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Abstract: Pulmonary defense mechanisms are critical for host integrity during pneumonia and sepsis. This defense is fundamentally dependent on the activation of neutrophils during the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet function, yet its role on neutrophil function within the lung is not well understood. This study aimed to identify the role of Sema7A during pulmonary inflammation and sepsis. In patients with acute respiratory distress syndrome (ARDS), we were able to show a correlation between Sema7A and oxygenation levels. During subsequent workup, we found that Sema7A binds to the neutrophil PlexinC1 receptor, increasing integrins, and L-selectin on neutrophils. Sema7A prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/-animals in the lung during pulmonary inflammation. This effect resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pulmonary sepsis. This finding was associated with significantly worse outcome of Sema7A-/- animals in a model of pulmonary sepsis. Sema7A has an immunomodulatory effect in the lung, affecting pulmonary sepsis and ARDS. This effect influences the response of neutrophils to external aggression and might influence patient outcome. This trial was registered at www.ClinicalTrials.gov as #NCT02692118., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Peritonitis is a life-threatening condition on intensive care units. Inflammatory cytokines and their receptors drive inflammation, cause the formation of platelet-neutrophil complexes (PNCs) and therefore the migration of polymorphonuclear neutrophils (PMNs) into the inflamed tissue. CX3CL1 and its receptor CX3CR1 are expressed in various cells, and promote inflammation. The shedding of CX3CL1 is mediated by a disintegrin and metalloprotease (ADAM) 17. The role of the CX3CL1-CX3CR1 axis in acute peritonitis remains elusive., Methods: In zymosan-induced peritonitis, we determined the formation of PNCs in the blood and the expression of PNC-related molecules on PNCs. PMN migration into the peritoneal lavage was evaluated in wild-type (WT) and CX3CR1-/- animals by flow cytometry. CX3CL1, ADAM17, and the expression of various inflammatory cytokines were detected. Further, we determined the inflammation-associated activation of the intracellular transcription factor extracellular signal-regulated kinase 1/2 (ERK1/2) by Western blot., Results: The PMN accumulation in the peritoneal lavage and the PNC formation in the circulation were significantly raised in CX3CR1-/- compared with WT animals. The expression of PNC-related selectins on PNCs was significantly increased in the blood of CX3CR1-/- animals, as well as cytokine levels. Further, we observed an increased activation of ERK1/2 and elevated ADAM17 expression in CX3CR1-/- during acute inflammation. Selective ERK1/2 inhibition ameliorated inflammation-related increased ADAM17 expression., Conclusions: A CX3CR1 deficiency raised the release of inflammatory cytokines and increased the PNC formation respectively PMN migration via an elevated ERK1/2 activation during acute peritonitis. Further, we observed a link between the ERK1/2 activation and an elevated ADAM17 expression on PNC-related platelets and PMNs during inflammation. Our data thus illustrate a crucial role of CX3CR1 on the formation of PNCs and regulating inflammation in acute peritonitis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by the Shock Society.)

Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A 2B knockout animals (A 2B -/-). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A 2B -/- animals, highlighting the impact of the A 2B -receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A 2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A 2B . The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro , CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A 2B -receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A 2B -receptor signaling., (Copyright © 2020 Ngamsri, Jans, Putri, Schindler, Gamper-Tsigaras, Eggstein, Köhler and Konrad.)

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.

Acute pulmonary inflammation is still a frightening complication in intensive care units and has a high mortality. Specific treatment is not available, and many details of the pathomechanism remain unclear. The recently discovered chemokine receptor CXCR7 and its ligand stromal cell-derived factor (SDF)-1 are known to be involved in inflammation. We chose to investigate the detailed role of CXCR7 in a murine model of LPS inhalation. Inflammation increased pulmonary expression of CXCR7, and the receptor was predominantly expressed on pulmonary epithelium and on polymorphonuclear neutrophil (PMNs) after transepithelial migration into the alveolar space. Specific inhibition of CXCR7 reduced transepithelial PMN migration by affecting the expression of adhesion molecules. CXCR7 antagonism reduced the most potent PMN chemoattractants CXCL1 and CXCL2/3. After inhibiting CXCR7, NF-κB phosphorylation was reduced in lungs of mice, tight junction formation increased, and protein concentration in the bronchoalveolar lavage diminished, showing the impact of CXCR7 on stabilizing microvascular permeability. In vitro studies with human cells confirmed the pivotal role of CXCR7 in pulmonary epithelium. Immunofluorescence of human lungs confirmed our in vivo data and showed an increase of the expression of CXCR7 in pulmonary epithelium. Highlighting the clinical potential of CXCR7 antagonism, nebulization of the agent before and after the inflammation showed impressive anti-inflammatory effects. Additional CXCR7 inhibition potentiated the effect of SDF-1 antagonism, most probably by downregulating SDF-1 and the second receptor of the chemokine (CXCR4) expression. In conclusion, our data identified the pivotal role of the receptor CXCR7 in pulmonary inflammation with a predominant effect on the pulmonary epithelium and PMNs., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Recruiting polymorphonuclear neutrophil granulocytes (PMNs) from circulation and bone marrow to the site of inflammation is one of the pivotal mechanisms of the innate immune system. During inflammation, the enzyme heme oxygenase 1 (HO-1) has been shown to reduce PMN migration. Although these effects have been described in various models, underlying mechanisms remain elusive. Recent studies revealed an influence of HO-1 on different cells of the bone marrow. We investigated the particular role of the bone marrow in terms of HO-1-dependent pulmonary inflammation. In a murine model of LPS inhalation, stimulation of HO-1 by cobalt (III) protoporphyrin-IX-chloride (CoPP) resulted in reduced segmented PMN migration into the alveolar space. In the CoPP group, segmented PMNs were also decreased intravascularly, and concordantly, mature and immature PMN populations were higher in the bone marrow. Inhibition of the enzyme by tin protoporphyrin-IX increased segmented and banded PMN migration into the bronchoalveolar lavage fluid with enhanced PMN release from the bone marrow and aggravated parameters of tissue inflammation. Oxidative burst activity was significantly higher in immature compared with mature PMNs. The chemokine stromal-derived factor-1 (SDF-1), which mediates homing of leukocytes into the bone marrow and is decreased in inflammation, was increased by CoPP. When SDF-1 was blocked by the specific antagonist AMD3100, HO-1 activation was no longer effective in curbing PMN trafficking to the inflamed lungs. In conclusion, we show evidence that the anti-inflammatory effects of HO-1 are largely mediated by inhibiting the release of segmented PMNs from the bone marrow rather than direct effects within the lung., (Copyright © 2014 the American Physiological Society.)

Extracellular adenosine and adenosine receptors are critically involved in various inflammatory pathways. Adenosine receptor A1 (A1AR) has been implicated in mediating transmigration of leukocytes to sites of inflammation. This study was designed to characterize the role of A1AR in a murine model of LPS-induced lung injury. LPS-induced transmigration of polymorphonuclear cells (PMNs) and microvascular permeability was elevated in A1AR(-/-) mice. Pretreatment of wild-type mice with the specific A1AR agonist 2'Me-2-chloro-N6-cyclopentyladenosine attenuated PMN accumulation in the interstitium and alveolar space as well as microvascular permeability. Lower PMN counts in the lungs of pretreated wild-type mice were associated with reduced amounts of the chemotactic cytokines TNF-α, IL-6, and CXCL2/3 in the bronchoalveolar lavage. Pretreatment was only effective when A1AR was expressed on hematopoietic cells as demonstrated in chimeric mice. These findings were confirmed by in vitro transmigration assays demonstrating that chemokine-induced transmigration of PMNs was reduced when PMNs but not when pulmonary endothelial or alveolar epithelial cells were pretreated. 2'Me-2-chloro-N6-cyclopentyladenosine prevented pulmonary endothelial but not epithelial cells from LPS-induced cellular remodeling and cell retraction. Our data reveal what we believe to be a previously unrecognized distinct role of A1AR for PMN trafficking and endothelial integrity in a model of acute lung injury.

Extracellular adenosine has been implicated as anti-inflammatory signaling molecule during acute lung injury (ALI). The main source of extracellular adenosine stems from a coordinated two-step enzymatic conversion of precursor nucleotides via the ecto-apyrase (CD39) and the ecto-5'-nucleotidase (CD73). In the present study, we hypothesized a critical role of CD39 and CD73 in mediating pulmonary neutrophil (PMN) transmigration during lipopolysaccharide (LPS) -induced lung injury. Initial studies revealed that pulmonary CD39 and CD73 transcript levels were elevated following LPS exposure in vivo. Moreover, LPS-induced accumulation of PMN into the lungs was enhanced in cd39(-/-) or cd73(-/-) mice, particularly into the interstitial and intra-alveolar compartment. Such increases in PMN trafficking were accompanied by corresponding changes in alveolar-capillary leakage. Similarly, inhibition of extracellular nucleotide phosphohydrolysis with the nonspecific ecto-nucleoside-triphosphate-diphosphohydrolases inhibitor POM-1 confirmed increased pulmonary PMN accumulation in wild-type, but not in gene-targeted mice for cd39 or cd73. Finally, treatment with apyrase or nucleotidase was associated with attenuated pulmonary neutrophil accumulation and pulmonary edema during LPS-induced lung injury. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in attenuating PMN trafficking into the lungs during LPS-induced lung injury and suggest treatment with their soluble compounds as a therapeutic strategy.

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils (PMNs) into the different compartments of the lung, passing an endothelial and epithelial barrier. Recent studies showed evidence that phosphodiesterase (PDE)4-inhibitors stabilized endothelial cells. PDE4B and PDE4D subtypes play a pivotal role in inflammation, whereas blocking PDE4D is suspected to cause gastrointestinal side effects. We thought to investigate the particular role of the PDE4-inhibitors roflumilast and rolipram on lung epithelium. Acute pulmonary inflammation was induced by inhalation of LPS. PDE4-inhibitors were administered i.p. or nebulized after inflammation. The impact of PDE4-inhibitors on PMN migration was evaluated in vivo and in vitro. Microvascular permeability, cytokine levels, and PDE4B and PDE4D expression were analyzed. In vivo, both PDE4-inhibitors decreased transendothelial and transepithelial migration even when administered after inflammation, whereas roflumilast showed a superior effect compared to rolipram on the epithelium. Both inhibitors decreased TNFα, IL6, and CXCL2/3. CXCL1, the strong PMN chemoattractant secreted by the epithelium, was significantly more reduced by roflumilast. In vitro assays with human epithelium also emphasized the pivotal role of roflumilast on the epithelium. Additionally, LPS-induced stress fibers, an essential requirement for a direct migration of PMNs into the alveolar space, were predominantly reduced by roflumilast. Expression of PDE4B and PDE4D were both increased in the lungs by LPS, PDE4-inhibitors decreased mainly PDE4B. The topical administration of PDE4-inhibitors was also effective in curbing down PMN migration, further highlighting the clinical potential of these compounds. In pulmonary epithelial cells, both subtypes were found coexistent around the nucleus and the cytoplasm. In these epithelial cells, LPS increased PDE4B and, to a lesser extend, PDE4D, whereas the effect of the inhibitors was prominent on the PDE4B subtype. In conclusion, we determined the pivotal role of the PDE4-inhibitor roflumilast on lung epithelium and emphasized its main effect on PDE4B in hyperinflammation. [ABSTRACT FROM AUTHOR]

Adenosine receptor A3 (A3) regulates directed movement of polymorphonuclear cells (PMNs) to sites of inflammation and has been implicated as a relevant mediator in models of inflammatory diseases. Here, we sought to characterize the role of A3 in a murine model of lung inflammation. Initial studies revealed that pulmonary A3 transcript levels were elevated following LPS exposure in vivo. In addition, inhalation of LPS increased the accumulation of PMNs in wild-type and A3-/- mice in all lung compartments. Pretreatment with the specific A3-agonist Cl-IB-MECA significantly decreased migration of PMNs into lung interstitium and alveolar air space of wild-type mice but not of A3-/- mice. Lower PMN counts were associated with reduced levels of TNF-α and IL-6 in the alveolar space of wild-type mice that received Cl-IB-MECA. In addition, Cl-IB-MECA attenuated LPS-induced microvascular permeability in wild-type mice as assessed by the extravasation of Evans blue. In pulmonary microvascular endothelial cells, Cl-IB-MECA reduced LPS-induced cytoskeletal remodeling and cell retraction, consistent with a specific role of A3 for maintaining endothelial integrity. Migratory activity of human PMNs across an endothelial or epithelial monolayer was reduced when A3 was activated on PMNs. Studies in chimeric mice, however, revealed that Cl-IB-MECA required A3 on both hematopoietic and nonhematopoietic cells to reduce transmigration in vivo. Together, our results shed new light on the role of A3 in LPS-induced PMN trafficking in the lung and suggest pharmacological modulation of A3-dependent pathways as a promising approach in lung inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]