Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes1,2. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α3. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway4. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells5, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.
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Work in R.M.T.’s laboratory is supported by US Public Health Service (USPHS) Grants AI112258 and AI109799. Work in A.J.B.’s laboratory was supported by USPHS Grants AI044170, AI076246 and AI096528. Work in S.J.M.’s laboratory is supported by USPHS Grants AI076278 and AI117303. S.A.C. was supported by USPHS Grant GM056765. A.M.K.-G. is supported by the American Heart Association Grant 12SDG12220022. N.S. was supported by a CAPES Science without Borders fellowship.
The authors declare no competing financial interests.
Extended data figures and tables
a, Model of how ER stress induces a NOD1/2-dependent pro-inflammatory response through a TUDCA/KIRA6-sensitive pathway, which differs from the TUDCA/KIRA6-resistant pathways induced by bacterial peptidoglycan fragments (MDP or C12-iE-DAP). b, NF-κB activation induced by ectopic expression of VceC in HEK293 cells transfected with a dominant negative form of TRAF2 or a vector control. c, NF-κB activation mediated by expression-induced auto-activation of NOD1, NOD2 or RIP2 in HEK293 cells that were transfected with a dominant negative form of TRAF2 or a vector control. Data are expressed as mean luciferase activity ± s.e.m. from five independent experiments.
a, b, BMDMs from Nod1/2−/− mice and wild-type littermates were stimulated with thapsigargin or MDP, and mRNA abundance for Hspa5 (a) and Chop (b) was quantified (n = 4). c, Expression of SGT1, HSP90 and TRAF2 was detected by western blot in lysates of thapsigargin-stimulated BMDMs from wild-type mice and Nod1/2−/− mice (n = 3 mice of each genotype). Detection of tubulin served as a loading control. A representative image for BMDMs from one wild-type and one Nod1/2−/− animal is shown. d, LDH release induced by treatment of BMDMs from wild-type mice and Nod1/2−/− mice (n = 5) with thapsigargin, DTT or KIRA6. e, Stimulation with MDP or DTT induced IL-6 production in BMDMs from C57BL/6 mice (wild type) but not in BMDMs from Nod1/2−/− mice (n = 8). f, IL-6 secretion induced by thapsigargin, but not by the canonical NOD2 ligand MDP, was significantly inhibited by ER stress inhibitor TUDCA in BMDMs (n = 8). BMDMs did not respond to stimulation with a canonical NOD1 ligand (C12-iE-DAP). g–k, BMDMs from wild-type mice and Nod1/2−/− mice (n = 4) were treated with the PERK inhibitor GSK2656157 (GSK) (g, i) or the IRE1α RNase inhibitor STF-083010 (STF) (h, k) and IL-6 synthesis measured by ELISA (g, h) or mRNA analysed by real-time PCR (i, j). Data are presented as mean ± s.e.m. n represents the number of independent assays (biologic replicates) performed for each experiment.
a–c, Groups (n = 5) of wild-type mice and Nod1/2−/− mice were treated with thapsigargin and received either vehicle control of TUDCA. Synthesis of IL-6 (a), KC (b) and MIP-1β (c) in the serum was determined using a Bio-plex cytokine assay. d, e, Wild-type (C57BL/6) mice and Nod1/2−/− mice (n = 4) were treated with thapsigargin and transcript levels of Il6 determined by quantitative real-time PCR. Data are expressed as fold-increases over vehicle control-treated animals. Data are presented as mean ± s.e.m. n represents the number of independent assays (biologic replicates) performed for each experiment.
Extended Data Figure 4 B. abortus-induced inflammatory responses in mice are blunted by TUDCA treatment.
a–g, Mice (n ≥ 4) were mock infected or infected with the B. abortus wild type and were treated with TUDCA or vehicle control. Three days after infection, circulating levels of IL-6 (a), IL-12p40 (b), IFNγ (c), KC (d), MIP-1β (e), G-CSF (f) and RANTES (g) were profiled in serum using a Bio-Plex cytokine assay. Data are presented as mean ± s.e.m.
a, c, e, g, i, Bacterial burden in the spleen and in BMDMs of wild-type and Nod1/2−/− mice (a, c, e, g) or Rip2−/− mice (i). No statistically significant differences in colony-forming units (CFU) recovered from the spleen (a, c, g, i) or from BMDMs (e) of wild-type and Nod1/2−/− or Rip2−/− mice (n ≥ 4) infected with B. abortus wild type or the vceC mutant were observed. b, d, f, h, Host responses elicited during B. abortus infection. b, Groups of mice (n = 5) were infected with the indicated B. abortus strains and treated with KIRA6. d, f, BMDMs from wild-type mice and Nod1/2−/− mice (d) or wild-type mice and Rip2−/− mice (n ≥ 4) (f) were infected with the indicated B. abortus strains. h, Groups (n = 5) of wild-type mice and Rip2−/− mice were infected with the indicated B. abortus strains. Il6 mRNA levels were determined by quantitative real-time PCR (b, d, h). IL-6 synthesis was determined by ELISA (f). Data are presented as mean ± s.e.m.
a, Bacterial numbers of wild-type B. abortus (strain 2308) recovered from in the spleen and placenta (n = 5 mice per group). b, c, Il6 mRNA expression (b) and total histopathology scores (c) in the placenta of mice at days 3, 7 and 13 after infection with B. abortus. d, Scoring criteria for blinded evaluation of haematoxylin and eosin (H&E)-stained sections from the placenta. e, Representative images of the histopathology observed in the placenta of B. abortus infected mice at days 3, 7 and 13 after infection. Arrow, neutrophil infiltration; N, necrosis.
a–c, No statistically significant differences in colony-forming units in the spleen and placenta of wild-type and Nod1/2−/− mice infected with B. abortus wild type or the vceC mutant at 13 days post-infection were observed. Data are presented as mean ± s.e.m. (n = 5 mice per group).
HeLa cells were stimulated with MDP, thapsigargin or infected with Chlamydia muridarum and treated with KIRA6 or transfected with RIP2DN (dominant negative form of RIP2). Expression of Il6 was determined by quantitative real-time PCR. Data are presented as mean ± s.e.m. from 4 independently performed assays.
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Keestra-Gounder, A., Byndloss, M., Seyffert, N. et al. NOD1 and NOD2 signalling links ER stress with inflammation. Nature 532, 394–397 (2016) doi:10.1038/nature17631
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