Abstract
Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.
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Change history
09 July 2014
Affiliation address 9, for author David Gilot, was given incorrectly and has been updated.
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Acknowledgements
This work was supported by funding from the Italian Association for Cancer Research (AIRC, to P.P.), Fondazione Italiana Sclerosi Multipla Project No. 2010/R/17 (to F.F.), Associazione Umbra Contro il Cancro (to G.S. & M.A.D.F.), Bayer Grants4Target Focus Grant no. 2012-03-0630 (to A.I., F.F. and D.M.), Bayer Early Career Investigator Award (to D.M.), Grant no. R01ES007685 from the US National Institutes of Environmental Health Sciences (to M.S.D.), the Specific Targeted Research Project FUNMETA (to L.R.), and the Italian Ministry of Health in association with Regione dell’Umbria (GR-2008-1138004 to C.O.) We thank D. Fuchs for serum kynurenine determinations. We also thank G. Andrielli for digital art and image editing and G. Ricci for histopathology.
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A.B. and M.G. designed and conducted all experiments unless otherwise indicated below; M.T.P., D.M. and C.V. analysed IDO and Src phosphorylation; S.B., E.M.C.M., D.P., M.P. and A.I. conducted bioinformatics studies and statistical analysis. A.M. performed homology modelling and docking studies. R.I., T.Z., M.A.D.F., L.R. and L.T. conducted the in vivo studies with Salmonella and GBS. C.V., M.L.B., C.O., G.S., C.B. and R.B. contributed to specific experimental designs; T.V.L., M.P., H.F. and T.N. made possible, and designed, the experiments with TDO2-deficient mice; J.B.D., G.C.P. and R.M. made possible, and designed, the experiments with IDO2-deficient mice; D.G., M.S.D., G.J.G., M.G., B.V., L.B. and U.G. provided conceptual help and reagents throughout experimentation; F.F. designed and supervised all experiments; P.P. supervised the overall study and wrote the manuscript. F.F. and P.P. share senior authorship on this paper.
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Extended data figures and tables
Extended Data Figure 1 Increased susceptibility to primary LPS challenge in mice treated with a TDO2 inhibitor.
a, Twelve hours before LPS challenge (10 mg per kg), WT mice were treated with vehicle, the IDO1 and IDO2 inhibitor 1-MT (200 mg per kg), or the TDO2 inhibitor 680C91 (10 mg per kg), control groups receiving 1-MT or 680C91 but no LPS. Survival was monitored every 24 h through day 8 of LPS challenge (n = 10 mice per group per experiment, in one out of three). **P < 0.001 (log-rank test). b, Estimation of LD50 (mg per kg) in mice treated with 1.25, 2.5, 5, 10, 20, 40, or 80 mg per kg LPS. n = 10 per group per dose. LD50 values were calculated by curve-fitting (r2 ≥ 0.95) in one experiment representative of two.
Extended Data Figure 2 Lack of endogenous IL-10 increases susceptibility to endotoxaemia.
a, Survival of WT mice exposed to 10 mg per kg LPS in the presence of anti–IL-10 (0.2 mg per mouse daily, for 4 d, commencing 6 h before challenge) or an isotype control. Data are from three independent experiments (mean ± s.d.). b, Survival of WT and Il10–/– mice treated with 10 mg per kg LPS. **P < 0.001 (log-rank test). c, Survival curves of mice of different genotypes challenged with 10 mg per kg LPS, with or without therapeutic subcutaneous IL-10 at 250 ng per mouse, daily, from challenge (day 0) through day 5. *P < 0.05 (IL-10 versus vehicle). The data show that exogenous IL-10 compensates for both the TDO2 and AhR defects at the lower LPS dosage. IL-10 is protective only in TDO2 knockouts when 20 mg per kg LPS is used.
Extended Data Figure 3 Mutation of Gln 377 to Ala in AhR PAS-B domain does not alter receptor half-life, and apparently results in increased TCDD ligand potency.
a, AhR-deficient cDCs were transfected with WT or AhR(Q377A). After 24 h, cells were incubated with cycloheximide (CXM) (10 µg ml−1) and harvested at different times, lysed, and analysed for AhR expression by immunoblotting, using a specific antibody. β-tubulin was used as a loading control. Data are from one experiment of three. b, Ratios (means ± s.d. of three experiments) of WT or AhR(Q377A) to β-tubulin in transfected cDCs at different times of CHX treatment. (No differences by Student’s t-test.)
Extended Data Figure 4 LPS tolerance potentiates IDO1 expression and AhR activation in splenic cDCs.
a, b, Real-time PCR analysis of Ido1 mRNA expression and immunoblot analysis of IDO1 protein in peritoneal exudate macrophages (MΦ) and neutrophils (Neu) (a), as well as in splenic conventional DCs (cDCs) or plasmacytoid DCs (pDCs) (b). Cells were harvested and purified at 24 h (a) or 72 h (b) of LPS rechallenge. For comparison, samples were included from mice on first exposure to 40 mg per kg LPS (unprimed), as opposed to tolerized mice (primed). Data of Ido1 mRNA fold induction are presented as means ± s.d. of three experiments; *P < 0.05 and **P < 0.001, Shapiro test. Immunoblotting data are from one experiment of three. c, Real-time PCR analysis of Ahr and Cyp1a1 transcript expressions in cDCs from the same mice as in b. **P < 0.001, Shapiro test.
Extended Data Figure 5 Absolute requirement for functional AhR, but not TDO2, in LPS tolerance manifestations.
a, Survival curves of WT and LPS-primed (0.5 mg per kg, day 0) WT (prWT) and AhR-deficient (prAhr–/–) mice after a second challenge (on day +7) with 40 mg per kg LPS. Survival was monitored every 24 h through day 8 of LPS challenge. n = 8–10 mice per group per experiment. One experiment of three. *P < 0.05, log-rank test. b, Survival curves of WT and LPS-primed (10 mg per kg, day 0) WT (prWT) and TDO2-deficient (prTdo2–/–) mice after a second challenge (on day +7) with 40 mg per kg LPS. Survival was monitored every 24 h through day 8 of LPS challenge. n = 8–10 mice per group per experiment. One experiment of three. **P < 0.001, log-rank test.
Extended Data Figure 6 Bioinformatic data from myeloid cDCs data sets.
a, Expression changes of tyrosine kinases in LPS-primed myeloid DCs compared to untreated counterparts. b, Log2 fold changes, depicted as mean values and standard errors.
Extended Data Figure 7 LPS tolerance modulates cytokine production and Foxp3 and Rorc transcription in S. Typhimurium infection.
a, IL-6, IL-1β, TNF-α, IL-10, and TGF-β were measured in caecum cell supernatants from LPS-tolerant mice infected with S. enterica Typhimurium. Data are from three independent experiments (means ± s.d.). *P < 0.05 and **P < 0.001 (Student’s t-test). b, RT–PCR expression of Foxp3 and Rorc transcripts in mesenteric lymph node cells from LPS-tolerant, Salmonella-infected mice. Data (mean ± s.d. of three experiments) are presented as normalized transcript expression in the samples relative to normalized transcript expression in control cultures (that is, cells from vehicle-treated mice, in which fold change = 1; dotted line). *P < 0.05; **P < 0.001 (Shapiro test).
Extended Data Figure 8 Ahr–/– and Ido–/– mice are more susceptible than WT mice to S. Typhimurium infection.
a, Naive mice of different genotypes were challenged intragastrically with S. Typhimurium. Mortality data were recorded (**P < 0.001, WT versus all other genotypes; log-rank test). b, Haematoxylin and eosin staining of mouse caeca was performed at 7 days of infection. Scale bars, 50 μm. One of three experiments. c, Transcript expressions of Il17a, Rorc, Il10, and Foxp3 were quantified in mesenteric lymph node cells. Data (mean ± s.d. of three experiments) are presented as normalized transcript expression in the samples relative to normalized transcript expression in cells from uninfected donors, in which fold change = 1. **P < 0.001 (Shapiro test).
Extended Data Figure 9 LPS tolerance modulates Foxp3 and Rorc transcription in GBS infection.
RT–PCR expression of Foxp3 and Rorc transcripts in joint-draining lymph node cells from LPS-tolerant, GBS-infected mice. Data (mean ± s.d. of three experiments) are presented as normalized transcript expression in the samples relative to normalized transcript expression in control cells (that is, cells from vehicle-treated mice, in which fold change = 1; dotted line). **P < 0.001 (Shapiro test).
Extended Data Figure 10 Ahr–/– and Ido–/– mice are more susceptible than WT mice to GBS immunopathology.
a, Naive mice of different genotypes were infected with GBS (107 c.f.u.). Mortality data were recorded (*P < 0.05 and **P < 0.001, WT versus all other genotypes; log-rank test). b, Haematoxylin and eosin staining of joints was performed at 10 days of infection. Scale bars, 100 μm. One of three experiments. c, Transcript expressions of Il17a, Rorc, Il10, and Foxp3 were quantified in joint-draining lymph nodes. Data (mean ± s.d. of three experiments) are presented as normalized transcript expression in the samples relative to normalized transcript expression in cells from uninfected donors, in which fold change = 1. **P < 0.001 (Shapiro test).
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Bessede, A., Gargaro, M., Pallotta, M. et al. Aryl hydrocarbon receptor control of a disease tolerance defence pathway. Nature 511, 184–190 (2014). https://doi.org/10.1038/nature13323
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DOI: https://doi.org/10.1038/nature13323
