Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis

Enhanced adrenergic stimulation and catecholamine release are important components of the pathophysiology of sepsis. Under physiological conditions, adrenergic stimulation has been shown to be a negative regulator of proinflammatory cytokine production through increasing IL-10 production. Here we have investigated if adrenergic stimulation similarly inhibits TNF- and IL-6 production by splenic macrophages isolated from a polymicrobial sepsis model. Male B₆D₂F₁ mice were subjected to sham (S), laparotomy (Lap), and cecal ligation and puncture (CLP) under anesthesia. Splenic macrophages were isolated 72 h after the initial injury and were stimulated with endotoxin (LPS) in the presence and absence of epinephrine. Compared with S and Lap, splenic macrophages from the CLP group produced significantly less TNF- and IL-6 and more IL-10 when stimulated with LPS. Macrophage cultures from CLP animals incubated with either epinephrine or IL-10 for 2 h had significantly reduced TNF- and IL-6 release in response to LPS. However, similar cultures pretreated with IL-10 antibody before the addition of exogenous epinephrine failed to reverse the attenuation of LPS-stimulated cytokines. Pretreatment of macrophage cultures with ₂- (ICI-118551) but not ₁-adrenergic (atenolol) receptor antagonists reversed the epinephrine-mediated cytokine attenuation following LPS treatment. Data are also presented that demonstrate the involvement of protein kinase A activation with adrenergic agonist but not with IL-10 stimulation. Taken together, these findings suggest that adrenergic mechanisms may influence peripheral tissue macrophage inflammatory cytokine response following trauma and sepsis, independent of the effects of IL-10. catecholamines; sympathetic; cecal ligation and puncture; bacterial endotoxin; -antagonist     

Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis

Physiological anti-inflammatory mechanisms can potentially be exploited for the treatment of inflammatory disorders. Here we report that the neurotransmitter acetylcholine inhibits HMGB1 release from human macrophages by signaling through a nicotinic acetylcholine receptor. Nicotine, a selective cholinergic agonist, is more efficient than acetylcholine and inhibits HMGB1 release induced by either endotoxin or tumor necrosis factor-alpha (TNF-alpha). Nicotinic stimulation prevents activation of the NF-kappaB pathway and inhibits HMGB1 secretion through a specific 'nicotinic anti-inflammatory pathway' that requires the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In vivo, treatment with nicotine attenuates serum HMGB1 levels and improves survival in experimental models of sepsis, even when treatment is started after the onset of the disease. These results reveal acetylcholine as the first known physiological inhibitor of HMGB1 release from human macrophages and suggest that selective nicotinic agonists for the alpha7nAChR might have therapeutic potential for the treatment of sepsis.     

The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1ΔC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1ΔC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.     

HMGB1 release promotes paclitaxel resistance in castration-resistant prostate cancer cells via activating c-Myc expression

Paclitaxel (PTX) is one of standard chemotherapy drug for patients with metastatic castration-resistant prostate cancer (mCRPC). However, PTX resistance leads to treatment failures, for which the underlying molecular mechanisms remain exclusive. In this study, we reported that PTX-induced constant HMGB1 expression and release confers to PTX resistance in mCRPC cells via activating and sustaining c-Myc signaling. PTX upregulated HMGB1 expression and triggered its release in human mCRPC cells. Silencing HMGB1 by RNAi and blocking HMGB1 release by glycyrrhizin or HMGB1 neutralizing antibody sensitized the response of PTX-resistant mCRPC cells to PTX. Release HMGB1 activated c-Myc expression. Inhibiting c-Myc expression by RNAi or c-MyC inhibitor significantly enhance the sensitivity of PTX-resistant CRPC cells to PTX. Therefore, HMGB1/c-Myc axis is critical in the development of PTX resistance, and targeting HMGB1/c-Myc axis would counteract PTX resistance in mCRPC cells.     

Interplay between in vitro acetylation and phosphorylation of tailless HMGB1 protein

The postsynthetic acetylation of HMGB1 protein and its truncated form affects significantly its properties as “architectural” factor - recognition of bent DNA and bending of short DNA fragments. We created mutants at the target sites (lysines 2 and 81) in the tailless HMGB1 modified by the histone acetyltransferase CBP. The results show that there is no preferential site for the enzymatic activity of CBP and both lysine moieties are modified independently. Our findings for the first time demonstrate the link between the acetylation and phosphorylation of HMGB1ΔC in vitro. The PKC phosphorylation prior to acetylation inhibits the CBP activity 40-60% for the truncated form and its mutants. The effect of the CBP acetylation on the phosphorylation level turns out to be much more prominent. In the case of HMGB1ΔC modified at Lys 2 and Lys 81 prior to PKC treatment background phosphorylation is detected. If only one of the lysines is modified the inhibitory effect decreases.     

高迁移率族蛋白1在脓毒症血管内皮细胞激活过程中的作用

血管内皮细胞激活是脓毒症病理生理过程的中心环节。活化的血管内皮细胞为炎症介质的聚集和迁移提供了重要的场所,是放大炎症反应的前提条件。高迁移率族蛋白1（high-mobility group box protein1,HMGB1）是脓毒症晚期致死性的促炎介质,维持并延长了脓毒症病理过程。HMGBl通过晚期糖基化终产物受体（advanced glycation end products receptor,RAGE）对血管内皮细胞有重要的激活作用。     

Microtubule acetylation promotes kinesin-1 binding and transport

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.     

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.     

Irradiation-induced osteocyte damage promotes HMGB1-mediated osteoclastogenesis in vitro

Irradiation-induced bone loss is widely reported, especially in radiotherapy-induced osteoporosis. In addition to the mechanism of osteogenesis inhibition and osteoclastogenesis promotion, the regulation effect of osteocytes, which also send signals to modulate osteoclastogenesis, should be elucidated. In this study, the effect of irradiation on osteocyte and its accommodation to osteoclastogenesis via the release of high mobility group box 1 (HMGB1) was explored. Furthermore, the control response of HMGB1 inhibitor on receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression in osteocyte and osteocyte-induced osteoclastogenesis was assessed. It was observed that irradiated osteocyte-like MLO-Y4 cells exhibited polygonal-shaped morphological changes and shortened dendrites, inhibited cell viability and induced cellular apoptosis, along with the reduction in dendritic E11 protein/messenger RNA expression at a doses of 4 Gy. Additionally, the secretion of HMGB1 in supernatants was promoted, accompanied by the decreased OPG and elevated RANKL expression. When the RAW264.7 cells were cocultured with irradiated MLO-Y4 cells or its conditioned medium, enhanced migration and differentiation of osteoclast precursor was observed, and this difference was alleviated with anti-HMGB1 neutralizing antibody. In conclusion, this study demonstrated that irradiation deteriorated osteocytes’ potential to promote recruitment and differentiation of osteoclast precursor via stimulating HMGB1 release and subsequent elevation of RANKL/OPG level. This study will assist in designing the intervention programs for irradiation-induced bone loss.     

Suppression of HMGB1 release by stearoyl lysophosphatidylcholine:an additional mechanism for its therapeutic effects in experimental sepsis

Stearoyl lysophosphatidylcholine (LPC) has recently been proven protective against lethal sepsis by stimulating neutrophils to eliminate invading pathogens through an H2O2-dependent mechanism. Here, we demonstrate that stearoyl LPC, but not caproyl LPC, significantly attenuates circulating high-mobility group box 1 (HMGB1) levels in endotoxemia and sepsis by suppressing endotoxin-induced HMGB1 release from macrophages/monocytes. Neutralizing antibodies against G2A, a potential cell surface receptor for LPC, partially abrogated stearoyl LPC-mediated suppression of HMGB1 release. Thus, stearoyl LPC confers protection against lethal experimental sepsis partly by facilitating the elimination of the invading pathogens and partly by inhibiting endotoxin-induced release of a late proinflammatory cytokine, HMGB1.     

Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.     

Cell death, BAX activation, and HMGB1 release during infection with Chlamydia

Infection by a number of Chlamydia species leads to resistance of the host cell to apoptosis, followed by induction of host-cell death. In a population of infected cells that displays protection against staurosporine-induced apoptosis among the adherent cells, we find that cells that had been recovered from the supernatant share characteristics of both apoptosis and necrosis, as assayed by the propidium iodide (PI)-annexin V double-labeling technique. Cell death was observed in both an epithelial cell line and primary fibroblasts, although the primary cells had a higher propensity to die through apoptosis than the immortalized cell line. Staurosporine-mediated activation of the pro-apoptotic BCL-2 family member, BAX, was inhibited in the epithelial cell line infected for 32 h with the lymphogranuloma venereum (LGV/L2) but not the murine pneumonitis (MoPn) strain of C. trachomatis, but inhibition of staurosporine-mediated BAX activation disappeared after 48 h of infection with the LGV/L2 strain. Conversely, infection with MoPn (C. muridarum) but not LGV/L2 led to BAX activation after 72 h, as previously reported for shorter (48 h) infection with the guinea pig inclusion conjunctivitis (GPIC) serovar of C. psittaci (C. caviae). These results suggest that the ability to inhibit staurosporine-mediated BAX activation or to activate BAX due to the infection itself may vary as a function of the chlamydial strain. Interestingly, both the epithelial cells and the fibroblasts also released high mobility group box 1 protein (HMGB1) during infection, although much less HMGB1 was released from fibroblasts, consistent with the higher level of apoptosis observed in the primary cells. HMGB1 is released preferentially by necrotic or permeabilized viable cells, but not apoptotic cells. In the extracellular space, HMGB1 promotes inflammation through interaction with specific cell-surface receptors. Higher levels of HMGB1 were also measured in the genital-tract secretions of mice infected vaginally with C. trachomatis, compared to uninfected controls. These results suggest that cells infected with Chlamydia release intracellular factors that may contribute to the inflammatory response observed in vivo.     

IL-10 mediation of activation-induced TH1 cell apoptosis and lymphoid dysfunction in polymicrobial sepsis

Recent studies suggest that increased activation-induced lymphocyte apoptosis (AICD) is detected in mouse splenocytes during polymicrobial sepsis which may contribute to lymphocyte immune dysfunction [i.e., decreased interleukin (IL-)2 and interferon-gamma (IFN-gamma) production] leading to the associated morbidity seen in those animals. Thus, we wanted to examine the hypothesis that immune suppressive agents, such as IL-4, IL-10 or prostaglandin E2(PGE2), known to be elevated in septic animals, also contribute to this increase in AICD. Here we demonstrate that the inclusion of monoclonal antibody (mAb) to IL-10, but not anti-IL-4 or ibuprofen (IBU), blunted this sepsis induced increase in splenocyte AICD. Additionally, septic mice deficient in the IL-10 gene product (-/-) showed neither an increase in AICD nor a loss of IL-2/IFN-gamma release capacity. Interestingly, mAb to IL-10 did not altered the extent of AICD in a Th2-cell line, but exogenous IL-10 did potentiate Th1-like cell line AICD. This was consistent with the finding that the increased AICD seen in septic mouse splenocytes was restricted largely to the CD4+ cells producing IL-2 (Th1-cells) and that mAb to IL-10 treatment suppressed this change. Furthermore, IL-10 appears to mediate its AICD effect by upregulation of the Fas receptor and Fas receptor signaling protein components, but not by altered expression of Bcl/Bax/Bad family members, in septic mouse splenocytes. To the extent that these processes contribute in a pathological fashion to the animal's capacity to survive sepsis we have previously observed that in vivo post-treatment of mice with mAb IL-10 markedly attenuated septic mortality. Collectively, these data indicate that in the septic mouse the Th2 cytokine IL-10 not only serves to actively induce Th1 lymphocyte immune dysfunction but also plays a role in their apoptotic depletion. These processes in turn appear to contribute to the animal's inability to ward off lethal septic challenge.     

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.     

Construction and characterization of the HMGB1 mutant as a competitive antagonist to HMGB1 induced cytokines release

Recent studies indicate that the High Mobility Group Box-1 protein (HMGB1) acts as a potent proinflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. The proinflammatory cytokine activity of HMGB1 has become a therapeutic target. In this study, we cloned the cDNA encoding human HMGB1 and constructed HMGB1 mutants using a one-step opposite direction PCR. The binding of the HMGB1 mutants to THP-1 cell and the cytokine activities of these HMGB1 mutants were observed. Results showed that the HMGB1 Mut (102-105), one of the HMGB1 mutants, in which amino acids 102-105 (FFLF) were replaced with two Glys, significantly decreased the full-length HMGB1 protein induced TNF-α release in human monocyte cultures. The results indicate that we have developed a novel recombinant HMGB1 mutant that competitively antagonizes the proinflammatory activity of HMGB1. This may be of significant importance in providing a new anti-inflammatory strategy for the treatment of severe sepsis and other inflammatory disorders.     

A small molecule binding HMGB1 inhibits caspase-11-mediated lethality in sepsis

Caspase-11, a cytosolic lipopolysaccharide (LPS) receptor, mediates lethal immune responses and coagulopathy in sepsis, a leading cause of death worldwide with limited therapeutic options. We previously showed that over-activation of caspase-11 is driven by hepatocyte-released high mobility group box 1 (HMGB1), which delivers extracellular LPS into the cytosol of host cells during sepsis. Using a phenotypic screening strategy with recombinant HMGB1 and peritoneal macrophages, we discovered that FeTPPS, a small molecule selectively inhibits HMGB1-mediated caspase-11 activation. The physical interaction between FeTPPS and HMGB1 disrupts the HMGB1-LPS binding and decreases the capacity of HMGB1 to induce lysosomal rupture, leading to the diminished cytosolic delivery of LPS. Treatment of FeTPPS significantly attenuates HMGB1- and caspase-11-mediated immune responses, organ damage, and lethality in endotoxemia and bacterial sepsis. These findings shed light on the development of HMGB1-targeting therapeutics for lethal immune disorders and might open a new avenue to treat sepsis.Subject terms: Cell death and immune response, Sepsis     

STIM1 promotes angiogenesis by reducing exosomal miR-145 in breast cancer MDA-MB-231 cells

Cancer cells secrete abundant exosomes, and the secretion can be promoted by an increase of intracellular Ca²⁺. Stromal interaction molecule 1 (STIM1) plays a key role in shaping Ca²⁺ signals. MicroRNAs (miRNAs) have been reported to be potential therapeutic targets for many diseases, including breast cancer. Recently, we investigated the effect of exosomes from STIM1-knockout breast cancer MDA-MB-231 cells (Exo-STIM1-KO), and from {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365-treated MDA-MB-231 cells (Exo-SKF) on angiogenesis in human umbilical vein endothelial cells (HUVECs) and nude mice. The exosomes Exo-STIM1-KO and Exo-SKF inhibited tube formation by HUVECs remarkably. The miR-145 was increased in {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 treated or STIM1-knockout MDA-MB-231 cells, Exo-SKF and Exo-STIM1-KO, and HUVECs treated with Exo-SKF or Exo-STIM1-KO. Moreover, the expressions of insulin receptor substrate 1 (IRS1), which is the target of miR-145, and the downstream proteins such as Akt/mammalian target of rapamycin (mTOR), Raf/extracellular signal regulated-protein kinase (ERK), and p38 were markedly inhibited in HUVECs treated with Exo-SKF or Exo-STIM1-KO. Matrigel plug assay in vivo showed that tumor angiogenesis was suppressed in Exo-STIM1-KO, but promoted when miR-145 antagomir was added. Taken together, our findings suggest that STIM1 promotes angiogenesis by reducing exosomal miR-145 in breast cancer MDA-MB-231 cells.Subject terms: Tumour angiogenesis, Cancer microenvironment