Immortalized Human Microglia-SV40 (HMC-3)

Mayaro and Una Viruses Efficiently Replicate in Human Microglia HMC3 Cells in a Time- and MOI-Dependent Manner

Mayaro (MAYV) and Una (UNAV) viruses are two alphaviruses that have recently emerged in the Americas. MAYV infection causes Mayaro fever, whereas the symptoms induced by UNAV remain unknown. In previous studies, MAYV was found to infect various types of human cells. However, the effect of MAYV on brain cells is not fully understood. Campos et al. investigated the susceptibility of two human brain cells, microglia HMC3 and brain microvascular endothelial HBEC-5i, to MAYV and UNAV infection using an inverted microscope, MTT assay, plaque-forming assays, and RT-qPCR.

To assess whether MAYV and UNAV can replicate in HMC3 or HBEC-5i cells, they performed a kinetic infection experiment. Immortalized human microglia HMC3 or HBEC-5i cells were infected with MAYV or UNAV at MOI 1 or 10, and viral progeny yields in the cell supernatants were quantified using plaque-forming assays at different time points. They found that MAYV and UNAV replicated in HMC3 cells in a time- and MOI-dependent manner (Fig. 1C and D). In contrast, only MAYV at a low level, but not UNAV, infected HBEC-5i cells (Fig. 1E). They confirmed these results by evaluating the expression of E1 and nsP1 viral proteins in infected cells using immunofluorescence assays and Western blot. Strong expression of both proteins was detected in MAYV- and UNAV-infected HMC3 cells at 48 hpi (Fig. 2A), and high protein levels were confirmed using Western blot (Fig. 2B and C). In contrast, low protein expression was detected in MAYV-infected HBEC-5i cells and no expression in UNAV-infected HBEC-5i cells (Fig. 2A, D, E). Taken together, these data show that MAYV and UNAV efficiently infect human microglia HMC3 cells.

Fig. 1. Mayaro and Una viruses efficiently infect human microglia HMC3 cells (Campos D, Sugasti-Salazar M, et al., 2024).

Fig. 2. Detection of viral E1 and nsP1 proteins in MAYV- or UNAV-infected HMC3 and HBEC-5i cells (Campos D, Sugasti-Salazar M, et al., 2024).

LAV-BPIFB4-infected STHdh Q^111/111 Displays an Anti-Inflammatory and Pro-Resolving M2-Polarizing Effect on HM-SV40

Huntington's disease (HD) is a neurodegenerative disorder characterized by neural death due to expanded CAG repeats in the huntingtin protein (Htt). Neuroinflammation plays a key role in HD progression. BPIFB4 is a longevity-associated variant (LAV) that is overrepresented in centenarians and has been demonstrated to have immunomodulatory properties. Pardo et al. investigated the potential of the LAV-BPIFB4 protein to counteract the progression of HD by improving striatal–microglia communication and thus explore a potential therapeutic approach against the disease.

Their previous work showed that LAV-BPIFB4-treated mice had elevated SDF-1 levels in peripheral blood and myocardium, which was crucial for LAV-BPIFB4-induced benefits. Since BPIFB4 had a macrophage M2-polarizing effect via a CXCR4-dependent mechanism, we next examined SDF-1 secretion after BPIFB4 infection. ELISA assays confirmed that STHdh Q^111/111 cells infected with LAV-BPIFB4, but not WT-BPIFB4, secreted higher SDF-1 levels compared with the untreated cells (Fig. 3a). Next, conditioned medium (CM) from these cells was used to treat immortalized human microglia-SV40 microglial cells in vitro. Flow cytometry analysis revealed that the normal STHdh Q^7/7 cells induced the upregulation of CD163 on HM-SV40 cells, while the mutant STHdh Q^111/111 cells failed to exert this effect. Interestingly, after 48-h treatment with CM from LAV-BPIFB4-infected STHdh Q^111/111 cells, CD163 expression and IL-10 release significantly increased (Fig. 3b - d). This effect was inhibited by the CXCR4 antagonist AMD3100 (20 μM), which confirmed the CXCR4-dependent mechanism (Fig. 3b - d).

Fig. 3. Analysis of anti-inflammatory and M2-polarizing action of CM from LAV-BPIFB4-infected striatal cells (Pardo A Di, Ciaglia E, et al., 2020).