Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation

Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects. The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes. In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium. E. coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72. YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule. Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH(2)-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed). The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS. LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine. The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25. In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.     

IL-6 protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS+IL-6.     

LPS/CD14 activation triggers SGLT-1-mediated glucose uptake and cell rescue in intestinal epithelial cells via early apoptotic signals upstream of caspase-3

Recent findings indicate that enhanced glucose uptake protects enterocytes from excessive apoptosis and barrier defects induced by LPS exposure. The aim of this study was to characterize the mechanisms responsible for increased sodium-dependent glucose cotransporter (SGLT)-1 activity in enterocytes challenged with LPS. SGLT-1-transfected Caco-2 cells were incubated with LPS in high glucose media. LPS increased SGLT-1 activity in dose- and time-dependent fashion, and is due to increased V(max) of the cotransporter. Elevated apical expression of SGLT-1 was also demonstrated. This LPS-induced effect was colchicine-inhibitable, suggesting microtubule-dependent translocation of SGLT-1 onto apical surface. Immunofluorescence staining showed expression of CD14 on the apical surface, but no TLR-4, on these cells. Neutralizing anti-CD14 decreased the LPS-induced upregulation of SGLT-1 activity, whereas anti-TLR-4 had no effect. Pharmacological studies indicated that signaling for LPS-mediated SGLT-1 glucose uptake depends on caspase-8 and -9 activation, but occurs independently of caspase-3. The findings describe a novel feedback mechanism within the apoptotic signaling pathway for SGLT-1-dependent cytoprotection. The observation suggests a new function for CD14 on enterocytes, involving the induction of the caspase-dependent SGLT-1 activity, which ultimately leads to cell rescue. The understanding of these signaling events may shed light on enterocytic cytoprotection and homeostasis mechanism upon pro-apoptotic challenges.     

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

Infection with Giardia duodenalis is one of the most common causes of waterborne diarrheal disease worldwide. Mechanisms of pathogenesis and host response in giardiasis remain incompletely understood. Previous studies have shown that exposure to G. duodenalis products induce apoptosis in enterocytes. We recently discovered that sodium-dependent glucose cotransporter (SGLT)-1-mediated glucose uptake modulates enterocytic cell death induced by bacterial lipopolysaccharide. The aim of this study was to examine whether enhanced epithelial SGLT-1 activity may constitute a novel mechanism of host defense against G. duodenalis-induced apoptosis. SGLT-1-transfected Caco-2 cells were exposed to G. duodenalis products in low (5mM) or high (25mM) glucose media. In low glucose environments, G. duodenalis-induced caspase-3 activation and DNA fragmentation in these cells. These apoptotic phenomena were abolished in the presence of high glucose. A soluble proteolytic fraction of G. duodenalis was found to upregulate SGLT-1-mediated glucose uptake in a dose- and time-dependent manner, in association with increased apical SGLT-1 expression on epithelial cells. Kinetic analysis showed that this phenomenon resulted from an increase in the maximal rate of sugar transport (V(max)) by SGLT-1, with no change in the affinity constant (K(m)). The addition of phloridzin (a competitive inhibitor for glucose binding to SGLT-1) abolished the anti-apoptotic effects exerted by high glucose. Together, the findings indicate that SGLT-1-dependent glucose uptake may represent a novel epithelial cell rescue mechanism against G. duodenalis-induced apoptosis.     

Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway

Early weaning usually causes intestinal disorders, enteritis, and diarrhea in young animals and human infants. Astragalus polysaccharides (APS) possesses anti-inflammatory activity. To study the anti-inflammatory mechanisms of APS and its potential effects on intestinal health, we performed an RNA sequencing (RNA-seq) study in lipopolysaccharide (LPS)-stimulated porcine intestinal epithelial cells (IPEC-J2) in vitro. In addition, LPS-stimulated BALB/c mice were used to study the effects of APS on intestinal inflammation in vivo. The results from the RNA-seq analysis show that there were 107, 756, and 5 differentially expressed genes in the control versus LPS, LPS versus LPS+APS, and control versus LPS+APS comparison groups, respectively. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways play significant roles in the regulation of inflammatory factors and chemokine expression by APS. Further verification of the above two pathways by using western blot and immunofluorescence analysis revealed that the gene expression levels of the phosphorylated p38 MAPK, ERK1/2, and NF-κB p65 were inhibited by APS, while the expression of IκB-α protein was significantly increased (p < .05), indicating that APS inhibits the production of inflammatory factors and chemokines by the inhibition of activation of the MAPK and NF-κB inflammatory pathways induced by LPS stimulation. Animal experiments further demonstrated that prefeeding APS in BALB/c mice can alleviate the expression of the jejunal inflammatory factors interleukin 6 (IL-6), IL-Iβ, and tumor necrosis factor-α induced by LPS stimulation and improve jejunal villus morphology.     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

A single dose of carbon monoxide intraperitoneal administration protects rat intestine from injury induced by lipopolysaccharide

Treatment with inhaled carbon monoxide (CO) has been shown to ameliorate intestinal injury induced by lipopolysaccharide (LPS) or ischemia-reperfusion in experimental animals. We hypothesized that CO intraperitoneal administration (i.p) might provide similar protection against inhaled gas. In the present study, 1 h after intravenously receiving 5 mg/kg LPS, rats were exposed to either room air or 2 ml/kg of 250 ppm CO i.p for 1, 3, and 6 h. Intestinal tissues were collected to determine the levels of platelet activator factor (PAF), intercellular adhesion molecule-1 (ICAM-1), interlukin-10 (IL-10), maleic dialdehyde (MDA), cell apoptotic rate and the phosphorylated p38 mitogen activated protein kinase (MAPK), as well as myeloperoxidase (MPO) and superoxide dismutase (SOD) activity. After CO i.p, the increase of PAF, ICAM-1, MDA, MPO, and cell apoptosis rate induced by LPS was markedly reduced (P < 0.05 or 0.01), while the decrease of IL-10 and SOD was significantly increased (P < 0.05). Western blotting showed that the effects of CO i.p were mediated by p38 MAPK pathway. Thus, the results of our study show that CO i.p exerts potent protection against LPS induced injury to the intestine via anti-oxidant, anti-inflammation and anti-apoptosis, which may involve the p38 MAPK pathway.     

Rapid co-release of interleukin 1beta and caspase 1 in spinal cord inflammation

Mounting evidence supports the hypothesis that pro-inflammatory cytokines secreted by astrocytes and microglia modulate nociceptive function in the injured CNS and following peripheral nerve damage. Here we examine the involvement of interleukin-1beta (IL-1beta) and microglia activation in nociceptive processing in rat models of spinal cord inflammation. Following application of lipopolysaccharide (LPS) to an ex vivo dorsal horn slice preparation, we observed rapid secretion of IL-1beta which was prevented by inhibition of glial cell metabolism and by inhibitors of either p38 mitogen-activated protein kinase (MAPK) or caspase 1. LPS superfusion also induced rapid secretion of active caspase 1 and apoptosis-associated speck-like protein containing a caspase recruitment domain from the isolated dorsal horn. Extensive microglial cell activation in the dorsal horn, as determined by immunoreactivity for phosphorylated p38 MAPK, was found to correlate with the occurrence of IL-1beta secretion. In behavioural studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in the rat hind-paws which was attenuated by concomitant injections of a p38 MAPK inhibitor, a caspase 1 inhibitor or the rat recombinant interleukin 1 receptor antagonist. These data suggest a critical role for the cytokine IL-1beta and caspase 1 rapidly released by activated microglia in enhancing nociceptive transmission in spinal cord inflammation.     

Inhibition of lipopolysaccharide-induced release of interleukin-8 from intestinal epithelial cells by SMA, a novel inhibitor of sphingomyelinase and its therapeutic effect on dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The use of SMase inhibitors may offer new therapies for the treatment of the LPS- and cytokines-related inflammatory bowel disease (IBD). We synthesized a series of difluoromethylene analogues of SM (SMAs). Here, we show that LPS efficiently increases the release of IL-8 from HT-29 intestinal epithelial cells by activating both neutral SMase and nuclear factor (NF)-kappaB in the cells. The addition of SMA-7 suppressed neutral SMase-catalyzed ceramide production, NF-kappaB activation, and IL-8 release from HT-29 cells caused by LPS. The results suggest that activation of neutral SMase is an underlying mechanism of LPS-induced release of IL-8 from the intestinal epithelial cells. Ceramide production following LPS-induced SM hydrolysis may trigger the activation of NF-kappaB in nuclei. Oral administration of SMA-7 (60 mg/kg) to mice with 2% dextran sulfate sodium (DSS) in their drinking water, for 21 consecutive days, reduced significantly the severity of colonic injury. This finding suggests a central role for SMase/ceramide signaling in the pathology of DSS-induced colitis in mice. The therapeutic effect of SMA-7 observed in mice may involve the suppression of IL-8 production from intestinal epithelial cells by LPS or other inflammatory cytokines.     

Endotoxin tolerance in rats: expression of TNF-alpha, IL-6, IL-10, VCAM-1 AND HSP 70 in lung and liver during endotoxin shock.

Endotoxin can induce a state of tolerance against its own pathological effects, commonly referred to as endotoxin tolerance. This phenomenon has been found to be associated with reduced serum levels of cytokines such as TNF-alpha, IL-1, IL-6 and IL-10. In the present study the expression of TNF-alpha, IL-6, IL-10, the adhesion molecule VCAM-1 and the heat shock protein 70 was determined in vivo in lung and liver of LPS-tolerant and naive rats by means of semiquantitative RT-PCR after i.v. LPS injection. TNFalpha, IL-6, IL-10, HSP 70 and VCAM-1 were induced in lung and liver after LPS injection. In liver and lung of endotoxin-tolerant rats TNF-alpha and IL-6 were induced to a lower degree after LPS treatment when compared to non-tolerant controls. The LPS-induced IL-10 expression was also slightly attenuated in the lung of tolerant rats, but in the liver no differences between tolerant and non-tolerant animals were observed. HSP 70 and VCAM-1 were expressed after systemic LPS treatment in liver and lung. The degree of induction, however, was the same in tolerant and untreated controls. The presented data show that endotoxin tolerance is reflected by a reduced cytokine expression in lung and liver in vivo. On the other hand, levels of expression of the adhesion molecule VCAM-1 and the stress protein HSP 70 do not appear to be changed by endotoxin tolerance.     

Rapid expression of IL-1beta by intestinal epithelial cells in vitro

Intestinal epithelial cells have been shown to produce IL-1beta in vivo. This gene expression is rapid and precedes most determinants of inflammation, suggesting a pivotal role for IL-1beta in the early events leading to inflammation. To better understand the mechanisms leading to this IL-1beta production, we have developed an in vitro model system employing a nontransformed intestinal epithelial cell line that does not constitutively express IL-1beta. Following detachment, these cells rapidly expressed IL-1beta mRNA. This expression was enhanced, but not induced, by LPS. IL-1beta protein was detected by immunoprecipitation in the culture medium from passaged IEC-18 but not intracellularly, suggesting an efficient secretion of the molecule following induction. Interestingly, culture supernatants from passaged cells were without IL-1 bioactivity, suggesting the presence of an inhibitor as well. RT-PCR and Western blot analysis showed expression of IL-1RII by IEC-18 following detachment, possibly explaining the observed lack of bioactivity. These results indicate a novel pathway for IL-1beta production and suggest that proinflammatory effects of IEC-derived IL-1 may be modulated by the simultaneous production of IL-1 antagonists.     

Cytokine patterns during dengue shock syndrome

OBJECTIVE: To investigate the patterns of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, interferon-gamma (IFN-gamma) and interleukin-1 receptor antagonist (IL-1Ra) during the course of dengue shock syndrome. DESIGN: Prospective clinical study. SETTING: Pediatric Intensive Care Unit, Dr. Kariadi Hospital, the university hospital of Diponegoro University, Semarang, Indonesia. PATIENTS: Fifty children with dengue shock syndrome. MEASUREMENTS: The plasma concentration and the ex vivo production, with and without lipopolysaccharide (LPS), of TNF-alpha, IL-1beta and IL-1Ra were measured in duplicate by nonequilibrium radioimmunoassay (RIA); IFN-gamma and IL-6 were measured by ELISA. RESULTS: During the acute phase, the plasma concentrations and the ex vivo production without LPS of IL-1Ra were considerably elevated and returned to normal on recovery. However, the ex vivo LPS-stimulated production of the proinflammatory cytokines TNF-alpha and IL-1beta were considerably depressed. Also, these concentrations returned towards normal on recovery. In non-survivors, the plasma concentrations of IL-6 and IL-1Ra were significantly higher than in survivors (p = 0.00001 and p = 0.0005, respectively). In addition, the ex vivo production of IL-1Ra in non-survivors was significantly higher than in survivors, both without LPS stimulation (p = 0.0008) and with LPS (p < 0.004). IL-1Ra was significantly associated with mortality (p = 0.007). CONCLUSION: Since IL-1Ra was significantly associated with mortality, this measurement may be used as an index of disease severity in dengue shock syndrome.     

Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia.

Cytokines have been studied intensively to delineate their role in the altered pathophysiology observed in septic shock. We studied the role of TNF in the lethality of two well characterized models of septic shock by inhibiting TNF's activity with a specific antibody. In the first model, sepsis was induced by cecal ligation and puncture (CLP), and in the second model sepsis was induced by either an i.p. or i.v. injection of LPS. After CLP, plasma endotoxin was detectable within 4 h and reached a peak at 8 h (136 +/- 109 ng/ml). TNF bioactivity peaked at 12 h (528 +/- 267 pg/ml) at a significantly higher level than sham-operated control mice (64 +/- 31 pg/ml). After i.p. LPS, TNF peaked much more quickly (90 min) compared with CLP and at a significantly higher level (107,900 +/- 25,000 pg/ml). Another cytokine studied in septic shock, IL-6, peaked at 12 h after CLP at 1011 +/- 431 pg/ml, and at 90 min after lethal LPS at 16,300 +/- 3,700 pg/ml. Mice were treated with an anti-TNF antibody that has been shown previously to inhibit in vivo TNF activity. Antibody treatment of mice subjected to CLP significantly reduced TNF bioactivity but did not reduce mortality or pulmonary neutrophilic infiltration. In the i.v. LPS model, anti-TNF antibody treatment concomitant with LPS injection reduced plasma TNF activity from 80,000 +/- 20,000 pg/ml to undetectable levels. However, anti-TNF treatment immediately before either i.v. or i.p. LPS did not reduce mortality. Additionally, when the antibody was administered 4 h before the lethal i.v. LPS, there was no reduction in lethality. These data show that in two separate models of septic shock blockade of TNF biologic activity will not prevent lethality.     

Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice

The mechanisms underlying the action of the potent anti-inflammatory interleukin-10 (IL-10) are poorly understood. Here we show that, in murine macrophages, IL-10 induces expression of heme oxygenase-1 (HO-1), a stress-inducible protein with potential anti-inflammatory effect, via a p38 mitogen-activated protein kinase-dependent pathway. Inhibition of HO-1 protein synthesis or activity significantly reversed the inhibitory effect of IL-10 on production of tumor necrosis factor-alpha induced by lipopolysaccharide (LPS). Additional experiments revealed the involvement of carbon monoxide, one of the products of HO-1-mediated heme degradation, in the anti-inflammatory effect of IL-10 in vitro. Induction of HO-1 by IL-10 was also evident in vivo. IL-10-mediated protection against LPS-induced septic shock in mice was significantly attenuated by cotreatment with the HO inhibitor, zinc protoporphyrin. The identification of HO-1 as a downstream effector of IL-10 provides new possibilities for improved therapeutic approaches for treating inflammatory diseases.     

Phospholipase C Activator m-3M3FBS Protects against Morbidity and Mortality Associated with Sepsis

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS-dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-γ and IL-12 while inhibiting proseptic TNF-α and IL-1β production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-α and IL-1β following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.