Tumor Necrosis Factor-alpha utilizes MAPK/NFκB pathways to induce cholesterol-25 hydroxylase for amplifying pro-inflammatory response via 25-hydroxycholesterol-integrin-FAK pathway.

Exaggerated inflammatory response results in pathogenesis of various inflammatory diseases. Tumor Necrosis Factor-alpha (TNF) is a multi-functional pro-inflammatory cytokine regulating a wide spectrum of physiological, biological, and cellular processes. TNF induces Focal Adhesion Kinase (FAK) for various activities including induction of pro-inflammatory response. The mechanism of FAK activation by TNF is unknown and the involvement of cell surface integrins in modulating TNF response has not been determined. In the current study, we have identified an oxysterol 25-hydroxycholesterol (25HC) as a soluble extracellular lipid amplifying TNF mediated innate immune pro-inflammatory response. Our results demonstrated that 25HC-integrin-FAK pathway amplifies and optimizes TNF-mediated pro-inflammatory response. 25HC generating enzyme cholesterol 25-hydroxylase (C25H) was induced by TNF via NFκB and MAPK pathways. Specifically, chromatin immunoprecipitation assay identified binding of AP-1 (Activator Protein-1) transcription factor ATF2 (Activating Transcription Factor 2) to the C25H promoter following TNF stimulation. Furthermore, loss of C25H, FAK and α5 integrin expression and inhibition of FAK and α5ß1 integrin with inhibitor and blocking antibody, respectively, led to diminished TNF-mediated pro-inflammatory response. Thus, our studies show extracellular 25HC linking TNF pathway with integrin-FAK signaling for optimal pro-inflammatory activity and MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an essential role to amplify TNF dependent pro-inflammatory response. Thus, we have identified 25HC as the key factor involved in FAK activation during TNF mediated response and further demonstrated a role of cell surface integrins in positively regulating TNF dependent pro-inflammatory response.

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.

Inflammasome Antagonism by Human Parainfluenza Virus Type 3 C Protein.

Human parainfluenza virus type 3 (HPIV3) is a negative-sense single-stranded RNA virus belonging to the Paramyxoviridae family. HPIV3 is a lung-tropic virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. The activation of the inflammasome by pathogens results in the production of proinflammatory cytokines such as interleukin-1ß (IL-1ß) during infection. Thus, the inflammasome-mediated proinflammatory response plays a critical role in regulating the immune response and virus clearance. The inflammasome is a multimeric protein complex triggering caspase-1 activation. Activated caspase-1 cleaves pro-IL-1ß into its mature (and active) secretory form. Our study revealed inflammasome activation in macrophages following HPIV3 infection. Specifically, the activation of the NLRP3/ASC inflammasome resulted in the production of mature IL-1ß from HPIV3-infected cells. Furthermore, Toll-like receptor 2 (TLR2) activation (first signal) and potassium efflux (second signal) constituted two cellular events mediating inflammasome activation following HPIV3 infection. During our studies, we surprisingly identified the HPIV3 C protein as an antagonist of inflammasome activation. The HPIV3 C protein is an accessory protein encoded by the open reading frame of the viral phosphoprotein (P) gene. The HPIV3 C protein interacted with the NLRP3 protein and blocked inflammasome activation by promoting the proteasomal degradation of the NLRP3 protein. Thus, our studies report NLRP3/ASC inflammasome activation by HPIV3 via TLR2 signaling and potassium efflux. Furthermore, we have identified HPIV3 C as a viral component involved in antagonizing inflammasome activation.IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a paramyxovirus that causes respiratory tract diseases during infancy and childhood. Currently, there is no effective vaccine or antiviral therapy for HPIV3. Therefore, in order to develop anti-HPIV3 agents (therapeutics and vaccines), it is important to study the HPIV3-host interaction during the immune response. Inflammasomes play an important role in the immune response. Inflammasome activation by HPIV3 has not been previously reported. Our studies demonstrated inflammasome activation by HPIV3 in macrophages. Specifically, HPIV3 activated the NLRP3/ASC inflammasome by TLR2 activation and potassium efflux. C proteins of paramyxoviruses are accessory proteins encoded by the viral phosphoprotein gene. The role of the C protein in inflammasome regulation was unknown. Surprisingly, our studies revealed that the HPIV3 C protein antagonizes inflammasome activation. In addition, we highlighted for the first time a mechanism utilized by paramyxovirus accessory proteins to block inflammasome activation. The HPIV3 C protein interacted with the NLRP3 protein to trigger the proteasomal degradation of the NLRP3 protein.

Autophagy, TGF-ß, and SMAD-2/3 Signaling Regulates Interferon-ß Response in Respiratory Syncytial Virus Infected Macrophages.

Human respiratory syncytial virus (RSV) is a lung tropic virus causing severe airway diseases including bronchiolitis and pneumonia among infants, children, and immuno-compromised individuals. RSV triggers transforming growth factor-ß (TGF-ß) production from lung epithelial cells and TGF-ß facilitates RSV infection of these cells. However, it is still unknown whether RSV infected myeloid cells like macrophages produce TGF-ß and the role of TGF-ß if any during RSV infection of these cells. Our study revealed that RSV infected macrophages produce TGF-ß and as a consequence these cells activate TGF-ß dependent SMAD-2/3 signaling pathway. Further mechanistic studies illustrated a role of autophagy in triggering TGF-ß production from RSV infected macrophages. In an effort to elucidate the role of TGF-ß and SMAD-2/3 signaling during RSV infection, we surprisingly unfolded the requirement of TGF-ß-SMAD2/3 signaling in conferring optimal innate immune antiviral response during RSV infection of macrophages. Type-I interferon (e.g., interferon-ß or IFN-ß) is a critical host factor regulating innate immune antiviral response during RSV infection. Our study revealed that loss of TGF-ß-SMAD2/3 signaling pathway in RSV infected macrophages led to diminished expression and production of IFN-ß. Inhibiting autophagy in RSV infected macrophages also resulted in reduced production of IFN-ß. Thus, our studies have unfolded the requirement of autophagy-TGF-ß-SMAD2/3 signaling network for optimal innate immune antiviral response during RSV infection of macrophages.

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.

Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G-Deficient Candidate Vaccine Strain.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

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.

Cross-protective immune responses between genotypically distinct lineages of infectious laryngotracheitis viruses.

Recent phylogenetic studies have identified different genotypic lineages of infectious laryngotracheitis virus (ILTV), and these lineages can recombine in the field. The emergence of virulent recombinant field strains of ILTV by natural recombination between commercial vaccines belonging to different genotypic lineages has been reported recently. Despite the use of attenuated ILTV vaccines, these recombinant viruses were able to spread and cause disease in commercial poultry flocks, raising the question of whether the different lineages of ILTV can induce cross-protective immune responses. This study examined the capacity of the Australian-origin A20 ILTV vaccine to protect against challenge with the class 8 ILTV recombinant virus, the genome of which is predominantly derived from a heterologous genotypic lineage. Following challenge, birds vaccinated via eyedrop were protected from clinical signs of disease and pathological changes in the tracheal mucosa, although they were not completely protected from viral infection or replication. In contrast, the challenge virus induced severe clinical signs and tracheal pathology in unvaccinated birds. This is the first study to examine the ability of a vaccine from the Australian lineage to protect against challenge with a virus from a heterologous lineage. These results suggest that the two distinct genotypic lineages of ILTV can both induce cross-protection, indicating that current commercial vaccines are still likely to assist in control of ILTV in the poultry industry, in spite of the emergence of novel recombinants derived from different genotypic lineages.

Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction with the gG-ve ILTV vaccine in a 'differentiating infected from vaccinated animals' approach to the control of ILT.