Fenton, M. | bioRxiv Science

bioRxiv2025

Impact of cell culture model systems on SARS-CoV-2 and MERS-CoV infection dynamics and antiviral responses

Cell cultures are widely used to study infectious respiratory diseases and to test therapeutics; however, they do not faithfully recapitulate the architecture and complexity of the human respiratory tract. Lung organoids have emerged as an alternative model that partially overcomes this key disadvantage. Lung organoids can be cultured in various formats that offer potential for studying highly pathogenic viruses. However, the effects of these different formats on virus infection remain unexplored, leaving their relative value unclear. In this study, we generated primary lung organoids from human donor cells and used them to derive monolayers and air-liquid interface (ALI) cultures with the goal of comparing the replication kinetics of two circulating highly pathogenic coronaviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV). Infection studies revealed that organoid-derived monolayers displayed limited infection and the innate immune response was impaired against bacterial lipopolysaccharide (LPS) but not against virus-like double-stranded dsRNA or poly(I:C). Meanwhile, organoids and organoid-derived ALI cultures retained viral permissivity, with ALI cultures displaying diverse antiviral immune responses against both coronaviruses. SARS-CoV-2 and MERS-CoV demonstrated differential replication kinetics in organoid and organoid-derived ALI cultures. Therefore, primary organoid-derived cells in two-dimensional monolayer or three-dimensional ALI formats influence virus infection and host antiviral responses. Our study informs the selection of culture conditions for organoid-based respiratory disease research and therapeutic testing. ImportanceThe COVID-19 pandemic heralded the upsurge in human-derived lung organoid based studies due to their cellular heterogeneity that emulates the cellular complexity of the respiratory tract. A major disadvantage of organoid models resides in their apical-in conformation that "hides" cells and proteins that are typically exposed to the air-liquid interface (ALI) in the airways and are targets of viruses. Here, we generated monolayers and ALI cultures to facilitate cell exposure to highly relevant pathogens and compared them to parental organoids. Organoids at the ALI captured infection and immune responses better than organoids and organoid-derived monolayer cultures. Organoids at the ALI are a viable approach to improve identification and characterization of virus infection, host responses, and therapeutic testing.

microbiology↗

bioRxiv2023

Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1{beta} from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1{beta} secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-{kappa}B and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1{beta} in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases. Author SummaryAcute myeloid leukaemia (AML) and myelodysplastic syndromes MDS) are related blood cancers that are associated with frequent infections because the cancers suppress normal immunity. These infections are therefore generally considered as medical complications arising as a result of but separate to the cancer. However, we provide evidence here that infections may promote or drive cancer progression. We and others previously demonstrated that a human protein called NM23-H1 promotes the survival of AML cells by eliciting survival signals from other cells. NM23-H1 belongs to a highly conserved family of proteins that also occur in bacteria and fungi that cause infections in AML and MDS patients. Here we demonstrate that these bacterial and fungal proteins recapitulate the pro-survival effect of NM23-H1 on AML cells. We also determine that these effects are mediated via mechanisms already known to be important in the development and progression of AML and MDS. This study is the first to identify NM23-H1 like proteins from pathogenic microorganisms as novel activators of these pathways. These findings have important implications for how we understand infections in AML and MDS patients and suggest that in addition to being the consequence of these diseases, infections may also drive the cancer process.

microbiology↗