IKKβ is required for the formation of the NLRP3 inflammasome

The rapid formation and activation of the NLRP3 inflammasome is induced by co‐stimulation with LPS and nigericin. It requires the LPS‐stimulated activation of IKKβ, which exerts its effects independently of de novo gene transcription, protein translation and other protein kinases activated by IKKβ. IKKβ is not required for the nigericin‐induced dispersion of the trans‐Golgi network (TGN), but to bring NLRP3 in proximity with TGN38. The nigericin‐induced dispersion of the Golgi is enhanced by co‐stimulation with LPS, and this enhancement is IKKβ‐dependent. Prolonged stimulation with LPS to increase the expression of NLRP3, followed by stimulation with nigericin, produced larger TGN38‐positive puncta, and the ensuing activation of the NLRP3 inflammasome was also suppressed by IKKβ inhibitors added prior to stimulation with nigericin. IKKβ therefore has a key role in recruiting NLRP3 to the dispersed TGN, leading to the formation and activation of the NLRP3 inflammasome

An important role for A20-binding inhibitor of nuclear factor-kB-1 (ABIN1) in inflammation-mediated endothelial dysfunction::an <i>in vivo</i> study in ABIN1 (D485N) mice

INTRODUCTION: The link between cardiovascular disease (CVD) and patients with chronic inflammation is not clearly understood. We examined a knock-in mouse expressing a poly-ubiquitin-binding-defective mutant of the protein ABIN1 (ABIN1(D485N)), which develops a systemic lupus erythematosus-like autoimmune disease because of the hyperactivation of IκB kinases (IκKs) and mitogen-activated protein kinases (MAPKs). These mice were used to determine the potential role of these signaling pathways in inflammation-mediated CVD development. METHODS: Laser Doppler imaging in combination with the iontophoresis of vasoactive chemicals were used to assess endothelium-dependent vasodilatation in vivo in ABIN1 (D485N)) mutant defective (n = 29) and wild-type (WT) control (n = 26) mice. Measurements were made at baseline, and animals were subdivided to receive either chow or a proatherogenic diet for 4 weeks, after which, follow-up assessments were made. Paired and unpaired t tests, and ANOVA with post hoc Bonferroni correction were used for statistical significance at P <0.05. RESULTS: Endothelium-dependent vasodilatation to acetylcholine was attenuated at 4 weeks in ABIN1(D485N)-chow-fed mice compared with age-matched WT-chow-fed mice (P <0.05). The magnitude of attenuation was similar to that observed in WT-cholesterol-fed animals (versus WT-chow, P <0.01). ABIN1(D485N)-cholesterol-fed mice had the poorest endothelium-dependent responses compared with other groups (P <0.001). ABIN1(D485N)-chow-fed mice had increased plasma interleukin-6 (IL-6) levels (versus WT-chow, P <0.001), and this was further elevated in ABIN1(D485N)-cholesterol-fed mice (versus ABIN1(D485N)-chow; P <0.05). IL-1α was significantly greater in all groups compared with WT-chow (P <0.01). ABIN1(D485N) mice showed significant cardiac hypertrophy (P <0.05). CONCLUSIONS: The ABIN(D485N) mice display endothelial dysfunction and cardiac hypertrophy, which is possibly mediated through IL-6 and, to a lesser degree, IL-1α. These results suggest that the ABIN1-mediated hyperactivation of IKKs and MAPKs might mediate chronic inflammation and CVD development

Why are the phenotypes of TRAF6 knock-in and TRAF6 knock-out mice so different?

The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4(+) and CD8(+) T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44(high)/CD62L(low) expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44(low)/CD62L(high) phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2–3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice

Suppression of IRAK1 or IRAK4 catalytic activity, but not type1 IFN signaling, prevents lupus nephritis in mice expressing a ubiquitin binding-defective mutant of ABIN1

Polymorphisms in the TNIP1 gene encoding A20-binding inhibitor of NF-κB1 (ABIN1) predispose to lupus and other autoimmune diseases in at least eight different human populations. We found previously that knock-in mice expressing a ubiquitin-binding-defective mutant of ABIN1 (ABIN1[D485N]) develop autoimmunity as they age and succumb to a disease resembling lupus nephritis in humans. Here we report that Flt3-derived dendritic cells from these mice overproduced Type1 IFNs upon stimulation with ligands that activate TLR7 or TLR9. However, crossing the ABIN1[D485N] mice to IFNAR1 knock-out mice that do not express the α-subunit of the Type1 IFN receptor, did not prevent splenomegaly, the appearance of high serum levels of auto-antibodies and other Igs, or liver inflammation, and only reduced kidney inflammation modestly. In contrast, crossing the ABIN1[D485N] mice to knock-in mice expressing catalytically inactive mutants of IRAK1 or IRAK4 prevented splenomegaly, autoimmunity, liver and kidney inflammation. Our results support the notion that IRAK1 and/or IRAK4 are attractive targets for the development of drugs to prevent, and perhaps treat, lupus nephritis and other auto-inflammatory diseases caused by decreased ability of ABIN1 or other proteins to restrict the strength of MyD88 signaling

Prevention and partial reversion of the lupus phenotype in ABIN1[D485N] mice by an IRAK4 inhibitor

OBJECTIVE: We have reported previously that the IRAK4 inhibitor PF06426779 given to ubiquitin-binding-defective ABIN1[D485N] mice at 6 weeks of age prevents the major facets of lupus that develop 10 weeks later. The present study was undertaken to investigate whether PF06426779 could reverse the lupus phenotype when administered to 13-week-old ABIN1[D485N] mice that had already developed symptoms of lupus. METHODS: Splenomegaly, the number of splenic neutrophils, T(FH) and Germinal Centre B (GCB) cells, serum levels of immunoglobulins, the extent of kidney, liver and lung pathology, and glomerular IgA and IgM were measured after feeding 13-week-old ABIN1[D485N] and wild-type mice for another 10 weeks with R&M3 diet with and without PF06426779 (4 g/kg). RESULTS: Following drug treatment, spleen size and weight, splenic neutrophil numbers, and serum IgA and glomerular IgA levels of ABIN1[D485N] mice returned to those seen in wild-type mice. The rise in splenic T(FH) and GCB numbers, the increase in kidney and liver pathology, and the concentrations of serum IgG1, IgG2A and IgE between 13 and 23 weeks were suppressed. There was no reduction in the level of anti-self double-stranded DNA, anti-self nuclear antigens or IgM during the drug treatment. CONCLUSIONS: The results demonstrate the therapeutic potential of IRAK4 inhibitors for the treatment of lupus and raise the possibility of monitoring efficacy by measuring decreases in the serum levels of IgA. Our results support the view that there may be a closer connection between lupus and IgA nephropathy than realised previously

Distinct signals and immune cells drive liver pathology and glomerulonephritis in ABIN1[D485N] mice

We report that TLR7, IL-6, and the adaptive immune system are essential for autoimmunity and glomerulonephritis but not for liver pathology in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant. The blood and organs of ABIN1[D485N] mice have exceptionally high numbers of patrolling monocytes (pMo), which develop independently of IL-6 and the adaptive immune system. They are detectable in the blood months before autoimmunity and organ pathology are seen and may have diagnostic potential. The splenic pMo, inflammatory monocytes (iMo), and neutrophils of ABIN1[D485N] mice expressed high levels of mRNAs encoding proteins released during NETosis, which together with the high numbers of monocyte-derived dendritic cells (MoDCs) may drive the liver pathology in ABIN1[D485N] mice, and contribute to the pathology of other organs. The splenic iMo of ABIN1[D485N] mice displayed high expression of mRNAs encoding proteins controlling cell division and were actively dividing; this may underlie the increased pMo and MoDC numbers, which are derived from iMo. An orally active IRAK4 inhibitor suppressed all facets of the disease phenotype and prevented the increase in pMo numbers

HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan