β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase,UCH-L1

We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.     

Autophagy Modulates Borrelia burgdorferi-induced Production of Interleukin-1β (IL-1β)

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease. Recent studies have shown that recognition of the spirochete is mediated by TLR2 and NOD2. The latter receptor has been associated with the induction of the intracellular degradation process called autophagy. The present study demonstrated for the first time the induction of autophagy by exposure to B. burgdorferi and that autophagy modulates the B. burgdorferi-dependent cytokine production. Human peripheral blood mononuclear cells treated with autophagy inhibitors showed an increased IL-1β and IL-6 production in response to the exposure of the spirochete, whereas TNFα production was unchanged. Autophagy induction against B. burgdorferi was dependent on reactive oxygen species (ROS) because cells from patients with chronic granulomatous disease, which are defective in ROS production, also produced elevated IL-1β. Further, the enhanced production of the proinflammatory cytokines was because of the elevated mRNA expression in the absence of autophagy. Our results thus demonstrate the induction of autophagy, which, in turn, modulates cytokine production by B. burgdorferi for the first time.     

Interleukin-1β (187–207)-Induced Hyperthermia is Inhibited by Interleukin-1β (193–195) in Rats

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine, which plays an important role in the immune response and signal transduction both in the periphery and the central nervous system (CNS). Various diseases of the CNS, including neurodegenerative disorders, vascular lesions, meningo-encephalitis or status epilepticus are accompanied by elevated levels of IL-1β. Different domains within the IL-lβ protein are responsible for distinct functions. The IL-lβ domain in position 208–240 has pyrogenic properties, while the domain in position 193–195 exerts anti-inflammatory effects. Previous studies provide little evidence about the effect of the domain in position 187–207 on the body temperature. Therefore, the aim of the present study was to investigate the action of IL-1β (187–207) and its interaction with IL-1β (193–195) on the body temperature. IL fragments were administered intracerebroventricularly and the body temperature was measured rectally in male Wistar rats. IL-1β (187–207) induced hyperthermia, while IL-1β (193–195) did not influence the core temperature considerably. In co-administration, IL-1β (193–195) completely abolished the IL-1β (187–207)-induced hyperthermia. The non-steroid anti-inflammatory drug metamizole also reversed completely the action of IL-1β (187–207). Our results provide evidence that the IL-lβ domain in position 187–207 has hyperthermic effect. This effect is mediated through prostaglandin E2 stimulation and other mechanisms may also be involved in the action of IL-1β (187–207). It also suggests that IL-lβ domain in position 187–207 and IL-1β (193–195) fragment may serve as novel target for treatment of disorders accompanied with hyperthermia.     

Oligomeric Amyloid-β Inhibits the Proteolytic Conversion of Brain-derived Neurotrophic Factor (BDNF), AMPA Receptor Trafficking,and Classical Conditioning

Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.     

Porphyromonas gingivalis culture supernatants differentially regulate Interleukin-1β and Interleukin-18 in human monocytic cells

Porphyromonas gingivalis is a major bacterial species implicated in chornic periodontitis, a disease characterized by inflammatory destruction of the tooth supporting tissues. Its main virulence factors are lipopolysaccharide (LPS) and gingipains, a group of cysteine proteinases. Interleukin (IL)-18 is a potent pro-inflammatory cytokine with structural similarities to IL-1β. This study aimed to investigate if P .gingivalis regulates IL-1β and IL-18 in monocytic cells. Monomac-6 cells were challenged with P. gingivalis culture supernatants. Quantitative real-time PCR and ELISA were used to investigate IL-1β and IL-18 mRNA expression and protein secretion, respectively. P. gingivalis enhanced IL-1β and IL-18 mRNA expression, the former being induced earlier, but transiently. IL-18 up-regulation was not affected by P. gingivalis heat-inactivation or chemical inhibition of its gingipains, whereas both treatments resulted in 50% reduction of IL-1β expression. Purified P. gingivalis LPS enhanced both IL-1β and IL-18 expression. However, only IL-1β, but not IL-18, secretion was detected, and was up-regulated by P. gingivalis. In conclusion, although IL-1β and IL-18 belong to the same cytokine family, their gene expression and secretion are differentially regulated in human monocytic cells in response to P. gingivalis. Therefore, cytokines of the IL-1 family may participate via different pathways in the complex pathogenesis of periodontitis.     

Interleukin-1β and mitochondria damage, and the development of diabetic retinopathy

Mitochondrial dysfunction is considered to play an important role in the development of diabetic retinopathy. Recent evidence has also shown many similarities between diabetic retinopathy and a low grade chronic inflammatory disease. The aim of this study is to understand the interrelationship between proinflammtory mediator, IL-1β and mitochondrial dysfunction in the accelerated loss of capillary cells in the retina. Using IL-1β receptor gene knockout (IL-1R1^?/^?) diabetic mice, we have investigated the effect of regulation of IL-1β on mitochondrial dysfunction and mtDNA damage, and increased retinal capillary cell apoptosis and the development of retinopathy. Retinal mitochondrial dysfunction and mtDNA damage were significantly ameliorated in IL-1R1^?/^? mice, diabetic for ~10 months, compared to the wild-type diabetic mice. This was accompanied by protection of accelerated capillary cell apoptosis and the development of acellular capillaries, histopathology associated with diabetic retinopathy. Thus, mitochondrial damage could be one of the key events via which increased inflammation contributes to the activation of the apoptotic machinery resulting in the development of diabetic retinopathy, and the possible mechanism via which inflammation contributes to the development of diabetic retinopathy includes continuous fueling of the vicious cycle of mitochondrial damage, which could be disrupted by inhibitors of inflammatory mediators.     

Stromal Cell-Derived Factor-1β Mediates Cell Survival through Enhancing Autophagy in Bone Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) hold great potential for cell-based therapy, yet the therapeutic efficacy remains uncertain. Transplanted BMSCs often fail to engraft within the bone marrow (BM), in part due to the poor survival of donor cells in response to inflammatory reactions, hypoxia, oxidative stress, or nutrient starvation. Two basic cell processes, apoptosis and autophagy, could potentially be responsible for the impaired survival of transplanted BMSCs. However, the functional relationship between apoptosis and autophagy in BMSC homeostasis is complex and not well understood. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling axis appears to be critical in maintaining proliferation and survival of BM stem cell populations through improving cell proliferation and survival in response to stress; however, the exact mechanisms remain unclear. We recently described novel genetically engineered Tet-Off-SDF-1β BMSCs, which over-express SDF-1β under tight doxycycline-control, thus providing an ideal model system to investigate the isolated effects of SDF-1β. In this study we tested the hypothesis that SDF-1β can mediate cell survival of BMSCs in vitro through increasing autophagy. We found that SDF-1β had no effect on BMSC proliferation; however, SDF-1β significantly protected genetically engineered BMSCs from H₂O₂-induced cell death through increasing autophagy and decreasing caspase-3-dependent apoptosis. Taken together, we provide novel evidence that the SDF-1/CXCR4 axis, specifically activated by the SDF-1β isoform, plays a critical role in regulating BMSC survival under oxidative stress through increasing autophagy.     

Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.     

Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1β, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1β. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1β processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.     

Induction of Autophagy by a Novel Small Molecule Improves Aβ Pathology and Ameliorates Cognitive Deficits

Growing evidence has demonstrated a neuroprotective role of autophagy in Alzheimer’s disease (AD). Thus, autophagy has been regarded as a potential therapeutic target, attracting increasing interest in pharmaceutical autophagy modulation by small molecules. We designed a two-cycle screening strategy on the basis of imaging high-throughout screening (HTS) and cellular toxicity assay, and have identified a novel autophagy inducer known as GTM-1. We further showed that GTM-1 exhibits dual activities, such as autophagy induction and antagonism against Aβ-oligomer toxicity. GTM-1 modulates autophagy in an Akt-independent and mTOR-independent manner. In addition, we demonstrated that GTM-1 enhances autophagy clearance and reverses the downregulation of autophagy flux by thapsigargin and asparagine. Furthermore, administration of GTM-1 attenuated Aβ pathology and ameliorated cognitive deficits in AD mice.     

Selective Attachment of Oligonucleotides to Interleukin-1β and Targeted Delivery to Cells

Abstract

Conjugates between oligodeoxyribonucleotides and an interleukin-1β mutant protein have been constructed using a heterobifunctional cross-linker. These protein-DNA conjugates had conserved binding activity to the interleukin-1 receptor. The oligonucleotide hybridization properties were unchanged.     

Molecular Characterization of Pro-Inflammatory Cytokines Interleukin-1β and Interleukin-8 in Asian Elephant (Elephas maximus)

Interleukin (IL)-1β and IL-8 are pro-inflammatory cytokines produced primarily by monocytes and macrophages in response to a variety of microbial and nonmicrobial agents. As yet, no molecular data have been reported for IL-1β and IL-8 of the Asian elephant. In the present study, we have cloned and sequenced the cDNA encoding IL-1β and IL-8 of the Asian elephant. The open reading frame (ORF) of Asian elephant IL-1β is 789 bp in length, encoded a propeptide of 263 amino acid polypeptide. The predicted protein revealed the presence of IL-1 family signature motif and an ICE cut site. Whereas, IL-8 contained 321 bp of open reading frame. Interestingly, the predicted protein sequence of 106 aa, contains an ELR motif immediately upstream of the CQC residues, common in all vertebrate IL-8 molecules. Identity levels of the nucleic acid and deduced amino acid sequences of Asian elephant IL-1β ranged from 68.48 (Squirrel monkey) to 98.57% (African elephant), and 57.78 (Sheep) to 98.47% (African elephant), respectively, whereas that of IL-8 ranged from 72.9% (Human) to 87.8% (African elephant), and 63.2 (human, gorilla, chimpanzee) to 74.5% (African elephant, buffalo), respectively. The phylogenetic analysis based on deduced amino acid sequenced showed that the Asian elephant IL-1β and IL-8 were most closely related to African elephant. Molecular characterization of these two cytokines, IL-1β and IL-8, in Asian elephant provides fundamental information necessary to progress the study of functional immune responses in this animal and gives the potential to use them to manipulate the immune response as recombinant proteins.     

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background

Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.

Methods and Results

IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.

Conclusions

Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.     

Autophagy suppresses interleukin-1β (IL-1β) signaling by activation of p62 degradation via lysosomal and proteasomal pathways

ATG16L1 is an essential component of the autophagasome. The T300A allele of ATG16L1 is associated with the increased susceptibility to Crohn disease. In this study, we identified a novel function of ATG16L1, which suppresses signaling of the pro-inflammatory cytokine IL-1β. Deletion of ATG16L1 in mouse embryonic fibroblasts significantly amplifies IL-1β signal transduction cascades. This amplification is due to elevated p62 levels in ATG16L1-deficient cells. We found that ATG16L1 regulates p62 levels via both autolysosomal and proteasomal pathways. For proteasomal degradation, we found that Cullin-3 (Cul-3) is a E3 ubiquitin ligase of p62 and that ATG16L1 is essential for neddylation of Cul-3, a step required for Cul-3 activation. Taken together our data indicate that loss-of-function of ATG16L1 results in a hyper-responsiveness to the IL-1β signaling because of the increased p62 level.