Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse

Abstract

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

Glycoprotein VI in securing vascular integrity in inflamed vessels

“…In this model, deletion of platelet‐CLEC‐2 resulted in an increased severity of sepsis that was associated with a dysregulation of inflammatory cytokine production, thus indicating a protective role of platelet CLEC‐2 . These findings showing a protective role of platelet CLEC‐2 in inflammation have since been published and are in line with those of a recent study demonstrating that platelet CLEC‐2 protects against LPS‐induced lung injury via its interaction with podoplanin on inflammatory alveolar macrophages . While it was previously shown that engagement of platelet CLEC‐2 by podoplanin on inflammatory macrophages triggers platelet activation, these new data indicate that this interaction induces reciprocal regulation between platelets and inflammatory macrophages.…”

“…63 These findings showing a protective role of platelet CLEC-2 in inflammation have since been published 64 and are in line with those of a recent study demonstrating that platelet CLEC-2 protects against LPS-induced lung injury via its interaction with podoplanin on inflammatory alveolar macrophages. 61 While it was previously shown that engagement of platelet CLEC-2 by podoplanin on inflammatory macrophages triggers platelet activation, 145 these new data indicate that this interaction induces reciprocal regulation between platelets and inflammatory macrophages. In a more general way, these results are consistent with a model where platelets and leukocytes regulate their recruitment and activation during inflammatory reactions in a mutual and reciprocal manner.…”

“…Those situations likely encompass most traumatic vascular injuries, which might explain why GPVI patients do not present severe bleeding. Indeed, besides lymphatic endothelial cells, several types of podoplanin‐expressing cells (inflammatory macrophages, fibroblasts) have been described in the vicinity of blood vessels and could therefore engage platelet CLEC‐2 through vascular breaches.…”

“…Studies based on the combination of mouse models of inflammation and severe thrombocytopenia (< less than 3% of normal mouse platelet count) have helped to demonstrate that platelets continuously secure the inflamed vasculature by preventing bleeding from the very onset of inflammation . This protective action of platelets was reported in many different models of acute inflammation including various models of dermatitis, IC‐mediated glomerulonephritis, endotoxin‐ and bacteria‐induced acute lung injury, and myocardial and cerebral ischemia‐reperfusion injury, as well as in models of viral infection and solid tumors . Strikingly, in several of these models, prevention of inflammatory bleeding was maintained in mice compromised for platelet aggregation at sites of traumatic vessel injury .…”

Lymphatic blood filling in CLEC-2-deficient mouse models

“…It has a broad expression profile including on lymphatic endothelial cells [2], alveolar epithelial type I cells [3,4], stromal cells and fibroblasts [5][6][7], inflammatory leukocytes [4,8,9] and specialized tissue structures such as in kidney podocytes [10] and on choroid plexuses in the brain [11,12]. CLEC-2 (as a consequence of its expression on platelets) and podoplanin have been proposed as novel targets in a wide range of disorders including sepsis, wound repair, infection, and cancer metastasis [4,8,[13][14][15][16][17]. If these novel therapy approaches are to become a reality, it is vital that the functions of CLEC-2-mediated platelet activation, and the ligand podoplanin, are fully understood in healthy adult individuals as well as in these disorders.…”