Autophagy, TGF-β, and SMAD-2/3 Signaling Regulates Interferon-β 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-beta(TGF-beta) production from lung epithelial cells and TGF-beta facilitates RSV infection of these cells. However, it is still unknown whether RSV infected myeloid cells like macrophages produce TGF-beta and the role of TGF-beta if any during RSV infection of these cells. Our study revealed that RSV infected macrophages produce TGF-beta and as a consequence these cells activate TGF-beta dependent SMAD-2/3 signaling pathway. Further mechanistic studies illustrated a role of autophagy in triggering TGF-beta production from RSV infected macrophages. In an effort to elucidate the role of TGF-beta and SMAD-2/3 signaling during RSV infection, we surprisingly unfolded the requirement of TGF-beta---SMAD2/3 signaling in conferring optimal innate immune antiviral response during RSV infection of macrophages. Type-I interferon (e.g. interferon-beta or IFN-beta) is a critical host factor regulating innate immune antiviral response during RSV infection. Our study revealed that loss of TGF-beta---SMAD2/3 signaling pathway in RSV infected macrophages led to diminished expression and production of IFN-beta. Inhibiting autophagy in RSV infected macrophages also resulted in reduced production of IFN-beta. Thus, our studies have unfolded the requirement of autophagy---TGF-beta---SMAD2/3 signaling network for optimal innate immune antiviral response during RSV infection of macrophages

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 α5β1 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

Nedd8 Regulates Inflammasome-Dependent Caspase-1 Activation

Caspase-1 is activated by the inflammasome complex to process cytokines like interleukin-1β (IL-1β). 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-1β 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

Nedd8 regulates inflammasome-dependent caspase-1 activation

Caspase-1 is activated by the inflammasome complex to process cytokines like interleukin-1β (IL-1β). 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-1β 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