Molecular interfaces between childhood respiratory virus infection and development of asthma

Viral respiratory tract infections (RTIs) occurring in the first few years of life have been suggested as independent risk factors for the development of asthma. Still, it is not clear whether viral infections directly affects the respiratory immune system in a way that results in asthma, or if viral infections uncover asthma to those predisposed to develop the disease. The current project aim to combine basic research and clinical studies with focus on identifying inflammatory mechanisms related to the development of asthma. The first part of the project has focused on the cytokine Thymic stromal lymphopoietin (TSLP). TSLP is associated with several allergic diseases including asthma. Two isoforms of TSLP exist in human, a long form (lfTSLP) and a short form (sfTSLP), displaying distinct immunological functions. Recently, TSLP was found to be upregulated in human airway cells upon human metapneumovirus (hMPV) infection, yet it remains unclear if the two isoforms are regulated differently during hMPV infection. Importantly, the molecular mechanisms underlying hMPV-mediated TSLP induction remain undescribed. We have characterized the expression and regulation of TSLP in hMPV-infected human airway cells. We demonstrated that hMPV strongly induced the expression of pro-inflammatory lfTSLP. The expression of lfTSLP was particularly pronounced in hMPV-infected lung fibroblasts, indicating that fibroblasts might be an important source of lfTSLP under pathophysiological conditions. Further, knockdown of pattern recognition receptors (PRR) retinoic acid-inducible gene I (RIG-I) or Toll-like receptor 3 (TLR3), as well as downstream signal transducers, abrogated hMPV-mediated lfTSLP induction. Importantly, silencing of TANK-binding kinase 1 (TBK1) also impaired hMPV-mediated lfTSLP induction, which could be attributed to compromised NF-kB activation. Overall, these results suggest that TBK1 may be instrumental for hMPV-mediated activation of NF-kB downstream RIG-I and TLR3, leading to a specific induction of lfTSLP in hMPV-infected human airway cells. During this work, unexpected turns lead us to focus on the antimicrobial peptide cathelicidin. We found that hMPV infection inhibits the expression of cathelicidin in human macrophages. This is a very interesting finding that could potentially have important implications in co-infections with bacteria and viruses. We also provided mechanistic insight into the intracellular processes behind this inhibition. In particular we found that hMPV infection strongly represses the expression of the transcription factor C/EBP?, which was crucial for Cathelicidin expression. We clearly demonstrated that this is independent on type-I interferon signalling (a cellular hallmark in the anti-viral response). However, importantly, the modulation could be reproduced by Toll-like receptor ligands, suggesting a common mechanism underlying pathogen-mediated downregulation of cathelicidin. This study provides novel understanding of altered antimicrobial response following infections. The second part of the project focuses on IL-1R8. IL-1R8 is a negative regulator of IL-receptor (ILR)- and PRR signaling, and thus a break in inflammation. Interestingly, it has been demonstrated that IL-1R8 targets the IL-33 receptor ST2, controlling the production of Th2 cytokines. Recently, collaborators at Humanitas University revealed a novel function of IL1-R8 as a checkpoint molecule for NK cell activity. However, the role of IL-1R8 in virus-triggered airway disease is unexplored. As a part of a research stay at Humanities University, Milano (August 2017-August2018), we assessed the role of IL-1R8 in hMPV-infected airways of mice. We found that IL1-R8 deficiency is associated with more efficient control of hMPV infection. Also, in mice lacking IL-1R8 the production of antiviral cytokines is significantly decreased. We further demonstrated that the control of hMPV in IL1-R8 deficient mice is dependent on NK cells, which show increased maturation status in the absence of IL-1R8, and on IFNgamma. A manuscript is ready for publication. The third part of the project focuses on the possible correlation between molecular findings and development of airway disease. For this we have initiated a multidisciplinary follow-up study, VIRAS, to assess lung function at age 6-12 years in children exposed to infant bronchiolitis caused by the "new" hMPV and compare to the more well-known airway viruses RS-virus (RSV) and rhinovirus (RV). Moreover, we have collected biological samples to identifying genetic variants (collaboration with Yale) and inflammatory mechanisms (including expression of lfTSLP and IL1-R8) in asthmatic children with a history of infant bronchiolitis. A PhD candidate granted by St. Olav`s Hospital and NTNU is responsible for the clinical study. We have in addition received a PhD position granted by NTNU to carry out the genetic analysis at Yale.

This project has provided increased understanding of the mechanisms involved in the initiation and regulation of TSLP production. Morover, this project has revealed that unleashing the NK cell checkpoint, IL1R8, provides more efficient control of hMPV infection. Such mechanistic insight can in the long term lead to new antivirals and novel strategies for prevention and treatment of asthma. Preliminary clinical data from the VIRAS study already indicates that 6-8-year-old children with a history of infant bronchiolitis often have undiagnosed asthma at school-age. Hence, the project also has important short-term impact on the emphasis on appropriate care of high-risk children. to reduce their risk of developing uncontrolled asthma. The project has led to ongoing international collaboration with researchers at Humanitas University and at Yale University. Importantly, the project has been instrumental in the establishment of a multidisciplinary research group at NTNU and St Olavs Hospital.

Viral respiratory tract infections (RTIs) occurring in the first few years of life have been suggested as independent risk factors for the subsequent development of asthma. hMPV is a newly characterized virus to which the immune response is largely enigma tic. Still, hMPV is recognized as a clinically important respiratory pathogen. As a part of a joint research initiative, including national and international collaborators, we apply for funding to assess the association between early hMPV infections and s ubsequent development of asthma. We will apply basic research, in vitro and in vivo models, advanced cellular imaging and unique biobanks to study mechanisms and characteristics of the host response to hMPV that trigger inflammation and respiratory diseas e. We will focus on the role of the allergic inflammatory cytokine TSLP in virus-triggered pathogenesis, and elucidate the molecular basis for its regulation and production during hMPV infection. The project addresses unanswered questions that are highly relevant to society, and it has the possibility to bring forward innovative knowledge through the use of highly unique and well-organized biobanks. If successful, the project could have a major impact worldwide through increased understanding of host resp onses to hMPV infections, which is fundamental for developing interventional and therapeutic strategies.