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Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages
Published on: April 20, 2018
OR2AT4 and OR1A2 counterregulate molecular pathophysiological processes of steroid-resistant inflammatory lung
Daniel Weidinger1, Kaschin Jamal Jameel1, Desiree Alisch1
1Medical Clinic III for Pneumology, Allergology and Sleep Medicine, Bergmannsheil University Hospital, Ruhr-University Bochum, Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany.
Background:
Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets.
Methods:
Human primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry.
Results:
We demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines.
Conclusion:
These are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.
Insights
Olfactory receptors (ORs) in alveolar macrophages can be targeted to treat steroid-resistant lung diseases. Stimulating specific ORs reduced inflammation and phagocytosis, offering new therapeutic avenues.
Area of Science:
- Immunology
- Pulmonology
- Cell Biology
Background:
- Steroid-resistant non-type 2 inflammation in obstructive lung diseases lacks effective treatments.
- Alveolar macrophages drive disease progression via proinflammatory cytokine release.
- Olfactory receptors (ORs) have uncharacterized roles in human primary alveolar macrophages.
Purpose of the Study:
- Investigate the presence and function of ORs in human alveolar macrophages.
- Determine if ORs can be targeted therapeutically for steroid-resistant lung diseases.
Main Methods:
- Isolated human primary alveolar macrophages from 50 patients.
- Validated OR expression using RT-PCR, immunocytochemistry, and Western blot.
- Assessed intracellular calcium, cAMP levels, cytokine secretion, and phagocytic capacity upon OR stimulation.
Main Results:
- Confirmed expression of functional OR2AT4 and OR1A2 on mRNA and protein levels in alveolar macrophages.
- Sandalore (OR2AT4 ligand) and Citronellal (OR1A2 ligand) increased intracellular calcium and cAMP.
- OR stimulation with Sandalore and Citronellal decreased phagocytic capacity and proinflammatory cytokine release.
Conclusions:
- Olfactory receptors (ORs) are present and functional in human alveolar macrophages.
- ORs represent potential therapeutic targets for non-type 2 inflammatory steroid-resistant lung diseases.
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