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The Novel Notch Ligand DNER Modulates IFNγ Levels in Recruited Macrophages by Enhancing Non-Canonical Notch During COPD Progression
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A7408 - The Novel Notch Ligand DNER Modulates IFNγ Levels in Recruited Macrophages by Enhancing Non-Canonical Notch During COPD Progression
Author Block: C. Ballester Lopez1, T. M. Conlon1, Z. Ertüz1, O. Eickelberg2, A. O. Yildirim1; 1iLBD/CPC, Helmholtz Zentrum, Munich, Germany, 2Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
RATIONALE: In the last decades it has become more evident that chronic inflammation plays a pivotal role in the progression of chronic obstructive pulmonary disease (COPD). Nevertheless, the underlying pathways that drive and exacerbate the disease remain unclear. A novel non-canonical Notch ligand, DNER (Delta/Notch-like Epidermal Growth Factor (EGF)-related Receptor), has been shown in a genome-wide association study (GWAS) to correlate with GOLD stages (Hancock et al. PLoS Genet 2012). Therefore, our aim is to unravel the role of DNER during COPD development using a CS exposed mouse model and patient samples.
METHODS: DNER expression was quantified in lung tissue from healthy and COPD patients. Immunofluorescence co-staining of DNER with pro-SPC (ATII cells), acta-2 (smooth muscle cells), Galectin-3 (macrophages), CD31 (endothelial cells), p63, Krt5 (airway cells), CD45 (immune cells) and CGRP (pneuroendocrine cells) were performed in COPD human and 6 month cigarette smoke (CS)-exposed murine lung tissue. WT, Dner-/- bone marrow-derived macrophages (BMDM) and human monocyte derived macrophages (MDM) were treated for 24h with LPS, LPS+IFNγ, LPS+CS extract (CSE) or IL-4. Inflammation and Notch pathway markers were quantified by qPCR and Western Blot. WT and Dner-/- mice were exposed to CS for 3 days or 2 months, and lung immune cell populations were analyzed by FACS.
RESULTS: DNER expression is increased in COPD patients compared to healthy lungs and mainly localized to macrophages. LPS-treated BMDM and LPS+CSE-treated MDM exhibited significantly upregulated DNER expression compared to non-treated controls (19.26±11.07 and 3.22±0.49 fold change (FC), respectively). Furthermore, LPS-treated Dner-/- BMDM demonstrated reduced NICD1 protein levels, especially in nuclear fractions, but transcription of canonical Notch pathway markers (Hes1, Hey1, Notch1) was not affected. Interestingly, LPS-treated Dner-/- BMDM showed decreased induction of Ifnγ (25.91±37.28 vs. 37.64±49.99 FC) compared to WT cells. In in vivo studies, immunofluorescence revealed lower nuclear translocation of NICD1 in 3 day CS-exposed Dner-/- vs WT murine alveolar macrophages. In line with in vitro results, a significantly lower F4/80+ SiglecF- IFNγ+ macrophage population was observed in 2 month CS-exposed Dner-/- mice compared to WT (1.25±0.1 vs. 1.6±0.3 FC).
CONCLUSION: Taken together, DNER is a novel protein induced in COPD patients and 6 months CS-exposed mice that regulates IFNγ secretion via non-canonical Notch signaling in pro-inflammatory recruited macrophages. These results provide a new pathway involved in COPD immunity that could contribute to the discovery of innovative therapeutic targets.
Author Block: C. Ballester Lopez1, T. M. Conlon1, Z. Ertüz1, O. Eickelberg2, A. O. Yildirim1; 1iLBD/CPC, Helmholtz Zentrum, Munich, Germany, 2Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
RATIONALE: In the last decades it has become more evident that chronic inflammation plays a pivotal role in the progression of chronic obstructive pulmonary disease (COPD). Nevertheless, the underlying pathways that drive and exacerbate the disease remain unclear. A novel non-canonical Notch ligand, DNER (Delta/Notch-like Epidermal Growth Factor (EGF)-related Receptor), has been shown in a genome-wide association study (GWAS) to correlate with GOLD stages (Hancock et al. PLoS Genet 2012). Therefore, our aim is to unravel the role of DNER during COPD development using a CS exposed mouse model and patient samples.
METHODS: DNER expression was quantified in lung tissue from healthy and COPD patients. Immunofluorescence co-staining of DNER with pro-SPC (ATII cells), acta-2 (smooth muscle cells), Galectin-3 (macrophages), CD31 (endothelial cells), p63, Krt5 (airway cells), CD45 (immune cells) and CGRP (pneuroendocrine cells) were performed in COPD human and 6 month cigarette smoke (CS)-exposed murine lung tissue. WT, Dner-/- bone marrow-derived macrophages (BMDM) and human monocyte derived macrophages (MDM) were treated for 24h with LPS, LPS+IFNγ, LPS+CS extract (CSE) or IL-4. Inflammation and Notch pathway markers were quantified by qPCR and Western Blot. WT and Dner-/- mice were exposed to CS for 3 days or 2 months, and lung immune cell populations were analyzed by FACS.
RESULTS: DNER expression is increased in COPD patients compared to healthy lungs and mainly localized to macrophages. LPS-treated BMDM and LPS+CSE-treated MDM exhibited significantly upregulated DNER expression compared to non-treated controls (19.26±11.07 and 3.22±0.49 fold change (FC), respectively). Furthermore, LPS-treated Dner-/- BMDM demonstrated reduced NICD1 protein levels, especially in nuclear fractions, but transcription of canonical Notch pathway markers (Hes1, Hey1, Notch1) was not affected. Interestingly, LPS-treated Dner-/- BMDM showed decreased induction of Ifnγ (25.91±37.28 vs. 37.64±49.99 FC) compared to WT cells. In in vivo studies, immunofluorescence revealed lower nuclear translocation of NICD1 in 3 day CS-exposed Dner-/- vs WT murine alveolar macrophages. In line with in vitro results, a significantly lower F4/80+ SiglecF- IFNγ+ macrophage population was observed in 2 month CS-exposed Dner-/- mice compared to WT (1.25±0.1 vs. 1.6±0.3 FC).
CONCLUSION: Taken together, DNER is a novel protein induced in COPD patients and 6 months CS-exposed mice that regulates IFNγ secretion via non-canonical Notch signaling in pro-inflammatory recruited macrophages. These results provide a new pathway involved in COPD immunity that could contribute to the discovery of innovative therapeutic targets.
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