Deficient Follistatin-like 3 Secretion by Asthmatic Airway Epithelium Impairs Fibroblast Regulation and Fibroblast-to-Myofibroblast Transition

Abstract

Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT), whereas asthmatic BECs do so less effectively, suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs, and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma, we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and α-smooth muscle actin (α-SMA) was quantified by qPCR, and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts, we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002), and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV/FVC ratio < 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92% (P = 0.001) and α-SMA expression by 88% (P = 0.02), and increased FMT by flow cytometry in cocultured HLFs, whereas shRNA depletion of activin A (n = 6) resulted in decreased α-SMA (22%; P = 0.01) expression and decreased FMT. Together, these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT.

Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history

“…Using a similar BEC/HLF co-culture model system, our group has previously reported that that HLFs co-cultured with BECs obtained from asthmatic donors drive co-cultured HLFs to produce greater amounts of important ECM constituents including types I and III collagen and HA 12 , and that HLFs co-cultured with BECs obtained from asthmatic donors displayed a greater degree of FMT compared to HLFs co-cultured with BECs obtained from healthy donors 13 . In recent studies we have provided both indirect and direct mechanistic evidence that airway epithelial TGFβ 2 , PGE 2 , and follistatin-like-3 (FSTL3)/activin A signaling influence FMT and HLF expression of ECM constituents 12 , 13 , 36 . In the present study, the positive association between PGE 2 concentrations in co-culture supernatant and measures on lung function among asthmatic BEC donors provides additional evidence in support of the hypothesis that epithelial PGE 2 may play a role in regulating FMT and HLF expression of ECM constituents, although future studies are needed to more directly test this potential mechanism by blocking epithelial PGE 2 signaling.…”

Precision medicine and phenotypes, endotypes, genotypes, regiotypes, and theratypes of allergic diseases

“…Follistatin-like 3 (FSTL3), a glycoprotein secreted by bronchial epithelial cells that binds to and inhibits activin A, is diminished in children with asthma, especially when airway obstruction is present. Decreased FSTL3 expression in the asthmatic bronchial epithelium led to subepithelial fibrosis (109).…”

Immunologic and Non-Immunologic Mechanisms Leading to Airway Remodeling in Asthma

“…This observation may further explain the effect of allergens and methacholine on the remodeling, which has been described earlier in patients with very mild asthma [25]. The decreased secretion of folistatin-like 3 protein by airway epithelium impaired the transition of fibroblasts to myo-fibroblasts [74]. The release of basic fibroblast growth factor (bFGF) from epithelial cells was triggered by rhinovirus infection, thereby stimulating sub-epithelial fibroblast remodeling [75].…”