UVM Theses and Dissertations
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Author:
Olson, Nels C.
Dept./Program:
Cell and Molecular Biology Program
Year:
2011
Degree:
Ph. D.
Abstract:
Enhanced production of nitric oxide (NO) by airway epithelial nitric oxide synthase 2 (NOS2) is a characteristic feature of allergic airway inflammation. NO can promote inflammation and injury by conversion to more reactive intermediates, whereas its anti-inflammatory properties include inhibition of nuclear factor (NF)-kB, a central regulator of allergic airway inflammation. NO can also regulate the activity of hypoxiainducible factor-1 (HIF-1), a transcription factor that plays important roles in inflammation and epithelial cell function.
Using immortalized human bronchial (16HBE14o- ) and primary mouse tracheal (MTE) epithelial cells, we first sought to elucidate the role of NOS2 on epithelial barrier integrity. We demonstrated that high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased epithelial permeability to FITC-dextran, but barrier disruption induced by inflammatory cytokines occurred independent of NOS2. Conversely, cellular pre-incubation with LPS was found to enhance epithelial wound repair and TER recovery in response to mechanical injury in a NOS2-dependent manner.
In order to determine the contributions of endogenous NOS2 on the regulation of NF-kB and inflammation during allergic airway disease, NOS2-/-mice were utilized in the ovalbumin (OVA) model. Activation of NF-kB was found to be increased in NOS2-/mice, however, no significant differences between markers of allergic inflammation were observed in comparison to OVA challenged WT mice. We also tested the hypothesis that the induction of NOS2 contributes to HIF-1 activation during allergic airway inflammation in vivo. Our results showed that while HIF-1 was activated in response to OVA sensitization and challenge, this response was independent of NOS2.
We next determined the consequences of HIF-1 activation on airway epithelial barrier function in an in vitro model of oxidant-induced epithelial injury. Cellular exposure to H₂O₂ resulted in significant loss of TER and increased permeability to FITC-dextran, which was associated with reduced levels of the tight junction protein occludin and increased overoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO₂H). Using short hairpin RNA (shRNA) to knock down HIF-1a, we demonstrated that pre-activation of HIF-1 by CoC1₂ delays or prevents barrier dysfunction and restores levels of occludin and reduced peroxiredoxin.
Finally, we tested the use of S-nitrosoglutathione (GSNO) as a therapeutic for allergic airway inflammation, hypothesizing it would inhibit pro-inflammatory signaling of NF-kB. Results from these studies demonstrated that instillation of GSNO inhibits NF-kB activity during allergic airway inflammation, but does not significantly effect overall markers of inflammation or mucus metaplasia. Collectively, these results demonstrated that induction of epithelial NOS2 is beneficial in conditions of epithelial injury by promoting wound repair and the restoration of epithelial integrity. Activation of NOS2 during allergic airway inflammation does not contribute to HIF-1 activation or to the overall inflammatory response, however it can serve to regulate the activation of NF-kB. Our results also demonstrated that airway epithelial HIF-1 serves a protective role against oxidant-induced epithelial injury which may have important consequences during airway inflammation.
Using immortalized human bronchial (16HBE14o- ) and primary mouse tracheal (MTE) epithelial cells, we first sought to elucidate the role of NOS2 on epithelial barrier integrity. We demonstrated that high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased epithelial permeability to FITC-dextran, but barrier disruption induced by inflammatory cytokines occurred independent of NOS2. Conversely, cellular pre-incubation with LPS was found to enhance epithelial wound repair and TER recovery in response to mechanical injury in a NOS2-dependent manner.
In order to determine the contributions of endogenous NOS2 on the regulation of NF-kB and inflammation during allergic airway disease, NOS2-/-mice were utilized in the ovalbumin (OVA) model. Activation of NF-kB was found to be increased in NOS2-/mice, however, no significant differences between markers of allergic inflammation were observed in comparison to OVA challenged WT mice. We also tested the hypothesis that the induction of NOS2 contributes to HIF-1 activation during allergic airway inflammation in vivo. Our results showed that while HIF-1 was activated in response to OVA sensitization and challenge, this response was independent of NOS2.
We next determined the consequences of HIF-1 activation on airway epithelial barrier function in an in vitro model of oxidant-induced epithelial injury. Cellular exposure to H₂O₂ resulted in significant loss of TER and increased permeability to FITC-dextran, which was associated with reduced levels of the tight junction protein occludin and increased overoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO₂H). Using short hairpin RNA (shRNA) to knock down HIF-1a, we demonstrated that pre-activation of HIF-1 by CoC1₂ delays or prevents barrier dysfunction and restores levels of occludin and reduced peroxiredoxin.
Finally, we tested the use of S-nitrosoglutathione (GSNO) as a therapeutic for allergic airway inflammation, hypothesizing it would inhibit pro-inflammatory signaling of NF-kB. Results from these studies demonstrated that instillation of GSNO inhibits NF-kB activity during allergic airway inflammation, but does not significantly effect overall markers of inflammation or mucus metaplasia. Collectively, these results demonstrated that induction of epithelial NOS2 is beneficial in conditions of epithelial injury by promoting wound repair and the restoration of epithelial integrity. Activation of NOS2 during allergic airway inflammation does not contribute to HIF-1 activation or to the overall inflammatory response, however it can serve to regulate the activation of NF-kB. Our results also demonstrated that airway epithelial HIF-1 serves a protective role against oxidant-induced epithelial injury which may have important consequences during airway inflammation.