Fractalkine Is Linked with the Necrosome Pathway in Acute Pulmonary Inflammation

Abstract

Background: Acute pulmonary inflammation affects more than 10% of ICU admissions and is associated with a high mortality. The chemokine fractalkine (CX3CL1) and its receptor CX3CR1 have been shown to influence pulmonary inflammation. Methods: In a murine model of acute lung inflammation, we determined the LPS-induced PMN infiltration and particular the accumulation of necroptotic PMNs in the lung tissue and alveolar space of wild type (WT) and CX3CR1-/- animals by flow cytometry. Chemokines and damage-associated molecular patterns (DAMPs) were detected by ELISA and RT-PCR. The activation of necrosome-related proteins receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3, and mixed lineage kinase domain-like protein (MLKL) in WT and CX3CR1-/- mice were evaluated by western blots. Findings: Acute pulmonary inflammation was significantly enhanced in CX3CR1-/- compared to WT-mice concerning neutrophil migration and microvascular permeability. Furthermore, CX3CR1 deficiency revealed an increase of necroptotic PMN counts and an augmented activation of RIPK1 and RIPK3. Both kinases enhanced the phosphorylation of MLKL, leading to membrane rupture and the release of DAMPs. DAMPs were significantly higher in CX3CR1-/- mice, explaining the increased inflammation in CX3CR1-/- mice. The necrosome led to activation of the signaling cascade AKT, ERK1/2, and NFκB, resulting in an increase of chemo- and cytokines. Pharmacologic inhibition of RIPK3 and MLKL improved the inflammatory response. In vitro studies confirmed our in vivo findings. Interpretation: In conclusion, we linked for the first time CX3CL1 with necroptosis in pulmonary inflammation and demonstrated a pivotal role of the necrosome complex in human PMNs. Funding Statement: This work was supported by the Else Kroner-Fresenius-Stiftung 2014_A148 (to FK), the Fortuene Program F1211382 (to FK), and the German Research Foundation Grant KO4280/2-1 (to FK). We acknowledge support by Open Access Publishing Fund of University of Tubingen. Declaration of Interests: There are no competing interests for any of the authors. Ethics Approval Statement: All animal protocols were approved by the Animal Care and Use Committee of the University of Tuebingen.