Lipopolysaccharide-induced Cytokine Expression Research Articles

Type II taste cells participate in mucosal immune surveillance.

The oral microbiome is second only to its intestinal counterpart in diversity and abundance, but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 (Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer's patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL), a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection.

LncRNA HOTAIR regulates lipopolysaccharide-induced cytokine expression and inflammatory response in macrophages

Long noncoding RNAs (lncRNAs) are emerging as major regulators of a variety of cell signaling processes. Many lncRNAs are expressed in immune cells and appear to play critical roles in the regulation of immune response. Here, we have investigated the potential role of a well-known lncRNA, HOTAIR, in inflammatory and immune response. Our studies demonstrate that HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS). Knockdown of HOTAIR reduces NF-κB-mediated inflammatory gene and cytokine expression in macrophages. Inhibition of NF-κB resulted in down-regulation of LPS-induced expression of HOTAIR as well as IL-6 and iNOS expression. We further demonstrated that HOTAIR regulates activation of NF-κB and its target genes (IL-6 and iNOS) expression via facilitating the degradation of IκBα. HOTAIR knockdown reduces the expression of NF-κB target gene expression via inhibiting the recruitment of NF-κB and associated cofactors at the target gene promoters. Taken together, our findings suggest that HOTAIR is a critical player in NF-κB activation in macrophages suggesting its potential functions in inflammatory and immune response.

Liver X receptors regulate hepatic F4/80+CD11b+ Kupffer cells/macrophages and innate immune responses in mice

The liver X receptors (LXRs), LXRα and LXRβ, are nuclear receptors that regulate lipid homeostasis. LXRs also regulate inflammatory responses in cultured macrophages. However, the role of LXRs in hepatic immune cells remains poorly characterized. We investigated the role of LXRs in regulation of inflammatory responses of hepatic mononuclear cells (MNCs) in mice. Both LXRα and LXRβ were expressed in mouse hepatic MNCs and F4/80+ Kupffer cells/macrophages. LXRα/β-knockout (KO) mice had an increased number of hepatic MNCs and elevated expression of macrophage surface markers and inflammatory cytokines compared to wild-type (WT) mice. Among MNCs, F4/80+CD11b+ cells, not F4/80+CD11b− or F4/80+CD68+ cells, were increased in LXRα/β-KO mice more than WT mice. Isolated hepatic MNCs and F4/80+CD11b+ cells of LXRα/β-KO mice showed enhanced production of inflammatory cytokines after stimulation by lipopolysaccharide or CpG-DNA compared to WT cells, and LXR ligand treatment suppressed lipopolysaccharide-induced cytokine expression in hepatic MNCs. Lipopolysaccharide administration also stimulated inflammatory cytokine production in LXRα/β-KO mice more effectively than WT mice. Thus, LXR deletion enhances recruitment of F4/80+CD11b+ Kupffer cells/macrophages and acute immune responses in the liver. LXRs regulate the Kupffer cell/macrophage population and innate immune and inflammatory responses in mouse liver.

DNA methylcytosine dioxygenase ten-eleven translocation 2 enhances lipopolysaccharide-induced cytokine expression in human dental pulp cells by regulating MyD88 hydroxymethylation.

Dental pulp inflammation is a bacterially driven inflammation process characterized by the local accumulation of cytokines/chemokines that participate in destructive processes in the pulp. Multiple mechanisms are involved in dental pulp inflammation, including epigenetic events, such as DNA methylation/demethylation. Ten-eleven translocation 2 (TET2) is a recently discovered DNA methylcytosine dioxygenase that plays important roles in inflammatory disease. However, its role in the inflammatory response of dental pulp is unknown. We observed elevated mRNA and protein levels of TET2 after lipopolysaccharide (LPS) stimulation in human dental pulp cells (hDPCs). To identify the effects of TET2 on cytokine expression, TET2 was knocked down and cytokines were detected using a cytokine antibody array after LPS stimulation. The protein expression of GM-CSF, IL-6, IL-8 and RANTES decreased in the LPS-induced hDPCs following TET2 knockdown. The downregulated expression levels of IL-6 and IL-8 were further confirmed by real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Additionally, the phosphorylation levels of IKK-α/β, p65 and IκBα of the NF-κB signaling pathway were decreased in the TET2-silenced group. Furthermore, the global 5-hydroxymethylcytosine (5hmC) level was significantly decreased and the genomic 5-methylcytosine (5mC) level was increased in the TET2-deficient hDPCs; TET2 depletion resulted in a decrease in the 5hmC level of the MyD88 promoter following LPS stimulation. These findings indicate that TET2 knockdown inhibits LPS-induced inflammatory response in hDPCs by downregulating MyD88 hydroxymethylation. Thus, TET2-dependent DNA demethylation might play an important role in dental pulp inflammation as an epigenetic regulator.

Lipopolysaccharide-induced cytokine expression pattern in peripheral blood mononuclear cells in childhood obesity.

Obesity is characterized by the abnormal or excessive fat accumulation that may impair health and extensive increase in body mass index (BMI). Childhood obesity may occur due to disturbances in metabolic regulation, which lead to metabolic syndrome and other diseases. Peripheral blood suspended immune cells are responsible for immune surveillance. The aim of the present study was to map the inflammatory cytokine expression pattern of isolated peripheral blood mononuclear cells (PBMCs) following lipopolysaccharide (LPS) stress invitro and clinical chemistry parameters in the plasma of subjects. PBMCs were isolated through density gradient ultracentrifugation of the blood from obese infants that would reflect the cytokine response. Isolated PBMCs were cultured invitro in RPMI‑1640 medium and stressed with 1µg LPS in order to investigate the expression pattern of cytokines, such as interleukin (IL)‑2, interferon (IFN)‑γ, IFN‑α, tumor necrosis factor‑α and IL‑6 using enzyme‑linked immunosorbent assays and reverse transcription‑quantitative polymerase chain reaction. Levels of NO, lipid levels, total protein, albumin, marker enzymes aspartate transaminase and alanine transaminase, malondialdehyde (MDA), and reduced glutathione in the plasma were detected. Reduction in the expression of the inflammatory cytokines in PBMCs, reduced protein and globulins, and altered MDA and GSH levels in the plasma were observed. Altered or compromised pro‑inflammatory signals from PBMCs invitro and the clinical chemistry parameters of the plasma suggested that there were compromised immunological responses in obese children compared with matched controls.

Alkaloids of Linderae Radix suppressed the lipopolysaccharide-induced expression of cytokines in cultured macrophage RAW 264.7 cells

Linderae Radix, the dry roots of Lindera aggregata (Sims) Kosterm, has long been used as traditional Chinese medicine for treatment of inflammatory diseases. The total alkaloids are believed to be the active components responsible for anti-inflammation of Linderae Radix. Here, the total alkaloids of Linderae Radix were extracted and isolated, including 12 isoquinoline alkaloids and 1 furan sesquiterpene. Within the alkaloids, norisoboldine, boldine, linderaline, isoboldine, reticuline, N-methyllaurotetanine, norjuziphine were found to be the major ingredients. In lipopolysaccharide-treated macrophage RAW 264.7 cells, application of Linderae Radix extract, or total alkaloids, suppressed the transcription of proinflammatory cytokines, interleukin-<TEX>$1{\beta}$</TEX> and interleukin-6. Out of the 12 alkaloids, norisoboldine, boldine, and isoboldine were tested in lipopolysaccharide-treated macrophages, and norisoboldine was the strongest alkaloid in suppressing the cytokine expressions. The current studies suggested that the identification of alkaloids from Linderae Radix could provide a plausible explanation for herbal therapeutic functions.

Impairment of hippocampal-dependent memory induced by juvenile high-fat diet intake is associated with enhanced hippocampal inflammation in rats

In addition to metabolic and cardiovascular disorders, obesity pandemic is associated with chronic low-grade inflammation as well as adverse cognitive outcomes. However, the existence of critical periods of development that differ in terms of sensitivity to the effects of diet-induced obesity remains unexplored. Using short exposure to a high-fat diet (HFD) exerting no effects when given to adult mice, we recently found impairment of hippocampal-dependent memory and plasticity after similar HFD exposure encompassing adolescence (from weaning to adulthood) showing the vulnerability of the juvenile period (Boitard et al., 2012). Given that inflammatory processes modulate hippocampal functions, we evaluated in rats whether the detrimental effect of juvenile HFD (jHFD) on hippocampal-dependent memory is associated with over-expression of hippocampal pro-inflammatory cytokines. jHFD exposure impaired long-term spatial reference memory in the Morris water maze without affecting acquisition or short-term memory. This suggests an effect on consolidation processes. Moreover, jHFD consumption delayed spatial reversal learning. jHFD intake did neither affect basal expression of pro-inflammatory cytokines at the periphery nor in the brain, but potentiated the enhancement of Interleukin-1-beta and Tumor Necrosis Factor-alpha expression specifically in the hippocampus after a peripheral immune challenge with lipopolysaccharide. Interestingly, whereas the same duration of HFD intake at adulthood induced similar weight gain and metabolic alterations as jHFD intake, it did neither affect spatial performance (long-term memory or reversal learning) nor lipopolysaccharide-induced cytokine expression in the hippocampus. Finally, spatial reversal learning enhanced Interleukin-1-beta in the hippocampus, but not in the frontal cortex and the hypothalamus, of jHFD-fed rats. These results indicate that juvenile HFD intake promotes exaggerated pro-inflammatory cytokines expression in the hippocampus which is likely to contribute to spatial memory impairment.

Hypoxia Augments Lipopolysaccharide-Induced Cytokine Expression in Periodontal Ligament Cells

Periodontitis is a chronic inflammatory disease characterized by the destruction of tooth supporting tissues. Hypoxia, the mainly changes of the plateau environment, can induce severe periodontitis by animal experiments. There is, however, very little information on hypoxia and lipopolysaccharide (LPS) induced cytokine expression in periodontal ligament (PDL) cells. In this article, we characterized hypoxia or P. gingivalis lipopolysaccharide (Pg LPS) induced tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 expression by human periodontal ligament (hPDL) cells. We found that hypoxia augmented Pg LPS induced TNF-α, IL-1β, and IL-6 expression in hPDL cells. We also demonstrated that nuclear factor kappa B pathway was involved in hypoxia augmenting Pg LPS induced cytokine expression in hPDL cells. Thus, our results suggest that the hypoxic environment may enhance the immune function of hPDL cells that is induced by Pg LPS.

Loss of class I MHC function alters behavior and stress reactivity

The importance of the classical immune molecule, class I major histocompatibility complex to central nervous system function is one of the most surprising discoveries related to neuroimmunology in the past decade. Mice lacking both β-2microglobulin and transporter associated with antigen processing (β2M−/−TAP−/−) showed differences in basal behavior. In response to saline injection, β2M−/−TAP−/− mice showed a significant hypothalamic pituitary adrenal activation that was not observed in wild type mice, while lipopolysaccharide-induced cytokine expression in the hypothalamus was similar in β2M−/−TAP−/− and wild type mice. Overall, these data show that class I MHC plays an important role in behavior and stress reactivity.

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: Role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.

Interaction of Melittin with Phospholipid- and Lipopolysaccharide- Containing Model Membranes

Up to now the details of the mechanisms of melittin action on biological bilayer model sys- tems in dependence on lipid composition, in particular on the kind of head groups, temperature, and ionic strength are not well understood. In particular, the influence of cholesterol present in most eu- caryotic cells and the influence of glycolipids present in bacterial membranes are far from being clear. Here, data are presented from investigations by small-angle X-ray scattering (SAXS), Fourier- transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy, Forster resonance electron transfer (FRET) spectroscopy, and calorimetric techniques (DSC: differential scanning calorimetry and ITC: isothermal titration calorimetry) on the interaction of melittin with different (glyco)lipids, in order to elucidate 1) the peptide secondary struc- ture during the melittin-lipid interaction, 2) to monitor the intercalation of the peptide into membranes, 3) to characterize the aggregate structure of the lipids, 4) to characterize the influence of lipid:melittin ratio with regard to the acyl chain melting behaviour as well as 5) to determine the peptide-lipid binding stoichiometry. These experiments are correlated with results from biological test systems, in which the inhibition of the lipopolysaccharide-induced cytokine expression in human immune cells by melittin was monitored. Furthermore, the findings are related to data found in literature with various membrane systems and different techniques. In this way, it is now possible to better understand the details of the melittin-membrane interactions, which is important with respect to the understanding of its anti-inflammatory and antimicrobial properties.

The effects of the inhibitor of nuclear factor on lipopolysaccharide-induced cytokine expression in cultured human corneal fibroblasts

To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on Lipopolysaccharide (LPS)-mediated activation of nuclear factor kappa B (NF-kappaB) and cytokine expression in cultured human corneal fibroblasts. It was a experimental study. A completely random design was employed in this research. Human corneal fibroblasts (HCFs) were obtained from human specimen. HCFs were divided into three groups: control group (group 0 h), LPS alone group and PDTC treatment group. Cells were incubated with PDTC for 30 min in the PDTC pretreatment group before LPS challenged. At different time after LPS challenged, the activities of NF-kappaB were assessed by Western Blot analysis, the secretion of IL-6 and IL-8 from cultured corneal fibroblasts was measured with enzyme-linked immunosorbent assays (ELISA); the mRNAs expression of IL-6 and IL-8 was determined by reverse transcription polymerase chain reaction (RT-PCR). The effects of PDTC on activation of NF-kappaB and the expression of IL-6 and IL-8 were also assessed in HCFs challenged with LPS. Compared with control group, NF-kappaB level was significantly enhanced in the nucleus in the LPS alone group, indicating that LPS mediated the activation of NF-kappaB in HCFs. The activation of NF-kappaB by LPS was markedly inhibited by PDTC (t1h = 9.3766, t2h = 15.9011, t4h = 12.5851, t8h = 10.8346, P < 0.01). Compared with control group, LPS increased IL-6 and IL-8 expression both mRNA and protein in HCFs. At the same time, PDTC partly inhibited the expression of IL-6 and IL-8 in corneal fibroblasts induced by LPS. These inhibitory effects were significant at both the mRNA and protein levels (protein of IL-6: t1h = 7.9154, t2h = 10.863, t4h = 8.2451, t8h = 13.5063. protein of IL-8: t1h = 8.5663, t2h = 20.5169, t4h = 25.1580, t8h = 34.8699. mRNA of IL-6: t1h = 12.0235, t2h = 13.2894, t4h = 24.0799, t8h = 27.2261. mRNA of IL-8: t1h = 20.9424, t2h = 24.1314, t4h = 29.8580, t8h = 47.9442. P < 0.01). These results suggest that LPS can activation of NF-kappaB in cultured HCFs and PDTC partly inhibits NF-kappaB activation.

Transcriptional upregulation of inflammatory cytokines in human intestinal epithelial cells following Vibrio cholerae infection

Coordinated expression and upregulation of interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, granulocyte-macrophage colony-stimulating factor, interleukin-8, monocyte chemotactic protein-1 (MCP-1) and epithelial cell derived neutrophil activator-78, with chemoattractant and proinflammatory properties of various cytokine families, were obtained in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection. These proinflammatory cytokines also showed increased expression in T84 cells, except for interleukin-6, whereas a striking dissimilarity in cytokine expression was observed in Caco-2 cells. Gene expression studies of MCP-1, granulocyte-macrophage colony-stimulating factor, interleukin-1alpha, interleukin-6 and the anti-inflammatory cytokine transforming growth factor-beta in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopolysaccharide and ctxA mutant demonstrated that, apart from cholera toxin and lipopolysaccharide, V. cholerae culture supernatant harbors strong inducer(s) of interleukin-6 and MCP-1 and moderate inducer(s) of interleukin-1alpha and granulocyte-macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-kappaB (p65 and p50) and cAMP response element-binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-kappaB and cAMP response element-binding protein, and as such the activation is mediated by cholera toxin-independent factors as well. We conclude that V. cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor-kappaB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and/or other secreted products of V. cholerae.

Chronic administration of tianeptine balances lipopolysaccharide-induced expression of cytokines in the spleen and hypothalamus of rats

The antidepressant tianeptine has been shown to protect the hippocampus against the deleterious consequences of stress and to attenuate the behavioral and neuroendocrine effects of the cytokine inducer lipopolysaccharide (LPS). Since sickness symptoms are linked to peripheral and brain production of cytokines and pro-inflammatory cytokines can promote neurotoxicity, the present study was undertaken to test the possibility that tianeptine attenuates production of pro-inflammatory cytokines. This hypothesis has been tested by studying the effects of a chronic intraperitoneal (ip) administration of tianeptine (10 mg/kg twice a day for 21 days) to rats on the induction by LPS (250 μg/kg, ip) of the production of pro- and anti-inflammatory cytokines, at the periphery (spleen, pituitary) and in the brain (hypothalamus, hippocampus). The expression of mRNAs coding for IL-1β, TNF-α, IL-6 or IL-10 (RT-PCR) and plasma levels of IL-1β, TNF-α and IL-10 (ELISA) were measured at various time intervals following LPS. Chronic tianeptine treatment attenuated LPS-induced expression of TNF-α in the spleen as well as plasma levels of this cytokine and altered the central balance between pro- and anti-inflammatory cytokines (IL-1β/IL-10). These results open new vistas in the pharmacological activity of tianeptine and provide further insights on the possible mechanisms of action involved in its neuroprotective properties.