HO-1 and NRF2 limit pro-inflammatory activation of macrophages. A role for IRF5

Macrophages are innate immune cells with pivotal role in inflammation. Through a plethora of pattern recognition receptors (PRRs)-among which TLRs- they recognize pathogens and tissue damage, driving the inflammatory response. TLR4 and TLR7/8 stimulation, through signalling pathways which are highly interrelated, activates NF-kB and interferon regulatory factor 5 (IRF5) leading to pro-inflammatory cytokine release (e.g. TNF-α). The activation of NRF2-dependent antioxidant response, instead, has been related to the resolution of inflammation. In particular, the induction of HO-1, among the NRF2-dependent genes, carries out an anti-inflammatory activity through its metabolites CO and bilirubin, and their inhibitory activity on NF-kB has been demonstrated. Considering the crucial role played by IRF5 dysregulation in the development of inflammatory diseases, we investigated the role played by NRF2/HO-1 pathway in the regulation of IRF5. We started with the analysis of the possible cross talk between IRF5 and NRF2/HO-1 in RAW 264.7 and THP-1 cells. Cells were treated with LPS and R848 to activate TLR4 and TLR7/8- dependent signalling, respectively. Sulforaphane (SFN) was used to activate NRF2/HO-1. IRF5 activity was evaluated measuring its dimerization. Proinflammatory activation of cells was evaluated measuring the TNF-α and type I interferon (IFN). We demonstrated that HO-1 is activated endogenously downstream TLR4 activation and through the intracellular production of bilirubin negatively regulates proinflammatory activation of cells. Moreover, we proved that IRF5 is activated by both LPS and R848, and that exogenous activation of NRF2 and HO-1 prevented it. Furthermore, we evaluated whether microglial cells recapitulated IRF5 and NRF2 interaction observed in RAW 264.7 cells and THP-1. By using a human microglia cell line (HMC3) exposed to LPS and R848, similar results were obtained. Moreover, in collaboration with Prof. Cristina d’Abramo and Prof. Luca Giliberto, from the Litwin-Zucker Center for the Study of Alzheimer’s Disease, Institute of Molecular Medicine-Feinstein Institutes for Medical Research, Northwell Health System, Manhasset, NYC, USA, we also tested the involvement of IRF5 in the proinflammatory activity of paired helical filaments (PHF), the aggregate form of Tau protein. Indeed, tau aggregation is linked to microglial activation that is crucially involved in favouring the progression of neurodegeneration. Our preliminary data showed that IRF5 was activated by cell exposure to PHF and that NRF2 activation reduced it. Lastly, we evaluated the relevance of IRF5 and HO-1 detection as possible clinical biomarkers. In collaboration with Prof. Giovanni Pratesi, Head of Vascular Surgery Division at IRCCS Ospedale Policlinico San Martino, Genova, and with the Laboratory of Vascular Biology at Department of Surgery at University of Genoa, that runs the Genoa-Tissue bank- Vascular division (GTB-VD), we analysed serum and PBMCs from 64 patients who undergone carotid endarterectomy (CEA). IRF5, HO-1 and NRF2 expression in PBMCs and HO-1 serum level were evaluated, proving that HO-1 is higher expressed in serum of symptomatic patients compared to asymptomatic ones and that its expression correlates with IRF5 expression in PBMCs, leading to hypothesise that these parameters could be take into consideration to identify patients with a major risk of plaque rupture.