Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10)

Abstract

Microglia respond to CNS injuries and diseases with complex reactions, often called “activation.” A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.

Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke

“…Intriguingly, Ym1 was significantly upregulated with M1 stimulation alone. In fact, a previous study has shown that LPS exposure increased the expression of Ym1 in rat primary microglia 24 h after stimulation, suggesting that Ym1 is not a typical M2 marker for these cells [45]. In addition, combined IL-13 and M1 stimulation increased MerTK and Gal3 levels, a receptor and its ligand known to be associated with the phagocytic activity of microglial cells [30,31].…”

Chemical composition of tetraploid Gynostemma pentaphyllum gypenosides and their suppression on inflammatory response by NF‐κB/MAPKs/AP‐1 signaling pathways

“…Furthermore, the intracellular nitric oxide (NO) release is also involved in the signal transduction of inflammatory responses (Sagar et al, ). It is important to inhibit the over‐production of NO in response to inflammatory stimuli, which can induce proinflammatory responses in inflammatory disorders (Lively & Schlichter, ). Compared to the blank, the NO level was significantly increased in LPS‐stimulated RAW264.7 macrophage cells ( p < .01) (Figure c), and the pretreatment of gypenosides inhibited the NO production in a dose‐dependent manner.…”

“…Furthermore, the intracellular nitric oxide (NO) release is also involved in the signal transduction of inflammatory responses (Sagar et al, 2017). It is important to inhibit the over-production of NO in response to inflammatory stimuli, which can induce proinflammatory responses in inflammatory disorders (Lively & Schlichter, 2018).…”

Effect of Glycine on BV-2 Microglial Cells Treated with Interferon-γ and Lipopolysaccharide

“…The present study was performed to reveal to what extent glycine affected the viability, activation state and secretory activity of microglial cells during inflammation. BV-2 cells were stressed with a combination of LPS and IFN-γ over 24 h. LPS is the most common pro-inflammatory stimulus and causes inflammatory reactions in primary microglia and BV-2 cells [17,18], and IFN-γ is released during CNS injury [18][19][20]. Approximately 90% of the genes induced in BV-2 cells after LPS exposure were also found in primary microglia and showed similar reaction patterns [21], which warrants this cell line as a suitable experimental model.…”