Commensal Bacteria Modulate Toll-like Receptor-3 and -7 Responses towards Human Norovirus-Like Particle Infection in Microfold Cells 3D Alcart Model

Human norovirus (HuNoV) is the leading cause of non-bacterial acute gastroenteritis, imposing a substantial global health and socioeconomic burden. Although commensal bacteria have been shown to influence viral infectivity by facilitating attachment and modulating host immune signalling, their role in HuNoV recognition at the intestinal interface remains poorly understood. This study investigated whether commensal Escherichia coli (E. coli) influences Toll-like receptor (TLR)-mediated recognition and transcriptional activation during HuNoV exposure using a microfold (M) cells 3D Alcart model. The set up was established by co-culturing Caco-2 colonic epithelial cells with Raji B cells within alginate hydrogel beads as a recapitulation of the intestinal environment. The model was inoculated with HuNoV virus-like particles (VLPs) in the presence or absence of E. coli, and the TLR3 and TLR7 levels of expression were analysed by flow cytometry. Expression levels of transcription factor IRF3, IRF7, NF-κB1, and NF-κB2 genes were quantified with Real-Time PCR at 24 and 48 hours post-inoculation to assess early immune signalling. Flow cytometry results showed that at 24 hours post-inoculation, co-exposure to HuNoV VLPs and E. coli enhanced TLR3 expression but transiently suppressed TLR7, whereas by 48 hours, both receptors were markedly upregulated, indicating a time-dependent immune activation. Transcription factor gene analysis revealed early but short-lived activation of IRF3, IRF7, NF-κB1, and NF-κB2, suggesting that TLR activation precedes downstream gene induction. By 48 hours, TLR3 and TLR7 remained elevated while transcription factor expression returned to baseline, indicating transient immune activation. These findings demonstrate that commensal bacteria enhanced TLR3-mediated recognition of HuNoV while transiently suppressing TLR7 signalling in the M cells 3D Alcart model. Evidently, the M cells 3D Alcart presents a physiologically relevant platform for HuNoV pathobiology study, particularly on the influence of commensal bacteria towards antiviral responses while limiting excessive inflammation.