Macrophage phagocytosis of HuNoV infected cells in an ex vivo human enteroid-macrophage co-culture model

Ngan Fung Li, Baylor College of Medicine

Ngan Fung Li1, Sue E. Crawford1, Sydney R. Mittiga1, Cristian Coarfa2,3, Hoa Nguyen-Phuc1, Budi Utama6, Sarah E. Blutt1,5, Sasirekha Ramani1, Mary K. Estes1,2,5#1Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA 2Department of Medicine, Baylor College of Medicine, Houston, Texas, USA3Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA4Center for Precision and Environmental Health, Baylor College of Medicine, Houston, Texas, USA5Texas Medical Center Digestive Disease Center Gastrointestinal Experimental Model Systems (GEMS) Core, Houston, Texas, USA6Shared Equipment Authority, Rice University, Houston, Texas, USA

Structure & Pathogenesis

Human norovirus (HuNoV) causes acute gastroenteritis in immunocompetent hosts and chronic infection in immunocompromised individuals. Many recent studies of replication and innate immune responses following HuNoV infection have utilized epithelium-only human intestinal enteroids (HIEs), which lack immune cells. Here, we utilized an ex vivo enteroid-macrophage co-culture model consisting of HIEs and different subtypes of human peripheral blood mononuclear cell-derived macrophages to better recapitulate in vivo gut biology and explore the role of macrophages in HuNoV replication and pathogenesis. We show that GII.4 HuNoV infection in HIEs polarized on Transwells leads to bilateral release of viral genome, with predominant apical virus release. Co-culture with naïve M0, pro-inflammatory M1 or anti-inflammatory M2 macrophages does not change levels of HuNoV replication. We found that macrophages respond to HuNoV infection by phagocytosis of virus-infected cells with pro-inflammatory M1 macrophages exhibiting the greatest phagocytic activity. Apical release of chemokines (IP-10, MIP-1α and RANTES), acute-phase inflammatory mediators (IL-1α, IL1-RA, IL-6 and TNF-), and anti-inflammatory mediators (IL-10 and IL-1RA) are increased following HuNoV infection only in HIEs co-cultured with activated macrophages. By employing an ex vivo human intestinal enteroid-macrophage coculture model, we advance our understanding of the cellular crosstalk that shapes the innate immune response to HuNoV. Our findings, particularly the demonstration that macrophages phagocytose infected epithelial cells and modulate chemokine and cytokine release, highlight the immunological complexity of early HuNoV infection and address an important limitation of prior epithelium-only enteroid studies.