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. 2012 May 1;52(9):2047-56.
doi: 10.1016/j.freeradbiomed.2012.01.027. Epub 2012 Feb 4.

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice

Maria C Seleme  1 , Weiqi Lei, Ashley R Burg, Kah Yong Goh, Allison Metz, Chad Steele, Hubert M Tse
Affiliations

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice

Maria C Seleme et al. Free Radic Biol Med. .

Abstract

In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic β cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1(m1J)) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-Mϕ) from NOD and NOD.Ncf1(m1J) mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1(m1J) BM-Mϕ exhibited a 2- and 10-fold decrease in TNF-α and IFN-β proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-Mϕ. Optimal expression of IFN-α/β is not solely dependent on superoxide synthesis, but requires p47(phox) to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A(g7) expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A(g7) downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-β proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1(m1J) mice.

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Figures

Figure 1
Figure 1. Poly(I:C) stimulation of NOD BM-Mϕ results in a rapid increase in superoxide generation
Luminol-oxidized chemiluminescence of 5×104 NOD and NOD.Ncf1m1J BM-Mϕ was observed kinetically at 2 minute intervals for an hour after 25μg/mL poly(I:C) stimulation. NOD BM-Mϕ exhibit a 5-fold increase in luminescent arbitrary units at the 8-minute time point in contrast to NOD.Ncf1m1J BM-Mϕ. Data shown is representative of 3 independent experiments performed with at least triplicates for each sample. *** p < 0.001 versus poly(I:C)-stimulated NOD.Ncf1m1J BM-Mϕ.
Figure 2
Figure 2. Superoxide is required for down-regulation of MHC-II I-Ag7 (signal 1) on NOD.Ncf1m1J BM-Mϕ
Flow cytometric surface expression of F4/80 - MHC-II I-Ag7 (A), CD11b - CD80 (B), CD11b - CD86 (C), or CD11b - CD40 (D) double positive cells after poly(I:C) stimulation for 0, 4, 24, and 48 hr. Bar graphs representing the average percent of double positive cells in 3 independent experiments. ** p < 0.005, * p < 0.05, and n.s. – not significant versus poly(I:C)-stimulated NOD BM-Mϕ.
Figure 3
Figure 3. Poly(I:C)-stimulated NOD.Ncf1m1J BM-Mϕ exhibit a decrease in pro-inflammatory cytokine expression
Supernatants from NOD and NOD.Ncf1m1J BM-Mϕ stimulated with 25 μg/ml of poly(I:C) for 24 hr were assayed for TNF-α (A), IL-1β (B), IL-6 (C), G-CSF (D), IFN-α (E), and IFN-β (F) cytokine expression by ELISA (TNF-α, IFN-α, IFN-β) or with a Bio-plex Multiplex kit (IL-1β, IL-6, G-CSF). qRT-PCR expression analysis of Ifna2 (G), Ifnb1 (H), and Isg15 (I) genes in NOD and NOD.Ncf1m1J BM-Mϕ after 8 hr stimulation with poly(I:C). Gene expression was calculated using the 2−ΔΔCt method and is represented as fold-change relative to the non-stimulated cells used as calibrator samples and arbitrarily set to 1, and Gapdh was used as an endogenous normalization control. Plotted data represent averages of 3 independent experiments, done in triplicates ** p < 0.005, * p < 0.05, and n.s. – not significant versus poly(I:C)-stimulated NOD BM-Mϕ.
Figure 4
Figure 4. Exogenous xanthine oxidase (XO) can restore NF-κB-dependent pro-inflammatory cytokines, but not Type I interferon synthesis in poly(I:C)-stimulated NOD.Ncf1m1J BM-Mϕ
Supernatants from NOD and NOD.Ncf1m1J BM-Mϕ stimulated with 25 μg/ml of poly(I:C) with or without 1mU/mL of xanthine oxidase for 24 hr were assayed for TNF-α (A) and IFN-β (B) cytokine expression by ELISA. Plotted data represent averages of 3 independent experiments, done in triplicates *** p < 0.001, * p < 0.05, and n.s. – not significant versus poly(I:C)-stimulated NOD BM-Mϕ or NOD.Ncf1m1J BM-Mϕ.
Figure 5
Figure 5. Scavenging of superoxide with a superoxide dismutase (SOD) mimetic inhibits TNF-α and IFN-β synthesis in NOD and NOD.Ncf1m1J BM-Mϕ after poly(I:C) stimulation
Supernatants from NOD and NOD.Ncf1m1J BM-Mϕ stimulated with 25 μg/ml of poly(I:C) with or without 68μM SOD mimetic for 24 hr were assayed for TNF-α (A) and IFN-β (B) cytokine expression by ELISA. Plotted data represent averages of 3 independent experiments, done in triplicates * p < 0.05 versus poly(I:C)-stimulated NOD BM-Mϕ or NOD.Ncf1m1J BM-Mϕ.
Figure 6
Figure 6. NOD.Ncf1m1J BM-Mϕ exhibit a decrease in mRNA accumulation and protein expression levels of TLR3 and TRIF after poly(I:C)-stimulation
qRT-PCR was performed on mRNA isolated from NOD and NOD.Ncf1m1J BM-Mϕ untreated or treated with poly(I:C) for 8 hours to assess the expression of Tlr3 (A) and the TRIF gene, Ticam1 (B). Western blot analysis of whole cell lysates from untreated or 25 μg/ml poly(I:C)-treated NOD and NOD.Ncf1m1J BM-Mϕ for 24 hours. Membranes were probed for TLR3, TRIF or β-actin as a loading control (C). Densitometry graph of TLR3 and TRIF protein expression as a ratio with β-actin with stimulated NOD BM-Mϕ arbitrarily set as 1. The results are representative of three independent experiments.
Figure 7
Figure 7. Impaired NF-κB activation in NOD.Ncf1m1J BM-Mϕ after poly(I:C)-stimulation
(A) Representative Western blot of phospho-IκB-α (S32/S36) and total IκB-α from NOD and NOD.Ncf1m1J BM-Mϕ untreated or treated with 25 μg/ml of poly(I:C) for 30′. (B) Densitometry bar graph of the average of 3 independent experiments of phospho-IκB-α (S32/S36) expression in poly(I:C)-stimulated NOD and NOD.Ncf1m1J BM-Mϕ. The phospho-IκB-α (S32/S36) expression level in stimulated NOD BM-Mϕ was arbitrarily set as 1. (C) Representative Western blot of NF-κB p50 and actin from NOD and NOD.Ncf1m1J BM-Mϕ nuclear extracts after 25 μg/ml poly(I:C) treatment for 60′. (D) Densitometry bar graph of the average of 3 independent experiments of NF-κB p50 expression in poly(I:C)-stimulated NOD and NOD.Ncf1m1J BM-Mϕ. NF-κB p50 expression level in stimulated NOD BM-Mϕ was arbitrarily set as 1. The results are representative of three independent experiments. * p < 0.05 versus poly(I:C)-stimulated NOD BM-Mϕ.
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Comment in

  • Radical innate regulation of autoimmune diabetes.
    Ostanin DV, Kevil CG. Ostanin DV, et al. Free Radic Biol Med. 2012 May 1;52(9):1698-9. doi: 10.1016/j.freeradbiomed.2012.02.024. Epub 2012 Feb 26. Free Radic Biol Med. 2012. PMID: 22373596 Free PMC article. No abstract available.

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