Super resolution microscopy analysis reveals increased Orai1 activity in asthma and cystic fibrosis lungs

Abstract

Question: In diseases such as asthma and cystic fibrosis (CF), the immune response is dysregulated and the lung is chronically inflamed. Orai1 activation is required for the initiation and persistence of inflammation. However, Orai1 expression in the lung is poorly understood. We therefore tested the hypothesis that Orai1 expression was upregulated in asthmatic and CF lungs.

Materials and methods: We used LungMAP to analyze single-cell RNAseq data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, asthma, and CF donors. We imaged sections by confocal and super resolution microscopy, and analyzed Orai1 and STIM1 expression in different pulmonary cell types.

Results: Orai1 was broadly-expressed, but expression was greatest in immune cells. At mRNA and protein levels, there were no consistent trends in expression levels between the three phenotypes. Orai1 must interact with STIM1 in order to activate and conduct Ca²⁺. We therefore used STIM1/Orai1 co-localization as a marker of Orai1 activity. Using this approach, we found significantly increased co-localization between these proteins in epithelia, interstitial and luminal immune cells, but not alveoli, from asthma and CF lungs. Orai1 also aggregates as part of its activation process. Using super resolution microscopy, we also found significantly increased Orai1 aggregation in immune cells from asthmatic and CF lungs.

Conclusion: We found evidence that Orai1 was more active in asthma and CF than normal lungs. These data suggest that Orai1 is a relevant target for reducing pulmonary inflammation.

Keywords: Asthma; Cystic fibrosis; Inflammation; Orai1/STIM1; RNAscope; Super resolution microscopy.

Figure 1:. Single cell RNAseq analysis indicates that Orai1 and STIM1 are broadly expressed in the lung.

(a) Reference UMAP (uniform manifold approximation and projection) depicting labeled cell type clusters. UMAP of (b) Orai1 and (c) STIM1 showing lung cell mRNA distribution. Violin plots of (d) Orai1 and (e) STIM1 mRNA frequency in select cell types. (f) UMAP showing Orai1/STIM1 co-expression in lung cell type clusters.

Figure 2:. Orai1 and STIM1 mRNA expression is upregulated in some regions of asthma and CF lung.

(a) Representative images of Orai1 (red) and STIM1 (yellow) mRNA from normal (NL), CF, and asthma lungs. I, interstitial; E, epithelia; IC, immune cell. Orai1 mRNA count in (b) epithelial, (c) interstitial, (d) alveolar, and (e) immune cells. STIM1 mRNA count in (f) epithelial, (g) interstitial, (h) alveolar, and (i) immune cells. *=p<0.05, ***=p<0.001, ****=p<0.0001. Each data point represents an analyzed region. Data shown as mean ± SD. Data were analyzed using Kruskal-Wallis ANOVA. N=8 subjects per group.

Figure 3:. Orai1 and STIM1 protein expression are not overly different between groups.

(a) Representative images of Orai1 (red) and STIM1 (yellow) protein expression in normal, asthma and CF lungs. I, interstitial; E, epithelia; IC, immune cell. Orai1 fluorescence intensity in (b) epithelial, (c) interstitial, (d) alveolar, and (e) immune cells. STIM1 fluorescence intensity in (f) epithelial, (g) interstitial, (h) alveolar, and (i) immune cells. *=p<0.05, ***=p<0.001, ****=p<0.0001. Each data point represents an analyzed region. Data shown as mean ± SD. Data were analyzed using Kruskal-Wallis ANOVA. N=8 subjects per group.

Figure 4:. Orai1/STIM1 colocalization is significantly increased in asthma and CF lungs.

(a) Representative image of the Leica SP8 colocalization software process. Orai1/STIM1 colocalization in (b) epithelial, (c) interstitial, (d) alveolar, and (e) immune cells. (f) Orai1/α-tubulin colocalization in ciliated cells. **=p<0.01, ***=p<0.001, ****=p<0.0001. Each data point represents an analyzed region. Data shown as mean ± SD. Data were analyzed using Kruskal-Wallis ANOVA. N=8 subjects per group.

Figure 5:. Orai1 puncta are significantly larger in CF and asthma immune cells, indicating increased Orai1 activation.

(a) Representative images of Orai1 puncta formation in vehicle-treated and thapsigargin-treated isolated human neutrophils from normal and CF blood. Blue, DAPI; red, Orai1. (b) Orai1 particle size (i.e. puncta) in isolated normal and CF blood neutrophils ± thapsigargin. N=5 subjects per group. (c) Representative images of Orai1 and STIM1 puncta formation in normal, asthma, and CF lung macrophages. (d) Orai1 and (e) STIM1 particle size in normal, asthma, and CF lung macrophages. (f) Orai1 particle size in NL and CF neutrophils. N=8 subjects per group. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p <0.0001. Each data point represents an individual cell. Data shown as mean ± SD. Data were analyzed using the Kruskal-Wallis ANOVA or a Mann Whitney test.