Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Hyperoxia impairs alveolar macrophage-mediated efferocytosis, and Nrf2-deficiency worsens it in vivo. Nrf2 +/+ (WT) and Nrf2 −/− mice ( n = 3 per group) were exposed to room air or hyperoxia for 48 h, and a set of hyperoxia-exposed mice were allowed to recover at room air for 72 as outlined in schema. ( a ) BAL from these mice was obtained, and macrophages were incubated on cover glasses with apoptotic neutrophils for 1 h. Macrophages were washed to remove unbound/non-internalized apoptotic cells and stained with Diff Quick stain. Images were captured to quantify apoptotic neutrophil binding, and internalization (indicated by red arrows). ( b ) Representative images of macrophages with neutrophils for each experimental condition are shown. ( c ) The number of apoptotic neutrophils either bound or internalized by macrophages from ~5–9 fields were quantified and expressed as % efferocytosis. Versus room air of respective genotypes, † Nrf2 −/− versus WT counterparts, § hyperoxia vs. recovery; †† p < 0.01; ****/§§§§/†††† p < 0.0001. Purple, room air group; Red, hyperoxia group; Green, hyperoxia and recovery.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: In Vivo, Incubation, Staining, Diff-Quik, Binding Assay

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 is required for optimal macrophage-mediated efferocytosis. BMDMФs from Nrf2 +/+ (WT) and Nrf2 −/− mice were cultured and exposed to hyperoxia for 24 h. ( a ) Macrophages (green) were then incubated with labeled apoptotic neutrophils (red) for 1 h, washed, and captured images. ( b ) Macrophages were incubated with green fluorescent beads. Representative images of macrophages with neutrophils or beads for each genotype and experimental condition are shown. Values represent from at least three independent samples. * Room air versus hyperoxia of respective genotypes, †, Nrf2 −/− versus WT counterparts. * p < 0.05; **/†† p < 0.01; **** p < 0.0001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Cell Culture, Incubation, Labeling

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2-deficiency in neutrophils does not affect their engulfment by macrophages. Neutrophils from the bone marrow of wild-type (WT) and Nrf2 −/− mice were isolated, labelled, and subjected to apoptosis as detailed in methods. Apoptotic neutrophils (red) were incubated with WT BMDMФs and Nrf2 −/− BMDMФs for 1 h, and efferocytosis was quantified. ( a ) Representative images of macrophages with apoptotic neutrophils (red). The blue color represents DAPI. ( b ) quantification of efferocytosis. Values are from 3–4 fields of two independent samples. † p < 0.05; Nrf2 −/− versus WT counterparts. Blue, room air group; pink hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Isolation, Incubation

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 regulates apoptotic neutrophil binding to the macrophages. Nrf2 +/+ (WT) and Nrf2 −/− BMDMФs were exposed to room air or hyperoxia and then incubated with cytochalasin D (CtyD) before adding the labeled apoptotic neutrophils. Efferocytosis was quantified. ( a ) Both bound/attached and internalized apoptotic cells in the presence and absence of CytD were enumerated, and data are represented % efferocytosis. * hyperoxia versus room air of respective genotypes, †, Nrf2 −/− versus WT counterparts; § DMSO versus CtyD of respective genotypes. */† p < 0.05; **/§§ p < 0.01; ***/†††/§§§ p < 0.001; §§§§ p < 0.0001. Blue, room air group; Red, hyperoxia group. ( b ) Both bound and internalized apoptotic cells were enumerated by comparing values of respective CytD (bound) and DMSO-treated (bound and internalized) samples, * Hyperoxia versus room air of respective genotypes, and §, Nrf2 −/− versus WT versus counterparts. Values are from three independent samples. * p < 0.05; **/§§ p < 0.01; §§§ p < 0.001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Binding Assay, Incubation, Labeling

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 activation augments efferocytosis in hyperoxia exposed BMDMΦs. Nrf2 +/+ and Nrf2 −/− BMDMΦs were pretreated with CDDO-Im (20 nM) for 6 h and exposed to hyperoxia for 24 h along with CDDO-Im. After hyperoxia, BMDMΦs were incubated with CellTracker Red stained apoptotic neutrophils for 1 hour. Images were captured, and % efferocytosis was enumerated. Values are at least from three independent samples. * Hyperoxia versus room air of respective genotypes, † versus WT counterparts, and § DMSO versus CDDO of respective genotypes. § p < 0.05; ****/††††/§§§§ p < 0.0001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Activation Assay, Incubation, Staining

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: Changes in body weight after neonatal hyperoxic exposure. The changes in body weight were evaluated in 4 and 14-day-old WT and TRX-Tg mice following normoxic or hyperoxic exposure ( n = 6 per group). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01. WT: wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Air: Normoxia; O 2 : Hyperoxia; SEM: Standard error of the mean; ANOV: Analysis of variance.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Transgenic Assay

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: Alveolar development on day four after neonatal hyperoxic exposure. ( A ) H&E-stained histological sections. Scale bar = 100 µm (magnification × 100). ( B ) Lm ( n = 6 per group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O 2 (filled bars). ( C ) Elastin-stained histological sections. The black arrows indicate elastin-positive secondary septa. Scale bar = 50 µm (magnification × 200). ( D ) Number of secondary septa ( n = 6 per group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O 2 (filled bars). Data are shown as the mean ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01. Air: Normoxia; O 2 : Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Lm: Mean linear intercept length; H&E: Hematoxylin and eosin; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Staining, Transgenic Assay

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: Alveolar development during recovery from neonatal hyperoxic exposure on day 14. ( A ) H&E-stained histological sections. Scale bar = 100 µm (magnification ×100). ( B ) Lm ( n = 6, per group). Lm was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O 2 (filled bars). ( C ) Elastin-stained histological sections. The black arrows indicate elastin-positive secondary septa. Scale bar = 50 µm (magnification × 200). ( D ) Number of secondary septa ( n = 6 per group). The number of secondary septa was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O 2 (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. Air: Normoxia; O 2 : Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; Lm: Mean linear intercept length; H&E: Hematoxylin and eosin; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Staining, Transgenic Assay

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: Changes in mRNA expression levels of Il-6 , Ho-1 , Mcp-1 , Il-1β , Tnf , Cxcl1 , and Cxcl2 in lungs. Quantitative RT-PCR was performed on ( A ) day 4 and ( B ) day 14 ( n = 6 per group). Animals were exposed to air (open bars) or O 2 (filled bars). Data are shown as means ± SEM. Comparison between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. IL: Interleukin; HO: Heme oxygenase; MCP-1: Monocyte chemotactic protein-1; TNF: Tumor necrosis factor; CXCL: Chemokine (C-X-C motif) ligand; Air: Normoxia; O 2 : Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: Immunohistochemistry for macrophages. Immunohistochemistry using the macrophage marker F4/80 was performed on lung sections on ( A ) day 4 and ( C ) day 14. The black arrows indicate F4/80-positive macrophages. Scale bar = 25 μm (magnification × 400). Number of macrophages on ( B ) day 4 and ( D ) day 14 ( n = 6 per group). The number of macrophages was assessed in six nonoverlapping fields of lung parenchyma in one tissue section per animal. Animals were exposed to air (open bars) or O 2 (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05; ** p < 0.01. Air: Normoxia; O 2 : Hyperoxia; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Immunohistochemistry, Marker, Transgenic Assay

Journal: Biomedicines

Article Title: Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression

doi: 10.3390/biomedicines8030066

Figure Lengend Snippet: ( A , B ) Mif mRNA expression levels. Quantitative RT-PCR for MIF was performed on ( A ) day 4 and ( B ) day 14 ( n = 6 per group). ( C , D ) MIF protein expression levels. Western blot analysis for MIF was performed on ( C ) day 4 and ( D ) day 14. Animals were exposed to air (open bars) or O 2 (filled bars). Data are shown as means ± SEM. Comparisons between groups were performed using one-way ANOVA followed by Tukey’s test. * p < 0.05. Air: Normoxia; O 2 : Hyperoxia; MIF: Macrophage migration inhibitory factor; WT: Wildtype; TRX: Thioredoxin-1; Tg: Transgenic; SEM: Standard error of the mean; ANOVA: Analysis of variance. ( E ) Immunoprecipitation of TRX with MIF. Immunoprecipitation using normal IgG and anti-TRX antibody was analyzed by Western blot analysis. The antibodies for Western blotting were anti-MIF, anti-hTRX, and anti-β-actin. hTRX: Human thioredoxin; MIF: Macrophage migration inhibitory factor.

Article Snippet: Exposure to hyperoxia was performed for 96 h in a chamber (BioSpherix, Redfield, NY, USA) that allowed for the continuous monitoring and regulation of O 2 and CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Migration, Transgenic Assay, Immunoprecipitation