Abstract
The respiratory tract is heavily populated with innate immune cells, but the mechanisms that control such cells are poorly defined. Here we found that the E3 ubiquitin ligase TRIM29 was a selective regulator of the activation of alveolar macrophages, the expression of type I interferons and the production of proinflammatory cytokines in the lungs. We found that deletion of TRIM29 enhanced macrophage production of type I interferons and protected mice from infection with influenza virus, while challenge of Trim29−/− mice with Haemophilus influenzae resulted in lethal lung inflammation due to massive production of proinflammatory cytokines by macrophages. Mechanistically, we demonstrated that TRIM29 inhibited interferon-regulatory factors and signaling via the transcription factor NF-κB by degrading the adaptor NEMO and that TRIM29 directly bound NEMO and subsequently induced its ubiquitination and proteolytic degradation. These data identify TRIM29 as a key negative regulator of alveolar macrophages and might have important clinical implications for local immunity and immunopathology.
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Change history
10 October 2016
In the version of this article initially published online, the immunoblots in Figures 1a, 5a, 5b, 5c, 5d, 5f, 6a, 6b, 7a, 7b, 8a and 8b were in color. These have been replaced with black and white immunoblots. The error has been corrected for the print, PDF and HTML versions of this article.
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Acknowledgements
We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for the mutant mouse line Trim29, and the European Mouse Mutant Archive (http://www.emmanet.org) partner from which the mouse line was received. Funding and associated primary phenotypic information is provided at the Sanger website (http://www.sanger.ac.uk/mouseportal). We also thank L. Minze for operational support. Supported by the US National Institutes of Health (R01AI080779 to X.C.L.).
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J.X. designed and performed most of the experiments; L.W., B.Y., Z.W., L.J., R.J. and H.L. helped with some of the experiments; X.C.L., Y.-J.L. and Z.Z. wrote the manuscript; and Z.Z. supervised the project.
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Integrated supplementary information
Supplementary Figure 1 Trim29 gene-targeting strategy.
(a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/−) and homozygous mice (−/−). (b) Immunoblot analysis of TRIM29 in lung primary AMs from wildtype mice (+/+) or TRIM29 knockout mice (−/−). Data are representative of three independent experiments with similar results.
Supplementary Figure 2 TRIM29 negatively regulates the production of type I interferons and proinflammatory cytokines in AMs.
(a-d) ELISA of IFN-α (a), IFN-β (b), TNF (c) and IL-6 (d) production by primary AMs from wildtype mice (+/+) and T rim29−/− mice (−/−) after 20 h infection with influenza PR8 virus. Virus was used at a multiplicity of infection (MOI) of 5. Individual circle represents the value from each independent experiment; small horizontal lines indicate the average of triplicates. Mock, wildtype cells without infection. (e) Flow cytometry analyzing CD11c and F4/80 expression in the primary AMs isolated from wildtype mice (+/+) and Trim29−/− mice (−/−) using isotype control antibodies (Control, Ctrl), CD11c-FITC and F4/80-APC antibodies. Flow cytometry data were acquired on a LSR-II flow cytometer (Beckton Dickinson) and analyzed using FlowJo v10 software (Tree Star). *P<0.005, **P<0.0005, ***P<0.0001 (unpaired t test). Data are representative of three independent experiments with similar results (a‒d) or two experiments (e).
Supplementary Figure 3 TRIM29 negatively regulates production of chemokines in responses to 5′pppRNA or viral infection.
(a, c) ELISA of MIP-1α (a) and IL-10 (c) production by primary AMs from wildtype mice (+/+) and Trim29−/− mice (−/−) after 12 h of stimulation with 5’pppRNA (5’ppp, 1 μg/ml) delivered by Lipofectamine 3000 or infection with influenza PR8 virus. Virus was used at a MOI of 5. Individual circle represents the value from each independent experiment; small horizontal lines indicate the average of triplicates. Mock, wildtype AMs without stimulation or infection. N.S., not significant, *P<0.005, **P<0.0005 (unpaired t test). (b,d) Quantification of the mRNA expression of chemokines including CCL-2, CCL-5 and CXCL-2 (b) and type II cytokines including IL-4, IL-5 and IL-13 (d) produced by primary AMs from wildtype mice (+/+) and Trim29−/− mice (−/−) after 12 h of stimulation with 5’pppRNA (5’ppp) or infection with influenza PR8 virus as shown in a and c. N.S., not significant, *P<0.05, **P<0.01, ***P<0.001 (unpaired t test). Data are representative three independent experiments with similar results (mean and s.d. in b,d).
Supplementary Figure 4 TRIM29 does not affect IL-6 production by peritoneal macrophages or splenic macrophages.
(a,b) ELISA of IL-6 production in peritoneal macrophages (a) or splenic macrophages (b) from wildtype mice (+/+) and Trim29−/− mice (−/−) after 12 h of stimulation with 5’pppRNA (5’ppp, 1 μg/ml) delivered by Lipofectamine 3000. Individual circle represents the value from each independent experiment; small horizontal lines indicate the average of triplicates. Mock, wildtype cells without stimulation. N.S., not significant (unpaired t test). Data are representative three independent experiments with similar results.
Supplementary Figure 5 TRIM29 negatively regulates the cytokines production in response to 5′pppRNA or viral infection.
(a,b) ELISA of IFN-α (a), IFN-β (b), TNF (c) and IL-6 (d) production in primary CD11c+ splenic dendritic cells from wildtype mice (+/+) and Trim29−/− mice (−/−) after 12 h of stimulation with 5’pppRNA (5’ppp, 1 μg/ml) delivered by Lipofectamine 3000 or infection with influenza PR8 virus. Virus was used at a MOI of 5. Mock, wildtype CD11c+ splenic dendritic cells without stimulation or infection. (e,f) ELISA of IL-6 in BALF samples from wildtype mice (+/+) and Trim29−/− mice (−/−) at day 2 (D2) or day 4 (D4) of intranasal infection with high dose 1×105 PFU (e) or low dose 1×102 PFU (f) of influenza PR8 virus. Mock, wildtype mice without infection. Individual circle represents the value from each independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P<0.01, ***P<0.001 (unpaired t test). Data are representative two independent experiments with similar results.
Supplementary Figure 6 TRIM29 has a minimal role in the production of type I interferons in response to LPS or H. influenzae infection.
(a) ELISA of IFN-α and IFN-β production in primary AMs from wildtype mice (+/+) and T rim29−/− mice (−/−) after 6 h or 12 h stimulation with LPS (10 ng/ml). Mock, wildtype cells without stimulation. (b,c) ELISA of MIP-1α (b) and IFN-α (c) production in BALF samples from wildtype mice (+/+) a nd Trim29−/− mice (−/−) at day 1 of intratracheal inoculation with H. influenzae infection (1×107 CFU per mouse). Mock, wildtype mice without H. influenzae infection. (d) ELISA of IFN-α and IFN-β production in BALF samples from wildtype mice (+/+) and T rim29−/− mice (−/−) at 12 h of intranasal inoculation with LPS. Mock, wildtype mice without LPS challenge. Individual circle represents the value from each independent experiment; small horizontal lines indicate the average of triplicates. N.S., not significant, *P<0.05, **P<0.0001 (unpaired t test). Data are representative two independent experiments with similar results.
Supplementary Figure 7 TRIM29 expression is not affected by LPS or reovirus infection in macrophages.
Quantification of TRIM29 mRNA expression in alveolar macrophages, peritoneal macrophages and splenic macrophages mock treated or treated with LPS (20 ng/ml) or reovirus infection (Reo) for 6h. Virus was used at a MOI of 5. Mock, cells without stimulation. Data are representative two independent experiments with similar results.
Supplementary Figure 8 TRIM29, but not TRIM29 BBOX, colocalizes with NEMO in the lysosome.
(a) Schematic diagram showing full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains (left margin). (b) Schematic diagram showing full-length NEMO and serial truncations of NEMO with deletion of various domains (left margin); numbers at ends indicate amino acid positions (top). (c‒e) Confocal microscopy of HEK293T cells co-transfected with HA-TRIM29 or HA-TRIM29 BBOX (T29 BBOX, lacking interaction with NEMO) and Myc-NEMO expression plasmids and mock (c and d) or infected with influenza PR8 virus for 4 h (e). TRIM29 was strained with Alexa Fluor 488–anti-HA (green), MitoTracker was used to probe the mitochondrion (red). LAMP1, TfR and LC3A served as the markers of lysosome, endosome and autophagosome (red). DAPI served as the nuclei marker (blue). Scale bars represent 10 µm for original images and 5 µm for enlarged images. Data are representative of two independent experiments with similar results.
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Xing, J., Weng, L., Yuan, B. et al. Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract. Nat Immunol 17, 1373–1380 (2016). https://doi.org/10.1038/ni.3580
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DOI: https://doi.org/10.1038/ni.3580
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