Irisin Attenuates Neuroinflammation Targeting the NLRP3 Inflammasome

Abstract

Neuroinflammation is defined as an immune response involving various cell types, particularly microglia, which monitor the neuroimmune axis. Microglia activate in two distinct ways: M1, which is pro-inflammatory and capable of inducing phagocytosis and releasing pro-inflammatory factors, and M2, which has anti-inflammatory properties. Inflammasomes are large protein complexes that form in response to internal danger signals, activating caspase-1 and leading to the release of pro-inflammatory cytokines such as interleukin 1β. Irisin, a peptide primarily released by muscles during exercise, was examined for its effects on BV2 microglial cells in vitro. Even at low concentrations, irisin was observed to influence the NLRP3 inflammasome, showing potential as a neuroprotective and anti-inflammatory agent after stimulation with lipopolysaccharides (LPSs). Irisin helped maintain microglia in their typical physiological state and reduced their migratory capacity. Irisin also increased Arg-1 protein expression, a marker of M2 polarization, while downregulating NLRP3, Pycard, caspase-1, IL-1β, and CD14. The results of this study indicate that irisin may serve as a crucial mediator of neuroprotection, thus representing an innovative tool for the prevention of neurodegenerative diseases.

Keywords: inflammasome; irisin; microglia; neuroinflammation.

Figure 1

Effect of irisin on cell viability (MTT assay). BV-2 cells were incubated with irisin at concentrations spanning a dose–response curve, ranging from 5 nM to 20 nM (A). Irisin at a concentration of 5 nM was used to treat cells, either in the absence or presence of 1 µg/mL LPSs (B). Data are reported as percentages compared to control values and are expressed as means ± SDs. ** p < 0.01 compared to the control. *** p < 0.001 compared to the control.

Figure 2

Morphological analysis of the BV2 cells following the administration of irisin, either alone or after LPS stimulation. The morphological analysis was conducted on BV2 cells in the control condition (A) and following the administration of 1 µg/mL LPSs (B), 5 nM irisin (C), or 5 nM irisin in the presence of 1 µg/mL LPSs (D). The scale bar is 100 µm (10× objective). The arrows in the images indicate cells that have undergone a morphological change. The cell areas (µm²) were quantified using the ImageJ 1.8.0 software, which was bound with Java 8 64-bit (E). The data are expressed as means ± standard deviations. A significant difference can be observed between the control and LPS groups (*** p < 0.001), as well as between the IRISIN + LPS and LPS groups (### p < 0.001).

Figure 3

The analysis of the migratory capacity of microglia after the administration of irisin in the presence or absence of LPSs. A wound was generated in a sub-confluent layer of BV2 cells, and the resulting space was captured at the wound site 0 and 24 h after the treatment: (A) BV2 cells at time 0, (B) 24 h after the cut in the control condition, (C) treated with 1 µg/mL LPSs, (D) with 5 nM irisin, and (E) with 5 nM irisin in the presence of 1 µg/mL LPSs. The images are representative of an experiment with three independent replicates. The percentage of the wound gap was analyzed using the ImageJ software and subsequently plotted and statistically analyzed as the percentage of wound closure compared to the 0 time condition (F). The values are presented as means ± standard deviations. Bar: 75 µm (20× objective). ** p < 0.01 compared to the control. # p < 0.05 compared to the LPS condition.

Figure 4

Evaluation of NLRP3 and Pycard expression following the administration of irisin, with or without LPSs. Western blotting detection and densitometric analysis of the expression of the pro-inflammatory NLRP3 (A) and Pycard (B) in control cells (CTR), BV2 cells treated with irisin, BV2 cells treated with LPSs (LPS), and BV2 cells treated with irisin + LPSs. The protein expression values are expressed in arbitrary units after normalization against β-actin. The data are presented as means ± SDs (* p < 0.05 vs. CTR; *** p < 0.001 compared to the control; and ### p < 0.001 compared to the condition of LPS-stimulated microglia).

Figure 5

Evaluation of caspase-1, IL1Beta, and CD-14 expression following the administration of irisin, with or without LPSs. Western blotting detection and densitometric analysis of the expression of pro-inflammatory caspase-1 (A), IL1B (B), and CD-14 (C) in control cells (CTR), BV2 cells treated with irisin, BV2 cells treated with LPSs (LPS), and BV2 cells treated with irisin + LPSs (*** p < 0.001 compared to the control condition; and ### p < 0.001 compared to the condition of LPS-stimulated cells).

Figure 6

Evaluation of Arginase 1 expression following the administration of irisin, with or without LPSs. Western blotting detection and densitometric analysis of the expression of the anti-inflammatory agent in control cells (CTR), BV2 cells treated with LPSs (LPS), BV2 cells treated with irisin, and BV2 cells treated with irisin + LPSs (*** p < 0.001 compared to the control condition; and ### p < 0.001 compared to the condition of LPS-stimulated cells).