key: cord-0320420-di0lzd7a authors: Dancer, Patrick; Pickard, Adam; Potocka, Wiktoria; Earle, Kayleigh; Fortune-Grant, Rachael; Kadler, Karl; Bertuzzi, Margherita; Gago, Sara title: Mutual inhibition of airway epithelial responses supports viral and fungal co-pathogenesis during coinfection date: 2022-04-14 journal: bioRxiv DOI: 10.1101/2022.04.13.488236 sha: ede3acf58e112511e58c455758d24223b078eb05 doc_id: 320420 cord_uid: di0lzd7a Awareness that fungal coinfection complicates viral respiratory infections causing worse disease outcome has recently emerged. The environmental fungus Aspergillus fumigatus (Af) has been reported as the main driver of fungal coinfection in patients suffering from viral infections caused by Cytomegalovirus, Influenza or more recently SARS-CoV2. The airway epithelium is the first common point of contact between inhaled pathogens and the host. Aberrant airway epithelial cell (AEC) responses against fungal challenge have been described in patients susceptible to aspergillosis. Therefore, it is likely that a dysregulation of AEC responses during fungal-viral coinfection represents a potent driver for the development of fungal disease. Here we used an in vitro model of Af-viral infection of AECs to determine outcomes of spore internalisation, killing and viral replication during coinfection. Our data indicate that viral stimulation, while boosting Af uptake by AECs, limits Af spore killing by those cells, favouring fungal persistence and growth. Type I viral-induced interferon release was significantly decreased in the presence of Af hyphal forms suggesting a possible role of Af secreted factors in modulating viral pathogenicity. We next explored the impact of Af challenge in SARS-CoV2 replication within airway epithelial cells using nano-luciferase as a measure of viral replication. We found that Af increased SARS-CoV2 pathogenicity in a strain-dependent manner. Collectively, our findings demonstrate a mutual inhibition of antifungal and antiviral AEC responses during Af-viral coinfection and also suggest that some fungal factors might be key regulators of co-pathogenicity during in lung infection. indicate that viral stimulation, while boosting Af uptake by AECs, limits Af spore killing by those 48 cells, favouring fungal persistence and growth. Type I viral-induced interferon release was 49 significantly decreased in the presence of Af hyphal forms suggesting a possible role of Af 50 secreted factors in modulating viral pathogenicity. We next explored the impact of Af challenge 51 in SARS-CoV2 replication within airway epithelial cells using nano-luciferase as a measure of 52 viral replication. We found that Af increased SARS-CoV2 pathogenicity in a strain-dependent 53 manner. Collectively, our findings demonstrate a mutual inhibition of antifungal and antiviral 54 AEC responses during Af-viral coinfection and also suggest that some fungal factors might be 55 key regulators of co-pathogenicity during in lung infection. Severe viral infections have emerged as a new risk factor for the development of pulmonary 58 aspergillosis. As mucosal responses are critical to prevent respiratory infections caused by a 59 variety of inhaled pathogens, we hypothesised that subversion of these responses during 60 coinfection is at the base of the increased susceptibility of patients suffering viral infections to 61 pulmonary aspergillosis. In this study, we discovered that in the presence of a viral mimicker, 62 airway epithelial cells (AECs) are less able to kill Aspergillus fumigatus (Af) spores and this 63 correlates with aberrant cytokine responses. Similarly, when AECs were exposed to SARS- CoV we observed Af can sustain viral pathogenesis in a strain-dependent manner. Understanding the mechanisms of AECs responses during fungal-viral coinfection will allow 66 to develop new strategies for the clinical management of these emerging coinfections. Severe bacterial pneumonia complicating Influenza or more recently COVID-19 have been 69 widely linked with worse disease outcome and increased mortality (1-3). However, awareness 70 that fungal coinfections complicate viral respiratory disease has only recently emerged. During 71 the H1N1 pandemic, influenza associated pulmonary aspergillosis doubled mortality risk (4, 5) . Similarly, a 30% reduction in survival has been reported in patients with coronavirus 73 associated pulmonary aspergillosis (CAPA) (6, 7). Cytomegalovirus infections are also known 74 to increase aspergillosis risk in transplant recipients (8) The airway epithelium is the first point of contact between inhaled pathogens and the host (13, (AECi) was determined using IFC. In the presence of FITC-Poly(I:C), Af spore uptake by AECs 110 increased 18-fold compared to Af A1160 +/tdT only challenges (P < 0.05) (Figure 1a ). Lipofectamine, which was used to deliver Poly(I:C) into the cells, did not influence Af 112 internalisation by AECs, nor did the presence of Af alter the interaction between AECs and 113 viral mimicker, as measured via mean fluorescence FITC Poly(I:C) (Supplementary Figure 114 1a-b). In vitro, in vivo and ex vivo studies by us and others have shown that AECs can kill internalised 116 Af spores (15) (16) (17) (18) (19) 23) . In order to measure Af viability, intracellular quenching and lack of during hyphal growth. These two Af reference strains were chosen as they have different protease activity (30, 31). We previously reported that SARS-CoV2 was able to infect A549 145 epithelial cells., but showed limited replicative capacity. Increased NLuc signal, indicating viral 146 replication (29), was detected when monolayers were exposed to Af culture filtrates from 147 Af293 but not A1160 strains (P < 0.05) (Figure 2c) . Interestingly, heightened NLuc activity 148 was detected when A549 cells were co-cultured with THP1 monocytes and Af293 culture All cells were maintained at 37°C, 5% CO2 and monolayers used when at ≥ 90% confluence. pandemic influenza A (H1N1): pathology 225 and pathogenesis of 100 fatal cases in the United States Predominant role of bacterial 227 pneumonia as a cause of death in pandemic influenza: implications for pandemic 228 influenza preparedness Invasive aspergillosis complicating pandemic influenza A (H1N1) 231 infection in severely immunocompromised patients Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 269 2009 virus in France The co-pathogenesis of influenza viruses with bacteria in the lung Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease Microbial uptake by the respiratory epithelium: 276 outcomes for hosst and pathogen Intracellular and extracellular 278 growth of Aspergillus fumigatus Cystic fibrosis 280 transmembrane conductance regulator regulates epithelial cell response to Aspergillus 281 and resultant pulmonary inflammation Characterisation of Aspergillus fumigatus Endocytic 284 Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative