Hyperglycemia is associated with increased susceptibility to atherothrombotic stimuli. The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects the glycocalyx, thereby increasing vascular vulnerability. The systemic glycocalyx volume was estimated by comparing the distribution volume of a glycocalyx permeable tracer (dextran 40) with that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy male subjects. Measurements were performed in random order on five occasions: two control measurements, two measurements during normoinsulinemic hyperglycemia with or without N-acetylcysteine (NAC) infusion, and one during mannitol infusion. Glycocalyx measurements were reproducible (1.7 ؎ 0.2 vs. 1.7 ؎ 0.3 l). Hyperglycemia reduced glycocalyx volume (to 0.8 ؎ 0.2 l; P < 0.05), and NAC was able to prevent the reduction (1.4 ؎ 0.2 l). Mannitol infusion had no effect on glycocalyx volume (1.6 ؎ 0.1 l). Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (from 70 ؎ 6 to 112 ؎ 16 ng/ml; P < 0.05) and coagulation activation (prothrombin activation fragment 1 ؉ 2: from 0.4 ؎ 0.1 to 1.1 ؎ 0.2 nmol/l; D-dimer: from 0.27 ؎ 0.1 to 0.55 ؎ 0.2 g/l; P < 0.05). Taken together, these data indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during hyperglycemia. Diabetes 55:480 -486, 2006 P atients with diabetes have increased vascular vulnerability to atherogenic insults, leading to accelerated atherogenesis. Although atherogenesis is in part due to the increased prevalence of traditional cardiovascular risk factors, these factors cannot fully explain the propensity toward vascular complications in diabetic patients (1). Hyperglycemia itself has been shown to induce a wide array of downstream effects that adversely affect the protective capacity of the vessel wall (2). Hyperglycemia has been associated with enhanced endothelial permeability, increased leukocyte-endothelium adhesion, and impaired nitric oxide (NO) bioavailability (3-5). Despite clear progress in understanding the underlying pathophysiological mechanisms contributing to this vascular dysfunction, it has proven difficult to unravel a final common pathway for the increased vascular vulnerability under hyperglycemic conditions (6).The glycocalyx covers the endothelium and consists of endothelial cell-derived proteoglycans, glycoproteins, and adsorbed plasma proteins. This layer has been shown to orchestrate vascular homeostasis (7). Its thickness (up to 1 m) may explain its potent antiadhesive effects on leukocytes and platelets (8,9). Hyaluronan glycosaminoglycans, one of the major constituents of the glycocalyx, are crucial for maintaining endothelial barrier properties for plasma macromolecules (10). The glycocalyx also serves as a mechanosensor of shear stress, mediating shear-induced release of NO by endothelial cells (11-13). In fact, selective perturbation of the g...

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In this study, the relative roles of Toll-like receptor (TLR)2 and TLR4 were investigated independently and together. Moreover, we studied the role of haematopoietic compartment in anti-Klebsiella host defence.We infected TLR2 and TLR4 single-, and TLR264 double knockout (KO) animals with different doses of Klebsiella pneumoniae. In addition, bone marrow chimeric mice were created and infected.TLR4 played a more prominent role in antibacterial defence than TLR2, considering that only TLR4 KO mice demonstrated enhanced bacterial growth in lungs and spleen 24 h after infection with 3610 3 colony-forming units of Klebsiella compared with wild-type (WT) mice. In late-stage infection or after exposure to a higher infectious dose, bacterial counts in lungs of TLR2 KO animals were elevated compared with WT mice and TLR264 KO animals were more susceptible to infection than TLR4 KO mice. TLR signalling in cells of haematopoietic origin is of primary importance in host defence against K. pneumoniae. These data suggest that: 1) TLR4 drives the antibacterial host response after induction of pneumonia with relatively low Klebsiella doses; 2) TLR2 becomes involved at a later phase of the infection and/or upon exposure to higher bacterial burdens; and 3) haematopoietic TLR2 and TLR4 are important for an adequate host response during Klebsiella pneumonia.

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.

MyD88- and TRIF-dependent signaling has a differential contribution to host defense in different cell types that changes from early- to late-stage gram-negative pneumonia.

Klebsiella pneumoniae is an important cause of sepsis. The common Toll-like receptor adapter myeloid differentiation primary response gene (MyD)88 is crucial for host defense against Klebsiella. Here we investigated the role of MyD88 in myeloid and endothelial cells during Klebsiella pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-Myd88−/−) and myeloid plus endothelial (Tie2-Myd88−/−) cells showed enhanced lethality and bacterial growth. Tie2-Myd88−/− mice reconstituted with control bone marrow, representing mice with a selective MyD88 deficiency in endothelial cells, showed an unremarkable antibacterial defense. Myeloid or endothelial cell MyD88 deficiency did not impact on lung pathology or distant organ injury during late stage sepsis, while LysM-Myd88−/− mice demonstrated a strongly attenuated inflammatory response in the airways early after infection. These data suggest that myeloid but not endothelial MyD88 is important for host defense during gram-negative pneumonia derived sepsis.

BackgroundThe inflammasome is an intracellular protein complex triggered by exposure to intracellular pathogens, its components or other endogenous proteins. It leads to the activation of and subsequent release of proinflammatory cytokines such as IL-1β and IL-18. S. Typhimurium is a Gram-negative intracellular bacterium, which is known to trigger inflammasome assembly via recognition by the cytosolic receptors, NLRP3 and NLRC4 (which act via the adaptor protein, ASC) to induce cell death and cytokine release. We sought to characterize the role of ASC and NLRP3 in two different murine models (typhoid and colitis) of systemic Salmonella infection.ResultsRelease of the inflammasome cytokine IL-18 was hampered in Asc−/− but not Nlrp3−/− mice (background C57BL/6) during S. Typhimurium infection. Unexpectedly, neither ASC nor NLRP3 played a significant role in host defense against S. Typhimurium infection, as reflected by equal bacterial counts in WT, Asc−/− and Nlrp3−/− mice at all time points, in both the typhoid and colitis models. Proinflammatory cytokine levels (TNF-α, IL-6) and the extent of hepatic and splenic pathology did not differ between groups in the typhoid model. In the colitis model small differences were seen with regard to splenic and hepatic inflammation, although this was IL-18 independent.ConclusionsIL-18 release was reduced in Asc−/− but not Nlrp3−/− mice during S. Typhimurium infection. Despite this reduction, bacterial counts, cytokine levels and histological inflammation did not differ between wild-type and knockout mice in either model. Our results reveal a limited role for ASC and NLRP3 during in vivo S. Typhimurium infection despite its role in cytokine maturation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0030-7) contains supplementary material, which is available to authorized users.

Streptococcus (S.) pneumoniae is the most common cause of community-acquired pneumonia. The Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, consisting of NLRP3, ASC (the adaptor apoptosis-associated speck-like protein containing a CARD) and caspase-1, has been implicated in protective immunity during pneumonia induced by high doses of S. pneumoniae serotype 2. Here we investigated the role of the NLRP3 inflammasome in the host response during lethal airway infection with a low dose of serotype 3 S. pneumoniae. Mice were euthanized at predefined endpoints for analysis or observed in survival studies. In additional studies, Tlr2 −/− /Tlr4 −/− mice and Myd88 −/− mice incapable of Toll-like receptor signaling were studied. In stark contrast with existing literature, both Nlrp3 −/− and Asc −/− mice showed a strongly improved host defense, as reflected by a markedly reduced mortality rate accompanied by diminished bacterial growth and dissemination. Host defense was unaltered in Tlr2 −/− /Tlr4 −/− mice and Myd88 −/− mice. These results show that the NLRP3 inflammasome impairs host defense during lethal pneumonia caused by serotype 3 S. pneumoniae. Our findings challenge the current paradigm that proximal innate detection systems are indispensable for an adequate host immune response against bacteria.