Potential neuroprotective effects of anti‐cytokine antibodies against lipopolysaccharide‐induced microglial inflammation

Abstract

Aims/Purpose: Microglia‐mediated neuroinflammation is involved in many neurodegenerative diseases, including glaucoma and diabetic retinopathy. This study examined the effects of anti‐cytokine antibodies on LPS‐induced inflammatory response of microglial cells, and explored the underlying molecular mechanisms by proteomic analysis.Methods: A lipopolysaccharide (LPS)‐induced neuroinflammation model was established in murine BV‐2 microglial cells in vitro. The expression of microglial cell markers (CD68 and CD206) were detected by immunocytochemistry. BV‐2 was stimulated with LPS (1 μg/mL) with or without anti‐tumour necrosis factor‐alpha (anti‐TNF‐α, 2 μg /ml) or anti‐interleukin‐1 beta (anti‐IL‐1β, 4 μg /ml) for 24 h. LPS‐induced inflammation level was analysed using the Griess assay to determine nitric oxide (NO) concentration. Proteome alterations were characterized employing the mass spectrometry‐based proteomics and bioinformatics analyses.Results: Immunocytochemistry confirmed the expression of microglial markers in BV‐2 cells. LPS stimulation induced excessive activation of BV‐2, with significant increase in NO generation (p < 0.0001). Anti‐IL‐1β significantly attenuated 34% of the LPS‐induced NO generation (p < 0.0001). Interestingly, anti‐TNF‐α showed a stronger anti‐inflammatory effect by reducing 57% of LPS‐induced NO generation (p < 0.0001). Bioinformatics analyses revealed that differentially expressed proteins involved in inflammation (p = 3.8 × 10−5) and apoptotic (p = 3.2 × 10−11) signalling pathways were significantly activated by LPS. Particularly, LPS‐mediated upregulation of STAT1 (p = 8.6 × 10−6) was restored by anti‐IL‐1β and anti‐TNF‐α (p = 3.1 × 10−4 and p = 2.7 × 10−3, respectively). Anti‐IL‐1β induced proteome alterations involved in anti‐inflammatory signalling pathways and promote glial cell polarization towards neuroprotective M2 phenotype (p = 5.3 × 10−6), while anti‐TNF‐α treatment significantly regulated proteins involved in immune response of macrophages (p = 2.7 × 10−8).Conclusions: In summary, anti‐TNF‐α and anti‐IL‐1β antibodies alleviated LPS‐induced neuroinflammation and altered proteins involved in neuroprotective pathways. Hence, anti‐cytokine antibodies have therapeutic potential as neuroprotective agents.