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Novel Roles for Notch3 and Notch4 Receptors in Gene Expression and Susceptibility to Ozone-Induced Lung Inflammation in Mice.

Verhein KC, McCaw Z, Gladwell W, Trivedi S, Bushel PR, Kleeberger SR - Environ. Health Perspect. (2015)

Bottom Line: We hypothesized that Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation.Relative to air-exposed controls, ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared with WT and Notch3-/- mice.These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Resources (DHHS), Research Triangle Park, North Carolina, USA.

ABSTRACT

Background: Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized that Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation.

Methods: Wild-type (WT), Notch3 (Notch3-/-), and Notch4 (Notch4-/-) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6-72 hr.

Results: Relative to air-exposed controls, ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared with WT and Notch3-/- mice. Significantly greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared with WT mice after ozone exposure. Expression of whole lung Tnf was significantly increased after ozone in Notch3-/- and Notch4-/- mice, and was significantly greater in Notch3-/- compared with WT mice. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member.

Conclusions: These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.

No MeSH data available.


Related in: MedlinePlus

Blocking TNF signaling with etanercept reduced airway neutrophilia in Notch3 knockout mice. Prior to 24‑hr ozone exposure, mice were treated with a TNF inhibitor (etanercept, 10 mg/kg i.p.) or vehicle (human IgG, 10 mg/kg i.p.). (A) Etanercept pretreatment did not affect BALF protein concentration. (B) Etanercept reduced the ozone-induced influx of neutrophils in Notch3–/– mice. n = 5–10 mice per group.*p < 0.05 compared with respective air controls, and #p < 0.05 between etanercept and vehicle after ozone exposure, by 2-way ANOVA with Tukey’s post hoc tests.
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f3: Blocking TNF signaling with etanercept reduced airway neutrophilia in Notch3 knockout mice. Prior to 24‑hr ozone exposure, mice were treated with a TNF inhibitor (etanercept, 10 mg/kg i.p.) or vehicle (human IgG, 10 mg/kg i.p.). (A) Etanercept pretreatment did not affect BALF protein concentration. (B) Etanercept reduced the ozone-induced influx of neutrophils in Notch3–/– mice. n = 5–10 mice per group.*p < 0.05 compared with respective air controls, and #p < 0.05 between etanercept and vehicle after ozone exposure, by 2-way ANOVA with Tukey’s post hoc tests.

Mentions: A potential mechanism through which Notch3 and Notch4 protect against ozone-induced inflammation is by modulation of Tnf expression. TNF-α is increased during ozone-induced airway inflammation, and TNF inhibition reduces neutrophilia (Cho et al. 2001; Shore et al. 2001). To determine whether TNF-α mediates exacerbated inflammation in Notch-deficient mice, we treated WT, Notch3–/–, and Notch4–/– mice with the TNF-α inhibitor etanercept before ozone exposure. Etanercept had no effect on BALF protein concentration (Figure 3A), as predicted because antibodies to TNF, as well as targeted deletion of TNF receptors and Tnf, also had no effect on lung permeability (Cho et al. 2001). In WT mice, neutrophils were significantly increased in BALF after ozone exposure with IgG/vehicle treatment and were not significantly increased after etanercept treatment (Figure 3B). However, in Notch3–/– mice, etanercept significantly reduced neutrophils compared with IgG controls (p = 0.042) but had no effect on neutrophil influx in Notch4–/–, even though Tnf gene expression was increased in these mice (Figure 3B). Our results therefore support a TNF-α–mediated role for Notch3 but not Notch4 in protection against ozone-induced inflammation, and suggest that the mechanism behind increased susceptibility is different for mice with targeted deletion of Notch3 and Notch4. The link between TNF-α and Notch signaling likely occurs through NF-κB in a cell type– and Notch receptor– specific manner. Positive and negative regulation of NF-κB by Notch receptors has been demonstrated previously. For example, Notch1 and Notch2 negatively regulate Toll-like receptor (TLR) signaling in macrophages by inhibiting NF-κB transcription activity (Zhang et al. 2012). In contrast, Notch1 signaling activates NF-κB by suppressing an inhibitor of IκB kinase in T-cell acute lymphoblastic leukemia cells (Espinosa et al. 2010). TNF-α also regulates Notch signaling. In rheumatoid synovial fibroblasts, TNF-α stimulation induced expression of Notch1 and Notch4 and nuclear translocation of the intracellular domain (Ando et al. 2003). Additional investigations are necessary to understand the specific mechanistic interaction of TNF-α and Notch3 signaling in response to ozone.


Novel Roles for Notch3 and Notch4 Receptors in Gene Expression and Susceptibility to Ozone-Induced Lung Inflammation in Mice.

Verhein KC, McCaw Z, Gladwell W, Trivedi S, Bushel PR, Kleeberger SR - Environ. Health Perspect. (2015)

Blocking TNF signaling with etanercept reduced airway neutrophilia in Notch3 knockout mice. Prior to 24‑hr ozone exposure, mice were treated with a TNF inhibitor (etanercept, 10 mg/kg i.p.) or vehicle (human IgG, 10 mg/kg i.p.). (A) Etanercept pretreatment did not affect BALF protein concentration. (B) Etanercept reduced the ozone-induced influx of neutrophils in Notch3–/– mice. n = 5–10 mice per group.*p < 0.05 compared with respective air controls, and #p < 0.05 between etanercept and vehicle after ozone exposure, by 2-way ANOVA with Tukey’s post hoc tests.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4529014&req=5

f3: Blocking TNF signaling with etanercept reduced airway neutrophilia in Notch3 knockout mice. Prior to 24‑hr ozone exposure, mice were treated with a TNF inhibitor (etanercept, 10 mg/kg i.p.) or vehicle (human IgG, 10 mg/kg i.p.). (A) Etanercept pretreatment did not affect BALF protein concentration. (B) Etanercept reduced the ozone-induced influx of neutrophils in Notch3–/– mice. n = 5–10 mice per group.*p < 0.05 compared with respective air controls, and #p < 0.05 between etanercept and vehicle after ozone exposure, by 2-way ANOVA with Tukey’s post hoc tests.
Mentions: A potential mechanism through which Notch3 and Notch4 protect against ozone-induced inflammation is by modulation of Tnf expression. TNF-α is increased during ozone-induced airway inflammation, and TNF inhibition reduces neutrophilia (Cho et al. 2001; Shore et al. 2001). To determine whether TNF-α mediates exacerbated inflammation in Notch-deficient mice, we treated WT, Notch3–/–, and Notch4–/– mice with the TNF-α inhibitor etanercept before ozone exposure. Etanercept had no effect on BALF protein concentration (Figure 3A), as predicted because antibodies to TNF, as well as targeted deletion of TNF receptors and Tnf, also had no effect on lung permeability (Cho et al. 2001). In WT mice, neutrophils were significantly increased in BALF after ozone exposure with IgG/vehicle treatment and were not significantly increased after etanercept treatment (Figure 3B). However, in Notch3–/– mice, etanercept significantly reduced neutrophils compared with IgG controls (p = 0.042) but had no effect on neutrophil influx in Notch4–/–, even though Tnf gene expression was increased in these mice (Figure 3B). Our results therefore support a TNF-α–mediated role for Notch3 but not Notch4 in protection against ozone-induced inflammation, and suggest that the mechanism behind increased susceptibility is different for mice with targeted deletion of Notch3 and Notch4. The link between TNF-α and Notch signaling likely occurs through NF-κB in a cell type– and Notch receptor– specific manner. Positive and negative regulation of NF-κB by Notch receptors has been demonstrated previously. For example, Notch1 and Notch2 negatively regulate Toll-like receptor (TLR) signaling in macrophages by inhibiting NF-κB transcription activity (Zhang et al. 2012). In contrast, Notch1 signaling activates NF-κB by suppressing an inhibitor of IκB kinase in T-cell acute lymphoblastic leukemia cells (Espinosa et al. 2010). TNF-α also regulates Notch signaling. In rheumatoid synovial fibroblasts, TNF-α stimulation induced expression of Notch1 and Notch4 and nuclear translocation of the intracellular domain (Ando et al. 2003). Additional investigations are necessary to understand the specific mechanistic interaction of TNF-α and Notch3 signaling in response to ozone.

Bottom Line: We hypothesized that Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation.Relative to air-exposed controls, ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared with WT and Notch3-/- mice.These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Resources (DHHS), Research Triangle Park, North Carolina, USA.

ABSTRACT

Background: Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized that Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation.

Methods: Wild-type (WT), Notch3 (Notch3-/-), and Notch4 (Notch4-/-) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6-72 hr.

Results: Relative to air-exposed controls, ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared with WT and Notch3-/- mice. Significantly greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared with WT mice after ozone exposure. Expression of whole lung Tnf was significantly increased after ozone in Notch3-/- and Notch4-/- mice, and was significantly greater in Notch3-/- compared with WT mice. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member.

Conclusions: These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation.

No MeSH data available.


Related in: MedlinePlus