Opposing effects of in vitro differentiated macrophages sub-type on epithelial wound healing

Abstract

Inappropriate repair responses to pulmonary epithelial injury have been linked to perturbation of epithelial barrier function and airway remodelling in a number of respiratory diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. We developed an in vitro mechanical scratch injury model in air-liquid interface differentiated primary human small airway epithelial cells that recapitulates many of the characteristics observed during epithelial wound injury in both human tissue and small animal models. Wound closure was initially associated with de-differentiation of the differentiated apical cells and rapid migration into the wound site, followed by proliferation of apical cells behind the wound edge, together with increases in FAK expression, fibronectin and reduction in PAI-1 which collectively facilitate cell motility and extracellular matrix deposition. Macrophages are intimately involved in wound repair so we sought to investigate the role of macrophage sub-types on this process in a novel primary human co-culture model. M1 macrophages promoted FAK expression and both M1 and M2 macrophages promoted epithelial de-differentiation. Interestingly, M2a macrophages inhibited both proliferation and fibronectin expression, possibly via the retinoic acid pathway, whereas M2b and M2c macrophages prevented fibronectin deposition, possibly via MMP expression. Collectively these data highlight the complex nature of epithelial wound closure, the differential impact of macrophage sub-types on this process, and the heterogenic and non-delineated function of these macrophages.

Fig 1

TEER development and Scratch width measurement. A TEER development measured in the cellZScope over time using no (black line), a 200 μL (orange line), a 20 μL (green line) and a 10 μL (violet line) pipet tip. B Microscopic image (CellIQ) of a scratch introduced with a 200 μL pipette tip (width (74 pts) of the scratch indicated by the red line) C scratch width (mean width 68.38 ± 5.39 pts) measured and plotted to demonstrate reproducibility of the scratch (n = 48) D time course of the closure of the scratch (n = 6)

Fig 2

IHC of scratched cells and fibronectin expression. A IHC microscopic images of scratched cells at t = 0h, 6h, 24h, 48h and 72h; DAPI in blue, KRT5 in yellow, Ki67 in red. B IHC microscopic images of scratched cells at t = 0h, 6h, 24h, 48h and 72h; DAPI in blue, FAK in yellow, FN1 in red. C ELISA of FN1 in the supernatant after 0h, 6h, 24h, 48h and 72h with and without scratch.

Fig 3

IHC of scratched cells and FN1 regulation/expression. A scratched epithelial cells incubated with different macrophage subtypes after 24h and 72h; DAPI stained in blue, FAK stained in yellow, FN1 stained in red, F-Actin stained in green. B FN1 mRNA regulation in the co-culture cell lysates measured after 24h and 72h Data are displayed as fold regulation compared to control medium containing macrophage maturation factors; Data is expressed as mean ± SD; n = 3 C FN1 expression measured in the supernatants via ELISA after 24h and 72h; Data is expressed as mean ± SD; n = 3 (**** = p>0.0001)

Fig 4. mRNA regulation in the co-cultures.

mRNA expression levels in lysates of the co-culture after 24h and 72h for MUC5AC, CLDN3, OCLDN, TFF3, SERPINE1 and MKI67/Ki67; Data are displayed as fold regulation compared to control medium containing macrophage maturation factors; Data is expressed as mean ± SD; n = 3

Fig 5

Retinoid acid amount and MMP3/MMP14 expression. A Retinoic acid concentration in supernatants of different macrophage subtypes was measured by ELISA B MMP3 and C MMP14 expression in different macrophage subtypes was measured by TaqMan RPKM = Reads per kilobase per million mapped reads; Data is expressed as mean ± SD; n = 3