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.

NLRP3 Is a Critical Regulator of Inflammation and Innate Immune Cell Response during Mycoplasma pneumoniae Infection.

Mycoplasma pneumoniae is an atypical bacterial respiratory pathogen known to cause a range of airway inflammation and lung and extrapulmonary pathologies. We recently reported that an M. pneumoniae-derived ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin is capable of triggering NLRP3 (NLR-family, leucine-rich repeat protein 3) inflammasome activation and interleukin-1ß (IL-1ß) secretion in macrophages. However, it is unclear whether the NLRP3 inflammasome is important for the immune response during M. pneumoniae acute infection. In the current study, we utilized in vitro and in vivo models of M. pneumoniae infection to characterize the role of the NLRP3 inflammasome during acute infection. M. pneumoniae-infected macrophages deficient for inflammasome components NLRP3, ASC (apoptosis speck-like protein containing a caspase activation and recruitment domain), or caspase-1 failed to process and secrete IL-1ß. The MyD88/NF-κB signaling pathway was found to be critical for proinflammatory gene expression in macrophages infected with M. pneumoniae C57BL/6 mice deficient for NLRP3 expression were unable to produce IL-1ß in the airways during acute infection, and lack of this inflammatory response led to deficient immune cell activation and delayed bacterial clearance. These findings are the first to report the importance of the NLRP3 inflammasome in regulating the inflammatory response and influencing the progression of M. pneumoniae during acute infection.

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.

ADP-ribosylation of NLRP3 by Mycoplasma pneumoniae CARDS toxin regulates inflammasome activity.

UNLABELLED: The inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1ß (pro-IL-1ß) into its mature form. IL-1ß is a critical proinflammatory cytokine that dictates the severity of inflammation associated with a wide spectrum of inflammatory diseases. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the current study, we uncovered a yet unknown mechanism of NLRP3 inflammasome activation by a pathogen-derived factor. We show that the unique bacterial ADP-ribosylating and vacuolating toxin produced by Mycoplasma pneumoniae and designated community-acquired respiratory distress syndrome (CARDS) toxin activates the NLRP3 inflammasome by colocalizing with the NLRP3 inflammasome and catalyzing the ADP-ribosylation of NLRP3. Mutant full-length CARDS toxin lacking ADP-ribosyltransferase (ADPRT) activity and truncated CARDS toxins unable to bind to macrophages and be internalized failed to activate the NLRP3 inflammasome. These studies demonstrate that CARDS toxin-mediated ADP-ribosylation constitutes an important posttranslational modification of NLRP3, that ADPRT activity of CARDS toxin is essential for NLRP3 inflammasome activation, and that posttranslational ADPRT-mediated modification of the inflammasome is a newly discovered mechanism for inflammasome activation with subsequent release of IL-1ß and associated pathologies. IMPORTANCE: Inflammation is a fundamental innate immune response to environmental factors, including infections. The inflammasome represents a multiprotein complex that regulates inflammation via its ability to activate specific proinflammatory cytokines, resulting in an effective host protective response. However, excessive release of proinflammatory cytokines can occur following infection that skews the host response to "hyperinflammation" with exaggerated tissue damage. Mycoplasma pneumoniae, a common bacterial airway pathogen, possesses a unique protein toxin with ADP-ribosyltransferase and vacuolating properties capable of reproducing the robust inflammation and cytopathology associated with mycoplasma infection. Here, we show that the toxin uniquely activates the NLRP3 inflammasome by colocalizing with and ADP-ribosylating NLRP3, possibly leading to "hyperinflammation" and thus uncovering a novel target for therapeutic intervention.

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.

KLF6 and iNOS regulates apoptosis during respiratory syncytial virus infection.

Human respiratory syncytial virus (RSV) is a highly pathogenic lung-tropic virus that causes severe respiratory diseases. Enzymatic activity of inducible nitric oxide (iNOS) is required for NO generation. Although NO contributes to exaggerated lung disease during RSV infection, the role of NO in apoptosis during infection is not known. In addition, host trans-activator(s) required for iNOS gene expression during RSV infection is unknown. In the current study we have uncovered the mechanism of iNOS gene induction by identifying kruppel-like factor 6 (KLF6) as a critical transcription factor required for iNOS gene expression during RSV infection. Furthermore, we have also uncovered the role of iNOS as a critical host factor regulating apoptosis during RSV 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.

Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles.

Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in the life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.

Oncolytic targeting of androgen-sensitive prostate tumor by the respiratory syncytial virus (RSV): consequences of deficient interferon-dependent antiviral defense.

BACKGROUND: Oncolytic virotherapy for cancer treatment utilizes viruses for selective infection and death of cancer cells without any adverse effect on normal cells. We previously reported that the human respiratory syncytial virus (RSV) is a novel oncolytic virus against androgen-independent PC-3 human prostate cancer cells. The present study extends the result to androgen-dependent prostate cancer, and explores the underlying mechanism that triggers RSV-induced oncolysis of prostate cancer cells. METHODS: The oncolytic effect of RSV on androgen-sensitive LNCaP human prostate cancer cells and on androgen-independent RM1 murine prostate cancer cells was studied in vitro in culture and in vivo in a xenograft or allograft tumor model. In vitro, cell viability, infectivity and apoptosis were monitored by MTT assay, viral plaque assay and annexin V staining, respectively. In vivo studies involved virus administration to prostate tumors grown in immune compromised nude mice and in syngeneic immune competent C57BL/6J mice. Anti-tumorogenic oncolytic activity was monitored by measuring tumor volume, imaging bioluminescent tumors in live animals and performing histopathological analysis and TUNEL assay with tumors RESULTS: We show that RSV imposes a potent oncolytic effect on LNCaP prostate cancer cells. RSV infectivity was markedly higher in LNCaP cells compared to the non-tumorigenic RWPE-1 human prostate cells. The enhanced viral burden led to LNCaP cell apoptosis and growth inhibition of LNCaP xenograft tumors in nude mice. A functional host immune response did not interfere with RSV-induced oncolysis, since growth of xenograft tumors in syngeneic C57BL/6J mice from murine RM1 cells was inhibited upon RSV administration. LNCaP cells failed to activate the type-I interferon (IFNα/ß)-induced transcription factor STAT-1, which is required for antiviral gene expression, although these cells could produce IFN in response to RSV infection. The essential role of IFN in restricting infection was further borne out by our finding that neutralizing IFN activity resulted in enhanced RSV infection in non-tumorigenic RWPE-1 prostate cells. CONCLUSIONS: We demonstrated that RSV is potentially a useful therapeutic tool in the treatment of androgen-sensitive and androgen-independent prostate cancer. Moreover, impaired IFN-mediated antiviral response is the likely cause of higher viral burden and resulting oncolysis of androgen-sensitive prostate cancer cells.

Activation of innate immune antiviral responses by Nod2.

Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs) and RIG-like helicase (RLH) receptors, are involved in innate immune antiviral responses. Here we show that nucleotide-binding oligomerization domain 2 (Nod2) can also function as a cytoplasmic viral PRR by triggering activation of interferon-regulatory factor 3 (IRF3) and production of interferon-beta (IFN-beta). After recognition of a viral ssRNA genome, Nod2 used the adaptor protein MAVS to activate IRF3. Nod2-deficient mice failed to produce interferon efficiently and showed enhanced susceptibility to virus-induced pathogenesis. Thus, the function of Nod2 as a viral PRR highlights the important function of Nod2 in host antiviral defense mechanisms.

Efficiency enhancement in GaAs solar cells using self-assembled microspheres.

In this study we develop an efficient light harvesting scheme that can enhance the efficiency of GaAs solar cells using self-assembled microspheres. Based on the scattering of the microspheres and the theory of photonic crystals, the path length can be increased. In addition, the self-assembly of microspheres is one of the simplest and the fastest methods with which to build a 2D periodic structure. The experimental results are confirmed by the use of a simulation in which a finite-difference time-domain (FDTD) method is used to analyze the absorption and electric field of the 2D periodic structure. Both the results of the numerical simulations and the experimental results show an increase in the conversion power efficiency of GaAs solar cell of about 25% when 1 microm microspheres were assembled on the surface of GaAs solar cells.

Role of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaB-mediated innate antiviral response against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-alpha/beta-independent, innate antiviral response against RSV that was dependent on activation of NF-kappaB. We demonstrated that NF-kappaB inducing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) confers potent antiviral function against RSV in an NF-kappaB-dependent fashion, independent of interferon-alpha/beta. During our efforts to study this pathway, we identified HBD2 (human beta-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-kappaB-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-kappaB-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine beta-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-kappaB-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.

Plasma antibodies against Trichomonas vaginalis and subsequent risk of prostate cancer.

BACKGROUND: Although several previous case-control studies have investigated associations between sexually transmitted infections (STI) and prostate cancer, most have focused on gonorrhea and syphilis, two well-recognized, symptomatic STIs. Another STI of interest for prostate carcinogenesis is trichomonosis, a less well recognized and frequently asymptomatic STI with known prostate involvement. We investigated this infection in relation to incident prostate cancer in a nested case-control study within the Health Professionals Follow-up Study. METHODS: Prostate cancer cases were men diagnosed with prostate cancer between the date of blood draw (1993-1995) and 2000 (n = 691). Controls were men who had had at least one prostate-specific antigen test and who were free of prostate cancer and alive at the time of case diagnosis. One control was individually matched to each case by age (n = 691). Serologic evidence of a history of trichomonosis was assessed by a recombinant Trichomonas vaginalis alpha-actinin IgG ELISA. RESULTS: Thirteen percent of cases and 9% of controls were seropositive for trichomonosis (adjusted odds ratio, 1.43; 95% confidence interval, 1.00-2.03). This association persisted after additional adjustment for such factors as a history of other STIs, and was strongest among men who used aspirin infrequently over the course of their lives (odds ratio, 2.05; 95% confidence interval, 1.05-4.02, P(interaction) = 0.11). CONCLUSIONS: Serologic evidence of a history of trichomonosis was positively associated with incident prostate cancer in this large, nested case-control study of male health professionals. As this study is the first, to our knowledge, to investigate associations between T. vaginalis serology and prostate cancer, additional studies are necessary before conclusions can be made.

Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis.

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high-iron conditions. Fluorescence and immuno-cytochemical experiments show that high-iron-grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low-iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti-AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two-dimensional electrophoresis and immunoblot of adhesins from surface-labelled parasites provided evidence that all members of the multigene family were co-ordinately expressed and placed on the trichomonal surface. Similar two-dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface-expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug-resistant isolate absent in hydrogenosome proteins and adhesins, were non-adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.