Muscarinic receptor M3 activation promotes COPD fibrocyte contraction - 09/03/22

Fibrocytes are circulating monocytes-derived cells, able to migrate to injured organs in order to differentiate into myofibroblasts-like cells. We have previously shown that their densities are increased in the lungs of Chronic Obstructive Pulmonary Disease (COPD) patients and associated to worse lung function (Eur Respir J 2019 54: 1802173). COPD is characterized by irreversible airflow obstruction, partly due to an increased cholinergic environment (increased muscarinic M3 signalling, targeted by short and long-acting muscarinic antagonists). We hypothesize that the contractile status of differentiated fibrocytes is under control of a cholinergic stimulation.

Fibrocytes were isolated from COPD patients’ blood and cultured for 14 days to reach full differentiation. Presence of M3 receptor was quantified by qPCR, flow cytometry and immunocytochemistry. Calcium signalling as well as contraction experiments using collagen gels were performed in presence of carbachol (cholinergic agonist)±tiotropium bromide (M2/M3 antagonist).

qPCR showed that differentiated fibrocytes expressed all isoforms of muscarinic receptors (M1 to M5). Moreover, 59,7% (±15) of fibrocytes were found to express the M3 receptor by flow cytometry. Immunocytochemistry showed the existence of cytoplasmic and membrane-associated pools of M3 (Fig. 1A). Stimulation with carbachol (10⁻⁴M) elicited an intracellular calcium response in 39% (±20) of fibrocytes, with a mean peak amplitude of 0,86 (±0,66). Such response was blunted in presence of tiotropium bromide (10^–6M): 28% (±4) of responding cells and mean peak amplitude of 0,2±0,04. Moreover, carbachol induced a significant contraction of fibrocytes embedded in collagen gels (86,4±0,3% of initial gel surface versus 97,5±4,1%; p<0,0001), which was prevented by prior tiotropium bromide addition (resulting in 95,9±2,7% of initial surface; p<0,0001, Fig. 1B).

Around 1/3 of COPD patient's fibrocytes express a functional muscarinic M3 receptor. Cholinergic-induced fibrocyte contraction could participate in airway diameter reduction and subsequent increase of airflow resistance in patients with COPD. The inhibition of these processes could participate to the beneficial effects of muscarinic antagonists for COPD treatment.