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Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING

Pediatric Research volume 96, pages 1666–1672 (2024)Cite this article

Abstract

Background

The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs.

Methods

We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21).

Results

We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs.

Conclusion

Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH.

Impact statement

We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

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Fig. 1: Nitrofen exposure leads to dysregulation of cGAS and STING and higher dsDNA concentration in human bronchial epithelial cells (BEAS-2B).
Fig. 2: cGAS and STING protein abundance is dysregulated in lungs of nitrofen treated rat pups.
Fig. 3: cGAS and STING mRNA expression is dysregulated in lungs of nitrofen treated rat pups.
Fig. 4: Potential relevance of DNA sensing on pathogenesis of CDH and other congenital malformations.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Funding

MM was the holder of a scholarship (MA 8982/1-1) from the “Deutsche Forschungsgemeinschaft e.V.” (DFG - German Research Foundation) during his work on this study; RK is supported by project grants (148797, 178347 and 178387) from the Canadian Institutes of Health Research and is the inaugural Thorlakson Chair in Surgical Research for the University of Manitoba. MJ is a holder of a scholarship (JA 3507/1-1) from the “Deutsche Forschungsgemeinschaft e.V.”.

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Authors and Affiliations

  1. Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany

    Moritz Markel, Martin Lacher & Richard Wagner

  2. Division of Pediatric Surgery, Departments of Surgery, Pediatrics & Child Health and Physiology & Pathophysiology, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, and Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada

    Moritz Markel, Wai Hei Tse, Nolan De Leon, Marietta Jank, Jaida Albrechtsen, Shana Kahnamoui Zadeh, Daywin Patel, Arzu Ozturk & Richard Keijzer

  3. Department of Pediatric Surgery, Medical Faculty Mannheim, University of Medical Center Mannheim, Heidelberg University, Heidelberg, Germany

    Marietta Jank

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Contributions

MM, WHT, RW and RK: substantial contributions to conception and design. MM, WHT, NDL, SKZ, JA, SH, DP, AO, MJ, RK: acquisition of data, or analysis and interpretation of data. MM, WHT, NDL, AO, MJ, RW, ML, RK: drafting the article or revising it critically for important intellectual content. All authors: Final approval of the version to be published.

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Correspondence to Richard Keijzer.

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Markel, M., Tse, W.H., De Leon, N. et al. Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING. Pediatr Res 96, 1666–1672 (2024). https://doi.org/10.1038/s41390-024-03277-2

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