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Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation

Abstract

Misdirected immunity gives rise to the autoimmune tissue inflammation of rheumatoid arthritis, in which excess production of the cytokine tumor necrosis factor (TNF) is a central pathogenic event. Mechanisms underlying the breakdown of self-tolerance are unclear, but T cells in the arthritic joint have a distinctive metabolic signature of ATPlo acetyl-CoAhi proinflammatory effector cells. Here we show that a deficiency in the production of mitochondrial aspartate is an important abnormality in these autoimmune T cells. Shortage of mitochondrial aspartate disrupted the regeneration of the metabolic cofactor nicotinamide adenine dinucleotide, causing ADP deribosylation of the endoplasmic reticulum (ER) sensor GRP78/BiP. As a result, ribosome-rich ER membranes expanded, promoting co-translational translocation and enhanced biogenesis of transmembrane TNF. ERrich T cells were the predominant TNF producers in the arthritic joint. Transfer of intact mitochondria into T cells, as well as supplementation of exogenous aspartate, rescued the mitochondria-instructed expansion of ER membranes and suppressed TNF release and rheumatoid tissue inflammation.

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Fig. 1: Mitochondrial insufficiency and ER expansion in RA.
Fig. 2: Mitochondrial insufficiency promotes ER expansion.
Fig. 3: Mitochondria-derived aspartate controls ER size.
Fig. 4: Aspartate is anti-inflammatory.
Fig. 5: Aspartate is required for NAD+ regeneration and ADP ribosylation of BiP.
Fig. 6: Expansion of the rough ER increases co-translational translocation in RA T cells.
Fig. 7: ERrich RA T cells are TNF superproducers.
Fig. 8: TNF-producing CD4+ T cells function as arthritogenic effector cells.

Data availability

All data in the present study are available within the article and its Supplementary files and from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institutes of Health (grant nos. R01AR042527, R01AI108906, R01HL142068 and P01HL129941 to C.M.W. and nos. R01AI108891, R01AG045779, U19AI057266 and R01AI129191 to J.J.G.).

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Authors and Affiliations

Authors

Contributions

C.M.W., J.J.G. and B.W. conceived the project. B.W., T.V.Z., Z.H. and K.J. formally analyzed and investigated the data. K.J.W. and M.P.A. recruited the patients. C.M.W., J.J.G. and B.W. wrote the original manuscript. C.M.W. and J.J.G. supervised the study. C.M.W. and J.J.G. acquired the funds.

Corresponding author

Correspondence to Cornelia M. Weyand.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Navdeep Chandel and George Tsokos for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer review reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Mitochondrial mass in healthy and RA T cells.

CD4+CD45RA+ T cells were stimulated for 72 h. Flow cytometric quantification of mitochondria mass (MitoTrack Green MFI); n = 6 healthy and 6 RA. Data are mean ± SEM. Two-tailed unpaired Mann-Whitney-Wilcoxon rank test.

Source data

Extended Data Fig. 2 Gating strategy to analyze ER and mitochondrial function on the single cell level.

CD4+CD45RA+ T cells were stimulated for 72 h. ER biomass was determined with ER tracker and mitochondrial function was assessed with the mitochondrial membrane potential. Gate #1: small cellular size. Gate #2: medium cellular size. Gate #3: large cellular size.

Extended Data Fig. 3 XBP1S overexpression induces ER expansion.

Naive CD4+ T cells were stimulated and transfected with control or XBP1S overexpression plasmid before the ER size was determined. (a, b) Flow cytometry for ER Tracker staining; n = 4. (c) Confocal microscopy imaging of the ER protein calnexin. Scale bar, 10 μm, n = 3 independent experiments. (d, e) Flow cytometry for calnexin expression; n = 3. (f) XBP1S expression in T cells from patients treated with or w/o Methotrexate (MTX) (MTX: n = 13; w/o MTX: n = 12). All data are mean ± SEM. Two-tailed paired t test (b, e). Unpaired Mann-Whitney-Wilcoxon rank test (f). *P < 0.05, ***P < 0.001.

Source data

Extended Data Fig. 4 Inhibitors of mitochondrial respiration promote ER expansion.

Healthy naive CD4+ T cells were stimulated for 72 h in the presence of the mitochondrial respiration inhibitors Piericidin A (10 pM), Antimycin A (10 nM) or Oligomycin (1 nM). ER size was determined by flow cytometry measuring ER tracker (n = 4). Data are mean ± SEM. One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method. **P < 0.01.

Source data

Extended Data Fig. 5 Experimental scheme for mitochondria transfer.

(a) Experimental scheme for mitochondria transfer into RA T cells. (b) Experimental scheme for mitochondria transfer in Jurkat T cells. Mitochondria were labeled with MitoTrackerRed and isolated, then transferred into recipient cells. (c) Flow cytometric analysis of MitoTracker Red intensity after mitochondria transfer. Ratio indicates donor cell number/recipient cell number. (d) Representative confocal imaging of exogenous mitochondria transferred into Jurkat T cells, n = 3 independent experiments, scale bar, 20 μm.

Extended Data Fig. 6 Expression of GOT1 and GOT2 in healthy and RA T cells.

Peripheral blood CD4+CD45RA+ T cells from patients with RA and age-matched healthy individuals were isolated and stimulated for 72 h. mRNA levels of GOT1 and GOT2 were determined by qPCR. n = 4 in each group. All data are mean ± SEM. Two-tailed unpaired Mann-Whitney-Wilcoxon rank test.

Source data

Extended Data Fig. 7 Mitochondrial complex I inhibitor Piercidin A inhibits ADP-ribosylation of BiP.

ADP-ribosylation of BiP in healthy CD4+ T cells treated with or w/o Piercidin A (10 pM) for 24 h. n = 2.

Source data

Extended Data Fig. 8 Isolation of Rough ER.

(a) Naïve CD4+ T cells were purified from peripheral blood mononuclear cells and stimulated with anti-CD3/CD28 for 72 h. The rough ER was isolated by calcium precipitation and the isolate was immunoblotted for the ER protein calnexin, the ribosomal protein L17 and the cytoplasmic protein a-actin. (b) Healthy CD4+ T cells were activated with PMA/Ionomycin for 2 h before isolation of the rough ER and immunoblotting of the ER protein calnexin, the ribosomal protein S7 and the cytosolic protein β-actin.

Source data

Extended Data Fig. 9 Effects of Tunicamycin, aspartate, asparagine, pyruvate, and α-ketobutyrate on ER size and TNF production.

Naïve CD4+ T cells were purified from PBMCs and stimulated with anti-CD3/CD28 beads for 72 h in the presence of the indicated molecules. ER size quantified by MFI of ER tracker staining and TNF production measured by intracellular staining of TNF after PMA/ION stimulation for 2 h in the presence of the secretion inhibitor BFA. (a) Fold change of ER size after Tunicamycin treatment compared to the control group, n = 6. (b) Fold change of TNF production after Tunicamycin treatment compared to the control group, n = 4. (c) ER size change after Aspartate (1 mM) or Asparagine (1 mM) treatment, n = 6. (d) TNF production after aspartate and asparagine treatment, n = 3. (e) ER size change after pyruvate (1 mM) or α-KB (1 mM) treatment, n = 6. (f) TNF production after pyruvate or α-KB treatment, n = 4. All data are mean ± SEM, one-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 10 Mitochondria transfer into CD4+ T cells protects synovial tissue from inflammation.

Mitochondria were isolated from healthy T cells and transferred into RA CD4+ T cells prior to their adoptive transfer into synovium-NSG chimeras. Explanted synovial grafts were analyzed by immunohistochemical staining and tissue transcriptomics (RT-PCR). 8 tissues in each group. (a) H&E staining of synovial tissue sections. Scale bar; 50 μm. (b) Immunofluorescence staining for CD3+ T cells in synovial infiltrates. Representative images. Scale bar; 10 μm. (c) Gene expression profiling (RT-PCR) of TRB, TBET, RORG and other key inflammatory markers (n = 8). All data are mean ± SEM. Two-tailed unpaired Mann-Whitney-Wilcoxon rank test. *P < 0.05, **P < 0.01, ***P < 0.001.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, and Supplementary Figs. 1–3.

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Wu, B., Zhao, T.V., Jin, K. et al. Mitochondrial aspartate regulates TNF biogenesis and autoimmune tissue inflammation. Nat Immunol 22, 1551–1562 (2021). https://doi.org/10.1038/s41590-021-01065-2

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