Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma
Sara C. Sebag, … , Mark E. Anderson, Isabella M. Grumbach
Sara C. Sebag, … , Mark E. Anderson, Isabella M. Grumbach
Published February 9, 2017
Citation Information: JCI Insight. 2017;2(3):e88297. https://doi.org/10.1172/jci.insight.88297.
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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

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Figure 1

CaMKII in mitochondria of respiratory epithelial cells regulates mitochondrial ROS and IL-5 expression.

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CaMKII in mitochondria of respiratory epithelial cells regulates mitocho...
(A) Representative images and quantification of mitochondrial ROS production in primary murine tracheal epithelial cells (MTBEC) isolated from WT mice after exposure to saline control (n = 6) or OVA (n = 15). Mitochondrial ROS production was determined with mitoSOX (red); MitoTracker (green) was used to colocalize mitochondria. Data were quantified for 10 images per treatment. Scale bar: 100 μm. (B) Immunoblots of active, oxidized, and total CaMKII in mitochondria from lungs of mice exposed to saline or OVA (n = 3–5). CF, cytoplasmic fraction; MF, mitochondrial fraction; MTCO2, mitochondrial protein marker; GAPDH, cytoplasmic protein marker. (C) Immunoblots of active oxidized and total CaMKII in mitochondria in mitochondrial and cytoplasmic fractions of primary human airway epithelial cells (HAEC) treated with media only (control) or IL-13 (10 ng/ml) for 48 hours (n = 3 independent experiments). COXIV, mitochondrial marker. (D) Representative images and quantification of mitochondrial ROS production in HAEC infected with Mt-GFP (empty) or Mt-CaMKIIN virus prior to exposure to IL-13 (10 ng/ml) for 2 days (n = 6 independent experiments; data were quantified for 10 images per treatment and represented as mean fluorescent intensity [MFI]). Scale bar: 100 μm. (E) ROS production by lucigenin assay in isolated mitochondria from HAEC treated as in D (n = 9 independent experiments). In F–I, triangles represent Mt-GFP, circles represent Mt-CaMKIIN. (F and G) IL-5 mRNA levels in HAEC or MTBEC (respectively) infected with Mt-GFP or Mt-CaMKIIN and treated as in D. Data in F are presented as the percentage change relative to Mt-GFP+IL-13 (n = 3 independent experiments). Data in G are presented as the fold change relative to Mt-GFP control (n = 3 independent experiments). (H) TSLP and (I) eotaxin mRNA levels in HAEC infected with Mt-GFP or Mt-CaMKIIN and treated as in D (n = 3–4 independent experiments). For A–C and F, Student’s 2-tailed t test; for D and E and G–I, 1-way ANOVA with Tukey post-hoc test. *P < 0.05 vs. control, #P < 0.05 vs. Mt-GFP+IL-13.