Fig 1: A1AT-releasing allogeneic HIP iECs prevent emphysema development in Serpina−/− B6 mice. (A) Serpina−/− B6 mice were challenged with LPS via their airways to trigger emphysematous lung disease. (B) Some Serpina−/− B6 mice were treated with MHC-engineered A1AT-secreting allogeneic (ealloS1e) or allogeneic HIP (ealloHIPS1e) iECs 7 d before the LPS challenge. Healthy WT B6 animals served as controls. (C) The study protocol included monitoring of graft survival, as well as functional and morphologic lung assessments. (D) Serum A1AT levels were quantified in all groups (mean ± SD, six animals in WT B6, five animals in Serpina−/− B6 LPS without cell injections at day 14, four animals in Serpina−/− B6 LPS ealloS1e iECs at day 14, four animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 14, and five animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 28; ANOVA with Bonferroni post hoc test for 14-d groups). (E and F) The survival of FLuc+ ealloS1e iECs (E, four animals) and ealloHIPS1e iECs (F, nine animals) in Serpina−/− B6 recipients was longitudinally followed by BLI. BLI signals of individual animals are plotted, representative pictures are shown. (G–M) FlexiVent lung physiology assessments were done after 14 and 28 d (scatter dot plots, mean ± SD, six animals in WT B6, five animals in Serpina−/− B6 LPS without cell injections at day 14, four animals in Serpina−/− B6 LPS ealloS1e iECs at day 14, four animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 14, and five animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 28; ANOVA with Bonferroni post hoc test for 14-d groups). (N–Q) Stereological lung assessments were done in all groups (mean ± SD, six animals in WT B6, five animals in Serpina−/− B6 LPS without cell injections at day 14, four animals in Serpina−/− B6 LPS ealloS1e iECs at day 14, four animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 14, and five animals in Serpina−/− B6 LPS ealloHIPS1e iECs at day 28; ANOVA with Bonferroni post hoc test for 14-d groups).
Fig 2: Humans with reduced AAT levels have an increased free cortisol fraction in parallel with increased adipose cortisol exposure.a–c Circulating glucocorticoid profile in subjects with heterozygous mutations in SERPINA1 (AAT +/-; orange symbols) and matched controls (Control; grey symbols), as part of a randomised, double-blind crossover study, using either a combination of RU486 and spironolactone (CRASH; white-filled symbols) or placebo (colour-filled symbols), including percent free cortisol (a), total cortisol (b), and CBG (c). d Cortisol levels in subcutaneous abdominal adipose from subjects with the indicated genotype and treatment. e Transcript expression in subcutaneous abdominal adipose from subjects with the indicated genotype in the placebo group. Data are presented as mean (dashed line) -/+ SD (dotted line). Control: Placebo n = 16, AAT + /-: Placebo n = 16, Control: CRASH n = 16, AAT + /-: CRASH n = 16. Data are analysed by Mixed-effects model (effects and corresponding P-values indicated on graph) (a–c), RM two-way ANOVA with Sidak’s post-hoc tests (d), or two-tailed unpaired t-tests (e). *P < 0.05.
Fig 3: The NE/AAT/CBG axis regulates glucocorticoid action in vitro.a NE activity is inhibited with indicated concentrations of AAT. Data are presented as mean −/+ SD. (A.U.; arbitrary units). b, c Treatment of 1:50 diluted mouse serum (b) and human serum (c) with either NE alone (4 U; green circle) or NE + AAT (100 µM; orange circle) for 10 min at 37 °C. Data are presented as individual points for n = 4 biological replicates. d, e Free glucocorticoid in diluted mouse serum (d) and human serum (e) treated with either NE alone (4 U; green circle) or NE + AAT (100 µM; orange circle). f Glucocorticoid-responsive luciferase reporter activity in human embryonic kidney (HEK293) cells -/+ transfection with human glucocorticoid receptor (GR). Cells were treated for 24 h with 1:50 diluted human serum -/+ NE (4 U, 10 min, 37 °C). Data are presented as individual points for n = 4 biological replicates. Data are presented as individual points for n = 4 biological replicates. Data analysed by RM one-way ANOVA with Tukey’s post-hoc tests (a–e), or two-tailed paired t-test (f). *P < 0.05, ***P < 0.001.
Fig 4: Overview of proposed NE/AAT/CBG control of local and systemic glucocorticoid action in mice and humans.Illustration of proposed NE/AAT/CBG mechanism of action in murine obesity and in humans with alterations in AAT levels. Created in BioRender (https://BioRender.com/b20b142).
Fig 5: Arterio-venous sampling across tissue reveals increased local glucocorticoid exposure in AAT heterozygotes.Arterio-venous sampling in subjects with heterozygous mutations in SERPINA1 (AAT +/-; orange triangles) and matched controls (Control; grey circles) with steady-state 9,11,12,12-[2H]4-cortisol (D4-cortisol) tracer infusion. a–c Plasma profile during D4-cortisol infusion, including endogenous cortisol levels (a), D4-cortisol/cortisol ratio (b), and D4-cortisol/D3-cortisol ratio (c). d–f Plasma glucocorticoid profile during steady state (180 – 270 min), including free cortisol (d), total cortisol (e), and CBG binding capacity (f). g–h Whole body rate of appearance (Ra) of cortisol (g) and D3-cortisol (h) during steady state. i Net balance of cortisol and D4-cortisol across skeletal muscle. j Percent free cortisol across skeletal muscle. k Net balance of free cortisol across skeletal muscle. Data are presented as mean (dashed line) -/+ SD (dotted line). Control n = 16, AAT + /- n = 16. Data analysed by Mixed-effects model (a–e), two-tailed unpaired t-tests (d–h, k), or RM two-way ANOVA with Sidak’s post-hoc tests (i, j). *P < 0.05, ***P < 0.001.
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