The Role of Ozone as an Nrf2-Keap1-ARE Activator in the Anti-Microbial Activity and Immunity Modulation of Infected Wounds

Abstract

Ozone is an allotrope of oxygen, widely known to exert an anti-oxidant potential. The ability of low, controlled and standardized doses of ozone in the ozone adjunct treatment of bacterial infections, which occur in wounds, is engaging clinical research to deepen the role of ozone in eradicating even multidrug-resistant bacteria. Ozone activates the nuclear factor erythroid 2-related factor 2 (Nrf2), and this activation triggers a complex cascade of events, which ultimately leads to macrophage training and an improvement in their ability to operate a clearance of bacteria in the patient's anatomical districts. In this review, we try to elucidate the recent evidence about the mechanisms with which ozone can actually remove bacteria and even multi-drug-resistant (MDR) bacteria, accounting on its complex ability in modulating immunity.

Keywords: Nrf2; immunity; macrophage; macrophage training; ozone; tolerance.

Figure 1

Cartoon showing the activity of ozone against bacterial infection in wounds. See text for details.

Figure 2

The main steps in the O₂-O₃-MAHT according to the SIOOT protocol. (A) A volume of 200 mL of peripheral blood is withdrawn and put in a sterile SANO3 disposable, certified bag; (B) ozone is prepared via a Multiossigen Medical 98 HCPS device (Gorle, Italy); its concentration measured in an O₂/O₃ mixture with an endowed spectrophotometer at 253.7 nm, reaching a final concentration of 40–50 μg/mL, depending on the clinical use; (C) ozone is collected in a 50 mL sterile, disposable syringe directly from the device and injected into the blood-collection bag, then gently mixed for 2 min; (D) the ozonated autologous blood is transferred to the patient.

Figure 3

The hypothetical mechanisms by which ozone, injected via a systemic approach (bloodstream, with O₂-O₃-MAHT), is able to drive immunity towards a possible complete clearance of bacteria in infected wounds, giving encouraging suggestions also for MDR bacteria and setting an anti-inflammatory and skilled M2-immune response. (A) A dose of 45 μg/mL O₃ in O₂/O₃ gas mixture, once injected into the blood, gives rise to about 3 μM 4-HNE, from ω6-PUFA peroxidation, a dose able to exert an anti-inflammatory action [29]. This dose promotes mitohormesis mitochondria turnover and biogenesis, via the contribution of also mitochondria-produced 4-HNE, and, moreover (B) it promotes the optimal mitochondria autophagy, which supports the M1-M2 skewing and the related immune regulation. (C) The alkenal 4-HNE inhibits, at 3 μM, the activation of the inflammasome NLRP3, therefore downregulating M1-driven inflammation and the subsequent maturation of the cytokines IL-1β and IL-18, alongside other pro-inflammatory cytokines. (D) The 4-HNE-mediated activation of Nrf2 triggers the HO-1/CO-mediated M1/M2 skewing and the Nrf2-caused upregulation on the M2 marker MARCO, which enhances the ability of these cells to phagocytize bacteria. (E) Finally, the NLRP3 inhibition blocks the activation of NF-κB, allowing only the activity of NF-κB p50, involving the recruitment of M2 macrophages and their immune training. Green circles: activation or promotion; Red circles: inhibition.