Fig 2: The impact of sevoflurane on heme oxygenase-1 (HO-1) modulation during acute pulmonary inflammation. (A) Gene, and (B) protein expression of HO-1 was evaluated in the pulmonary tissue three and 24 h after lipopolysaccharide (LPS) exposure. The effects of sevoflurane on HO-1 gene expression were examined by real-time polymerase chain reaction (RT-PCR). HO-1 protein expression (green) in the lung tissue of wild-type animals was evaluated by immunofluorescence staining at indicated conditions (original magnification ×63; one representative image of three independent experiments is shown; n = 3). DAPI (blue) was used as nuclear marker. Mean fluorescence intensity (MFI) of HO-1 with or without LPS exposure was measured by using ImageJ (n = 6–8). (C) Adenosine receptor A2B (Adora2b) gene expression was determined in the lung tissue after sevoflurane treatment, respectively, sevoflurane and hemin combination three hours after LPS exposure (n = 6–8). (D) Immunofluorescence staining of Adora2b surface expression (green) in lung tissue of wild-type animals was evaluated at indicated conditions (original magnification ×63; one representative image of three independent experiments is shown; n = 3). DAPI (blue) was used as a nuclear marker. MFI of Adora2b surface expression was measured at indicated conditions by using ImageJ (n = 6–8). (E) The impact of HO-1 activation or inhibition on sevoflurane effects on polymorphonuclear neutrophil (PMN) migration 24 h after lipopolysaccharide (LPS) stimulation was detected (n = 8). (F) Surface expression of CD11a/CD18 (also known as lymphocyte function-associated antigen-1; LFA-1), and (G) CD162 (also called P-selectin glycoprotein 1; PSGL1) was evaluated on human PMNs after LPS exposure. The impact of sevoflurane and heme oxygenase modulation on the presence of CD11a/CD18 and CD162 (mean fluorescence intensity; MFI) on human PMNs was assessed by flow cytometry (n = 6–8). Statistical analyses were performed by one-way ANOVA + Bonferroni correction or Kruskal-Wallis test. Data are presented as mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 3: CD18 activation mechanisms are Duox-independent. Duoxa knockout mice (−/−) and wild-type mice (+/+) were subjected to injurious positive pressure ventilation (PPV) or instilled intratracheally with lipopolysaccharide (LPS) followed by isolation of neutrophils from bronchoalveolar lavage (BAL) by flow cytometry using Ly6G antibody and subsequently phenotyped using CD18/CD11b antibodies. (A) Percentage of CD11b+ neutrophils isolated from BAL fluid in wild-type mice with no exposure (NV), injurious positive pressure ventilation (PPV), or intratracheal LPS (LPS IT) (*, p = .029; **, p = .0009). (B) Mean fluorescence intensity (MFI) of Ly6G+, CD18/CD11b+ neutrophils in PPV- or LPS-exposed mice compared to non-ventilated, untreated controls (*, p = .025, **, p = .003). (C) Populations of Ly6G+ (y-axis), CD11b+ (x-axis) neutrophils in Duoxa knockout (−/−) compared to wild-type (+/+) mice after exposure to PPV or LPS. (D) MFI of BAL neutrophils gated for Ly6G+ and CD18/CD11b+ by flow cytometry isolated from wild-type (+/+) and Duoxa knockout (−/−) mice in non-ventilated, untreated (NV) controls compared to PPV- and LPS-exposed mice. (***, p = .002; ****, p < .001).

Fig 4: Injurious positive pressure ventilation induces CD18-independent mechanisms that is Duox-dependent. Duoxa knockout mice (−/−) and wild-type mice (+/+) were subjected to injurious positive pressure ventilation (PPV) or instilled intratracheally with lipopolysaccharide (LPS) followed by isolation of neutrophils from bronchoalveolar lavage (BAL) by flow cytometry using Ly6G antibody and subsequently phenotyped using CD49d antibodies. (A) Mean fluorescence intensity (MFI) of BAL neutrophils gated for Ly6G+ and CD49b by flow cytometry isolated from wild-type (+/+) mice in non-ventilated, untreated (NV) controls compared to PPV- and LPS-exposed mice. (**, p = .005). (B) Fold change of MFI for Ly6G+, CD49d+ neutrophils isolated from BAL fluid in non-ventilated, untreated wild-type (+/+) and Duoxa knockout (−/−) mice compared to PPV- and LPS-treated mice. CD49d+ expression was higher in wild-type versus knockout mice only in the PPV group (p = .0079). (C) MFI of Ly6G+ CD49d+ cells was correlated to absolute neutrophils in bronchoalveolar lavage in mice exposed to PPV. There was a strong correlation seen in wild-type mice (R 2 = 0.94, p = .006) that was not observed in Duoxa knockout mice.