Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4–7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A–sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s–deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed–specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.
Takashi Minami, Kiichiro Yano, Mai Miura, Mika Kobayashi, Jun-ichi Suehiro, Patrick C. Reid, Takao Hamakubo, Sandra Ryeom, William C. Aird, Tatsuhiko Kodama
Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ecto-nucleotidases, including CD73 and ecto–nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.
Thomas Weissmüller, Eric L. Campbell, Peter Rosenberger, Melanie Scully, Paul L. Beck, Glenn T. Furuta, Sean P. Colgan
Inflammation plays a critical role in the development of cardiovascular diseases. Infiltration of leukocytes to sites of injury requires their exit from the blood and migration across basement membrane; this process has been postulated to require remodeling of the ECM. Plasminogen (Plg) is a protease that binds to the ECM and, upon conversion to plasmin, degrades multiple ECM proteins. In addition, plasmin directly activates MMPs. Here, we used Plg–/– mice to investigate the role of Plg in inflammatory leukocyte migration. After induction of peritonitis by thioglycollate injection, we found that Plg–/– mice displayed diminished macrophage trans-ECM migration and decreased MMP-9 activation. Furthermore, injection of the active form of MMP-9 in Plg–/– mice rescued macrophage migration in this model. We used periaortic application of CaCl2 to induce abdominal aortic aneurysm (AAA) and found that Plg–/– mice displayed reduced macrophage infiltration and were protected from aneurysm formation. Administration of active MMP-9 to Plg–/– mice promoted macrophage infiltration and the development of AAA. These data suggest that Plg regulates macrophage migration in inflammation via activation of MMP-9, which, in turn, regulates the ability of the cells to migrate across ECM. Thus, targeting the Plg/MMP-9 pathway may be an attractive approach to regulate inflammatory responses and AAA development.
Yanqing Gong, Erika Hart, Aleksey Shchurin, Jane Hoover-Plow
Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.
Kai Kessenbrock, Leopold Fröhlich, Michael Sixt, Tim Lämmermann, Heiko Pfister, Andrew Bateman, Azzaq Belaaouaj, Johannes Ring, Markus Ollert, Reinhard Fässler, Dieter E. Jenne
In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapse-associated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8–/– mice in response to these treatments. In addition, although ZGs accumulated in Vamp8–/– acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8–/– acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion.
Laura I. Cosen-Binker, Marcelo G. Binker, Cheng-Chun Wang, Wanjin Hong, Herbert Y. Gaisano
Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-α, and IL-6, than typical intestinal resident macrophages (CD14–CD33+ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-α compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14+ macrophages contributed to IFN-γ production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-α. Furthermore, the IFN-γ produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23–hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-γ–positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.
Nobuhiko Kamada, Tadakazu Hisamatsu, Susumu Okamoto, Hiroshi Chinen, Taku Kobayashi, Toshiro Sato, Atsushi Sakuraba, Mina T. Kitazume, Akira Sugita, Kazutaka Koganei, Kiyoko S. Akagawa, Toshifumi Hibi
In the liver, the JNK cascade is induced downstream of TNF receptors (TNFRs) in response to inflammatory, microbial, and toxic challenges. Sustained activation of JNK triggers programmed cell death (PCD), and hepatocyte survival during these challenges requires induction of the NF-κB pathway, which antagonizes this activation by upregulating target genes. Thus, modulation of JNK activity is crucial to the liver response to TNFR-mediated challenge. The basis for this modulation, however, is unknown. Here, we investigated the role of the NF-κB target Gadd45b in the regulation of hepatocyte fate during liver regeneration after partial hepatectomy. We generated Gadd45b–/– mice and found that they exhibited decreased hepatocyte proliferation and increased PCD during liver regeneration. Notably, JNK activity was markedly increased and sustained in livers of Gadd45b–/– mice compared with control animals after partial hepatectomy. Furthermore, imposition of a Jnk2-null mutation, attenuating JNK activity, completely rescued the regenerative response in Gadd45b–/– mice. Interestingly, Gadd45β ablation did not affect hepatotoxic JNK signaling after a TNFR-mediated immune challenge, suggesting specificity in the inducible hepatic program for JNK restraint activated during distinct TNFR-mediated challenges. These data provide a basis for JNK suppression during liver regeneration and identify Gadd45β as a potential therapeutic target in liver diseases.
Salvatore Papa, Francesca Zazzeroni, Yang-Xin Fu, Concetta Bubici, Kellean Alvarez, Kathryn Dean, Peter A. Christiansen, Robert A. Anders, Guido Franzoso
Septic shock results from an uncontrolled inflammatory response, mediated primarily by LPS. Cholesterol transport plays an important role in the host response to LPS, as LPS is neutralized by lipoproteins and adrenal cholesterol uptake is required for antiinflammatory glucocorticoid synthesis. In this study, we show that scavenger receptor B-I (SR-BI), an HDL receptor that mediates HDL cholesterol ester uptake into cells, is required for the normal antiinflammatory response to LPS-induced endotoxic shock. Despite elevated plasma HDL levels, SR-BI–null mice displayed an uncontrollable inflammatory cytokine response and a markedly higher lethality rate than control mice in response to LPS. In addition, SR-BI–null mice showed a lack of inducible glucocorticoid synthesis in response to LPS, bacterial infection, stress, or ACTH. Glucocorticoid insufficiency in SR-BI–null mice was due to primary adrenal malfunction resulting from deficient cholesterol delivery from HDL. Furthermore, corticosterone supplementation decreased the sensitivity of SR-BI–null mice to LPS. Plasma from control and SR-BI–null mice exhibited a similar ability to neutralize LPS, whereas SR-BI–null mice showed decreased plasma clearance of LPS into the liver and hepatocytes compared with normal mice. We conclude that SR-BI in mice is required for the antiinflammatory response to LPS-induced endotoxic shock, likely through its essential role in facilitating glucocorticoid production and LPS hepatic clearance.
Lei Cai, Ailing Ji, Frederick C. de Beer, Lisa R. Tannock, Deneys R. van der Westhuyzen
Sepsis is characterized by a systemic response to severe infection. Although the inflammatory phase of sepsis helps eradicate the infection, it can have detrimental consequences if left unchecked. Therapy directed against inflammatory mediators of sepsis has shown little success and has the potential to impair innate antimicrobial defenses. Heme oxygenase-1 (HO-1) and the product of its enzymatic reaction, CO, have beneficial antiinflammatory properties, but little is known about their effects on microbial sepsis. Here, we have demonstrated that during microbial sepsis, HO-1–derived CO plays an important role in the antimicrobial process without inhibiting the inflammatory response. HO-1–deficient mice suffered exaggerated lethality from polymicrobial sepsis. Targeting HO-1 to SMCs and myofibroblasts of blood vessels and bowel ameliorated sepsis-induced death associated with Enterococcus faecalis, but not Escherichia coli, infection. The increase in HO-1 expression did not suppress circulating inflammatory cells or their accumulation at the site of injury but did enhance bacterial clearance by increasing phagocytosis and the endogenous antimicrobial response. Furthermore, injection of a CO-releasing molecule into WT mice increased phagocytosis and rescued HO-1–deficient mice from sepsis-induced lethality. These data advocate HO-1–derived CO as an important mediator of the host defense response to sepsis and suggest CO administration as a possible treatment for the disease.
Su Wol Chung, Xiaoli Liu, Alvaro A. Macias, Rebecca M. Baron, Mark A. Perrella
Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib–) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin αMβ2 binding motif (Fibγ390–396A) or the αIIbβ3 platelet integrin-binding motif (FibγΔ5), were challenged with collagen-induced arthritis (CIA). Fib– mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibγ390–396A mice, which retain full clotting function. In contrast, arthritis in FibγΔ5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib– and Fibγ390–396A mice with CIA displayed reduced local expression of TNF-α, IL-1β, and IL-6, which suggests that αMβ2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-α expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to αMβ2-mediated inflammatory processes.
Matthew J. Flick, Christine M. LaJeunesse, Kathryn E. Talmage, David P. Witte, Joseph S. Palumbo, Malinda D. Pinkerton, Sherry Thornton, Jay L. Degen
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