Preterm ovine respiratory epithelial cell responses to mechanical ventilation, lipopolysaccharide, and interleukin-13

Abstract

Mechanical ventilation causes airway injury, respiratory epithelial cell proliferation, and lung inflammation in preterm sheep. Whether preterm epithelial cells respond similarly to adult epithelial cells or are altered by mechanical ventilation is unknown. We test the hypothesis that mechanical ventilation alters the responses of preterm airway epithelium to stimulation in culture. Respiratory epithelial cells from the trachea, left mainstem bronchi (LMSB), and distal bronchioles were harvested from unventilated preterm lambs, ventilated preterm lambs, and adult ewes. Epithelial cells were grown in culture or on air-liquid interface (ALI) and challenged with combinations of either media only, lipopolysaccharide (LPS; 10 ng/mL), bronchoalveolar fluid (BALF), or interleukin-13 (IL-13). Cell lysates were evaluated for mRNA changes in cytokine, cell type markers, Notch pathway, and acute phase markers. Mechanical ventilation altered preterm respiratory epithelium cell types. Preterm respiratory epithelial cells responded to LPS in culture with larger IL-8 induction than adults, and mechanical ventilation further increased cytokines IL-1β and IL-8 mRNA induction at 2 h. IL-8 protein is detected in cell media after LPS stimulation. The addition of BALF from ventilated preterm animals increased IL-1β mRNA to LPS (fivefold) in both preterm and adult cells and suppressed IL-8 mRNA (twofold) in adults. Preterm respiratory epithelial cells, when grown on ALI, responded to IL-13 with an increase in goblet cell mRNA. Preterm respiratory epithelial cells responded to LPS and IL-13 with responses similar to adults. Mechanical ventilation or exposure to BALF from mechanically ventilated animals alters the responses to LPS.NEW & NOTEWORTHY Preterm lamb respiratory epithelial cells can be extracted from the trachea and bronchi and frozen, and the preterm cells can respond in culture to stimulation with LPS or IL-13. Brief mechanical ventilation changes the distribution and cell type of preterm respiratory cells toward an adult phenotype, and mechanical ventilation alters the response to LPS in culture. Bronchoalveolar lavage fluid from preterm lambs receiving mechanical ventilation also alters unventilated preterm and adult responses to LPS.

Keywords: air-liquid interface; culture; interleukin-13; preterm; respiratory epithelium.

Graphical abstract

Figure 1.

Schematic for methods used for experiments. [Image created with BioRender.com and published with permission.]

Figure 2.

Comparison of baseline mRNA levels for tracheal (open symbols) and LMSB cells (filled symbols) at time of harvest. Median level for unventilated preterm lambs (n = 5) was set at one and other groups compared to them. A: SPDEF mRNA increased with mechanical ventilation in preterm lambs (n = 8). B: CCSP is more abundant in adult ewe (n = 4) and did not change with mechanical ventilation. C: Fox J1 was similar between groups. p63 (D) and KRT5 (E) were lower in adult ewes than unventilated preterm lambs. F: KRT14 was increased in ventilated preterm lambs and lower in adult ewe. G: Hes1 decreased with mechanical ventilation to adult ewe levels. H: Hey 1 is higher in adult ewe without change from mechanical ventilation. I: EGFR is lower in adult ewe than preterm unventilated lambs. J: CTGF increases with mechanical ventilation in preterm lambs. Means ± SE. *P < 0.05 vs. unventilated controls. CCSP, club cell secretory protein; CTGF, connective tissue growth factor; EGFR, epithelial growth factor receptor; FOX J1, Forkhead box protein J1; Hes1, Hes family BHLH transcription factor 1; KRT5, keratin 5; LMSB, left mainstem bronchus; SPDEF, SAM-pointed domain-containing ETS transcription factor.

Figure 3.

Cytokine mRNA responses in tracheal cells exposed to 10 ng/mL LPS for 2 h and 24 h. A: IL-1β mRNA increased in all groups with LPS exposure at 2 h. B: IL-6 mRNA increased with LPS exposure at 2 h, but was highest in the preterm animals exposed to a brief ventilation. C: IL-8 mRNA increased more in preterm cells than adult cells, and fetal mechanical ventilation increased the IL-8 mRNA response to LPS at 2 h. D: IL-1β remained slightly elevated at 2 h compared with media. E: IL-6 returned to baseline in all groups except unventilated preterm cells. F: IL-8 was higher at 24 h in the unventilated preterm cells than adult cells. Values are means ± SE; *P < 0.05 vs. media; &P < 0.05 vs. adult ewe; #P < 0.05 vs. unventilated preterm. LPS, lipopolysaccharide.

Figure 4.

Effects of bronchoalveolar lavage fluid (BALF) on response to LPS in ewe and unventilated preterms. A and B: when exposed to BALF from lambs receiving gentle ventilation (GV) or injurious ventilation (IV), IL-1β mRNA increased compared with LPS 10 ng/mL or unventilated control BALF (UVC). C and D: IL-8 mRNA was decreased in the adult cells exposed to BALF compared with LPS alone. Preterm cells had increased IL-8 mRNA when exposed to GV and IV. *P < 0.05 vs. media; &P < 0.05 vs. LPS only; %P < 0.05 vs. UVC + LPS. LPS, lipopolysaccharide.

Figure 5.

Tracheal and LMSB epithelial cell responses to IL-13 on ALI. A: SPDEF mRNA in with IL-13 exposures in all groups, with a larger increase in ventilated preterm cells. B: MUC5B mRNA increased in unventilated and ventilated preterm cells, but no change was found in adult cells. C: variable responses in CCSP mRNA with overall increases in preterm cells. D: FoxJ1 mRNA was decreased in preterm cells. No change in p63 mRNA was found (E), though KRT14 activation was decreased (F) in preterm cells. *P < 0.05 vs. media; #P < 0.05 vs. adult ewe. ALI, air-liquid interface; CCSP, club cell secretory protein; LMSB, left mainstem bronchus; MUC5B, Mucin 5B; SPDEF, SAM-pointed domain-containing ETS transcription factor.