Fig 1: Alpha-1 antitrypsin reduces NET-driven barrier dysfunction. HBE were exposed to PBS, 5µg/ml NETs, NETs+A1AT or A1AT for 18h in triplicate wells. (A–C) Activity of neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) were reduced in the apical supernatants of HBE exposed to NETs+A1AT. Data analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test (experiments=3, HBE donors=4, NET donor=2). (D, E) Representative western blot and analysis of E-cadherin protein from lysates of HBE demonstrates that NETs decrease full-length E-cadherin and increases cleaved E-cadherin peptides compared to PBS control. A1AT limits NET-driven reductions in full-length E-cadherin protein. (F, G) Representative western blot and analysis demonstrate decreased cleaved caspase 9 (less activation of apoptosis) in HBE exposed to NETs with A1AT. Western blots were normalized to C4-actin and analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test (experiments=2, HBE donors=2, NET donors=3). (H) A1AT maintained HBE TEER with exposure to NETs (representative graph). Data analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test (experiments=3, HBE donors=3, NETs=3). *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig 2: NET effect on bronchial epithelial barrier function. (A) Pseudostratified bronchial epithelia form a monolayer with cell-cell contact via junctional proteins. This epithelial barrier tightly controls the paracellular flux of molecules and cells. (B) Neutrophils expel NETs, which contain biologically active proteases that degrade adherens junction protein E-cadherin creating gaps in the monolayer. A1AT limits NET degradation of E-cadherin to help restore barrier function. (C) NETs also disrupt barrier function by causing epithelial cell death via apoptosis, which is limited by A1AT.
Fig 3: Alpha-1 antitrypsin binds serine proteases in NET exposed HBE. HBE grown at ALI were exposed to PBS, 5µg/ml NETs, NETs + A1AT or A1AT for 18h in triplicate wells. (A) Western blot of HBE apical supernatants probed with A1AT antibody demonstrate the colocalization of A1AT (51-55kDa) with a 29kDa protein in NETs. (B) Western blot of HBE apical supernatants probed with NE antibody demonstrate the colocalization of A1AT with NE (29kDa) in NETs forming an 80kDa complex. (C) Representative mass spectrometry results from bands shown on western blot in 7A & B confirm that HBE exposed to NETs have peptide spectral matches (PSMs) that correspond to CG, PR3, and NE at the expected 17-31kDa sizes. HBE exposed to NETs + A1AT demonstrate PSMs corresponding to NE, CG, PR3 and A1AT at 70-102kDa indicating formation of a A1AT:NE, A1AT:CG and A1AT:PR3 complexes (mass spec run=3, HBE donors=2, NET donors=3).
Fig 4: Alpha-1 antitrypsin preserves epithelial cell junction integrity disrupted by NETs. Representative images from transmission electron microscopy of HBE exposed to PBS, 5µg/ml NETs, NETs + A1AT or A1AT for 18 h. NET exposure resulted in partial disruption of epithelial tight junctions. A1AT helped maintain tight junctions in HBE exposed to NETs. (M=mitochondria, MV=microvilli, SG=secretory granules in a goblet cell, (→) =cell junction, experiments=1, HBE donors=1, NET donors=1).
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