The Properties of Circulating Fibrocytes in Severe Asthma

Abstract

Inflammation associated with asthma mainly affects large airways and is accompanied by extensive structural changes, termed airway remodelling. 5-10% of patients with asthma suffer from severe or refractory asthma which is difficult to control despite receiving high doses of inhaled and sometimes oral corticosteroids (CS). These patients show more prominent characteristics of airway remodelling, specifically sub-epithelial fibrosis and airway smooth muscle (ASM) thickening. My project focuses on one of the cells implicated in airway remodelling, circulating fibrocytes, which are bone marrow-derived peripheral blood mesenchymal progenitors expressing both leukocyte markers, such as CD45, and mesenchymal proteins including collagen I (Col I). Fibrocytes migrate to the sites of disease under the guidance of chemokine receptors such as CC chemokine receptor type 7 (CCR7), and differentiate into α–smooth muscle actin (α-SMA)–expressing myofibroblasts, a process that is facilitated by a variety of pro-inflammatory cytokines and growth factors. Myofibroblasts can promote subepithelial fibrosis as well as contribute to ASM thickening. Indeed, the number of fibrocytes in peripheral blood is correlated with the decline rate of forced expiratory volume in 1s (FEV1) in patients with chronic obstructive asthma. Most importantly, there is increased recruitment of fibrocytes to the airway wall of patients with severe asthma. However, the mechanisms driving the accumulation of fibrocytes in the airways of these patients are currently unclear. I hypothesised that in severe asthma there are increased numbers of circulating fibrocytes that have an increased capacity to differentiate into myofibroblasts and have differential responses to pro-inflammatory mediators and asthma therapeutic agents compared to non-severe asthma. Fibrocytes were isolated from the non-adherent non-T (NANT) cell fraction of peripheral blood mononuclear cells (PBMC) of healthy subjects and patients with non-severe or severe asthma. The number of fibrocytes (Col I+/CD45+ cells) and differentiating fibrocytes (α-SMA+ cells), as well as the expression of CCR7 and glucocorticoid receptor (GR) in fibrocytes were determined by flow cytometry. Apoptosis was determined by Annexin V/propidium iodide staining. Messenger ribonucleic acid (mRNA) expression was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Fibrocytes were also isolated from the adherent fraction of PBMC. Severe asthmatic patients had a higher number of circulating fibrocytes with a greater capacity to differentiate into myofibroblasts in culture compared to healthy subjects and patients with non-severe asthma. Severe asthmatic fibrocytes did not have a heightened responsiveness to either interleukin (IL)-4, IL-13, nerve growth factor or brain-derived neurotrophic factor. Dexamethasone induced apoptosis in NANT cells, including fibrocytes and differentiating fibrocytes, from healthy subjects and patients with non-severe asthma, but not in the cells from patients with severe asthma. Dexamethasone also reduced CCR7 expression in fibrocytes from patients with non-severe asthma but not in those from patients with severe asthma. The relative CS insensitivity in severe asthmatic fibrocytes may be related to the lower expression of the GR or the heightened c-Jun N-terminal kinase activity. Salmeterol xinafoate, a long-acting β2-adrenoceptor agonist (LABA), reduced the number, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and patients with non-severe asthma. Salmeterol did not improve the suppressive effect of dexamethasone, although it was not detrimental to dexamethasone’s effect either. In contrast, tiotropium bromide, a long-acting muscarinic antagonist (LAMA), did reduce the number of fibrocytes and differentiating fibrocytes from patients with severe asthma. Increasing intracellular 3’,5’-cyclic adenosine monophosphate (cAMP), the downstream signalling molecule of β2-adrenoceptor and muscarinic M2 receptor, by phosphodiesterase type IV inhibitor (rolipram) and cAMP analogue (8-bromoadenosine-3’,5’-cyclic monophosphate) could reduce fibrocytes from patients with severe asthma. Patients with severe asthma have elevated numbers of circulating fibrocytes showing enhanced myofibroblastic differentiation and are less responsive to the suppressive effect of CS and LABA, but can be inhibited by LAMA. This study provides insight into a novel target for the treatment of airway remodelling in severe asthma.