The Anti-Inflammatory Effects of the Small Molecule Pifithrin-µ on BV2 Microglia

Abstract

Neonatal encephalopathy (NE) is a leading cause of childhood death and disability in term infants. Treatment options for perinatal brain injury are limited and developing therapies that target multiple pathways within the pathophysiology of NE are of great interest. Pifithrin-µ (PFT-µ) is a drug with striking neuroprotective abilities in a preclinical model of hypoxia-ischemia (HI)-induced NE wherein cell death is a substantial cause of injury. Work from neurons and tumor cells reports that PFT-µ is able to inhibit p53 binding to the mitochondria, heat shock protein (HSP)-70 substrate binding and activation of the NF-kB pathway. The purpose of this study is to understand whether the neuroprotective effects of PFT-µ also include direct effects on microglia. We utilized the microglial cell line, BV2, and we studied the dose-dependent effect of PFT-µ on M1-like and M2-like phenotype using qRT-PCR and Western blotting, including the requirement for the presence of p53 or HSP-70 in these effects. We also assessed phagocytosis and the effects of PFT-µ on genes within metabolic pathways related to phenotype. We noted that PFT-µ robustly reduced the M1-like (lipopolysaccharide, LPS-induced) BV2 response, spared the LPS-induced phagocytic ability of BV2 and had no effect on the genes related to metabolism and that effects on phenotype were partially dependent on the presence of HSP-70 but not p53. This study demonstrates that the neuroprotective effects of PFT-µ in HI-induced NE may include an anti-inflammatory effect on microglia and adds to the evidence that this drug might be of clinical interest for the treatment of NE.

Pifithrin‐μ modulates microglial activation and promotes histological recovery following spinal cord injury

“…Together, these data indicate that PFT‐μ polarizes cultured primary microglia to “M2‐like” state under pro‐inflammatory conditions. These data are in agreement with those reported using BV2 cells and a model of diabetic neuropathy …”

“…In our experiments, PFT‐μ results in a shift of the polarization of microglia/macrophages from an “M1‐like” to an “M2‐like” phenotype, consistent with studies in peripheral models demonstrating that PFT‐μ reduces the expression of inflammatory markers, such as TNF‐α, IL‐6, and iNOS, through inhibition of Hsp70 . These results also corroborate the findings that PFT‐μ has direct anti‐inflammatory effects on the BV2 microglia cell line . Although PFT‐μ is canonically described as being neuroprotective by inhibiting p53‐mediated mitochondrial‐induced apoptosis, these studies were conducted in neuron‐specific injury models .…”

“…The mechanism of action of PFTμ has been associated with both the inhibition of mitochondria translocation of p53, and the inhibition of heat shock protein 70 (Hsp70). 17,31,46,47 In our experiments, PFTμ results in a shift of the polarization of microglia/macrophages from an "M1-like" to an "M2-like" phenotype, consistent with studies in peripheral models demonstrating that PFTμ reduces the expression of inflammatory markers, such as TNFα, IL-6, and iNOS, through inhibition of Hsp70. 48 These results also corroborate the findings that PFTμ has direct anti-inflammatory effects on the BV2 microglia cell line.…”

“…A previous report on PFT‐μ has shown that it exerts anti‐inflammatory effects on the cultured BV2 microglia cell line . We examined a potential direct effect of PFT‐μ on primary microglia, as they represent more reliably native cell activity.…”

“…A previous report on PFTμ has shown that it exerts anti-inflammatory effects on the cultured BV2 microglia cell line. 31 We examined a potential direct effect of PFTμ on primary microglia, as they represent more reliably native cell activity. Microglia were treated with PFTμ at different concentrations, and LPS was used as a positive control for microglial activation.…”