CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet–Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet–neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b−/− mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b−/− animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation

The role of adenosine receptor A1 in LPS-induced acute pulmonary inflammation

Die Migration neutrophiler Granulozyten (PMNs) in die Lunge ist ein zentraler pathophysiologischer Mechanismus in der frühen Phase der akuten respiratorischen Insuffizienz (Acute Respiratory Distress Syndrome, ARDS). Dabei vermittelt extrazelluläres Adenosin über vier Adenosinrezeptoren (AR) (A1, A2a, A2b und A3) wichtige pro- und anti-inflammatorische Effekte. In diesen Arbeiten untersuchten wir im murinen ARDS-Model, die Rolle von Adenosinrezeptor A1 bei der LPS-induzierten pulmonalen Inflammation. Mittels durchflusszytometrischer Verfahren bestimmten wir die Migration von PMNs in der Lunge. Desweiteren untersuchten wir die LPS-induzierten mikrovaskuläre Permeabilität, Freisetzung inflammatorischer Mediatoren und Effekte von pharmakologischen Modulatoren. Für unsere in vivo Versuche nutzten wir Wildtyp- (WT, C57/Bl6) und A1-gendefiziente Mäuse (A1AR-/-). Wir haben auch chimäre Mäuse generiert, welche A1AR entweder auf hämatopoetischen oder auf nicht hämatopoetischen Zellen exprimierten. Die Rolle des A1AR in der PMN-Migration wurde ebenfalls in vitro untersucht und auch deren Effekte bei dem LPS-induzierten zytoskelettalen Remodelling. Unsere Daten zeigen, dass die pharmakologische Aktivierung von A1AR zu einer reduzierten PMN-Migration, verringerter mikrovaskulärer Permabilität und geringeren Ausschüttung von inflammatorischen Mediatoren in der Lunge führt. Interessanterweise, führte die pharmakologische Aktivierung von A1AR nur bei den chimären Mäusen, welche den A1AR nur hämatopoietischen Zellen exprimierten, zur einen signifikanten Reduktion der PMN-Migration im Alveolarraum. Die pharmakologische Modulation, der von uns untersuchter Adenosinrezeptor, greift in entscheidende pathophysiologischen Schlüsselstellen der akuten Phase des ARDS ein und untermauert die Rolle von A1AR als wichtigen Adenosinrezeptor in der akuten pulmonalen Inflammation. Pharmakologische Modulatoren des A1 Adenosinrezeptors könnten zukünftig neue kausale Therapiestrategien für die Behandlung der akuten pulmonalen Inflammation darstellen.The migration of neutrophils (PMNs) into the lung is a central pathophysiological mechanisms in the early phase of acute respiratory failure (Acute Respiratory Distress Syndrome, ARDS). Extracellular adenosine acts through four adenosine receptors (AR) (A1, A2a, A2b and A3) and exhibits important pro-and anti-inflammatory effects. In this work, we investigated the role of A1AR in a murine model of LPS-induced pulmonary inflammation. Using flow cytometry method, we determined the migration of PMNs into the different lung compartiments. Furthermore, we investigated LPS-induced microvascular permeability, release of inflammatory mediators and effects of pharmacological compounds. For our in vivo experiments, we used wild-type (WT C57/Bl6) and A1-gene deficient mice (A1AR-/-). We have also generated chimeric mice, which A1AR expressing either hematopoietic or non-hematopoietic cells. The role of A1AR in PMN transmigration was also studied in vitro and their effects on the LPS-induced cytoskeletal remodeling on human pulmonary endothelial and epithelial cells were assesed. Our data show that A1AR exhibits anti-inflammatory effects in LPS-induced ARDS-model. Pharmacological activation of A1AR leeds to reduction of PMN migration, decreased microvascular permability and reduced the release of inflammatory mediators in the lung. A1AR Knockout mice compared to wildtype mice showed higher LPS-induced PMN-accumulation and destruction of the pulmonary architecture. The pharmacological activation and its effects play a key role in the acute phase of ARDS. Our results support the role of A1AR as one important adenosine receptors in the acute pulmonary inflammation. Pharmacological modulators of the A1AR could represent a novel causal future therapeutic strategy for the treatment of acute pulmonary inflammation

Anti-inflammatory Effects of Heme Oxygenase-1 Depend on Adenosine A2A- and A2B-Receptor Signaling in Acute Pulmonary Inflammation

Acute pulmonary inflammation is still a frightening complication in intensive care units. In our previous study, we determined that heme oxygenase (HO)-1 had anti-inflammatory effects in pulmonary inflammation. Recent literature has emphasized a link between HO-1 and the nucleotide adenosine. Since adenosine A2A- and A2B-receptors play a pivotal role in pulmonary inflammation, we investigated their link to the enzyme HO-1. In a murine model of pulmonary inflammation, the activation of HO-1 by hemin significantly decreased polymorphonuclear leukocyte (PMN) migration into the lung. This anti-inflammatory reduction of PMN migration was abolished in A2A- and A2B-knockout mice. Administration of hemin significantly reduced chemokine levels in the BAL of wild-type animals but had no effects in A2A-/- and A2B-/- mice. Microvascular permeability was significantly attenuated in HO-1-stimulated wild-type mice, but not in A2A-/- and A2B-/- mice. The activity of HO-1 rose after LPS inhalation in wild-type animals and, surprisingly, also in A2A-/- and A2B-/- mice after the additional administration of hemin. Immunofluorescence images of animals revealed alveolar macrophages to be the major source of HO-1 activity in both knockout strains—in contrast to wild-type animals, where HO-1 was also significantly augmented in the lung tissue. In vitro studies on PMN migration further confirmed our in vivo findings. In conclusion, we linked the anti-inflammatory effects of HO-1 to functional A2A/A2B-receptor signaling under conditions of pulmonary inflammation. Our findings may explain why targeting HO-1 in acute pulmonary inflammation has failed to prove effective in some patients, since septic patients have altered adenosine receptor expression

How Adhesion Molecule Patterns Change While Neutrophils Traffic through the Lung during Inflammation

In acute pulmonary inflammation, polymorphonuclear cells (PMNs) pass a transendothelial barrier from the circulation into the lung interstitium followed by a transepithelial migration into the alveolar space. These migration steps are regulated differentially by a concept of adhesion molecules and remain—despite decades of research—incompletely understood. Current knowledge of changes in the expression pattern of adhesion molecules mainly derives from in vitro studies or from studies in extrapulmonary organ systems, where regulation of adhesion molecules differs significantly. In a murine model of lung inflammation, we determined the expression pattern of nine relevant neutrophilic adhesion molecules on their way through the different compartments of the lung. We used a flow cytometry-based technique that allowed describing spatial distribution of the adhesion molecules expressed on PMNs during their migration through the lung in detail. For example, the highest expression of CD29 was found in the intravascular compartment, highlighting its impact on the initial adhesion to the endothelium. CD47 showed its peak of expression on the later phase of transendothelial migration, whereas CD11b and CD54 expression peaked interstitial. A pivotal role for transepithelial migration was found for the adhesion molecule CD172a. Thereby, expression may correlate with functional impact for specific migration steps. In vitro studies further confirmed our in vivo findings. In conclusion, we are the first to determine the changes in expression patterns of relevant adhesion molecules on their migration through the different compartments of the lung. These findings may help to further understand the regulation of neutrophil trafficking in the lung