TNF-α and IL-1β-activated human mesenchymal stromal cells increase airway epithelial wound healing in vitro via activation of the epidermal growth factor receptor

Abstract

Background: Mesenchymal stromal cells (MSCs) are investigated for their potential to reduce inflammation and to repair damaged tissue. Inflammation and tissue damage are hallmarks of chronic obstructive pulmonary disease (COPD) and MSC infusion is a promising new treatment for COPD. Inflammatory mediators attract MSCs to sites of inflammation and affect their immune-modulatory properties, but little is known about their effect on regenerative properties of MSCs. This study investigates the effect of the pro-inflammatory cytokines TNF-α and IL-1β on the regenerative potential of MSCs, using an in vitro wound healing model of airway epithelial cells.

Methods: Standardized circular wounds were created by scraping cultures of the airway epithelial cell line NCI-H292 and primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC), and subsequently incubated with MSC conditioned medium (MSC-CM) that was generated in presence or absence of TNF-α/IL-1β. Remaining wound size was measured up to 72 h. Phosphorylation of ERK1/2 by MSC-CM was assessed using Western blot. Inhibitors for EGFR and c-Met signaling were used to investigate the contribution of these receptors to wound closure and to ERK1/2 phosphorylation. Transactivation of EGFR by MSC-CM was investigated using a TACE inhibitor, and RT-PCR was used to quantify mRNA expression of several growth factors in MSCs and NCI-H292.

Results: Stimulation of MSCs with the pro-inflammatory cytokines TNF-α and IL-1β increased the mRNA expression of various growth factors by MCSs and enhanced the regenerative potential of MSCs in an in vitro model of airway epithelial injury using NCI-H292 airway epithelial cells. Conditioned medium from cytokine stimulated MSCs induced ERK1/2 phosphorylation in NCI-H292, predominantly via EGFR; it induced ADAM-mediated transactivation of EGFR, and it induced airway epithelial expression of several EGFR ligands. The contribution of activation of c-Met via HGF to increased repair could not be confirmed by inhibitor experiments.

Conclusion: Our data imply that at sites of tissue damage, when inflammatory mediators are present, for example in lungs of COPD patients, MSCs become more potent inducers of repair, in addition to their well-known immune-modulatory properties.

Figures

Fig. 1

Stimulation of MSCs with TNF-α and IL-1β increases the expression of several growth factors. MSCs were stimulated with TNF-α and IL-1β 20 ng/ml each and harvested for RNA extraction after 6 h. mRNA expression of various growth factors (AREG, EGF, HBEGF (all EGFR ligands), FGF2, HGF, IL6, PDGFA, and VEGF) was determined by qPCR, which showed a significant increase of FGF2, HBEGF, HGF and of IL6, and an increase of AREG and EGF. Values were normalized to ACTB and GAPDH reference genes. Box and whiskers represent median, interquartile range and minimum and maximum for n = 4 obtained from three different donors; (*) p < 0.05

Fig. 2

MSC-CM increases wound closure. a, b: NCI-H292 cells were injured by making a circular wound with a diameter of 3 mm, and subsequently incubated with MSC-CMCTRL, MSC-CMSTIM, DMEMCTRL and DMEMSTIM, and a negative control (NC, RPMI only) and positive control (RPMI supplemented with TGF-α 20 ng/ml). The wound size was measured at 0, 24, 48 and 72 h after wounding. MSC-CMSTIM significantly increased wound closure. The effect of MSC-CMSTIM was dose-dependent. Error bars represent standard error of the mean (SEM). n = 4-6; (*) p < 0.05 TGF-α compared to NC and (**) compared to DMEM; (***) p < 0.05 MSC-CM compared to NC and (****) compared to DMEM; (*****) p < 0.05 DMEMSTIM compared to NC. c: Morphology of the closure of the wound: photos in the upper panel are taken at t = 0 h, the lower panel shows the same wounds photographed 48 h later. The two photos on the left side are obtained from MSC-CMSTIM stimulated cells, whereas photos at the right represent its control, DMEMSTIM. d: Dose response. Box and whiskers represent median, interquartile range and minimum and maximum. n = 4-6; (*) p < 0.05. e: Wound closure in ALI-PBEC measured at 6 and 24 h (at 48 h all wounds were closed). At 24 h, MSC-CMSTIM significantly enhanced wound healing compared to the NC, but no significant differences were observed compared to its control, DMEMSTIM. Error bars represent SEM, n = 4; (*) p < 0.05 for MSC-CMSTIM compared to NC. f. Wound closure in ALI-PBEC at 24 h. Box and whiskers represent median, interquartile range and minimum and maximum, for n = 4 as in Fig. 2f; (*) p < 0.05

Fig. 3

MSC-CMSTIM increases ERK1/2 phosphorylation. NCI-H292 cells were incubated with MSC-CMSTIM, DMEMSTIM, DMEMCTRL or TGF-α 20 ng/ml (pos ctrl). ERK1/2 phosphorylation and total ERK was determined in cell lysates using Western blot. a: After 15 min incubation time MSC-CMSTIM increased ERK1/2 phosphorylation compared to its control. b: Densitometry for Fig. 3a. Error bars represent SEM, n = 6; (*) p < 0.05. c: Time course experiment, demonstrating that the effect of MSC-CMSTIM was most prominent up to 30 min, but could still be observed up to 6 h. DMEMCTRL was obtained at 15 min. d: Densitometry for Fig. 3c. Error bars represent SEM, n = 4-5; (*) p < 0.05

Fig. 4

MSC-CMSTIM induced ERK1/2 phosphorylation is mediated via (trans)activation of EGFR. a: 1 h pre-incubation of NCI-H292 with 0.05 μM PF04217903 and/or 10 μM AG1478, followed by 15 min stimulation with MSC-CMSTIM or DMEMSTIM, or DMEMCTRL showed that inhibition of EGFR decreased ERK1/2 phosphorylation as determined using Western blot. b: Densitometry for Fig. 4a. Error bars represent SEM, n = 5; (*) p < 0.05. c: 1 h pre-incubation with 2 μg/ml neutralizing anti-EGFR antibodies and 10 μM TAPI-1 followed by incubation with MSC-CMSTIM during 15 min showed that these agents decreased ERK1/2 phosphorylation. d: Densitometry for Fig. 4c. Error bars represent SEM, n = 3; (*) p < 0.05. e: NCI-H292 cells were incubated with MSC-CMSTIM or DMEMSTIM and harvested after 9 h for mRNA analysis. mRNA expression of the EGFR ligands AREG, HBEGF and TGFA was determined by qPCR. MSC-CMSTIM significantly increased the expression of AREG and HBEGF. Expression of TGFA increased but this was not significant. f: mRNA expression of the proliferation marker CCDN1 was significantly increased. Values were normalized against RPL13A and ATP5B reference genes. Box and whiskers represent median, interquartile range and minimum and maximum. n = 4; (*) p < 0.05

Fig. 5

Blocking of EGFR reduces the stimulatory effect of MSC-CMSTIM on wound closure. a: NCI-H292 wounded cell layers were stimulated with MSC-CMSTIM or DMEMSTIM, DMEMCTRL, or DMEM supplemented with TGF-α 20 ng/ml or HGF 20 ng/ml (positive controls). Inhibitors of EGFR (0.2 μM AG1478) or c-Met (0.05 μM PF04217903) were added during the full culture period in assigned conditions. At 48 h, HGF and TGF-α both significantly enhanced wound healing in NCI-H292 cells, which was averted to a level comparable to the negative control by their respective inhibitors. b: In the presence of the EGFR inhibitor, the wound healing capacity of MSC-CMSTIM decreased to values below those observed in the negative control as determined after 48 h. Inhibition of c-Met alone had no effect on wound healing induced by MSC-CMSTIM. Box and whiskers represent median, interquartile range and minimum and maximum. n = 4-7; (*) p < 0.05

Fig. 6

Proposed model of enhanced wound closure in airway epithelial cells by MSC-CMSTIM. In MSCs, exposure to TNF-α and IL-1β increases the mRNA expression of various growth factors. Conditioned medium from these MSCs contributes to wound healing of airway epithelial cells via three mechanisms: direct activation of EGFR; activation of matrix metalloproteases which results in shedding of membrane bound EGFR ligands; induction of mRNA expression of EGFR ligands by the airway epithelium. Activation of the downstream MAP kinase ERK1/2, which is known as a regulator of cell proliferation (a.o. assessed by increased Cyclin D1) and differentiation, subsequently results in increased wound healing