Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus–sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype.
Since the advent of high-throughput screening (HTS), there has been an urgent need for methods that facilitate the interrogation of large-scale chemical biology data to build a mode of action (MoA) hypothesis. This can be done either prior to the HTS by subset design of compounds with known MoA or post HTS by data annotation and mining. To enable this process, we developed a tool that compares compounds solely on the basis of their bioactivity: the chemical biological descriptor "high-throughput screening fingerprint" (HTS-FP). In the current embodiment, data are aggregated from 195 biochemical and cell-based assays developed at Novartis and can be used to identify bioactivity relationships among the in-house collection comprising ~1.5 million compounds. We demonstrate the value of the HTS-FP for virtual screening and in particular scaffold hopping. HTS-FP outperforms state of the art methods in several aspects, retrieving bioactive compounds with remarkable chemical dissimilarity to a probe structure. We also apply HTS-FP for the design of screening subsets in HTS. Using retrospective data, we show that a biodiverse selection of plates performs significantly better than a chemically diverse selection of plates, both in terms of number of hits and diversity of chemotypes retrieved. This is also true in the case of hit expansion predictions using HTS-FP similarity. Sets of compounds clustered with HTS-FP are biologically meaningful, in the sense that these clusters enrich for genes and gene ontology (GO) terms, showing that compounds that are bioactively similar also tend to target proteins that operate together in the cell. HTS-FP are valuable not only because of their predictive power but mainly because they relate compounds solely on the basis of bioactivity, harnessing the accumulated knowledge of a high-throughput screening facility toward the understanding of how compounds interact with the proteome.
While signal transducer and activator of transcription (STAT) 3 signaling has been linked to multiple pathways influencing immune function and cell survival, the direct influence of this transcription factor on innate immunity and tissue homeostasis during pneumonia is unknown. Human patients with dominant-negative mutations in the Stat3 gene develop recurrent pneumonias, suggesting a role for STAT3 in pulmonary host defense. We hypothesized that alveolar epithelial STAT3 is activated by IL-6 family cytokines and is required for effective responses during gram-negative bacterial pneumonia. STAT3 phosphorylation was increased in pneumonic mouse lungs and in murine lung epithelial (MLE)-15 cells stimulated with pneumonic bronchoalveolar lavage fluid (BALF) through 48 hours of Escherichia coli pneumonia. Mice lacking active STAT3 in alveolar epithelial cells (Stat3 D/D ) had fewer alveolar neutrophils and more viable bacteria than control mice early after intratracheal E. coli. By 48 hours after E. coli infection, however, lung injury was increased in Stat3 D/D mice. Bacteria were cleared from lungs of both genotypes, albeit more slowly in Stat3 D/D mice. Of the IL-6 family cytokines measured in lungs from infected C57BL/6 mice, IL-6, oncostatin M, leukemia inhibitory factor (LIF), and IL-11 were significantly elevated. Neutralization studies demonstrated that LIF and IL-6 mediated BALF-induced STAT3 activation in MLE-15 cells. Together, these results indicate that during E. coli pneumonia, select IL-6 family members activate alveolar epithelial STAT3, which functions to promote neutrophil recruitment and to limit both infection and lung injury.Keywords: lung; neutrophils; STAT3; pneumonia; cytokines Lung infections account for a tremendous burden of disease worldwide and are a leading cause of acute lung injury (1, 2). While Streptococcus pneumoniae is the most common agent in patients with community-acquired pneumonia (3), gram-negative rods such as Escherichia coli are a frequent cause of nosocomial pneumonia (4). Elimination of these and other pathogens from the lower respiratory tract is made possible by an effective innate immune response (5), which is necessary yet potentially dangerous to the infected host. For this reason, cytokine networks, neutrophil emigration, plasma extravasation, and other characteristics of acute inflammation must be precisely regulated to maintain tissue homeostasis.The STAT3 transcription factor influences both immunity and inflammatory injury, but the importance of STAT3 signaling during pneumonia is unknown. STAT3 activity has been attributed both inflammatory (6-9) and anti-inflammatory (10-12) roles. Likewise, the cytokine interleukin (IL)-6, which largely signals through STAT3 (13, 14), has also been described as both pro-(15-19) and anti-inflammatory (16, 20-22), depending on the biological context. During E. coli pneumonia, neutrophil recruitment and bacterial clearance are impaired in IL-6-deficient mice (15). While the mechanisms through which IL-6 functions during this i...
Pulmonary inflammation is an essential component of the host defense against Streptococcus pneumoniae infection of the lungs. The early response cytokines, TNF-α and IL-1, are rapidly induced upon microbial exposure. Mice deficient in all TNF- and IL-1-dependent signaling receptors were used to determine the roles of these cytokines during pneumococcal pneumonia. The deficiency of signaling receptors for TNF and IL-1 decreased bacterial clearance. Neutrophil recruitment to alveolar air spaces was impaired by receptor deficiency, as was pulmonary expression of the neutrophil chemokines KC and MIP-2. Because NF-κB mediates the expression of both chemokines, we assessed NF-κB activation in the lungs. During pneumococcal pneumonia, NF-κB proteins translocate to the nucleus and activate gene expression; these functions were largely abrogated by the deficiency of receptors for TNF-α and IL-1. Thus, the combined deficiency of TNF and IL-1 signaling reduces innate immune responses to S. pneumoniae in the lungs, probably due to essential roles for these receptors in activating NF-κB.
The recruitment and activation of neutrophils at infected tissues is essential for host defense against invading microorganisms. However, excessive neutrophil recruitment or activation can also damage the surrounding tissues and cause unwanted inflammation. Hence, the responsiveness of neutrophils needs to be tightly regulated. In this study, we have investigated the functional role of tumor suppressor PTEN in neutrophils by using a mouse line in which PTEN is disrupted only in myeloid-derived cells. Chemoattractant-stimulated PTEN ؊/؊ neutrophils displayed significantly higher Akt phosphorylation and actin polymerization. A larger fraction of these neutrophils displayed membrane ruffles in response to chemoattractant stimulation. In addition, chemoattractant-induced transwell migration and superoxide production were also augmented. Single-cell chemotaxis assays showed that PTEN ؊/؊ neutrophils have a small (yet statistically significant) defect in directionality. However, these neutrophils also showed an increase in cell speed. As a result, overall chemotaxis, which depends on speed and directionality, was not affected. Consistent with the increased responsivenessof PTEN ؊/؊ neutrophils, the in vivo recruitment of these cells to the inflamed peritoneal cavity was significantly enhanced. Thus, as a physiologic-negative regulator, PTEN should be a promising therapeutic target for modulating neutrophil functions in various infectious and inflammatory dis- IntroductionNeutrophils are the most abundant cell type among circulating white blood cells. The recruitment and activation of neutrophils are important components of the innate immune system. In response to inflammatory stimuli, neutrophils migrate from the blood to infected tissues, where they protect their host by engulfing, killing, and digesting invading bacterial and fungal pathogens. Conversely, excessive neutrophil accumulation or hyperresponsiveness of neutrophils can also be detrimental to the system. The toxic reactive oxygen species and granule enzymes (eg, proteases) released by neutrophils can damage surrounding tissues and cause unwanted and exaggerated tissue inflammation. Hence, the response of neutrophils to inflammatory stimuli needs to be well controlled.Neutrophils get recruited to the site of infection by responding to a variety of chemokines, leukotrienes, complement peptides, and some chemicals released by bacteria directly, such as peptides bearing the N-formyl group (formyl-peptides). 1-3 All these responses are mediated by heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)-coupled receptors (GPCRs). One essential downstream target of GPCRs is PtdIns(3,4,5)P3, an inositol phospholipid which has been implicated in a variety of neutrophil functions such as polarization, chemotaxis, and superoxide generation. [4][5][6] PtdIns(3,4,5)P3 exerts its function by mediating protein translocation via pleckstrin homology (PH) domains on the protein. 7,8 This membrane translocation is crucial for these proteins to fulfill the...
Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation. To determine the roles of the NF-κB subunit RelA in facilitating these events, we infected RelA-deficient mice (generated on a TNFR1-deficient background) with Streptococcus pneumoniae. RelA deficiency decreased cytokine expression, alveolar neutrophil emigration, and lung bacterial killing. S. pneumoniae killing was also diminished in the lungs of mice expressing a dominant-negative form of IκBα in airway epithelial cells, implicating this cell type as an important locus of NF-κB activation during pneumonia. To study mechanisms of epithelial RelA activation, we stimulated a murine alveolar epithelial cell line (MLE-15) with bronchoalveolar lavage fluid (BALF) harvested from mice infected with S. pneumoniae. Pneumonic BALF, but not S. pneumoniae, induced degradation of IκBα and IκBβ and rapid nuclear accumulation of RelA. Moreover, BALF-induced RelA activity was completely abolished following combined but not individual neutralization of TNF and IL-1 signaling, suggesting either cytokine is sufficient and necessary for alveolar epithelial RelA activation during pneumonia. Our results demonstrate that RelA is essential for the host defense response to pneumococcus in the lungs and that RelA in airway epithelial cells is primarily activated by TNF and IL-1.
Interleukin (IL)-6 concentrations are positively associated with the severity of pneumonia, and this cytokine is essential to surviving experimental pneumococcal pneumonia. The role that IL-6 plays during pneumonia and its impact during gram-negative bacterial pneumonia remain to be determined. During Escherichia coli pneumonia, IL-6-deficient mice had increased bacterial burdens in their lungs, indicating compromised host defenses. Decreased neutrophil counts in alveolar air spaces, despite normal blood neutrophil counts and survival of emigrated neutrophils, suggested that defective neutrophil recruitment was responsible for exacerbating the infection. Neutrophil recruitment requires nuclear factor (NF)- kappa B, but IL-6 was neither sufficient nor essential to induce NF- kappa B-mediated gene expression in the lungs. In contrast, IL-6 induced the phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3 in the lungs, and STAT1 and STAT3 phosphorylation during E. coli pneumonia was decreased by IL-6 deficiency. Thus, IL-6 plays essential roles in activating STAT transcription factors, enhancing neutrophil recruitment, and decreasing bacterial burdens during E. coli pneumonia.
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