Gaining insight into COVID-19 neuropathogenesis: a closer look to SARS-Cov-2 infection and driven neuroinflammation in the neuron/microglia axis

Background: SARS-CoV-2, though primarily a respiratory virus, often causes neurological symptoms, which may persist and evolve in the long-COVID syndrome. The virus may spread to the central nervous system, using ACE2 receptor and TMPRSS2 protease to infect brain cells. Infection triggers a “cytokine storm”,which activate transcription factors like NF-κB and drive neuroinflammation. Microglia, the brain’s resident immune cells, play a central role in modulating neuroinflammation, initially acting as protectors but also potentially contributing to neuronal damages when chronically activated. Aim of the study: To develop an in vitro model of neuron–microglia interaction to study microglial role in SARS-CoV-2-induced neuroinflammation. The project focused on differentiating and characterizing iMGs and iNeu from hESCs, analyzing their susceptibility to viral infection and antiviral responses. Co-cultures of iNeu and iMGs were used to assess microglial contribution to virus-mediated neuronal damage. Material and methods: iMGs were differentiated from H9-CEBPA-hSPI1 human embryonic stem cells (hESCs), following a modified version of the protocol described by Professor Yu-Hui Wong. Cells were characterized by real time RT-PCR, immunofluorescence staining and flow cytometry analysis of expression MG-specific markers. Cortical neurons were differentiated from H9-AVVS1-TRE3G-NGN2 human embryonic stem cells. Co-culture systems with cortical neurons were established starting from different stages of microglial differentiation. To investigate about viral neurotropism and anti-inflammatory responses, induced cortical neurons (iNeu) along with induced microglia cells (iMGs) were infected with Wuhan and Omicron BA.1.1.529 SARS-CoV-2 variants, at a multiplicity of infection (MOI) of 0.1. Viral infection and replication was assessed by qRT-PCR, immunofluorescence analysis, and TCID50 assay in cell supernatant in time course experiments. Results: hESCs H9-AVVS1-TRE3G-NGN2 stem cells were differentiated into cortical neurons (iNeu) and, after 14 days, iNeu were infected with Wuhan and Omicron BA.1.1.529 SARS-CoV-2 strains at MOI 0.1. Viral titration I cell supernatant and immunostaining showed that iNeu were permissive to both viral strains. Human embryonic stem cells (H9-CEBPA-hSPI1 hESCs) were differentiated into induced microglia cells (iMGs) over a 14-day protocol. Morphological analysis revealed a transition from round, clustered cells to ramified forms, characteristic of surveillant microglia. Real-time RT-PCR and immunofluorescence assays confirmed the expression of microglial markers such as CD68, IBA1, and TGF-β1. Flow cytometry analysis showed a marked increase of difference microglial genes expression between day 0 and day 14 of differentiation. These results confirmed a successful differentiation of hESCs into functional iMGs with key phenotypic and molecular signatures. Furthermore, a novel protocol for co-culturing induced neurons (iNeu) and induced microglia (iMGs) was developed. After testing various conditions, optimal results were achieved by combining iNeu and iMGs at day 10 of differentiation. Co-cultures were infected with Wuhan and Omicron BA.1.1.529 SARS-CoV-2 strains. Following infection, iMGs exhibited a morphological shift from a resting state to an ameboid-activated form, indicating microglial activation. Additionally, iNeu showed clear signs of infection as demonstrated by Real Time PCR, TCID50 assay and immunofluorescence results. Conclusion and discussion: this study successfully generated and characterized iMGs from hESCs, showing clear signs of microglial maturation. In co-culture with hESC-derived neurons, these iMGs were used to model SARS-CoV-2-induced neuroinflammation. Preliminary results showed that infection triggered a morphological shift in iMGs from a resting to an ameboid form, indicating microglial activation.