated plates with or without TNF, and ROS production was measured. Accumulated O 2 production was summarized as area under the curve (n = 3–8 mice in each group). ( I ) LTB4 was measured in the supernatants by ELISA after BM-derived neutrophils were incubated on IC-coated plates for 6 h. Albumin (Alb)-coated plates were used as controls (n = 3–6 mice in each group). All data are presented as mean +/− SEM, one and two-way ANOVA, ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ELISAs of C5a and LTB4 (R&D Systems, Minneapolis, MN, USA) were performed according to the manufacturer’s protocols.

Techniques: Injection, Binding Assay, Flow Cytometry, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Cells

Article Title: CD45 and CD148 Are Critically Involved in Neutrophil Recruitment and Function During Inflammatory Arthritis in Mice

doi: 10.3390/cells14151169

Figure Lengend Snippet: SFK phosphorylation is differentially affected by CD45 and CD148. BM-derived neutrophils were left untreated or stimulated with CXCL1 or LTB4 for 2 min or TNF for 15 min. Additionally, BM-derived neutrophils were incubated on IC-coated plates for 15 min. Alb-coated plates were used as controls. Cells were lysed and immunoblotted with Ab against total-Src, phospho-Src Y416, or Y529. Quantification of the phosphorylated proteins as a relative density of total protein, normalized to untreated or Alb-coated lysates, was performed. Representative Western blots of total lysates and quantification from CD45KO ( A ), CD148KO ( B ), and DKO ( C ) neutrophils, each compared to WT, showing the phosphorylation of SFKs (n = 5–7 mice in each group). All data are presented as mean +/− SEM, two-way ANOVA followed by Fisher’s LSD test, ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ELISAs of C5a and LTB4 (R&D Systems, Minneapolis, MN, USA) were performed according to the manufacturer’s protocols.

Techniques: Phospho-proteomics, Derivative Assay, Incubation, Western Blot

Journal: Cells

Article Title: CD45 and CD148 Are Critically Involved in Neutrophil Recruitment and Function During Inflammatory Arthritis in Mice

doi: 10.3390/cells14151169

Figure Lengend Snippet: CD45 and CD148 are differentially involved in GPCR- and Fc-mediated signaling. BM-derived neutrophils were left untreated or stimulated with CXCL1 or LTB4 for 2 min or TNF for 15 min. Additionally, BM-derived neutrophils were incubated on IC-coated plates for 15 min. Therefore, Alb-coated plates were used as controls. Cells were lysed and immunoblotted with Ab against total-Syk, phospho-Syk, total-P38, phospho-P38, total-ERK1/2, and phospho-ERK1/2. Quantification of the phosphorylated proteins as a relative density of total protein, normalized to untreated or Alb-coated lysates, is shown for CD45KO ( A ), CD148KO ( B ), and DKO ( C ) neutrophils, each compared to WT (n = 4–10 mice in each group). All data are presented as mean +/− SEM, two-way ANOVA followed by Fisher’s LSD test, ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ELISAs of C5a and LTB4 (R&D Systems, Minneapolis, MN, USA) were performed according to the manufacturer’s protocols.

Techniques: Derivative Assay, Incubation

Journal: Cells

Article Title: CD45 and CD148 Are Critically Involved in Neutrophil Recruitment and Function During Inflammatory Arthritis in Mice

doi: 10.3390/cells14151169

Figure Lengend Snippet: Schematic model of the role of CD45 and CD148 during inflammatory arthritis. ( A ) After injection of K/BxN serum into CD45KO, CD148KO, DKO, as well as C57BL/6J WT mice, CD45 and CD148 feature distinct regulative properties regarding clinical and histological parameters, presence of cytokines, as well as neutrophil recruitment and functionality. Based on these properties, CD45KO and, in particular, DKO lead to an overall reduction in the development of inflammatory arthritis, whereas CD148KO results in reduced ROS production and cartilage damage but unaltered cytokine release and overall neutrophil infiltration. ( B ) On a cellular level, upon GPCR-mediated stimulation, Mac-1 activation and selectin engagement are positively (+) mediated by CD45 and both positively and negatively (−) mediated by CD148, whereas LFA-1 activation is independent of CD45 or CD148. Additionally, CD45 and CD148 are required for ROS production and LTB4 release after TNF- and Fc-mediated stimulation. ( C ) At a molecular level, signaling pathways are regulated in distinct ways. Activation of SFKs (detected by pY416) and Syk is positively regulated by both CD45 and CD148, while dephosphorylation of inactivated SFKs (pY529) is only dependent on CD45. The activation of ERK1/2 is positively regulated by CD45 and negatively by CD148, with particularly strong regulation after stimulation with CXCL1. P38 is only positively regulated by CD45 in a stimulus-dependent manner after TNF- and Fc-mediated activation.

Article Snippet: ELISAs of C5a and LTB4 (R&D Systems, Minneapolis, MN, USA) were performed according to the manufacturer’s protocols.

Techniques: Injection, Activation Assay, Protein-Protein interactions, De-Phosphorylation Assay