Ruxana T. Sadikot

Early Identification of Patients at Risk of Acute Lung Injury: Evaluation of Lung Injury Prediction Score in a Multicenter Cohort Study

RATIONALEnAccurate, early identification of patients at risk for developing acute lung injury (ALI) provides the opportunity to test and implement secondary prevention strategies.nnnOBJECTIVESnTo determine the frequency and outcome of ALI development in patients at risk and validate a lung injury prediction score (LIPS).nnnMETHODSnIn this prospective multicenter observational cohort study, predisposing conditions and risk modifiers predictive of ALI development were identified from routine clinical data available during initial evaluation. The discrimination of the model was assessed with area under receiver operating curve (AUC). The risk of death from ALI was determined after adjustment for severity of illness and predisposing conditions.nnnMEASUREMENTS AND MAIN RESULTSnTwenty-two hospitals enrolled 5,584 patients at risk. ALI developed a median of 2 (interquartile range 1-4) days after initial evaluation in 377 (6.8%; 148 ALI-only, 229 adult respiratory distress syndrome) patients. The frequency of ALI varied according to predisposing conditions (from 3% in pancreatitis to 26% after smoke inhalation). LIPS discriminated patients who developed ALI from those who did not with an AUC of 0.80 (95% confidence interval, 0.78-0.82). When adjusted for severity of illness and predisposing conditions, development of ALI increased the risk of in-hospital death (odds ratio, 4.1; 95% confidence interval, 2.9-5.7).nnnCONCLUSIONSnALI occurrence varies according to predisposing conditions and carries an independently poor prognosis. Using routinely available clinical data, LIPS identifies patients at high risk for ALI early in the course of their illness. This model will alert clinicians about the risk of ALI and facilitate testing and implementation of ALI prevention strategies. Clinical trial registered with www.clinicaltrials.gov (NCT00889772).

Targeted Immunomodulation of the NF-κB Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-κB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-κB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-κB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-κB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-κB. The TNF-α concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-κB up-regulation were abrogated in TNF-α-deficient mice. In contrast, NF-κB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-κB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

Bacterial clearance of Pseudomonas aeruginosa is enhanced by the inhibition of COX-2

Prostanoids generated by COX‐2 are involved in the regulation of inflammation but their exact role in the innate immune response has not been defined. We investigated whether COX‐2 is involved in host defense against Pseudomonas aeruginosa pneumonia. In vitro studies, in a macrophage cell line, showed that cytotoxic strain of Pu2004aeruginosa (PA103) induced significant COX‐2 protein expression and enzymatic function. In vivo data showed that infection with PA103 increased COX‐2 protein production in whole lung tissue compared to mice that were infected with mutant bacteria that lack ExoU (ΔU) or ExoU and ExoT (ΔUT). COX‐2–/– mice had accentuated clearance of cytotoxic P.u2004aeruginosa from the lungs. We further tested the effects of COX‐2 products such as prostaglandinu2004E2 on the function of phagocytic cells. Our studies indicate that prostaglandinu2004E2 may be involved through interacting with the EP2 receptors in modulating the host response because treatment of macrophages with prostaglandinu2004E2 suppressed production of reactive oxygen species. Furthermore there was enhanced bacterial clearance in EP2 receptor–/– mice compared to the wild‐type controls. Thus it is possible that inhibition of COX‐2 or EP2 receptors could be an effective adjunctive treatment for severe or resistant P.u2004aeruginosa pneumonia.

FoxM1 mediates the progenitor function of type II epithelial cells in repairing alveolar injury induced by Pseudomonas aeruginosa

Mice lacking FoxM1 specifically in progenitor-like type II alveolar epithelial cells exhibit defective alveolar barrier repair after microbe-induced lung injury.

TREM-1 expression in macrophages is regulated at transcriptional level by NF-κB and PU.1

Triggering receptor expressed on myeloid cells (TREM)‐1 is a recently identified immunoglobulin receptor that is expressed on neutrophils and monocytes where it amplifies the acute inflammatory response to bacteria. We examined the transcriptional regulation of TREM‐1 in macrophages. Treatment of RAW cells with Escherichia coli LPS or Pseudomonas aeruginosa led to the induction of TREM‐1 within 1u2004h with an expression lasting up to at least 24u2004h in vitro as detected by RT‐PCR. Since the promoter of TREM‐1 has multiple binding sites for NF‐κB and PU.1 (one of the members of the ets family of transcription factors), we investigated the role of these transcription factors in the induction of TREM‐1. Treatment of cells with NF‐κB inhibitors abolished the expression of message of TREM‐1 induced by LPS and P.u2004aeruginosa. In contrast, the expression of TREM‐1 was increased after stimulation with LPS or P.u2004aeruginosa in cells that had gene of PU.1 silenced. Additionally, over‐expression of PU.1 led to inhibition of TREM‐1 induction in response to LPS and P.u2004aeruginosa. These data suggest that both these transcription factors are involved in the expression of TREM‐1. NF‐κB functions as a positive regulator whereas PU.1 is a negative regulator of the TREM‐1 gene.

A novel peptide nanomedicine against acute lung injury: GLP-1 in phospholipid micelles.

ABSTRACTPurposeTreatment of acute lung injury (ALI) observed in Gram-negative sepsis represents an unmet medical need due to a high mortality rate and lack of effective treatment. Accordingly, we developed and characterized a novel nanomedicine against ALI. We showed that when human glucagon-like peptide 1(7–36) (GLP-1) self-associated with PEGylated phospholipid micelles (SSM), the resulting GLP1-SSM (hydrodynamic size, ~15xa0nm) exerted effective anti-inflammatory protection against lipopolysaccharide (LPS)-induced ALI in mice.MethodsGLP1-SSM was prepared by incubating GLP-1 with SSM dispersion in saline and characterized using fluorescence spectroscopy and circular dichroism. Bioactivity was tested by in vitro cAMP induction, while in vivo anti-inflammatory effects were determined by lung neutrophil cell count, myeloperoxidase activity and pro-inflammatory cytokine levels in LPS-induced ALI mice.ResultsAmphipathic GLP-1 interacted spontaneously with SSM as indicated by increased α-helicity and fluorescence emission. This association elicited increased bioactivity as determined by in vitro cAMP production. Correspondingly, subcutaneous GLP1-SSM (5–30xa0nmol/mouse) manifested dose-dependent decrease in lung neutrophil influx, myeloperoxidase activity and interleukin-6 in ALI mice. By contrast, GLP-1 in saline showed no significant anti-inflammatory effects against LPS-induced lung hyper-inflammatory responses.ConclusionsGLP1-SSM is a promising novel anti-inflammatory nanomedicine against ALI and should be further developed for its transition to clinics.

Induction and Function of Lipocalin Prostaglandin D Synthase in Host Immunity

Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD2 produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD2 enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD2 against Pseudomonas pneumonia.

Functional PU.1 in macrophages has a pivotal role in NF-κB activation and neutrophilic lung inflammation during endotoxemia

Although the role of ETS family transcriptional factor PU.1 is well established in macrophage maturation, its role in mature macrophages with reference to sepsis- related animal model has not been elucidated. Here, we report the in vivo function of PU.1 in mediating mature macrophage inflammatory phenotype by using bone marrow chimera mice with conditional PU.1 knockout. We observed that the expression of monocyte/macrophage-specific markers CD 11b, F4/80 in fetal liver cells, and bone marrow-derived macrophages were dependent on functional PU.1. Systemic inflammation as measured in terms of NF-κB reporter activity in lung, liver, and spleen tissues was significantly decreased in PU.1-deficient chimera mice compared with wild-type chimeras on lipopolysaccharide (LPS) challenge. Unlike wild-type chimera mice, LPS challenge in PU.1-deficient chimera mice resulted in decreased lung neu-trophilic inflammation and myeloperoxidase activity. Similarly, we found attenuated inflammatory gene expression (cyclooxygenase-2, inducible nitric-oxide synthase, and TLR4) and inflammatory cytokine secretion (IL-6, MCP-1, IL-1β, TNF-α, and neutrophilic chemokine keratinocyte-derived chemokine) in PU.1-deficient mice. Most importantly, this attenuated lung and systemic inflammatory phenotype was associated with survival benefit in LPS-challenged heterozygotic PU.1-deficient mice, establishing a novel protective mechanistic role for the lineage-specific transcription factor PU.1.

Bioluminescent Detection of Endotoxin Effects on HIV-1 LTR-driven Transcription in Vivo

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor κB (NF-κB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5–6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3–6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.

MYD88-dependent and -independent activation of TREM-1 via specific TLR ligands

Triggering receptor expressed on myeloid cells (TREM)‐1 plays an important role in myeloid cell‐activated inflammatory responses. Although TLR ligands such as LPS and lipoteichoic acid have been shown to upregulate TREM‐1 expression in macrophage and neutrophils, the role of specific TLR in inducing the expression of TREM‐1 remains unclear. In this study, we investigated whether the presence of TLR is necessary for the expression of TREM‐1. We show that BM‐derived macrophages from TLR4 and TLR2 KO mice failed to induce expression of TREM‐1 message and protein in response to their specific ligands. Interestingly, the expression of TREM‐1 in response to LPS is not altered in myeloid differentiation factor 88 (MyD88) KO macrophages, suggesting that downstream of TLR a MyD88‐independent pathway induces the expression of TREM‐1. Inhibiting toll/IL‐1R domain‐containing adaptor‐inducing IFN‐β (TRIF) expression by siRNA decreased TREM‐1 expression in response to LPS, suggesting that the expression of TREM‐1 in response to LPS was mediated by the TRIF signaling pathway. On the other hand, the expression of TREM‐1 in response to lipoteichoic acid is dependent on MyD88 expression. These data indicate that the expression of TREM‐1 in response to TLR ligands occurs secondary to downstream signaling events and that the presence of TLR is necessary for the expression of TREM‐1 in response to their specific ligands. However, the downstream signaling required for the expression of TREM‐1 is dependent on the stimulus and the surface receptor through which the signaling is initiated.