Leukocyte transcriptional signatures dependent on LPS dosage in human endotoxemia.

The host immune response is characterized by a complex interplay of signal-specific cellular transcriptional responses. The magnitude of the immune response is dependent on the strength of the external stimulus. Knowledge on leukocyte transcriptional responses altered in response to different stimulus dosages in man is lacking. Here, we sought to identify leukocyte transcriptional signatures dependent on LPS dose in humans. Healthy human volunteers were administered 1 ng/kg (n = 7), 2 ng/kg (n = 6), or 4 ng/kg (n = 7) LPS intravenously. Blood was collected before (pre-LPS) and 4 h after LPS administration. Total RNA was analyzed by microarrays and generalized linear models. Pathway analysis was performed by using Ingenuity pathway analysis. Leukocyte transcriptomes altered per LPS dosage were predominantly shared, with 47% common signatures relative to pre-LPS. A univariate linear model identified a set of 3736 genes that exhibited a dependency on differing LPS dosages. Neutrophil, monocyte, and lymphocyte counts explained 38.9% of the variance in the LPS dose-dependent gene set. A multivariate linear model including leukocyte composition delineated a set of 295 genes with a dependency on LPS dose. Evaluation of the 295 gene signature in patients with sepsis due to abdominal infections showed significant correlations. Promoter regions of the LPS dose gene set were enriched for YY1, EGR1, ELK1, GABPA, KLF4, and REL transcription factor binding sites. Intravenous injection of 1, 2, or 4 ng/kg LPS was accompanied by both shared and distinct leukocyte transcriptional alterations. These data may assist in assessing the severity of the insult in patients with abdominal sepsis.

Neutrophils mitigate the systemic host response during endotoxemia in mice.

Recent studies have suggested that neutrophils can exert anti-inflammatory effects. To determine the role of neutrophils in the acute response to lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, we challenged neutrophil-depleted and control mice with LPS and analyzed the plasma concentrations of biomarkers indicative of the cytokine and chemokine network, activation of coagulation and the vascular endothelium, and cellular injury. We here show that neutrophils serve an anti-inflammatory role upon LPS administration, as reflected by sustained elevations of multiple cytokines and chemokines, and enhanced release of nucleosomes in mice depleted of neutrophils, compared with control mice.

Neutrophil extracellular traps in patients with pulmonary tuberculosis.

Tuberculosis is a devastating infectious disease causing many deaths worldwide. Recent investigations have implicated neutrophil extracellular traps (NETs) in the host response to tuberculosis. The aim of the current study was to obtain evidence for NETs release in the circulation during human tuberculosis. For this we measured the plasma concentrations of nucleosomes in conjunction with neutrophil elastase, in 64 patients with active pulmonary tuberculosis and 32 healthy controls. Patients with active tuberculosis had elevated plasma levels of nucleosomes and elastase when compared with local healthy blood donors. Furthermore nucleosome and elastase levels showed a positive correlation. These findings provide the first evidence for the release of NETs in the circulation of patients with active pulmonary tuberculosis.

Toll-like receptor 9 enhances bacterial clearance and limits lung consolidation in murine pneumonia caused by methicillin resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in pneumonia, associated with severe lung damage. Tissue injury causes release of Damage Associated Molecular Patterns (DAMPs), which may perpetuate inflammation. DNA has been implicated as a DAMP that activates inflammation through Toll-like receptor (TLR)9. The aim of this study was to evaluate the role of TLR9 in MRSA pneumonia. Wild-type (Wt) and TLR9 knockout (tlr9-/-) mice were infected intranasally with MRSA USA300 (BK 11540) (5E7CFU) and euthanized at 6,24,48 or 72 hours for analyses. MRSA pneumonia was associated with profound release of cell-free host DNA in the airways, as reflected by increases in nucleosome and DNA levels in bronchoalveolar lavage fluid (BALF), accompanied by transient detection of pathogen DNA in MRSA-free BALF supernatants. In BALF, as compared to Wt -mice tlr9-/- mice showed reduced TNFα and IL-6 levels at 6 hours and reduced bacterial clearance at 6 and 24 hours post infection. Furthermore, tlr9-/- mice exhibited a greater influx of neutrophils in BALF and increased lung consolidation at 24 and 48 hours. This study demonstrates the release of host- and pathogen-derived TLR9 ligands (DNA) into the alveolar space after infection with MRSA via the airways and suggests that TLR9 has pro-inflammatory effects during MRSA pneumonia associated with enhanced bacterial clearance and limitation of lung consolidation.

High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia.

INTRODUCTION: Staphylococcus (S.) aureus has emerged as an important cause of necrotizing pneumonia. Lung injury during S. aureus pneumonia may be enhanced by local release of damage associated molecular patterns such as high-mobility group box 1 (HMGB1). In the current study we sought to determine the functional role of HMGB1 and its receptors, toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), in the injurious host response to S. aureus pneumonia. METHODS: Pneumonia was induced in wild type (Wt), TLR4 deficient (tlr4-/-) and RAGE deficient (rage-/-) mice by intranasal inoculation of 1 × 107 colony-forming units (CFU) of a USA300 S. aureus. In a separate set of experiments, Wt mice were injected intraperitoneally with a monoclonal anti-HMGB1 antibody or an isotype matched control antibody immediately before and every 24 hours after intranasal infection of S. aureus. Mice were sacrificed at 6, 24, 48 or 72 hours after infection for harvesting of blood and organs. RESULTS: S. aureus pneumonia was associated with HMGB1 release in the bronchoalveolar compartment peaking after 24 hours. Anti-HMGB1 attenuated lung pathology and protein leak and reduced interleukin-1ß release 6 hours after infection, but not at later time points. RAGE deficiency more modestly attenuated lung pathology without influencing protein leak, while TLR4 deficiency did not impact on lung injury. CONCLUSION: These data suggest that HMGB1 and RAGE, but not TLR4, contribute to lung injury accompanying the early phase of S. aureus pneumonia.

Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control.

Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell-cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell-cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell-cell contacts.

Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining.

OBJECTIVE: Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to be produced and released during surgery, the effects of ROS on the liver vascular lining and tumour cell adhesion were investigated. METHODS: Human endothelial cell monolayers (human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells of the lung (HMEC-1s)) were exposed to ROS production, after which electrical impedance, cellular integrity and tumour cell adhesion were investigated. Furthermore, surgery-induced tumour cell adhesion as well as the role of ROS and liver macrophages (Kupffer cells) in this process were studied in vivo. RESULTS: Production of ROS decreased cellular impedance of endothelial monolayers dramatically. Moreover, formation of intercellular gaps in endothelial monolayers was observed, exposing subendothelial extracellular matrix (ECM) on which colon carcinoma cells adhered via integrin molecules. Endothelial damage was, however, prevented in the presence of ROS-scavenging enzymes. Additionally, surgery induced downregulation of both rat and human liver tight junction molecules. Treatment of rats with the ROS scavenger edaravone prevented surgery-induced tumour cell adhesion and downregulation of tight junction proteins in the liver. Interestingly, depletion of Kupffer cells prior to surgery significantly reduced the numbers of adhered tumour cells and prevented disruption of expression of tight junction proteins. CONCLUSIONS: In this study it is shown that surgery-induced ROS production by macrophages damages the vascular lining by downregulating tight junction proteins. This leads to exposure of ECM, to which circulating tumour cells bind. In light of this, perioperative therapeutic intervention, preventing surgery-induced inflammatory reactions, may reduce the risk of developing liver metastases, thereby improving the clinical outcome of patients with colorectal cancer.