Abstract
The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1β, TNFα, COX2, CD14, TLR2, and IFNAR, but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.
Highlights
Elevated levels of hydrogen peroxide have been reported in various cell types inter alia in some cancer cells and pro-inflammatory—M1 macrophages
RosetteSepTM Monocyte Enrichment Cocktail was purchased from STEMCELL Technologies (Grenoble, France), cell culture media were from Biowest (CytoGen, Zgierz, Poland), granulocyte–macrophage colony-stimulating factor (GM-CSF) from PeproTech (London, UK), anti-rabbit IgG (A0545) and anti-mouse IgG (A4416) peroxidase-labeled antibodies produced in goat, BLUeye prestained protein marker (#94964), SIGMAFASTTM Protease Inhibitor Tablets (PIC), sodium butyrate, C646, ML385 and oligonucleotides for real-time PCR were from Sigma-Aldrich (Poznan, Poland)
Bearing in mind that pro-inflammatory polarization of mouse macrophages isolated from the bone marrow as well as monocytic THP1 and Mono Mac6 cancer cells cause substantial reduction in catalase expression, we tested first if the same effects could be observed in human macrophages derived from monocytes, which were incubated with granulocyte– macrophage colony-stimulating factor (GM-CSF) for 6 days
Summary
Elevated levels of hydrogen peroxide have been reported in various cell types inter alia in some cancer cells and pro-inflammatory—M1 macrophages. A similar effect was observed in a monocyte-to-macrophage differentiation model of THP1 and Mono Mac 6, monocytic cell lines, where the cells were first differentiated with phorbol 12-myristate 13-acetate and polarized with TLR1/2 ligand Pam3CSK4, a synthetic triacylated lipopeptide (LP) that mimics the acylated amino terminus of bacterial lipopeptides [3]. Both TLR ligands act as potent activators of the pro-inflammatory transcription factor NF-κB. Redox-relevant enzymes control macrophage pro-inflammatory phenotype and M1 response to damaging agents, as was reported in our recent paper, where inhibition of termination of SOD2 transcription upon TLR4 activation increased the protein level of the enzyme and rendered pro-inflammatory macrophages even more resistant to oxidative stress [7]
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