ABSTRACT
Type-I interferons (IFN) are key antiviral factors that induce several cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity. Apart from ISGylation, unconjugated ISG15 is also released from cells during immuno-stimulation and virus infection. The role of extracellular ISG15 during virus infection was unknown. We now show that extracellular ISG15 acts as a soluble antiviral factor to restrict virus infection in human epithelial cells via an IFN-independent mechanism. Additionally, we demonstrated that extracellular ISG15 induces expression of intracellular ISG15 and ISGylation in the absence of IFN. Mechanistic studies revealed that extracellular ISG15 interacts with cell surface integrin (α5ß1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified ISG15 protein as a new soluble factor that confers IFN-independent antiviral activity by inducing ISGylation. Additionally, we have unfolded an IFN-independent non-canonical ISGylation by extracellular ISG15 that operates via the integrin-FAK pathway.
ABSTRACT
The study of S100A9 in viral infections has seen increased interest since the COVID-19 pandemic. S100A8/A9 levels were found to be correlated with the severity of COVID-19 disease, cytokine storm, and changes in myeloid cell subsets. These data led to the hypothesis that S100A8/A9 proteins might play an active role in COVID-19 pathogenesis. This review explores the structures and functions of S100A8/9 and the current knowledge on the involvement of S100A8/A9 and its constituents in viral infections. The potential roles of S100A9 in SARS-CoV-2 infections are also discussed.
Subject(s)
COVID-19 , Calgranulin A , Calgranulin B , Inflammation , SARS-CoV-2 , Humans , COVID-19/immunology , SARS-CoV-2/immunology , Inflammation/immunology , Cytokine Release Syndrome/immunology , Virus Diseases/immunologyABSTRACT
Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects the respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccines to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Cytoplasmic stabilization of ß-catenin protein results in activation of canonical Wingless (Wnt)/ß-catenin signaling pathway that culminates in transcriptional activation of various genes regulated by T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This pathway is involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering ß-catenin protein stabilization and induction of ß-catenin mediated transcriptional activity. Functionally, the activated ß-catenin pathway promoted a pro-inflammatory response during RSV infection of lung epithelial cells. Studies with ß-catenin inhibitors and A549 cells lacking optimal ß-catenin activity demonstrated a significant loss of pro-inflammatory chemokine interleukin-8 (IL-8) release from RSV-infected cells. Mechanistically, our studies revealed a role of extracellular human beta defensin-3 (HBD3) in interacting with cell surface Wnt receptor LDL receptor-related protein-5 (LRP5) to activate the non-canonical Wnt independent ß-catenin pathway during RSV infection. We showed gene expression and release of HBD3 from RSV-infected cells and silencing of HBD3 expression resulted in reduced stabilization of ß-catenin protein during RSV infection. Furthermore, we observed the binding of extracellular HBD3 with cell surface localized LRP5 protein, and our in silico and protein-protein interaction studies have highlighted a direct interaction of HBD3 with LRP5. Thus, our studies have identified the ß-catenin pathway as a key regulator of pro-inflammatory response during RSV infection of human lung epithelial cells. This pathway was induced during RSV infection via a non-canonical Wnt-independent mechanism involving paracrine/autocrine action of extracellular HBD3 activating cell surface Wnt receptor complex by directly interacting with the LRP5 receptor.
ABSTRACT
A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer's disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvß3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvß3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway.
Subject(s)
Hydroxycholesterols , Integrin alphaVbeta3 , Computer Simulation , Humans , Integrin alphaVbeta3/chemistry , Integrin alphaVbeta3/metabolism , Hydroxycholesterols/chemistry , Hydroxycholesterols/metabolism , Inflammation/metabolism , Signal Transduction , Macrophages/metabolism , Models, Molecular , Thermodynamics , Protein Conformation , Surface Plasmon Resonance , Cholesterol 24-Hydroxylase/metabolismABSTRACT
Critically ill COVID-19 patients display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We inoculated and treated human macrophage cell line THP-1 with SARS-CoV-2 and purified, glycosylated, soluble SARS-CoV-2 spike protein S1 subunit (S1) to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication or viral entry, virus exposure resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that extracellular soluble S1 protein is a key viral component inducing pro-inflammatory responses in macrophages, independent of virus replication. Thus, virus- or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Spike Glycoprotein, Coronavirus/genetics , Tumor Necrosis Factor-alpha , Inflammation , MacrophagesABSTRACT
Paramyxoviruses such as respiratory syncytial virus (RSV) are the leading cause of pneumonia in infants, the elderly, and immunocompromised individuals. Understanding host-virus interactions is essential for the development of effective interventions. RSV induces autophagy to modulate the immune response. The viral factors and mechanisms underlying RSV-induced autophagy are unknown. Here, we identify the RSV nonstructural protein NS2 as the virus component mediating RSV-induced autophagy. We show that NS2 interacts and stabilizes the proautophagy mediator Beclin1 by preventing its degradation by the proteasome. NS2 further impairs interferon-stimulated gene 15 (ISG15)-mediated Beclin1 ISGylation and generates a pool of "hypo-ISGylated" active Beclin1 to engage in functional autophagy. Studies with NS2-deficient RSV revealed that NS2 contributes to RSV-mediated autophagy during infection. The present study is the first report to show direct activation of autophagy by a paramyxovirus nonstructural protein. We also report a new viral mechanism for autophagy induction wherein the viral protein NS2 promotes hypo-ISGylation of Beclin1 to ensure availability of active Beclin1 to engage in the autophagy process. IMPORTANCE Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. Here, we identify the RSV nonstructural protein 2 (NS2) as the virus component involved in autophagy induction. NS2 promotes autophagy by interaction with and stabilization of the proautophagy mediator Beclin1 and by impairing its ISGylation to overcome autophagy inhibition. To the best of our knowledge, this is the first report of a viral protein regulating the autophagy pathway by modulating ISGylation of autophagy mediators. Our studies highlight a direct role of a paramyxovirus nonstructural protein in activating autophagy by interacting with the autophagy mediator Beclin1. NS2-mediated regulation of the autophagy and ISGylation processes is a novel function of viral nonstructural proteins to control the host response against RSV.
Subject(s)
Respiratory Syncytial Virus, Human , Aged , Humans , Infant , Autophagy , Beclin-1/metabolism , Interferons/metabolism , Respiratory Syncytial Virus, Human/genetics , Viral Nonstructural Proteins/geneticsABSTRACT
Respiratory syncytial virus, or RSV, is a leading cause of viral pneumonia and bronchiolitis in children and other susceptible populations. RSV infection dysregulates the immune response leading to exaggerated inflammation in the airway. Among other responses, RSV induces macroautophagy/autophagy, a key process that regulates immune response during infection. We investigated the molecular mechanisms underlying RSV-induced autophagy and showed that the RSV nonstructural NS2 protein promotes autophagy using a dual mechanism. First, NS2 interacts with and stabilizes the autophagy regulator BECN1 (beclin 1), augmenting its intracellular availability for autophagy induction. Second, NS2 interferes with BECN1 ISGylation, thus restricting the intracellular pool of the anti-autophagy ISGylated form of BECN1. Thus, the viral protein (i.e., NS2)-autophagy-ISGylation axis represents a yet unknown regulatory network for viruses. As many viruses induce autophagy that shapes virus-associated immune responses including inflammation, exploring viral protein-autophagy-ISGylation regulatory networks can aid in developing interventions to curb exaggerated immune responses such as inflammation for treating virus-associated inflammatory diseases.
ABSTRACT
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.
Subject(s)
Signal Transduction/drug effects , Steroid Hydroxylases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Activating Transcription Factor 2/metabolism , Animals , Cells, Cultured , Chemokine CCL3/metabolism , Female , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Hydroxycholesterols/metabolism , Integrin alpha5/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Protein Binding , Steroid Hydroxylases/deficiency , Steroid Hydroxylases/genetics , Up-Regulation/drug effectsABSTRACT
Critically ill COVID-19 patients infected with SARS-CoV-2 display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We used SARS-CoV-2 infected and glycosylated soluble SARS-CoV-2 Spike S1 subunit (S1) treated THP-1 human-derived macrophage-like cell line to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication, virus infection resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that S1 is a key viral component inducing inflammatory response in macrophages, independently of virus replication. Thus, virus-infected or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.
ABSTRACT
The symptoms of infectious diarrheal disease are mediated by a combination of a pathogen's virulence factors and the host immune system. Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide due to its near-ubiquitous zoonotic association with poultry. One of the outstanding questions is to what extent the bacteria are responsible for the diarrheal symptoms via intestinal cell necrosis versus immune cell initiated tissue damage. To determine the stepwise process of inflammation that leads to diarrhea, we used a piglet ligated intestinal loop model to study the intestinal response to C. jejuni. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine. We found that the abundance of neutrophil related proteins increased in the intestinal lumen during C. jejuni infection, including proteins related to neutrophil migration (neutrophil elastase and MMP9), actin reorganization (Arp2/3), and antimicrobial proteins (lipocalin-2, myeloperoxidase, S100A8, and S100A9). The appearance of neutrophil proteins also corresponded with increases of the inflammatory cytokines IL-8 and TNF-α. Compared to infection with the C. jejuni wild-type strain, infection with the noninvasive C. jejuni ∆ciaD mutant resulted in a blunted inflammatory response, with less inflammatory cytokines and neutrophil markers. These findings indicate that intestinal inflammation is driven by C. jejuni virulence and that neutrophils are the predominant cell type responding to C. jejuni infection. We propose that this model can be used as a platform to study the early immune events during infection with intestinal pathogens.
Subject(s)
Campylobacter Infections/immunology , Campylobacter jejuni/immunology , Cytokines/immunology , Intestine, Small/immunology , Intestine, Small/microbiology , Neutrophils/immunology , Animals , Campylobacter Infections/microbiology , Campylobacter jejuni/genetics , Campylobacter jejuni/metabolism , Campylobacter jejuni/pathogenicity , Cell Line , Cytokines/metabolism , Disease Models, Animal , Epithelial Cells/immunology , Gastrointestinal Microbiome , Inflammation/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Intestine, Small/pathology , Macrophages/immunology , Proteome/analysis , Swine , Swine, Miniature , Transcriptome , Virulence/genetics , Virulence Factors/metabolismABSTRACT
Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages.
Subject(s)
Macrophages/pathology , Necroptosis , Pyroptosis , Respiratory Syncytial Virus, Human/pathogenicity , CARD Signaling Adaptor Proteins/metabolism , Caspase 1/metabolism , Cell Death , Humans , Inflammasomes/metabolism , L-Lactate Dehydrogenase/metabolism , Macrophages/metabolism , Macrophages/virology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , THP-1 CellsABSTRACT
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.
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Hydroxycholesterols/metabolism , Immunity, Innate/immunology , Integrin alpha5beta1/immunology , Integrin alphaVbeta3/immunology , Macrophages/immunology , Animals , Focal Adhesions , Inflammation , Integrin alpha5beta1/metabolism , Integrin alphaVbeta3/metabolism , Interleukin-6/immunology , Macrophages/metabolism , Mice , Mice, Knockout , Receptors, Pattern Recognition/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/immunologyABSTRACT
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.
Subject(s)
Inflammasomes/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Parainfluenza Virus 3, Human , Viral Proteins/physiology , Caspase 1/immunology , HEK293 Cells , Humans , Immunity, Innate , Interleukin-1beta/immunology , Macrophages/immunology , Potassium/metabolism , Signal Transduction , Toll-Like Receptor 2/immunology , Virus ReplicationABSTRACT
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.
Subject(s)
Immunity, Innate/immunology , Inflammation/metabolism , Mycoplasma pneumoniae/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pneumonia, Mycoplasma/immunology , Pneumonia, Mycoplasma/metabolism , Animals , Apoptosis Regulatory Proteins/immunology , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins/immunology , CARD Signaling Adaptor Proteins/metabolism , Caspase 1/immunology , Caspase 1/metabolism , Inflammasomes/immunology , Inflammasomes/metabolism , Inflammation/immunology , Inflammation/microbiology , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/immunology , NF-kappa B/metabolism , Pneumonia, Mycoplasma/microbiology , Signal Transduction/immunologyABSTRACT
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.
Subject(s)
Interferon-beta/immunology , Macrophages/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Antiviral Agents , Autophagy/physiology , Beclin-1/metabolism , Disease Models, Animal , Immunity, Innate , Interferon Type I/immunology , Interferon Type I/metabolism , Interferon-beta/metabolism , Macrophages/metabolism , Macrophages/virology , Mice , Mice, Inbred C57BL , RAW 264.7 Cells , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Calgranulin B/immunology , Macrophages/immunology , RNA Viruses/immunology , Toll-Like Receptor 3/immunology , Animals , Blotting, Western , Calgranulin B/genetics , Calgranulin B/metabolism , Cell Line , Cell Line, Tumor , Cells, Cultured , Female , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Interferon-beta/genetics , Interferon-beta/immunology , Interferon-beta/metabolism , Macrophages/metabolism , Macrophages/virology , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Poly I-C/immunology , Poly I-C/pharmacology , Protein Transport/drug effects , Protein Transport/immunology , RNA Interference , RNA Viruses/physiology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 3/metabolismABSTRACT
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.
Subject(s)
Caspase 1/metabolism , Inflammasomes/metabolism , Influenza A virus/isolation & purification , Ubiquitins/metabolism , Animals , Carrier Proteins , Enzyme Activation , Humans , Interleukin-1beta/biosynthesis , Macrophages/metabolism , Macrophages/virology , Mice, Inbred C57BL , NEDD8 ProteinABSTRACT
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.
Subject(s)
Adenosine Diphosphate/metabolism , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Carrier Proteins/metabolism , Host-Pathogen Interactions , Inflammasomes/metabolism , Mycoplasma pneumoniae/physiology , Protein Processing, Post-Translational , Animals , Cells, Cultured , Humans , Macrophages/immunology , Macrophages/metabolism , Mice , Mycoplasma pneumoniae/pathogenicity , NLR Family, Pyrin Domain-Containing 3 ProteinABSTRACT
How intestinal epithelial cells (IECs) recognize pathogens and activate inflammasomes at intestinal surfaces is poorly understood. We hypothesized that IECs use integrin receptors to recognize pathogens and initiate inflammation within the intestinal tract. We find that IECs infected with Yersinia enterocolitica, an enteric pathogen, use ß1 integrins as pathogen recognition receptors detecting the bacterial adhesin invasin (Inv). The Inv-integrin interaction provides the first signal for NLRP3 inflammasome activation with the type three secretion system translocon providing the second signal for inflammasome activation, resulting in release of IL-18. During infection, Yersinia employs two virulence factors, YopE and YopH, to counteract Inv-mediated integrin-dependent inflammasome activation. Furthermore, NLRP3 inflammasome activation in epithelial cells requires components of the focal adhesion complex signaling pathway, focal adhesion kinase, and rac1. The binding of Inv to ß1 integrins rapidly induces IL-18 mRNA expression, suggesting integrins provide a first signal for NLRP3 inflammasome activation. These data suggest integrins function as pathogen recognition receptors on IECs to rapidly induce inflammasome-derived IL-18-mediated responses.
Subject(s)
Epithelial Cells/immunology , Inflammasomes/immunology , Inflammasomes/metabolism , Integrin alpha5beta1/physiology , Intestinal Mucosa/immunology , Signal Transduction/immunology , Yersinia enterocolitica/immunology , Adhesins, Bacterial/metabolism , Adhesins, Bacterial/physiology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Toxins/pharmacology , Caco-2 Cells , Carrier Proteins/metabolism , Cell Line, Tumor , Epithelial Cells/metabolism , Humans , Integrin alpha5beta1/metabolism , Interleukin-18/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , NLR Family, Pyrin Domain-Containing 3 Protein , Protein Binding/immunology , Protein Tyrosine Phosphatases/deficiency , Protein Tyrosine Phosphatases/genetics , Virulence Factors/physiology , Yersinia enterocolitica/geneticsABSTRACT
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.