lipoteichoic-acid and Pneumonia

The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition.. Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN.. Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1-10 nM.. TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an important component of endothelial cell retraction. The data suggest that inhibition of NSF in pneumonia and sepsis may be beneficial to prevent the pulmonary microvascular and hemodynamic instability associated with ARDS.

Topics: Angiopoietin-1; Animals; Bacterial Infections; Blood Vessels; Cell Line; Cell Wall; Cytoskeleton; Disease Models, Animal; Exocytosis; Gram-Positive Bacteria; Humans; Inflammation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; Microcirculation; N-Ethylmaleimide-Sensitive Proteins; Oxygen; Peptidoglycan; Phosphorylation; Pneumonia; Respiratory Distress Syndrome; Sepsis; Teichoic Acids; Vascular Diseases

Acute lung injury (ALI) is a significant source of morbidity and mortality in critically ill patients. Age is a major determinant of clinical outcome in ALI. The increased ALI-associated mortality in the older population suggests that there are age-dependent alterations in the responses to pulmonary challenge. The objective of this observational study was to evaluate age-dependent differences in the acute (within 6 hours) immunological and physiological responses of the heart and lung, to pulmonary challenge, that could result in increased severity.. Male C57Bl/6 mice (young: 2-3 months, old: 18-20 months) were challenged intratracheally with cell wall components from Gram-positive bacteria (lipoteichoic acid and peptidoglycan). After 6 hours, both biochemical and physiological consequences of the challenge were assessed. Alveolar infiltration of inflammatory cells and protein, airspace and blood cytokines, cardiac function and myocardial proteasome activity were determined.. In young mice, there was a dose-dependent response to pulmonary challenge resulting in increased airspace neutrophil counts, lung permeability, and concentrations of cytokines in bronchoalveolar lavage fluid and plasma. A midrange dose was then selected to compare the responses in young and old animals. In comparison, the old animals displayed increased neutrophil accumulation in the airspaces, decreased arterial oxygen saturation, body temperatures, plasma cytokine concentrations, and a lack of myocardial proteasome response, following challenge.. Age-dependent differences in the onset of systemic response and in maintenance of vital functions, including temperature control, oxygen saturation, and myocardial proteasome activation, are evident. We believe a better understanding of these age-related consequences of ALI can lead to more appropriate treatments in the elderly patient population.

Topics: Acute Lung Injury; Age Factors; Aging; Animals; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Hemodynamics; Inflammation Mediators; Lipopolysaccharides; Lung; Male; Mice, Inbred C57BL; Myocardium; Neutrophil Infiltration; Peptidoglycan; Pneumonia; Proteasome Endopeptidase Complex; Risk Factors; Severity of Illness Index; Teichoic Acids; Time Factors

The aging lung is increasingly susceptible to infectious disease. Changes in pulmonary physiology and function are common in older populations, and in those older than 60 years, pneumonia is the major cause of infectious death. Understanding age-related changes in the innate and adaptive immune systems, and how they affect both pulmonary and systemic responses to pulmonary challenge are critical to the development of novel therapeutic strategies for the treatment of the elderly patient. In this observational study, we examined age-associated differences in inflammatory responses to pulmonary challenge with cell wall components from Gram-positive bacteria. Thus, male Sprague-Dawley rats, aged 6 months or greater than 18 months (approximating humans of 20 and 55-65 years), were challenged, intratracheally, with lipoteichoic acid and peptidoglycan. Cellular and cytokine evaluations were performed on both bronchoalveolar lavage fluid (BAL) and plasma, 24 h post-challenge. The plasma concentration of free thyroxine, a marker of severity in non-thyroidal illness, was also evaluated. The older animals had an increased chemotactic gradient in favor of the airspaces, which was associated with a greater accumulation of neutrophils and protein. Furthermore, macrophage migration inhibitory factor (MIF), an inflammatory mediator and putative biomarker in acute lung injury, was increased in both the plasma and BAL of the older, but not young animals. Conversely, plasma free thyroxine, a natural inhibitor of MIF, was decreased in the older animals. These findings identify age-associated inflammatory/metabolic changes following pulmonary challenge that it may be possible to manipulate to improve outcome in the older, critically ill patient.

Topics: Adult; Age Factors; Aged; Animals; Chemotaxis; Humans; Lipopolysaccharides; Macrophage Migration-Inhibitory Factors; Male; Middle Aged; Neutrophils; Peptidoglycan; Pneumonia; Rats; Rats, Sprague-Dawley; Teichoic Acids; Thyroxine; Young Adult

Gram-positive and gram-negative bacteria are common causative agents of respiratory tract infection. Lipopolysaccharide (LPS) is a component of the gram-negative cell wall and a strong inducer of inflammation. The main proinflammatory component of the gram-positive bacterial cell wall is lipoteichoic acid (LTA). The protein kinase p38 mitogen activated protein kinase (MAPK) plays an important role in the inflammatory process induced by these two bacterial structures.. We here sought to establish the impact of local p38 MAPK inhibition on lung inflammatory responses induced by LPS and LTA. We investigated the effects of direct intrapulmonary delivery of a p38 MAPK inhibitor on local LPS and LTA induced airway inflammation in mice.. In vitro, BIRB 796 reduced LPS induced p38 MAPK phosphorylation in alveolar macrophage and respiratory epithelial cell lines and diminished cytokine/chemokine release. In vivo, BIRB 796 circumvented p38 MAPK phosphorylation in both LPS and LTA induced inflammation. Cellular influx was not affected. Lung TNFα, IL-6, MIP-2 and LIX production was reduced in LPS induced inflammation but not in lung inflammation by LTA. BIRB 796 reduced total protein and IgM in bronchoalveolar lavage fluid after LTA instillation, while enhancing TATc and d-dimers in LPS- and LTA induced inflammation.. These results taken together with earlier studies on systemic administration of p38 MAPK inhibitors in rodents and humans suggest that direct intrapulmonary delivery of a p38 MAPK inhibitor is less effective in inhibiting inflammation and is associated with unexpected procoagulant effects in the bronchoalveolar space.

Topics: Animals; Cell Line; Cytokines; Female; Humans; Immunoglobulin M; Lipopolysaccharides; Macrophages, Alveolar; Male; Mice; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pneumonia; Protein Kinase Inhibitors; Pulmonary Alveoli; Pyrazoles; Teichoic Acids

Adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK) is a highly conserved kinase that plays a key role in energy homeostasis. Activation of AMPK was shown to reduce inflammation in response to lipolysaccharide in vitro and in vivo. 5-Aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) is intracellularly converted to the AMP analog ZMP, which activates AMPK. Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria that can trigger inflammatory responses. In contrast to lipopolysaccharide, little is known on the effects of AMPK activation in LTA-triggered innate immune responses. Here, we studied the potency of AMPK activation to reduce LTA-induced inflammation in vitro and in lungs in vivo. Activation of AMPK in vitro reduced cytokine production in the alveolar macrophage cell line MH-S. In vivo, AMPK activation reduced LTA-induced neutrophil influx, as well as protein leak and cytokine/chemokine levels in the bronchoalveolar space. In conclusion, AMPK activation inhibits LTA-induced lung inflammation in mice.

Topics: Acetyl-CoA Carboxylase; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Line; Chemokines; Enzyme Activation; Female; Hypoglycemic Agents; Interleukin-6; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Neutrophils; Phosphorylation; Pneumonia; Ribonucleotides; Teichoic Acids; Tumor Necrosis Factor-alpha

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bronchoalveolar Lavage Fluid; Caspase 3; Cell Line; Chemokines; Cyclin-Dependent Kinases; Female; Humans; Inflammation Mediators; Leukocyte Count; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neutrophils; Pneumonia; Purines; Roscovitine; Streptococcus pneumoniae; Teichoic Acids

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in leukocyte recruitment to sites of infection and inflammation. In addition, MCP-1 may attenuate inflammation by virtue of its capacity to inhibit the production of proinflammatory cytokines. We here investigated the role of MCP-1 in lung inflammation induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA), constituents of the gram-negative and gram-positive bacterial cell wall, respectively. Healthy humans demonstrated elevated MCP-1 concentrations in their bronchoalveolar lavage fluid (BALF) 6h after inhalation of LPS. Similarly, intranasal administration of LPS or LTA to mice resulted in a rise in BALF MCP-1 levels. Murine alveolar macrophage-like cells released significant amounts of MCP-1 upon stimulation with LPS or LTA in vitro. Compared to Wt mice, MCP-1(-/-) mice demonstrated lower TNF-α levels and a diminished neutrophil influx into their bronchoalveolar space after either LPS or LTA instillation. After intrapulmonary delivery of LPS MCP-1(-/-) mice had decreased interleukin-6 and KC concentrations and less severe lung inflammation upon histopathological examination. Remarkably, MCP-1 deficiency was associated with an early enhancement of interleukin-10 release in BALF after both LPS and LTA instillation. These data suggest that MCP-1 is a proinflammatory mediator during pulmonary inflammation induced by either LPS or LTA.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Chemokine CCL2; Chemotaxis, Leukocyte; Cytokines; Female; Humans; Interleukin-10; Interleukin-6; Lipopolysaccharides; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neutrophils; Pneumonia; Teichoic Acids; Tumor Necrosis Factor-alpha; Young Adult

Pulmonary infection is a major cause of mortality and morbidity, and the magnitude of the lung inflammatory response correlates with patient survival. Previously, we have shown that neutrophil migration into joints is regulated by arthritis severity quantitative trait loci (QTLs). However, it is unclear whether these QTLs contribute to the regulation of lung inflammation in pneumonias. Therefore, to more clearly define the factors regulating acute inflammatory responses in the lung, we examined two inbred rat strains, DA and F344, that differ in these QTLs and their susceptibility to joint inflammation. Staphylococcal cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN), administered intratracheally, significantly increased the numbers of neutrophils retrieved in the bronchoalveolar lavage fluid (BALF). F344 had approximately 10-fold more neutrophils in the BALF compared with DA (P < 0.001) and higher BALF concentrations of total protein, tumor necrosis factor-α and macrophage inflammatory protein 2. LTA/PGN administration in DA×F344 congenic strains (Cia3d, Cia4, Cia5a, and Cia6) resulted in inflammation similar to that in DA, demonstrating that the genes responsible for the differences in pulmonary inflammation are not contained within the chromosomal intervals carried by these congenic strains. Alveolar macrophages (AMs) isolated from naïve F344 stimulated in vitro with LTA/PGN produced significantly higher levels of keratinocyte-derived chemokine and macrophage inflammatory protein 2 than alveolar macrophages from DA rats. The differences were related to differential mitogen-activated protein kinase phosphorylation. We conclude that the factors contributing to inflammation can be site and challenge dependent. A better understanding of site-specific inflammation may lead to more effective treatment of acute lung inflammation and injury.

Topics: Animals; Animals, Congenic; Arthritis, Experimental; Blotting, Western; Bronchoalveolar Lavage Fluid; Chemokine CXCL2; Chemokines; Female; Imidazoles; Lipopolysaccharides; Lung; Macrophages, Alveolar; Male; Mitogen-Activated Protein Kinases; Neutrophil Infiltration; Neutrophils; Peptidoglycan; Phosphorylation; Pneumonia; Pyridines; Quantitative Trait Loci; Rats; Rats, Inbred F344; Species Specificity; Teichoic Acids

Ethyl pyruvate (EP) is a stable pyruvate derivative that has been shown to exert anti-inflammatory effects in various models of systemic inflammation including endotoxemia. We here sought to determine the local effects of EP, after intrapulmonary delivery, in models of lung inflammation induced by instillation via the airways of either lipopolysaccharide (LPS, a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA, a component of the gram-positive bacterial cell wall). For this, we first established that EP dose dependently reduced the responsiveness of mouse MH-S alveolar macrophages and mouse MLE-15 and MLE-12 respiratory epithelial cells to stimulation with LPS or LTA in vitro. We then showed that intranasal administration of EP dose dependently inhibited tumor necrosis factor alpha release in bronchoalveolar lavage fluid of mice challenged with either LPS or LTA via the airways. Moreover, EP reduced the recruitment of neutrophils into the bronchoalveolar space after either LPS or LTA administration. These data suggest that intrapulmonary delivery of EP diminishes lung inflammation induced by LPS or LTA, at least in part by targeting alveolar macrophages and respiratory epithelial cells.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Line; Dose-Response Relationship, Drug; Epithelial Cells; Female; Flavoring Agents; Lipopolysaccharides; Macrophages, Alveolar; Mice; Pneumonia; Pyruvates; Teichoic Acids; Tumor Necrosis Factor-alpha

We studied the interaction effects of a single intratracheal instillation of ultrafine carbon black (CB) particles and staphylococcal lipoteichoic acid (LTA) on early pulmonary inflammation in male BALB/c mice. We examined the cellular profile, cytokine and chemokine levels in the bronchoalveolar lavage (BAL) fluid, and expression of chemokine and toll-like receptor (TLR) mRNAs in lungs. LTA produced a dose-related increase in early pulmonary inflammation, which was characterized by (1) influx of polymorphonuclear neutrophils (PMNs) and (2) induction of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha/CCL3, but no effect on monocyte chemoattractant protein (MCP)-1/CCL2 at 24 h after instillation. Levels of some proinflammatory indicators and TLR2-mRNA expression were significantly increased by 14 nm or 95 nm CB (125 microg) and low-dose LTA (10 microg) treatment compared to CB or LTA alone at 4 h after instillation. Notably, PMN levels and production of IL-6 and CCL2 in the 14 nm CB + LTA were significantly higher than that of 95 nm CB + LTA at 4 h after instillation. However, at 24 h after instillation, only PMN levels were significantly higher in the 14 nm CB + LTA than 95 nm CB + LTA but not the cytokines and chemokines. These data show additive as well as synergistic interaction effects of 14 nm or 95 nm ultrafine CB particles and LTA. We suggest that early pulmonary inflammatory responses in male BALB/c mice may be induced in a size-specific manner of the CB particles used in our study.

Topics: Animals; Bronchoalveolar Lavage Fluid; Carbon; Cytokines; Drug Synergism; Gene Expression Regulation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; Neutrophils; Particle Size; Pneumonia; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4