Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice

Abstract

Mice deficient in the hemochromatosis gene, Hfe, have attenuated inflammatory responses to Salmonella infection associated with decreased macrophage TNF-alpha and IL-6 biosynthesis after exposure to LPS. In this study, we show that the abnormal cytokine production is related to impaired TLR4 signaling. Despite their abnormal response to LPS, Hfe KO macrophages produced amounts of TNF-alpha similar to those in WT cells after TLR2 stimulation. Consistent with this finding, LPS-induced activation of Mal/MyD88-dependent events was normal in the mutant macrophages. However, LPS-induced IFN-beta expression, a TRAM/TRIF-dependent response activated by TLR4, was reduced by Hfe deficiency. This reduction could be replicated in WT macrophages with the use of iron chelators. In contrast, TLR3-activated expression of IFN-beta, a TRIF-dependent response, was normal in Hfe KO macrophages and was unaffected by iron chelation. Our data suggest that low intracellular iron selectively impairs signaling via the TLR4/TRAM/TRIF pathway proximal to TRIF and results in reduced LPS-induced cytokine expression. Furthermore, by mimicking the altered iron metabolism associated with Hfe deficiency, we found that 3 different inhibitors of hepcidin attenuated Salmonella-induced and noninfectious enterocolitis. Thus, manipulation of iron homeostasis could represent a new therapeutic approach to controlling inflammation.

Figure 1. Hfe KO macrophages have an abnormal cytokine response to stimulation with TLR4, but not TLR2.

Peritoneal macrophages from WT and Hfe KO mice were stimulated with 100 ng/ml LPS (n = 4) or 1 μg/ml Pam3CSK4 (n = 3) for 6 hours. Supernatants were collected and analyzed by ELISA for TNF-α. Data represent mean ± SD. Similar results were obtained in 2 separate experiments. *P < 0.0001.

Figure 2. LPS-induced activation of ERK and p38 kinase pathways is intact in Hfe KO macrophages.

WT and Hfe KO macrophages were stimulated for the indicated times with 100 ng/ml LPS. Cell lysates were prepared, and Western blotting was performed to determine levels of total and phosphorylated kinases. Similar results were obtained in 2 separate experiments.

Figure 3. Induction of IFN-β by activation of TLR4, but not TLR3, is abnormal in Hfe KO macrophages.

WT and Hfe KO macrophages were stimulated with (A) 100 ng/ml LPS or (B) 10 μg/ml poly(I:C) for 6 hours (n = 3 per group). Supernatants were collected and analyzed by ELISA for IFN-β. Data represent mean ± SD. Similar results were obtained in 2 separate experiments. *P = 0.026; **P = 0.039.

Figure 4. SIH inhibits LPS-induced, but not poly(I:C)-induced, expression of IFN-β in WT macrophages.

WT macrophages were stimulated with 100 ng/ml LPS or 10 μg/ml poly(I:C) for 6 hours in the presence of the indicated concentrations of SIH (n = 3 per group). Supernatants were collected and analyzed by ELISA for IFN-β. Data represent mean ± SD. Similar results were obtained in 2 separate experiments. *P = 0.03.

Figure 5. Effects of dorsomorphin on Salmonella-induced changes in hepcidin expression and serum iron levels.

(A and B) Liver hepcidin mRNA was measured by quantitative RT-PCR in (A) control or Salmonella-infected WT mice 48 hours after infection (n = 5 per group) and in (B) Salmonella-infected WT mice treated with vehicle (n = 7) or dorsomorphin (n = 6). (C) Serum iron levels were measured at the same time (n = 5 [control]; 7 [vehicle]; 5 [dorsomorphin]). Data represent mean ± SEM. *P = 0.04; **P = 0.01; ***P < 0.0001.

Figure 6. Effects of dorsomorphin treatment on Salmonella-induced intestinal inflammation in WT mice.

(A–C) Groups of WT mice treated with vehicle or dorsomorphin were infected with Salmonella, and intestinal inflammation was evaluated 48 hours later by (A) gross appearance of cecum, (B) cecal histopathology (original magnification, ×40), and (C) cecal TNF-α mRNA levels (mean ± SEM). (D) Numbers of Salmonella present in the liver were assessed at the same time; data points represent individual mice. n = 5 (uninfected); 7 (vehicle); 6 (dorsomorphin). *P = 0.024.

Figure 7. LDN-193189 suppresses hepcidin upregulation and intestinal inflammation in piroxicam-induced colitis.

Il10 KO mice were treated with piroxicam for 2 weeks and then injected with vehicle or LDN-193189 for an additional week. The animals were sacrificed, and (A) liver hepcidin mRNA (n = 3 [untreated and vehicle]; 4 [LDN-193189]) and (B) colon IL-6 mRNA (n = 9 [vehicle]; 12 [LDN-193189]) were measured by quantitative RT-PCR. Data represent mean ± SEM. *P = 0.005; **P = 0.0007.

Figure 8. Influence of low intracellular iron on LPS-induced cytokine expression.

Based on the effects of Hfe deficiency and iron chelation on responses to TLR2, TLR3, and TLR4 stimulation, low intracellular iron is likely to impair TLR4 signaling at a step in the TRAM/TRIF pathway proximal to TRIF. IRF3, IFN regulatory factor 3.