Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

Abstract

Lung epithelial cells can influence immune responses to airway allergens. Airway epithelial cells also undergo apoptosis after encountering environmental allergens; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells. Administration of exogenous IL-10 'rescued' the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens.

Figure 1. Rac1-dependent engulfment and anti-inflammatory cytokine production by airway epithelial cells

a, Uptake of CypHer5-labelled apoptotic epithelial cells by viable epithelial cells (CFSE). b, Internalization is blocked by annexin (anx) V (n = 5). AC, airway epithelial cells. c, TGF-β (n = 5) and PGE₂ (n = 3) production by BEAS-2B epithelial cells. d, Phagocytosis and TGF-β production in MLE-12 epithelial cells transfected with Rac1^T17N (n = 3). e, Schematic for generating CCSP-Cre/Rac1^fl/fl mice and Dox-induced (‘Tet-On’) Rac1 deletion in airway epithelial cells. f, Left, YFP⁺ epithelial cells from control and Rac1-deficient mice. Right, Rac1 mRNA expression in epithelial cells (n = 10). g, Loss of Rac1 in the airways of Dox-treated CCSP-Cre/Rac1^fl/fl mice (open arrowheads). h, Left, engulfment by airway epithelial cells from control and Rac1-deficient mice (n = 5). **P < 0.01, representative of three experiments. Middle and right, IL-10 and TGF-β in BAL fluid of control and Rac1-deficient mice after intranasal administration of apoptotic cells (f, h, n = 5–10 mice from three experiments). *P < 0.05, **P < 0.01, unpaired Student’s t-test with Welch’s correction (b, c, d, h), Mann–Whitney test (h). Error bars, s.e.m.

Figure 2. Mice lacking Rac1 in airway epithelial cells show increased allergic inflammation

a, Protocol for inducing airway inflammation. b, Representative haematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining of lung sections from control or CCSP-Cre/Rac1^fl/fl mice. c, Infiltrating CD4⁺ T cells, eosinophils and Th2-type cytokines in BAL fluid of control or Rac1-deficient mice (n = 12–18 mice per group from more than four experiments). d, Cytokine production by mediastinal lymphnode (LN) cells restimulated in vitro for 5 days with HDM (n = 8–10 mice per group from three experiments). e, Airway resistance, compliance and pleural pressure in control or in Rac1-deficient mice (n = 5 mice from two experiments). * P < 0.05, **P < 0.01, *** P < 0.001, unpaired Student’s t-test with Welch’s correction (c–e).

Figure 3. Allergic airway inflammation in Rac1-deficient mice is rescued by rIL-10

a, Schematic of inducing airway inflammation and IL-10 treatment. b, H&E and PAS staining of lung sections from CCSP-Cre/Rac1^fl/fl mice treated with or without rIL-10 (1 µg), representative of several experiments. c, BAL fluid analysis showing IL-4, IL-5 and IL-13 production by Rac1-deficient mice treated with or without rIL-10 (n = 8–10 mice per group, from three experiments). d, Image of mediastinal lymph nodes (n = 3 mice per group). e, Cytokine production by lymph node cells re-stimulated in vitro for 5 days with HDM (n = 5 mice per group). f, Airway resistance, compliance and pleural pressure in Rac1-deficient mice treated with or without rIL-10 (n = 5) (d–f, representative of three experiments). *P < 0.05, unpaired Student’s t-test with Welch’s correction (c, e, f).

Figure 4. Airway inflammation with Ova after Rac1 deletion

a, Schematic of intranasal priming and challenge with Ova. b, H&E staining of lung sections from Dox-treated control and CCSP-Cre/Rac1^fl/fl mice (×20 magnification). c, Infiltrating CD4⁺ T cells and eosinophils in BAL fluid of indicated mice. d, Th2-cytokines in the BAL fluid. (c, d, n = 10 mice per group). e, Cytokine production by mediastinal lymph node T cells re-stimulated in vitro for 5 days with Ova (n = 6 mice per group). (c–e, Representative of three experiments). f–h, Rac1-deficient mice primed and challenged with Ova with IL-10. f, H&E staining of lung sections. Arrowheads indicate leukocyte infiltration (×20 magnification). g, Image of mediastinal lymph nodes (f, g, representative of three experiments). h, BAL fluid analysis of infiltrating T-cells, eosinophils and Th2 cytokines (n = 6 mice combined from two experiments). *P < 0.05, **P < 0.01, unpaired Student’s t-test with Welch’s correction.

Figure 5. IL-33 upregulation in Rac1-deficient epithelial cells correlates with airway inflammation

a, IL-33 and TSLP in BAL fluid of control or Rac1-deficient mice. b, Strategy for gating nuocyte-like cells and their numbers in lungs after HDM priming (a, b, n = 6 or 7 mice from two experiments). c, IL-33 or TGF-β expression in Rac1-siRNA-treated MLE-12 cells with apoptotic cells (n = 3) (c, representative of three experiments). d, e, In vivo IL-33 induction after intranasal HDM or apoptotic cells in CCSP-Cre/Rosa26^YFP mice (d), or in purified YFP⁺ EpCam⁺ epithelial cells (n = 5 or 6 mice, from three experiments) (e). f, IL-33 mRNA in human nasal epithelial cells stimulated with HDM or apoptotic cells (n = 3); *P < 0.05, **P < 0.01, unpaired Student’s t-test with Welch’s correction. Error bars, s.e.m.