DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells
Dan Li, Danique Lobee, Melissa Van der Veen, Hoeke.A. Baarsma, Reinoud Gosens, Martina Schmidt 

Background:
The acute inflammatory response to injury or pathogens protects the host from systemic infection and initiates tissues repair. Recent research showed that resolution of acute inflammation is an active process which is initiated by the omega 3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Here we aim to investigate in the role of DHA and EPA in models of inflammation in the lung epithelium. 
Method:
Human bronchial epithelial (BEAS-2B) cells and human alveolar epithelial (A549) cells were exposed to different concentrations and the indicated time periods (up to 24h) to DHA and EPA (up to 100 μM), followed by stimulation with lipopolysaccharide (LPS) (0.01-100 μg/ml). Cell viability was measured by MTT. The inflammatory markers interleukin-8 (IL-8, IL-6) were measured by ELISA. Expression of IkkB, IkB-a, p65 expression has been detected by western blot and immunofluorescence. To translate the findings from the 2D cell cultrues to the 3D cell cultures, a mouse organoid model comprised of a co-culture of primary mouse CD31-CD45-Epcam+ cells with CCL206 fibroblasts was used. 
Results:
DHA and EPA alone and in combination with LPS did not affect the cell viability in both BEAS-2B and A549 cells. In BEAS-2B cells, DHA and EPA did not reduce the LPS-stimulated IL-8 and IL-6 release. On the contrary, in A549 cells DHA (60μM) but not EPA significantly reduced the LPS-induced IL-8 release, leaving the IL-6 release unchanged. Total IKKB and p65 expression was not changed by DHA, EPA, measured alone or in combination with LPS. The p-IKKa/b, IkB-a expression and p-65 nuclear translocation are being researched now by Western blot and immunofluorescence. Importantly, we found in the organoid model that LPS induces mouse KC release, a process significantly reduced by DHA but not EPA. 
Conclusion: 
DHA but not EPA may reduce the inflammatory response in lung alveolar epithelial cells by suppressing IL-8 (KC) release. The mechanism involved still needs to be studied further. 


DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells

Chronic Obstructive Pulmonary Disease (COPD) patients frequently develop osteoporosis, severely limiting life quality and increasing hospitalisation. Accelerated bone degradation is traditionally explained by elevated Receptor Agonist of NFKB signalling Ligand (RANKL) levels in the bone, resulting in osteoclast activity, however, COPD patients with dominant emphysema also presented with elevated serological levels of RANKL (Bai et al, 2011). Recent data from Woldhuis et al shows that RANKL is produced by lung fibroblasts, and that in COPD, compared to a plethora of secreted cytokines, RANKL secretion has the highest fold increase. Also, local production of the inflammatory cytokine IL1β is known to be a potent inducer of RANKL in osteoblasts, which in the inflamed COPD lung could translate to increased RANKL production. However, it is currently unclear whether IL1β also induces local RANKL production in the lung. We hypothesize that IL1β induces RANKL expression in lung fibroblasts, resulting in higher production and secretion of RANKL. To test this, we first explored a single-cell sequencing dataset generated by Lee et al. comprising IL1β treated murine lung organoids. Re-analysis showed that stromal cells in this model strongly upregulated RANKL in response to IL1β. To further explore the effects of IL1b, primary fibroblasts from COPD lung were treated with il1b for 48 hours. Although free RANKL was not detected in culture supernatants in either condition, levels of the RANKL/osteoprotegerin (OPG) complex were increased under influence of IL1β, whereas OPG levels remained constant, indicating that RANKL production indeed increased. Combined, these results show that RANKL production is regulated by IL1β in lung fibroblasts. Also, these data mark the importance of measuring both free- and OPG-bound RANKL, as OPG might obscure any effects of RANKL. Building on this, future research should focus on exploring whether this regulatory effect is altered in COPD by checking both free- and OPG-bound RANKL in lung tissue and serum and examining the possible role of RANKL in lung tissue.

IL1β induces RANKL secretion in human lung fibroblasts

Background: COPD involves emphysema and small airway wall thickening. Chronic inflammation and aberrant repair processes play a role in both, but the exact mechanisms are unknown. Pulmonary fibroblasts are key regulators of repair and are affected by chronic inflammation. So far, a comprehensive analysis comparing COPD and control fibroblasts on protein level is lacking.
Aim: To identify COPD-related protein changes in lung fibroblasts using a shotgun proteomics approach.
Methods: Lung fibroblasts were isolated from 13 stage IV COPD patients and 10 matched controls. Isolated proteins were subjected to shotgun proteomic analysis using LC-MS/MS. Differential protein expression comparing COPD patients and controls was assessed using linear regression modeling correcting for age and sex. Differentially expressed proteins were cross-referenced with literature via high-throughput text-mining with TenWise KMAP-API to determine associations with COPD.
Results: After removal of lowly expressed proteins, we detected the expression of 774 proteins in primary lung fibroblasts of which 40 proteins were significantly differentially expressed between COPD and control fibroblasts, with the most significant being HNRNPA2B1 and FHL1 . 20 of the 40 proteins had significantly higher expression while the other 20 had lower expression in COPD fibroblasts compared to controls. The proteins were enriched for pathways relating to the immune system and protein folding, among others. Of the 40 proteins 13 were previously associated with COPD in literature cross-referencing, including FHL1 and GSTP1.
Conclusions: We identified 40 differentially expressed proteins in COPD stage IV compared to control fibroblasts, who are enriched for immune system pathways that suggest a role in chronic inflammation in COPD. These proteins include previously described COPD related proteins (e.g. FHL1 and GSTP1), validating their altered expression in COPD lung fibroblasts. But they also include new COPD research targets like HNRNPA2B1 ad CAPN2. Thus this study demonstrates the robustness of the proteomic approach and provides several intriguing new research foci for further functional studies in COPD.

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DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells

IL1β induces RANKL secretion in human lung fibroblasts

Proteomics approach to identify COPD-related changes in pulmonary fibroblasts

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