Human Astrocytes (HA)

Human Primary Astrocytes Differently Respond to Pro- and Anti-Inflammatory Stimuli

Astrocytes, once regarded as passive brain cells, are now active members of the central nervous system (CNS) with different functional identities. Szpakowski's team aimed to examine whether pro-inflammatory or anti-inflammatory human astrocytes produce inflammatory chemokines and neurotrophic factors that contribute to CNS pathologies.

GFAP was increased in astrocytes that were exposed to the microglia proinflammatory cytokine cocktail (TNF-α/IL-1a/C1q) compared with unstimulated cells assessed by flow cytometry. The levels of surface receptor expression were also not changed in the CD80 or CD86 densities. However, the CD83 level in cells treated with TNF-α/IL-1a/C1q was significantly higher than in controls (Fig. 1). The stimulation with TNF-α/IL-1a/C1q resulted in high levels of the chemokines CCL1, CXCL1, CXCL10 and CXCL13 and proinflammatory IL-1, which were not present in unstimulated or anti-inflammatory cytokine-treated astrocytes (Fig. 2). The unstimulated astrocytes generated an abundance of PDGF-A, and were not impacted by IL-4 or IL-10. BDNF was detected but unaffected by the conditions (Fig. 3). GDNF appeared in one donor culture, and β-NGF was undetected. These results confirm that astrocytes differentially respond to pro- and anti-inflammatory stimuli, especially to inflammatory cytokines TNF-α, IL-1a, and C1q, suggesting their role in leukocyte recruitment.

Fig. 1. Expression of surface co-stimulatory CD86, CD83, CD80, and MHC class II (HLA-DR) molecules on astrocyte cells in response to cytokine environment (Szpakowski P, Winiarek DK, et al., 2022).

Fig. 2. CCL1 (A), CXCL1 (B), CXCL10 (C), CXCL13 (D), and IL-1β (E) production in human astrocyte cultures (Szpakowski P, Winiarek DK, et al., 2022).

Fig. 3. PDGF-AA (A) and BDNF (B) production in human astrocyte cultures (Szpakowski P, Winiarek DK, et al., 2022).

Levels of Viral Infection Differ between the Asian and African ZIKV Strains in Human Astrocytes

Zika virus (ZIKV), a flavivirus, is known for causing neurodevelopmental disorders and has notably emerged in regions like French Polynesia and Brazil. It's transmitted mainly by Aedes mosquitoes, with astrocytes as primary targets in the CNS. However, the mechanisms of ZIKV's entry and pathology remain unclear. Ojha et al. compared the level of infectivity and the release of inflammatory molecules among three isolated strains of ZIKV representing the African and Asian lineages and determine the mechanisms of their responses in primary human astrocytes.

ZIKV infection in human astrocytes was assessed using immunofluorescence, RT-PCR, and western blot. In astrocytes, different infectivity levels were found among ZIKV strains, with Asian strains (PRVABC59 and R103451) showing higher infectivity than the African strain (MR766) (Fig. 4A-C, Fig. 5A). Infected microglia showed similar patterns. Plaque assays revealed peak viral titers at 48 hpi, while intracellular RNA levels were higher than viral progeny in supernatants, highlighting differences between RNA presence and active virus release (Fig. 4D). Despite high infectivity, astrocytes released fewer viral particles than microglia (Fig. 5D). To explore viral entry in human glial cells, they studied TAM receptors Tyro3, AXL, and MERTK in astrocytes. ZIKV infection didn't change AXL levels but slightly increased Tyro3 expression with Asian and African strains (Fig. 4E-G). The AXL receptor's role was tested using R428, which decreased ZIKV (MR766 and PRVABC59) cell entry. For the Honduras strain (R103451), entry decreased only at high inhibitor concentrations (Fig. 4H, Fig. 5E and F). siRNA silencing of AXL showed similar results (Fig. 5H and I). These findings suggest strain-dependent AXL role differences. Despite decreased IFN-β signaling and increased IP-10 in PRVABC59-infected astrocytes treated with R428, ZIKV entry persisted, implying alternative pathways. Tyro3 inhibition with BMS777607 had no effect, indicating ZIKV entry is Tyro3-independent (Fig. 5J and K).

Fig. 4. Asian and African strains of ZIKV have differential levels of infectivity in human astrocytes (Oiha CR, Rodriguez M, et al., 2019).

Fig. 5. ZIKV infects human microglial cells and utilizes AXL receptor for cell entry (Oiha CR, Rodriguez M, et al., 2019).