HMGB1 Secretion Research Articles

Cardiomyocyte–fibroblast interaction contributes to diabetic cardiomyopathy in mice: Role of HMGB1/TLR4/IL-33 axis

Diabetic cardiomyopathy (DiCM) is characterized by myocardial fibrosis and dysfunction. In rodent models of diabetes myocardial HMGB1 increases while IL-33 decreases. The major cardiac cell type expressing HMGB1 is the myocyte while the primary IL-33 expressing cell is the fibroblast. The aim of this study was to delineate the extracellular communication pathway(s) between cardiomyocytes and fibroblasts that contributes to murine DiCM. The streptozotocin (STZ)-induced murine model of diabetes and a cardiomyocyte/fibroblast co-culture challenged with high glucose were used. In STZ mice, myocardial HMGB1 expression was increased while IL-33 expression decreased (immunofluorescence and Western blot). In addition, STZ mice had an increased myocardial collagen deposition and myocardial dysfunction (pressure-volume loop analysis). An HMGB1 inhibitor (A-box) or exogenous IL-33 prevented the myocardial collagen deposition and dysfunction. In the cardiomyocyte/fibroblast co-culture model, HG increased cardiomyocyte HMGB1 secretion, decreased fibroblast IL-33 expression, and increased fibroblast collagen I production. Further, using A-box and HMGB1 shRNA transfected myocytes, we found that cardiomyocyte-derived HMGB1 dramatically potentiated the HG-induced down-regulation of IL-33 and the increase in collagen I expression in the fibroblasts. The potentiating effects of the cardiomyocytes was diminished when toll-like receptor 4 deficient (TLR4−/−) fibroblasts were co-cultured with wild-type myocytes. Finally, TLR4−/− mice with diabetes had increased myocardial expression of HMGB1, but failed to down-regulate IL-33. The diabetes-induced myocardial collagen deposition and cardiac dysfunction were significantly attenuated in TLR4−/− mice. In conclusion, our findings support a role for “cardiomyocyte HMGB1–fibroblast TLR4/IL-33 axis” in the development of myocardial fibrosis and dysfunction in a murine model of diabetes.

IL-1β enhances vascular smooth muscle cell proliferation and migration via P2Y2 receptor-mediated RAGE expression and HMGB1 release

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessel walls, and their proliferation and migration play important roles in the development of atherosclerosis. Recently, it has been reported that IL-1β mediates the inflammatory response through the upregulation of the P2Y2 receptor (P2Y2R). Thus, we examined the role of P2Y2R in IL-1β-mediated proliferation and migration of VSMCs and the underlying molecular mechanisms. VSMCs were pretreated with IL-1β for 24h to upregulate P2Y2R expression. The cells were then stimulated with UTP or ATP for the indicated times, and cell proliferation and migration and the related signaling pathways were examined. The equipotent P2Y2R agonists ATP and UTP enhanced proliferation, RAGE expression and HMGB1 secretion in IL-1β-pretreated VSMCs. Additionally, pretreatment with IL-1β enhanced UTP-mediated VSMC migration and MMP-2 release, but these effects were not observed in the P2Y2R-siRNA- or RAGE-siRNA-transfected VSMCs. Next, the signaling molecules involved in P2Y2R-mediated cell proliferation and migration were determined. The ERK, AKT, PKC, Rac-1 and ROCK2 pathways were involved in UTP-induced cell proliferation and migration, MMP-2 and HMGB1 secretion and RAGE expression in the IL-1β-pretreated VSMCs. UTP induced the phosphorylation of ERK, AKT and PKC and the translocation of Rac-1 and ROCK2 from cytosol to membrane as well as stress fiber formation, which were markedly increased in the IL-1β-pretreated VSMCs but not in the P2Y2R-siRNA-transfected VSMCs. These results demonstrate that pro-inflammatory cytokines associated with atherosclerosis, such as IL-1β, can accelerate the process of atherosclerosis through the upregulation of P2Y2R.

Sodium arsenic and ultraviolet-radiation synergistically induce cell injury in lens and retinal epithelial cells occurring migration of HMGB1 (IRM11P.639)

Abstract Although sodium arsenic (As) and ultraviolet radiation (UVR) can induce cell injury through either DNA damage or reactive oxidative species (ROS) in various epithelial cells, whether damaged-associated molecular pattern (DAMP) involved in cell injury is still unclear. To determine the association of HMGB1, one of DAMP molecules that can be secreted by oxidative stress, we investigated cell injury in human lens epithelial cell (HLE B3) and human retinal epithelial cell (ARPE-19) using cell viability assay, western blot, confocal microscopy and flow cytometry (FACS analysis). In both HLE B3 and ARPE-19 cells, DNA damage marker, cleaved PARP and cell viability assay showed that UVR synergistically increased As-caused cell death(injury). During UVR and As-caused cell injury, HMGB1 was translocated from nucleus to cytosol and then secreted to extracellular region. ROS scavenger N-acetylcystein (NAC) and mito-tempo inhibited both translocation and secretion of HMGB1 in dose-dependent manner, implicating that migration of HMGB1 is closely associated with ROS generation in mitochondria. Taken together, we concluded that HMGB1 is associated in cell injury caused by UVR and As. Nevertheless how migrated HMGB1 plays a role in UVR-As caused cell injury of HLE B3 and arpe-19 cells is still unclear.

N-linked glycosylation plays a crucial role in the secretion of HMGB1.

HMGB1 protein is a delayed mediator of sepsis that is secreted to the extracellular milieu in response to various stimulants, inducing a pro-inflammatory response. HMGB1 is devoid of an endoplasmic reticulum (ER)-targeting signal peptide; hence, the mechanism of extracellular secretion is not completely understood, although HMGB1 is secreted after being subjected to post-translational modifications. Here, we identified the role of N-glycosylation of HMGB1 in extracellular secretion. We found two consensus (N37 and N134) and one non-consensus (N135) residues that were N-glycosylated in HMGB1 by performing liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzing for N-glycan composition and structure. Inhibition of N-glycosylation with tunicamycin resulted in a molecular shift of HMGB1 as assessed by gel electrophoresis. Non-glycosylated double mutant (N→Q) HMGB1 proteins (HMGB1(N37Q/N134Q) and HMGB1(N37Q/N135Q)) showed localization to the nuclei, strong binding to DNA, weak binding to the nuclear export protein CRM1 and rapid degradation by ubiquitylation. These mutant proteins had reduced secretion even after acetylation, phosphorylation, oxidation and exposure to pro-inflammatory stimuli. Taken together, we propose that HMGB1 is N-glycosylated, and that this is important for its DNA interaction and is a prerequisite for its nucleocytoplasmic transport and extracellular secretion.

HMGB1 modulates Lewis cell autophagy and promotes cell survival via RAGE-HMGB1-Erk1/2 positive feedback during nutrient depletion

Autophagy is a self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by lysosomes. It also allows cells to survive during nutrient depletion and/or in the absence of growth factors. High-mobility group protein 1 (HMGB1) is a highly-conserved nuclear protein that has been associated with cell autophagy; however, the mechanisms responsible for this role remain unclear. Many reports have demonstrated that autophagy represents a survival strategy for tumor cells during nutrient depletion, oxidative stress and DNA damage. In the present study, we explored the mechanisms whereby HMGB1 regulates tumor cell autophagy during nutrient depletion (the cells were cultured in Hank's balanced salt solution, HBSS). HMGB1 expression in Lewis cells increased and the protein was shuttled from the nucleus to the cytoplasm and was secreted, coincident with up-regulation of autophagy. Prevention of HMGB1 binding to the receptor for advanced glycation end products (RAGE) or knock-down of HMGB1 expression led to inhibition of autophagy and increased apoptosis. These results demonstrated a positive feedback pathway whereby starvation of Lewis cells promoted HMGB1 secretion, allowing cells to survive by regulating autophagy via a RAGE–HMGB1-extracellular signal-regulated kinase1/2-dependent pathway. These results also implicate HMGB1 as a potential risk factor for cancer growth and metastasis.

HMGB1 secretion during cervical carcinogenesis promotes the acquisition of a tolerogenic functionality by plasmacytoid dendritic cells.

Acquisition of an impaired functionality by plasmacytoid dendritic cells (pDCs) contributing to cancer progression has been documented in different types of cancers. In the present study, we postulate that molecules secreted by (pre)neoplastic epithelial cells of the genital tract (cervix/vulva) might attract pDCs but also modify their proper functionality, allowing these cells to initiate a tolerogenic response interfering with antitumor immunity. We demonstrated that pDCs are recruited during the cervical metaplasia-dysplasia-cancer sequence, through the action of their chemoattractant, chemerin. We showed that stimulated-pDCs exposed to cervical/vulvar tumor microenvironment display an altered phenotype. We also demonstrated that cervical/vulvar neoplastic keratinocytes inhibit the proper function of pDCs by decreasing their IFNα secretion in response to CpG oligonucleotides. In parallel, we observed that (pre)neoplastic areas of the cervix are infiltrated by FoxP3(+) Treg cells which colocalize with pDCs. Accordingly, pDCs cocultured with cervical/vulvar neoplastic keratinocytes have the capacity to induce a Treg cell differentiation from naïve CD4(+) T cells, which is in agreement with the development of a tolerogenic response. We identified HMGB1 as a soluble factor produced by neoplastic keratinocytes from the genital tract involved in pDCs functional alteration. Indeed, this molecule inhibited pDC maturation, decreased IFNα secretion following TLR9 stimulation and forced these cells to become tolerogenic. In contrast, inhibition of HMGB1 restored pDC phenotype. Our findings indicate that the use of inhibitory molecules notably directed against HMGB1 in cervical/vulvar (pre)neoplastic lesions might prevent alterations of pDCs functionality and represent an attractive therapeutic strategy to overcome immune tolerance in cancers.

MLL-Rearranged Infant ALL, but Not Standard-Risk ALL, Induces Inflammasome Signaling and Immune Activation

T cell-based therapy for infants with MLL-rearranged B-ALL (MLL-ALL) represents a promising approach in this high-risk, poor-prognosis subset. Factors in the immune microenvironment that influence T-cell function, however, are poorly understood in MLL-ALL. We have observed complete inhibition of healthy donor T-cell proliferation by ALL cells from MLL-ALL, but not standard risk ALL (SR-ALL) patients. T-cell function is restored in the presence of bone marrow macrophages. In this study, we hypothesized that MLL-ALL cells selectively induce the inflammasome- a cytokine signaling complex formed by macrophages in response to damage-associated molecular patterns (DAMPs) released by leukemic cells. In the canonical inflammasome pathway, DAMPs engage macrophage Toll-like receptors, which activate caspase-1 and drive production of T-cell activating cytokines such as IL1-β.First, we determined whether ALL cells obtained from MLL-ALL vs. SR-ALL patients could produce high mobility group box 1 (HGMB1), a prototypical DAMP associated with tumor cell stress and macrophage PRR activation, particularly after anthracycline therapy. CD19+ cells were isolated from the diagnostic bone marrow specimens of patients with MLL-ALL (n=1), SR-ALL (n=4), and healthy donor PBMC (n=2) and treated with either control media or 2uM doxorubicin. Prior to treatment both MLL-ALL and SR-ALL, but not healthy donor cells, expressed HMGB1 protein as measured by Western immunoblot (29 kDa, GADPH internal control). Following doxorubicin treatment, HMGB1 protein was increased in MLL-ALL but not SR-ALL. Soluble HMGB1, measured by ELISA, was significantly increased in MLL-ALL (12.6 + 3.7 ng/ml untreated vs. 51.4 + 1.9 treated) compared to SR-ALL cells (5.0 + 0.8 ng/ml untreated, 4.3 + 1.5 treated, p<0.05). Soluble HMGB1 was not detectable in healthy donor cells before or after treatment.Next, we determined whether the increased HMGB1 secretion observed in MLL-ALL vs. SR-ALL cells was associated with macrophage caspase-1 activation. Control media and 2 uM doxorubicin treated-and-washed MLL-ALL, SR-ALL and healthy control CD19+ cells were cultured alone or 1:1 with allogeneic primary human macrophages for 24 hours. The active isoform of caspase-1 (20 kD tetramer) was detectable by immunoblot only in macrophages exposed to doxorubicin-treated MLL-ALL. Mean levels of IL1-β, measured by ELISA, were significantly increased in cocultures of doxorubicin-treated MLL-ALL + macrophages (1255 pg/ml + 322) compared to untreated MLL-ALL (820 pg/ml + 183, p=<0.05) and doxorubicin-treated SR-ALL + macrophages (494 pg/ml + 85 p<0.01). Macrophages cultured alone did not produce significant levels of IL1-β before or after doxorubicin treatment.Finally, we wished to determine whether expression of Toll-like receptor 4, a surface receptor for HMGB1 known to signal caspase-1, was increased on macrophages exposed to doxorubicin-treated MLL-ALL vs. SR-ALL. Folowing coculture with untreated and doxorubicin treated MLL-ALL and SR-ALL cells, macrophage TLR4 mRNA and surface expression was quantified by qPCR and flow cytometry, respectively. Both mRNA and surface expression of TLR4 was high at baseline, and did not change significantly after coculture. Unexpectedly, however, the MLL-ALL cells themselves displayed a significant increase in TLR4 mRNA (20-fold compared to endogenous beta-glucuronidase, p<0.0001; 12-fold compared to untreated MLL-ALL, p<0.05 by ΔΔCt) and surface expression (median fluorescence intensity 1005 + 89 vs. 367 + 32 respectively, p<0.05) following doxorubicin treatment. This phenomenon was not observed in SR-ALL or healthy CD19+ cells and is consistent with other tumor models of autocrine immune activation by HMGB1.Taken together, these data indicate that primary MLL-ALL cells, but not SR-ALL cells, are capable of inducing a caspase-1 associated inflammasome that enhances both innate and T-cell immune activation following anthracycline therapy. Studies are currently underway with additional primary MLL-ALL samples to determine how strategic augmentation of this pathway may impact the anti-leukemia killing capacity of therapeutic T-cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract 632: Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

Abstract Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. Citation Format: Sofia R. Gameiro, Momodou L. Jammeh, Max M. Wattenberg, Kwong Y. Tsang, Soldano Ferrone, James W. Hodge. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2014-632

Abstract 202: Involvement of neuronal high mobility group box 1 (HMGB1) in Angiotensin II-mediated neuronal-microglial interaction

We have demonstrated that activation of microglia in the autonomic brain regions, primarily in the paraventricular nucleus (PVN), is critical in the development and establishment of hypertension. Neuroimmune synaptic communication is implicated in activating microglia and inducing neuroinflammation in many neuropathophysiological diseases. Specifically, HMGB1 secreted by neurons is a critical mediator in neuroimmune crosstalk. Thus we hypothesized that HMGB1 is a key regulator involved in microglia activation in Ang II-induced hypertension. Neuronal and microglial cells in primary culture were established for this study. Effect of Ang II on microglial movement was recorded and quantified by time-lapse microscopy and video analysis. In addition, the expression, translocation and secretion of HMGB1 in neurons were measured by qPCR, Western blot and immunocytochemistry. Ang II treatment of neuronal-microglial co-culture resulted in a 5-fold increase of microglial migration towards neurons, which was completely abolished by treatment with 1 μM Losartan, an Ang II receptor type 1 antagonist. The presence of neurons was found to be essential for microglial movement since Ang II failed to induce these effects in the absence of neurons. Neurons express high levels of HMGB1, which was predominantly localized in the nucleus. Ang II treatment resulted in a time-dependent translocation of nuclear hMGB1 into the cytoplasm and ultimately into the medium. Both translocation and release of Ang II-induced HMGB1 was blocked by Losartan. Furthermore treatment of microglia with 500ng/ml recombinant HMGB1 caused ~2 fold increase of proinflammatory cytokines (TNF-α and IL-1β). The importance of HMGB1 is further underscored by our observation that its protein level was increased ~2 fold in the PVN but not in the nucleus of the solitary tract (NTS) or rostral ventrolateral medulla (RVLM) of the spontaneously hypertensive rats (SHR) compared with the normotensive WKY rats. Taken together, these observations suggest that Ang II primarily activates the neuronal AT1R to increase the expression, cytosolic translocation and release of HMGB1. The released HMGB1 primes microglial activation and release of cytokines, two key events in neuroinflammation in hypertension.

2-O, 3-O-Desulfated Heparin Inhibits Neutrophil Elastase–Induced HMGB-1 Secretion and Airway Inflammation

Neutrophil elastase (NE) is a major inflammatory mediator in cystic fibrosis (CF) that is a robust predictor of lung disease progression. NE directly causes airway injury via protease activity, and propagates persistent neutrophilic inflammation by up-regulation of neutrophil chemokine expression. Despite its key role in the pathogenesis of CF lung disease, there are currently no effective antiprotease therapies available to patients with CF. Although heparin is an effective antiprotease and anti-inflammatory agent, its anticoagulant activity prohibits its use in CF, due to risk of pulmonary hemorrhage. In this report, we demonstrate the efficacy of a 2-O, 3-O-desulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity, to inhibit NE activity and to block NE-induced airway inflammation. Using an established murine model of intratracheal NE-induced airway inflammation, we tested the efficacy of intratracheal ODSH to block NE-generated neutrophil chemoattractants and NE-triggered airway neutrophilic inflammation. ODSH inhibited NE-induced keratinocyte-derived chemoattractant and high-mobility group box 1 release in bronchoalveolar lavage. ODSH also blocked NE-stimulated high-mobility group box 1 release from murine macrophages in vitro, and inhibited NE activity in functional assays consistent with prior reports of antiprotease activity. In summary, this report suggests that ODSH is a promising antiprotease and anti-inflammatory agent that may be useful as an airway therapy in CF.

Inhibition of high-mobility group box 1 improves myocardial fibrosis and dysfunction in diabetic cardiomyopathy

BackgroundHigh-mobility group box 1 (HMGB1) is an important mediator of the inflammatory response. Its expression is increased in diabetic cardiomyopathy (DCM), but its role is unclear. We investigated the potential role and mechanism of HMGB1 in diabetes-induced myocardial fibrosis and dysfunction in mice. MethodsIn vivo, type 1 diabetes was induced by streptozotocin (STZ) in mice. HMGB1 expression was knocked down by lentivirus-mediated short-hairpin RNA (shRNA). Cardiac function was assessed by echocardiography. Total collagen deposition was assessed by Masson's trichrome and Picrosirius red staining. HMGB1, collagen I and III, and transforming growth factor β1 (TGF-β1) expression was quantified by immunostaining and western bolt analysis. In vitro, isolated neonatal cardiac fibroblasts were treated with high glucose (HG) or recombinant HMGB1 (rHMGB1). Pharmacologic (neutralizing anti-HMGB1 antibody) or genetic (shRNA-HMGB1) inhibition of HMGB1 was used to investigate the role of HMGB1 in HG-induced functional changes of cardiac fibroblasts. ResultsIn vivo, HMGB1 was diffusely expressed in the myocardium of diabetic mice. HMGB1 silencing ameliorated left ventricular dysfunction and remodeling and decreased collagen deposition in diabetic mice. In vitro, HG induced HMGB1 translocation and secretion in both viable cardiomyocytes and fibroblasts. Administration of rHMGB1 dose-dependently increased the expression of collagens I and III and TGF-β1 in cardiac fibroblasts. HMGB1 inhibition reduced HG-induced collagen production, matrix metalloproteinase (MMP) activities, proliferation, and activated mitogen-activated protein kinase signaling in cardiac fibroblasts. ConclusionsHMGB1 inhibition could alleviate cardiac fibrosis and remodeling in diabetic cardiomyopathy. Inhibition of HMGB1 might have therapeutic potential in the treatment of the disease.

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone.

LPS potentiates nucleotide-induced inflammatory gene expression in macrophages via the upregulation of P2Y2 receptor

Sepsis is a severe systemic inflammatory response that is associated with high morbidity and mortality. A previous study using an animal model of sepsis showed that survival was significantly lower in WT mice than in P2Y(2) receptor (P2Y(2)R)-deficient mice, suggesting that P2Y(2)R plays a role in septic death. We therefore investigated the role of P2Y(2)R in the inflammatory responses of RAW264.7 murine macrophages to LPS. LPS time-dependently upregulated P2Y(2)R mRNA levels, with a prominent increase observed at 4 h. In addition, LPS increased ATP release in a time dependent manner (5-120 min post LPS treatment). Accordingly, we pretreated cells with LPS for 4 h to induce P2Y(2)R expression and then stimulated the cells with UTP or ATP for 16 h. Interestingly, ATP- or UTP-dependent P2Y(2)R activation in LPS-pretreated cells resulted in dramatically enhanced HMGB1 secretion, COX-2 and iNOS expression, and furthermore PGE2 and NO production compared to LPS treatment alone (4 h) or ATP or UTP treatment alone (16 h), an effect that was inhibited by P2Y(2)R silencing. In addition, these increases in HMGB1 secretion, COX-2 and iNOS expression and PGE(2) and NO production commonly involved the JNK, PKC and PDK pathways. Taken together, these data demonstrate that LPS-dependent upregulation of P2Y(2)R plays a critical role in facilitating the inflammatory responses induced by LPS.

Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes.

P35 Deficiency Accelerates HMGB-1-mediated Neuronal Death in the Early Stages of an Alzheimer’s disease Mouse Model

The activities of CDK5 and p35 are thought to be important in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). We studied the effect of p35 deletion in Tg2576 mice, which is an AD animal model. To obtain the desired mice, we crossed p35(-/-) with Tg2576 mice. The resulting p35(-/-)/Tg2576 (KO/Tg) mice displayed higher mortality rates and exhibited impaired spatial learning and memory at 6 months of age. Using immunohistochemical and biochemical approaches, we observed a reduction in the expression of pre- and post-synaptic markers such as NMDAR1, synaptophysin and GluR1. In addition, the intensity of MAP-2-positive dendrites extending from neuronal cell bodies was significantly decreased in KO/Tg mice compared with KO/WT and WT/Tg mice. We also detected increased neuronal cell death in the hippocampus, along with thinned and collapsed morphological changes in the alveus region and a dramatic increase in the number of microglial cells. Microglial infiltration in the hippocampus could result in the increased secretion of the soluble high mobility group box-1 protein (HMGB-1). The secretion of HMGB-1 is increased by Aβ, and secretion of HMGB-1 promotes neuronal cell death. Moreover, we found that HMGB-1 secretion induced by Aβ in KO/Tg mice gave rise to ER-mediated cell death. In summary, during the stages of KO/Tg mice model, the microglial infiltration and secretion of soluble HMGB-1 were significantly increased in the hippocampus. These conditions promote neuronal death, synaptic destruction and behavioral deficits.

Endogenous HMGB1 is required in endotoxin tolerance

BackgroundHigh-mobility group box 1 protein (HMGB1), a downstream inflammatory response modifier in sepsis and endotoxemia, alters endotoxin tolerance by affecting cellular hyporesponsiveness and tumor necrosis factor α and interleukin 1 production. ObjectiveEndogenous HMGB1 signaling mechanisms during low-dose lipopolysaccharide (LPS)-induced endotoxin tolerance were investigated. MethodsBALB/c mice were preconditioned with either 0.1 mL low-dose LPS (0.2 mg/kg) or phosphate-buffered saline (PBS) (control) followed by treatment with three consecutive injections of anti-HMGB1, IgY (an nonspecific antibody), or PBS, at 2, 12, and 22 h, respectively, Mice were then subjected to 0.1 mL high-dose LPS (10 mg/kg) or PBS at 24 h. Serum and hepatic tissue samples were obtained 1 or 3 h after final treatments. Signaling mechanisms were further investigated in the serum and hepatic tissues of mice preconditioned with 0.1 mL HMGB1 (1 mg/kg), low-dose LPS (0.2 mg/kg), or PBS for 1 h, and then high-dose LPS treatment for 3 h. ResultsThe signaling mechanisms involved in low-dose LPS preconditioning required enhanced endogenous HMGB1 expression and secretion. Neutralizing endogenous HMGB1 with anti-HMGB1 antibodies following low-dose LPS preconditioning altered endotoxin tolerance by increasing serum tumor necrosis factor α, reducing hepatic interleukin-1R-associated kinase M expression, and partially restoring nuclear factor κB in vivo. The translocation from nucleus to cytoplasm of endogenous HMGB1 in RAW264.7 cells was also observed during low-dose LPS–induced endotoxin tolerance. ConclusionsIncreased interleukin-1R-associated kinase M and decreased nuclear factor κB activity in endotoxin tolerance is associated with endogenous HMGB1 expression after low-dose LPS preconditioning. These findings provide a basis for a better mechanistic understanding and the development of safer clinical therapeutics utilizing induced endotoxin tolerance.

Targeted delivery of HMGB1 siRNA to macrophages and dendritic cells for sepsis treatment in humanized mouse model (P6223)

Abstract Over expression cytokine by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient NOD/SCID/IL2Rγ-/- mice and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, as compared to immunocompetent WT mice, NOD/SCID/IL2Rγ-/- mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ-/- mice transplanted with human hematopoietic stem cells (humanized BLT mice) showed high serum levels of HMGB1 as well as multiple human, but not murine proinflammatory cytokines and uniformly succumbed, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short AchR binding peptide (RVG-9R) effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis and rescued humanized mice from CLP-induced mortality. SiRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that: CLP in humanized mice provides a model to study human sepsis; HMGB1 siRNA might provide a treatment strategy for human sepsis; and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used suppress a variety of human inflammatory diseases.

Neuropeptide Y induces secretion of high-mobility group box 1 protein in mouse macrophage via PKC/ERK dependent pathway

Despite increasing evidence highlighting the role of NPY in the modulation of inflammatory reaction, surprisingly little is known about the direct effects of NPY on the release of proinflammatory mediators. In the present work, we have evaluated the effects of NPY on the release of TNF-α, IL-1β, IL-6 and HMGB1 mediators in peritoneal macrophages. Our results demonstrate for the first time that NPY can directly induce active HMGB1 release and cytoplasmic translocation, while the production of TNF-α, IL-1β and IL-6 is not affected. PKC and ERK pathway inhibitors can abolish the promotive effect of NPY on HMGB1 secretion. Thus, our results indicate that NPY might impact on the innate immune system by directly potentiating the HMGB1 release from the macrophage.

Abstract 1676: Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.

Abstract Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from immunogenic cell death. Docetaxel treatment of tumor cells did not induce ATP or HMGB1 secretion, or cell death. However, calreticulin exposure was observed in all cell lines examined after chemotherapy treatment. Killing by CEA, MUC-1, or PSA-specific CD8+ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by calreticulin membrane translocation, as determined by functional knockdown of calreticulin, PERK, or calreticulin-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1+, CD133+) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to immunogenic cell death and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy. Citation Format: James W. Hodge, Charlie T. Garnett, Benedetto Farsaci, Claudia Palena, Kwong-Yok Tsang, Soldano Ferrone, Sofia R. Gameiro. Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1676. doi:10.1158/1538-7445.AM2013-1676

Abstract 3588: Common and different effects induced in primary human mesothelial cells and mice exposed to chrysotile or crocidolite asbestos.

Abstract Crocidolite is considered the most oncogenic asbestos type. Chrysotile accounts for &amp;gt;90% of asbestos used worldwide, however its capacity to induce MM is still debated. We linked crocidolite carcinogenesis to the release of High-Mobility Group Box-1 protein (HMGB1) and activation of downstream genes (TNF-α and NF-κB). We compared the carcinogenicity of crocidolite and chrysotile in primary human mesothelial cells (HM). Microarray analyses of HM exposed to crocidolite or chrysotile identified 57 genes that were significantly induced or repressed. Forty-eight hours after exposure we observed induction of HMGB1 and NF-κB targeted genes and E-cadherin and SFRP4 down-regulation. In vitro, both crocidolite and chrysotile induced HMGB1 and TNF-α secretion, induced β-catenin nuclear translocation and caused HM death and epithelial-mesenchymal transition in surviving cells. Five weeks from exposure these effects were sustained in HM exposed to crocidolite; the expression of these genes had returned to normal in HM exposed to chrysotile. Soon after injection, serum HMGB1 levels were elevated in mice injected with either crocidolite or chrysotile, and remained elevated 10 weeks post-injection only in mice injected with crocidolite. Our results show that chrysotile induces the same intracellular signaling elicited by crocidolite, however these effects are transient. Accordingly, HM exposure to chrysotile in the presence of TNF-α or in co-culture with macrophages, induced foci formation with a lower transforming efficiency compared to crocidolite. Citation Format: Fang Qi, Gordon Okimoto, Sandro Jube, Harvey I. Pass, Rozalia Laczko, Richard M. DeMay, Ghazal Khan, Maarit Tiirikainen, Haining Yang, Giovanni Gaudino, Michele Carbone. Common and different effects induced in primary human mesothelial cells and mice exposed to chrysotile or crocidolite asbestos. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3588. doi:10.1158/1538-7445.AM2013-3588