Brain-derived neurotrophic factor and airway fibrosis in asthma

Abstract

Airway remodeling in asthma driven by inflammation involves proliferation of epithelial cells and airway smooth muscle (ASM), as well as enhanced extracellular matrix (ECM) generation and deposition, i.e., fibrosis. Accordingly, understanding profibrotic mechanisms is important for developing novel therapeutic strategies in asthma. Recent studies, including our own, have suggested a role for locally produced growth factors such as brain-derived neurotrophic factor (BDNF) in mediating and modulating inflammation effects. In this study, we explored the profibrotic influence of BDNF in the context of asthma by examining expression, activity, and deposition of ECM proteins in primary ASM cells isolated from asthmatic vs. nonasthmatic patients. Basal BDNF expression and secretion, and levels of the high-affinity BDNF receptor TrkB, were higher in asthmatic ASM. Exogenous BDNF significantly increased ECM production and deposition, especially of collagen-1 and collagen-3 (less so fibronectin) and the activity of matrix metalloproteinases (MMP-2, MMP-9). Exposure to the proinflammatory cytokine TNFα significantly increased BDNF secretion, particularly in asthmatic ASM, whereas no significant changes were observed with IL-13. Chelation of BDNF using TrkB-Fc reversed TNFα-induced increase in ECM deposition. Conditioned media from asthmatic ASM enhanced ECM generation in nonasthmatic ASM, which was blunted by BDNF chelation. Inflammation-induced changes in MMP-2, MMP-9, and tissue inhibitor metalloproteinases (TIMP-1, TIMP-2) were reversed in the presence of TrkB-Fc. These novel data suggest ASM as an inflammation-sensitive source of BDNF within human airways, with autocrine effects on fibrosis relevant to asthma.

Keywords: airway smooth muscle; collagen; extracellular matrix; neurotrophin; tropomyosin-related kinase.

Fig. 1.

Brain-derived neurotrophic factor (BDNF) expression in nonasthmatic and asthmatic airway smooth muscle (ASM). Gene expression of BDNF (A) and the full-length version of its high-affinity receptor tropomyosin-related kinase (TrkB-FL; B) was significantly increased in ASM cells obtained from asthmatic patients compared with cells from nonasthmatic patients. Protein expression of BDNF (C) and TrkB-FL (D) was correspondingly increased in whole cell lysates of asthmatic ASM. Epithelium-denuded ASM tissues from asthmatic patients also demonstrated significant increase in BDNF (E) and TrkB-FL (F) expression compared with ASM tissue from nonasthmatic patients. Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. *Significant difference between nonasthmatics and asthmatics (P < 0.05). Asthmatics indicated in gray for clarity.

Fig. 2.

BDNF increases extracellular matrix (ECM) expression and deposition by ASM: Forty-eight-hour exposure to BDNF significantly increased expression of major ECM proteins collagen 1, -3, and fibronectin in nonasthmatic ASM cells compared with vehicle alone (A). ECM protein deposition was measured via modified In-Cell Western technique (B; essentially a semiquantitative immunofluorescence measurement; see materials and methods). BDNF exposure induced increases in collagen-1, -3, and fibronectin deposition in both nonasthmatic and asthmatic ASM cells, with a relatively greater effect of BDNF in asthmatic ASM. Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. #Significant BDNF effect; *significant difference between nonasthmatics and asthmatics (P < 0.05). Asthmatics indicated in gray for clarity.

Fig. 3.

BDNF effects on ECM regulatory enzymes. Nonasthmatic human ASM cells treated with BDNF showed no significant increase in matrix metalloproteinases-2 or -9 (MMP-2, MMP-9) levels or decreases in corresponding tissue inhibitor of metalloproteinases-1 or -2 (TIMP-1, TIMP-2) in conditioned medium (A, left panel of B). However, MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios significantly increased with BDNF treatment (right panel of B), suggesting increased overall enzyme activity. Gelatin zymography correspondingly demonstrated an increase in MMP-2 and MMP-9 activity with BDNF treatment in conditioned media (C, D). Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. #Significant BDNF effect (P < 0.05).

Fig. 4.

Proinflammatory cytokines induces BDNF secretion in ASM. Asthmatic ASM secrete more BDNF at baseline compared with nonasthmatic ASM measured from concentrated conditioned medium using ELISA and normalized to cell lysate protein concentration. Furthermore, exposure of TNFα significantly increased BDNF secretion in ASM, whereas IL-13 did not significantly increase BDNF secretion in nonasthmatic or asthmatic ASM. Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. #Significant TNFα effect; *significant difference between nonasthmatics and asthmatics (P < 0.05). Asthmatics indicated in gray for clarity.

Fig. 5.

BDNF mediates inflammation-induced ECM deposition in ASM. Exposure to TNFα for 72 h significantly increased ECM protein (collagen-1, -3, and fibronectin) deposition in both nonasthmatic (A, C, E) and asthmatic (B, D, F) ASM cells. Attenuation of BDNF through chelation of extracellular BDNF using the chimeric protein TrkB-Fc blunted TNFα-mediated increase in collagen-1 (A, B), collagen-3 (C, D), and fibronectin deposition (E, F). Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. #Significant TNFα effect; %significant TrkB-Fc effect (P < 0.05). Asthmatics indicated in gray for clarity.

Fig. 6.

BDNF mediates inflammation-induced MMP regulation in ASM: MMP and TIMP expression in conditioned medium from nonasthmatic human ASM cells was significantly altered by TNFα exposure. Furthermore, MMP/TIMP ratios also increased significantly with TNFα exposure (A, C). Gelatin zymography of conditioned media was used to determine MMP activity. TNFα exposure significantly increased MMP-9 and MMP-2 activity (B, D). BDNF chelation via TrkB-Fc significantly reduced TNFα-mediated effect on MMP activity. Means ± SD with individual patient data points [n values (patient numbers)] specified in individual panels. #Significant TNFα effect; %significant TrkB-Fc effect (P < 0.05).

Fig. 7.

Conditioned medium from ASM potentiates BDNF-induced ECM expression. ECM protein (collagen-1, -3, and fibronectin) expression was measured from nonasthmatic ASM cells treated for 72 h with asthmatic conditioned medium (ACM) with and without TrkB-Fc, with the idea that if asthmatic ASM produces/secretes more BDNF that modulates ECM, then BDNF chelation with TrkB-Fc should attenuate the effects of the ACM on production of ECM proteins. Indeed, on the one hand ACM increased ECM production in nonasthmatic ASM cells compared with baseline levels (black dotted lines in A, C, E). Conversely, TrkB-Fc attenuated such increases in ECM protein expression in nonasthmatic ASM induced by the ACM. In a parallel set of studies using asthmatic ASM cells, treatment with nonasthmatic conditioned media (NCM) also enhanced ECM production (B, D, F) compared with baseline production by such asthmatic ASM cells (gray dotted line), supporting the idea of nonasthmatic cells also producing BDNF that can act on TrkB-FL in asthmatic ASM cells (which are higher compared with nonasthmatic cells; Fig. 2). Indeed, TrkB-Fc also attenuated NCM-induced increases in production of collagens and fibronectin. Means ± SD with individual patient data points [n values (patient numbers] specified in individual panels. @Significant ACM or NCM effect; %significant TrkB-Fc effect (P < 0.05). Asthmatics indicated in gray for clarity. Note different y-axis scales for the different panels.

Fig. 8.

ASM-derived BDNF mediates/modulates inflammation effects on airway fibrosis. ASM is a potentially major source of BDNF in the airway. Inflammation and asthma enhance BDNF release by ASM, which can contribute to stimulating a positive-feedback loop of airway fibrosis via effects on collagen-1, -3, fibronectin, MMPs, and TIMPs. Accordingly, targeting ASM BDNF may be a novel approach attenuating remodeling in diseases such as asthma.