Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response.

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

Longitudinal perceptions of the side effects of chemotherapy in patients with gynecological cancer.

PURPOSE: This study aimed to assess the incidence and difference of side effects among six courses of chemotherapy (C/T) in gynecological cancer patients. METHODS: The study period was from Sep. 2010 to Dec. 2011 at the Kaohsiung Veterans General Hospital in Taiwan. The treating protocols, courses, and drugs of C/T in patient were considered according to the different malignant cancers and clinical conditions. The patient data of age, marriage status, education, religion, and experiences of C/T were collected. The patients' or their families' reported side effects of C/T were recorded daily from the beginning of C/T to the 10th day after C/T in each cycle and every course of C/T. RESULTS: Total 89 patients enrolled into the study received total 450 courses of C/T. The mean age was 54.52 ± 11.02. Ovarian cancer was the most common malignant disease (64.0%). The most often combination of drugs used was Taxol and carboplatin (40.9%). Patients complained peripheral numbness of limbs, with the highest incidence of 58.6%. The side effects with incidence about 50% were decreased fatigue (55.0%) and hair loss (49.9%). Other side effects with different levels of incidence were also noticed, such as lack of appetite, changes in taste, and muscle ache. The incidences of peripheral limb numbness and hair loss were increased with following courses of C/T. The high incidence of fatigue did not show variation between different courses of C/T. CONCLUSION: This study revealed the incidence of side effects and occurrence timing during C/T in patients with gynecological cancer. These data provide substantial information to patients and their families to understand the potential side effects of C/T courses, which might increase their compliance in receiving adjuvant C/T. Relieving the side effects in C/T would be important to improve their quality of daily life and treatment willingness.

Regulation of TLR3 Activation by S100A9.

Recognition of viral dsRNA by endosomal TLR3 activates innate immune response during virus infection. Trafficking of TLR3 to the endolysosomal compartment arising from fusion of late endosome (LE) with lysosome is required for recognition and detection of pathogen associated molecular patterns, which results in activation of the TLR3-dependent signaling cascade. Existing knowledge about the mechanism(s) and cellular factor(s) governing TLR3 trafficking is limited. In the current study, we identified intracellular S100A9 protein as a critical regulator of TLR3 trafficking. S100A9 was required for maturation of TLR3 containing early endosome (EE) into LE, the compartment that fuses with lysosome to form the endolysosomal compartment. A drastic reduction in cytokine production was observed in S100A9-knockout (KO) primary macrophages following RNA virus infection and treatment of cells with polyinosinic-polycytidylic acid (polyIC; a dsRNA mimetic that acts as a TLR3 agonist). Mechanistic studies revealed colocalization and interaction of S100A9 with TLR3 following polyIC treatment. S100A9-TLR3 interaction was critical for maturation of TLR3 containing EE into LE because TLR3 could not be detected in the LE of polyIC-treated S100A9-KO macrophages. Subsequently, TLR3 failed to colocalize with its agonist (i.e., biotin-labeled polyIC) in S100A9-deficient macrophages. The in vivo physiological role of S100A9 was evident from loss of cytokine production in polyIC-treated S100A9-KO mice. Thus, we identified intracellular S100A9 as a regulator of TLR3 signaling and demonstrated that S100A9 functions during pre-TLR3 activation stages by facilitating maturation of TLR3 containing EE into LE.

Nedd8 regulates inflammasome-dependent caspase-1 activation.

Caspase-1 is activated by the inflammasome complex to process cytokines like interleukin-1ß (IL-1ß). Pro-caspase-1 consists of three domains, CARD, p20, and p10. Association of pro-caspase-1 with the inflammasome results in initiation of its autocatalytic activity, culminating in self-cleavage that generates catalytically active subunits (p10 and p20). In the current study, we show that Nedd8 is required for efficient self-cleavage of pro-caspase-1 to generate its catalytically active subunits. Nedd8 silencing or treating cells with the neddylation inhibitor MLN4924 led to diminished caspase-1 processing and reduced IL-1ß maturation following inflammasome activation. Coimmunoprecipitation and mass spectrometric analysis of 293 cells overexpressing pro-caspase-1 (and CARD) and Nedd8 suggested possible neddylation of caspase-1 CARD. Following inflammasome activation in primary macrophages, we observed colocalization of endogenous Nedd8 with caspase-1. Similarly, interaction of endogenous Nedd8 with caspase-1 CARD was detected in inflammasome-activated macrophages. Furthermore, enhanced autocatalytic activity of pro-caspase-1 was observed following Nedd8 overexpression in 293 cells, and such activity in inflammasome-activated macrophages was drastically diminished upon treatment of cells with MLN4924. Thus, our studies demonstrate a role of Nedd8 in regulating caspase-1 activation following inflammasome activation, presumably via augmenting autoprocessing/cleavage of pro-caspase-1 into its corresponding catalytically active subunits.

DAMP molecule S100A9 acts as a molecular pattern to enhance inflammation during influenza A virus infection: role of DDX21-TRIF-TLR4-MyD88 pathway.

Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous "inflammatory mediators" called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 (also known as Calgranulin B or MRP-14), as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection.

Transactivation of inducible nitric oxide synthase gene by Kruppel-like factor 6 regulates apoptosis during influenza A virus infection.

Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice.

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection.

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1ß (IL-1ß) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1ß is synthesized as an immature pro-IL-1ß form. It is cleaved by activated caspase-1 to yield mature IL-1ß that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1ß release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1ß secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1ß production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1ß and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1ß release during RSV infection.