Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

Semaphorin 7A coordinates neutrophil response during pulmonary inflammation and sepsis.

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.

Semaphorin 7A is protective during inflammatory peritonitis through integrin receptor signaling.

Introduction: The study explores the role of endothelial Semaphorin 7A (SEMA7A) in inflammatory processes. SEMA7A is known for enhancing inflammation during tissue hypoxia and exhibiting anti-inflammatory properties in the intestinal system during colitis. This research extends the understanding of SEMA7A's function by examining its role in inflammatory peritonitis and intestinal inflammation. Methods: The research involved inducing peritonitis in SEMA7A knockout (SEMA7A-/-) and wild-type (WT) animals through Zymosan A (ZyA) injection. The inflammatory response was assessed by measuring cell count and cytokine release. In parallel, the study investigated the expression of SEMA7A in intestinal epithelial cells under inflammatory stimuli and its impact on interleukin 10 (IL-10) production using an in vitro co-culture model of monocytes and epithelial cells. Additionally, the distribution of SEMA7A target receptors, particularly ITGAV/ITGB1 (CD51/CD29), was analyzed in WT animals. Results: The results revealed that SEMA7A-/- animals exhibited increased inflammatory peritonitis compared to the WT animals. Inflammatory conditions in intestinal epithelial cells led to the induction of SEMA7A. The co-culture experiments demonstrated that SEMA7A induced IL-10 production, which depended on integrin receptors and was independent of PLXNC1 expression. Furthermore, ITGAV/ITGB1 emerged as the predominant SEMA7A receptor in the intestinal area of WT animals. Discussion: These findings underscore the multifaceted role of SEMA7A in inflammatory processes. The differential responses in peritonitis and intestinal inflammation suggest that SEMA7A's function is significantly influenced by the expression and distribution of its target receptors within different organ systems. The study highlights the complex and context-dependent nature of SEMA7A in mediating inflammatory responses.

Platelets and the Cybernetic Regulation of Ischemic Inflammatory Responses through PNC Formation Regulated by Extracellular Nucleotide Metabolism and Signaling.

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet-neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y12, ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet-neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Inhibition of CXCR4 and CXCR7 Is Protective in Acute Peritoneal Inflammation.

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 A2B knockout animals (A2B-/-). 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 A2B-/- animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. 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 A2B-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 A2B-receptor signaling.

Red blood cell-derived semaphorin 7A promotes thrombo-inflammation in myocardial ischemia-reperfusion injury through platelet GPIb.

Myocardial ischemia is one of the leading health problems worldwide. Therapy consists of the restitution of coronary perfusion which is followed by myocardial inflammation. Platelet-neutrophil interaction is a crucial process during inflammation, yet its consequences are not fully understood. Here, we show that platelet-neutrophil complexes (PNCs) are increased in patients with acute myocardial infarction and that this is associated with increased levels of neuronal guidance protein semaphorin 7A (SEMA7A). To investigate this further, we injected WT animals with Sema7a and found increased infarct size with increased numbers of PNCs. Experiments in genetically modified animals identify Sema7a on red blood cells to be crucial for this condition. Further studies revealed that Sema7a interacts with the platelet receptor glycoprotein Ib (GPIb). Treatment with anti-Sema7a antibody protected from myocardial tissue injury. In summary, we show that Sema7a binds to platelet GPIb and enhances platelet thrombo-inflammatory activity, aggravating post-ischemic myocardial tissue injury.