IL-6 In Supernatants Research Articles

IL-10-producing B cells play important role in the pathogenesis of recurrent pregnancy loss

Interleukin (IL)-10-producing B cells are recently known for their regulatory function in several disorders. However, the possible role of these cells remains unclear in recurrent pregnancy loss (RPL) pathogenesis. Total B cells from 24 RPL patients with cellular immune abnormalities, as well as that of 25 normal pregnant women were cultured and stimulated by Toll Like Receptor (TLR) agonists (CpG oligodeoxynucleotides (ODN) and imiquimod). Then, the frequency of IL-10+ CD19+ B cells was found out using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of IL-10 in supernatant medium and serum of stimulated B cells, as well as those of several serum autoantibodies. Real-Time PCR method was carried out for determining the IL-10 expression level and specific genes transcripts. RPL patients indicated a lower proportion of IL-10+ CD19+ B cells, and reduced levels of IL-10 in both serum and supernatant of the culture medium of the stimulated B cells. According to the results, total IgG levels was greater in serum of RPL patients in comparison with healthy pregnant women. Similarly, the percentage of these cells was negatively correlated with serum total IgG levels and the number of miscarriages. The expression levels of the mRNA of programmed death-ligand 1 (PD-L1) and IL-10 were lower in RPL patients, while those of x binding protein 1 (XBP-1), interferon regulatory factor 4 (IRF4), and B lymphocyte-induced maturation protein 1 (BLIMP1) were significantly increased. These observations indicated that the reduction in the population of peripheral blood IL-10-synthesizing B cells may prompt RPL pathogenesis, suggesting suppressive effects of these cells on autoantibody production and successful pregnancy outcomes.

Over-expression of miR-223 induces M2 macrophage through glycolysis alteration and attenuates LPS-induced sepsis mouse model, the cell-based therapy in sepsis.

The attenuation of hyper-inflammation in sepsis with the administration of anti-inflammatory macrophages is an interesting adjuvant therapy for sepsis. Because the induction of anti-inflammatory macrophages by microRNA (miR), a regulator of mRNA, has been mentioned, the exploration on miR-induced anti-inflammatory macrophages was performed. The over-expression of miR-223 and miR-146a in RAW264.7 induced M2 macrophage-polarization (anti-inflammatory macrophages) as evaluated by the enhanced expression of Arginase-1 and Fizz. However, miR-223 over-expressed cells demonstrated the more potent anti-inflammatory property against LPS stimulation as lesser iNOS expression, lower supernatant IL-6 and higher supernatant IL-10 compared with miR-146a over-expressed cells. Interestingly, LPS stimulation in miR-223 over-expressed cells, compared with LPS-stimulated control cells, demonstrated lower activity of glycolysis pathway and higher mitochondrial respiration, as evaluated by the extracellular flux analysis, and also down-regulated HIF-1α, an important enzyme of glycolysis pathway. In addition, the administration of miR-223 over-expressed macrophages with IL-4 pre-conditioning, but not IL-4 stimulated control cells, attenuated sepsis severity in LPS injected mice as evaluated by serum creatinine, liver enzymes, lung histology and serum cytokines. In conclusion, miR-223 interfered with the glycolysis pathway through the down-regulation of HIF-1α, resulting in the anti-inflammatory status. The over-expression of miR-223 in macrophages prevented the conversion into M1 macrophage polarization after LPS stimulation. The administration of miR-223 over-expressed macrophages, with IL-4 preconditioning, attenuated sepsis severity in LPS model. Hence, a proof of concept in the induction of anti-inflammatory macrophages through the cell-energy interference for sepsis treatment was proposed as a basis of cell-based therapy in sepsis.

CPEB3 inhibits epithelial-mesenchymal transition by disrupting the crosstalk between colorectal cancer cells and tumor-associated macrophages via IL-6R/STAT3 signaling

BackgroundCrosstalk between cancer cells and tumor-associated macrophages (TAMs) mediates tumor progression in colorectal cancer (CRC). Cytoplasmic polyadenylation element binding protein 3 (CPEB3) has been shown to exhibit tumor-suppressive role in CRC.MethodsThe expression of CPEB3, CD68, CD86 and CD163 was determined in CRC tissues. SW480 or HCT116 cells overexpressing CPEB3 and LoVo or RKO cells with CPEB3 knockdown were constructed. Stably transfected CRC cells were co-cultured with THP-1 macrophages to determine the malignant phenotype of CRC cells, macrophage polarization, and secretory signals. The inhibition of CPEB3 on tumor progression and M2-like TAM polarization was confirmed in nude mice.ResultsDecreased CPEB3 expression in CRC was associated with fewer CD86+ TAMs and more CD163+ TAMs. CPEB3 knockdown in CRC cells increased the number of CD163+ TAMs and the expression of IL1RA, IL-6, IL-4 and IL-10 in TAM supernatants. TAMs enhanced CRC cell proliferation and invasion via IL-6, and then activated the IL-6R/STAT3 pathway in CRC cells. However, CPEB3 reduced the IL-6R protein levels by directly binding to IL-6R mRNA, leading to decreased phosphorylated-STAT3 expression in CRC cells. CCL2 was significantly increased in CPEB3 knockdown cells, while CCL2 antibody treatment rescued the effect of CPEB3 knockdown in promoting CD163+ TAM polarization. Eventually, we confirmed that CPEB3 inhibits tumor progression and M2-like TAM polarization in vivo.ConclusionsCPEB3 is involved in the crosstalk between CRC cells and TAMs by targeting IL-6R/STAT3 signaling.

MicroRNA-21-5p promotes the inflammatory response after spinal cord injury by targeting PLAG1.

The aim of this study was to investigate the expression level of microRNA-21-5p and its target gene PLAG1 in the serum of patients with spinal cord injury (SCI), as well as the role and mechanism of microRNA-21-5p in lipopolysaccharide-induced primary microglial inflammation. The levels of microRNA-21-5p and PLAG1 in the peripheral blood of 24 patients with spinal cord injury and 24 healthy people were examined by quantitative Polymerase Chain Reaction (qPCR) analysis. The expressions of inflammatory factors, including IL-1, IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). After microglial BV2 cells were treated with different concentrations of LPS, microRNA-21-5p, and PLAG1 expressions were examined by qPCR, while IL-1, IL-6, and TNF-α levels in cell supernatant were measured by ELISA. Finally, the binding relationship between microRNA-21-5p and its target gene was analyzed by Luciferase reporter assay. The data showed that microRNA-21-5p was upregulated in the serum of SCI patients, and also increased in LPS-treated cells, with a certain concentration dependence. On the contrary, PLAG1 was remarkably downregulated, suggesting that the above two genes were negatively correlated in SCI patients. ELISA results indicated that the knockdown of microRNA-21-5p or overexpression of PLAG1 reduced the levels of IL-1, IL-6 and TNF-α in BV2 cells. Meanwhile, microRNA-21-5p might be involved in regulating the inflammatory response of BV2 cells by modulating PLAG1. MicroRNA-21-5p promotes the inflammatory response after SCI by targeting PLAG1.

Irisin alleviates renal injury caused by sepsis via the NF-κB signaling pathway.

Renal injury caused by sepsis is a difficult point in the field of critical care medicine today, which seriously endangers the health of patients. The aim of our paper was to study the role of irisin in the inflammation and apoptosis of renal injury caused by sepsis and its potential mechanism of action. Lipopolysaccharide (LPS) was utilized to establish an acute kidney injury model. HK-2 cells were divided into 3 groups: control group, LPS group, LPS+irisin group. The expression of TNF-α, IL-1β, Bcl-2, and Bax were detected using Western blot. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect the levels of TNF-α, IL-6, and IL-1β in the cell supernatant. The LDH content was detected to observe cell damage. TUNEL staining and flow cytometry were to investigate the apoptosis in three groups. The viability of HK-2 cells was detected using Cell Counting Kit-8 (CCK-8) assay. After HK-2 cells were treated with LPS, the LDH content in the cell supernatant was greatly increased, and the expression of TNF-α, IL-6, and IL-1β was also significantly increased. However, after treatment with irisin, LDH content and expression of inflammatory factors were significantly suppressed. Similarly, LPS treatment greatly elevated the levels of TNF-α, IL-1β, Bax, p65 and IκKα, as well as inhibited the expression of Bcl-2 and IκB-α. However, irisin treatment reversed these situations. In addition, the number of TUNEL-positive cells and the apoptotic rate were also greatly decreased in LPS+irisin group compared with those in LPS group. Irisin could inhibit inflammation and apoptosis of HK-2 cells treated with LPS via the NF-κB pathway.

Klotho Alleviates Lung Injury Caused by Paraquat via Suppressing ROS/P38 MAPK-Regulated Inflammatory Responses and Apoptosis.

Acute lung injury (ALI) induced by paraquat (PQ) progresses rapidly with high mortality; however, there is no effective treatment, and the specific mechanism is not well understood. The antiaging protein klotho (KL) has multiple functions and exerts significant influences on various pathophysiological processes. This work evaluated the impact of KL on PQ-induced ALI and investigated its underlying mechanisms. As for in vivo research, C57BL/6 mice were treated with PQ (30 mg/kg) intraperitoneal (IP) injection to create a toxicity model of ALI (PQ group). The mice were divided into control group, KL group, PQ group, and PQ+KL group. For in vitro experiment, A549 cells were incubated with or without KL and then treated in the presence or absence of PQ for 24 h. In vivo result indicated that KL reduced the mortality, reduced IL-1β and IL-6 in the bronchoalveolar lavage fluid (BALF), attenuated ALI, and decreased apoptosis in situ. In vitro result revealed that KL significantly improved cell viability, reduced the levels of IL-1β and IL-6 in culture supernatants, suppressed cell apoptosis, inhibited caspase-3 activation, and enhanced mitochondrial membrane potential (ΔΨm) after PQ treatment. Besides, KL effectively abated reactive oxygen species (ROS) production, improved GSH content, and lowered lipid peroxidation in PQ-exposed A549 cells. Further experiments indicated that phosphorylated JNK and P38 MAPK was increased after PQ treatment; however, KL pretreatment could significantly lower the phosphorylation of P38 MAPK. Suppression of P38 MAPK improved cell viability, alleviated inflammatory response, and reduced apoptosis-related signals; however, it had no obvious effect on the production of ROS. Treatment with N-acetylcysteine (NAC), a classic ROS scavenger, could suppress ROS production and P38 MAPK activation. These findings suggested that KL could alleviate PQ-caused ALI via inhibiting ROS/P38 MAPK signaling-regulated inflammatory responses and mitochondria-dependent apoptosis.

Effect of deletion of SUMOylation on dendritic cell function in septic mice and its role in sepsis.

To investigate the effect of deletion of small ubiquitin-like modifier (SUMO) on the function of dendritic cells (DC) in septic mice and its role in sepsis. Septic models of DC-specific ubiquitin-conjugating enzyme 9 (UBC9) deficient (UBC9ΔDC) mice and wild type (WT) mice with cecal ligation and puncture (CLP) were established. The differences in 7-day mortality of the mice were analyzed. Bacteria loads of blood, liver, and spleen were tested. ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α in plasma and culture medium of bone marrow-derived dendritic cells (BMDC). The levels of cytokine IFN-γ and IL-4 in supernatant of spleen mononuclear cells were detected by ELISA.The expressions of MHC II, CD54, and CD80 on the cell surface of DC were analyzed by flow cytometry. The percentages of Th1, and Th2 cells in spleen mononuclear cells were analyzed by flow cytometry. Compared with the WT septic mice, the 7-day mortality of UBC9ΔDC septic mice was higher (P<0.05). Bacterial loads in blood (P<0.01), liver (P<0.01), and spleen (P<0.05) were significantly increased in UBC9ΔDC septic mice. Levels of IL-1β and IL-18 in plasma and culture supernatant of BMDC were also significantly increased in UBC9ΔDC septic mice (all P<0.01). There was no significant difference in the number of DC and the expression of cell surface molecules in DC of UBC9ΔDC septic mice (all P>0.05).The percentage of Th2 cells was significantly increased in UBC9ΔDC septic mice (P<0.05). The ratio of Th1 to Th2 was decreased in UBC9ΔDC septic mice but the difference was not significant (P>0.05). Levels of IFN-γ and IL-4 were increased in UBC9ΔDC septic mice, and the ratio of IFN-γ to IL-4 were significantly decreased in UBC9ΔDC septic mice (all P<0.05). Deletion of SUMOylation may increase the mortality of mice with sepsis through regulating the release of inflammatory factors from DC and abnormal activation of T cells by DC.

Combination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells in Vitro.

Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.

Amelioration of experimental autoimmune encephalomyelitis by Rhodiola rosea, a natural adaptogen

Adaptogens are stress-response modifiers which have been considered to exert important influences on cytokines activities and immune response. Here, we investigated the effects of Rhodiola rosea (R. rosea), a natural adaptogen on the experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that R. rosea alleviated the severity of EAE as evaluated by clinical and histopathology scores. R. rosea decreased IL-6, sIL-6R, IFN-γ and IL-17A in both serum and splenocytes supernatants while increased IL-4 in splenocytes supernatants of EAE mice. R. rosea also modulated the T cell response by inhibiting Th1 and Th17 cells, restoring Treg cells, combining with regulated Th17/Th1, Th17/Th2 and Th17/Treg ratios in the spleen, inguinal lymph nodes, brain and spinal cord of EAE mice. Furthermore, R. rosea regulated the expression of JAK1, JAK2, STAT3 and RORγt in the spinal cord of EAE mice. Together, our findings suggested the adaptogenic effects of R. rosea on EAE, which may help to deepen the understanding of potential therapeutic values of traditional Chinese medicines for autoimmune diseases of CNS.

Astragalus propinquus Schischkin and Panax notoginseng (A&P) compound relieved cisplatin-induced acute kidney injury through inhibiting the mincle maintained macrophage inflammation

Ethnopharmacological relevanceAcute kidney injury (AKI) is a common disease in hospitalized patients, especially in critically ill patients. It is characterised with high morbidity and mortality, and is also an important cause of chronic kidney disease and chronic renal failure. Astragalus propinquus Schischkin and Panax notoginseng (A&P) compound, a famous traditional Chinese medicine, consists of Astragalus propinquus Schischkin, Panax notoginseng, Angelica sinensis, Achyranthes bidentata, and Ecklonia kurome, has been widely used for the treatment of various kidney diseases in the southwest of China. However, the effects of A&P on treatment of AKI and its underlying mechanism are needed to be uncovered. Aim of the studyRecent researches reported that Mincle (Macrophage-inducible C-type lectin) plays a key role in renal injury of AKI by regulating the expression and secretion of inflammatory cytokines on macrophage through modulating NF-κB signaling pathway. Here, we aimed to investigate the renoprotective effect of A&P on AKI and whether by inhibiting Mincle. Materials and methodsWe established a lipopolysaccharide (LPS)-induced Bone Marrow-Derived Macrophage (BMDM) inflammatory cell model and a cisplatin-induced mouse AKI model in vitro and in vivo. Renal histopathology staining was performed to observe kidney morphology. The expression and secretion of inflammatory cytokines were detected by real-time PCR and Enzyme-linked immunosorbent assay. Western blotting was used to detect the protein levels and Flow cytometry performed to detect polarization of macrophage. ResultsThe results showed that A&P significantly reduced the mRNA expression of IL-1β, IL-6, TNFα and MCP-1 in LPS-stimulated BMDM cells, and secretion of IL-1β and IL-6 in supernatant. The same results were found in Cisplatin-induced AKI kidney and serum after treatment with A&P. The data also showed that A&P strongly reduced the mRNA and protein levels of Mincle in vitro and vivo, and also inhibited the activation of Syk and NF-κB. Notably, A&P down-regulated the M1 macrophage marker iNOS, which may relate to the inhibition of Mincle. Interestingly, both overexpression of Mincle by transfection of pcDNA3.1-Mincle plasmid and administration of TDB (a ligand of Mincle) can significantly abolished the A&P-inhibited inflammation in BMDM, suggesting Mincle pathway play a key role in macrophage inflammation in AKI. ConclusionOur findings indicated that A&P protected kidney from inhibiting inflammation through down-regulating of Mincle pathway in macrophage in AKI. It provides a potential medicine compound for the treatment of AKI.

Curcumin Suppresses Apoptosis and Inflammation in Hypoxia/Reperfusion-Exposed Neurons via Wnt Signaling Pathway.

BackgroundCerebral ischemia/reperfusion (I/R) injury contributes to mortality and morbidity in preterm infants. Curcumin has been shown to exert neuro-protective effects in the central nervous system (CNS). The aim of this study was to investigate the neuro-protective activity of curcumin and the possible underlying molecular mechanisms.Material/MethodsA hypoxia/reoxygenation (H/R) protocol was used to simulate I/R injury in vitro. Isolated neonatal neurons were pre-treated with curcumin at serially diluted concentrations and exposed to H/R injury. Cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. Contents of TNFα and IL6 in supernatant of cell culture medium were detected by ELISA. Protein expression, phosphorylation, and nuclear translocation levels were studied by Western blotting.ResultsH/R reduced cell viability and increased apoptosis of neurons. H/R significantly increased Wnt5a expression, JNK1 phosphorylation, and NF-κB nuclear translocation. Moreover, expression levels of cleaved caspase3, TNFα, and IL6 were elevated in H/R-exposed neurons. Curcumin pre-treatment significantly increased cell viability and inhibited apoptosis of neurons exposed to H/R, in a concentration-dependent manner. Moreover, curcumin pre-treatment significantly decreased expression levels of Wnt5a, IL6, TNFα, and phosphorylation level of JNK1, as well as the nuclear translocation level of NF-κB in H/R-exposed neurons, in a concentration-dependent manner.ConclusionsCurcumin exerted neuro-protective effects against H/R-induced neuron apoptosis and inflammation by inhibiting activation of the Wnt/JNK1 signaling pathway.

HDAC6 inhibitor Cay10603 inhibits high glucose-induced oxidative stress, inflammation and apoptosis in retinal pigment epithelial cells via regulating NF-κB and NLRP3 inflammasome pathway.

The present study aimed to investigate the effects of histone deacetylase 6 (HDAC6) inhibitor Cay10603 (Cay) on high glucose (HG)-stimulated human retinal pigment epithelium (RPE) cells and its underlying mechanisms. ARPE-19 cells were cultured under normal glucose (NG) or high glucose (HG) conditions. The results revealed that HDAC6 was upregulated in HG-stimulated ARPE-19 cells. Cay treatment caused a decrease in intracellular reactive oxygen species (ROS). The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were reduced accompanied by increase in the activities of superoxide dismutase (SOD) and catalase (CAT) after treatment with Cay. Besides, Cay decreased the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and monocyte chemoattractant protein-1 (MCP-1) in supernatant. Meanwhile, the apoptotic rate in Cay-treated ARPE-19 cells notably reduced, coupled with an upregulation in Bcl-2 expression and a downregulation in cleaved caspase-3 and cleaved caspase-9 expression. Cay decreased the expression of phospho (p)-NF-κB p65, p-IκB-α, NLRP3, cleaved caspase-1 and ASC while increased the expression of NF-κB p65 (cytoplasm). Taken together, these findings demonstrated that Cay suppressed HG-induced oxidative stress, inflammation and apoptosis via regulating NF-κB and NLRP3 inflammasome pathway in HG-induced ARPE-19 cells, suggesting that Cay might be a therapeutic agent for the treatment of diabetic retinopathy.

Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte.

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host.OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated.METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry.FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines.MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Ethanol extract of Elephantopus scaber Linn. Attenuates inflammatory response via the inhibition of NF-κB signaling by dampening p65-DNA binding activity in lipopolysaccharide-activated macrophages.

Elephantopus scaber Linn. (E.scaber) is a widely-used traditional herb whose use has been documented for various inflammatory diseases such as fever, sore throat, dysentery, carbuncle and so on. However, the effect and mechanism of E.scaber in LPS-activated macrophages remain unclear. This study aims to investigate the anti-inflammatory mechanism of the ethanol extract of E.scaber (ESE) in lipopolysaccharide (LPS)-induced inflammatory models. Griess reagent was used to determine NO production, and the levels of TNF-α, IL-6, MCP-1 and IL-1β were determined by ELISA kits. The molecular mechanism research was performed by RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA). LPS-induced endotoxemia mouse model was used for evaluating the in vivo anti-inflammatory action of ESE. ESE suppressed LPS-induced iNOS, TNF-α, IL-6, MCP-1 and IL-1β transcription as well as supernatant NO, TNF-α, IL-6, MCP-1 and IL-1β production in macrophages. Although ESE inhibited NF-κB activation, it did not affect the IκBα phosphorylation and degradation and the NF-κB p65 nuclear translocation. The result of EMSA revealed that ESE inhibited the NF-κB p65-DNA binding activity. Additionally, ESE also decreased the proinflammatory cytokines in serum and peritoneal lavage fluid of LPS-induced endotoxemic mice. ESE has a potently anti-inflammatory effect through inhibiting the NF-κB p65-DNA binding activity in LPS-activated macrophages.

Long non-coding RNA NKILA inhibited angiogenesis of breast cancer through NF-κB/IL-6 signaling pathway

ObjectiveThe relationship between NF-κB Interacting lncRNA (NKILA) and angiogenesis in breast cancer has never been studied. Our study aimed to investigate effect of NKILA on proliferation, migration, apoptosis, as well as angiogenesis in breast cancer. MethodsNKILA was over-expressed in MDA-MB-231 cells by transfection of pcDNA3.1-NKILA vector. Cell viability, apoptosis and migration were measured by MTT, flow cytometry and wound healing assays, respectively. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was measured using tube formation assay. The expression levels of NKILA, IL-6, VEGFA, VEGFR, apoptosis and epithelial-mesenchymal transition (EMT) and NF-κB/IL-6 signaling-related markers were determined using qRT-PCR or Western blotting. ResultsCell viability and migration of MDA-MB-231 cells were significantly inhibited, while cell apoptosis was obviously promoted by overexpression of NKILA. Overexpression of NKILA could also inhibit the phosphorylation of IκBα and the nuclear transposition of p65, as well as induce cell apoptosis-related proteins and inhibit epithelial-mesenchymal transition-related proteins. Cell viability and migration of HUVEC were also significantly inhibited when treated with supernatant of cells overexpressed NKILA or treated with BAY11-7028. Exogenous IL-6 significantly increased the cell viability and migration of HUVEC, and overexpression of NKILA could reverse these effects induced by IL-6. Overexpression of NKILA significantly inhibited the protein levels of IL-6 and VEGFA in supernatant, as well as VEGFR in HUVEC, thus inhibited the angiogenesis of HUVEC. NKILA also reversed the above effects on protein levels of IL-6 and VEGFA in supernatant and angiogenesis induced by exogenous IL-6. ConclusionOverexpression of NKILA could inhibit cell proliferation, migration and promote apoptosis of breast cancer cells. It could also inhibit cell proliferation, migration and angiogenesis of HUVEC through inhibiting IL-6 secretion via NF-κB signaling pathway.

Neutralizing effects of anti-infliximab antibodies on synergistically-stimulated human coronary artery endothelial cells

Background and aimsPatients with rheumatic diseases have an increased risk of atherosclerosis with up-regulated serum amyloid A (SAA), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which were reported to activate human coronary artery endothelial cells (HCAEC). We aimed to investigate the effects of TNF-α inhibitor infliximab and anti-infliximab antibodies on the TNF-α/IL-1β/SAA activated HCAEC. MethodsHCAEC were incubated with TNF-α, IL-1β, SAA, infliximab, anti-infliximab antibodies and their combinations. The protein levels of pro- and anti-atherogenic analytes were measured in supernatants using ELISA and multiplex assays, while mRNA expression was determined by RT-PCR. Anti-infliximab antibodies were purified from sera samples by affinity chromatography. ResultsIL-6, IL-8, GM-CSF and GRO-α were synergistically up-regulated in triple stimulation with TNF-α, IL-1β and SAA, while their levels in solely SAA- or TNF-α-stimulated HCAEC did not increase. IL-1Ra, IL-1α, VCAM-1, MCP-1, IL-10 and IL-17A were increased, but no synergistic responses were observed in triple stimulation. Infliximab was effective in lowering the synergistic effect of IL-6, IL-8, GM-CSF and GRO-α in triple stimulation, while anti-infliximab antibodies restored the levels. The changes were confirmed at the mRNA expression level for IL-6, IL-8 and GM-CSF. ConclusionsTriple stimulation with TNF-α, IL-1β and SAA synergistically elevated IL-6, IL-8, GM-CSF and GRO-α release in supernatants of HCAEC, with infliximab substantially inhibiting their levels. An isolated, enriched fraction of polyclonal anti-infliximab antibodies was capable of neutralizing infliximab, in the presence of TNF-α/IL-1β/SAA. The long-term presence of anti-infliximab antibodies in the circulation of patients with chronic rheumatic diseases is potentially important for promoting the atherosclerotic process.

Relationship between TIPE2 and caspase-11 during pyroptosis in macrophages of mice

Objective To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) and caspase-11 during pyroptosis in macrophages of mice. Methods J774A.1 macrophages of mice were divided into 4 groups (n=6 each) using a random number table method: non-specific siRNA (Scr-siRNA) group (S group), Scr-siRNA plus LPS/ATP group (S+ LPS/ATP group), TIPE2-siRNA group (T group) and TIPE2-siRNA plus LPS/ATP group (T+ LPS/ATP group). The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group, and transfection was performed for 24-48 h, and in addition LPS 1.0 μg/ml was then added, cells were incubated for 5 h, then ATP 5.0 mmol/L was added, and cells were incubated for 1 h in S+ LPS/ATP and T+ LPS/ATP groups.Cells were collected to detect the expression of TIPE2, caspase-11, NOD-like receptor familypyrin domain containing 3 (NLRP3) and caspase-1 (by Western blot). The supernatant was collected for determination of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) activities and concentrations of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results Compared with group S, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group S+ LPS/ATP (P<0.05). Compared with group T, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Compared with group S+ LPS/ATP, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Conclusion TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice. Key words: Pyroptosis; Macrophages; Caspases; TIPE2

Changes of endotoxin tolerant dendritic cell immune function and its effect on sepsis in mouse model

Objective To study the changes of immune function of endotoxin tolerant dendritic cell (ETDC) and to observe its effect on sepsis in mouse model. Methods ETDC were prepared by pretreated bone marrow dendritic cells derived from BALB/c mice with lipopolysaccharide stimulation. The cells were collected and the expressions of surface markers including major histocompatibility complex (MHC)Ⅱ, CD86 and CD11c were detected by flow cytometry. The proliferation rate of T lymphocytes was evaluated by cell counting kit-8 and the concentrations of cytokines in the supernatant were detected by enzyme linked immuno sorbent assay. Afterwards, 36 mice were randomly assigned into 4 groups. The blank control group did not receive any treatment, the sham-operated group underwent simple incision suture, the sepsis group and ETDC reinfusion group underwent cecal ligation and puncture to establish sepsis. Before sepsis model establishment, 0.9% sodium chloride solution or suspension of ETDC and 0.9% sodium chloride were reinfused by tail vein. The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), tumor necrosis factor (TNF)-α, interleukin(IL)-6 and IL-10, and the proportion of help T cell (Th) 17/regulatory T cell (Treg) in spleen of each group were detected. The pathological manifestations of liver, kidney and ileum in each group were observed. T test and χ2 test were used for comparisons between groups. Results The results of flow cytometry showed that MHCⅡ, CD86 and CD11c of ETDC were 70.4%, 43.1%, and 73.1%, respectively, which were significantly lower than those of mature dendritic cell (mDC) (96.1%, 89.5%, and 84.6%, respectively) (χ2=56.47, 83.78, and 23.29, respectively, all P<0.01). The concentrations of IL-10, TNF-α and IL-6 in the supernatant of ETDC were (978.04±56.70), (980.34±111.96) and (12 743.03±865.81) ng/L, respectively, and those of mDC were (741.35±99.23), (1 703.11±117.00) and (19 052.28±1 145.84) ng/L, respectively. The differences were all statistically significant (t=5.073, 10.93, and 10.76, respectively, all P<0.01). The proliferation rates of T lymphocytes co-cultured with ETDC in 1∶5 and 1∶10 ratio group were (676.95±85.99)% and (514.00±106.39)%, respectively, which were lower than those of the mDC group (956.25±127.12)% and (772.07±214.08)%, respectively. The pathological injuries of liver, kidney and ileum in ETDC treatment group were significantly lighter than those in sepsis group. Serum ALT and AST levels in the ETDC reinfusion group were (299.71±36.91) and (690.39±154.92) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.067±0.005), (0.428±0.051) and (0.058±0.005) ng/L, respectively. Serum ALT and AST levels in the sepsis group were (620.67±69.27) and (1 430.17±134.05) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.085±0.007), (0.774±0.088) and (0.036±0.005) ng/L, respectively. The differences were all statistically significant (t=11.60, 10.96, 5.991, 8.657, and 8.04, respectively, all P <0.01). The proportion of Treg/Th17 in the ETDC reinfusion group was 23.4%, and that in the sepsis group was 60.8% (χ2=28.69, P<0.01). Conclusion The reinfusion of ETDC has a protective effect on sepsis in mouse model, which may play a negative immune regulatory role by regulating the differentiation of T cells. Key words: Sepsis; Dendritic cells; Endotoxin tolerance

Multi-parametric Cell-Based Inflammation Assays for Cytokines and Cell Surface Antigens

Abstract Macrophages and vascular cells upregulate expression of cytokines and adhesion molecules in response to inflammation stimuli. Monitoring these molecules can provide a physiological read-out for inflammation. We present results from a multiparametric assays that used a combination of low-volume ELISA for secreted cytokines and automated cell imaging for cell surface markers to evaluate the effect of different mediators on inflammatory response with two cell models. First, human monocytic THP-1 cells were differentiated to macrophages and activated by LPS. Phenotypic changes in cell morphology were quantified by cell area and number of adherent cells using TL imaging. Secretion of IL-8, IL-1β and TNF-α was quantified by an automated low-volume ELISA (PuMA System). Second, primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β), then PuMA System was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence from antibody-stained cells using an ImageXpress Pico system. Concentration dependent inhibition of inflammation by the compounds AG126 and MG132, SB202190 and PDTC were measured and EC50s calculated. Clear differences in cytokine expression were seen between the compounds consistent with reported mechanism of actions of the kinase inhibitors. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can test the efficacy of anti-inflammatory compounds versus toxicity and provide insight into their MoA by selective inhibition of markers triggered by different signaling pathways.

Fluoxetine suppresses inflammatory reaction in microglia under OGD/R challenge via modulation of NF-κB signaling.

We aimed to investigate the anti-inflammatory role of fluoxetine, a selective serotonin reuptake inhibitor, in microglia (MG) and the mechanisms under oxygen glucose deprivation/reoxygenation (OGD/R). An OGD/R model on BV-2 cells was used for the study of microglia under ischemia/reperfusion injury in ischemic stroke. Lentiviral transfection was applied to knock down IκB-α. Enzyme-linked immunosorbent assay (ELISA) was used for detecting levels of TNF-α, IL-1β, and IL-6, and real-time PCR was used to assess the expression of IκB-α protein. Western blotting was applied to analyze NF-κB-signaling related proteins and Cell Counting Kit-8 (CCK-8) was used for assessing cell viability. Molecular docking and drug affinity responsive target stability (DARTS) assay were used for the detection of the interaction between IκB-α and fluoxetine. We found that fluoxetine decreased the levels of TNF-α, IL-1β, and IL-6 in supernatant as well as NF-κB subunits p65 and p50 in BV-2 cells under OGD/R. Fluoxetine significantly increased the level of IκB-α through the inhibition of IκB-α ubiquitylation and promoted the bonding of IκB-α and fluoxetine in BV-2 cells under OGD/R. Knocking down IκB-α attenuated the decreasing effect of TNF-α, IL-1β, and IL-6 as well as p65 and p50 in BV-2 cells under OGD/R led to by fluoxetine. In conclusion, our present study demonstrated the anti-inflammatory role of fluoxetine and its mechanisms related to the modulation of NF-κB-related signaling in MG under ischemia/reperfusion challenge.