Carla Jennewein

MicroRNA-27b Contributes to Lipopolysaccharide-mediated Peroxisome Proliferator-activated Receptor γ (PPARγ) mRNA Destabilization

Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.

Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis?

The endothelial integrity, as mechanical barrier against microorganisms and as natural “anticoagulant”, is crucial for physiologic organ function. Systemic activation of the endothelium upon inflammation, sepsis, and septic shock is always ending in blood–tissue barrier disruption. With increasing dysfunction, uncontrolled clotting activation, capillary microthrombi formation, tissue edema, local hypoxia, and ischemia are initiated. This in turn enhances a vicious circle leading to multiple organ failure and death. Therefore, biomarkers reflecting this special compartment may help in the early detection of systemic inflammation and its complications. This review provides an overview of the most important endothelial biomarkers and their possible use in sepsis.

Sumoylation of peroxisome proliferator-activated receptor gamma by apoptotic cells prevents lipopolysaccharide-induced NCoR removal from kappaB binding sites mediating transrepression of proinflammatory cytokines.

Efficient clearance of apoptotic cells (AC) by professional phagocytes is crucial for tissue homeostasis and resolution of inflammation. Macrophages respond to AC with an increase in antiinflammatory cytokine production but a diminished release of proinflammatory mediators. Mechanisms to explain attenuated proinflammatory cytokine formation remain elusive. We provide evidence that peroxisome proliferator-activated receptor γ (PPARγ) coordinates antiinflammatory responses following its activation by AC. Exposing murine RAW264.7 macrophages to AC before LPS stimulation reduced NF-κB transactivation and lowered target gene expression of, that is, TNF-α and IL-6 compared with controls. In macrophages overexpressing a dominant negative mutant of PPARγ, NF-κB transactivation in response to LPS was restored, while macrophages from myeloid lineage-specific conditional PPARγ knockout mice proved that PPARγ transmitted an antiinflammatory response, which was delivered by AC. Expressing a PPARγ-Δaa32–250 deletion mutant, we observed no inhibition of NF-κB. Analyzing the PPARγ domain structures within aa 32–250, we anticipated PPARγ sumoylation in mediating the antiinflammatory effect in response to AC. Interfering with sumoylation of PPARγ by mutating the predicted sumoylation site (K77R), or knockdown of the small ubiquitin-like modifier (SUMO) E3 ligase PIAS1 (protein inhibitor of activated STAT1), eliminated the ability of AC to suppress NF-κB. Chromatin immunoprecipitation analysis demonstrated that AC prevented the LPS-induced removal of nuclear receptor corepressor (NCoR) from the κB site within the TNF-α promoter. We conclude that AC induce PPARγ sumoylation to attenuate the removal of NCoR, thereby blocking transactivation of NF-κB. This contributes to an antiinflammatory phenotype shift in macrophages responding to AC by lowering proinflammatory cytokine production.

The liaison between apoptotic cells and macrophages--the end programs the beginning.

Abstract The efficient execution of apoptotic cell death with the clearance of apoptotic debris by phagocytes is a key regulatory mechanism ensuring tissue homeostasis. Failure in this execution program contributes to the pathogenesis of many human diseases. In this review, we describe the current knowledge regarding the interaction of apoptotic cells with their professional ‘captors’, the macrophages, with special emphasis on the immunological outcome. Removal of apoptotic cells must be considered as a process that actively delivers signals to polarize macrophages, which are fundamental for the resolution of inflammation. However, the sculpting of macrophage responses by apoptotic cells can be misused under certain inflammatory disease conditions, including tumor development.

Casein-kinase-II-dependent phosphorylation of PPARγ provokes CRM1-mediated shuttling of PPARγ from the nucleus to the cytosol

PPARγ exerts significant anti-inflammatory signaling properties in monocytes and macrophages, which are affected by its intracellular localization. Based on our previous report, which showed that cytosolic localization of PPARγ attenuates PKCα signaling in macrophages, we elucidated the molecular mechanisms provoking cytosolic PPARγ localization. Using the DsRed-tagged PPARγ deletion constructs PPARγ1 Δ1-31 and PPARγ1 Δ407-475, we observed an exclusive nuclear PPARγ1 Δ1-31 localization in transfected HEK293 cells, whereas PPARγ1 Δ407-475 did not alter its cytosolic or nuclear localization. The casein kinase II (CK-II) inhibitor 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole (DRB) prevented cytosolic PPARγ localization. Mutation of two possible CK-II phosphorylation sites at serine 16 and serine 21 of PPARγ into alanine (PPARγ S16A/S21A) inhibited cytosolic PPARγ localization. Moreover, a PPARγ S16E/S21E mutant that mimicks constitutive phosphorylation of residues 16 and 21, predominantly resides in the cytosol. The CRM1 inhibitor leptomycin B abolished cytosolic PPARγ localization, suggesting that this is a CRM1-dependent export process. CRM1-mediated PPARγ export requires Ran and phosphorylated RanBP3. Finally, co-immunoprecipitation studies demonstrated that DRB blocks PPARγ binding to CRM1, whereas PD98059 inhibits RanBP3 binding to CRM1 and concomitant shuttling from nucleus to cytosol, but does not alter PPARγ binding to CRM1. We conclude that CK-II-dependent PPARγ phosphorylation at Ser16 and Ser21 is necessary for CRM1/Ran/RanBP3-mediated nucleocytoplasmic translocation of PPARγ.

Linking inflammation and coagulation: novel drug targets to treat organ ischemia.

Purpose of review Activation of the coagulation system during ischemia/reperfusion injury is an unavoidable event and even further augmented during cardiovascular surgery. Clotting not only leads to disturbance of blood rheology but also enhances the inflammatory response. We aim to highlight the inflammatory properties of the coagulation system and novel potential therapeutic approaches targeting both features. Recent findings Heparin, a thrombin inhibitor, is still the drug of choice for preventing coagulation following, for example, cardiovascular surgery. On the contrary, much effort is done to evaluate the utilization of direct thrombin inhibitors to prevent ischemia/reperfusion injury. Furthermore, targeting the inflammatory potential of the coagulation system seems to be very promising. Fibrin(ogen) and its degradation products modulate the inflammatory response, especially by inducing leukocyte migration. Inhibiting these pro-inflammatory effects, for example, by administration of B&bgr;15–42 was recently shown to be beneficial under various inflammatory conditions. Summary Ischemia and reperfusion are common activators of coagulation that is also accompanied by inflammation. Therefore, targeting this well orchestrated system might be of therapeutic benefit, as its mode of action is dual: clotting inhibition and anti-inflammation. This novel therapeutic approach might at least be of benefit in the treatment of systemic inflammatory syndromes following, that is, cardiovascular surgery.

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

Contribution of Ninjurin1 to Toll-Like Receptor 4 Signaling and Systemic Inflammation

Nerve injury-induced protein (Ninjurin [Ninj]) 1 is an adhesion molecule originally identified in Schwann cells after nerve injury, whereas it is also expressed in leukocytes, epithelium, endothelium, and various organs, and is induced under inflammatory conditions. Its contribution to inflammation was so far restricted to the nervous system and exclusively attributed to its role during leukocyte migration. We hypothesized a proinflammatory role for Ninj1 also outside the nervous system. To elucidate its impact during inflammation, we analyzed expression levels and its contribution to inflammation in septic mice and studied its effect on inflammatory signaling in vitro. The effect on inflammation was analyzed by genetic (only in vitro) and pharmacologic repression in septic mice (cecal ligation and puncture) and cell culture, respectively. Repression of Ninj1 by an inhibitory peptide or small interfering RNA attenuated LPS-triggered inflammation in macrophages and endothelial cells by modulating p38 phosphorylation and activator protein-1 activation. Inhibition of Ninj1 in septic mice reduced systemic and pulmonary inflammation as well as organ damage, and ameliorated survival after 24 hours. Ninj1 is elevated under inflammatory conditions and contributes to inflammation not only by mediating leukocyte migration, but also by modulating Toll-like receptor 4-dependent expression of inflammatory mediators. We assume that, owing to both mechanisms, inhibition reduces systemic inflammation and organ damage in septic mice. Our data contribute to a better understanding of the complex inflammatory mechanisms and add a novel therapeutic target for inflammatory conditions such as sepsis.

The Small Fibrinopeptide Bβ15–42 as Renoprotective Agent Preserving the Endothelial and Vascular Integrity in Early Ischemia Reperfusion Injury in the Mouse Kidney

Disruption of the renal endothelial integrity is pivotal for the development of a vascular leak, tissue edema and consequently acute kidney injury. Kidney ischemia amplifies endothelial activation and up-regulation of pro-inflammatory mechanisms. After restoring a sufficient blood flow, the kidney is damaged through complex pathomechanisms that are classically referred to as ischemia and reperfusion injury, where the disruption of the inter-endothelial connections seems to be a crucial step in this pathomechanism. Focusing on the molecular cell-cell interaction, the fibrinopeptide Bβ15–42 prevents vascular leakage by stabilizing these inter-endothelial junctions. The peptide associates with vascular endothelial-cadherin, thus preventing early kidney dysfunction by preserving blood perfusion efficacy, edema formation and thus organ dysfunction. We intended to demonstrate the early therapeutic benefit of intravenously administered Bβ15–42 in a mouse model of renal ischemia and reperfusion. After 30 minutes of ischemia, the fibrinopeptide Bβ15–42 was administered intravenously before reperfusion was commenced for 1 and 3 hours. We show that Bβ15–42 alleviates early functional and morphological kidney damage as soon as 1 h and 3 h after ischemia and reperfusion. Mice treated with Bβ15–42 displayed a significantly reduced loss of VE-cadherin, indicating a conserved endothelial barrier leading to less neutrophil infiltration which in turn resulted in significantly reduced structural renal damage. The significant reduction in tissue and serum neutrophil gelatinase-associated lipocalin levels reinforced our findings. Moreover, renal perfusion analysis by color duplex sonography revealed that Bβ15–42 treatment preserved resistive indices and even improved blood velocity. Our data demonstrate the efficacy of early therapeutic intervention using the fibrinopeptide Bβ15–42 in the treatment of acute kidney injury resulting from ischemia and reperfusion. In this context Bβ15–42 may act as a potent renoprotective agent by preserving the endothelial and vascular integrity.