Macrophage ALOX15B restricts CIITA-dependent antigen presentation via MHC II through lipid peroxide-driven NRF2 activation 2308504

Abstract

Abstract Introduction Macrophage major histocompatibility complex (MHC) II-dependent antigen presentation is essential for activating CD4+ T cells. Transcriptionally, macrophage (Mφ) MHC II genes hinge on the constitutively expressed transactivator CIITA. While IFNγ strongly boosts CIITA and thereby MHC II gene expression, intracellular signals controlling constitutive CIITA expression remain largely unknown. Methods Human CD4+ T cells and monocytes were isolated from blood of anonymous donors. Genes of interest were silenced in monocyte-derived Mφ using siRNA. Changes in gene expression and protein abundance were quantitatively analyzed using RNA-seq, RT-qPCR, Western analysis and flow cytometry. Intracellular protein localization was assessed by confocal microscopy. Pull-down of thiol-oxidized proteins was achieved through BIAM (biotinylated iodoacetamide) switch assays. Following Mφ-CD4+ T cell co-culture, T cell proliferation and cytokine release were investigated via flow cytometry. Results The lipid peroxidizing enzyme ALOX15B (Arachidonate 15-Lipoxygenase Type B) is constitutively expressed in primary human Mφ and drives lipid peroxide-mediated nuclear translocation and thus activation of NRF2 (NFE2 Like BZIP Transcription Factor 2), the master regulator of cellular antioxidant response. We noticed that under resting conditions, ALOX15B dampens the constitutive expression of CIITA through NRF2 activation. Following ALOX15B KD, an increase in CIITA expression enhanced total and cell surface MHC II proteins as well as MHC II-dependent CD4+ T cell activation. While MHC II upregulation in ALOX15B KD Mφ was attenuated through NRF2 stabilization, MHC II-dependent CD4+ T cell activation increased significantly upon NRF2 KD. Investigations into its relevance in lung pathologies are ongoing. Conclusion In Mφ, MHC II-dependent antigen presentation along with CD4+ T cell activation is controlled through ALOX15B-driven repression of CIITA via lipid peroxide-mediated NRF2 activation. Funding Source Deutsche Forschungsgemeinschaft SFB1039 TP B06 Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)