(450) Pro-inflammatory Orai1 activity is elevated in CF patients regardless of Trikafta treatment

Background: Orai1 is a plasma membrane Ca²⁺ channel that is involved in store operated calcium entry (SOCE). In pulmonary cells, SOCE regulates gene expression and stimulates cytokine, mucin, and protease secretion. Activation of Orai1/SOCE results in the recruitment of neutrophils to the lungs. Orai1 activation is also upstream of transcription factors like Nuclear Factor of Activated T cells, which facilitate the onset of inflammation. In cystic fibrosis (CF), the immune response is dysregulated and the lung is chronically inflamed. However, Orai1 expression in the CF lung is poorly understood. Orai1 is a promising target for drug development, so we tested the hypothesis that Orai1 was upregulated in CF lungs.

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, CF and asthma (disease control) donors (4M/4F per group). We imaged sections by confocal and super resolution microscopy, and analyzed Orai1 and STIM1 expression, colocalization, and particle size in different pulmonary cell types.

Results: Orai1 was broadly-expressed throughout the lung, but expression was greatest in immune cells. At mRNA and protein levels, there were no consistent trends in expression levels between the three groups. Orai1 must interact with STIM1 in order to activate SOCE. We therefore used STIM1/Orai1 colocalization as a marker of Orai1 activity. Using this approach, we found significantly increased colocalization between these proteins in both CF and asthma lung epithelia (CF, 50%; asthma, 54%; normal, 15%), interstitia (CF, 57%; asthma, 49%; normal, 16%) and luminal immune cells (CF, 66%; asthma, 70%; normal, 38%). Orai1 also aggregates as part of its interaction process. Using super resolution microscopy, we found significantly more Orai1 and STIM1 aggregation in immune cells from CF and asthmatic lungs (average Orai1 particle size: CF, 52 nm; asthma, 63 nm; normal, 28 nm; average STIM1 particle size: CF, 77 nm; asthma, 59 nm; normal, 14 nm). We also looked at Orai1 in peripheral blood neutrophils from normal and CF donors (5 per group). All CF subjects took Trikafta, but under baseline conditions, there were significantly bigger puncta in CF neutrophils (CF, 10 nm; normal, 6 nm), suggesting that these patients continued to have significant inflammation despite taking Trikafta. Indeed, the mean % predicted FEV₁ in our CF cohort was 55±22%, indicating that these patients had persistent lung disease.

Conclusions: This is the first comprehensive analysis of Orai1 and STIM1 expression in lungs from normal and CF donors. We found evidence that Orai1 was more active in CF than normal lungs. This novel application of super resolution microscopy has the potential to be used in clinical settings for the analysis of ex vivo patient samples and to evaluate inflammation in CF patients. Although traditional biomarkers of inflammation, such as serum cytokine levels, are useful for rapid detection of systemic inflammation, our technique allows for the precise localization of upstream inflammatory signaling biomarkers at the cellular level and in fixed samples. Therefore, these data suggest Orai1 has a key role in CF lung inflammation and attest to the potential of anti-inflammatory therapeutics that target Orai1.

Acknowledgements: This work was supported by Cystic Fibrosis Foundation grants TARRAN02I0 and WRENNA21F0.

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