Tiago Granja

Vagus nerve controls resolution and pro-resolving mediators of inflammation

Axonal guidance molecule netrin-1 promotes resolution of inflammation, with netrin-1 and resolvin D1 mutually inducing each other’s expression.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury

Objective:To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. Design:C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01–1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. Setting:University hospital research laboratory. Subjects:Anesthetized male mice and murine cardiomyocytes. Measurements and Main Results:Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. Conclusions:Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

The neuroimmune guidance cue netrin-1 controls resolution programs and promotes liver regeneration.

Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti‐inflammatory properties of the axonal guidance cue netrin‐1 were reported. Here, we demonstrate that netrin‐1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin‐1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin‐1 deficiency (Ntn1+/−) or wild‐type C57BL/6 treated with exogenous netrin‐1 to examine the endogenous and therapeutically administered impact of netrin‐1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin‐1 transcript and protein in murine liver tissue. Subsequent studies in netrin‐1‐deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic‐specific injury enzymes. Conversely, mice treated with exogenous netrin‐1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin‐1‐mediated protection during hepatic I/R injury. Conclusion: The present study indicates a previously unrecognized role for netrin‐1 in liver protection and its contribution to tissue homeostasis and regeneration. (Hepatology 2016;63:1689‐1705)

Toll-like receptor 2 signaling triggers fatal arrhythmias upon myocardial ischemia-reperfusion

Objective:Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors. Toll-like receptor 2 deficiency is associated with a reduced infarct size after myocardial ischemia and reperfusion. Because a marked mortality was observed in C3HeN wild-type mice, which was absent in TLR2−/− mice, we tested whether cardiac arrhythmias are the underlying pathology and aimed to elucidate how toll-like receptor 2 ligation might prevent lethal arrhythmias. Design:Experimental animal model. Setting:University hospital research laboratory. Subjects:Male C3HeN mice. Interventions:Myocardial ischemia and reperfusion was surgically induced by ligation of the left anterior descending coronary artery for 20 mins followed by 24 hrs of reperfusion. Electrocardiography was continuously recorded during the observation period through an implantable telemetry transmitter to detect cardiac arrhythmias during reperfusion. Measurements and Main Results:Toll-like receptor 2 expression was associated with a 51% mortality rate (23 of 45 mice died) after myocardial ischemia and reperfusion. Absence of toll-like receptor 2 improved survival toward 100% (17 of 17 mice survived). Electrocardiography diagnostics in conscious animals and histologic analysis revealed that absence of toll-like receptor 2 signaling prevented the formation of pathologic heart rate turbulence after myocardial ischemia and reperfusion and modulated the density of connexin 43-positive gap junctions in the ischemic area compared with wild-type hearts, indicating arrhythmia as the cause underlying the observed mortality. Conclusions:The results presented here indicate toll-like receptor 2 as a novel target for the prevention of lethal arrhythmic complications after myocardial ischemia and reperfusion.

The plexin C1 receptor promotes acute inflammation.

Acute inflammation is the pathophysiological basis of important clinical conditions associated with organ failure. The initial inflammatory response is controlled by the chemokine system, yet recent data have indicated that the neuronal guidance cues are significantly involved in the orchestration of this process. Previous work has shown the proinflammatory capacity of the guidance cue semaphorin (Sema) 7a, but the role of one of its target receptors, the plexin C1 (PLXNC1) receptor is to date unknown. We report here that PLXNC1 is expressed outside the nervous system and induced during acute inflammation. PLXNC1−/− mice with C57BL/6 background demonstrated decreased inflammatory responses during zymosan A (ZyA)‐induced peritonitis. Subsequent in vivo studies revealed altered rolling, adhesion, and transmigration properties of PLXNC1−/− leukocytes. Blockade of PLXNC1 was associated with attenuated chemotactic transendothelial migration properties in vitro. Studies in chimeric mice revealed that hematopoietic PLXNC1−/− animals demonstrated an attenuated inflammatory response. To probe the therapeutic potential of PLXNC1 we treated C57BL/6 WT mice with an anti‐PLXNC1 antibody and a PLXNC1 binding peptide. Both of these interventions significantly dampened ZyA‐induced peritonitis. These results implicate an important role of PLXNC1 during an acute inflammatory response and indicate PLXNC1 as a potential target for the control of conditions associated with acute inflammation.

Crucial role of Plexin C1 for pulmonary inflammation and survival during lung injury

Acute pulmonary inflammation during lung injury is initiated by the migration of neutrophils into the alveolar space. The severity of these inflammatory changes within the pulmonary tissue determines the severity of lung injury and ultimately patient outcome. Recent work has demonstrated that the guidance protein Semaphorin 7A propagates the infiltration of neutrophils into an hypoxic tissue site, yet the role of its target receptor Plexin C1 (PLXNC1) during lung injury is to date unknown. We demonstrate here that PLXNC1+ neutrophils are present within the alveolar space and that PLXNC1 is induced in vitro and in vivo during lung injury. In a model of high-pressure ventilation PLXNC1−/− animals show decreased signs of alveolar inflammation and improved survival compared with wild-type controls. Studies employing chimeric animals identified the hematopoietic expression of PLXNC1 to be of crucial importance for the observed results. Functional inhibition of PLXNC1 resulted in improved survival and ameliorated the signs of inflammation within the lung. Furthermore, the injection of a peptide binding to PLXNC1 resulted in improved survival and attenuated pulmonary inflammation. As such we demonstrate here, that previously unknown PLXNC1 holds significant importance for degree of pulmonary inflammation and determines outcome during experimental lung injury.

Gαi2- and Gαi3-Deficient Mice Display Opposite Severity of Myocardial Ischemia Reperfusion Injury

G-protein-coupled receptors (GPCRs) are the most abundant receptors in the heart and therefore are common targets for cardiovascular therapeutics. The activated GPCRs transduce their signals via heterotrimeric G-proteins. The four major families of G-proteins identified so far are specified through their α-subunit: Gαi, Gαs, Gαq and G12/13. Gαi-proteins have been reported to protect hearts from ischemia reperfusion injury. However, determining the individual impact of Gαi2 or Gαi3 on myocardial ischemia injury has not been clarified yet. Here, we first investigated expression of Gαi2 and Gαi3 on transcriptional level by quantitative PCR and on protein level by immunoblot analysis as well as by immunofluorescence in cardiac tissues of wild-type, Gαi2-, and Gαi3-deficient mice. Gαi2 was expressed at higher levels than Gαi3 in murine hearts, and irrespective of the isoform being knocked out we observed an up regulation of the remaining Gαi-protein. Myocardial ischemia promptly regulated cardiac mRNA and with a slight delay protein levels of both Gαi2 and Gαi3, indicating important roles for both Gαi isoforms. Furthermore, ischemia reperfusion injury in Gαi2- and Gαi3-deficient mice exhibited opposite outcomes. Whereas the absence of Gαi2 significantly increased the infarct size in the heart, the absence of Gαi3 or the concomitant upregulation of Gαi2 dramatically reduced cardiac infarction. In conclusion, we demonstrate for the first time that the genetic ablation of Gαi proteins has protective or deleterious effects on cardiac ischemia reperfusion injury depending on the isoform being absent.

Inhibition of neogenin dampens hepatic ischemia-reperfusion injury.

Objective:Liver ischemia and reperfusion injury is a common source of significant morbidity and mortality following liver transplantation, hemorrhagic shock, or major hepatic surgery. Based on studies showing a critical role for the neuronal guidance receptor neogenin (Neo1) outside the nervous system in mediating tissue adaption during acute inflammation, we hypothesized that Neo1 enhances hepatic ischemia and reperfusion injury. Design:Animal study. Setting:University-based experimental laboratory. Subjects:Wid-type, neogenin deficient and chimeric mice. Interventions:Neogenin expression was evaluated during inflammatory stimulation in vitro and during ischemia and reperfusion injury in vivo, intravital microscopy performed to study intravascular flow characteristics. The extent of liver injury was evaluated using histology, serum levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. The functional role of Neo1 during liver IR was evaluated in mice with gene targeted repression of neogenin (Neo1-/-), bone marrow chimeric animals and controls. In addition, functional inhibition of neogenin was performed using antibody injection. Measurements and Main Results:We observed an induction of Neo1 during inflammation in vitro and ischemia and reperfusion in vivo. Intravital microscopy demonstrated a decreased ability of Neo1−/− leukocytes to attach to endothelial vascular wall during inflammation. Subsequent studies in Neo1−/− mice showed attenuated serum levels of lactate dehydrogenase, aspartate, alanine, and proinflammatory cytokines during hepatic ischemia and reperfusion injury. This was associated with improved hepatic histology scores. Studies in chimeric animals demonstrated that the hematopoietic Neo1 expression to be crucial for the observed results. Treatment with an anti-Neo1 antibody resulted in a significant reduction of experimental hepatic ischemia and reperfusion injury, involving attenuated variable of lactate dehydrogenase, alanine, aspartate, and cytokine levels. Conclusions:These data provide a unique role for Neo1 in the development of hepatic ischemia and reperfusion injury and identified Neo1 as a potential target to prevent liver dysfunction in the future.

The Axonal Guidance Receptor Neogenin Promotes Acute Inflammation

Neuronal guidance proteins (NGP) were originally described in the context of axonal growth and migration. Yet recent work has demonstrated that NGPs also serve as guidance cues for immune competent cells. A crucial target receptor for NGPs during embryonic development is the neogenin receptor, however its role during acute inflammation is unknown. We report here that neogenin is abundantly expressed outside the nervous system and that animals with endogenous repression of neogenin (Neo1−/−) demonstrate attenuated changes of acute inflammation. Studies using functional inhibition of neogenin resulted in a significant attenuation of inflammatory peritonitis. In studies employing bone marrow chimeric animals we found the hematopoietic presence of Neo1−/− to be responsible for the attenuated inflammatory response. Taken together our studies suggest that the guidance receptor neogenin holds crucial importance for the propagation of an acute inflammatory response and further define mechanisms shared between the nervous and the immune system.

Semaphorin 7A Aggravates Pulmonary Inflammation during Lung Injury.

The extent of pulmonary inflammation during lung injury ultimately determines patient outcome. Pulmonary inflammation is initiated by the migration of neutrophils into the alveolar space. Recent work has demonstrated that the guidance protein semaphorin 7A (SEMA7A) influences the migration of neutrophils into hypoxic tissue sites, yet, its role during lung injury is not well understood. Here, we report that the expression of SEMA7A is induced in vitro through pro-inflammatory cytokines. SEMA7A itself induces the production of pro-inflammatory cytokines in endothelial and epithelial cells, enhancing pulmonary inflammation. The induction of SEMA7A facilitates the transendothelial migration of neutrophils. In vivo, animals with deletion of SEMA7A expression showed reduced signs of pulmonary inflammatory changes following lipopolysaccharide challenge. We define here the role of SEMA7A in the development of lung injury and identify a potential pathway to interfere with these detrimental changes. Future anti-inflammatory strategies for the treatment of lung injury might be based on this finding.