NF-κB exists in a complex with dynein/dynactin <i>in vivo.</i>

<p>NF-κB p65 co-immunoprecipitates with dynactin and cytoplasmic dynein. 10 mg soluble rat brain extract were cross-linked with DSP to stabilize transient protein-protein interactions and subsequent immunoprecipitated with an anti-p65 antibody. Immunoprecipitates were analyzed by western blotting with an anti- NF-κB p65 or NF-κB p50 antibody or with antibodies against dynein intermediate chain (IC 74) or the motor-associated proteins dynamitin and p150^Glued. Control IP was done with a non-immune monoclonal antibody.</p

Microtubule-perturbing drugs inhibit neuronal transport of NF-κB.

<p>(A) Effect of microtubule-perturbing drugs on the organization of neuronal microtubule network. Microtubule network was visualized by α−tubulin immunostaining. Treatment of hippocampal neurons with 200 nM colchicine or 100 nM vincristine resulted in efficient depolymerisation, as shown by the diffuse tubulin staining and the loss of the well-organized microtubule patterns seen in untreated cells.(B) Hippocampal neurons treated with 300 µM glutamate for 5 min, either alone or after pre-treatment with 200 nM colchicine or 100 nM vincristine for 30 min were fixed (90 min after glutamate exposure) and visualised by SYTOX nuclear staining (green) and anti-NF-κB p65 immunofluorescence (red) to monitor neuronal transport of NF-κB (C) Quantification of nuclear/dendritic ratio of α-p65 fluorescence in neurons with functional or disrupted microtubules. Note that microtubule-perturbing drugs impaired dendritic to nuclear redistribution of NF-κB p65 after glutamate stimulation. (D) Reporter gene assay showed reduced NF-κB-dependent transcription activity in neurons with not functional microtubules. (E) In order to confirm the data obtained by microscopy, the subcellular localization of p65 was examined by cell fractionation and Western blotting. 250 µg of soluble protein from cytoplasmic extracts were immunoblotted for p65 protein level. In agreement, with the above experiments, pre-treatment with vincristine or colchicines before exposure to glutamate resulted in reduced relocation of p65 from cytoplasm to the nucleus.</p

Inhibition of dynein function by overexpression of dynamitin impairs nuclear accumulation of NF-κB and reduces NF-κB-dependent transcription activity.

<p>(A) Cultured hippocampal neurons transfected with dynamitin or mock vector were left untreated or incubated with 300 µM glutamate for 5 min. After 90 min neurons were fixed and analyzed for subcellular NF-κB p65 destribution. Nuclei were stained with SYTOX (green). Anti-p65 immunoreactivity is depicted in red. (B) Quantification of nuclear/dendritic ratio of α-p65 fluorescence in dynamitin-transfected and control-transfected neurons with and without glutamate stimulation. Note that transfection with dynamitin impaired redistribution of NF-κB p65 from distal sites to the nucleus following glutamate stimulation. Fluorescence measurements were made from 20 to30 neurons in each experimental condition. (C) Reporter gene assay showed reduced NF-κB-dependent transcription activity in neurons overexpressing dynamitin.</p

The functional nuclear localization signal (NLS) of p65 is essential for its interaction with dynein/dynactin <i>in vitro.</i>

<p>(A) Nuclear localization signal (NLS) of wild-type NF-κB p65 and its NLS mutant (NLSmut). The NLS of NF-κB p65 consists of a stretch of a basic amino acids, arginines and lysines. The three point mutations were introduced into the NLS (depicted in yellow). The construct was tagged with polyhistidine to enable purification from bacterial extracts and pull-down assays. (B) Polyhistidine-tagged wild-type p65 and p65 with mutant NLS (NLSmut) purified from bacterial extracts were immobilized separately on nickel-coated matrix and incubated with the rat brain extract. The bound material was eluted from the matrices, fractionated by SDS–PAGE and examined by Western blot with anti-IC74 and anti-dynactin p50 antibodies. As expected, wild-type p65 associates with dynein/dynactin <i>in vitro.</i> In contrast, p65 with mutant NLS failed to form this complex.</p

Schematic presentation for the NF-κB activation by synaptic activity and its dynein-mediated retrograde transport to the nucleus along microtubules.

<p>Upon stimulation of primary neurons with glutamate, different signalling pathways (represented here by CaMKII) originating from intracellular Ca²⁺ elevation induces phosphorylation of IκB, which subsequently leads to its degradation within the proteasome. Thereby, the nuclear localisation signals (NLS) of NF-κB subunits are unmasked, allowing its binding to importin α/β heterodimer. This complex is then transported retrogradely towards the nucleus via an association with motor protein dynein/dynactin, where it activates NF-κB target genes.</p

Cultured human nasal slices show unimpaired epithelium containing mucus-filled goblet cells.

<p><b>A,B</b>: Overview images of the established nasal slice culture system showing sliced nasal tissue cultured in culture plate inserts within a 12-well-plate. <b>C,D</b>: Immunohistochemical staining revealed the presence of acetyl-α-tubulin-positive cilia in nasal slice cultures. <b>E</b>: Hematoxylin and eosin-staining displayed the integrity of the <i>ex vivo</i> cultured epithelium containing ciliated epithelial cells (arrowheads), goblet cells (arrows) and a basal membrane (BM). Scale Bar: 20 μm. <b>F, G</b>: Mucin-filled goblet cells (arrows) were detected in cultivated nasal slices by Alcian Blue-staining and Periodic acid-Schiff stain. Scale Bar: 20 μm.</p

1,8-Cineol Reduces Mucus-Production in a Novel Human <i>Ex Vivo</i> Model of Late Rhinosinusitis

<div><p>Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established <i>ex vivo</i> cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the <i>ex vivo</i> cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.</p></div

Schematic view on nasal epithelium containing mucus-filled goblet cells during LPS-induced rhinosinusitis and after co-treatment with 1,8-cineol.

<p>Co-treatment with LPS and 1,8-cineol leads to reduced production of mucin and decreased expression levels of mucin genes closely associated with attenuated NF-κB-activity.</p

Increased number of mucus-filled cells in LPS-treated nasal slice cultures is significantly reduced by co-treatment with 1,8-cineol.

<p><b>A</b>: Representative Alcian Blue-staining of an untreated nasal slice culture revealed no increased amount of mucus-filled goblet cells (arrows). <b>B</b>: Representative Alcian Blue-staining of LPS-treated nasal slices showed highly increased numbers of mucus-filled goblet cells (Arrows). <b>C</b>: Representative Alcian Blue-staining of cultured nasal slices co-treated with LPS and 1,8-cineol displayed a highly decreased number of mucus-filled goblet cells (Arrows). Scale Bar: 20 μm. <b>D</b>: Quantification of total areas of Alcian Blue-stained slice cultures from four independent donors revealed a significantly increased number of mucin-filled goblet cells in LPS-treated nasal slice cultures, which was significantly decreased after co-treatment with 1,8-cineol. *p < 0.5, **p < 0.01 were considered significant (t-test); ns: not significant (t-test).</p

Primer Sequences.

<p>Primer Sequences.</p