Fosfomycin alters lipopolysaccharide-induced inflammatory cytokine production in mice

To determine the mechanisms of immunomodulating action of fosfomycin (FOF), we examined its effect on the production of inflammatory cytokines in mice injected with lipopolysaccharide (LPS). Treatment with FOF significantly lowered the peak serum levels of tumor necrosis factor alpha and interleukin-1 beta, indicating that FOF alters inflammatory cytokine production after LPS stimulation.     

Interleukin 1 receptor signaling regulates DUBA expression and facilitates Toll-like receptor 9-driven antiinflammatory cytokine production

The interleukin 1 receptor (IL-1R) and the Toll-like receptors (TLRs) are highly homologous innate immune receptors that provide the first line of defense against infection. We show that IL-1R type I (IL-1RI) is essential for TLR9-dependent activation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and for production of the antiinflammatory cytokines IL-10 and type I interferon (IFN). Noncanonical K63-linked ubiquitination of TRAF3, which is essential for type I IFN and IL-10 production, was impaired in Il1r1(-/-) CD11c(+) dendritic cells. In contrast, degradative ubiquitination of TRAF3 was not affected in the absence of IL-1R1 signaling. Deubiquitinating enzyme A (DUBA), which selectively cleaves K63-linked ubiquitin chains from TRAF3, was up-regulated in the absence of IL-1R1 signaling. DUBA short interference RNA augmented the TLR9-dependent type I IFN response. Mice deficient in IL-1RI signaling showed reduced expression of IL-10 and type I IFN and increased susceptibility to dextran sulphate sodium-induced colitis and failed to mount a protective type I IFN response after TLR9 ligand (CpG) administration. Our data identifies a new molecular pathway by which IL-1 signaling attenuates TLR9-mediated proinflammatory responses.     

A novel DNA vaccine-targeting macrophage migration inhibitory factor improves the survival of mice with sepsis

Sepsis is a common and frequently fatal condition and there is an urgent need for new therapies that will further reduce sepsis-induced mortality. Macrophage migration inhibitory (MIF) factor is important in the regulation of innate and adaptive immunity and is believed to play a key regulatory role in sepsis and autoimmune disease. As MIF deficiency or immunoneutralization protects mice or rats from fatal endotoxic shock or other inflammatory diseases, we examined whether DNA vaccination against this molecule would also be protective. DNA vaccines can stimulate both humoral and cellular immunity simultaneously and have been shown to be effective against a variety of pathogens or cytokine-driven pathologies. Mice were immunized with a MIF/tetanus toxin (TTX) DNA vaccine and sepsis was then induced by lipopolysaccharide or cecal ligation and puncture. The MIF/TTX DNA-vaccinated mice were protected from the lethal effect of sepsis compared with control-vaccinated mice in both models. Compared with the control-vaccinated mice, the MIF/TTX DNA-vaccinated mice also showed significantly lower serum tumor necrosis factor (TNF)-alpha protein levels and reduced mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, macrophage inflammatory protein-2 and Toll-like receptor-4 in the lungs. Thus, the MIF/TTX DNA vaccine may be useful for the prophylaxis of septic shock.     

脂氧素A4对内毒素诱导小鼠肺内炎症反应的影响

目的 探讨脂氧素A4对脂多糖诱导小鼠急性肺内炎症反应的影响。方法 36只雄性C57BL／6小鼠，随机分为对照组、脂氧素A4组、ZnPP组、内毒素组、脂氧素A4治疗组和ZnPP＋脂氧素A4治疗组，每组6只。雾化吸入脂多糖8h后，测定肺泡灌洗液总蛋白浓度和TNF—α含量，测定肺组织湿干质量比值、丙二醛（MDA）活性和一氧化氮（NO）浓度，免疫组化测定肺组织HO-1的蛋白表达。结果 与LPS组比较，脂氧素A4治疗组肺水肿减轻，肺泡灌洗液总蛋白浓度、TNF—α含量、肺组织MDA活性、NO含量均降低（P〈0.01），而HO-1的蛋白表达明显升高，肺组织损伤减轻。HO-1抑制剂ZnPP减弱脂氧素A4保护作用。结论 脂氧素A4能减轻内毒素诱导的肺部急性炎症反应，HO-1介导其保护作用。     

Fraxin inhibits lipopolysaccharide-induced inflammatory cytokines and protects against endotoxic shock in mice

Fraxin, the effective component isolated from Cortex Fraxini, has been reported to have anti-inflammation effects. The aim of this study was to explore the effect of fraxin on lipopolysaccharide (LPS)-induced endotoxic shock in mice. We used Kunming male mice to establish the model, and we found that fraxin could improve the survival rate of the LPS-induced mice. Histopathological study showed that fraxin could mitigate the injuries in LPS-induced lung and liver tissues. The levels of tumour necrosis factor-α and interleukin-6 both in serum and lung, liver tissues, and the productions of nitric oxide (NO), aspartate transaminase and alanine transaminase in serum were decreased by fraxin. Western blot assay demonstrated that the pretreatment with fraxin could downregulate LPS-induced protein expressions of nuclear factor-kappa B (NF-κB) and NLRP3 inflammatory corpuscle signalling pathways. Overall, fraxin had protective effects on LPS-induced endotoxic shock mice and the possible mechanisms might activate through NF-κB and NLRP3 inflammatory corpuscle signalling pathways.     

Modulation of atherosclerosis in mice by Toll-like receptor 2

Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led to striking protection against Pam3CSK4-mediated atherosclerosis, suggesting a role for BM-derived cell expression of TLR2 in transducing the effects of an exogenous TLR2 agonist. These studies support the concept that chronic or recurrent microbial infections may contribute to atherosclerotic disease. Additionally, these data suggest the presence of host-derived endogenous TLR2 agonists.     

SREBP-2 regulates gut peptide secretion through intestinal bitter taste receptor signaling in mice

Bitter taste-sensing G protein-coupled receptors (type 2 taste receptors [T2Rs]) are expressed in taste receptor cells of the tongue, where they play an important role in limiting ingestion of bitter-tasting, potentially toxic compounds. T2Rs are also expressed in gut-derived enteroendocrine cells, where they have also been hypothesized to play a role in limiting toxin absorption. In this study, we have shown that T2R gene expression in both cultured mouse enteroendocrine cells and mouse intestine is regulated by the cholesterol-sensitive SREBP-2. In addition, T2R stimulation of cholecystokinin (CCK) secretion was enhanced directly by SREBP-2 in cultured cells and in mice fed chow supplemented with lovastatin and ezetimibe (L/E) to decrease dietary sterol absorption and increase nuclear activity of SREBP-2. Low-cholesterol diets are naturally composed of high amounts of plant matter that is likely to contain dietary toxins, and CCK is known to improve dietary absorption of fats, slow gastric emptying, and decrease food intake. Thus, these studies suggest that SREBP-2 activation of bitter signaling receptors in the intestine may sensitize the gut to a low-fat diet and to potential accompanying food-borne toxins that make it past the initial aversive response in the mouth.     

Alterations in the cardiac inflammatory response to burn trauma in mice lacking a functional Toll-like receptor 4 gene

Our group and others have previously shown that Toll-like receptor 4 (TLR-4) inactivation prevents burn-induced myocardial contractile dysfunction; however, the molecular mechanisms that are involved in this cardioprotection are not well defined. This present study examines the involvement of TLR-4 in the cardiac inflammatory response to thermal insult. C3H/HeJ (TLR-4 mutant mice) and C3H/HeN wild-type (WT) mice were subjected to either a sham burn or 40% full-thickness burn injury and were fluid resuscitated with lactated Ringer using the Parkland formula. Mice (n = 7-9 per group) were killed at 2, 4, or 24 h postsham or burn, and heart tissue was harvested. Immunoblotting was performed to evaluate phosphorylated p38 mitogen-activated protein kinase (MAPK), nuclear p50, and cytoplasmic p50. Nuclear factor-kappaB was also characterized via electrophoretic mobility shift assay. Systemic and cardiac myocyte secretion of TNF-alpha, IL-1 beta, IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay. Burn injury in WT mice promoted myocardial inflammatory signaling that included increased expression of phosphorylated p38 MAPK, nuclear p50, and increased cardiac myocyte secretion of cytokines. Systemic cytokines were also increased in WT animals, although not to the extent of the myocardial cytokine expression. Toll-like receptor 4 inactivation resulted in an attenuation of several burn-induced responses, including phosphorylation of p38 MAPK, nuclear translocation of nuclear factor-kappaB, and cytokine secretion. These data suggest that burn injury initiates an inflammatory response via Toll/IL-1 signaling in the heart, which contributes to cardiac injury and contractile dysfunction.     

Toll-like receptor 7 regulates pancreatic carcinogenesis in mice and humans

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-β, PPARγ, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-κB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.     

A novel class of pyranocoumarin anti-androgen receptor signaling compounds

Androgen and the androgen receptor (AR)-mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti-androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC(50) of approximately 1 mumol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G(0/1) arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects.     

Implication of Toll-like receptor and tumor necrosis factor alpha signaling in septic shock

Septic shock is initiated by a systemic inflammatory response to microbial infection that frequently leads to impaired perfusion and multiple organ failure. Because of its high risk of death, septic shock is a major problem particularly for patients in the intensive care unit. In general, bacterial lipopolysaccharide (LPS) is a strong activator of various immune responses and stimulates monocytes/macrophages to release a variety of inflammatory cytokines. However, overproduction of inflammatory factors in response to bacterial infections is known to cause septic shock, similar to that induced by LPS. Studies of LPS-signaling pathways and downstream inflammatory cytokines may have critical implications in the treatment of sepsis. In recent years, there has been significant progress in understanding the signaling pathways activated by LPS and its receptor Toll-like receptor 4 (TLR4), as well as by tumor necrosis factor alpha (TNFalpha), a potent inflammatory cytokine induced by LPS stimulation. This review briefly summarizes our current knowledge of these signaling pathways and critical signal transducers. Characterization of key signal transducers may allow us to identify tractable, novel targets for the therapeutic interventions of sepsis.     

Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2

Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.     

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr^–/– osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer.     

HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.     

霉酚酸酯对小鼠肾脏缺血再灌注损伤早期单核细胞TLR4信号传导途径的影响

目的探讨在小鼠肾脏缺血再灌注免疫损伤早期过程中霉酚酸酯对单核细胞TLR4信号传导途径的影响。方法采用雄性Bal B/C小鼠,随机分为2组。肾脏缺血再灌注损伤(IRI)组:采用双肾蒂阻断,缺血时间为45 min,分别于6 h、12 h、24 h、48 h再灌注。每个时间点6只小鼠;霉酚酸酯(MMF)药物组:缺血前2 d开始灌胃给MMF 40 mg?kg-1?d-1,其余同肾脏缺血再灌注损伤组。两组均设假手术对照各6只;观察不同时间点各组动物肾功能、肾组织学变化;流式细胞学检测单核细胞表面TLR4的表达及CBA法检测血浆细胞因子(IL-6、MCP-1、TNF-α)浓度;ELISA法检测血浆中TLR4配体HMGB-1的浓度。结果 MMF组较IRI组血肌酐水平明显降低(再灌注6 h、12 h、24 h、48 h,均P<0.05),病理组织学观察提示MMF组肾脏损伤程度较轻;而血浆中TLR4配体HMGB-1浓度无显著性差异(P>0.05),单核细胞表面TLR4表达及血浆细胞因子(IL-6、MCP-1、TNF-α)浓度均明显降低(P<0.05)。结论霉酚酸酯可减轻小鼠肾脏缺血再灌注损伤后肾功能和肾组织的损伤,可抑制小鼠肾脏缺血再灌注损伤早期过程中单核细胞TLR4信号转导途径的活化。提示单核细胞TLR4信号转导途径在小鼠肾脏缺血再灌注损伤早期过程中起着重要的调控作用,而霉酚酸酯可以通过抑制该途径对肾脏起到保护作用。     

Mechanisms of increased survival after lipopolysaccharide-induced endotoxic shock in mice consuming olive oil-enriched diet

We examined the impact of dietary fatty acid intake on lipopolysaccharide (LPS)-induced endotoxic shock. C57Bl/6J mice were fed for 6 weeks with a commercial laboratory chow (CC) or with test chows containing 7% (w/w) canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). The increase in body weight and energy consumption were similar for all diets tested. In the sixth week, mice were injected intraperitoneally with 400 microg of bacterial LPS to induce endotoxic shock. LPS induced a massive neutrophil infiltration into the peritoneal cavity and an increase in lipid body (LB) formation in leukocytes recovered from the peritoneal fluid of mice fed with CC, CO, SeO, or SO. In addition, there were increases in prostaglandin E(2) (PGE(2)), leukotriene B4 (LTB(4)), and cytokines IL-6, IL-10, and MCP-1 in peritoneal lavage, as well as in plasma TNF-alpha. In contrast, mice fed with OO exhibited reduced neutrophil accumulation and LB formation, and also had lower levels of PGE(2), LTB(4), MCP-1, and TNF-alpha. All mice fed with CC, CO, SeO, or SO died within 48 to 72 h after LPS injection. Interestingly, mice fed with the OO diet were resistant to endotoxic shock, with 60% survival at 168 h. These data indicate that intake of OO may have a beneficial role, reducing the magnitude of the inflammatory process triggered by endotoxic shock through modulation of LB formation and of the production of inflammatory mediators.     

Effect of rifampin on production of inflammatory mediators in HepG2 liver epithelial cells

Rifampin, a potent antibacterial agent, is one of the main drugs used in the treatment of mycobacterial infections. Hepatotoxicity is a well-documented adverse event. The aim of this study was to investigate the effect of rifampin on the production of inflammatory mediators in human epithelial HepG2 liver cells in the absence or presence of proinflammatory cytokines. Incubation of HepG2 cells with a cytokine mix plus rifampin was associated with a significant dose-dependent increase in the production of nitric oxide compared to incubation with the cytokine mix alone (P < 0.05) as well as with an increase in inducible nitric oxide synthase protein and mRNA expression. Rifampin significantly increased the secretion of interleukin 8 (IL-8) in both untreated cells (P < 0.001) and cytokine-treated cells (P < 0.006). An array screening assay revealed that rifampin stimulated the production of IL-1β and gamma interferon-induced protein-10 (IP-10) in untreated cells and increased the secretion of RANTES in cytokine-treated cells. Together, these results indicate that rifampin may exert proinflammatory effects on liver cells.     

骨髓间充质干细胞下调类风湿性关节炎小鼠脾脏单个核细胞TLR8及TLR9的表达

背景:间充质干细胞能够缓解类风湿性关节炎小鼠的症状,但是其机制还不清楚.目的:观察骨髓间充质干细胞对类风湿性关节炎小鼠脾脏单个核细胞表达TLR8及TLR9等的影响.方法:DBA/1J小鼠随机分3组,非造模组不造模,阳性对照组和实验组制备Ⅱ型胶原诱导的小鼠类风湿性关节炎模型.实验组尾静脉注射移植大鼠骨髓间充质干细胞.结果与结论:与阳性对照组比较,实验组小鼠关节直径明显减小,关节炎症细胞浸润程度明显降低,但比非造模组略高;小鼠脾脏单个核细胞表达TLR8、TLR9和白细胞介素1β的水平较阳性对照组明显降低(P < 0.01或P < 0.05);阳性对照组与实验组中TLR8与TLR9的表达均无明显相关性(P > 0.05).说明骨髓间充质干细胞下调了类风湿关节炎小鼠脾脏单个核细胞表达TLR8及TLR9的水平,但TLR8与TLR9的表达无相关性.