• Airway epithelial cells generate pro-inflammatory tenascin-C and small extracellular vesicles in response to TLR3 stimuli and rhinovirus infection

      Mills, Jake; Schwenzer, Anja; Marsh, Elizabeth; Edwards, Michael; Midwood, Kim; Sabroe, Ian; Parker, Lisa; University of Sheffield; University of Oxford; University of Derby; et al. (Frontiers, 2019-08-21)
      Viral infections are a common cause of asthma exacerbations, with human rhinoviruses (RV) the most common trigger. RV signals through a number of different receptors, including toll-like receptor (TLR)3. Tenascin-C (TN-C) is an immunomodulatory extracellular matrix protein present in high quantities in the airway of people with asthma, and expression is also upregulated in nasal lavage fluid in response to RV infection. Respiratory viral infection has been demonstrated to induce the release of small extracellular vesicles (sEV) such as exosomes, whilst exosomal cargo can also be modified in the bronchoalveolar lavage fluid of people with asthma. These sEVs may potentiate airway inflammation and regulate the immune response to infection. This study characterises the relationship between RV infection of bronchial epithelial cells and the release of TN-C, and the release of sEVs following stimulation with the TLR3 agonist and synthetic viral mimic, poly(I:C), as well as the function of the released protein / vesicles. The BEAS-2B airway epithelial cell line and primary human bronchial epithelial cells (PBECs) from asthmatic and non-asthmatic donors were infected with RV or treated with poly(I:C). TN-C expression, release and localisation to sEVs was quantified. TN-C expression was also assessed following intra-nasal challenge of C57BL/6 mice with poly(I:C). BEAS-2B cells and macrophages were subsequently challenged with TN-C, or with sEVs generated from BEAS-2B cells pre-treated with siRNA targeted to TN-C or control. The results revealed that poly(I:C) stimulation induced TN-C release in vivo, whilst both poly(I:C) stimulation and RV infection promoted release in vitro, with elevated TN-C release from PBECs obtained from people with asthma. Poly(I:C) also induced the release of TN-C-rich sEVs from BEAS-2B cells. TN-C, and sEVs from poly(I:C) challenged cells, induced cytokine synthesis in macrophages and BEAS-2B cells, whilst sEVs from control cells did not. Moreover, sEVs with approximately 75% reduced TN-C content did not alter the capacity of sEVs to induce inflammation. This study identifies two novel components of the inflammatory pathway that regulates the immune response following RV infection and TLR3 stimulation, highlighting TN-C release and pro-inflammatory sEVs in the airway as relevant to the biology of virally induced exacerbations of asthma.
    • DUSP10 negatively regulates the inflammatory response to Rhinovirus through IL-1β signalling.

      Manley, Grace C. A; Stokes, Clare A; Marsh, Elizabeth K.; Sabroe, Ian; Parker, Lisa C; University of Sheffield (American Society for Microbiology, 2018-10-17)
      Rhinoviral infection is a common trigger of the excessive inflammation observed during exacerbations of asthma and chronic obstructive pulmonary disease. Rhinovirus (RV) recognition by pattern recognition receptors activates the MAPK pathways, common inducers of inflammatory gene production. A family of dual-specificity phosphatases (DUSPs) can regulate MAPK function, but their roles in rhinoviral infection are not known. We hypothesised that DUSPs would negatively regulate the inflammatory response to RV infection. Our results revealed that p38 and JNK MAPKs play key roles in the inflammatory response of epithelial cells to RV infection. Three DUSPs previously shown to have roles in innate immunity, 1, 4 and 10, were expressed in primary bronchial epithelial cells, one of which, DUSP10, was down regulated by RV infection. Small interfering-RNA knock down of DUSP10 identified a role for the protein in negatively regulating inflammatory cytokine production in response to IL-1β alone and in combination with RV, without any effect on RV replication. This study identifies DUSP10 as an important regulator of airway inflammation in respiratory viral infection.Importance Rhinoviruses are one of the causes of the common cold. In patients with asthma or chronic obstructive pulmonary disease, viral infections, including rhinovirus, are the commonest cause of exacerbations. Novel therapeutics to limit viral inflammation are clearly required. The work presented here identifies DUSP10 as an important protein involved in limiting the inflammatory response in the airway without affecting immune control of the virus.
    • Pellino-1 regulates immune responses to Haemophilus influenzae in models of inflammatory lung disease.

      Hughes, Bethany; Burton, Charlotte; Reese, Abigail; Jabeen, Maisha; Wright, Carl; Khoshaein, Nika; Marsh, Elizabeth; Peachell, Peter; Sun, Shao-Cong; Dockrell, David; et al. (Frontiers Media, 2019-07-31)
      Nontypeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signalling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defence against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.
    • Pellino-1 regulates the responses of the airway to viral infection

      Marsh, Elizabeth K; Prestwich, Elizabeth C; Marriott, Helen M; Williams, Lynne; Hart, Amber R; Muir, Claire F; Parker, Lisa C; Jonker, Marnix R; Heijink, Irene H; Timens, Wim; et al. (Frontiers, 2020-08-31)
      Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pellino-1 is an E3 ubiquitin ligase known to regulate virally-induced inflammation. We wished to determine the role of Pellino-1 in the host response to respiratory viruses in health and disease. Pellino-1 expression was examined in bronchial sections from patients with GOLD stage 2 COPD and healthy controls. Primary bronchial epithelial cells (PBECs), in which Pellino-1 expression had been knocked down, were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with clinically-relevant respiratory viruses; rhinovirus (RV1B) and influenza A. We find that Pellino-1 is expressed in the airways of normal subjects and those with COPD, and that Pellino-1 regulates TLR3 signalling and responses to airways viruses. In particular we observed that knockout of Pellino‐1 in the murine lung resulted in increased production of proinflammatory cytokines IL‐6 and TNFα upon viral infection, accompanied by enhanced recruitment of immune cells to the airways, without any change in viral replication. Pellino-1 therefore regulates inflammatory airway responses without altering replication of respiratory viruses.