Loss of neutrophil Shp1 produces hemorrhagic and lethal acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Radiolabelling and immunohistochemistry reveal platelet recruitment into lungs and platelet migration into airspaces following LPS inhalation in mice.

INTRODUCTION: Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS. OBJECTIVES: Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques. RESULTS: Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces. CONCLUSIONS: Intranasal administration of LPS activates the innate immune response which includes a fall in peripheral blood platelet counts with subsequent platelet recruitment to the lung, spleen and liver, measured by immunohistochemistry and radiolabelling techniques.

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice.

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.

The application of local hypobaric pressure - A novel means to enhance macromolecule entry into the skin.

The local application of controlled hypobaric stress represents a novel means to facilitate drug delivery into the skin. The aims of this work were to understand how hypobaric stress modified the properties of the skin and assess if this penetration enhancement strategy could improve the percutaneous penetration of a macromolecule. Measurements of skin thickness demonstrated that the topical application of hypobaric stress thinned the tissue (p<0.05), atomic force microscopy showed that it shrunk the corneocytes in the stratum corneum (p<0.001) and the imaging of the skin hair follicles using multiphoton microscopy showed that it opened the follicular infundibula (p<0.001). Together, these changes contributed to a 19.6-fold increase in in vitro percutaneous penetration of a 10,000 molecular weight dextran molecule, which was shown using fluorescence microscopy to be localized around the hair follicles, when applied to the skin using hypobaric stress. In vivo, in the rat, a local hemodynamic response (i.e. a significant increase in blood flow, p<0.001) was shown to contribute to the increase in follicular transport of the dextran to produce a systemic absorption of 7.2±2.81fg·mL(-1). When hypobaric stress was not applied to the rat there was no detectable absorption of dextran and this provided evidence that this novel penetration enhancement technique can improve the percutaneous penetration of macromolecules after topical application to the skin.