Data independent acquisition mass spectrometry of irradiated mouse lung endothelial cells reveals a STAT-associated inflammatory response

Abstract

Purpose: Pulmonary inflammation is an adverse consequence of radiation therapy in breast cancer. The aim of this study was to elucidate biological pathways leading to this pathology.

Materials and methods: Lung endothelial cells were isolated 24 h after thorax-irradiation (sham or 10 Gy X-ray) from female C57Bl/6 mice and cultivated for 6 days.

Results: Quantitative proteomic analysis of lung endothelial cells was done using data independent acquisition (DIA) mass spectrometry. The data were analyzed using Ingenuity Pathway Analysis and STRINGdb. In total, 4220 proteins were identified using DIA of which 60 were dysregulated in the irradiated samples (fold change ≥2.00 or ≤0.50; q-value <0.05). Several (12/40) upregulated proteins formed a cluster of inflammatory proteins with STAT1 and IRF3 as predicted upstream regulators. The several-fold increased expression of STAT1 and STAT-associated ISG15 was confirmed by immunoblotting. The expression of antioxidant proteins SOD1 and PRXD5 was downregulated suggesting radiation-induced oxidative stress. Similarly, the phosphorylated (active) forms of STING and IRF3, both members of the cGAS/STING pathway, were downregulated.

Conclusions: These data suggest the involvement of JAK/STAT and cGas/STING pathways in the genesis of radiation-induced lung inflammation. These pathways may be used as novel targets for the prevention of radiation-induced lung damage.

Keywords:

• cGAS/STING pathway
• endothelial cell
• ionizing radiation
• pulmonary inflammation
• STAT1

Acknowledgments

The authors thank Dr. Herbert Braselmann for help and discussion in the statistics.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was funded by the Federal Ministry of Education and Research of Germany (BMBF) [02NUK038A (Wolfgang Sievert) and 02NUK038B (Jos Philipp)].

Notes on contributors

Jos Philipp

Jos Philipp (MSc) is a PhD student in the group of Radiation Proteomics at the Institute of Radiation Biology at HMGU. He investigates the effect of radiation-induced damages on endothelial cells of different origin.

Wolfgang Sievert

Wolfgang Sievert (PhD) is a research scientist in the group of tumor imaging/radio-oncology/immunotherapy at Technical University Munich. His focus is radiation-induced endothelial damage.

Omid Azimzadeh

Omid Azimzadeh (PhD) is an Adjunct Professor (Docent) of Radiation Biology and a research scientist at the Institute of Radiation Biology at HMGU. His research interests focus on the effects of radiation exposure on normal tissue with a specific emphasis on the myocardial and endothelial proteome response.

Christine von Toerne

Christine von Toerne (PhD) is a group leader in the Research Unit Protein Science at HMGU, with a research focus on discovery proteomics for investigating cohort-based biomarker studies in the context of diabetes and complex diseases. Further, she is responsible for project management and quality controlled LC-MS/MS measurements in the Core Facility Proteomics at HMGU.

Fabian Metzger

Fabian Metzger (PhD) is a postdoctoral researcher with the focus on targeted proteomics. He is responsible for project management and quality control at the Core Facility Proteomics, HMGU.

Anton Posch

Anton Posch (PhD) is a guest scientist at the Institute of Radiation Biology (HMGU) and Federal Office for Radiation Protection with research interest in development of proteomics and immunoblotting.

Daniela Hladik

Daniela Hladik (MSc) is a PhD student in the group of Radiation Proteomics at the Institute of Radiation Biology at HMGU. She investigates radiation-induced damages on the hippocampus.

Prabal Subedi

Prabal Subedi (PhD) is a research scientist in the group of Radiation Proteomics at the Institute of Radiation Biology at HMGU. He investigates the role of extracellular vesicles in propagating radiation-induced leukemia.

Gabriele Multhoff

Gabriele Multhoff (PhD) is the head of the group tumor imaging/radio-oncology/immunotherapy at Technical University Munich. Her research goal is the development of innovative immunological diagnostic and therapeutic methods related to heat shock proteins to target in combination with irradiation different cancer tissues.

Michael J. Atkinson

Michael J. Atkinson (PhD) is the director of the Institute of Radiation Biology at the Helmholtz Zentrum München and Chair of Radiation Biology at the Technical University of Munich. His research goal is the improvement of radiation therapy by modulating the actions of radiation on tumor cells and non-cancerous tissues.

Soile Tapio

Soile Tapio (PhD) is an Adjunct Professor (Docent) of Radiation Biology. She leads the group of Radiation Proteomics at the Institute of Radiation Biology at HMGU. Her research topic is radiation-induced normal tissue damage in heart and brain.