Myocardial injury modulates the innate immune system and changes myocardial sensitivity

Objective Transverse aortic constriction (TAC) results in a transient increase of proinflammatory cytokines, which return to baseline levels within 3 d. In contrast to cytokine baseline levels, the myocardium remains capable to respond even stronger to a new stimulus. As the molecular mechanisms for this phenomenon are unknown, we tested whether TAC modulates the innate immune system in mice and changes the inflammatory reaction to a new stimulus. Methods Following 3 d of TAC or sham-operation procedure (SOP), LPS (20 mg/kg) or PBS (control) were administered intraperitoneal for 10 min as well as for 6 h. Hemodynamic parameters were recorded to measure the effects of TAC and LPS. After TAC/SOP alone CD14 expression was monitored and after additional 6 h of LPS/PBS the expression of CD14, TLR4 and proinflammatory cytokines were determined by western-blot, ELISA and RNase protection assay, respectively. Following TAC/SOP and 10 min of LPS/PBS, NFκB activation was investigated by EMSA. Results TAC induced cardiac hypertrophy and elevated blood pressure. LPS application led to hypotension and other symptoms of sepsis. CD14 expression increased after TAC alone and even further after additional LPS challenge. However, we did not detect changes of TLR4 expression. Also NFκB activation increased after LPS challenge higher in the TAC than in the SOP group. LPS-stimulation induced also higher cytokine expression in the TAC than in the SOP group. Conclusion TAC modulates innate immunity by regulating the expression of CD14 and changes the myocardial tissue to respond more powerful to LPS. Returned for 1st revision: 30 November 2005 1st revision received: 28 March 2006     

Diminished Reactive Hematopoiesis and Cardiac Inflammation in a Mouse Model of Recurrent Myocardial Infarction

BackgroundRecurrent myocardial infarction (MI) is common in patients with coronary artery disease and is associated with high mortality. Long-term reprogramming of myeloid progenitors occurs in response to inflammatory stimuli and alters the organism’s response to secondary inflammatory challenges.ObjectivesThis study examined the effect of recurrent MI on bone marrow response and cardiac inflammation.MethodsThe investigators developed a surgical mouse model in which 2 subsequent MIs affected different left ventricular regions in the same mouse. Recurrent MI was induced by ligating the left circumflex artery followed by the left anterior descending coronary artery branch. The study characterized the resulting ischemia by whole-heart fluorescent coronary angiography after optical organ clearing and by cardiac magnetic resonance imaging.ResultsA first MI-induced bone marrow “memory” via a circulating signal, reducing hematopoietic maintenance factor expression in bone marrow macrophages. This dampened the organism’s reaction to subsequent events. Despite a similar extent of injury according to troponin levels, recurrent MI caused reduced emergency hematopoiesis and less leukocytosis than a first MI. Consequently, fewer leukocytes migrated to the ischemic myocardium. The hematopoietic response to lipopolysaccharide was also mitigated after a previous MI. The increase of white blood count in 28 patients was lower after recurrent MI compared with their first MI.ConclusionsThe data suggested that hematopoietic and innate immune responses are shaped by a preceding MI.     

Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway

Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.The Gram-negative bacterial cell envelope is a remarkably complex structure with critical functions for cellular growth and viability. It protects the cell from rapidly changing and potentially harmful environments and must do so while also allowing the selective import of nutrients and export of waste (1). Structurally, the Gram-negative cell envelope consists of an inner membrane (IM) and an outer membrane (OM) that delimit an aqueous compartment known as the periplasm (1, 2). Within the periplasmic space is a mesh-like network of peptide-crosslinked glycan chains, known as the peptidoglycan cell wall (1, 3, 4). This structure shapes the cell and provides mechanical resistance to turgor pressure-driven expansion (3). After inoculation into fresh medium, cells use nutrients in the medium to carry out processes essential to growth. Once these nutrients are depleted, cells enter stationary phase, during which they undergo gross morphological and physiological changes and stop growing (5). Throughout these growth phases and during septum formation and cytokinesis, synthesis of the various layers of the cell envelope must remain coordinated.The Escherichia coli OM is an asymmetric bilayer that contains phospholipids (PLs) in the inner leaflet and LPS in the outer leaflet (6). This structure functions as a robust, highly selective permeability barrier that protects the cell from harmful agents such as detergents, bile salts, and antibiotics (1). The effectiveness of the OM can be attributed to the hydrophobicity of and strong lateral interactions between LPS molecules (6); E. coli must properly synthesize and transport LPS to the outer leaflet of the OM to survive (7). Many proteins contribute to LPS biosynthesis and assembly (for a review, see refs. 8 and 9). By contrast with LPS, how lipids are transported to the OM is virtually unknown.When LPS biosynthetic or transport proteins are compromised, PLs are flipped from the inner to the outer leaflet of the OM to accommodate the reduction in LPS abundance (10). In the outer leaflet, it is thought that PLs form rafts (11), creating patches in the membrane that are more susceptible to the influx of hydrophobic, toxic molecules. To prevent damage resulting from surface-exposed PLs in wild-type E. coli cells, several mechanisms destroy or remove these PLs from the outer leaflet. The OM β-barrel protein PagP is a palmitoyltransferase that removes a palmitate from the sn-1 position of a surface-exposed PL and transfers it to lipid A or phosphatidylglycerol (12, 13). Another OM β-barrel phospholipase, PldA, removes both sn-1 and sn-2 palmitate moieties from PLs and lyso-PLs (14).The Mla (maintenance of lipid asymmetry) ABC transport system is a third mechanism for maintaining lipid asymmetry. Mla proteins are present in all compartments of the cell envelope and facilitate retrograde phospholipid transport from the OM back to the IM (15). MlaA is the lipoprotein component that interacts with OmpC in the OM (16) and is thought to remove PLs from the outer leaflet of the OM and shuttle them to MlaC, the soluble periplasmic component. MlaC delivers the PLs to the IM MlaFEDB complex, which is presumed to aid in the reintegration of PLs into the IM. Null mutations in any mla gene increase the permeability of the OM, rendering cells susceptible to detergent by an increase in surface-exposed PLs (15).Here we show that a dominant mutation in mlaA disrupts the lipid balance of the OM by a mechanism that does not require the other mla gene products but does require active PldA. Cells carrying this mutation are sensitized to the transition to stationary phase in medium with low divalent cation concentrations. This transition triggers an unexpected cell-death trajectory in starving cells in which death is correlated with increased OM blebbing at sites of cell division concomitant with decreased cytoplasmic volume and IM surface area, suggesting that lipid flow from the IM to the OM compensates for lipid loss by OM blebs and vesicles. Thus, our data may provide insights into the long-standing question of how lipids are transported to the OM.     

Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis

BACKGROUND AND AIMS: Cholestasis is one of the principal manifestations of liver disease and often results from disorders involving bile duct epithelia rather than hepatocytes. A range of disorders affects biliary epithelia, and no unifying pathophysiologic event in these cells has been identified as the cause of cholestasis. Here we examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R)/Ca(2+) release channel in Ca(2+) signaling and ductular secretion in animal models of cholestasis and in patients with cholestatic disorders. METHODS: The expression and distribution of the InsP3R and related proteins were examined in rat cholangiocytes before and after bile duct ligation or treatment with endotoxin. Ca(2+) signaling was examined in isolated bile ducts from these animals, whereas ductular bicarbonate secretion was examined in isolated perfused livers. Confocal immunofluorescence was used to examine cholangiocyte InsP3R expression in human liver biopsy specimens. RESULTS: Expression of the InsP3R was selectively lost from biliary epithelia after bile duct ligation or endotoxin treatment. As a result, Ca(2+) signaling and Ca(2+)-mediated bicarbonate secretion were lost as well, although other components of the Ca(2+) signaling pathway and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated bicarbonate secretion both were preserved. Examination of human liver biopsy specimens showed that InsP3Rs also were lost from bile duct epithelia in a range of human cholestatic disorders, although InsP3R expression was intact in noncholestatic liver disease. CONCLUSIONS: InsP3-mediated Ca(2+) signaling in bile duct epithelia appears to be important for normal bile secretion in the liver, and loss of InsP3Rs may be a final common pathway for cholestasis.     

Chronic ethanol consumption impairs cellular immune responses against HCV NS5 protein due to dendritic cell dysfunction

BACKGROUND & AIMS: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. METHODS: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. RESULTS: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. CONCLUSIONS: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.     

TLR4基因表达及TNF-α在老年大鼠多器官功能障碍综合征中作用的研究

目的探讨Toll样受体4基因（TLR4）与TNF-α在老年大鼠多器官功能障碍综合征（MODSE）发病机制中的作用.方法20月龄和3月龄SD大鼠各40只,分别设为老年组及青年组,予油酸（OA）0.25ml/kg和脂多糖（LPS）3.5mg/kg分次静脉注射（间隔4h）,建立二次打击MODSE和青年多器官功能障碍综合征（MODSY）模型.观察对照组及伤后2、6、24h肺、心、肝、肾的病理改变及功能变化,检测肺、心、肝和肾组织中TLR4 mRNA表达及TNF-α含量.结果OA/LPS二次致伤能够导致青年及老年鼠MODS,其中肺脏损害出现最早最重,在同一时相点,老年鼠脏器损害重于青年鼠（P＜0.05～0.01）.致伤后肺、心、肝和肾组织中TLR4 mRNA表达均显著升高（P＜0.05～0.01）,以肺组织中表达最高,2h达峰值,心、肝、肾组织中于6h达峰值;各组织中TNF-α含量均显著升高（P＜0.05～0.01）,以肺组织中升幅最大.在相同时相点老年鼠各组织中TLR4 mRNA表达及TNF-α含量高于青年鼠（P＜0.05～0.01）.结论OA/LPS致伤后老年鼠肺组织中TLR4 mRNA表达及TNF-α水平迅速升高,在MODS发病机制中起重要作用,有可能是MODSE过程中肺损伤出现最早最重的重要原因.     

Expression of inducible nitric oxide synthase in cultured smooth muscle cells from rat mesenteric lymphatic vessels

OBJECTIVE: The objective was to devise a method for establishing cultures of rat mesenteric lymphatic vessel smooth muscle cells (LSMC) and to investigate if inducible nitric oxide synthase (iNOS) expression could be activated in LSMC treated with bacterial lipopolysaccharide (LPS). METHODS: LSMC were successfully grown from explanted rat lymphatic microvessels and maintained by subculture. Treatment of LSMC for 24 h with LPS (1-100 microg/mL) activated iNOS protein induction, associated with (1) assay of increased nitrite concentrations in the medium representing cellular nitric oxide synthesis, and (2) demonstration of iNOS in cell extracts by Western blotting. RESULTS: The protein synthesis inhibitor cycloheximide (10 microM) blocked both LPS-induced nitrite formation and iNOS protein expression in LSMC. 1400 W (1 microM), a selective iNOS inhibitor, prevented LPS-induced nitrite formation but not iNOS expression. As well as induction of iNOS by LPS, "constitutive" iNOS was present in some cultures, producing nitrite in amounts that were also subsequently reduced after cell treatment with 1400 W. CONCLUSION: Rat mesenteric LSMC produce nitrite and express iNOS in response to bacterial LPS. Cultured LSMC may provide a useful model for studying mechanisms of iNOS induction in relation to possible influences of iNOS upon lymphatic vessel function.     

Anti‐Inflammatory and Osteoprotective Effects of Cannabinoid‐2 Receptor Agonist HU‐308 in a Rat Model of Lipopolysaccharide‐Induced Periodontitis

Background: Anti‐inflammatory and immunologic properties of cannabinoids have been reported in several tissues. Expression of cannabinoid receptor Type 2 was reported in osteoblasts and osteoclasts, suggesting a key role in bone metabolism. The aim of this study is to assess the effect of treatment with cannabinoid‐2 receptor agonist HU‐308 in the oral health of rats subjected to lipopolysaccharide (LPS)‐induced periodontitis. Methods: Twenty‐four rats were distributed in four groups (six rats per group): 1) control rats; 2) sham rats; 3) rats submitted to experimental periodontitis (LPS); and 4) rats submitted to experimental periodontitis and treated with HU‐308 (LPS+HU). In groups LPS and LPS+HU, periodontitis was induced by LPS (1 mg/mL) injected into the gingival tissue (GT) of maxillary and mandibular first molars and into the interdental space between the first and second molars, 3 days per week for 6 weeks. In group LPS+HU, HU‐308 (500 ng/mL) was applied topically to the GT daily. Results: Alveolar bone loss resulting from LPS‐induced periodontitis was significantly attenuated with HU‐308 treatment (LPS+HU), measured by macroscopic and histologic examination. Treatment also reduced gingival production of inflammatory mediators augmented in LPS‐injected rats, such as: 1) inducible nitric oxide (iNOS) activity (LPS: 90.18 ± 36.51 pmol/minute/mg protein versus LPS+HU: 16.37 ± 4.73 pmol/minute/mg protein; P <0.05); 2) tumor necrosis factor alpha (LPS: 185.70 ± 25.63 pg/mg protein versus LPS+HU: 95.89 ± 17.47 pg/mg protein; P <0.05); and 3) prostaglandin E₂ (PGE₂) (LPS: 159.20 ± 38.70 pg/mg wet weight versus LPS+HU: 71.25 ± 17.75 pg/mg wet weight; P <0.05). Additionally, HU‐308 treatment prevented the inhibitory effect of LPS‐induced periodontitis on the salivary secretory response to pilocarpine. Moreover, iNOS activity and PGE₂ content, which were increased by LPS‐induced periodontitis in the submandibular gland, returned to control values after HU‐308 treatment. Conclusion: This study demonstrates anti‐inflammatory, osteoprotective, and prohomeostatic effects of HU‐308 in oral tissues of rats with LPS‐induced periodontitis.     

TREM-2对小鼠肺成纤维细胞凋亡的影响

目的:初步探讨髓样细胞触发受体-2(triggering receptors expressed on myeloid cells-2,TREM-2)过表达对小鼠肺成纤维细胞凋亡的影响.方法:将小鼠肺成纤维细胞分成脂多糖( lipopolysaccharide,LPS)组、LPS+转染试剂组、LPS+空质粒组和LPS+...     

Bacterial endotoxin: a trigger factor for alcoholic pancreatitis? Evidence from a novel, physiologically relevant animal model

BACKGROUND & AIMS: This study examined the possible role of endotoxinemia (from increased gut permeability) as an additional trigger factor for overt pancreatic disease and as a promoter of chronic pancreatic injury in alcoholics by using a rat model of chronic alcohol feeding and in vitro experiments with cultured pancreatic stellate cells (PSCs), the key mediators of pancreatic fibrosis. METHODS: In the in vivo model, Sprague-Dawley rats fed isocaloric Lieber-DeCarli liquid diets +/- alcohol for 10 weeks were challenged with a single dose or 3 repeated doses of the endotoxin lipopolysaccharide (LPS) and the pancreas was examined. In the in vitro studies, rat PSCs were assessed for activation on exposure to LPS +/- ethanol. The expression of LPS receptors TLR4 and CD14 also was assessed in rat and human PSCs. RESULTS: In the in vivo model, single or repeated LPS challenge resulted in significantly greater pancreatic injury in alcohol-fed rats compared with rats fed the control diet without alcohol. Notably, repeated LPS injections caused pancreatic fibrosis in alcohol-fed rats, but not in rats fed the control diet. In the in vitro studies, PSCs were activated by LPS. Alcohol + LPS exerted a synergistic effect on PSC activation. Importantly, both rat and human PSCs expressed TLR4 and CD14. CONCLUSIONS: This study describes, for the first time, a clinically relevant animal model of alcohol-related pancreatic injury and provides strong in vivo and in vitro evidence that suggests that LPS is a trigger factor in the initiation and progression of alcoholic pancreatitis.