LPS-induced Inflammatory Cytokine Expression Research Articles

Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells

Inflammation is a complex process involving cytokine production to regulate host defense cascades in order to clear pathogenic agents. Upregulation of inflammatory cytokines, such as IL-6 and IL-8 by bacteria infection, occurs in pulmonary tissues and has been demonstrated to be critical to the lung inflammatory response. Glucosamine, primarily identified as an anti-arthritis supplement, has been also regarded as a potential anti-inflammatory agent. Thus we hypothesized that lipopolysaccharide (LPS) would activate IL-6 and IL-8 expressions in human primary bronchial epithelial cells and glucosamine could attenuate such an effect. The RT-PCR, real-time PCR, and ELISA analyses demonstrated that LPS-induced mRNAs encoding IL-6 and IL-8 and the subsequent secretion of IL-6 and IL-8 were inhibited by glucosamine treatment. MTT, alamarBlue, and annexin V apoptosis assays all suggested that this inhibition effect was not due to a cytotoxic effect mediated by glucosamine. Using the inhibitors of the MAP kinases and NFκB, it was revealed that p38, JNK and ERK, as well as NFκB, are all involved in LPS-induced IL-8 secretion; however only p38 is involved in LPS-induced IL-6 secretion. Immunoblot analysis further demonstrated that LPS-mediated phosphorylation of JNK and ERK, but not the LPS-induced NFκB translocation, was inhibited by glucosamine. Altogether, our results indicate that glucosamine can potently suppress LPS-induced inflammatory cytokine expression, at least in part via attenuation of MAPK activation.

Functional linkage of cirrhosis‐predictive single nucleotide polymorphisms of toll‐like receptor 4 to hepatic stellate cell responses†‡

In a recent study, a single nucleotide polymorphism (SNP) of the Toll-like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild-type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4(-/-) mice and a human HSC line (LX-2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hyporesponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS-induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein-1, interleukin-6), down-regulation of bone morphogenic protein and the activin membrane-bound inhibitor expression (an inhibitory transforming growth factor beta pseudoreceptor), and activation of a nuclear factor kappaB (NF-kappaB)-responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF-kappaB, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase were greatly increased in HSCs from either TLR4(-/-) or myeloid differentiation factor 88(-/-) (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; increased apoptosis in these lines was accompanied by decreased phospho-ERK and Bcl-2. TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4-mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression.

Galectin-3 Is a Negative Regulator of Lipopolysaccharide-Mediated Inflammation

Galectin-3 is a beta-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, beta-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.

Novel iminobenzoxathiolone compound inhibits nuclear factor-κB activation targeting inhibitory κB kinase β and down-regulating interleukin-1β expression in lipopolysaccharide-activated macrophages

Benzoxathiolone derivatives have been reported to show pharmacological potentials in the psoriasis and acne. However, molecular basis for these pharmacological properties is little known. We postulated that the derivatives could mediate some of their pharmacological actions by modulating nuclear factor (NF)-κB activation, which is closely linked to the inflammatory and immune disorders. In this study, a novel iminobenzoxathiolone LYR-71 of 6-methyl-2-propylimino-6,7-dihydro-5 H-benzo[1,3]oxathiol-4-one has been demonstrated to inhibit in vitro catalytic activity of inhibitory κB (IκB) kinase β (IKKβ), a key enzyme required for NF-κB activation, with an IC 50 value of 7 μM. LYR-71 inhibited IKKβ-mediated phosphorylation of cytoplasmic IκBα in lipopolysaccharide (LPS)-activated macrophages, and sequentially preventing IκBα degradation as well as transcriptional activation of NF-κB. Furthermore, LYR-71 down-regulated LPS-induced transcription of interleukin (IL)-1β or other cytokines in the cells, and inhibited expression vector IKKβ-elicited IL-1β promoter activity. Taken together, LYR-71 was an efficient inhibitor of IKKβ, preventing NF-κB activation in macrophages, and this mechanism of action could contribute its down-regulatory effect on LPS-induced expression of inflammatory cytokines at the transcription level.

111. The curcumin analog, UBS-109 decreases peripheral expression of LPS-induced inflammatory cytokines

111. The curcumin analog, UBS-109 decreases peripheral expression of LPS-induced inflammatory cytokines

A p38 Mitogen‐Activated Protein Kinase Inhibitor Arrests Active Alveolar Bone Loss in a Rat Periodontitis Model

Gram-negative bacterial species, such as Actinobacillus actinomycetemcomitans, contain lipopolysaccharide (LPS) that initiates the innate immune system, resulting in inflammatory alveolar bone loss. LPS activates Toll-like receptors on membrane surfaces, stimulating many intracellular signaling cascades, including the p38 mitogen-activated protein kinase (MAPK). Activation of p38 signaling mediates inflammatory cytokine expression, contributing toward osteoclastogenesis and bone loss. The aim of this study was to determine whether the novel, orally active p38 MAPK inhibitor SD282 could arrest progression of LPS-induced alveolar bone destruction in rats. Three groups of female Sprague-Dawley rats received LPS injections to the palatal molar gingiva three times per week for 4 weeks to establish periodontitis. From weeks 5 through 8, two groups received the drug SD282 (N = 14) or 1% polyethylene glycol drug vehicle (N = 14) via oral gavage in addition to LPS injections. The third group continued to receive only LPS injections (N = 8). Microcomputed tomography was used to measure volumetric alveolar bone loss, expressed as bone volume fraction (BVF). Expression of interleukin (IL)-1 and -6 and tumor necrosis factor-alpha (TNF-alpha) was assessed by immunohistochemistry, and osteoclasts were enumerated by tartrate-resistant acid phosphatase staining. By 4 weeks, severe alveolar bone resorption was seen in LPS-injected animals. Administration of SD282 significantly blocked additional volumetric bone loss in the LPS-only versus LPS + SD282 groups (0.37 +/- 0.01 BVF versus 0.43 +/- 0.01 BVF; P < 0.01). Significant reductions in IL-1beta (P < 0.01 ), TNF-alpha (P < 0.05), and osteoclast formation (P < 0.01) occurred in the presence of SD282. An orally active p38 MAPK inhibitor reduced LPS-induced inflammatory cytokine expression, osteoclastogenesis, and alveolar bone loss in rats. Within the limits of the current study, SD282 arrested periodontal disease progression, thus highlighting the therapeutic potential of this novel class of inhibitors.

Activated protein C reduces endotoxin-induced white matter injury in the developing rat brain

Periventricular leukomalacia (PVL), the dominant form of brain injury in premature infants, is characterized by white matter injury (WMI) and is associated with cerebral palsy. The pathogenesis of PVL is complex and likely involves ischemia/reperfusion, free radical formation, excitotoxicity, impaired regulation of cerebral blood flow, a procoagulant state, and inflammatory mechanisms associated with maternal and/or fetal infection. Using an established animal model of human PVL, we investigated whether activated protein C (APC), an anti-coagulant factor with anti-inflammatory, anti-apoptotic, anti-oxidant, and cytoprotective activities, could reduce endotoxin-induced WMI in the developing rat brain. Intraperitoneal injections of lipopolysaccharide (LPS) (0.5 mg/kg body weight) were given at embryonic days 18 (E18) and 19 (E19) to pregnant Sprague–Dawley rats; control rats were injected with sterile saline. A single intravenous injection of recombinant human (rh) APC (0.2 mg /kg body weight) was given to pregnant rats following the second LPS dose on embryonic day 19 (E19). Reduced cell death in white matter and hypomyelination were shown on TUNEL and myelin basic protein (MBP) staining, respectively, on late postnatal days (P7) in APC-treated groups. There were significantly fewer TUNEL + nuclei in the periventricular WM in the APC + LPS group than in the untreated LPS group. Compared to the APC + LPS and control group, MBP expression was weak in the LPS group on P7, indicating endotoxin-induced hypomyelination in the developing rat brain. APC attenuated the LPS-induced protein expression of inflammatory cytokines, tumor necrosis factor-alpha, and interleukin-6, as evaluated by ELISA in neonatal rat brains. A single intraperitoneal injection of rhAPC (0.2 mg/kg body weight) to neonatal rats on P1 also had similar protective and anti-inflammatory effects against maternally administered LPS. Collectively, these data support the hypothesis that APC may provide protection against an endotoxin-evoked inflammatory response and WMI in the developing rat brain. Moreover, our results suggest that the possible use of APC in treatment of preterm infants and pregnant women with maternal or placental infection may minimize the risk of PVL and cerebral palsy.

Inhibitory effect of chroman carboxamide on interleukin-6 expression in response to lipopolysaccharide by preventing nuclear factor-κB activation in macrophages

6-Hydroxy-7-methoxychroman-2-carboxylic acid (3-nitrophenyl)amide (CP-1158) is a synthetic chroman carboxamide with trolox-like chemical structure. In the present study, CP-1158 was found to inhibit interleukin (IL)-6 production in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. The CP-1158 attenuated LPS-induced synthesis of IL-6 transcript but also inhibited LPS-induced IL-6 promoter activity. Further, CP-1158 attenuated LPS-induced syntheses of tumor necrosis factor (TNF)-α, IL-1β, interferon-inducible protein (IP)-10 and macrophage inflammatory protein (MIP)-1β transcripts. Nuclear factor (NF)-κB has been evidenced to play a major mechanism in LPS-induced expression of IL-6 or other inflammatory cytokines. CP-1158 prevented LPS-induced nuclear translocation of NF-κB complex and subsequently inhibited DNA binding activity of NF-κB complex as well as NF-κB transcriptional activity in macrophages RAW 264.7. However, CP-1158 did not affect LPS-induced phosphorylation and degradation of inhibitory κB (IκB). In another experiment, CP-1158 inhibited IL-6 promoter activity elicited by expression vectors encoding NF-κB p50 or p65 subunit. Taken together, CP-1158 inhibited LPS-induced expression of inflammatory cytokines including IL-6, targeting NF-κB activating pathway downstream IκB degradation, and thus could provide an anti-inflammatory potential of chroman carboxamide.

Xylitol inhibits inflammatory cytokine expression induced by lipopolysaccharide from Porphyromonas gingivalis.

Porphyromonas gingivalis is one of the suspected periodontopathic bacteria. The lipopolysaccharide (LPS) of P. gingivalis is a key factor in the development of periodontitis. Inflammatory cytokines play important roles in the gingival tissue destruction that is a characteristic of periodontitis. Macrophages are prominent at chronic inflammatory sites and are considered to contribute to the pathogenesis of periodontitis. Xylitol stands out and is widely believed to possess anticaries properties. However, to date, little is known about the effect of xylitol on periodontitis. The aim of the present study was to determine tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) expression when RAW 264.7 cells were stimulated with P. gingivalis LPS (hereafter, LPS refers to P. gingivalis LPS unless stated otherwise) and the effect of xylitol on the LPS-induced TNF-alpha and IL-1beta expression. The kinetics of TNF-alpha and IL-1beta levels in culture supernatant after LPS treatment showed peak values at 1 h (TNF-alpha) and 2 to 4 h (IL-1beta), respectively. NF-kappaB, a transcription factor, was also activated by LPS treatment. These cytokine expressions and NF-kappaB activation were suppressed by pretreatment with pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB). Pretreatment with xylitol inhibited LPS-induced TNF-alpha and IL-1beta gene expression and protein synthesis. LPS-induced mobilization of NF-kappaB was also inhibited by pretreatment with xylitol in a dose-dependent manner. Xylitol also showed inhibitory effect on the growth of P. gingivalis. Taken together, these findings suggest that xylitol may have good clinical effect not only for caries but also for periodontitis by its inhibitory effect on the LPS-induced inflammatory cytokine expression.

Risk factors that modulate periodontal diseases

Periodontal diseases now is a chronic inflammatory disease caused by Gram negative bacterial infection. Recently, it is of interest to evaluate risk factors that modulate initiation and development of this disease. A recent study have suggested that obesity is one of the risk factors. In this regard, most recently, we found that leptin, a regulator of food intake and whole-body energy metabolism, acts as a negative regulator of LPS-induced expression of inflammatory cytokines of macrophages via inhibition of NF-kB.

Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression.

The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-beta1 (TGF-beta1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-beta1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-beta1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fos and c-jun oligonucleotides. Since TGF-beta1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-beta1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1beta and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-beta1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-beta1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-beta1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-beta1 inhibition of LPS-induced septic shock.