Klemens König

The neuronal guidance protein netrin-1 reduces alveolar inflammation in a porcine model of acute lung injury

IntroductionAcute lung injury (ALI) is an inflammatory disorder of pulmonary or extrapulmonary origin. We have previously demonstrated that netrin-1 dampens murine ALI, and in an attempt to advance this finding into future clinical practice we evaluated whether netrin-1 would reduce alveolar inflammation during porcine ALI.MethodsThis was a controlled in vivo experimental study in pigs. We induced ALI through lipoploysaccharide (LPS) infusion (50 μg/kg) for 2 hours. Following this, we exposed animals to either vehicle, intravenous netrin-1 (netrin-1 i.v.) or inhaled netrin-1 (netrin-1 inh.). Serum samples and bronchoalveolar lavage (BAL) were obtained to determine levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, interleukin-6 and interleukin-8 at baseline and 6 hours following treatment. Myeloperoxidase activity (MPO) and protein levels were determined in the BAL, and tissue samples were obtained for histological evaluation. Finally, animals were scanned with spiral CT.ResultsFollowing LPS infusion, animals developed acute pulmonary injury. Serum levels of TNF-α and IL-6 were significantly reduced in the netrin-1 i.v. group. BAL demonstrated significantly reduced cytokine levels 6 hours post-netrin-1 treatment (TNF-α: vehicle 633 ± 172 pg/ml, netrin-1 i.v. 84 ± 5 pg/ml, netrin-1 inh. 168 ± 74 pg/ml; both P < 0.05). MPO activity and protein content were significantly reduced in BAL samples from netrin-1-treated animals. Histological sections confirmed reduced inflammatory changes in the netrin-1-treated animals. Computed tomography corroborated reduced pulmonary damage in both netrin-1-treated groups.ConclusionsWe conclude that treatment with the endogenous anti-inflammatory protein netrin-1 reduces pulmonary inflammation during the initial stages of ALI and should be pursued as a future therapeutic option.

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.

The guidance receptor neogenin promotes pulmonary inflammation during lung injury

Lung injury is marked by a persistent self‐propagating inflammation within the pulmonary tissue that is initiated by the migration of leukocytes into the alveolar space. Recent work has demonstrated that neuronal guidance proteins are involved into the orchestration of leukocyte migration. Neogenin is a crucial guidance receptor for axonal migration, yet its role during leukocyte migration and acute inflammation is to date unknown. Here, we report that neogenin influences neutrophil migration across endothelial HMEC‐1 and alveolar A549 monolayers in vitro. In vivo, Neo1‐/‐ mice demonstrated 59% reduced cell count, 41% reduced TNF‐α, and 76% reduced IL‐6 levels within the alveolar space during lung injury. In studies employing chimeric animals, the presence of Neo1‐/ bone marrow was associated with a 42% reduction of cell count and reduced inflammatory changes within pulmonary tissue during lung injury. The functional inhibition of neogenin through antibody injection confirmed these results and the role of neogenin for the inflammatory changes within the alveolar space. Previously unappreciated, the guidance receptor neogenin has a significant effect on the orchestration of leukocyte migration and the control of acute inflammation.—Mirakaj, V., Jennewein, C., König, K., Granja, T., Rosenberger, P. The guidance receptor neogenin promotes pulmonary inflammation during lung injury. FASEB J. 26, 1549‐1558 (2012). www.fasebj.org

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.

Inhibition of Plexin C1 Protects Against Hepatic Ischemia-Reperfusion Injury.

Objectives:Hepatic ischemia-reperfusion injury is a disease pattern that is associated with an acute inflammatory reaction. It is well known that neutrophils play an essential role in the early phase of hepatic ischemia-reperfusion injury and determine the extent of tissue damage. Hepatic ischemia-reperfusion injury can result in organ failure, which is linked to high mortality. Recent data indicate that the neuronal guidance receptor Plexin C1 is involved in the control of the acute inflammatory response and, as such, modulates the transmigration of neutrophils. Hence, we investigated the functional role of Plexin C1 in a mouse model of early hepatic ischemia-reperfusion injury. Design:Animal study. Setting:University experimental laboratory. Subjects:Wild-type, PLXNC1−/− and chimeric mice. Interventions:Hepatic ischemia-reperfusion injury or sham operation. Measurements and Main Results:We found that the functional inhibition of Plexin C1 in wild-type mice treated with an anti-Plexin C1 antibody and a Semaphorin 7A peptide reduced hepatic ischemia-reperfusion injury, as measured by the levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. This reduction in ischemia-reperfusion injury was accompanied by reduced numbers of neutrophils in ischemic hepatic tissue and reduced serum levels of inflammatory cytokines. Experiments using Plexin C1 receptor–deficient (PLXNC1−/−) mice also demonstrated decreased hepatic ischemia-reperfusion injury. Studies of chimeric mice revealed that the hematopoietic Plexin C1 knockout is crucial for reducing the extent of hepatic ischemia-reperfusion injury. Conclusions:These results describe a role for Plexin C1 during ischemia-reperfusion injury, highlight the role of hematopoietic Plexin C1 in the development of hepatic ischemia-reperfusion injury, and suggest that Plexin C1 is a potential drug target.

The uncoordinated-5 homolog B (UNC5B) receptor increases myocardial ischemia-reperfusion injury.

The UNC5 receptor family are chemorepulsive neuronal guidance receptors with additional functions outside the central nervous system. Previous studies have implicated that the UNC5B receptor influences the migration of leukocytes into sites of tissue inflammation. Given that this process is a critical step during the pathophysiology of myocardial ischemia followed by reperfusion (IR) we investigated the role of UNC5B during myocardial IR. In initial in-vitro experiments, the functional inhibition of UNC5B resulted in a significant reduction of chemotactic migration of neutrophils. In-vivo, using a model of acute myocardial ischemia in UNC5B+/− and wild type (WT) animals, we found a significant reduction of infarct sizes in UNC5B+/− animals. This was associated with significantly reduced levels of troponin-I and IL-6 in UNC5B+/− mice. The repression of UNC5B using siRNA and the functional inhibition of UNC5B significantly dampened the extent of myocardial IR injury. Following depletion of neutrophils, we were not able to observe any further reduction in infarct size through functional inhibition of UNC5B in WT and UNC5B+/− mice. In summary our studies demonstrate an important role for UNC5B during myocardial IR injury, and that UNC5B might be a potential therapeutic target to control reperfusion injury in the future.

The uncoordinated-5 homolog B receptor affects hepatic ischemia reperfusion injury

Recent evidence has demonstrated additional roles for the neuronal guidance protein receptor UNC5B outside the nervous system. Given the fact that ischemia reperfusion injury (IRI) of the liver is a common source of liver dysfunction and the role of UNC5B during an acute inflammatory response we investigated the role of UNC5B on acute hepatic IRI. We report here that UNC5B+/− mice display reduced hepatic IRI and neutrophil (PMN) infiltration compared to WT controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate- (AST) and alanine- (ALT) aminotransferase, the presence of PMN within ischemic hepatic tissue, and serum levels of inflammatory cytokines. Moreover, injection of an anti-UNC5B antibody resulted in a significant reduction of hepatic IR injury. This was associated with reduced parameters of liver injury (LDH, ALT, AST) and accumulation of PMN within the injured hepatic tissue. In conclusion our studies demonstrate a significant role for UNC5B in the development of hepatic IRI and identified UNC5B as a potential drug target to prevent liver dysfunction in the future.

Aggressive versus conservative initiation of antibiotics

www.thelancet.com/infection Vol 13 May 2013 389 2 Kumar A, Roberts D, Wood KE, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006; 34: 1589–96. 3 Gaieski DF, Mikkelsen ME, Band RA, et al. Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department. Crit Care Med 2010; 38: 1045–53. 4 Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008; 36: 296–327. 5 Ranieri VM, Thompson BT, Barie PS, et al. Drotrecogin alfa (activated) in adults with septic shock. N Engl J Med 2012; 366: 2055–64. than 2 h. It is unclear why an ICU should be different, especially when the starting point is the drawing of blood cultures. By treating their aggressive group very late, the potential benefit of early antibiotics might have waned, while the hypothesised detrimental effects of empirical antibiotics remained. If true, a high case-fatality rate would be expected in the aggressive group. A high rate was indeed reported: overall 40% died in the aggressive group, with 66% mortality in the subset with septic shock. By contrast, the case fatality rate in the recent PROWESS-SHOCK study, in which all patients had septic shock, was only 24·2–26·4%. Waiting for positive cultures before starting antibiotics might only be effective when compared with very delayed empirical therapy. The aggressive protocol required antibiotics to be discontinued at day 3 if no positive cultures had been obtained. All the patients in Hranjec and colleagues’ study were in a surgical ICU, so presumably most received intraoperative prophylactic antibiotics, which might have increased the chance of a negative culture result. Mortality in the aggressive group might have been artificially high because of this policy. These substantial concerns warrant the investigators’ interpretation that practice should only change (if at all) in a context in which outcomes are rigorously and frequently analysed.