Abstract
Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors.
Highlights
Of efferocytic receptors initiates signaling events modulated by two main complexes, CrkII/ELMO/Dock18019 or ABCA1/GULP20, both resulting in activation of Rac[1], which facilitates cytoskeletal rearrangement for engulfment[18]
Treatment with conditioned medium derived from a murine macrophage cell line (RAW 264.7) exposed to apoptotic Jurkat cells for 20 h (ApoJ-exposed Conditioned medium (CM)) inhibited transforming growth factor (TGF)-β 1-induced epithelial-mesenchymal transition (EMT) in LA-4 cells, based on morphologic cellular alteration (Supplementary Fig. S1a) and EMT marker expression profiles at both the protein (Fig. 1a) and mRNA level (Fig. 1b–d)
We confirmed the inhibitory effect of the ApoJ-exposed CM on TGF-β 1-induced EMT in primary mouse alveolar type II epithelial (AT II) cells (Fig. 1f) as well as HEK-293 human embryonic kidney epithelial cells (Supplementary Fig. S2a)
Summary
Of efferocytic receptors initiates signaling events modulated by two main complexes, CrkII/ELMO/Dock18019 or ABCA1/GULP20, both resulting in activation of Rac[1], which facilitates cytoskeletal rearrangement for engulfment[18]. Apoptotic cell recognition by macrophages actively leads to production of anti-inflammatory mediators, such as transforming growth factor (TGF)-β , interleukin (IL)-10, and prostaglandin E2 (PGE2)[16,25]. We demonstrated that in vivo apoptotic cell exposure resulted in enhanced HGF and cyclooxygenase (COX)-2 expression and PGE2 secretion until the late fibrotic phase in bleomycin-induced lung injury[30,31]. The impact of the COX-2 and HGF pathways on the prevention of EMT progression in the context of enhanced apoptotic cell recognition and clearance has not been studied. We used in vitro co-incubation assays to demonstrate that macrophages programmed by apoptotic cells modulate EMT in lung epithelial cells. We determined how COX-2-derived PGE2 and PGD2, as well as RhoA-dependent HGF secretion from macrophages in response to apoptotic cells, contribute to EMT inhibition. We provided in vivo evidence that apoptotic cell instillation after bleomycin treatment inhibits EMT in primary mouse alveolar type II epithelial (AT II) cells, suggesting a potential therapeutic option for IPF treatment
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