Role of Semaphorin3F in Neutrophilic inflammation
Item statusRestricted Access
Embargo end date06/07/2020
Plant, Tracie Marie
Effective host responses to injury and infection require both rapid recruitment of neutrophils into tissues and timely inflammation resolution. Research efforts have primarily focused separately on the initiation and resolution phases of inflammation and on how neutrophil survival responses determine the duration and extent of the inflammatory response. Less focus has been placed on mechanisms retaining viable neutrophils at inflamed sites and thus contributing to ongoing inflammation. This may be of therapeutic importance given the numerical dominance of viable over apoptotic neutrophils in inflamed tissue, even during inflammation resolution. Semaphorins were originally identified as chemo-repulsive molecules for axonal growth cones. They have since been implicated in cell motility in the context of vascular growth, tumour progression and in immune signalling and immune synapse formation. Recently the class 3 Semaphorin, Sema3A, was shown to act as an attractant for tumour-associated macrophages (TAM), regulating their localization and retention within hypoxic tumour areas. Previous unpublished work from our group found neutrophils express the class 3 Semaphorin, Sema3F, with differential expression observed in hypoxia. Whether neutrophil expression of Sema3F can directly regulate recruitment to inflamed sites is currently unknown. Semaphorins can interact both directly with Plexins and in complexes with Neuropilin co-receptors, leading to activation of protein kinase and (guanosine triphosphate-ase) GTPase signalling pathways. This affects actin cytoskeletal re-organisation following oxidation of actin filaments leading to modifications of neutrophil shape and migration. In light of emerging evidence that neutrophils also undergo a process of reverse migration away from inflamed tissue, I propose neutrophil expression of Sema3F regulates neutrophil retention within the tissues. Where autocrine production of neutrophil Sema3F induces neutrophil F-actin disassembly, acting as a cytoskeletal brake to cell movement and is thus a determinant of both the magnitude and duration of the innate immune response. To investigate this, in vivo experiments were performed in murine models of acute lung injury, using both wild type and neutrophil specific Sema3F knockout mice. Complementary in vitro experiments were performed in human peripheral blood neutrophils. Inflammatory neutrophils express sema3F. Sema3F protein expression is seen in the recruited neutrophils found in tissue from patients with chronic inflammatory lung disease (COPD). In vivo modelling showed Sema3F expression is regulated at a transcriptional level during inflammation, with induction of Sema3F mRNA in neutrophils recruited to the airways following Lipopolysaccharide (LPS) challenge and a parallel increase in Sema3F protein expression in Ly6G positive cells within the alveoli. Similarly, the surface expression of Neuropilin 2 (NRP2), the obligatory co-receptor to Sema3F, is increased in both human and murine neutrophils following an inflammatory stimulus. In a murine model of LPS-induced lung injury, intra-tracheal instillation of Sema3F at 24 hours increases retention of airway recruited neutrophils at 48 hours. Furthermore, neutrophil specific knockout of murine Sema3F in an LPS-induced lung injury model increases both recruitment and clearance of neutrophils found in the bronchoalveolar lavage but does not alter apoptotic responses. The Sema3F deficient neutrophils migrate, to and away from the site of tissue injury rapidly and are not retained within inflamed murine lung tissue. The retention phenotype seen in the Sema3F knockout mouse appears neutrophil specific as macrophage numbers were unchanged throughout the inflammatory response. In human peripheral blood neutrophils Sema3F treatment inhibits neutrophil chemotaxis to the bacterial product N-Formyl-Met-Leu-Phe (fMLF). Using a microfluidic chip assay, neutrophils responded to Sema3F in a dose dependent manner. Following Sema3F treatment there was a reduction in neutrophil migration. This was observed in neutrophils migrating up an increasing Leukotriene B4 (LTB4) gradient (chemotaxis). After the neutrophils had reached the chamber and the highest concentration of LTB4, the rate of neutrophil migration out of the chamber and against the gradient (retrotaxis) was reduced. Effectively the neutrophils were retained within the chamber for 90 minutes. Sema3F treated neutrophils have preserved functions including phagocytosis of opsonised Zymosan granules and fluorescently labelled Escherichia coli (E. coli) particles. Following Sema3F treatment neutrophils produce increased intra-cellular levels of radical oxygen species (ROS). Increased ROS has been shown to promote F-actin disassembly and result in cytoskeleton rearrangement. Sema3F not only inhibits neutrophil chemotaxis, but also affects neutrophil rounding and regulates exogenous myeloperoxidase and elastase activity. It is evident that Sema3F signalling could selectively modulate diverse neutrophil functions related to neutrophil cytoskeletal dynamics. Using cultured murine lung slices and real-time confocal imaging neutrophils treated with Sema3F are slower than controls. On delivery of intra-tracheal (IT) Sema3F to the murine ALI model the retained neutrophils are found in the alveoli space and are not retained by a rate-limiting interaction with the barriers to neutrophil lung transmigration. Following IT Sema3F, these neutrophils retained in the murine airway are more spherical and undergo increased F-actin disassembly. These results for the first time identify a role for Sema3F as a neutrophil specific retention signal in acute lung injury. Thus, revealing a novel mechanism by which neutrophils are recruited to and maintained at the site of tissue injury. I therefore propose that Sema3F and the co-receptor NRP2 are potential therapeutic targets in acute lung injury and lung diseases caused by exaggerated and persistent neutrophilic inflammation.