Lipid peroxidation causes endosomal antigen release for cross-presentation

Abstract

Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer.

Figure 1. NOX2-produced ROS cause endosomal lipid peroxidation.

(a) H₂O₂ production by DCs cultured in absence (no LPS; blue curve) or presence of LPS (+LPS; black) measured with Amplex Red. (b) Sensor Bodipy581/591-C11 (structure) increases fluorescence at 510 nm upon peroxidation. Histograms show fluorescence intensity distributions from a typical experiment of DCs labeled with Bodipy581/591-C11 and incubation with LPS for the times indicated. (c–d) The mean fluorescence intensities (MFI) measured by flow cytometry of DCs cultured for 60 min with Bodipy581/591-C11 in presence or absence of LPS (c), or with LPS in absence (Ctrl) or presence of α-tocopherol (α-Toc) or phenylarsine oxide (PAO) (d). (e) Quantification of knock-down efficiency for gp91phox (NOX2^KD) by Western blot (NT: non-targeting siRNA; GAPDH: loading control). (f) Similar to c, but now for NOX2^KD DCs. (g) Similar to d, but now for mouse BMDCs. (h) Confocal microscope images of DCs stained for gp91phox (green) after treatment with OVA conjugated with Alexa fluor 647 (OVA-AF647; red). (i) Detection of peroxidation-modified proteins on endosomes. DCs were incubated with OVA-AF647 and linoleamide alkyne (LAA). After incubation, cells were fixed and stained with Alexa fluor 488 azide (LAA-AF488). The fluorescence threshold was set on the OVA-AF647 channel and the green fluorescence was quantified. BF: bright field. (j) Difference between green fluorescence of the whole cell and the fluorescence thresholded on OVA-AF647. (k) LAA assay for DCs cultured in the absence or presence of LPS. LAA-AF488 signal was quantified for at least 10 cells. (l) Same as panel (k), but now for LPS-treated DCs in the absence or presence of α-Toc. Results show individual results for at least 3 donors/mice. Results from the same donor/mouse are connected by a black line. Scale bars, 10 μm.

Figure 2. Lipid peroxidation causes antigen release from endosomes.

(a) Liposomes with lysozyme encapsulated were treated with a combination of H₂O₂ and Fe(II)SO₄ (ROS) or Triton X-100 (TX100; positive control) followed by incubation with trypsin. Samples were analyzed by SDS-PAGE. Graph shows semi-quantifications of band intensities from 4 independent experiments (mean ± S.E.M). (b) Scheme and confocal microscope images of a CCF4 assay. The cytosolic FRET probe CCF4 was cleaved by exogenous β-lactamase (β-lac) resulting in a decreased ratio of fluorescein (acceptor fluorophore; green) over coumarin (donor; blue) fluorescence. BF: bright field. (c–d) CCF4 cleavage efficiencies for DCs cultured in the absence or presence of LPS or α-tocopherol (α-Toc) (c) and for NOX2^KD DCs (d). NT: non-targeting siRNA. (e–f) Cell viability by the MTT assay of cells cultured with exogenous cytochrome C (Cyt C) and in presence or absence of α-tocopherol (α-Toc) (e) as well as for NOX2^KD DCs (f). Each donor is plotted in a different color. (g) Confocal micrographs of an unstimulated (no LPS) and stimulated (+LPS) DC expressing galectin-3-mAzami (green), incubated with OVA conjugated to Alexa fluor 647 (OVA-AF647; red). (h) Fluorescence intensities of galectin-3-mAzami thresholded on OVA-AF647, relative to cytosolic fluorescence intensity, in DCs in the presence or absence of LPS and α-Toc. Results show individual results for at least 3 donors. Scale bars, 10 μm.

Figure 3. Lipid peroxidation promotes cross-presentation.

(a) Scheme of the cytosolic and lysosomal pathways leading to antigen cross-presentation. (b) Time line of the Jurkat T cell activation assay with DCs presenting gp100. (c) Jurkat T cell activation assay with DCs incubated with α-tocopherol (α-Toc), MG132 or lactacystin and with NOX2^KD DCs after incubation with long (residues 272–300) or short peptide (residues 280–288). On the y-axes the percentages of CD69-positive T cells for long (residues 272–300) over short peptide are depicted. (d) B3Z T cell activation assay with BMDCs incubated with α-tocopherol (α-Toc) after incubation with complete OVA protein or OVA peptide (SIINFEKL). On the y-axis the percentages of β-galactosidase producing T cells for OVA protein over OVA peptide. Results show individual results for at least 3 donors. Raw FACS data, and full short and long peptide T cell activation controls are in Suppl. Figs 3 and 4.

Figure 4. Direct induction of endosomal antigen release and impaired release in CGD.

(a) Confocal micrographs of a DC expressing VAMP8-KillerRed (green) and incubated with OVA conjugated to Alexa fluor 647 (OVA-AF647; red). BF: bright field. Scale bar, 10 μm. (b) Linoleamide alkyne lipid peroxidation assay as in fig. 1i, but now for exposed or non-exposed DCs with or without expression of KillerRed. (c) CCF4 endosomal antigen leakage assay as in fig. 2b, but now for exposed or non-exposed DCs with or without expression of KillerRed. (d–e) Similar as panels (b,c) but now for HEK293T cells. (f) p47phox and gp91phox expression in lymphocytes from three p47phox-/- (CGD1-3; blue) and one gp91phox-/- (CGD4; green) CGD patients by Western blot (GAPDH: loading control). (g) CCF4 endosomal antigen release assay as in fig. 2b, but now with DCs derived from monocytes from the CGD patients from panel (d) (Ctrl: DCs from healthy donors). (h) Uptake of BSA conjugated to Alexa fluor 488 (BSA-AF488) by DCs of CGD patients, presented as percentage BSA-AF488-positive cells and MFI. Results show individual results of at least 3 donors/experiments. (i) Model scheme of NOX2-induced antigen leakage from endosomes. Superoxide anions produced by NOX2 and protons provided by the V-ATPase form H₂O₂ and hydroxyl radicals (Fenton reaction). These ROS induce lipid peroxidation, which disrupts endosomal membranes and causes the release of antigen (purple spheres) into the cytosol.