HMGB1 Production Research Articles

An Amino Acids and Dipeptide Injection Inhibits the TNF-α/HMGB1 Inflammatory Signaling Pathway to Reduce Pyroptosis and M1 Microglial Polarization in POCD Mice: the Gut to the Brain.

Peripheral surgery-induced neural inflammation is a key pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the mechanism underlying neuroinflammation and associated neural injury remains elusive. Surgery itself can lead to gut damage, and the occurrence of POCD is accompanied by high levels of TNF-α in the serum and blood‒brain barrier (BBB) damage. Reductions in stress, inflammation and protein loss have been emphasized as strategies for enhanced recovery after surgery (ERAS). We designed an amino acids and dipeptide (AAD) formula for injection that could provide intestinal protection during surgery. Through the intraoperative infusion of AAD based on the ERAS concept, we aimed to explore the effect of AAD injection on POCD and its underlying mechanism from the gut to the brain. Here, we observed that AAD injection ameliorated neural injury in POCD, in addition to restoring the function of the intestinal barrier and BBB. We also found that TNF-α levels decreased in the ileum, blood and hippocampus. Intestinal barrier protectors and TNF-α inhibitors also alleviated neural damage. AAD injection treatment decreased HMGB1 production, pyroptosis, and M1 microglial polarization and increased M2 polarization. In vitro, AAD injection protected the impaired gut barrier and decreased TNF-α production, alleviating damage to the BBB by stimulating cytokine transport in the body. HMGB1 and Caspase-1 inhibitors decreased pyroptosis and M1 microglial polarization and increased M2 polarization to protect TNF-α-stimulated microglia in vitro. Collectively, these findings suggest that the gut barrier-TNF-α-BBB-HMGB1-Caspase-1 inflammasome-pyroptosis-M1 microglia pathway is a novel mechanism of POCD related to the gut-brain axis and that intraoperative AAD infusion is a potential treatment for POCD.

Imp7 siRNA nanoparticles protect against mechanical ventilation-associated liver injury by inhibiting HMGB1 production and NETs formation

Mechanical ventilation (MV) has the potential to induce extra-pulmonary organ damage by adversely affecting the lungs and promoting the secretion of inflammatory cytokines. High-mobility group box 1 protein (HMGB1) is a pro-inflammatory mediator in ventilator-induced lung injury (VILI), but its effect on MV-associated liver injury and the mechanisms are poorly understood. In the present study, mice were subjected to high-volume MV (20 ml/kg) to induce VILI. MV-induced HMGB1 prompted neutrophil extracellular traps (NETs) formation and PANoptosis within the liver. Inhibiting NETs formation by DNase I or PAD4 inhibitor, or by HMGB1 neutralizing ameliorated the liver injury. HMGB1 activated neutrophils to form NETs through TLR4/MyD88/TRAF6 pathway. Importantly, Importin7 siRNA nanoparticles inhibited HMGB1 release and protected against MV-associated liver injury. These data provide evidence of MV-induced HMGB1 prompted NETs formation and PANoptosis in the liver via the TLR4/MyD88/TRAF6 pathway. HMGB1 is a potential therapeutic target for MV-associated liver injury.

The p75 neurotrophin receptor inhibitor, LM11A-31, ameliorates acute stroke injury and modulates astrocytic proNGF

The precursor form of nerve growth factor (proNGF) is essential to maintain NGF survival signaling. ProNGF is also among endogenous ligands for p75 neurotrophin receptor (p75ntr). Mounting evidence implies that p75ntr signaling contributes to neural damage in ischemic stroke. The present study examines the therapeutic effect of the p75ntr modulator LM11A-31. Adult mice underwent transient distal middle cerebral artery occlusion (t-dMCAO) followed by LM11A-31 treatment (25 mg/kg, i.p., twice daily) either for 72 h post-injury (acute phase) or afterward till two weeks post-stroke (subacute phase). LM11A-31 reduced blood-brain barrier permeability, cerebral tissue injury, and sensorimotor function in the acute phase of stroke. Ischemic brain samples showed repressed proNGF/P75ntr signaling and Caspase 3 activation in LM11A-31 treated mice, where we observed less reactive microglia and IL-1β production. LM11A-31 (20–80 nM) also mitigated neural injury induced by oxygen-glucose deprivation (OGD) in sandwich co-cultures of primary cortical neurons (PCN) and astrocytes. This concurred with JNK/PARP downregulation and reduced caspase-3 cleavage in the PCNs and was associated with repressed proNGF generation in astrocytes. Further in vitro experiments indicated human proNGF suppresses the pro-inflammatory phenotype in microglial cultures, as determined by a sharp decline in HMGB-1 production and moderate arginase-1 upregulation. Despite significant protection in acute stroke, LM11A-31 treatment did not improve cortical atrophy and sensorimotor function in the subacute phase. Our findings provide preclinical evidence supporting LM11A-31 as a promising therapy for acute stroke injury. Further investigations may elucidate if reduced astrocytic proNGF, an endogenous reservoir of pro-neurotrophins, may restrict the therapeutic window.

Abstract 4244: Using lysosomal immunogenic cell death to target cancer via xanthine oxidase

Abstract Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence - a gap which can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells which can later present those tumorigenic antigens to T cells. We demonstrate that the oxidative agent native bovine xanthine oxidase can induce early markers of lysosome-induced immunogenic cell death in an in vitro human cancer cell line (CCL2). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of cells dosed with doxorubicin (a known inducer of LIICD). We looked at each marker at various time points after cancer cells were treated with xanthine oxidase, doxorubicin, and an untreated group. Upregulated biomarkers after treatment with the protein indicate an immunogenic response. Further, we used confocal imaging to show lysosomal membrane permeabilization after treatment with Native Bovine Xanthine Oxidase using Lucifer Yellow staining. We thus show the potential for this protein to induce an anticancer effect paired with an adaptive immune response against tumor cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method in patients and serve as the gateway to developing a new treatment approach against breast cancer. Citation Format: Iulianna Taritsa, Eric Fossel, Kuldeep Neote. Using lysosomal immunogenic cell death to target cancer via xanthine oxidase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4244.

Research on the Mechanism of HMGB1 Regulating Autoimmune Encephalomyelitis by Regulating NF-κB.

Background Autoimmune encephalomyelitis is a clinical condition in which memory and cognition is affected badly and is also associated with lower levels of consciousness or even coma in worse scenarios. It is a noninfectious condition which involves immune oriented inflammation. Objective The study's goal was to figure out what was causing the problem HMGB1 involved in regulating the autoimmune encephalomyelitis by regulating NF-κB. Materials and Methods The expressions of HMGB1, miR-129-5p, and TLR4/NF-κB signalling pathway-related proteins were measured by qRT-PCR. To explore the differences among its control, models, and all groups, histopathology, immunohistochemistry, and immunofluorescence tests were performed. Results According to the findings, miR-129-5p is in charge of suppressing HMGB1 production and inhibiting the TLR4/NF-κB signalling pathway. On development of autoimmune encephalomyelitis, neurons in the hippocampus area got injured in the miR-129-5p inhibitors class. In the miR-129-5p inhibitor class, expression of miR-129-5p reduced and HMGB1 elevated, increasing neuronal inflammation and damage. Impairment in the hippocampus, on the either side, was shown to be reduced in HMGB1 shRNA, miR-129-5p mimics, and TLR4/NF-κB classes. Conclusion According to the study's findings, there is indeed a link among increased miR-129-5p and decreased HMGB1 expression and also suppression of the TLR4/NF-κB signal transduction pathway in autoimmune encephalomyelitis in the miR-129-5p inhibitors group. As a result, we may assume the autoimmune disease illness has progressed once concentrations of HMGB1, TLR4/NF-κB, and miR-129-5p have decreased.

Inhibition of HMGB1/TLR4 Signaling Pathway by Digitoflavone: A Potential Therapeutic Role in Alcohol-Associated Liver Disease.

Digitoflavone (DG) is a natural flavonoid abundant in many fruits, vegetables, and medicinal plants. We investigated whether DG inhibits lipid accumulation and inflammatory responses in alcoholic liver disease (ALD) in vivo and in vitro. The mouse ALD model was established by chronically feeding male C57BL/6 mice an ethanol-containing Lieber-DeCarli liquid diet. In vitro, mouse peritoneal macrophages (MPMs) and mouse bone marrow-derived macrophages (BMDMs) were stimulated with LPS/ATP, whereas HepG2 cells and mouse primary hepatocytes were treated with ethanol. DG reduced the serum levels of transaminase and serum and hepatic levels of triglycerides and malondialdehyde in ALD mice. DG downregulated SREBP1 and its target genes and upregulated PPARα and its target genes in the liver of mice with ALD. DG inhibited TLR4-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of HMGB1, IL-1β, and IL-36γ, as well as the infiltration of macrophages and neutrophils. DG blocked NLRP3/ASC/caspase-1 inflammasome activation and HMGB1 release in LPS/ATP-stimulated MPMs. When Tlr4 was knocked in LPS/ATP-stimulated BMDMs, HMGB1 production and release were blocked, and NLRP3-mediated cleavage and release of IL-1β was suppressed in Hmgb1-silenced BMDMs. DG amplified these inhibitory effects in Tlr4 or Hmgb1 knockdown BMDMs. In ethanol-exposed hepatocytes, DG reduced lipogenesis and promoted lipid oxidation by inhibiting the HMGB1-TLR4 signaling pathway while suppressing the inflammatory response induced by ethanol exposure. Our data demonstrated that DG inhibited the occurrence of lipid accumulation and the inflammatory response via the HMGB1-TLR4 axis, underscoring a promising approach and utility of DG for the treatment of ALD.

P. aeruginosa Mediated Necroptosis in Mouse Tumor Cells Induces Long-Lasting Systemic Antitumor Immunity.

Necroptosis is a form of programmed cell death (PCD) characterized by RIP3 mediated MLKL activation and increased membrane permeability via MLKL oligomerization. Tumor cell immunogenic cell death (ICD) has been considered to be essential for the anti-tumor response, which is associated with DC recruitment, activation, and maturation. In this study, we found that P. aeruginosa showed its potential to suppress tumor growth and enable long-lasting anti-tumor immunity in vivo. What’s more, phosphorylation- RIP3 and MLKL activation induced by P. aeruginosa infection resulted in tumor cell necrotic cell death and HMGB1 production, indicating that P. aeruginosa can cause immunogenic cell death. The necrotic cell death can further drive a robust anti-tumor response via promoting tumor cell death, inhibiting tumor cell proliferation, and modulating systemic immune responses and local immune microenvironment in tumor. Moreover, dying tumor cells killed by P. aeruginosa can catalyze DC maturation, which enhanced the antigen-presenting ability of DC cells. These findings demonstrate that P. aeruginosa can induce immunogenic cell death and trigger a robust long-lasting anti-tumor response along with reshaping tumor microenvironment.

N-(2'-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Inhibits HDAC1 and Increases the Translocation of HMGB1 Levels in Human Cervical Cancer Cells.

N-(2′-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a VPA derivative designed to be a histone deacetylase (HDAC) inhibitor. HO-AAVPA has better antiproliferative effect than VPA in cancer cell lines. Therefore, in this work, the inhibitory effect of HO-AAVPA on HDAC1, HDAC6, and HDAC8 was determined by in silico and in vitro enzymatic assay. Furthermore, its antiproliferative effect on the cervical cancer cell line (SiHa) and the translocation of HMGB1 and ROS production were evaluated. The results showed that HO-AAVPA inhibits HDAC1, which could be related with HMGB1 translocation from the nucleus to the cytoplasm due to HDAC1 being involved in the deacetylation of HMGB1. Furthermore, an increase in ROS production was observed after the treatment with HO-AAVPA, which also could contribute to HMGB1 translocation. Therefore, the results suggest that one of the possible antiproliferative mechanisms of HO-AAVPA is by HDAC1 inhibition which entails HMGB1 translocation and ROS increased levels that could trigger the cell apoptosis.

Hyperoxia results in HMGB1 production and subsequent inflammatory signaling in human pulmonary microvascular endothelial cells

We have previously shown that neonatal mice exposed to 85% oxygen have substantial elevations in bronchoalveolar lavage levels of HMGB1 protein and that inhibiting TLR4 signaling using TAK‐242, which prevents TLR4 interaction with adaptor proteins like HMGB1, attenuated hyperoxia‐induced lung injury. To begin to understand the molecular and cellular mechanisms underlying these in vivo findings we studied HMGB1 mediated signaling in cultured cells. We hypothesized that hyperoxia would induce HMGB1 production in cells key to lung injury and that HMGB1 would interact with TLR4 to cause pro‐inflammatory cytokine production. We exposed human pulmonary microvascular endothelial cells (hPMVEC), A549 cells (a human lung epithelial cell line), and RAW264.7 cells (a murine macrophage‐like cell line) to hyperoxia, and examined the effect on HMGB1 levels. We found that exposure to 85% oxygen for 48 hours resulted in 16‐fold, 2‐fold, and 14‐fold increases in HMGB1 protein levels in these cells, respectively, relative to the same cells in normoxia. When HMGB1 (100 ng/ml) was added to the media of hPMVEC, there was significant (p<0.005) induction of IL‐8 and MCP‐1 protein levels. Furthermore, HMBG1 added to the media of hPMVEC resulted in significant induction of cleaved caspase‐1 and IL‐1β protein levels compared to vehicle treated hPMVEC. The addition of HMGB1 to the media of hPMVEC also resulted in a significant time‐dependent induction of pNFκB, pIKKα/β, and pIκBα. Hyperoxia exposed hPMVEC had a 3‐fold greater expression of phosphorylated p38 then did normoxic cells and this pp38 induction was significantly attenuated by TAK‐242. In hPMVEC hyperoxia resulted in 2.5‐fold greater IL‐8 protein levels than in normoxia exposed cells (p<0.05) and this effect was completely blocked by TAK‐242. In hPMVEC exposure to hyperoxia resulted in 8‐fold greater levels of cleaved caspase‐1 than in normoxia exposed cells (p<0.05), and in 4‐fold greater levels of cleaved IL‐1β than in normoxia exposed cells (p<0.005). In conclusion, our results suggest that hyperoxic exposure results in HMGB1 mediated TLR4 activation, leading to caspase‐1 activation and induction of pro‐inflammatory cytokines including cleaved IL‐1β. These results suggest that targeting HMGB1‐TLR4 interactions may confer protection from hyperoxia‐induced injury.

Protective effects of curcumin against neuroinflammation induced by Aβ25-35 in primary rat microglia: modulation of high-mobility group box 1, toll-like receptor 4 and receptor for advanced glycation end products expression.

Activated microglia induced by amyloid-beta (Aβ) release proinflammatory cytokines that can induce neurotoxicity. High-mobility group box 1 protein (HMGB1) and HMGB1-mediated inflammatory responses have been attributed with memory impairment in patients with Alzheimer's disease (AD). There is accumulating evidence to suggest curcumin is a potent anti-inflammatory polyphenol. However, whether curcumin could effectively inhibit inflammation through the suppression of HMGB1 production or HMGB1-mediated inflammatory responses in Aβ-activated microglia is still unclear. Primary microglia were prepared from the cerebral cortices of one- to three-day-old Sprague Dawley rats. The microglia were cultured and treated with Aβ25-35 50 µM for 24 h to prove a toxic effect. Curcumin 10 µM was administrated 1 h before Aβ25-35 treatment. The levels of HMGB1, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the culture medium were analyzed by ELISA. Western blotting was conducted to assess the expression level of toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE). In addition, PC12 cells were treated with conditioned medium from microglia treated with Aβ25-35 or Aβ25-35 and curcumin, and cell viability was subsequently assessed by MTT. Curcumin was found to significantly inhibit HMGB1 expression and release in Aβ25-35-stimulated microglia. Pretreatment with curcumin reduced TLR4 and RAGE expression. Proinflammatory cytokines such as IL-1β and TNF-α were also remarkably reduced by curcumin. In addition, curcumin protected neurons from indirect toxicity mediated by Aβ25-35-treated microglia. Curcumin effectively inhibits Aβ25-35-induced neuroinflammation in microglia, partly by suppressing the expression of HMGB1, TLR4, and RAGE.

Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity

Polyhexamethyleneguanidine phosphate (PHMG-P) is a polymeric biocide with a guanidine group. It has multiple positive charges in physiological conditions due to nitrogen atom in the guanidine and this cationic property contributes antimicrobial effect by disrupting cell membranes. To determine whether the cationic nature of PHMG-P results in cytotoxicity in human cell lines, anionic compounds were treated with PHMG-P. The cytotoxic effect was evaluated with ROS production and HMGB1 release into media. To verify the protection effect of anion against PHMG-P-induced cell death in vivo, a zebrafish assay was adopted. In addition, membrane disruption by PHMG-P was evaluated using fluorescein diacetate and propidium iodine staining. As a result, anionic substances such as DNA and poly-l-glutamic acids, decreased PHMG-P induced cell death in a dose-dependent manner. While HMGB1 and ROS production increased with PHMG-P concentration, the addition of anionic compounds with PHMG-P reduced the ROS production and HMGB1 release. The mortality of the zebrafish increased with PHMG-P concentration and co-treatment of anionic compounds with PHMG-P decreased mortality in a dose-dependent manner. In addition, FDA and PI staining confirmed that PHMG-P disrupts plasma membrane. Taken together, a cationic property is considered to be one of the main causes of PHMG-P-induced mammalian cell toxicity.

PO-429 Identification of the right immunostimulatory chemotherapeutic partner for anti-CD70 immunotherapy in non-small cell lung cancer

IntroductionCD70 has emerged as a promising immunotherapeutic target in various haematological and solid malignancies. Its overexpression on tumour cells is associated with immunosuppression in the tumour microenvironment. We have previously demonstrated constitutive CD70 overexpression on tumour cells in a subset of primary non-small cell lung cancer (NSCLC) biopsies. More importantly, preliminary findings from our group demonstrated the ability of cisplatin to induce CD70 overexpression on NSCLC cells, which broadens the therapeutic window of anti-CD70 immunotherapy. On the other hand, certain chemotherapeutic agents can elicit immunostimulatory changes in the tumour microenvironment by triggering immunogenic cell death and enabling tumor-specific cytotoxic T cell responses. In this regard, strategically combining such a chemotherapeutic agent with anti-CD70 immunotherapy has the potential to achieve enhanced anti-tumour effects.Material and methodsThe immunostimulatory potential of different chemotherapeutics (cisplatin, oxaliplatin, cyclophosphamide, docetaxel and carboplatin) was evaluated on several NSCLC cell lines, varying in genetic aberrations, by assessing the most important hallmarks of immunogenic cell death: IFNα and HMGB1 production (ELISA), membrane calreticulin expression (flow cytometry) and ATP secretion (bioluminescence). Furthermore, the potential of these different chemotherapeutics to induce CD70 overexpression on NSCLC cells was explored (flow cytometry).Results and discussionsCytotoxicity experiments demonstrated different mechanisms of action for all chemotherapeutics with divergent sensitivity to NSCLC cell lines. At least three out of five chemotherapeutic agents demonstrated ATP secretion and ecto-calreticulin induction on various NSCLC cell lines. In addition, initial experiments showed the highest induction of CD70 expression on NSCLC cells upon low doses of cisplatin and carboplatin. In vitro data on the immunogenic effects of clinically relevant combination strategies in different cell lines are currently being analysed.ConclusionThese in vitro results demonstrate the immunostimulatory effects of clinically relevant chemotherapeutics, making it worthwhile to further investigate the potential of a novel combination strategy of chemotherapy and anti-CD70 immunotherapy in NSCLC.

Activation of NLRP3 Inflammasomes in Non‐alcoholic Steatohepatitis: A Novel Molecular Target of FTZ Remedy for Metabolic Syndrome

NLRP3 inflammasome activation not only serves as an intracellular machinery triggering inflammation, but also produces uncanonical effects beyond inflammation such as changing cell metabolism, inducing cell transformation and increasing cell membrane permeability. The present study was designed to test whether this NLRP3 inflammasome activation plays a crucial role in the ‘two‐hit’ injury during non‐alcohilic steatohepatitis (NASH) and whether this inflammasome can be a therapeutic target for the action of Fufang Zhenzhu Tiaozhi (FTZ), a widely used herbal remedy for metabolic syndrome in China. By confocal microscopy, oil red O staining and immunohistochemistry, we demonstrated that NLRP3 inflammasomes formation and activation as well as lipid deposition occurred in the liver of mice on the high fat diet (HFD), as shown by increased NLRP3 components colocalization, enhanced production of IL‐1b and HMGB1, and remarkable lipid deposition in liver cells. FTZ extracts were found to not only significantly reduce the NLRP3 inflammasome formation and activation (from 1.46&1.81 to 1.01&1.29 folds of control), but also attenuated the liver steatosis (from 41.13% to 17.96%). In in vitro studies, palmitic acid (PA) was used to induce NLRP3 inflammasome activation and lipid deposition in hepatic stellate cells (HSCs). It was found that PA increased colocalization of NLRP3 components and enhanced cellular caspase‐1 activity (to 2.28 folds of control), indicating the formation and activation of NLRP3 inflammasomes. PA also increased HMGB1 production and enhance the lipid deposition in these cells. In FTZ treated cells, not only inflammasome activation was attenuated (from 1.78&1.81 to 1.01&1.07 folds of control, respectively), but also lipid deposition in HSCs was blocked, which was similar to the effects of GLY, an HMGB1 inhibitor (from 18.87% to 0.46%&4.29%, respectively). Mechanistically, we found that PA stimulated O2.− production (to 2.71 folds of control) to activate NLRP3 inflammasomes in HSCs, which was blocked by FTZ treatment as did by NAC, a O2.− scavenger (to 0.86 and 1.33 folds of control, respectively). It is concluded that NLRP3 inflammasomes formation and activation may contribute to the development of both lipid deposition and inflammation during NASH and that FTZ inhibits NASH by its action on both inflammatory response and liver lipid metabolism associated with NRLP3 inflammasome activation.Support or Funding Informationsupported by NSFC grants (81530102 to J. G) and NIH grants (HL057244 and HL075316 to P.L.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curcumin longa extract-loaded nanoemulsion improves the survival of endotoxemic mice by inhibiting nitric oxide-dependent HMGB1 release.

BackgroundHigh mobility group box 1 (HMGB1) is a well-known damage-related alarmin that participates in cellular inflammatory responses. However, the mechanisms leading to HMGB1 release in inflammatory conditions and the therapeutic agents that could prevent it remain poorly understood. This study attempted to examine whether the Curcumin longa herb, which is known to have anti-inflammatory property, can modulate cellular inflammatory responses by regulating HMGB1 release.MethodsThe murine macrophage RAW264.7 cells were treated with lipopolysaccharide (LPS) and/or a C. longa extract-loaded nanoemulsion (CLEN). The levels of released HMGB1, nitric oxide (NO) production, inducible NO synthase (iNOS) expression, and phosphorylation of mitogen-activated protein kinases were analyzed in RAW264.7 macrophages. The effects of CLEN on survival of endotoxemic model mice, circulating HMGB1 levels, and tissue iNOS expression were also evaluated.ResultsWe have shown that a nanoemulsion loaded with an extract from the C. longa rhizome regulates cellular inflammatory responses and LPS-induced systemic inflammation by suppressing the release of HMGB1 by macrophages. First, treatment of RAW264.7 macrophages with the nanoemulsion significantly attenuated their LPS-induced release of HMGB1: this effect was mediated by inhibiting c-Jun N-terminal kinase activation, which in turn suppressed the NO production and iNOS expression of the cells. The nanoemulsion did not affect LPS-induced p38 or extracellular signal-regulated kinase activation. Second, intraperitoneal administration of the nanoemulsion improved the survival rate of LPS-injected endotoxemic mice. This associated with marked reductions in circulating HMGB1 levels and tissue iNOS expression.DiscussionThe present study shows for the first time the mechanism by which C. longa ameliorates sepsis, namely, by suppressing NO signaling and thereby inhibiting the release of the proinflammatory cytokine HMGB1. These observations suggest that identification of agents, including those in the herb C. longa, that can inhibit HMGB1 production and/or activity may aid the treatment of endotoxemia.

Synthetic anti-endotoxin peptides inhibit cytoplasmic LPS-mediated responses

Toll-like receptor (TLR) 4-independent recognition of lipopolysaccharide (LPS) in the cytosol by inflammatory caspases leads to non-canonical inflammasome activation and induction of IL-1 secretion and pyroptosis. The discovery of this novel mechanism has potential implications for the development of effective drugs to treat sepsis since LPS-mediated hyperactivation of caspases is critically involved in endotoxic shock. Previously, we demonstrated that Pep19-2.5, a synthetic anti-endotoxin peptide, efficiently neutralises pathogenicity factors of Gram-negative and Gram-positive bacteria and protects against sepsis in vivo. Here, we report that Pep19-2.5 inhibits the effects of cytoplasmic LPS in human myeloid cells and keratinocytes. In THP-1 monocytes and macrophages, the peptide strongly reduced secretion of IL-1β and LDH induced by intracellular LPS. In contrast, the TLR4 signaling inhibitor TAK-242 abrogates LPS-induced TNF and IL-1β secretion, but not pyroptotic cell death. Furthermore, Pep19-2.5 suppressed LPS-induced HMGB-1 production and caspase-1 activation in THP-1 monocytes. Consistent with this observation, we found impaired IL-1β and IL-1α release in LPS-stimulated primary monocytes in the presence of Pep19-2.5 and reduced LDH release and IL-1B and IL-1A expression in LPS-transfected HaCaT keratinocytes. Additionally, Pep19-2.5 completely abolished IL-1β release induced by LPS/ATP in macrophages via canonical inflammasome activation. In conclusion, we provide evidence that anti-endotoxin peptides inhibit the inflammasome/IL-1 axis induced by cytoplasmic LPS sensing in myeloid cells and keratinocytes and activation of the classical inflammasome by LPS/ATP which may contribute to the protection against bacterial sepsis and skin infections with intracellular Gram-negative bacteria.

307 UVB-induced HMGB1 and extracellular ATP increase Langerhans cell production of IL-23 implicated in ILC3 activation

307 UVB-induced HMGB1 and extracellular ATP increase Langerhans cell production of IL-23 implicated in ILC3 activation

Improved prescription of taohechengqi-tang alleviates D-galactosamine acute liver failure in rats.

To investigate the hepatoprotective effect of improved prescription of Taohechengqi-tang (IPTT) against acute liver failure (ALF) in rats. Seventy specific pathogen free male Wistar rats were randomly divided into four groups: control group (normal rats, n = 10), ALF group (ALF model, n = 20), Stronger Neo-Minophagen C (SNMC) group (ALF model + SNMC, n = 20), and IPTT group (ALF model + IPTT, n = 20). The ALF model group was administered an intraperitoneal injection of D-galactosamine (1.4 g/kg), and the control group received normal saline intraperitoneally. The SNMC and IPTT groups were treated with SMMC (15.6 mg/kg) or IPTT (28.6 g/kg) by gavage at 24 h intervals, and the ALF and control groups were treated with normal saline. At 36 h after injection, serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, and cholinesterase and prothrombin time were determined, and liver histopathological scores were observed by microscopy after hematoxylin and eosin staining. mRNA expression of high mobility group box (HMGB) 1, toll-like receptor (TLR) 4, nuclear factor kappa B (NF-κB) and caspase-3 were analyzed via fluorescence quantitative reverse transcriptase polymerase chain reaction. Proliferating cell nuclear antigen (PCNA) immunohistochemistry in liver tissue was also performed. D-galactosamine notably decreased the biochemical and coagulation profiles in serum. IPTT not only improved liver function and histopathology but also normalized the gene expression levels in liver tissue. Compared with the model group, in the IPTT and SNMC groups, HMGB1 mRNA/β-actin (0.06 ± 0.03, 0.11 ± 0.04 vs 0.25 ± 0.04, P < 0.05); TLR4 mRNA/β-actin (0.07 ± 0.02, 0.22 ± 0.08 vs 0.41 ± 0.22, P < 0.05); NF-κB mRNA/β-actin (0.74 ± 0.41, 1.78 ± 0.64 vs 2.68 ± 1.35, P < 0.05); and caspase-3 mRNA/β-actin levels were all significantly reduced (1.61 ± 0.45, 2.57 ± 1.04 vs 3.41 ± 0.85, P < 0.05). The gene expression levels were significantly lower in the IPTT group than in the SNMC group (P < 0.05). Compared with the model group, the PCNA expression in liver tissue was significantly enhanced in the IPTT and SNMC groups (36.34 ± 4.91, 25.57 ± 2.94 vs 17.55 ± 2.40, P < 0.05). IPTT attenuates inflammation in ALF via inhibition of HMGB1 production, which may contribute to limited liver regeneration.

Abstract C112: Cabozantinib eradicates advanced murine prostate cancer by activating anti-tumor innate immunity

Abstract Several kinase inhibitors targeting aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in early phase clinical trials, particularly in cancer patients with bone metastases. Here we show that cabozantinib rapidly eradicates invasive, poorly-differentiated PTEN/p53 deficient murine prostate cancer. This was associated with increased neutrophil chemotactic factor expression, including CXCL12 and HMGB1 production by tumor cells, and robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor, plerixafor. Collectively, these results demonstrate that cabozantinib triggers neutrophil-mediated anti-tumor innate immune response that results in tumor clearance. (Manuscript submitted to Science) Citation Format: Akash Patnaik, Kenneth Swanson, Sabina Signoretti, Huihui Ye, Eva Csizmadia, Jesse Novak, Marina Gehring, Katja Helenius, Athalia Pyzer, Laleh Montaser, Lily Wang, Olivier Elemento, Elena Levantini, John Clohessy, John Asara, Kathleen Kelly, Pier Paolo Pandolfi, Jacalyn Rosenblatt, David Avigan, Steven Balk, Lewis Cantley. Cabozantinib eradicates advanced murine prostate cancer by activating anti-tumor innate immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C112.

Phosphatidylinositol 3-kinases pathway mediates lung caspase-1 activation and high mobility group box 1 production in a toluene-diisocyanate induced murine asthma model

We have previously demonstrated that downregulating HMGB1 decreases airway neutrophil inflammation in a toluene-diisocyanate (TDI)-induced murine asthma model, yet how HMGB1 is regulated in the lung remains uncertain.In this study, we intended to explore whether PI3K signaling pathway mediates pulmonary HMGB1 production in TDI-induced asthma model and the possible roles of NLRP3 inflammasome and caspase-1 in this process.BALB/c mice were sensitized and challenged with TDI to establish a TDI-induced asthma model. LY294002, a specific inhibitor of PI3K, was given intratracheally 1h before each challenge.Here we showed that airway hypersensitivity, airway infiltration of neutrophils and eosinophils, serum IgE and IL-4 in supernatant of cervical lymphocytes in TDI induced asthmatic mice were all markedly decreased by LY294002, accompanied by suppressed pulmonary expression of HMGB1. At the same time, we observed elevated protein levels of cleaved caspase-1 and IL-1β after TDI challenge, as well as increased immunoreactivity in lung, all of which were significantly recovered by LY294002. While both the protein expression and immunodistribution of NLRP3 in the lung stayed unchanged.These data suggest that PI3K mediates lung caspase-1 activation and HMGB1 production in TDI-induced murine asthma model.

Higenamine promotes M2 macrophage activation and reduces Hmgb1 production through HO-1 induction in a murine model of spinal cord injury.

Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to the activation of diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Higenamine (HG) (1-[(4-hydroxyphenyl) methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol), an active ingredient of Aconiti Lateralis Radix Praeparata, has been traditionally used as a heart stimulant and anti-inflammatory agent in oriental countries. However, the function and related mechanism of HG on SCI have never been investigated. In our current study, HG treatment displayed increased myelin sparring and enhanced spinal cord repair process. The numbers of CD4(+) T cells, CD8(+) T cells, Ly6G(+) neutrophils and CD11b(+) macrophages were all significantly lower in the HG-treated group than that in the control group after SCI. HG administration increased the expression of IL-4 and IL-10 and promoted M2 macrophage activation. Significantly reduced Hmgb1 expression was also observed in HG-treated mice with SCI. Furthermore, HG treatment promoted HO-1 production. The increased number of M2 macrophages, decreased expression of Hmgb1 and promoted locomotor recovery induced by HG were all reversed with additional HO-1 inhibitor treatment. In conclusion, HG promotes M2 macrophage activation and reduces Hmgb1 expression dependent on HO-1 induction and then promotes locomotor function after SCI.