Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: 12,13-diHOME promotes inflammatory macrophage polarization and reduces phagocytic capacity, (a) Representative flow cytograms of IL-1 β (CD68 + IL-1 β Hi ; upper panel) and CD206 (CD68 + CD206 Hi ; lower panel) THP-1 macrophages following treatment with 37.5 or 75 μ M 12,13-diHOME for 24 or 48 hr (gating strategy provided in Supplementary figure ). As controls, cells were stimulated 48 hr with 20 ng ml −1 IFN γ and 100 ng ml −1 LPS together to promote an IL-1 β phenotype or 20 ng ml −1 IL-4 to induce a CD206 phenotype. (b) IL-1 β + /CD206 + THP-1 macrophage ratio following treatment with 37.5 or 75 μ M 12,13-diHOME or vehicle (DMSO; control) for 24 or 48 hr (Brown–Forsythe and Welch ANOVA test). (c) Temporal gene expression (1, 2, 4, 8, 12, 24, and 48 hr) of macrophage cytokines associated with inflammatory functional states ( IL-1β , TNFα , IL-6 , and NFκB ( RELA )) and anti-inflammatory functional states ( IL-10 , TGFβ ) following exposure of primary human macrophages to 12,13-diHOME. Mean gene expression of triplicate reactions quantitated by qRT-PCR followed with 2 − ΔΔ CT calculation; each follow-up timepoint was compared to baseline. Significant ( p < 0.05) findings are indicated with an asterisk (linear mixed model, Supplementary table ). Dose-dependent reduction in phagocytosis of fluorescently labeled E. coli following exposure to 12,13-diHOME (50 or 100 μ M) exposure is observed in (d) Raw264.7 (blue) and THP-1 (red) macrophages and (e) confirmed using a gentamicin protection assay (Kruskal–Wallis test). All box plots indicate the interquartile range (IQR) and median.

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Control, Expressing, Functional Assay, Quantitative RT-PCR, Labeling

Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: 12,13-diHOME enhances inflammatory macrophages, IgE production, and cytokine secretion in the presence of antigenic stimulation. (a) 12,13-diHOME enhances allergen-induced a skew in IL-1 β + /CD206 + cell ratio in primary human monocyte-derived macrophages from four adult donors (A, B, C, and D). Cells were treated with individual allergens, CRA, LPS, OVA, or peanut extract, in the absence or presence of 12,13-diHOME (37.5 µ M). Data represent the mean ± standard deviation. A linear mixed model was used to determine significant differences. In primary human macrophage/T/B cell (from two adult donors) coculture assays, macrophage stimulation with peanut allergen (f171) in the presence of 12,13-diHOME (50 µ M) increases (b) the elevated ratio of IL-1 β + /CD206 + macrophages, secreted (c) IL-1 β , and (d) IL-6 concentrations (pg ml −1 ) in the supernatant after 5 days of coculture (e) the ratio of memory-B to naïve-B cell populations, and (f) the ratio of secreted IgE to IgG (data listed in Supplementary table , and the bar plots of both absolute IgE and IgG concentrations listed as Supplementary figure ) in the coculture supernatant (all box plots indicate the IQR and median; linear mixed model).

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Derivative Assay, Standard Deviation

Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: Gut microbial derived 12,13-diHOME exposure in context of airway allergen stimulation induces pathways related to inflammatory polarization and epigenetic modifications. (a) Principal components analysis of the normalized expression for all genes between mice treated with and without the combinatorial 3 epoxide hydrolase (3EH)-expressing E. coli and cockroach antigen (CRA) distinguishes the four treatment groups: no treatment (vehicle; red), 3EH alone (green), CRA alone (blue), and the combination of 3EH + CRA (purple). PC1 and PC2 were significantly associated with the four 3EH and CRA treatment groups (ANOVA, p = 4.9 × 10 −5 and 5.8 × 10 −4 , respectively). (b) Five upregulated GSEA pathways after treatment with combinatorial 3EH + CRA compared with vehicle-group. (c) IL-1 signaling pathway. (d) One-carbon metabolism pathway. Genes in orange and blue show relatively higher and lower expression in mice treated with 3EH + CRA combinatorial group, respectively. NES, normalized enrichment score.

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Derivative Assay, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

doi:

Figure Lengend Snippet: Binding of the recombinant VHDJH13/VκJκ13 (A), VH13G12-DJH13/Vκ13K20-Jκ13 (B), Lys52/VκJκ13 (C), and Arg58/VκJκ13 (D) IgG1 Abs to human recombinant insulin (●), ssDNA (△), tetanus toxoid (■), human recombinant IL-1β (▲), E. coli β-galactosidase (◇), and BSA (□). The Ag-binding activity of each Ab to solid-phase Ag is expressed as optical absorbance at 492 nm.

Article Snippet: Eluates were brought to pH 7.5 by addition of neutralizing buffer (pH 9.0), dialyzed against PBS, and stored in aliquots at 4°C. mAb concentration and binding to human recombinant insulin (Eli Lilly Research Laboratories, Indianapolis, IN), ssDNA, human recombinant IL-1 β (BASF Biotech Corp., Worcester, MA), tetanus toxoid, and Escherichia coli β -galactosidase were measured using appropriate ELISA, as previously described ( 12 , 20 , 21 ).

Techniques: Binding Assay, Recombinant, Activity Assay