Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma
Sara C. Sebag, Olha M. Koval, John D. Paschke, Christopher J. Winters, Omar A. Jaffer, Ryszard Dworski, Fayyaz S. Sutterwala, Mark E. Anderson, Isabella M. Grumbach
Sara C. Sebag, Olha M. Koval, John D. Paschke, Christopher J. Winters, Omar A. Jaffer, Ryszard Dworski, Fayyaz S. Sutterwala, Mark E. Anderson, Isabella M. Grumbach
View: Text | PDF
Research Article Inflammation Pulmonology

Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma

Abstract

Excessive ROS promote allergic asthma, a condition characterized by airway inflammation, eosinophilic inflammation, and increased airway hyperreactivity (AHR). The mechanisms by which airway ROS are increased and the relationship between increased airway ROS and disease phenotypes are incompletely defined. Mitochondria are an important source of cellular ROS production, and our group discovered that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is present in mitochondria and activated by oxidation. Furthermore, mitochondrial-targeted antioxidant therapy reduced the severity of allergic asthma in a mouse model. Based on these findings, we developed a mouse model of CaMKII inhibition targeted to mitochondria in airway epithelium. We challenged these mice with OVA or Aspergillus fumigatus. Mitochondrial CaMKII inhibition abrogated AHR, inflammation, and eosinophilia following OVA and A. fumigatus challenge. Mitochondrial ROS were decreased after agonist stimulation in the presence of mitochondrial CaMKII inhibition. This correlated with blunted induction of NF-κB, the NLRP3 inflammasome, and eosinophilia in transgenic mice. These findings demonstrate a pivotal role for mitochondrial CaMKII in airway epithelium in mitochondrial ROS generation, eosinophilic inflammation, and AHR, providing insights into how mitochondrial ROS mediate features of allergic asthma.

Authors

Sara C. Sebag, Olha M. Koval, John D. Paschke, Christopher J. Winters, Omar A. Jaffer, Ryszard Dworski, Fayyaz S. Sutterwala, Mark E. Anderson, Isabella M. Grumbach

×

Figure 2

Mitochondrial CaMKII inhibition in respiratory epithelium attenuates airway hyperreactivity.

Options: View larger image (or click on image) Download as PowerPoint
Mitochondrial CaMKII inhibition in respiratory epithelium attenuates air...
(A–C) Immunofluorescence for GFP (green), HA-tagged Mt-CaMKIIN (red), and nuclei (TO-PRO-3, blue) in lung sections from (A) control CC10.CreRT2 mice (WT), (B) Mt-CaMKIIN mice without tamoxifen (TAM) treatment, or (C) Mt-CaMKIIN mice after tamoxifen treatment. Scale bar: 100 μm. (D) Immunoblots for HA-tagged Mt-CaMKIIN in mitochondrial and cytoplasmic fractions from lungs of WT and Mt-CaMKIIN mice without and with tamoxifen treatment. Lungs were harvested 10 days after the last administration of tamoxifen. (E) Immunoblots for ox-CaMKII in mitochondrial fractions from lungs of WT and Mt-CaMKIIN mice treated with OVA or saline control. Densitometric analysis of signal was normalized to COXIV control and then represented as percentage ox-CaMKII from WT-OVA (n = 4) versus Mt-CaMKIIN OVA (n = 3). Student’s 2-tailed t test used. *P < 0.05 vs. WT-OVA. (F) Representative images and quantification of mitochondrial ROS production in primary murine tracheal epithelial cells (MTBEC) isolated from WT or Mt-CaMKIIN mice after exposure to saline control (n = 3) or OVA (n = 6). Mitochondrial ROS production was determined with mitoSOX (red); MitoTracker (blue) was used to colocalize mitochondria. Data were quantified for 5–10 images per treatment. Scale bar: 100 μm. Two-way ANOVA with Tukey post-hoc test was used. *P < 0.05 vs. control; #P < 0.05 vs. WT mice with OVA. (G) Airway hyperreactivity (AHR) in mice exposed to A. fumigatus (Asp) or control (Freund adjuvant alone) and challenged with increasing doses of methacholine (n = 6 WT control; 6 Mt-CaMKIIN control; 8 WT A. fumigatus; 8 Mt-CaMKIIN A. fumigatus–treated mice). (H) AHR in mice exposed to OVA or saline control and challenged with increasing doses of methacholine (n = 8 WT control; 9 Mt-CaMKIIN control; 10 WT OVA; and 12 Mt-CaMKIIN OVA–treated mice). Two-way ANOVA with Tukey post-hoc test was used. *P < 0.05 vs. control; #P < 0.05 vs. WT mice with A. fumigatus or OVA exposure.