Abstract
Acute pulmonary inflammation affects over 10% of intensive care unit (ICU) patients and is associated with high mortality. Fractalkine (CX3CL1) and its receptor, CX3CR1, have been shown to affect pulmonary inflammation, but previous studies have focused on macrophages. In a murine model of acute pulmonary inflammation, we identified inflammatory hallmarks in C57BL/6J and CX3CR1−/− mice. Pulmonary inflammation was significantly enhanced in the CX3CR1−/− animals compared to the C57BL/6J animals, as assessed by microvascular permeability, polymorphonuclear neutrophil (PMN) migration into lung tissue and alveolar space. The CX3CR1−/− mice showed increased levels of apoptotic PMNs in the lungs, and further investigations revealed an increased activation of necrosome-related receptor-interacting serine/threonine-protein kinases 1 (RIPK1), 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). Phosphorylated MLKL leads to membrane rupture and damage-associated molecular pattern (DAMP) release, which further enhance inflammation. The release of DAMPs was significantly higher in the CX3CR1−/− mice and led to the activation of various cascades, explaining the increased inflammation. RIPK3 and MLKL inhibition improved the inflammatory response in human PMNs in vitro and confirmed our in vivo findings. In conclusion, we linked CX3CL1 to the necrosome complex in pulmonary inflammation and demonstrated a pivotal role of the necrosome complex in human PMNs.
Highlights
Inflammation is a tightly regulated process that is characterized by the synchronized activation and orchestration of immune cells
In a murine model of LPS-induced acute pulmonary inflammation, we investigated the role of the chemokine CX3CL1 and its receptor, CX3CR1
In human polymorphonuclear neutrophil (PMN), the specific depletion of fractalkine reduced the gene levels of the necrosome-related receptor-interacting serine/threonine-protein kinases 1 (RIPK1) and RIPK3 (Figure 7F) and ameliorated the LPS-dependent release of the alarmins IL-33 and high motility group box 1 (HMGB1) (Figure 7G), confirming our in vivo results and linking fractalkine to the necrosome complex. We summarized these findings in a diagram of the LPS-mediated activation of FIGURE 7 | detected in human PMNs after LPS. (D) Effects of specific RIPK1 and RIPK3 inhibition on mixed-lineage kinase domain-like pseudokinase (MLKL) phosphorylation in human PMNs after LPS exposure were evaluated. (E) Human IL-33 and HMGB1 were determined in the supernatant of stimulated and treated PMNs as indicated (n = 6–8). (F) Effects of fractalkine depletion on necrosome-related RIPK1 and RIPK3 expression in human PMNs (n = 6–8) and on the release of human alarmins IL-33 and HMGB1 (G) were evaluated (n = 8–12)
Summary
Inflammation is a tightly regulated process that is characterized by the synchronized activation and orchestration of immune cells. Inflammation is caused by either tissue injury or infection, and its main function is eliminating the pathogenic insult or the damaged tissue. Both injury and infection induce the release of chemotactic factors to recruit polymorphonuclear neutrophils (PMNs) to the site of inflammation [1]. PMNs are the first cells to be recruited to the site of infection. PMNs are necessary for defense, their excessive migration into inflamed tissue even exacerbates tissue damage [2], through their release of reactive oxygen species (ROS) as part of their defense mechanisms [3]. The inefficient clearance of affected cells maintains inflammation and disrupts tissue homeostasis [4]
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