Aryl hydrocarbon receptor control of a disease tolerance defence pathway

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.

Figure 1. Increased susceptibility of Tdo2^−/− and Ahr^−/− mice to primary LPS challenge

a, Survival data of challenged mice (n = 10). One of three experiments. *P < 0.05; **P < 0.001 (log-rank test). b, Kyn/trp ratios. Mean ± s.d. of three experiments. *P < 0.05; **P < 0.001 (two-tailed Student’s t-test). c, Estimation of LD₅₀ (n = 10). d,e, Histopathology in lungs and liver, respectively. Scale bar, 100 μm. f, Cytokine measurements (means ± s.d.) from three experiments (n = 6; **P < 0.001; two-tailed Student’s t-test). g, Gene transcript expressions. Means ± s.d. (three experiments; **P < 0.001; Shapiro test). h, Immunoblotting data. One experiment of three. i, Kyn/trp ratios (means ± s.d. of three experiments). *P < 0.05; **P < 0.001 (two-tailed Student’s t-test). j, Cyp1a1 transcript expression; three experiments; **P < 0.001 (Shapiro test).

Figure 2. l-kynurenine (kyn) is an endogenous AhR ligand

a, Kyn competes with [³H]TCDD for binding cytosolic AhR. b, Proposed binding mode of TCDD into the homology model of PAS-B of AhR. c, Proposed binding mode of kyn (orange carbon atoms) into the homology model of PAS-B of AhR. Hydrophobic residues involved in the binding of kyn are shown as yellow sticks. Gln377 is shown in sticks with carbon atoms in gray. d, Transactivation activity of WT or G377A AhR by kyn or TCDD. e, Competition by kyn for specific binding of [³H]TCDD to cytosolic AhR from reconstituted Ahr^−/− DCs, allowing for an estimated kyn IC₅₀ value of 36.2 μM and of 21.6 μM for K_i. n = 3; means ± s.d. in one experiment of three. f–h, Ido1, Il10 and Tgfb1 transcripts. Means ± s.d. of three experiments; ** P < 0.001 (Shapiro test). i, Cyp1a1 transcripts. Compiled data (means ± s.d.) from three experiments. **P < 0.001 (Shapiro test). j, Survival curves of variously treated mice (n = 10; one of three experiments). **P < 0.001 (log-rank test).

Figure 3. Absolute requirement for AhR, functional IDO1 and TGF-β in LPS tolerance

a, Survival of WT and LPS-primed WT (prWT), IDO1-deficient (prIdo1^−/−) and TDO2-deficient (prIdo2^−/−) mice after rechallenge. n = 8–10; one experiment of three. **P < 0.001; log-rank test. b, Endotoxin LD₅₀ in tolerized mice. n = 10. c,d, Histopathology in lungs and liver, respectively; scale bar, 100 μm. One experiment of two. e, Survival of variously treated mice (n = 10; one of three experiments). **P < 0.001 (log-rank test). f, Cytokine measurements (mean ± s.d. of three experiments; n = 6). **P < 0.001 (two-tailed Student’s t-test). g, Neutralization of TGF-β upon LPS rechallenge (n = 10). Three experiments (mean ± s.d.); **P < 0.001 (log-rank test). h, Kyn/trp ratios (mean ± s.d. of three experiments). **P < 0.001 (two-tailed Student’s t-test). i, Survival of variously treated mice. n = 8–10; one experiment of three. **P < 0.001 (log-rank test).

Figure 4. LPS tolerance induces AhR- and Src-dependent IDO1 phosphorylation

a, Phosphorylation of IDO1 in total splenocytes isolated from LPS-tolerant mice in one experiment of three. Ratios are mean values from the three experiments. *P < 0.001 (15’ vs. single LPS exposure; two-tailed Student’s t-test). b, Splenic immunofluorescent staining. Scale bar, 100 μm. c, Phosphorylation of IDO1’s ITIM2 in WT and AhR-deficient cDCs. One experiment of three, ratios being means from the three experiments. *P < 0.001 (30’ vs. single LPS exposure; two-tailed Student’s t-test). d, Immunoblot analysis of Src and phosphorylated Src-Y416 (pSrc) in WT and AhR-deficient cDCs in one experiment of three. Ratios are means of the three experiments. *P < 0.001 (5’ and 15’ vs. single LPS exposure; two-tailed Student’s t-test). e, Production of TGF-β by Src-competent cDCs in response to single or double LPS exposure. Means ± s.d. from three experiments; **P < 0.001 (two-tailed Student’s t-test).

Figure 5. LPS tolerance ameliorates S. Typhimurium infection

a, Mice, treated once or twice with LPS, were challenged with S. Typhimurium, and bacterial counts assessed in cecum (CFU); **P < 0.001; two-tailed Student’s t-test; b, H&E staining of cecal tissues; c, cytokines in cecum cell supernatants. Data are means (± s.d.) from three experiments; *P < 0.05, **P < 0.01; two-tailed Student’s t-test. d, Cecal CFU and cecal weights at 14 days post infection in tolerant mice. Three experiments; means ± s.d., with *P < 0.05 and **P < 0.001; two-tailed Student’s t-test. e, Gross histopathology of cecum and distal colon (1 cm ruler scale).

Figure 6. LPS tolerance protects against Streptococcus immunopathology

a, Survival of variously treated, infected mice (*P < 0.05; log-rank test). b, Arthritis index. c, CFU enumeration in joints and kidneys. In b and c, values are means ± s.d. of three experiments; *P < 0.05 and **P < 0.001; two-tailed Student’s t-test. d, Histopathologic evaluation of arthritis in joints from LPS-tolerant, GBS-infected mice. Representative data from one of three experiments. Scale bar, 1mm. (e) Supernatants from joint homogenates were assayed kinetically for IL-6, IL-1β, IL-17A, IL-10, and TGF-β content. Values are the mean ± s.d. of three experiments. *P < 0.05 and **P < 0.001, two-tailed Student’s t-test.