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N-myristoyltransferase deficiency impairs activation of kinase AMPK and promotes synovial tissue inflammation

Nature Immunology volume 20, pages 313–325 (2019)Cite this article

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Abstract

N-myristoyltransferase (NMT) attaches the fatty acid myristate to the N-terminal glycine of proteins to sort them into soluble and membrane-bound fractions. Function of the energy-sensing AMP-activated protein kinase, AMPK, is myristoylation dependent. In rheumatoid arthritis (RA), pathogenic T cells shift glucose away from adenosine tri-phosphate production toward synthetic and proliferative programs, promoting proliferation, cytokine production, and tissue invasion. We found that RA T cells had a defect in NMT1 function, which prevented AMPK activation and enabled unopposed mTORC1 signaling. Lack of the myristate lipid tail disrupted the lysosomal translocation and activation of AMPK. Instead, myristoylation-incompetent RA T cells hyperactivated the mTORC1 pathway and differentiated into pro-inflammatory TH1 and TH17 helper T cells. In vivo, NMT1 loss caused robust synovial tissue inflammation, whereas forced NMT1 overexpression rescued AMPK activation and suppressed synovitis. Thus, NMT1 has tissue-protective functions by facilitating lysosomal recruitment of AMPK and dampening mTORC1 signaling.

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Fig. 1: N-myristoyltransferase 1 (NMT1) deficiency in T cells from patients with RA.
Fig. 2: NMT1 controls pro-inflammatory T cell differentiation.
Fig. 3: N-myristoylation protects against synovial inflammation.
Fig. 4: Impaired activation of the energy sensor AMPK in RA T cells.
Fig. 5: AMPK activation is myristoylation dependent.
Fig. 6: NMT1 controls lysosomal recruitment of AMPK.
Fig. 7: NMT1 deficiency promotes mTORC1 hyperactivity in RA T cells.
Fig. 8: AMPK activation corrects arthrogenic T cell effector functions in vitro and in vivo.

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The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the National Institutes of Health (grant nos. R01 AR042527, R01 HL 117913, R01 AI108906 and P01 HL129941 to C.M.W. and nos. R01 AI108891, R01 AG045779, U19 AI057266 and I01 BX001669 to J.J.G.).

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  1. These authors contributed equally: Ke Jin, Yi Shen.

Authors and Affiliations

  1. Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA

    Zhenke Wen, Ke Jin, Yi Shen, Zhen Yang, Yinyin Li, Bowen Wu, Stanford Shoor, Jorg J. Goronzy & Cornelia M. Weyand

  2. Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, CA, USA

    Lu Tian

  3. Arthritis Center, Stanford Health Care-ValleyCare, Pleasanton, CA, USA

    Niall E. Roche

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Contributions

Z.W., C.M.W., and J.J.G. designed the study and analyzed the data. Z.W., K.J., Y.S., Z.Y., Y.L., and B.W. performed experiments. S.S., N.E.R., C.M.W., and J.J.G. were responsible for patient selection, evaluation, and recruitment. L.T. supervised all statistical analyses. C.M.W., Z.W., and J.J.G. wrote the manuscript.

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Correspondence to Cornelia M. Weyand.

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Integrated supplementary information

Supplementary Figure 1 Expression of NMT1 and NMT2.

(a) CD4+CD45RA+ T cells were isolated from patients with rheumatoid arthritis (RA), patients with psoriatic arthritis (PsA) or age-matched healthy individuals and stimulated for 72 h. NMT2 expression was analyzed by flow cytometry. Data (mean ± SEM) from 10 RA patients, 8 PsA patients and 10 healthy individuals. One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method. (b) CD4+CD45RA+ T cells were stimulated for 72 h and NMT1 mRNA expression was measured by RT-PCR. Mean ± SEM from 10 samples in each group. Unpaired Mann-Whitney-Wilcoxon rank test. (c-h) PBMCs were collected from RA patients, PsA patients and healthy individuals. Multicolor flow cytometry was applied to quantify NMT1 expression in CD4+CD45RA+ T cells, CD4+CD45RA– T cells, CD8+CD45RA+ T cells, CD8+CD45RA– T cells, CD19+ B cells and CD14+ monocytes. Representative histograms and collective MFIs (mean ± SEM) for each of the subpopulations from 11 individuals in each donor cohort. One-way ANOVA and post-ANOVA pair-wise two-group comparisons conducted with Tukey’s method. **p < 0.01. ***p < 0.001

Supplementary Figure 2 Genetic manipulation of NMT1 expression in CD4+ T cells.

(a) CD4+CD45RA+ T cells from RA patients were transfected with a NMT1 expression vector or a control vector. (b) CD4+CD45RA+ T cells from healthy subjects were transfected with NMT1 siRNA or control siRNA. 24 h later, NMT1 protein expression was analyzed with flow cytometry. Representative histograms and collective data from 6 individuals in each group. Paired Mann-Whitney-Wilcoxon rank test. *p < 0.05

Supplementary Figure 3 NMT1 regulates lipid droplet accumulation in T cells.

CD4+CD45RA+ T cells from RA patients and healthy donors were stimulated for 72 h. Neutral lipid droplets were stained with Bodipy 493/503 and analyzed by flow cytometry. (a) Comparison of neutral lipid expression in control and RA T cells. Representative histograms and results from 6 patient-control pairs. Mean ± SEM. **p < 0.01 by paired Mann-Whitney-Wilcoxon rank test. (b) NMT1 activity was restored in T cells from RA patients by transfecting a NMT1-expressing vector. Lipid droplets were detected by staining with Bodipy. Representative histograms are shown. (c) NMT1 activity was inhibited by transfecting T cells from healthy individuals with NMT1 siRNA. Histograms of Bodipy staining for lipid droplets are shown.

Supplementary Figure 4 NMT1 and NMT2 expression in CD4+ T cells is independent of metabolic signals.

Naïve CD4 T cells from 6 healthy donors were stimulated for 72 h in the absence or presence of C75 (20 μM), 3PO (200 nM), ML265 (10 μM), Shikonin (250 nM), pyruvate (1 mM), succinate (1 mM), malic acid (1 mM), A769662 (10 μM), Compound C (1 μM) or rapamycin (10 μM), respectively. Protein expression of NMT1 and NMT2 in CD4 T cells was determined by flow cytometry. One-way ANOVA and post-ANOVA pair-wise two-group comparisons were conducted with Tukey’s method

Supplementary Figure 5 N-myristoylation protects against synovial inflammation.

(a) Scheme of experimental design. NSG mice were engrafted with human synovial tissue. Seven days later, CD4 T cells were FACS sorted from CD4+CD45RO– PBMCs of RA patients or healthy individuals, manipulated for NMT1 expression by transfecting with a NMT1 vector, NMT1 siRNA or controls respectively, and added back to the CD4-depleted PBMCs for adoptive transfer into the chimeras. In other experiments, CD45RO– PBMCs were transfected. At day 14, synovial tissues were harvested for transcriptome analysis and immunohistochemical staining. (b-e) NMT1 overexpression inhibits synovial inflammation. NSG mice were engrafted with human synovial tissue. Seven days after engraftment, CD45RO– PBMCs from RA patients were transfected with a NMT1-expressing or control vector and transferred into the chimeric mice. Seven days later, synovial tissues were harvested for transcriptome analysis and immunostaining. (b) Tissue sections stained with anti-IFN-γ (green) and anti-CD3 (red). Nuclei marked with DAPI. Representative images from 6 grafts. Scale bars 20 μm. (c–d) Frequencies of CD3+ T cells and of CD3+IFN-γ+ T cells in tissue sections. (e) Heat map presentation of gene transcripts measured in tissue extracts by qPCR. Data are mean ± SEM from 6 synovial grafts. *p < 0.05 by paired Mann-Whitney-Wilcoxon rank test. (f-i) NMT1 knockdown promotes synovial inflammation. Human synovial tissue was transplanted into NSG mice. After engraftment, CD45RO– PBMCs from healthy donors were transfected with NMT1 siRNA or control siRNA and adoptively transferred into the chimeric mice. Tissue transcriptome and immunohistochemical stains were analyzed in synovial explants after 7 days. (f) Tissue-infiltrating human T cells evaluated by dual-color immunostaining of CD3 (red) and IFN-γ (green). Nuclei marked with DAPI. Representative images from 6 grafts. Scale bars 20 μm. (g-h) Frequencies of tissue CD3+ T cells and of CD3+IFN-γ+ T cells. (i) Tissue transcriptome of inflammation-associated genes shown as a heat map. Data are mean ± SEM from 6 synovial grafts. *p < 0.05 by paired Mann-Whitney-Wilcoxon rank test

Supplementary Figure 6 AMPK activity and transcriptional expression in healthy individuals and RA patients.

CD4+CD45RA+ T cells were purified from RA patients or healthy individuals and stimulated for 72 h. (a) AMPK activity was determined by analyzing inhibitory phosphorylation of acetyl-CoA carboxylase (ACC). Protein expression of phospho-ACC was determined by Western blotting. β-actin served as loading control. Representative immunoblots and relative intensities (mean ± SEM) from 5 RA-healthy pairs. Each dot represents the data from one donor. *p < 0.05 by unpaired Mann-Whitney-Wilcoxon rank test. (b) Transcripts for AMPK complex components were analyzed by qPCR. Data are mean ± SEM from 7 RA-healthy pairs.

Supplementary Figure 7 AMPK regulates mTORC1 activation but not its lysosomal localization.

(a) Naïve CD4+ T cells were isolated from healthy individuals or RA patients and stimulated for 72 h. Lysosomes were identified with antibodies to human LAMP1 (red). mTOR was stained with anti-mTOR (green). LAMP1-mTOR co-localization was analyzed by confocal microscopy. Each dot represents one T cell. Scale bars 20 μm. Unpaired Mann-Whitney-Wilcoxon rank test. (b, c) Naïve CD4+ T cells from healthy donors were transfected with AMPKa siRNA or control siRNA and stimulated for 72 h. (b) AMPKα protein expression was analyzed by flow cytometry after 24 hrs. ***p < 0.001 by paired student’s t-test. (c) mTOR expression was quantified in lysosomal fractions and in whole cell lysates. Representative immunoblots from 4 individuals in each group. (d) AMPK-dependent inhibition of mTORC1 activity in T cells. Naïve CD4+ T cells from 6 healthy individuals were stimulated for 72 h in the absence or presence of the AMPK inhibitor Compound C (1 μM). Phospho-S6RP in CD4+ T cells was determined by phosflow cytometry. Paired Mann-Whitney-Wilcoxon rank test. *p < 0.05

Supplementary Figure 8 Anti-inflammatory effects of AMPK activation and mTORC1 inhibition.

(a) The AMPK activator A769662 does not affect Th2 and Treg lineage markers in vivo. NSG mice were implanted with human synovial tissues, reconstituted with CD45RO– PBMCs from RA patients, and randomly assigned to control arm (vehicle) or treatment arm (A769662, 30 mg/kg/mouse, twice a day). Synovial grafts were explanted and analyzed for mRNA expression of Th2 and Treg lineage markers (GATA3, IL4, FOXP3) after 7 days of treatment. Data are mean ± SEM from 6 grafts in each group (paired Mann-Whitney-Wilcoxon rank test). (b) The mTORC1 inhibitor rapamycin does not affect Th2 and Treg lineage markers in vivo. NSG mice were implanted with human synovial tissues, reconstituted with CD45RO– PBMCs from RA patients, and randomly assigned to control arm (vehicle) and treatment arm (rapamycin, 5 mg/kg/mouse, every other day). Synovial grafts were explanted and analyzed for mRNA expression of Th2 and Treg lineage markers (GATA3, IL4, FOXP3) after 9 days of treatment. Data are mean ± SEM from 6 grafts in each group. Paired Mann-Whitney-Wilcoxon rank test. (c) mTORC1 inhibition abrogates differentiation of RA T cells into pro-inflammatory effector cells. Naïve CD4+ T cells from 6 RA patients were stimulated in the absence or presence of the mTORC1 inhibitor rapamycin (Rapa, 10 μM). Lineage-determining transcription factors and signature cytokines were analyzed. Each dot represents the data from one patient. Paired Mann-Whitney-Wilcoxon rank test. *p < 0.05. (d) Effect of A769662 on CD4+ Treg cell induction in vitro. CD4+CD45RA+ T cells from RA patients were stimulated in the presence of an increasing dose of the AMPK activator A769662 for 4 days and expression of the Treg lineage-determining transcription factor FoxP3 was analyzed by flow cytometry. Data (mean ± SEM) from 6 RA patients. One-way ANOVA and post-ANOVA pair-wise two-group comparisons were conducted with Tukey’s method. ***p < 0.001

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Wen, Z., Jin, K., Shen, Y. et al. N-myristoyltransferase deficiency impairs activation of kinase AMPK and promotes synovial tissue inflammation. Nat Immunol 20, 313–325 (2019). https://doi.org/10.1038/s41590-018-0296-7

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