Cytosolic DNA Sensors as a New Pharmacological Target in EVLP

Abstract

<h3>Purpose</h3> Innate immunity is a key component of the inflammatory responses ensuing ischemia/reperfusion (I/R) injury. Cell damage and death caused by I/R promotes the release of danger-associated molecular patterns (DAMPs) recognized by specific pattern recognition receptors (PRRs). DNA released from nucleus and mitochondria of injured cells is a known DAMP, which sustains inflammation and cell death when recognized by specific PRRs like cytosolic DNA (cDNA) sensors cGAS and AIM2. The cGAS/STING pathway induces the production of type I interferons (IFNs) and IFN stimulated genes (ISGs). The role of cDNA sensors in reperfused lung is currently not characterized. Thus, using the rat <i>ex vivo</i> lung perfusion (EVLP) and lung transplantation (LTx) models, we begin to investigate the role of cGAS/STING in the early inflammatory events of lung I/R. <h3>Methods</h3> Heart-Lung blocks from male Sprague-Dawley rats were cannulated in the pulmonary artery and left atrium and assigned to four groups (n=5/6 rats/group). Cold ischemia (CI): under mechanical ventilation, the lungs were flushed with 4°C Perfadex. The heart-lung block was stored inflated (FiO2 0.5) for 7h in 4°C Perfadex. Warm ischemia (WI): After cannulation, the lungs were deflated for 1h at room temperature, flushed and stored as above for 6h. CI+EVLP: lungs were harvested as above, stored at 4°C for 2h, followed by 3-hour EVLP, and stored again (2h) in 4°C Perfadex. WI+EVLP: after 1 hour WI, lungs were flushed and stored at 4°C for 1 hour, followed by 3h EVLP, and stored again (2h) in 4°C Perfadex. Right lungs were used as controls and left lungs were transplanted and sampled 2h after LTx. We analyzed by RT-PCR and western blot the expression of the cytosolic DNA sensors cGAS, STING and AIM2 and by RT-PCR the expression of the downstream gene <i>Ifnb</i>, and the ISGs <i>Ifit1</i> and <i>Isg15</i> in whole lung tissue<i>.</i> <h3>Results</h3> cGAS, STING and AIM2 proteins are expressed in lung after the ischemic period. Their mRNA expression is strongly induced by the reperfusion (EVLP, LTx or EVLP+LTx) regardless of the conditions of preservation (CI or WI). We observed a similar behavior for the mRNA expression of <i>Ifnb</i> and the ISGs <i>Ifit1</i> and <i>Isg15</i>. <h3>Conclusion</h3> These preliminary data provide evidence that cytosolic DNA sensors, especially the cGAS-STING axis, may play a prominent role in the early inflammation of the reperfused lung regardless of the preservation conditions.