LPS-induced mRNA Expression Of IL-1β Research Articles

Stimulation of Angiotensin II Type 2 Receptor Modulates Pro-Inflammatory Response in Microglia and Macrophages: Therapeutic Implications for the Treatment of Stroke.

Sustained microglial activation contributes to the development of post-stroke cognitive impairment (PSCI). Compound 21 (C21), an angiotensin II type 2 receptor agonist, has shown some neurovascular protection after stroke. This study aimed to investigate the direct anti-inflammatory effects of C21 on macrophages, as well as brain innate immune cells. Murine microglial cell line (C8-B4) and RAW 264.7 macrophages were exposed to lipopolysaccharide (LPS) and co-treated with C21. Pro-inflammatory mediators were assessed via RT-qPCR and ELISA. Cellular reactive oxygen species (ROS) were evaluated via CellROXGreen staining, and nitrate production was assessed using Griess assay. C21 suppressed LPS-induced inflammation and ROS generation in both cells. In microglia, C21 blunted LPS-induced mRNA expression of IL-1β, IL-12b, COX-1, iNOS, and IL-6. A similar pattern was observed in macrophages, where C21 suppressed LPS-induced IL-1β, TNF-α, and CXCL1 expression. These anti-inflammatory effects in microglia and macrophages were associated with increased neuroprotective gene expression, including GDNF and BDNF, in a dose-dependent manner. Our findings suggest a protective effect of C21 against the inflammatory response, in both macrophages and microglia, via suppression of the release of pro-inflammatory cytokines/chemokines and the generation of ROS while stimulating the production of neurotrophic factors.

Chicken cathelicidin-2 promotes IL-1β secretion via the NLRP3 inflammasome pathway and serine proteases activity in LPS-primed murine neutrophils

Cathelicidins have antimicrobial and immunomodulatory activities. Previous studies have shown that chicken cathelicidin-2 (CATH-2) exerts strong anti-inflammatory activity through LPS neutralization. However, it is still unclear whether other intracellular signaling pathways are involved in CATH-2 immunomodulation. Therefore, the CATH-2-meadiated immune response was investigated in LPS-primed neutrophils. Firstly, inflammatory cytokines release was determined in LPS-primed neutrophils. The results showed that CATH-2 significantly promoted secretion of IL-1β and IL-1α while IL-6 and TNF-α were not affected. IL-1β is the key indicator of inflammasome activation. Next, NLRP3 inflammasome signaling pathway was explored using neutrophils of Nlrp3−/−, Asc−/− and Casp1−/− mice and the results showed that the CATH-2-enhanced IL-1β release was completely abrogated, indicating it is NLRP3-dependent. Moreover, CATH-2 significantly induced activation of caspase-1 and gasdermin D (GSDMD) but did not affect LPS-induced mRNA expression of IL-1β and NLRP3, demonstrating that CATH-2 serves as the second signal activating the NLRP3 inflammasome. Furthermore, CATH-2-mediated IL-1β secretion and caspase-1 activation is dependent on potassium efflux but independent of P2X7R. In addition, other signaling pathways including JNK, ERK and SyK were investigated using different inhibitors and the results showed that these signaling pathway inhibitors partially attenuated CATH-2-enhanced IL-1β secretion, especially the JNK inhibitor. Finally, the role of serine protease in CATH-2-mediated NLRP3 inflammasome activation was investigated in neutrophils and the results showed that serine protease activity is involved in CATH-2-enhanced IL-1β secretion and caspase-1 activation. In conclusion, after LPS priming in neutrophils, CATH-2 can be an agonist of the NLRP3 inflammasome. Our study increases the understanding on immunomodulatory effects of chicken cathelicidins and provides new insight on chicken cathelicidins-mediated immune response.

Chemical composition of cold-pressed blackberry seed flour extract and its potential health-beneficial properties.

The blackberry seed flour was cold‐extracted using 50% acetone and examined for its phytochemical composition and health‐beneficial properties including in vitro gut microbiota modulatory, free radical scavenging, anti‐inflammatory, and antiproliferative capacities. Among identified thirteen components of blackberry seed flour extract through UHPLC‐MS analysis, sanguiin H6 was the primary component and followed by ellagic acid and pedunculagin. For health‐beneficial properties, the blackberry seed flour extract increased the total number of gut bacteria and shifted the abundance of specific bacterial phylum, family, or genus. The extract had RDSC, ORAC, HOSC, and ABTS•+ scavenging capacities of 362, 304, 2,531, and 267 μmol Trolox equivalents (TE)/g, respectively. In addition, the blackberry seed flour extract showed capacities for anti‐inflammation and antiproliferation by suppressing LPS induced IL‐1β mRNA expressions in the cultured J774A.1 mouse macrophages and the proliferation of LNCaP prostate cancer cells. The results suggest potential health benefits and further utilization of blackberry seed flour as functional foods.

Anti-Inflammatory Activity of Adenosine 5'-Trisphosphate in Lipopolysaccharide-Stimulated Human Umbilical Vein Endothelial Cells Through Negative Regulation of Toll-Like Receptor MyD88 Signaling.

Activation of TLR4-MyD88-NF-κB signaling by lipopolysaccharide (LPS) evokes a proinflammatory immune response, and plays a pivotal role in initiation and progression of atherosclerosis (AS). ATP (adenosine 5'-trisphosphate), a powerful extracellular signal transduction molecule, functions to regulate immune inflammatory responses depending on the type of P2 receptors and cell lines. In this study, we first performed RT-PCR to detect the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1β induced by different concentrations of LPS in human umbilical vein endothelial cells (HUVECs). Protein level of TLR4 signaling including TLR4, myeloid differentiation factor (MyD88), and CD14 induced by LPS (1 μg/mL) at different times (0, 10, 30, 60, 120 min) was analyzed by Western blot. Then, RT-PCR was performed to detect the effect of different concentrations of ATP on mRNA expression of IL-1β and MCP-1 induced by LPS (1 μg/mL) and the TLR4 signaling pathway. Western blot was performed to detect the effect of low concentrations of ATP on phosphorylation of p65 induced by 1 μg/mL LPS. Finally, we used P2Y receptor blocker Suramin to verify whether the role of ATP on LPS-induced inflammatory cytokine expression was through P2Y receptors. The results showed that LPS upregulated the expression of MCP-1, IL-8, and IL-1β in a dose-dependent manner accompanied by the activation of TLR4-MyD88 signaling in HUVECs. Only low concentration ATP (1, 10 μM) inhibited LPS-induced mRNA expression of IL-1β and MCP-1. ATP at low concentrations also downregulated the mRNA expression of TLR4, CD14, and MyD88 and inhibited LPS-induced phosphorylation of p65. Furthermore, Suramin, a nonspecific P2Y receptor antagonist, did not attenuate the inhibition of ATP on LPS-induced IL-1β and MCP-1 expression. Taking this together, low concentration ATP inhibited LPS-induced inflammation in HUVECs by negatively regulating TLR4-MyD88 signaling, and P2Y receptors were not involved in this process, which might provide new ideas for prevention and treatment of inflammatory diseases such as AS.

Propofol Suppresses LPS-Induced Inflammation in Amnion Cells via Inhibition of NF-κB Activation.

Preterm labor is a leading risk factor for neonatal death and long-term impairment and linked closely with inflammation. Non-obstetric surgery is occasionally needed during pregnancy and the anesthetic drugs or surgery itself can give rise to inflammation. Here, we examined the influence of propofol pretreatment on the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) after lipopolysaccharide (LPS) stimulation. In addition, we evaluated the expression of pro-inflammatory cytokines and nuclear factor kappa B (NF-κB). Human amnion-derived WISH cells were used to investigate the effect of propofol on the LPS-induced expression of inflammatory substances involved in preterm labor. For the experiment, WISH cells were pretreated with various concentrations propofol (0.01-10μg/ml) for 1h and then treated with LPS (1μg/ml) for 24h. Cytotoxicity was evaluated using MTT assay. PGE2 concentration was assessed by ELISA. Protein expressions of COX-2, PGE2 and NF-κB were analyzed by western blotting analysis. RT-PCR was used for analysis of mRNA expression of COX-2, PGE2, interlukin (IL)-1β and tumor necrosis factor (TNF)-α. Propofol showed no cytotoxicity on the WISH cells. LPS-induced PGE2 production and COX-2 and PGE2 expression were decreased after propofol pretreatment. Propofol also attenuated the LPS-induced mRNA expression of IL-1β and TNF-α. Moreover, the activation of NF-κB was inhibited by propofol pretreatment on LPS-stimulated WISH cells. We demonstrated that propofol suppresses the expression of inflammatory substances enhanced by LPS stimulation. Furthermore, this inhibitory effect of propofol on the inflammatory substance expression is mediated by suppression of NF-κB activation.

Effects of ATP on expression of inflammatory factors in endothelial progenitor cells induced by LPS and the mechanisms

To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway. Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05). Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.

The anti-inflammatory pathway regulated via nicotinic acetylcholine receptors in rat intestinal mesothelial cells.

Regulation of inflammation in intestinal mesothelial cells in the abdominal cavity is important for the pathogeny of clinical conditions, such as postoperative ileus, peritonitis and encapsulating peritoneal sclerosis. Here we have examined the inflammatory effect of lipopolysaccharide (LPS) and the anti-inflammatory effect of nicotinic acetylcholine receptor stimulation in rat intestinal mesothelial cells. LPS upregulated mRNA expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS). The α7, α9 and α10 subunits of nicotinic acetylcholine receptor were detected in intestinal mesothelial cells. Nicotine (10 nM) significantly inhibited LPS-induced mRNA expression of IL-1β and iNOS, but not TNF-α and MCP-1. In addition, the α7 nicotinic acetylcholine receptor selective agonist, PNU-282987 (10 nM), significantly inhibited LPS-induced mRNA expression of IL-1β but not TNF-α, iNOS and MCP-1. Finally, we found that enteric nerves adhered to intestinal mesothelial cells located under the ileal serosa. In conclusion, intestinal mesothelial cells react to LPS to induce the production of nitric oxide from iNOS. The anti-inflammatory action of intestinal mesothelial cells expressing α7nAChR may be mediated via their connectivity with enteric nerves.

Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes.

BackgroundSurfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes.MethodsHighly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1β, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry.ResultsNeither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1β mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes.ConclusionThe new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.

Cucurbitacin D is a new inflammasome activator in macrophages

We previously reported that cucurbitacin D isolated from Trichosanthes kirilowii has anti-tumor roles to leukemia cells. However, the effect of cucurbitacin D on immune cells is not fully understood although there is no toxic activity to normal cells. In this study, immunomodulating activities of cucurbitacin D were investigated in macrophages. Cucurbitacin D could increase LPS-induced interleukin (IL)-1β production in culture supernatant of THP-1 cells, peritoneal exudate cells (PECs), bone marrow derived macrophages (BMDMs), and RAW264 cells. At the transcriptional level, cucurbitacin D enhanced LPS-induced IL-1β mRNA expression through activation of ERK1/2 mitogen-activated protein kinases (MAPKs). At the posttranscriptional level, the activation of caspase-1 induced by cucurbitacin D has also been demonstrated following treatment with a caspase-1 inhibitor and siRNA. Importantly, cucurbitacin D has further been shown to induce inflammasome activation independent of ERK1/2 activation. Western blotting showed interaction of NOD-like receptor family, pyrin domain containing 3 (NALP3) and apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain (ASC), suggesting activation of the inflammasome and a possible reason for activation of caspase-1. Taken together, these results suggest that cucurbitacin D could initiate immunomodulating activity in macrophages to lead to inflammasome activation as well as enhancement of LPS signaling.

GATA-2 transduces LPS-induced il-1β gene expression in macrophages via a toll-like receptor 4/MD88/MAPK-dependent mechanism.

Lipopolysaccharide (LPS) is a critical factor for inducing acute lung injury. GATA-2, a transcription factor, contributes to the control of cell activity and function. Exposure of RAW 264.7 cells to LPS induced interleukin (IL)-1β mRNA and protein expression and GATA-2 translocation from the cytoplasm to nuclei in concentration- and time-dependent manners. A bioinformatic search revealed that GATA-2-specific binding elements exist in the 5’-promoter region of the il-1β gene. LPS could enhance the transactivation activity of GATA-2 in macrophages. Knocking-down translation of GATA-2 mRNA using RNA interference significantly alleviated LPS-induced IL-1β mRNA and protein expression. As to the mechanism, transfection of toll-like receptor (TLR) 4 small interfering (si)RNA into macrophages concurrently decreased LPS-caused increases in nuclear GATA-2 levels. Sequentially, treatment with myeloid differentiation factor 88 (MyD88) siRNA decreased LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) kinase 1/2 and subsequent translocation of GATA-2. Reducing MAPK activities using specific inhibitors simultaneously decreased GATA-2 activation. Furthermore, exposure of primary macrophages to LPS significantly increased the transactivation activities of GATA-2 and IL-1β mRNA and protein expression. Transfection of GATA-2 siRNA inhibited LPS-induced IL-1β mRNA expression. Results of this study show that LPS induction of il-1β gene expression in macrophages is mediated by GATA-2 via activation of TLR4, MyD88, and MAPKs.

IL-21 Modulates Release of Proinflammatory Cytokines in LPS-Stimulated Macrophages through Distinct Signaling Pathways

The aim of this study was to investigate the anti-inflammatory effect of IL-21 on LPS-induced mouse peritoneal macrophages. The results showed that IL-21 significantly inhibited LPS-induced mRNA expression of IL-1β, TNF-α, and IL-6 in macrophages, but not of IFN-γ, IL-10, CCL5, or CXCL2. ELISA analysis showed that IL-21 also suppressed LPS-induced production of TNF-α and IL-6 in culture supernatants. Western blot analysis showed that IL-21 clearly inhibited ERK and IκBα phosphorylation and NF-κB translocation in LPS-stimulated macrophages, but it increased STAT3 phosphorylation. Flow cytometric and Western blot analysis showed that IL-21 decreased M1 macrophages surface markers expression of CD86, iNOS, and TLR4 in LPS-stimulated cells. All results suggested that IL-21 decreases IL-6 and TNF-α production via inhibiting the phosphorylation of ERK and translocation of NF-κB and promotes a shift from the M1 to M2 macrophage phenotype by decreasing the expression of CD86, iNOS, and TLR4 and by increasing STAT3 phosphorylation in LPS-stimulated cells.

Aldosterone synergizes with peripheral inflammation to induce brain IL-1β expression and depressive-like effects

Recent findings have shown that the physiological functions of the hormone aldosterone go far beyond its well-known role in blood-pressure regulation and salt/water homeostasis. Aldosterone is for example involved in the regulation of inflammation, and also binds directly to mineralocorticoid receptors in specific brain regions. Interestingly, depressive symptoms appear to correlate with alterations of the aldosterone system but the underlying mechanisms have not been elucidated.In this study aldosterone (2μg/100g body weight/day) was continuously administered via osmotic minipumps for 5days. Lipopolysaccharide (LPS) was administered once a day for 5days in a dose of 1mg/kg ip. The rats were tested for depressive-like behavior 24h after the last LPS injection. Protein levels of cytokines were measured in serum and cerebrospinal fluid (CSF). mRNA expression of interleukin (IL)-1β and IL-6 in the prefrontal cortex (PFC) was analyzed using reverse transcriptase qPCR.We found that aldosterone treatment increased LPS-induced IL-1β mRNA expression in the PFC and CSF. Moreover, there was a positive correlation between IL-1β in CSF and depressive-like behaviors. These findings suggest that IL-1β is affected by the renin–aldosterone–angiotensin system (RAAS) activity and connected to symptoms of depression.

Glucagon-like peptide-1 inhibits LPS-induced IL-1β production in cultured rat astrocytes

Glia play an important role in neurotoxicity in neurodegenerative diseases. In this study, we investigated the expression of glucagon-like peptide-1 (GLP-1) and its receptor, and the effects of GLP-1 on lipopolysaccharide (LPS)-induced IL-1β mRNA expression and IL-1β production in glia. GLP-1-like immunoreactivity was observed in amoeboid microglia, but not ramified microglia or astrocytes. GLP-1 binding and GLP-1 receptor mRNA expression were observed in both astrocytes and microglia. GLP-1-induced morphological changes in microglia from the ramified type to the amoeboid type, suggesting an increase in production and release of endogenous GLP-1. GLP-1 prevented the LPS-induced IL-1β mRNA expression, which effect was, in turn, inhibited by pretreatment with SQ22536, an adenylate cyclase inhibitor. GLP-1 also increased cAMP concentration and cAMP response element-binding protein phosphorylation in astrocytes. These results suggest that GLP-1 may be a modulator of inflammation in the central nervous system.

2-Acetylaminofluorene inhibits interleukin-1β production in LPS-stimulated macrophages by blocking NF-κB/Rel activation

In the present study, we demonstrate the inhibitory effect of 2-acetylaminofluorene (AAF) on interleukin-1β (IL-1β) gene expression in lipopolysaccharide (LPS)-stimulated macrophages. Acetylaminofluorene inhibited IL-1 production in LPS-stimulated splenic macrophages and RAW 264.7 cells. Additionally, AAF also suppressed LPS-induced mRNA expression of IL-1β in macrophages. To further characterize the molecular mechanism responsible for AAF-mediated suppression of IL-1β, we investigated the effect of AAF on LPS-mediated activation of transcription factors, such as NF-κB, AP-1, CRE and NF-IL6, which are known to be important for LPS-induced gene expression of IL-1β. Treatment of AAF caused a dose-related inhibition of LPS-induced NF-κB/Rel transcriptional activation, while the transcriptional activation of AP-1, CRE and NF-IL6 was not affected by AAF. Furthermore, LPS-induced NF-κB/Rel DNA binding was also suppressed by AAF treatment. These results suggest that AAF inhibits IL-1β gene expression by blocking NF-κB/Rel activation.

Dexamethasone inhibits IL-1β gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-κB/Rel and AP-1 activation

In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1β gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1β mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1β gene contains binding motifs for NF-κB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1β induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-κB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-κB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1β production by DEX is not related to p38, which is important in the IL-1β induction. These results suggest that DEX may inhibit IL-1β gene expression by a mechanism involving the blocking of LPS-induced NF-κB/Rel and AP-1 activation.