Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.     

Scaffold compound L971 exhibits anti-inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune-related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high-throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli-dependent activation of STAT1, STAT3 and IκBα and could significantly down-regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high-throughput RNA sequencing, and significant differentially up-regulated and down-regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti-inflammatory effects of L971. Finally, L971 anti-inflammatory character was further verified in LPS-induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down-regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.     

The effect of anti-inflammatory properties of ferritin light chain on lipopolysaccharide-induced inflammatory response in murine macrophages

Ferritin light chain (FTL) reduces the free iron concentration by forming ferritin complexes with ferritin heavy chain (FTH). Thus, FTL competes with the Fenton reaction by acting as an antioxidant. In the present study, we determined that FTL influences the lipopolysaccharide (LPS)-induced inflammatory response. FTL protein expression was regulated by LPS stimulation in RAW264.7 cells. To investigate the role of FTL in LPS-activated murine macrophages, we established stable FTL-expressing cells and used shRNA to silence FTL expression in RAW264.7 cells. Overexpression of FTL significantly decreased the LPS-induced production of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), nitric oxide (NO) and prostaglandin E2 (PGE2). Additionally, overexpression of FTL decreased the LPS-induced increase of the intracellular labile iron pool (LIP) and reactive oxygen species (ROS). Moreover, FTL overexpression suppressed the LPS-induced activation of MAPKs and nuclear factor-κB (NF-κB). In contrast, knockdown of FTL by shRNA showed the reverse effects. Therefore, our results indicate that FTL plays an anti-inflammatory role in response to LPS in murine macrophages and may have therapeutic potential for treating inflammatory diseases.     

TIS21/BTG2/PC3 inhibits interleukin-6 expression via downregulation of STAT3 pathway

Cancer cell growth was increased when co-cultured with fibroblasts, however, no effect was observed when co-cultured with TIS21-overexpressed fibroblast. Therefore, the role of TIS21 played in cancer microenvironment was investigated. TIS21 decreased interleukin-6 (IL-6) expression in human dermal fibroblast (HDF). Adenoviral transduction of TIS21 gene to HDF decreased the secretion of IL-6, whereas knockdown of the gene increased IL-6 expression. Furthermore, TIS21 overexpression inhibited STAT3 binding to IL-6 promoter region as well as JAK2–STAT3 signaling by inhibiting reactive oxygen species (ROS) generation by being localized in mitochondria. Mitochondria-target TIS21 (MT-TIS21) also inhibited IL-6 expression by downregulating STAT3 phosphorylation, whereas NF-κB pathway was not influenced by TIS21 expression. These results indicate that TIS21 negatively regulated cancer cell growth by inhibiting IL-6 expression through downregulation of STAT3 activation.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.     

In Vitro Investigation of the Immunomodulatory Potential of Probiotic Lactobacillus casei

The current study investigated the immunomodulatory potential of ethyl acetate soluble supernatant of Lactobacillus casei (LC-EAS) in vitro. The effect of LC-EAS on nitric oxide release was analyzed in RAW 264.7 cells, wherein, an inhibition in nitric oxide production through suppression of inducible nitric oxide synthase mRNA expression was observed. Evaluation of LC-EAS on LPS-induced peripheral blood mononuclear cells showed a down-regulation in TNF-α and IL-6 genes and an upregulation of IL-10. An inhibition in the protein expression of NF-κB, ERK1/2 and STAT3 phosphorylation confirms the immunomodulatory potential of LC-EAS. The effect of LC-EAS on in vitro intestinal epithelial cells was investigated using HT-29 human colon adenocarcinoma cancer cells. LC-EAS exhibited an inhibition of NF-κB and ERK1/2 phosphorylation, whereas STAT3 phosphorylation was unregulated. To evaluate the downstream target of STAT3 upregulation, expression of the intestinal trefoil factor TFF3 which is a NF-κB regulator and STAT3 downstream target was studied. LC-EAS was observed to elevate TFF3 mRNA expression. Overall the study shows that the anti-inflammatory potential of LC-EAS is through inhibition of NF-κB in different cell types.     

MicroRNA-146 protects A549 and H1975 cells from LPS-induced apoptosis and inflammation injury

Pneumonia is an inflammatory condition affecting the lungs, in which pro-inflammatory cytokines are secreted. It has been shown that microRNA-146 (miR-146) is involved in the regulation of immune and inflammatory responses. The present study explored the protective effects of miR-146 overexpression on lipopolysaccharide (LPS)-mediated injury in A549 and H1975 cells. In this study, A549 and H1975 cells were transfected with miR-146 mimic or inhibitor, and then were subjected with LPS. Thereafter, cell viability, colony formation capacity, apoptosis, the release of proinflammatory factors, Sirt1 expression, and the expression of NF-κB and Notch pathway proteins were respectively assessed. As a result, miR-146 overexpression exerted protective functions on LPS-damaged A549 and H1975 cells, as evidenced by the increases in cell viability and colony number, the decrease in apoptotic cell rate, as well as the down-regulations of IL-1, IL-6, and TNF-α. Sirt1 can be positively regulated by miR-146. Furthermore, miR-146 overexpression blocked NF-κB and Notch pathways, while these blocking effects were abolished when Sirt1 was silenced. The findings in the current study indicated that miR-146 protected A549 and H1975 cells from LPS-induced apoptosis and inflammation injury. miR-146 exerted protective functions might be via up-regulation of Sirt1 and thereby blocking NF-κB and Notch pathways.     

Increased autocrine interleukin-6 production is significantly associated with worse clinical outcome in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) remains incurable with current standard therapy. We have previously reported that an increased expression of interleukin-6 (IL-6) receptor CD126 leads to resistance of CLL cells to chemotherapy and worse prognosis for patients with CLL. In this study, we determine whether autocrine IL-6 production by CLL B cells is associated with poor clinical outcome and explore IL-6-mediated survival mechanism in primary CLL cells. Our results demonstrate that higher levels of autocrine IL-6 are significantly associated with shorter absolute lymphocyte doubling time, patients received treatment, without complete remission, advanced Binet stages, 17p/11q deletion, and shorter time to first time treatment and progression-free survival. IL-6 activated both STAT3 and nuclear factor kappa B (NF-κB) in primary CLL cells. Blocking IL-6 receptor and JAK2 inhibited IL-6-mediated activation of STAT3 and NF-κB. Our study demonstrates that an increased autocrine IL-6 production by CLL B-cells are associated with worse clinical outcome for patients with CLL. IL-6 promotes CLL cell survival by activating both STAT3 and NF-κB through diverse signaling cascades. Neutralizing IL-6 or blocking IL-6 receptor might contribute overcoming the resistance of CLL cells to chemotherapy. We propose that the measurement of autocrine IL-6 could be a useful approach to predict clinical outcome.     

人参皂苷Rg1对游离脂肪酸诱导非酒精性脂肪肝细胞炎症的改善作用及机制研究

该研究探讨人参皂苷Rg1对非酒精性脂肪性肝细胞炎症反应的作用及其分子机制。用1 mmol/L游离脂肪酸处理HepG2和L02细胞24 h,再用20μg/mL或40μg/mL人参皂苷Rg1处理6 h;设置对照组、模型组、低剂量Rg1组、高剂量Rg1组。全自动生化仪检测各组细胞上清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的含量;酶联免疫吸附法测定细胞上清IL-1β、IL-6、TNF-α。RT-qPCR及Western blot检测NF-κB通路相关基因及蛋白的改变。免疫荧光染色观察NF-κB核转移;Western blot检测各组胞质与胞核内的NF-κB P65蛋白的表达。与对照组相比,模型组培养上清炎症指标明显增加(P<0.05);Rg1能降低炎症指标的表达(P<0.05)。Rg1能减少游离脂肪酸诱导的NF-κB磷酸化及其下游IL-1β、IL-6、TNF-α的表达,减少NF-κB P65从胞质向胞核的转移(P<0.05)。Rg1可通过抑制NF-κB活化减少NASH细胞模型炎症反应,为非酒精性脂肪性肝炎的治疗提供了可能的靶点。     

γ-Tocotrienol inhibits lipopolysaccharide-induced interlukin-6 and granulocyte colony-stimulating factor by suppressing C/EBPβ and NF-κB in macrophages

Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) β appears to be involved in IL-6 formation because LPS induces C/EBPβ up-regulation, which parallels IL-6 production, and knockdown of C/EBPβ with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPβ and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPβ without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPβ target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBβ and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.     

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Mitochondrial DNA depleted (ρ⁰) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ⁺ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ⁰ cells compared to ρ⁺ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ⁺ HSF, but this response was substantially decreased in ρ⁰ HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ⁺ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ⁰ HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.