| dc.description.abstract | 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Previous unpublished work from our group found neutrophils express the class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. | en |
