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

Physiological strength electric fields modulate human T cell activation and polarisation

Acknowledgements: This work was supported by grants from an NHS Grampian Endowment Fund (Grant number 10/19). C.E.A was supported by an Institution of Medical Science University studentship. The authors acknowledge and are grateful to all volunteers for donating blood for T cell isolation. The authors also thank the University of Aberdeen Iain Fraser Cytometry Centre for their assistance.Peer reviewedPublisher PD

25-Hydroxycholesterol Regulates Inflammation by Activating Integrin-FAK Signaling Pathway

Inflammation is part of host innate immune response constituting the first line of defense against invading pathogens. Regulated inflammation is required for tissue homeostasis and host defense however, dysregulated inflammation is detrimental as it leads to tissue damage and development of various inflammatory diseases like arthritis, diabetes, Alzheimer’s etc. The cells of innate immune system like macrophages play a pivotal role during inflammation. Various receptors like Pathogen Recognition Receptors (PRRs) and Tumor Necrosis Factor-α receptor (TNFR) play important roles in triggering inflammatory response during infectious and non-infectious diseases. The activation of PRRs and TNFR results in the activation of various downstream signaling cascade which includes NFB, MAPK (mitogen activated protein kinase) and type I interferon pathways. Specifically, activation of NFB ad MAPK pathways results in the trans-activation of pro-inflammatory genes and subsequent production of pro-inflammatory cytokines and chemokines that shapes the innate immune inflammatory response. Our study has identified a lipid oxysterol 25 hydroxycholesterol (25HC) as a key positive regulator of PRR and TNFR mediated inflammatory response. 25HC is generated from the enzymatic conversion of cholesterol to 25HC by cholesterol 25-hydroxylase (C25H). Here, in we report that expression of C25H and extracellular production of 25HC from a cytosolic PRR nucleotide oligomerization domain protein 2 (Nod2) activated cells, TNFR activated cells and virus (Respiratory syncytial virus and influenza virus) infected macrophages. During these events, extracellular 25HC binds to cell surface integrin to activate integrin- FAK (focal adhesion kinase)-NFB signaling pathway for optimal pro-inflammatory response. Furthermore, our study also reveals that C25H expression in TNFR activated cells require NFB and MAPK pathways. Thus, our study has identified MAPK/NFB-C25H-25HC-integrin-FAK-NFB signaling network as a novel cellular mechanism involved in amplification of the pro-inflammatory response following Nod2 (PRR) and TNFR activation. Furthermore, we showed that a lipid oxysterol like 25HC can act as a “bridge” to link PRR and TNFR pathways with integrin-FAK signaling for optimal pro-inflammatory response

Lytic Cell Death Mechanisms in Human Respiratory Syncytial Virus-Infected Macrophages: Roles of Pyroptosis and Necroptosis

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

Molecular basis for the recognition of 24-(S)-hydroxycholesterol by integrin αvβ3

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 αvβ3, 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 αvβ3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway