Fibroblast-myofibroblast transition is differentially regulated by bronchial epithelial cells from asthmatic children

Abstract

Background: Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs.

Methods: BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition.

Results: Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone.

Conclusion: These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.

Figure 1

Time course of α-SMA expression in HLF cultures. The expression of α-SMA by HLF cultures is shown following initial seeding through Day 7 (start of co-culture exposures). Expression of α-SMA at Day 7 of culture was 25 fold higher than Day 1 (p < 0.0001).

Figure 2

Expression of α-SMA mRNA following 96 hours of co-culture. Expression of α-SMA by HLFs cultured alone was 1.9 fold higher than by HLFs co-cultured with healthy BECs (p = 0.008; *). α-SMA expression by HLFs co-cultured with asthmatic BECs was 2-fold higher than by HLFs co-cultured with healthy BECs (p = 0.002; **). There was no difference in α-SMA expression between HLFs cultured alone and HLFs co-cultured with asthmatic BECs.

Figure 3

Immunostaining for α-SMA in HLF alone or co-cultured with healthy or asthmatic BECs. Immunostaining for α-SMA in HLF Alone (Panel A), HLF Co-Cultured with heathy BECs (Panel B), and HLF Co-Cultured with asthmatic BECs (Panel C) following 96 hr exposure. All sections are counterstained for DAPI.

Figure 4

Quantification of α-SMA positive cells by flow cytometry. Flow cytometry for α-SMA (% positive) in HLF Alone, HLF Co-Cultured with Heathy BECs, and HLF Co-Cultured with Heathy BECs following 96 hr exposure. Flow cytometry revealed 3.6% α-SMA positive cells in the healthy co-culture group compared to 8.1% α-SMA positive cells in the asthmatic co-culture group (p < 0.03) and 11.6% α-SMA positive cells in the HLF alone (p < 0.03).

Figure 5

Quantification of total number of HLF cells in HLF cultured alone compared to co-culture with healthy or asthmatic BECs. Cell counts as demonstrated by software enumeration of DAPI positive nuclei. No significant differences were observed among the three HLF groups (n = 10 per group).

Figure 6

Expression of α-SMA mRNA in HLF following exposure to TGFβ _2, TGFβ ₂ monoclonal neutralizing antibody, and PGE _2. . TGFβ₂ exposure lead to a 17-fold increase in α-SMA expression (p < 0.001). This effect was decreased by nearly 50% with addition of TGFβ₂ neutralizing antibody (p < 0.01). Addition of PGE₂ lead to an 80% reduction in α-SMA expression compared to untreated HLF (p < 0.01).

Figure 7

Expression of α-SMA in HLF co-cultured with either healthy BEC or asthmatic BEC in the presence of TGFβ ₂ monoclonal neutralizing antibody. Expression of α-SMA by HLF was greater than both HLF-healthy BEC and HLF asthmatic BEC co-cultures. HLF-asthmatic BEC co-cultures demonstrated 40% greater expression of α-SMA compared to HLF-healthy BEC co-cultures (p < 0.01). Addition of TGFβ₂ neutralizing antibody did not significantly change α-SMA expression in HLF-healthy BEC co-cultures; however, similar treatment lead to a 30% reduction in the expression of α-SMA in HLF-asthmatic co-cultures (p < 0.01).

Figure 8

Expression of tropomyosin-I following 96 hours of co-culture. Expression of tropomyosin-I by HLFs cultured alone was not significantly different than by HLFs co-cultured with healthy BECs. Tropomyosin expression by HLFs co-cultured with asthmatic BECs was 4.3-fold higher than by HLFs co-cultured with healthy BECs (p = 0.03) and 6.2-fold greater than HLF alone (p < 0.004).