The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation (original) (raw)

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Acknowledgements

This work was supported by grants from the US National Institutes of Health (NIH; grants R37 DK057665, R37 AI048638, U19 AI090023, U19 AI057266) and from the Bill & Melinda Gates Foundation to B.P. R.J.K. was supported by NIH grants R01 DK088227, R37 DK042394, R01 DK103185 and the Crohn’s & Colitis Foundation of America Senior Fellow Award 3800. We thank B. Cervasi and K. P. Gill for help with sorting and D. Levesque, D. Hampton, S. Weismann and colleagues for animal husbandry and veterinary support. We thank the Center for AIDS Research core at Yerkes Primate Research Center for help with qPCR (grant P30-AI-50409) and the Integrated Cellular Imaging Center at Winship Cancer Center, Emory University for help with confocal microscopy. We also thank the Yerkes Pathology Core for performing the immunofluorescence and histology analysis. We thank S. Virgin for his advice and review of the autophagy experiments.

Author information

Author notes

  1. Rajesh Ravindran and Jens Loebbermann: These authors contributed equally to this work.

Authors and Affiliations

  1. Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road, Atlanta, 30329, Georgia, USA
    Rajesh Ravindran, Jens Loebbermann, Hualing Ma, Paul Hakimpour, Yi-chong Wang, Shuzhao Li & Bali Pulendran
  2. School of Pharmaceutical Sciences, University of São Paulo, São Paulo, 05508, Brazil
    Helder I. Nakaya & Leonardo Gama
  3. Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India
    Nooruddin Khan
  4. Division of Pathology, Yerkes National Primate Research Center, 954 Gatewood Road, Atlanta, 30329, Georgia, USA
    Deepa K. Machiah & Prachi Sharma
  5. Virology Core, Emory Vaccine Center and Yerkes National Primate Research Center, 954 Gatewood Road, Atlanta, 30329, Georgia, USA
    Benton Lawson
  6. Degenerative Disease Program, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, California, 92037, USA
    Randal J. Kaufman
  7. National Institute of Environmental Health Sciences, Mail Drop D2-01, Research Triangle Park, 27709, North Carolina, USA
    Jennifer Martinez

Authors

  1. Rajesh Ravindran
  2. Jens Loebbermann
  3. Helder I. Nakaya
  4. Nooruddin Khan
  5. Hualing Ma
  6. Leonardo Gama
  7. Deepa K. Machiah
  8. Benton Lawson
  9. Paul Hakimpour
  10. Yi-chong Wang
  11. Shuzhao Li
  12. Prachi Sharma
  13. Randal J. Kaufman
  14. Jennifer Martinez
  15. Bali Pulendran

Contributions

R.R., J.L. and B.P. designed experiments and wrote the manuscript. R.R., J.L., N.K., D.K.M., H.M., S.L. and B.L. conducted the experiments. H.I.N. and L.G. performed bioinformatics analysis of the public databases. P.H. and Y.-c.W. genotyped mice. P.S. performed the histology analysis. J.M. provided critical insight and advice about the autophagy experiments. R.J.K. provided reagents and edited the manuscript.

Corresponding author

Correspondence toBali Pulendran.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 eIF2α kinases are expressed in human and murine gut cells.

a, Analysis of p-eIF2α expression in APCs and epithelial cells in large intestine of naive and 2% DSS-treated mice by flow cytometry. DCs, dendritic cells; LI, large intestine. b, Comparison of immunohistological analysis of phosphorylated PKR, PERK, eIF2α and GNC2 in healthy and inflamed human colon tissue (n = 1). c, Expression levels of HRI, PKR, PERK and GCN2 in human organs quantified based on information from a public database (http://www.ebi.ac.uk). d, Expression intensity of various eIF2α kinases plotted from known published microarray data from colonic biopsies of patients with either ulcerative colitis or Crohn’s disease compared to healthy controls. Data are from one experiment that is representative of three separate experiments. *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 2 GCN2 deficiency does not affect the proliferation or differentiation of intestinal epithelial cells.

a–d, Immunohistology analysis (a–c) and quantification (d) of colons and jejenums from _Gcn2_−/− and wild-type (littermate) mice for chromagranin A (a), Ki67 (b) and lysozyme (c). KO, knockout; WT, wild type. Data are from one experiment that is representative of three separate experiments (n = 3). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 3 GCN2 expression protects mice from DSS-induced colitis.

a, H&E staining of colon sections before and after DSS in wild-type versus _Gcn2_−/− mice, Gcn2 fl/fl versus _Gcn2_Δvillin and Gcn2 fl/fl versus _Gcn2_ΔAPC. b–d, IL-17 levels in large intestinal (b) and small intestinal (c) CD4+ T cells measured by flow cytometry; _Gcn2_−/− mice show increased intestinal permeability after DSS treatment as evidenced by higher levels of fluorescein isothiocyanate (FITC)-conjugated dextran in the serum (d). SI, small intestine. e, Expression of antimicrobial defensins in wild-type and _Gcn2_−/− mice via quantitative polymerase chain reaction (qPCR). f, IL-17 production by flow cytometry and enzyme-linked immunosorbent assay (ELISA) of OTII-CD4 T cells after culturing with different large intestinal APC subsets. Data are from one experiment that is representative of three separate experiments (n = 4–5). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 4 PERK expression in epithelial cells has only a minor role in controlling mucosal homeostasis after DSS challenge.

a–d, The body weight (a), colon length (b), histology by H&E (c) and TH17 responses (d) both in the colon (large intestine; LI) and small intestine (SI) of _Perk_Δvillin and control wild-type littermates treated with DSS. e–h, The body weight (e), colon length (f), histology by H&E and histology score (g), and TH17 responses (h) both in the colon (LI) and small intestine (SI) of _Perk_ΔAPC and control wild-type littermates treated with DSS. Data are representative of two separate experiments (n = 5). *P < 0.05; **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 5 eIF2α expression in epithelial cells and APCs control weight loss and partially control TH17 responses after DSS challenge.

a–e, The body weight (a), colon length (b), histology by H&E and histology score (c), and TH17 responses in both the colon (LI) (d) and small intestine (SI) (e) of _Eif2a_Δvillin and control wild-type littermates treated with DSS. f–j, The body weight (f), colon length (g), histology by H&E and histology score (h), and TH17 responses in both the colon (LI) (i) and small intestine (SI) (j) of _Eif2a_ΔAPC and control wild-type littermates treated with DSS. Data are representative of three separate experiments (n = 5). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 6 Intestinal APCs and epithelial cells reveal high expression of LC3.

a, Expression of LC3–GFP in APC subsets and epithelial cells of naive LC3–GFP mice by flow cytometry (n = 3). Data are from a single experiment. b, Kinetic MFI comparison of LC3B and p62 expression with and without chloroquine on individual APC subsets and epithelial cells by flow cytometry after 12 and 24 h of single DSS administration. c, Western blot detection of LC3-I and II on lamina propria APCs before and after digitonin. d, LC3B staining of individual APCs and epithelial cells 12 h after they were treated with DSS. The portion of the lamina propria cells were subjected to digitonin before intracellular staining with the LC3B antibody. Data are representative of two separate experiments (n = 4–5). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 7 Atg5 and Atg7 expression in APCs partially protects mice from DSS challenge.

a–c, Colon length (a), histology by H&E and histology score (b), and TH17 responses (c) in both the colon (LI) and small intestine (SI) of _Atg5_ΔAPC and control wild-type littermates treated with DSS. d–f, Colon length (d), histology by H&E and histology score (e), and TH17 responses (f) in both the colon (LI) and small intestine (SI) of _Atg7_ΔAPC and control wild-type littermates treated with DSS. Data are representative of three separate experiments (n = 5). *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 8 GCN2-induced autophagy protects the intestinal tissue from the effects of excess oxidation and inflammation.

a–f, _Gcn2_−/− and littermate wild-type mice were treated with 2% DSS in the drinking water for 5 days. a, MFIs of ROS and mitochondrial ROS (MitoSOX) in individual APC subsets and epithelial cells of the small intestine isolated from wild-type and _Gcn2_−/− mice were analysed by flow cytometry (n = 5). b, MFIs of pro-IL-1β in individual APC subsets and epithelial cells of the small intestine isolated from wild-type or _Gcn2_−/− mice (day 5 after DSS) were analysed by flow cytometry (n = 3). c, Histology analysis of _Gcn2_−/− mice that were treated with neutralizing antibody. d, Effects of low protein diet on DSS-induced colitis. e, Colon length. f, Frequencies of CD4+, CD4+IFNγ+ and CD4+Foxp3+ T cells. Data are from one experiment that is representative of two or three separate experiments. *P < 0.05, **P < 0.005, ***P < 0.0005. Error bars indicate mean ± s.e.m.

Extended Data Figure 9 Mechanisms by which GCN2 contributes to protection in the gut against acute colitis.

a, Amino acid starvation induced by an inflamed colon activates GCN2, which triggers autophagy, which is important in inhibiting oxidative stress and pro-IL-1β. Furthermore, levels of IL-1β dictate the magnitude of IL-17A-producing CD4 T cells in the colon. b, A hypothetical model for the evolutionary significance of coupling amino acid starvation with control of inflammation.

Extended Data Figure 10 Mouse phenotyping by flow cytometry and western blot.

a, b, In addition to molecular genotyping via tail DNA, we analysed the protein levels in various mucosal subsets by flow cytometry in various APC-specific (a) and epithelial-specific (b) conditional knockouts. c, Western blot to show selective depletion in APC populations in _Atg5_ΔAPC and _Atg7_ΔAPC mice. DC, dendritic cell; Ep, epithelial cell.

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Ravindran, R., Loebbermann, J., Nakaya, H. et al. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation.Nature 531, 523–527 (2016). https://doi.org/10.1038/nature17186

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