bafilomycin-a1 and Inflammation

The release of interleukin (IL)-1β from primary human monocytes in response to extracellular LPS occurs through the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. In primary monocytes, in response to LPS, NLRP3 inflammasome activation is characterized by an independence of K

Topics: Caspase 1; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydrogen-Ion Concentration; Inflammasomes; Inflammation; Interleukin-1beta; Lipopolysaccharides; Lysosomes; Macrolides; Monocytes; Nigericin; NLR Family, Pyrin Domain-Containing 3 Protein; Primary Cell Culture; Proton-Translocating ATPases; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; THP-1 Cells

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Topics: Animals; Autophagy; Azepines; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Inflammasomes; Inflammation; Intervertebral Disc Degeneration; Macrolides; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Nuclear Proteins; Nucleus Pulposus; Rats; Signal Transduction; Transcription Factor RelA; Transcription Factors; Triazoles

Damage-associated molecular pattern molecules (DAMPs) are endogenous danger signals that alert the innate immune system and shape the inflammation response to cell death. However, the release and activity of DAMPs in ferroptosis, a recently identified form of regulated necrosis characterized by iron overload and lipid peroxidation, still remain poorly understood. Here, we demonstrate that HMGB1 is a DAMP released by ferroptotic cells in an autophagy-dependent manner. Both type I and II ferroptosis activators, including erastin, sorafenib, RSL3, and FIN56, induce HMGB1 release in cancer and noncancer cells. In contrast, genetic ablation (using ATG5

Topics: Animals; Autophagy; Carbolines; Cell Death; Cell Line, Tumor; Chloroquine; Ferritins; Fibroblasts; HMGB1 Protein; Humans; Immunity, Innate; Inflammation; Iron Overload; Lipid Peroxidation; Macrolides; Mice; Neoplasms; Oximes; Piperazines; Sorafenib; Sulfonamides; Toll-Like Receptor 4

Major depression is a multifactorial disease. Emerging evidence has suggested that autophagy is involved in the pathological process of depressive disorders. Bafilomycin A1 (Baf A1), is an inhibitor of vacuolar H+‑ATPase that is frequently used at high concentrations to block late‑phase autophagy. However, whether Baf A1 has antidepressant effects remains to be elucidated. The current study aimed to evaluate the antidepressant effects of Baf A1 in rats with chronic unpredictable mild stress (CUMS) and its potential mechanism. The CUMS animal model was established. The sucrose preference test, open‑field test (OFT) and forced swim test (FST) were applied to evaluate the depressive behavior. Synaptic plasticity‑associated proteins synaptophysin and postsynaptic density protein 95 were measured by western blotting and immunofluorescence. Apoptosis‑ and autophagy‑associated proteins in addition to pro‑inflammatory cytokines, including interleukin‑1β and tumor necrosis factor‑α, were detected by western blotting, reverse transcription‑quantitative polymerase chain reaction or ELISA. A 4‑week treatment period with Baf A1 markedly ameliorated CUMS‑induced behavioral abnormalities, including increasing sucrose intake, improving locomotor activity in the OFT, and decreasing immobility time in the FST. In addition, treatment with Baf A1 restored the dysregulation of synaptic plasticity and inhibited neuroinflammation in rats exposed to CUMS. Furthermore, Baf A1 decreased the levels of apoptosis‑ and autophagy‑associated proteins induced by CUMS. The present study demonstrated that Bafilomycin A1 resulted in antidepressant effects in rats, which may be mediated by the reversal of apoptosis, autophagy and neuroinflammation in the hippocampus.

Topics: Animals; Antidepressive Agents; Apoptosis; Autophagy; Depression; Disease Models, Animal; Hippocampus; Humans; Inflammation; Macrolides; Neuronal Plasticity; Rats; Stress, Psychological; Swimming; Vacuolar Proton-Translocating ATPases

Co ions released due to corrosion of Co nanoparticles (CoNPs) in the lysosomes of macrophages may be a factor in the particle-induced cytotoxicity and aseptic inflammation accompanying metal-on-metal (MOM) hip prosthesis failure. Here, we show that CoNPs are easily dissolved under a low pH, simulating the acidic lysosomal environment. We then used bafilomycin A1 to change the pH inside the lysosome to inhibit intracellular corrosion of CoNPs and then investigated its protective effects against CoNP-induced cytotoxicity and aseptic inflammation on murine macrophage RAW264.7 cells. XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} assays revealed that bafilomycin A1 can significantly decrease CoNP-induced cytotoxicity in RAW264.7 cells. Enzyme-linked immunosorbent assays showed that bafilomycin A1 can significantly decrease the subtoxic concentration of CoNP-induced levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), but has no effect on anti-inflammatory cytokines (transforming growth factor-β and interleukin-10) in RAW264.7 cells. We studied the protective mechanism of bafilomycin A1 against CoNP-induced effects in RAW264.7 cells by measuring glutathione/oxidized glutathione (GSH/GSSG), superoxide dismutase, catalase, and glutathione peroxidase levels and employed scanning electron microscopy, transmission electron microscopy, and energy dispersive spectrometer assays to observe the ultrastructural cellular changes. The changes associated with apoptosis were assessed by examining the pAKT and cleaved caspase-3 levels using Western blotting. These data strongly suggested that bafilomycin A1 can potentially suppress CoNP-induced cytotoxicity and aseptic inflammation by inhibiting intracellular corrosion of CoNPs and that the reduction in Co ions released from CoNPs may play an important role in downregulating oxidative stress in RAW264.7 cells.

Topics: Animals; Apoptosis; Cell Line; Cobalt; Inflammation; Interleukin-1beta; Interleukin-6; Macrolides; Macrophages; Mice; Nanoparticles; Tumor Necrosis Factor-alpha

Obesity-induced insulin resistance and diabetes are significantly associated with infiltrates of inflammatory cells in adipose tissue. Previous studies recognized the involvement of autophagy in the regulation of metabolism in multiple tissues, including β-cells, hepatocytes, myocytes, and adipocytes. However, despite the importance of macrophages in obesity-induced insulin resistance, the role of macrophage autophagy in regulating insulin sensitivity is seldom addressed. In the present study, we show that macrophage autophagy is important for the regulation of systemic insulin sensitivity. We found that macrophage autophagy is downregulated by both acute and chronic inflammatory stimuli, and blockade of autophagy significantly increased accumulation of reactive oxygen species (ROS) in macrophages. Macrophage-specific Atg7 knockout mice displayed a shift in the proportion to pro-inflammatory M1 macrophages and impairment of insulin sensitivity and glucose homeostasis under high-fat diet conditions. Furthermore, inhibition of ROS in macrophages with antioxidant recovered adipocyte insulin sensitivity. Our results provide evidence of the underlying mechanism of how macrophage autophagy regulates inflammation and insulin sensitivity. We anticipate our findings will serve as a basis for development of therapeutics for inflammatory diseases, including diabetes.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue; Animals; Autophagy; Autophagy-Related Protein 7; Diet, High-Fat; Disease Models, Animal; Enzyme Inhibitors; Glucose; Inflammation; Insulin; Insulin Resistance; Macrolides; Macrophages; Male; Mice; Mice, Knockout; Obesity; RAW 264.7 Cells; Reactive Oxygen Species

Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure.

Topics: Adenine; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Beclin-1; Bronchi; Cell Line, Tumor; Cell Transformation, Neoplastic; Epithelial Cells; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Macrolides; Male; Mice, Inbred C57BL; Nickel; Sequestosome-1 Protein; Signal Transduction; TOR Serine-Threonine Kinases; Transcription, Genetic; Tumor Necrosis Factor-alpha; Up-Regulation

A hallmark feature of cystic fibrosis (CF) is progressive pulmonary obstruction arising from exaggerated host proinflammatory responses to chronic bacterial airway colonization. The mechanisms for these heightened inflammatory responses have been only partially characterized, hampering development of effective anti-inflammatory therapies. The aim of this study was to identify and validate novel dysfunctional processes or pathways driving the hyperinflammatory phenotype of CF cells using systems biology and network analysis to examine transcriptional changes induced by innate defense regulator (IDR)-1018, an anti-inflammatory peptide. IDR-1018 selectively attenuated hyperinflammatory cytokine production from CF airway cells and PBMCs stimulated with multiple bacterial ligands, including flagellin (FliC). Network analysis of CF cell transcriptional responses to FliC and IDR-1018 identified dysfunctional autophagy as the target of the peptide via modulation of upstream adenosine monophosphate-activated protein kinase (AMPK)-Akt signaling. After treatment with FliC, CF cells were found to have elevated levels of the autophagosome marker LC3-II, and GFP-LC3-transfected CF airway cells showed abnormal perinuclear accumulation of GFP(+) structures. In both instances, treatment of CF cells with IDR-1018 abolished the accumulation of LC3 induced by FliC. Furthermore, inhibition of autophagosome-lysosome fusion with bafilomycinA1 attenuated the anti-inflammatory and autophagosome-clearing effects of IDR-1018, as did a chemical inhibitor of Akt and an activator of AMPK. These findings were consistent with hypotheses generated in silico, demonstrating the utility of systems biology and network analysis approaches for providing pathway-level insights into CF-associated inflammation. Collectively, these data suggest that dysfunctional autophagosome clearance contributes to heightened inflammatory responses from CF transmembrane receptor mutant cells and highlight autophagy and AMPK-Akt signaling as novel anti-inflammatory targets in CF.

Topics: AMP-Activated Protein Kinase Kinases; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Autophagy; Cell Line; Child; Cystic Fibrosis; Cytokines; Epithelial Cells; Escherichia coli Proteins; Flagellin; Gene Expression Regulation; Humans; Inflammation; Leukocytes, Mononuclear; Lung; Lysosomes; Macrolides; Microtubule-Associated Proteins; Models, Immunological; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Systems Biology; Transcription, Genetic

Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.

Topics: Caco-2 Cells; Cell Line, Tumor; Chemokines; Culture Media; Cytokines; Dendritic Cells; Endosomes; Epithelial Cells; HIV; HIV Infections; Humans; Inflammation; Interferons; Macrolides; Phenotype

Bone disorders with increased osteoclastic bone resorption are frequently associated with bone pain and inhibitors of osteoclasts reduce bone pain. Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, creating acidic microenvironments. Because acidosis is a well-known cause of pain, we reasoned that osteoclasts cause pain through proton secretion. We explored this using an animal model in which a single subcutaneous injection of the complete Freund's adjuvant (CFA) in the hind-paw caused inflammatory hyperalgesia (hyper-responsiveness to noxious stimuli). Osteoclastic bone resorption was increased in the metatarsal bones in the CFA-injected hind-paws. CFA-induced hyperalgesia was significantly suppressed by the bisphosphonates, zoledronic acid (ZOL) and alendronate and osteoprotegerin. c-src-deficient mice in which osteoclasts are inherently dysfunctional exhibited reduced CFA-induced hyperalgesia. Repeated subcutaneous injections of parathyroid hormone-related protein into the hind-paw also induced hyperalgesia with increased osteoclastic bone resorption. The hyperalgesia was associated with increased mRNA expression of acid-sensing ion channel (ASIC) 1a, 1b and 3 in the ipsi-lateral dorsal root ganglions (DRGs) by RT-PCR and c-Fos in the ipsi-lateral spinal dorsal horn by immunohistochemistry. Of note, ZOL decreased the ASIC1a mRNA expression and c-Fos. Treatment of the DRG cell line F-11 with acid (pH5.5) increased ASIC1a, 1b and 3 mRNA expression and nuclear c-Fos expression. The ASIC blocker amiloride inhibited acid-induced c-Fos expression in F-11 cells. Moreover, F-11 cells transfected with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) showed increased acid-induced nuclear c-Fos expression compared with parental F-11 cells. Finally, bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, reversed the hyperalgesia and down-regulated ASIC1a mRNA expression in the DRGs. These results led us to propose that osteoclasts play a part in CFA-induced inflammatory pain through an activation of the acid-sensing receptors including ASICs and TRPV1 by creating acidosis.

Topics: Acid Sensing Ion Channels; Animals; Bone and Bones; Bone Resorption; Cell Line; Diphosphonates; Freund's Adjuvant; Genes, src; Hyperalgesia; Inflammation; Injections, Intravenous; Injections, Spinal; Injections, Subcutaneous; Macrolides; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Osteoclasts; Osteoprotegerin; Pain; Parathyroid Hormone; Posterior Horn Cells; Reverse Transcriptase Polymerase Chain Reaction; Sodium Channels; TRPV Cation Channels; Vacuolar Proton-Translocating ATPases