Intestinal IL-33 promotes platelet activity for neutrophil recruitment during acute inflammation

The peripheral serotonin (5-HT) is mainly generated from the gastrointestinal tract, and taken up and stored by platelets in the circulation. While the gut is recognized as a major immune organ, how intestinal local immune responses control whole body physiology via 5-HT is yet unclear. Here, we show that intestinal inflammation enhances systemic platelet activation and blood coagulation. Intestinal epithelium damage induces the elevated level of alarm cytokine interleukin-33 (IL-33), leading to platelet activation via promoting gut-derived 5-HT release. More importantly, we found that loss of intestinal epithelial-derived IL-33 dampens peripheral 5-HT level, resulting in compromised platelet activation and hemostasis. Functionally, intestinal IL-33 contributes to the recruitment of neutrophils to the sites of acute inflammation by enhancing platelet activities. Genetical deletion of intestinal IL-33 or neutralization of peripheral IL-33, protects the animals from lipopolysaccharide endotoxic shock owing to attenuated neutrophil extravasation. Therefore, our data establish a distinct role of intestinal IL-33 in activating platelet by promoting 5-HT release for systemic physiology and inflammation.

The LTB4–BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation

The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4–BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

Platelets docking to VWF prevent leaks during leukocyte extravasation by stimulating Tie-2

Neutrophil extravasation requires opening of the endothelial barrier but does not necessarily cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis pore, keeping this opening tightly closed around the transmigrating neutrophils. We have identified the receptor system that is responsible for this. We show that silencing, or gene inactivation, of endothelial Tie-2 results in leak formation in postcapillary venules of the inflamed cremaster muscle at sites of neutrophil extravasation, as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, because it was absent upon neutrophil depletion. We identified the Cdc42 GTPase exchange factor FGD5 as a downstream target of Tie-2 that is essential for leakage prevention during neutrophil extravasation. Looking for the Tie-2 agonist and its source, we found that platelet-derived angiopoietin-1 (Angpt1) was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie-2 by secreting Angpt1, thereby preventing diapedesis-induced leakiness.

Circulating BMP9 protects the pulmonary endothelium during inflammation-induced lung injury in mice

ABSTRACTRationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.

Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

Acute lung inflammation has severe morbidity, as seen in COVID-19 patients. Lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries (“margination”). We sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. We designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. We found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. Specifically, nanoparticles with agglutinated protein (NAPs) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. We validated this finding by engineering protein-conjugated liposomes that recapitulate NAP targeting to neutrophils in inflamed lungs. We show that NAPs can diagnose acute lung injury in SPECT imaging and that NAP-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. Finally, we demonstrate that ischemic ex vivo human lungs selectively take up NAPs, illustrating translational potential. This work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and NAPs have significant potential in detecting and treating respiratory conditions arising from injury or infections.

Abstract WMP80: Live Animal Imaging Reveals Delayed But Sustained Neutrophil Recruitment to Both Ischemic and Non-Ischemic Cortical Cerebrovasculature in a Mouse Transient Stroke Model

Background: Treatment for acute ischemic stroke (AIS) relies on early restoration of blood flow; however, injury persists despite reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and timing of neutrophil recruitment to the cerebrovasculature (CBV) during AIS remain unclear. We sought to characterize the neutrophil response in the CBV following ischemia/reperfusion using a transient middle cerebral artery occlusion (tMCAO) AIS model. Methods: Fluorescent reporter mice underwent 90-minute tMCAO confirmed with LSCI or surgery without occlusion (sham). Real-time confocal intravital microscopy of the CBV was performed at 24 and 72 hrs post-reperfusion through cranial windows in ischemic and non-ischemic hemispheres. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo over both hemispheres. Neutrophil extravasation was further quantified using whole mount sections. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride staining. Results: An increase in neutrophil rolling, adhesion and extravasation was observed within the ischemic hemisphere compared to sham (p < 0.01) at 24 and markedly 72 hrs. Non-ischemic hemispheres showed increased neutrophil rolling and adhesion, but not extravasation, compared to sham surgery (p < 0.05). Whole mount sections demonstrated increased cortical and subcortical neutrophil extravasation in the ischemic hemisphere only (p < 0.01). Conclusions: Early neutrophil recruitment to the ischemic hemisphere following tMCAO was present at both the CBV and within the infarcted parenchyma at 72 hours. Acute neutrophil recruitment at the CBV in the non-ischemic hemisphere, but not the cortex, suggests that AIS triggers a global inflammatory response within the CBV. Targeted neutrophil-specific therapies during reperfusion may reduce global inflammation at the CBV, and potentially mitigate secondary neuronal injury.

ROLE OF NEUTROPHILS IN THE DELIVERY OF NANOPARTICLE INTO THE TUMOR

Mechanisms of liposomes and magnetic nanoparticles accumulation in the tumor tissue were shown to be associated with neutrophils. The intravital microscopy allowed to visualize the transfer of magnetic nanoparticles by neutrophils that captured these nanoparticles, while liposomes accumulate in the tissue via micro- and macroleakages appearing at sites of neutrophil extravasation.