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NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis
Ke Jin, … , Jorg J. Goronzy, Cornelia M. Weyand
Ke Jin, … , Jorg J. Goronzy, Cornelia M. Weyand
Published September 22, 2020
Citation Information: J Clin Invest. 2021;131(1):e136042. https://doi.org/10.1172/JCI136042.
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Research Article Autoimmunity Immunology Article has an altmetric score of 4

NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis

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Abstract

The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.

Authors

Ke Jin, Zhenke Wen, Bowen Wu, Hui Zhang, Jingtao Qiu, Yanan Wang, Kenneth J. Warrington, Gerald J. Berry, Jorg J. Goronzy, Cornelia M. Weyand

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Figure 7

NOTCH4 signaling regulates exosome production.

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NOTCH4 signaling regulates exosome production.
(A) Gene expression for t...
(A) Gene expression for the late endosome marker RAB7A in CD8+ Tregs (RT-PCR, n = 10). (B) Quantification of CD8+ Treg–produced exosomes (n = 10 controls, 7 patients). (C) NOX2 protein expressed on secreted exosomes. Representative histograms and FACS results from 8 samples each. (D–F) GCA CD8+ Tregs were transfected with NOTCH4 or control siRNA. (D) RAB7A transcripts. RT-PCR from 6 patients. (E) Secreted exosomes from n = 7 samples. (F) Exosomal NOX2 protein. Flow cytometry from 6 samples. (G–I) Healthy CD8+ Tregs were transfected with an N4ICD plasmid or empty vector. (G) RAB7A transcripts quantified by RT-PCR in 6 samples. (H) Secreted exosomes determined in 8 samples. (I) Exosomal NOX2 protein. Flow cytometry from 6 samples. (J and K) Control CD8+ Tregs (J) and GCA CD8+ Tregs (K) were transfected as indicated. (J) HES5-containing or empty vector; (K) HES5 or control siRNA. RT-PCR for RAB7A transcripts in n = 6 samples. (L) ChIP assays targeting HES5 or control IgG were performed on the promoter of RAB7A or a negative site. Signal normalized to 10% of input. Data from 6 GCA CD8+ Treg samples. (M) GCA CD8+ Tregs were transfected with HES5 or control siRNA. Occupancy of HES5 on the RAB7A promoter was examined by ChIP assay. Signal normalized to 10% of input. n = 6 samples. (N) GCA CD8+ Tregs were treated with the lysosomal inhibitors chloroquine (CQ) and leupeptin (Leu), the proteasome inhibitor MG132, or vehicle. Cell surface NOX2 was evaluated by FACS. Representative histograms and results from 5 samples. (O) GCA CD8+ Tregs were transfected with a RAB5DN-containing, a RAB7DN-containing, or control vector. Lysosome intensity was measured by LysoTracker. Representative histograms and FACS results from 6 samples. CD8+ Treg cells were induced ex vivo. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired (A–C) and paired (D–K and M) Mann-Whitney-Wilcoxon rank test, or ANOVA and post-ANOVA pairwise 2-group comparisons conducted with Tukey’s method (N and O).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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