Toll-like receptor-4 mediates cigarette smoke-induced cytokine production by human macrophages

Abstract Background The major risk factor for the development of COPD is cigarette smoking. Smoking causes activation of resident cells and the recruitment of inflammatory cells into the lungs, which leads to release of pro-inflammatory cytokines, chemotactic factors, oxygen radicals and proteases. In the present study evidence is found for a new cellular mechanism that refers to a link between smoking and inflammation in lungs. Methods Employing human monocyte-derived macrophages, different techniques including FACS analysis, Cytometric Bead Array Assay and ELISA were achieved to evaluate the effects of CS on pro-inflammatory cytokine secretion including IL-8. Then, Toll-like receptor neutralization was performed to study the involvement of Toll-like receptor-4 in IL-8 production. Finally, signaling pathways in macrophages after exposure to CS medium were investigated performing ELISA and Western analysis. Results We demonstrate that especially human monocytes are sensitive to produce IL-8 upon cigarette smoke stimulation compared to lymphocytes or neutrophils. Moreover, monocyte-derived macrophages produce high amounts of the cytokine. The IL-8 production is dependent on Toll-like receptor 4 stimulation and LPS is not involved. Further research resolved the cellular mechanism by which cigarette smoke induces cytokine production in monocyte-derived macrophages. Cigarette smoke causes subsequently a concentration-dependent phosphorylation of IRAK and degradation of TRAF6. Moreover, IκBα was phosphorylated which suggests involvement of NF-κB. In addition, NFκB -inhibitor blocked cigarette smoke-induced IL-8 production. Conclusion These findings link cigarette smoke to inflammation and lead to new insights/therapeutic strategies in the pathogenesis of lung emphysema.</p

Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)

The cytokine interleukin 1(IL-1) initiates a wide range of proinflammatory cascades and its inhibition has been shown to decrease inflammation in a variety of diseases. IL-1 receptor accessory protein (IL-1RAcP) is an indispensible part of the IL-1R complex that stabilizes IL-1/IL-1R interaction and plays an important role in the signal transduction of the receptor complex. The soluble form of IL-1RAcP (sIL-1RAcP) contains only the extracellular domain and serves as a natural inhibitor of IL-1 signaling. Therefore, increasing sIL-1RAcP levels might be an attractive therapeutic strategy to inhibit IL-1–driven inflammation. To achieve this we designed specific antisense oligonucleotides (AON), to redirect pre-mRNA IL-1RAcP splicing by skipping of the transmembrane domain encoding exon 9. This would give rise to a novel Δ9IL-1RAcP mRNA encoding a soluble, secreted form of IL-1RAcP, which might have similar activity as natural sIL-1RAcP. AON treatment resulted in exon 9 skipping both in vitro and in vivo. A single dose injection of 10 mg AON/kg body weight induced 90% skipping in mouse liver during at least 5 days. The truncated mRNA encoded for a secreted, soluble Δ9IL-1RAcP protein. IL-1RAcP skipping resulted in a substantial inhibition of IL-1 signaling in vitro. These results indicate that skipping of the transmembrane encoding exon 9 of IL-1RAcP using specific AONs might be a promising therapeutic strategy in a variety of chronic inflammatory diseases

A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

<div><p>Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.</p></div

Dystrophin signal specificity using MANDYS106.

<p>Immunofluorescence intensities measured in pre-treatment biopsies of different patients with DMD at microscope settings suitable for imaging DMD samples (7% laser intensity). <b>A.</b> (Left): Dystrophin staining (AlexaFluor488). All fibers exhibit varying intensities of dystrophin (trace dystrophin) and certain fibers termed “revertants” express higher levels of dystrophin. (Middle): Spectrin staining (AlexaFluor594): Fibers with equivalent spectrin intensities exhibited varying levels of dystrophin staining. (Right): Histogram of the mean dystrophin intensity in the fiber population depicting the fibers expressing trace dystrophin and revertant fibers. The mean dystrophin intensity of the fiber population expressing trace dystrophin is indicated by the arrow. <b>B.</b> Specificity of MANDYS106 antibody compared with isotype control for measuring dystrophin expression. Immunofluorescence images of MANDYS106 (left) or negative control isotype staining (right) of patients DMD 4 (deletion exon 45) and DMD 6 (deletion exon 43), and the corresponding histograms of mean dystrophin intensity (cumulative graphs). <b>C.</b> Dystrophin intensities in patients with DMD with different exon deletions (DMD patient 1: deletion exon 48–50; DMD patient 3: deletion exon 50; DMD patient 5: deletion exon 45; DMD patient 6: deletion exon 45). These samples were analyzed in the same experiment. (au: arbitrary units; del: deletion; DMD: Duchenne muscular dystrophy).</p

Dystrophin intensity in control muscle samples, and pre-treatment DMD and BMD muscle biopsies.

<p><b>A.</b> Representative immunofluorescence images (1 of 10 images) from the tibialis anterior muscle samples (from one control subject (CTRL2), two patients with DMD and four patients with BMD. Analysis was performed in the same experiment using the MANDYS106 antibody and ‘control’ imaging settings (1% laser intensity). <b>B.</b> Corresponding dystrophin membrane intensity distribution in the fiber populations analyzed (cumulative graphs). (au: arbitrary units; BMD: Becker muscular dystrophy; CTRL: control; DMD: Duchenne muscular dystrophy).</p

Long-Term Efficacy, Safety, and Pharmacokinetics of Drisapersen in Duchenne Muscular Dystrophy: Results from an Open-Label Extension Study

<div><p>Background</p><p>Drisapersen induces exon 51 skipping during dystrophin pre-mRNA splicing and allows synthesis of partially functional dystrophin in Duchenne muscular dystrophy (DMD) patients with amenable mutations.</p><p>Methods</p><p>This 188-week open-label extension of the dose-escalation study assessed the long-term efficacy, safety, and pharmacokinetics of drisapersen (PRO051/GSK2402968), 6 mg/kg subcutaneously, in 12 DMD subjects. Dosing was once weekly for 72 weeks. All subjects had a planned treatment interruption (weeks 73–80), followed by intermittent dosing (weeks 81–188).</p><p>Results</p><p>Subjects received a median (range) total dose of 5.93 (5.10 to 6.02) mg/kg drisapersen. After 177 weeks (last efficacy assessment), median (mean [SD]) six-minute walk distance (6MWD) improved by 8 (-24.5 [161]) meters for the 10 subjects able to complete the 6MWD at baseline (mean age [SD]: 9.5 [1.9] years). These statistics include 2 subjects unable to complete the test at later visits and who scored “zero”. When only the 8 ambulant subjects at week 177 were taken into account, a median (mean [SD]) increase of 64 (33 [121]) meters in 6MWD was observed. Of 7 subjects walking ≥330 m at extension baseline, 5 walked farther at week 177. Of 3 subjects walking <330 m, 2 lost ambulation, while 1 declined overall but walked farther at some visits. Over the 188 weeks, the most common adverse events were injection-site reactions, raised urinary α₁-microglobulin and proteinuria. Dystrophin expression was detected in all muscle biopsies obtained at week 68 or 72.</p><p>Conclusion</p><p>Drisapersen was generally well tolerated over 188 weeks. Possible renal effects, thrombocytopenia and injection-site reactions warrant continued monitoring. Improvements in the 6MWD at 12 weeks were sustained after 3.4 years of dosing for most patients. For a small, uncontrolled study, the outcomes are encouraging, as natural history studies would anticipate a decline of over 100 meters over a 3-year period in a comparable cohort.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01910649?term=NCT01910649&rank=1" target="_blank">NCT01910649</a></p></div