Oenothein B, a Cyclic Dimeric Ellagitannin Isolated from <em>Epilobium angustifolium</em>, Enhances IFNγ Production by Lymphocytes

<div><p>Oenothein B is a polyphenol isolated from <em>Epilobium angustifolium</em> and other plant sources, which has been reported to exhibit immunomodulatory properties. Oenothein B is known to activate myeloid cells and induce the production of IL-1 and other cytokines. However, its effects on lymphocytes are unknown. In this report, we show that oenothein B stimulated innate lymphocytes, including bovine and human γδ T cells and NK cells, resulting in either increased CD25 and/or CD69 expression. We also demonstrate that oenothein B enhanced the production of interferon-γ (IFNγ) by bovine and human NK cells alone and in combination with interleukin-18 (IL-18), a response not observed with other commonly studied polyphenols. Furthermore, we demonstrate that oenothein B enhanced the production of IFNγ by human T cells. Since IFNγ contributes to antitumor, antibacterial, and antiviral cell responses, these data suggest an additional mechanism that could account, at least in part, for the immune enhancing properties of oenothein B.</p> </div

IFNγ production by human lymphocytes in response to oenothein B.

<p>(A) Human PBMCs (10⁵ cells/well) were treated with the indicated concentrations of oenothein B or X-VIVO medium alone for 48 hrs, and soluble IFNγ levels in supernatant fluids were measured by ELISA. The graph represents data from ten individuals, with each sample plated in triplicate. Statistical significance was measured by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001 (B) Human PBMCs (10⁵ cells/well) were treated with oenothein B or X-VIVO medium alone for 6 hrs in the presence of brefeldin A. The percent of total CD3+ T cells, γδ T cells, CD8+ T cells, and NK cells positive for IFNγ staining was then determined by flow cytometry. The graphs represent data for five individuals, with each treatment analyzed in triplicate. Statistical significance was determined by paired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001 (C) Representative examples of two-color flow cytometry plots comparing IFNγ staining on oenothein B-treated and untreated human lymphocytes.</p

Priming of human NK cells to K562 cells by oenothein B.

<p>(A) Human PBMCs (10⁵ cells/well) were treated with 20 µg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and co-cultured with or without K562 cells at effector:target (E:T) ratios of 10∶1 and 1∶1 for approximately 42 hrs. After incubation, soluble IFNγ levels were measured by ELISA. The data represent pooled results from three donors and are expressed as mean +/− SEM. Samples were analyzed in duplicate. Statistical significance was measured by Two-way ANOVA with Bonferroni post-test. (B) Human PBMCs (10⁵ cells/well) were treated with 20 µg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and co-cultured with or without K562 cells at an effector:target (E:T) ratio of 1∶1 for approximately 18 hrs. After incubation, brefeldin A was added to the culture for 6 hrs. IFNγ expression by NK cells (CD3−/CD56+), T cells (CD3+), and others (CD3−/CD56-) was then measured by intracellular flow cytometry. The data represent pooled results from two donors and are expressed as mean +/− SEM. Samples were analyzed in duplicate. Statistical significance was measured by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Oenothein B induces IL-2Rα or CD69 on bovine and human lymphocyte subsets.

<p>(A) Bovine PBMCs (10⁵ cells/well) were treated with the indicated concentrations of oenothein B in X-VIVO medium for 24 hrs, and IL-2Rα expression on γδ T cells and NK cells was measured by multi-color flow cytometry. NK cells were defined as non-γδ T cells that expressed CD335. The graphs represent pooled data from 3 individuals. Each treatment was analyzed in triplicate and error bars indicate SEM. Significance compared to untreated cells (0 µg/ml) was determined by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001 (B) Human PBMCs (10⁵ cells/well) were treated with the indicated concentrations of oenothein B in cRPMI medium for 48 hrs. CD69 expression on lymphocytes, which included CD3+ T cells, CD8+ T cells, γδ T cells, and NK cells, was then measured by flow cytometry. The graphs represent pooled data from 5 individuals. Each treatment was analyzed in triplicate and error bars indicate SEM. Significance compared to untreated cells (0 µg/ml) was determined by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Oenothein B primes bovine PBMCs to respond to IL-18.

<p>Bovine PBMCs (10⁵ cells/well) were treated with oenothein B (40 µg/ml and 20 µg/ml), EGCG (40 µg/ml and 20 µg/ml), resveratrol (50 µg/ml and 25 µg/ml), curcumin (40 µg/ml and 20 µg/ml), theaflavin digallate (50 µg/ml), or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and treated with 10 ng/ml rhu IL-18, 100 ng/ml rhu IL-18, or X-VIVO medium alone for approximately 24 hrs. After incubation, soluble IFNγ levels were measured by ELISA. The data are expressed as mean +/− SEM of three independent experiments.</p

Oenothein B Primes bovine CD335+ cells to respond to IL-18.

<p>(A) Bovine PBMCs (10⁵ cells/well) were depleted of CD335+ cells and treated with 20 µg/ml oenothein B or X-VIVO medium alone for 24 hrs. Cells were then washed and treated with 10 ng/ml rhu IL-18 or medium alone for 18 hrs. After incubation, IFNγ levels in the supernatant fluids were measured by ELISA. The data are expressed as mean +/− SEM of three independent experiments comparing depleted PBMCs to un-depleted controls tested concurrently. All samples were tested in duplicate or triplicate. Statistical significance was measured by Two-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001 (B) Bovine PBMCs (10⁵ cells/well) from a new calf were treated with the indicated amounts of oenothein B or X-VIVO medium alone for 24 hrs. Cells were washed and treated with 10 ng/ml rhu IL-18 or X-VIVO medium alone for 6 hrs in the presence of brefeldin A. IFNγ production was measured by intracellular flow cytometry. The data are expressed as mean+/− SEM. All samples were tested in triplicate. Statistical significance was measured by Two-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001 (C) Representative examples of two-color flow cytometry plots comparing IFNγ staining on CD335+ cells.</p

Oenothein B directly primes purified human NK cells to produce IFNγ.

<p>Human NK cells (5×10⁴ cells/well) were sorted and treated with 20 µg/ml oenothein B, 100 ng/ml rhu IL-18, both, or medium alone. After 24 hrs, soluble IFNγ was measured by ELISA. The graph represents soluble IFNγ levels in culture supernatant fluids from three separate experiments with three different donors. Error bars indicate SEM. Each sample was analyzed in triplicate. Significance was determined by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Oenothein B induces CD25 on human T cells.

<p>Human PBMCs (10⁵ cells/well) were treated with the indicated concentrations of oenothein B in X-VIVO medium for 42 hrs. CD25 expression on lymphocytes, which included γδ T cells (CD3+/γδ TCR+), NK cells (CD3−/CD56+), and αβ T cells (CD3+/γδ TCR-), was then measured by flow cytometry. The graph represents pooled data from 5 individuals. Each treatment was analyzed in duplicate and error bars indicate SEM. Significance compared to untreated cells (0 µg/ml) was determined by One-way ANOVA with Bonferroni post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Cinnoline derivatives as human neutrophil elastase inhibitors

<p>Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared to <i>N</i>-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound, <b>18a</b>, had a good balance between HNE inhibitory activity (IC₅₀ value = 56 nM) and chemical stability (<i>t</i>_1/2 = 114 min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1<i>H</i>)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195.</p

Optimization of <i>N</i>‑Benzoylindazole Derivatives as Inhibitors of Human Neutrophil Elastase

Human neutrophil elastase (HNE) is an important therapeutic target for treatment of pulmonary diseases. Previously, we identified novel <i>N</i>-benzoylindazole derivatives as potent, competitive, and pseudoirreversible HNE inhibitors. Here, we report further development of these inhibitors with improved potency, protease selectivity, and stability compared to our previous leads. Introduction of a variety of substituents at position 5 of the indazole resulted in the potent inhibitor <b>20f</b> (IC₅₀ ∼10 nM) and modifications at position 3 resulted the most potent compound in this series, the 3-CN derivative <b>5b</b> (IC₅₀ = 7 nM); both derivatives demonstrated good stability and specificity for HNE versus other serine proteases. Molecular docking of selected <i>N</i>-benzoylindazoles into the HNE binding domain suggested that inhibitory activity depended on geometry of the ligand–enzyme complexes. Indeed, the ability of a ligand to form a Michaelis complex and favorable conditions for proton transfer between Hys57, Asp102, and Ser195 both affected activity