Figure 2. Activated neutrophils rapidly deplete O₂ and induce HIF-1α stabilization via respiratory burst.

(A) pO₂ values were recorded in hypoxia chamber set to 4% O₂, using a OxoProbe at indicated time points in the presence of 1×10⁶ PMN, activated with fMLF. Data are representative of three independent experiments and presented as means ± SD (n = 3; P < 0.001; two-way ANOVA). (B) Consumption of dissolved O₂ in normoxia was monitored in real-time using OxoDishes mounted on an SDR O₂ sensor reader in the presence of increasing numbers of PMN ± activation with fMLF set up with the “SDR real-time O₂” (see Figure S1d) model. Data is representative of three independent experiments and presented as means ± SD (n = 3; P < 0.001 throughout time course for ± PMN; P < 0.001 throughout time course for 1×10⁶ PMN ± chemoattractant; P < 0.001 between 46 and 948 seconds for 5×10⁶ PMN ± chemoattractant and P < 0.001 between 46 and 407 seconds for 1×10⁶ versus 5×10⁶ with chemoattractant; two-way ANOVA). (C) Immunofluorescent staining of T84 IECs exposed to PMN using the “Co-culture” model (see Figure S1c) for 0min and (D) 60min, representative images from two independent experiments. Hypoxyprobe-1 adduct staining (red), ZO-1 (green) and nuclei (blue). Scale bar = 200μm. (E) Nuclear accumulation of HIF-1α protein was assayed by Mesoscale ELISA, following exposure of epithelia to activated PMN by “Co-culture” model. Data are represented as means ± SEM and are pooled from three independent experiments (n = 3; P < 0.001 by two-way ANOVA). (F) IECs transfected with HRE-firefly and SV40-renilla Luciferase reporters and subsequently exposed to activated PMN, indicated increased HIF activity Data are represented as means ± SEM and are pooled from three independent experiments (n = 3; P < 0.001 by two-way ANOVA). (G) IECs transfected with HRE-firefly and SV40-renilla luciferase reporters and subsequently exposed to pre-treated PMN in the presence of fMLF. Data are represented as means ± SEM and are pooled from three independent experiments (n = 3; P < 0.05 for ± PMN; P < 0.01 for PMN versus PMN+DPI; one-way ANOVA; DPI = diphenyleneiodonium). (H) HRE-transfected Caco-2 IECs, with subsequent exposure to activated PMN from wild-type or CGD mice. Data are represented as means ± SEM and are pooled from three independent experiments (n = 3; P < 0.001 for no PMN versus wild-type PMN; P > 0.05 for no PMN versus PMN+DPI; P > 0.05 for no PMN versus CGD PMN; one-way ANOVA). See also Figure S2.