Abstract 197: Emerging Role of Toll-like Receptor 4 and Heat Shock Protein 60&70 in Ischaemia-Induced Skeletal Muscle Damage In Vitro

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Ali Navi ◽  
Rebekah Yu ◽  
Xu Shi-Wen ◽  
Sidney Shaw ◽  
Daryll Baker ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Inflammatory Cytokine ◽  
Caspase 3 ◽  
Neutralizing Antibody ◽  
Cytokine Release ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Skeletal Muscle Damage ◽  
Tnf Α

OBJECTIVES The innate immune response contributes to the skeletal muscle damage in patients with critical limb ischaemia (CLI); however, the detailed signaling mechanisms are not fully understood. We hypothesized that simulated ischaemia induces inflammatory cytokine release from skeletal myotubes, via a mechanism that involves heat shock protein (HSP) 60&70, known endogenous ligands of Toll-like receptor 4 (TLR4), in vitro. METHODS Human gastrocnemius muscle biopsies were taken from patients with CLI undergoing major lower limb amputation and from patients with no peripheral arterial disease (PAD). Human myoblasts were isolated, cultured to myotubes and then pre-treated with TLR4 neutralizing antibody prior to exposure to simulated ischaemia. Fluorescent immunostaining was carried out to confirm cell differentiation; ELISA analysis were carried out to quantify IL6 and TNF-α release; and Western blot was used to assess expression of HSP60&70, TLR4 and cleaved caspase-3 as a marker of apoptosis. RESULTS Myotubes from patients with CLI expressed greater levels of cleaved caspase-3 and TLR4 as compared to those from patients with no PAD. When exposed to ischaemic conditions, increased IL6 and TNF-α release and upregulation of HSP60&70, cleaved caspase-3 and TLR4 were observed in myotubes from both groups of patients compared to culturing in normoxic conditions (P<0.05). Pre-treatment of myotubes from patients with CLI with TLR4 neutralizing antibody prior to simulated ischaemia was associated with reduced expression of HSP60&70, IL6, TNF-α and cleaved caspase-3 (P<0.05). CONCLUSIONS Increased cytokine release, apoptosis and expression of HSP60&70 and TLR4 occur in ischaemic skeletal muscle in vitro. TLR4 antagonism was associated with reduced apoptosis and inflammatory cytokine release and down-regulation of HSP60&70 expression. This suggests a potential pathway where TLR4 and its endogenous ligands contribute to a positive feedback loop to maintain a proinflammatory environment during ischaemia.

scholarly journals Detrimental Role of Heat Shock Protein 60&70 and Toll-like Receptor 4 in Skeletal Muscle Ischaemia In Vitro

2013 ◽  
Vol 57 (5) ◽  
pp. 77S
Author(s):  
Ali Navi ◽  
Rebekah Yu ◽  
Xu Shi-Wen ◽  
Sidney Shaw ◽  
George Hamilton ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 60 ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Muscle Ischaemia

scholarly journals TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells

2016 ◽  
Vol 38 (6) ◽  
pp. 2139-2151 ◽  
Author(s):  
Won Seok Yang ◽  
Nam Jeong Han ◽  
Jin Ju Kim ◽  
Mee Jeong Lee ◽  
Su-Kil Park
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
High Mobility ◽  
Neutralizing Antibody ◽  
High Mobility Group ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Aortic Endothelial Cells ◽  
Tnf Α ◽  
Human Aortic Endothelial Cells

Background/Aims: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. Methods: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. Results: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. Conclusion: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.

P0528RENOPROTECTIVE EFFECT OF IL-34 INHIBITION ON CISPLATIN-INDUCED NEPHROTOXICITY IN MICE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Yukihiro Wada ◽  
Masayuki Iyoda ◽  
Kei Matsumoto ◽  
Taihei Suzuki ◽  
Ken Iseri ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Kidney Injury ◽  
Neutralizing Antibody ◽  
Inhibitory Effects ◽  
Tubulointerstitial Injury ◽  
Cisplatin Nephrotoxicity ◽  
Physiological Properties ◽  
Tnf Α ◽  
Vehicle Treatment

Abstract Background and Aims Interleukin (IL)-34, a macrophage (Mø) mediator, is expressed by tubular epithelial cells (TECs). However, the influence of IL-34 on TECs injury has not been fully elucidated. We investigated the physiological properties of IL-34 on TECs damage caused by cisplatin-nephrotoxicity (CP-N). Method 7-week-old male C57BL/6 (B6) mice (n=16) were fasted for 8 hours and then induced CP-N by intraperitoneal injection (IP) of CP (25 mg/kg) on day 0. Groups of animals were given either anti-mouse IL-34 antibody (CP+anti-IL-34 Ab, 400 ng/kg, n=8) or vehicle (CP+V, equal volume of saline, n=8) daily by IP from day -1 to day 2. Three age-matched male B6 mice were used as normal control (NC). All mice were sacrificed on day 3. In addition, mouse renal proximal TECs (MRTEpiC) were cultured to analyze the inhibitory effects of IL-34 on CP-induced TEC apoptosis. Cells were stimulated with CP (2 μg/mL), then treated with or without anti-IL-34 Ab (1000 pg/mL). Results Compared to the NC, CP+V mice exhibited marked acute kidney injury (AKI) and upregulated expression of IL-34 and its two receptors, cFMS and PTP-ζ. Compared to the vehicle treatment, anti-IL-34 Ab treatment significantly suppressed the intrarenal expression levels of IL-34 and its two receptors in CP-N mice; it also significantly suppressed serum IL-34 levels (72.1 ± 5.6 vs. 40.4 ± 7.5 pg/mL, p=0.013). Additionally, treatment with anti-IL-34 Ab significantly improved serum Cr levels (1.3 ± 0.2 vs. 0.7 ± 0.1 mg/mL, p=0.033), ameliorated tubulointerstitial injury (numbers of casts/HPF: 11.9 ± 2.6 vs. 6.5 ± 1.8, p=0.048), and suppressed the number of F4/80+ Mø (17.5 ± 2.7 vs. 11.1 ± 1.1/HPF, p=0.041) and TUNEL+ apoptotic cells (29.2 ± 4.9 vs. 16.7 ± 2.7/HPF, p=0.036) in CP-N mice. The renal cortical transcript levels of Kim-1, MIP-1/CCL3, TNF-α, and Bax were significantly lower in the CP+anti-IL-34 Ab mice than in the CP+V mice. Furthermore, the CP+anti-IL-34 Ab mice showed significantly less renal infiltration of CD11b+F4/80+TNF-α+ cells. In vitro, stimulation with CP induced the expression of IL-34 and its two receptors in MRTEpiC. Treatment with anti-IL-34 Ab significantly suppressed CP-induced caspase-3 and Bax expression with degradation of ERK1/2 phosphorylation in the damaged MRTEpiC. Conclusion IL-34 secreted from damaged TECs was involved in the progression of CP-N. Inhibition of IL-34 with neutralizing antibody directly prevented CP-induced TEC apoptosis by inhibiting the phosphorylation of ERK1/2. Blocking of IL-34 might suppressed proliferation of cytotoxic Mø, which indirectly led to the attenuation of CP-N. Thus, IL-34 represents a potential as therapeutic target for AKI with TECs injury.

scholarly journals Micrococcus luteus Teichuronic Acids Activate Human and Murine Monocytic Cells in a CD14- and Toll-Like Receptor 4-Dependent Manner

2001 ◽  
Vol 69 (4) ◽  
pp. 2025-2030 ◽  
Author(s):  
Shuhua Yang ◽  
Shunji Sugawara ◽  
Toshihiko Monodane ◽  
Masahiro Nishijima ◽  
Yoshiyuki Adachi ◽  
...  
Keyword(s):  
Lipid A ◽  
Micrococcus Luteus ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Necrosis Factor Alpha ◽  
Monocytic Cells ◽  
Tnf Α

ABSTRACT Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerlyMicrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4–12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-α) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-α-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IVA, an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

scholarly journals Cytoprotective Chaperone Proteins Are Novel Anti-Inflammatory Targets in Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1292-1292
Author(s):  
Sanjiv Kumar ◽  
Ciprian Anea ◽  
Itia Lee ◽  
Aluya Oseghale ◽  
Julia Brittain
Keyword(s):  
Inflammatory Response ◽  
Inflammatory Cytokine ◽  
Chaperone Proteins ◽  
Cytokine Release ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Proof Of Principle

Abstract Sickle cell disease (SCD) is a pro-inflammatory condition. Levels of TNF-α, IL-6, IL-8, and IL-10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non-crisis "steady state" condition. Additional insult, either via infection or vaso-occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro-inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro-coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti-inflammatory, anti-coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY-922 could block the bacterial toxin lipopolysaccharide (LPS) - induced TF expression and pro-inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY-922 in a pre-clinical study. Townes mice with SCD or without were administered AUY-922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS-mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro-inflammatory response in the AA mice (n=24). However, LPS-induced an exaggerated response in the SS mice. Levels of TNF-α, IL-6, IL-8, and IL-10 were elevated up to 40,000 fold over control treated SS mice. Pre-treatment with AUY-922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY-922 doubled the amount of the anti-inflammatory chaperone molecule HSP70 in the livers of the SS mice. This particular result suggested that the function of HSP90 could be spared, and the induction of HSP70 was potentially sufficient to protect against the LPS-induced insult. Of note, the main function of HSP70 is cytoprotection in response to oxidative and febrile stress. Therefore, we next sought to determine, in a proof of principle in vitro study, whether induction of HSP70 alone was sufficient to block LPS-induced cytokine release and coagulation activation. We treated human monocytes with the HSP70 inducer, celastrol for 24h, followed by treatment with LPS (1µg/ml). We observed a significant release of the cytokines IL-6 and TNF-α with LPS treatment. However, induction of HSP70 via celastrol was sufficient to block this inflammatory response. Furthermore, we observed that celastrol blocked the LPS-induced, TF-specific clotting of plasma in vitro. Interestingly, we also observed that conditioned media from celastrol treated monocytes could block LPS-induced IL-6 release in an HSP70 dependent manner. Thus, secreted HSP70 was an active participant in cellular protection from LPS-induced insult. Initial studies suggest that secreted HSP70 levels may be lower in patients with SCD than in unaffected individuals. Therefore, replacement of this chaperone may be of significant benefit as therapeutic. Thus, taken together, our data demonstrate in both a pre-clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals THP-1 Cells and Pro-inflammatory Cytokine Production: An in Vitro Tool for Functional Characterization of NOD1/NOD2 Antagonists

2019 ◽  
Vol 20 (17) ◽  
pp. 4265 ◽  
Author(s):  
Jakopin ◽  
Corsini
Keyword(s):  
Inflammatory Cytokine ◽  
Functional Characterization ◽  
Toll Like Receptor 4 ◽  
Inflammatory Cytokine Production ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor ◽  
Oligomerization Domain

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.

scholarly journals Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-α/JNK

2011 ◽  
Vol 300 (3) ◽  
pp. H913-H921 ◽  
Author(s):  
Hu Xu ◽  
Yongwei Yao ◽  
Zhaoliang Su ◽  
Yunbo Yang ◽  
Raymond Kao ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Myocardial Inflammation ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Myocardial Apoptosis ◽  
Apoptosis Inhibition ◽  
Tnf Α ◽  
Myocyte Apoptosis ◽  
Jnk Activation

High-mobility group box 1 (HMGB1) is a nuclear protein that has been implicated in the myocardial inflammation and injury induced by ischemia-reperfusion (I/R). The purpose of the present study was to assess the role of HMGB1 in myocardial apoptosis induced by I/R. In vivo, myocardial I/R induced an increase in myocardial HMGB1 expression and apoptosis. Inhibition of HMGB1 (A-box) ameliorated the I/R-induced myocardial apoptosis. In vitro, isolated cardiac myocytes were challenged with anoxia-reoxygenation (A/R; in vitro correlate to I/R). A/R-challenged myocytes also generated HMGB1 and underwent apoptosis. Inhibition of HMGB1 attenuated the A/R-induced myocyte apoptosis. Exogenous HMGB1 had no effect on myocyte apoptosis. However, inhibition of HMGB1 attenuated myocyte TNF-α production after the A/R was challenged; surprisingly, HMGB1 itself did not induce myocyte TNF-α production. Exogenous TNF-α induced a moderate proapoptotic effect on the myocytes, an effect substantially potentiated by coadministration of HMGB1. It is generally accepted that apoptosis induced by TNF-α is regulated by the balance of activation of c-Jun NH2-terminal kinase (JNK) and NF-κB. Indeed, in the present study, TNF-α increased the phosphorylation status of JNK and p65, a subunit of NF-κB; HMGB1 greatly potentiated TNF-α-induced JNK phosphorylation. Furthermore, inhibition of JNK (SP-600125) prevented the myocyte apoptosis induced by a TNF-α/HMGB1 cocktail. Finally, A/R increased HMGB1 production in both wild-type and toll-like receptor 4-deficient myocytes; however, deficiency in toll-like receptor 4 diminished A/R-induced myocyte apoptosis, TNF-α, and JNK activation. Our results indicate that myocyte-derived HMGB1 and TNF-α work in concert to promote I/R-induced myocardial apoptosis through JNK activation.

scholarly journals Lactoferrin retargets adenoviruses to TLR4 to induce an abortive NLRP3-associated pyroptotic response in human dendritic cells

2020 ◽  
Author(s):  
Coraline Chéneau ◽  
Karsten Eichholz ◽  
Tuan Hiep Tran ◽  
Thi Thu Phuong Tran ◽  
Océane Paris ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Membrane Integrity ◽  
Human Adenovirus ◽  
Mononuclear Phagocyte ◽  
Cytokine Release ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Human Dendritic Cells ◽  
Antigen Presenting

AbstractDespite decades of investigations, we still poorly grasp the immunogenicity of human adenovirus (HAdV)-based vaccines in humans. In this study, we explored the role of lactoferrin, which belong to the alarmin subset of antimicrobial peptides that provide immediate direct and indirect activity against a range of pathogens following a breach in tissue homeostasis. Lactoferrin is a globular, iron-sequestering, glycoprotein that can increase HAdV infection and maturation of antigen-presenting cells. However, the mechanism by which HAdV-lactoferrin complexes induce maturation is unknown. We show that lactoferrin redirects HAdVs from species B, C, and D to toll-like receptor 4 (TLR4) complexes on human mononuclear phagocyte. TLR4-mediated internalization induces an abortive NLRP3-associated pyroptotic response inducing pro-inflammatory cytokine release and disrupting plasma membrane integrity without cell death. These data impact our understanding of the immunogenicity of HAdV-based vaccines and may provide ways to increase their efficacy.

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