Age-related oxidative stress compromises endosomal proteostasis

Abstract

A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.

Figure 1. Detection of oxidatively modified proteins in DC purified from primary and secondary lymphatic organs

a,b,c,d,e) Western blot analysis of carbonylated proteins detected in a) purified CD34⁺ bone marrow precursors b) bone marrow dendritic cells (BMDC) cultured in GM-CSF for 7 days and c,d,e) conventional CD11c⁺ DC freshly purified from primary and secondary lymphatic organs from 3, 12, and 22 month old mice. One representative experiment out of four is shown. f) Western blot analysis of lipoxidated proteins (probing for malondialdehyde) detected in conventional CD11c⁺ freshly purified splenic DC. Lanes marked as (d) report derivatized proteins and (nd) report non-derivatized proteins (specificity control). Loading controls are reported in Supplement Figure 1. g) Pie chart reporting the subcellular distribution of the oxidative proteome immunoprecipitate from conventional CD11c⁺ splenic DC purified from 22 month old mice.

Figure 2. Accumulation of micro aggregates of oxidatively damaged proteins in splenic DC purified from aging mice

a) FPLC separation of micro-aggregates of carbonylated proteins derived from CD11c⁺ splenic DC purified from 3, 12, and 22 month old mice. One preparation, out of two is shown. b) Western blot analysis of the FPLC high molecular weight fractions to detect micro-aggregates of oxidized proteins. Total cell lysates were prepared from CD11c⁺ splenic DC purified from the spleen of 3, 12 and 22 month old mice. One preparation, out of two is shown. c) Western blot analysis of carbonylated proteins detected in late endosomal compartments, gradient-purified from splenic DC of 3, 12 and 22 month old mice. LAMP1 immunoblot is shown as a loading control. d) Ultrastructural morphology of late endosomal multivesicular bodies (MVBs) from CD11c⁺ splenic DC purified from a 22 month old mice. Immunogold labeling for MHC class II molecules (Ab clone AF120.6 is 5 nm gold and Ab clone M5-114 is 10 nm gold).

Figure 3. Decreased endosomal/lysosomal degradation and compromised macroautophagy in DC from aging mice

a, b) CD11c⁺ splenic DC purified from 3, 12 and 22-month mice were labeled for 2 days with [³H]leucine. During the chase cells were maintained for 24 hours in the presence or absence of NH₄Cl/leupeptine (a) or 3Methyl-adenine (b) to block all lysosomal proteolysis or macroautophagy, respectively. Proteolysis rates for long lived proteins after a 20h case are shown. Values are the mean ± S.D. of three different experiments with triplicate wells. * indicate a p value< 0.05. c) Immunofluorescence analysis of LC3 distribution in CD11c⁺ splenic DC from 3, 12 and 22 month old mice. d) Quantification of the number of LC3-positive puncta per cell. * indicate a p value< 0.05 e,f,g) LC3 flux in CD11c⁺ splenic DC from 3, 12 and 22-month mice. Cells were incubated in the presence or absence of lysosomal protease inhibitors (PI) for 2h, collected and subjected to SDS-PAGE and immunoblot for LC3. e) Representative immunoblot. f) Quantification of steady-state levels of LC3-II (content of autophagic vacuoles (AV)) and g) LC3 flux (ratio of LC3-II in the presence and absence of protease inhibitors. * indicate a p value< 0.05. h, i) Immunofluorescence of CD11c⁺ splenic DC from 3, 12 and 22-month mice h) incubated with BSA-biotin and mitotracker or i) incubated with BSA and immunostained for LC3 . Panels show individual channels and merged images. Arrows indicate points of convergence of the different fluorophores.

Figure 4. Decreased in vivo endosomal processing and MHC class II restricted presentation in conventional CD11c⁺ DC from aging mice

a) FACS analysis of CD11c⁺ splenic DC, purified from 3, 12 and 22 months old C57/Bl6 mice, following in vivo injection of Eα–RFP protein. Mice were injected with 50 μg/ml of Eα-RFP. CD11c⁺ DC were purified from the popliteal node and analyzed at different time points for RFP fluorescence, to quantify Eα–RFP processing, and Y-Ae staining to quantify I-Ab loading with the processed Eα 52-68 peptide. One representative experiment, out of three is shown. b, c) Bar graph of the mean fluorescence index and standard deviation of b) total surface MHC class II protein (I-A^b) and c) CLIP detected on the same CD11c⁺ DC population. d) Bar graph and standard deviation of the percentage of CD11c⁺ cells, which stained with the Y-Ae antibody (specific for I-A^b/Eα 52-68 complex). * indicate a p value< 0.05. Lymph nodal CD11c⁺ DC were purified from 3, 12 and 22 month old mice, immunized with 100 μg of Eα-RFP in CFA, two weeks earlier. e) T cell proliferative response from lymph nodes harvested from 3, 12 and 22 month old mice, previously immunized with 100 μg of Eα in CFA (one out of four experiments is shown). f) Quantitative MS scan of the immunodominant Ea 52-68 peptide eluted from nodal CD11c⁺ DC purified from 3 month old mice previously immunized with 100 μg of Eα–RFP in CFA. Isotopically labeled Eα 52-68 peptide was spiked in the eluate for comparative quantification. One out of two quantification is reported. g) MS/MS fragmentation of Eα 52-68 peptide eluted from nodal CD11c⁺ DC purified from 3 month old mice previously immunized with 100 μg of Eα–RFP in CFA.

Figure 5. Decreased in vitro endosomal processing and MHC class II restricted presentation in conventional DC from aging mice

a) FACS analysis of CD11c⁺ splenic DC purified from 3, 12 and 22 months old CBA mice, following phagocytosis of HEL-FITC protein. DC were incubated with 20 μg/ml of HEL-FITC protein (time 0). Cells were then chased at different time points for FITC fluorescence, to quantify HEL-FITC processing and AW3.1 staining to quantify I-A^k loading with the processed HEL 48-62 peptide. One representative experiment, out of four is shown. b) Ultrastructural analysis of MVBs purified from CD11c⁺ splenic DC by a 10/27 percoll gradient. c) Silver stained SDS-PAGE, of gradient purified late endosomal compartments (3, 12 and 22-month mice) incubated for the indicated time points with 5 μg of recombinant Eα–RFP protein. Bands correspond to the amount of undigested Eα–RFP at the indicated time points. d) Bar graph and standard deviation of the densitometric analysis of three independent endosomal processing experiments as reported in c. Data indicate the amount of Eα–RFP protein still unprocessed at different time points, calculated as percentage of total Eα–RFP (time 0). e) Peptide sequences, identified by MS/MS analysis, following endosoma Eα–RFP in vitro processing. Data are collected from two sets of separate mass spectrometry analysis. f) Bar graph of the mean fluorescence index of Y-Ae surface staining of CD11c⁺ splenic DC harvested from 3, 12 and 22 month old mice following incubation with or without Eα 52-68 peptide. One experiment out of three is shown.

Figure 6. In vivo antioxidant treatment ameliorates MHC class II restricted immune response to immunizing antigen

a) Silver staining of protein micro-aggregates present in CD11c⁺ DC purified from the inguinal lymph nodes of 3 and 22 month old mice untreated (-) or treated (+) with the antioxidant agent PDTC. One out of three experiments is reported. b) Western blot analysis of carbonylated proteins present in CD11c⁺ DC purified from the inguinal lymph nodes of 3 and 22 month old mice, untreated (-) or treated (+) with the antioxidant agent PDTC. One out of three experiments is reported. c) Y-Ae surface staining of CD11c⁺ splenic DC harvested from 22 month old mice untreated (-) or treated (+) with the antioxidant agent PDTC for 2 weeks. After purification DC were pulsed for one hour with 20 μg of Eα–RFP and chased overnight before staining with Y-Ae. d) Bar graph depicting the average and standard deviation of Y-Ae staining collected as in (c) by four independent experiments). * indicate a p value< 0.05. e) T cell proliferative response from inguinal lymph nodes harvested from 22 month old mice, previously immunized with 100 μg of Eα in CFA, untreated (-) or treated (+) with the antioxidant agent PDTC for 2 weeks following immunization. One of four experiments is shown.