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

Abstract

In Type 1 diabetes (T1D), reactive oxygen species (ROS) and pro-inflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic β-cells while promoting autoreactive T cell maturation. Superoxide-deficient Non-Obese Diabetic mice (NOD.Ncf1m1J) 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.Ncf1m1J 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.Ncf1m1J BM-Mϕ exhibited a 2- and 10-fold decrease in TNF-α and IFN-β pro-inflammatory cytokine synthesis, respectively, in contrast to NOD BM-Mϕ. Optimal expression of IFN-α/β is not solely dependent on superoxide synthesis, but requires p47phox to function in a NOX-independent manner to mediate Type I interferon synthesis. Interestingly, MHC-II I-Ag7 expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-Ag7 down-regulation. These findings suggest that defective innate immune pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-β pro-inflammatory cytokine synthesis necessary for autoreactive T cell maturation, may contribute to the T1D protection observed in NOD.Ncf1m1J mice.

TLR3-Triggered Reactive Oxygen Species Contribute to Inflammatory Responses by Activating Signal Transducer and Activator of Transcription-1

“…phox KO mice as compared with those from WT mice (28). Additionally, it was shown that dual oxidase 1 is involved in TLR ligand-induced IL-8 and VEGF production in airway epithelial cells (44).…”

“…TLR3 signaling activates the transcription factors NF-kB and IRF-3, inducing production of the proinflammatory cytokines TNF-a and IFN-b, respectively (27,28). We next examined whether ROS generation is involved in the nuclear translocation and activation of NF-kB and IRF-3 after stimulation with poly(I:C).…”

Loss of NADPH Oxidase–Derived Superoxide Skews Macrophage Phenotypes to Delay Type 1 Diabetes

“…Relative gene expression levels were calculated using the 2 −ΔΔCt method; Gapdh or Emr1 was set to 1. Serum levels of chemokine (C-C motif) ligand (CCL) 5 were quantified using a mouse Cytokine/Chemokine Magnetic Bead Panel Immunoassay (Merck Millipore), while CCL17 production was measured using a DuoSet ELISA Kit (R&D Systems) (18). For Western blotting, whole-cell lysates were probed with antibodies against iNOS (Novus Biologicals), Arg-1, STAT6 (Cell Signaling), P-STAT6 (Y641) (BD Bioscience), or β-actin (Sigma-Aldrich) as described (18).…”

“…Ncf1 m1J bone marrow–derived macrophages (BM-Mϕs) were generated (18) and polarized to an M1 phenotype, by treating them with 20 ng/mL r-mIFN-γ (PeproTech) and 100 ng/mL LPS (Sigma), or to an M2 phenotype by treating them with 20 ng/mL r-mIL-4 (PeproTech) (16). Nitrite production was measured by Griess assay (23), while IL-1β, TNF-α, IL-12p70, CCL5, and CCL17 were measured using a DuoSet ELISA kit (R&D Systems).…”

“…We recently demonstrated that NOX-deficient NOD (NOD. Ncf1 m1J ) mice were protected against autoimmune T1D (11,17) due, in part, to decreased activation of Toll-like receptor 3 and Toll-like receptor 4 signaling pathways in macrophages, and, subsequently, a robust diminution in IL-12p70, TNF-α, IL-1β, and type I IFN synthesis (11,17,18). Because oxidative stress is tightly linked with inflammation (19,20), and ROS synthesis and inducible nitric oxide synthase (iNOS) are characteristic M1 markers (1), we investigated the role of NOX-derived superoxide as a contributing proinflammatory molecule involved in M1 macrophage differentiation in T1D.…”