变形链球菌对EAhy926细胞TLR2 mRNA表达的影响

目的:观察变形链球菌细胞壁及培养上清对人血管内皮细胞EAhy926细胞TLR2 mRNA表达影响.方法:不同剂量的变形链球菌细胞壁或培养上清作用于EAhy926细胞,RT-PCR法检测EAhy926细胞TLR2 mRNA的表达.结果:变形链球菌细胞壁作用于EAhy926细胞后,TLR2 mRNA的表达量随着作用时间延长而逐渐增高,于16～24 h达到高峰,以后又逐渐下降(P<0.01);变形链球菌培养上清作用于EAhy926细胞后,TLR2 mRNA的表达量不随着作用时间和作用浓度而发生变化.结论:变形链球菌细胞壁可明显上调EAhy926细胞TLR2 mRNA的表达,并呈时间和剂量依赖性;变形链球菌培养上清不能刺激EAhy926细胞TLR2 mRNA的表达.     

Xiao-Xu-Ming decoction extract alleviates LPS-induced neuroinflammation associated with down-regulating TLR4/MyD88 signaling pathway in vitro and in vivo

In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract (XXM) on lipopolysaccaride (LPS)-induced neuroinflammation invitro and invivo. Invitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. Invivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. Invitro, XXMsignificantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressedexpressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. Invivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, andinhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.     

Role of TLR ligands in epicutaneously induced contrasuppression

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4⁺8⁺ T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-γ production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLRligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.     

Toll样受体9在中耳胆脂瘤中的表达及其与白细胞介素-1α的相关性

目的研究Toll样受体9(TLR9)在中耳胆脂瘤上皮细胞的表达,并分析与白细胞介素1α(IL-1α)表达的关系。方法采用免疫组织化学二步法检测23例慢性中耳炎合并获得性胆脂瘤手术标本与10例胆脂瘤患者正常外耳道皮肤中TLR9、IL-1α的表达。结果在23例慢性中耳炎合并获得性胆脂瘤手术标本与10例胆脂瘤患者正常外耳道皮肤中,TLR9的表达指数分别为0.34±0.14、0.17±0.07,两者比较差异有统计学意义(P<0.01);IL-1α的表达指数分别为0.53±0.12,0.20±0.08,两者比较差异有统计学意义(P<0.01);胆脂瘤上皮TLR9与IL-1α的表达呈显著正相关(r=0.730,P<0.01)。结论 TLR9与IL-1α表达密切相关,表明TLR9可能上调IL-1α的表达,TLR9的信号转导参与了中耳胆脂瘤的骨质破坏过程。     

白细胞介素7基因转染口腔癌细胞株的免疫调节效应的研究

目的：探讨IL-7基因转染人舌鳞癌Tca8113细胞株的免疫调节效应。方法：将已构建的原核／真核表达载体pBK—CMV／IL-7转染到癌细胞株中，采用MTT法检测转染前后肿瘤细胞培养上清对鼠脾细胞增殖的影响；ELISA法检测细胞培养上清转移生长因子β1（TGF-β1）、血管内皮生长因子（VEGF）和IL-10的浓度。将pPK—CMV／IL-7癌细胞株移植于小鼠腹腔，观察肿瘤细胞的成瘤性；并采用MTT法检测其NK杀伤活性。结果：IL-7基因转染癌细胞株的培养上清对鼠脾细胞的增殖抑制作用与对照组比较显著下降（P〈0．05），并且IL-7基因转染的肿瘤细胞产生TGF-β1、VEGF和IL-10三种免疫抑制因子明显降低（P〈0．05）；IL-7基因转染组鼠腹腔瘤结节明显小于对照组（P〈0．05）；NK的杀伤活性与对照组比较，具统计学差异（P〈0．05）。结论：IL-7基因转染人舌鳞癌细胞株具有抑瘤作用，其机理可能是通过降低肿瘤细胞产生免疫抑制因子，进而促进淋巴细胞增殖，提高NK的杀伤活性．     

Induction of VEGF and MMP-9 expression by toll-like receptor 2/4 in human endothelial cells infected with Chlamydia pneumoniae

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules, and, in particular, it is demonstrated that the 92 KDa gelatinase MMP-9 is often expressed in atherosclerotic plaques by macrophages and smooth muscle cells. Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerosis lesions. In this study, we analyzed the TLR2/TLR4 expression in HUVEC infected with C. pneumoniae and correlated it to the production of VEGF and MMP-9. The results obtained showed an increased VEGF and MMP-9 production correlated with a time-dependent increase in cellular proliferation in HUVEC infected with C. pneumoniae at a multiplicity of infection (MOI) of 2 IFU/cell. HUVEC preincubated with VEGF antibody did not release MMP-9, as detected by zymography assessment and ELISA assay. In addition, we demonstrated that TLR2/TLR4 are expressed in HUVEC infected with viable microorganisms (25% and 17%, respectively), while UV-inactivated microorganisms induced a lesser expression (20% and 11%, respectively) compared to control cells and HUVEC exposed to heat-killed bacteria showed a percentage of TLR-expressing cells similar to the control cells. In addition, the cells preincubated for 60 min with TLR2/TLR4 neutralizing antibodies showed a decrease in C. pneumonia-induced VEGF and MMP-9 production.     

RhoA/ROCK-dependent pathway is required for TLR2-mediated IL-23 production in human synovial macrophages: Suppression by cilostazol

IL-23 is produced by antigen presenting cells and plays critical roles in immune response in rheumatoid arthritis. In this study, we investigated whether the RhoA/Rho-kinase pathway is required to elevate TLR2-mediated IL-23 production in synovial macrophages from patients with rheumatoid arthritis (RA), and then examined the suppressive effect of cilostazol on these pathways. IL-23 production was elevated by lipoteichoic acid (LTA), a TLR2 ligand, and this elevation was more prominent in RA macrophages than in those from peripheral blood of normal control. LTA increased the activation of RhoA in association with increased the nuclear translocation of NF-κB and its DNA-binding activity. Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA), Y27632 (Rho-kinase) or BAY11-7082 (NF-κB) inhibited IL-23 production by LTA. Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-κB activation. Cilostazol suppressed the TLR2-mediated activation of RhoA, decreased NF-κB activity with down-regulated IL-23 production, and these effects were reversed by Rp-cAMPS, as an inhibitor of cAMP-dependent protein kinase. The expression of IL-23, which colocalized with CD68(+) cells in knee joint of CIA mice, was significantly attenuated by cilostazol along with the decreased severity of arthritis. Taken together, the RhoA/Rho-kinase pathway signals TLR2-stimulated IL-23 production in synovial fluid macrophages via activation of NF-κB. Thus it is summarized that cilostazol suppresses TLR2-mediated IL-23 production by suppressing RhoA pathway via cAMP-dependent protein kinase activation.     

Involvement of TLR2 and TLR4 and Th1/Th2 shift in inflammatory responses induced by fine ambient particulate matter in mice

Epidemiologic studies have reported the association between fine particles (aerodynamic diameter ≤ 2.5 μm; PM2.5) and health effects, but the immunological mechanisms are not clear. To investigate the dose and time-dependent role of toll-like receptor (TLR) and Th1/Th2 shift in local and systemic inflammation induced by PM2.5, mice were subjected to intratracheal instillation of 2.5, 5, or 10?mg/kg PM2.5 in this study. After 24?h, 72?h, 7 days, and 14 days, mice were sacrificed to measure TLR2 and TLR4 expressions and Th1/Th2 related cytokines in bronchoalveolar lavage fluid (BALF) and peripheral blood. Histopathological changes in lung were also examined. Inflammatory infiltration and macrophages with engulfed particles were found by lung histopathology after PM2.5 exposure. TLR4 positive cells decreased in BALF but increased in blood at 24?h after the exposure. The low percentage of TLR4 positive cells continued to day 14 in BALF, but recovered at day 7 and decreased further to lower than the control value at day 14 in blood. TLR2 positive cell changed similar to TLR4 in BALF on the dose effects. In BALF at 24?h after the exposure, the Th2 related cytokines IL-5 and IL-10 increased dose-dependently; and in blood, the Th2 related cytokines IL-4, IL-5, and IL-10 also increased. These results suggest that acute exposure of PM2.5 leads to acute inflammatory responses locally and systemically in mice. TLR2 and TLR4 are involved in this process and PM2.5 can drive a Th2-biased immune response.     

Fibroblast Growth Factor 21 dependent TLR4/MYD88/NF-κB signaling activation is involved in lipopolysaccharide-induced acute lung injury

Fibroblast Growth Factor 21 (FGF21) has been reported to reduce inflammation and apoptosis. Inflammation and apoptosis are both the essential mechanisms during development of acute lung injury. This study evaluated whether pre-treatment of FGF21 could alleviate acute lung injury. Mice were pre-treated with FGF21 prior to lipopolysaccharide (LPS) treatment. 24 h later, the lung tissues and BALF were obtained to detect H&E pathology, W/D ratio, pro-inflammatory factors (TNF-α, IL-1β and IL-6) and apoptosis. In vitro, Human BEAS-2B and THP-1 cells were overexpressed with TLR4 or MYD88 or NF-κB plasmid to detect the inflammation or apoptosis. Data showed that FGF21 was proved to be beneficial for inhibiting inflammation and apoptosis in the LPS- induced Balb/c mice or LPS induced BEAS-2B or THP-1 cells. Furthermore, the data showed that FGF21 suppressed inflammation and apoptosis via inhibition of TLR4/MYD88/NF-κB signaling pathway. Therefore, FGF21 provides a possibility for the treatment of LPS induced acute lung injury.     

FK506 inhibits prostaglandin E2 production from synovial cells by suppressing peripheral blood mononuclear cells

FK506, an immunosuppressive drug for T cells, reduces pain in patients with rheumatoid arthritis. However, the mechanism for pain reduction remains uncharacterized. In this study, we investigated the effect of FK506 on prostaglandin E(2) (PGE(2)) production from synovial cells in vitro. Human synovial cells were cultured with supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD28 antibodies. Cultured synovial cells with PBMC supernatant produced a high amount of PGE(2) and FK506 inhibited PGE(2) induction from synovial cells. Culture supernatant contained interleukin-1beta (IL-1beta) and TNFalpha, and FK506 suppressed both in PBMC supernatant. Anti-IL-1beta neutralizing antibody, but not anti-TNFalpha neutralizing antibody, completely inhibited PGE(2) induction by PBMC supernatant. These results suggest that FK506 suppresses inflammation by inhibiting PGE(2) production from synovial cells through suppression of IL-1beta production from leukocytes.     

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells.     

LOX-1 and TLR4 affect each other and regulate the generation of ROS in A. fumigatus keratitis

PurposeTo explore the relationship between LOX-1 and TLR4 in Aspergillus fumigatus (A. fumigatus) keratitis. To determine LOX-1 and TLR4 can affect each other and regulate inflammation through regulation of the generation of reactive oxygen species (ROS) in A. fumigatus keratitis.MethodsThe cornea and abdominal cavity extracted neutrophils of susceptible C57BL/6 mice were infected with A. fumigatus. The cornea and neutrophils were pretreated with LOX-1 neutralizing antibody, Polyinosinic acid (Poly(I)) (the inhibitor of LOX-1) or CLI-095 (the inhibitor of TLR4) separately before infection. LOX-1, TLR4 and IL-1β expression were detected in normal and infected cornea by PCR and Western Blot, while ROS was detected in the neutrophils by flow cytometry.ResultsLOX-1, TLR4, IL-1β mRNA and protein levels were up-regulated in C57BL/6 cornea after infection. LOX-1 neutralizing antibody or Poly(I) pretreatment decreased the expression of LOX-1, TLR4 and IL-1β in C57BL/6 cornea after infection and CLI-095 pretreatment decreased the expression of LOX-1, TLR4 and IL-1β in C57BL/6 cornea after infection. ROS generation was increased in C57BL/6 neutrophils after infection, however, ROS generation was decreased in C57BL/6 neutrophils after infection by LOX-1 neutralizing antibody or Poly(I) or CLI-095 pretreatment.ConclusionLOX-1, TLR4 and IL-1β expression and ROS generation are increased after infection. LOX-1 and TLR4 can affect each other and regulate the generation of ROS in A. fumigatus keratitis. Inhibition of LOX-1 and TLR4 can reduce ROS generation.     

Inhibition of in vitro tumor cell proliferation by cytokines induced by combinations of TLR or TLR and TCR agonists

The objective of this study was to learn from in vitro studies how to better utilize Toll-like receptor (TLR) agonists in controlling tumor growth. One of the primary effects of TLR agonists is induction of cytokine and chemokine production. In order to identify combinations of cytokines or chemokines with optimal ability to inhibit in vitro tumor cell proliferation, a panel of 17 recombinant human or mouse cytokines that have minimal effect on primary cell survival, were tested individually or in combinations of 2, 3 or 4 on a panel of human and mouse chemotherapy sensitive and resistant tumor cell lines. A combination of high (>10 ng/ml) levels of IFNgamma with moderate concentrations of TNFalpha>IFNalpha>IL-6=IL-8 was most effective at inhibiting in vitro tumor cell viability and proliferation with minimal effect on primary cells. We also observed that similar cytokine profile could be induced in vitro PBMC culture by using certain combinations of TLR-TLR and TLR-TCR agonists. Thus, concomitant activation of TLR7/8 with TLR4 or TLR 7/8 with T cell receptor (TCR) in PBMC, amongst all possible paired TLR-TLR and TLR-TCR agonist combinations, produced cytokine mix high in IFNgamma, in combination with IFNalpha, IL-6, IL-8, TNFalpha. Such cytokine mix was equal or more effective tumor cell killing and inhibition of tumor cell proliferation than the best rec-cytokine mixture tested. These results suggest that, TLR and/or TCR agonists combinations generate an optimal mixture of cytokines and chemokines competent in regulating in vitro tumor growth, and imply that realizing such "right cytokine induction" in vivo might be more efficacious than that with individual cytokines or TLR agonists induced cytokine mix.     

Toll样受体4在棕榈酸诱导RAW264.7巨噬细胞 炎症反应中的作用及机制

目的 探讨 Toll 样受体 4（TLR4）在棕榈酸诱导 RAW264.7 巨噬细胞炎症反应中的作用及可能机制。方 法 观察正常对照组（正常饮食）和高脂饲料组（高脂饮食诱导肥胖小鼠模型）C57BL/6J 小鼠血清自由脂肪酸（FFA） 水平及内脏脂肪组织炎症细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和单核细胞趋化蛋白-1（MCP- 1）的表达;采用棕榈酸（150 μmol/L 组和 300 μmol/L 组）刺激小鼠 RAW264.7 巨噬细胞,观察这 2 组和空白对照 （Control）组、无 FFA 牛血清白蛋白（BSA）组的 TNF-α、IL-6 和 MCP-1 的表达和分泌,同时检测 TLR4 的蛋白表达和 核因子-κB（NF-κB）的激活情况;利用 siRNA 干扰实验抑制 TLR4,观察在 Control 组、BSA 组、棕榈酸（300 μmol/L） 组、棕榈酸（300 μmol/L）+Control siRNA 组、棕榈酸（300 μmol/L）+TLR4 siRNA 组中棕榈酸诱导巨噬细胞炎症因子的 表达和分泌。结果 高脂饲料组体质量和 Lee’s 指数高于正常对照组,血清中 FFA 水平升高,内脏脂肪组织中 TNF-α、IL-6 和 MCP-1 的表达明显增加（P＜0.05）。与 BSA 组比较,棕榈酸 150 μmol/L 组和棕榈酸 300 μmol/L 组 炎症因子 TNF-α、IL-6 和 MCP-1 的表达和分泌均明显增加,TLR4 和 NF-κB p65 磷酸化蛋白水平均增加（P＜0.05）。 与 BSA 组比较,棕榈酸 300 μmol/L 组 NF-κB p65 的核转运水平和细胞内水平明显升高（P＜0.05）。TLR4 被抑制 后,棕榈酸+TLR4 siRNA 组的 TLR4、TNF-α、IL-6 和 MCP-1 的 mRNA 表达和分泌水平明显低于棕榈酸组（P＜ 0.05）。结论 TLR4 可能参与棕榈酸诱导的 RAW264.7 巨噬细胞炎症反应,诱导炎症因子 TNF-α、IL-6 和 MCP-1 的释放,且其介导的炎症反应与 NF-κB 的激活有关。     

Inhibition of P2X7R modulates ECM deposition in HSCs and crosstalk with macrophages in liver fibrosis

OBJECTIVE Modulation of immune response and reduction of extracellular matrix(ECM)deposition are both essential in the therapy of liver fibrosis.Regulating P2 X7 R might be a potential therapeutic strategy to treat liver fibrosis, we investigated whether the blockade of P2 X7 R could reverse liver fibrosis and how P2 X7 R is involved in fibrogenesis during hepatic stellate cells(HSCs) and macrophages crosstalk. METHODS In vivo,liver fibrosis model was established by thioacetamide(TAA) intraperitoneal administration in male C57 BL/6 mice. In vitro, LX-2 cells were treated with TGF-β and LPS/ATP respectively. Supernatant from LPS/ATP stimulated THP-1 macrophages were supplemented to LX-2 cells to mimic cellular crosstalk between HSCs and macrophages. RESULTS Blockade of P2 X7 R with its selective antagonist A438079, not only decreased liver injury and ECM deposition but also ameliorated inflammation by inhibiting NLRP3 inflammasome, NF-κB activation and IL-1β production in TAA-induced liver fibrosis. And the recruitment of macrophages, monocytes and granulocytes were also inhibited. In TGF-β-stimulated LX-2 cells,ECM deposition was reduced by the inhibiting of P2 X7 RTLR4-NLRP3 axis. Protein synthesis and cleavage of IL-1β and its m RNA level was dramatically increased by LPS 4 h combined with ATP 30 min than those in HSC treated with LPS or ATP alone. Additionally, LX-2 cells primed with LPS/ATP greatly increased m RNA and protein expression of caspase-1, NLRP3 and P2 x7 R, as well as liver fibrosis markers, α-SMA and typeⅠcollagen.These events were remarkably suppressed by A438079 pretreatment. si RNA against P2 x7 R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type Ⅰ collagen induced by LPS/ATP. Ectopic overexpression of P2 X7 R reduced the threshold of ECM deposition in HSCs induced by TGF-β.Inhibiting upstream receptors of NLRP3 inflammasome,P2 X7 receptor-selective antagonist(A438079), TLR4 inhibitor(CLI-095) reduced the fibrotic markers in both models of TGF-β and LPS/ATP-activated HSCs. The cultured medium of the THP-1 macrophages by LPS/ATP aggravated ECM deposition in LX-2 cells. The decreased IL-1β by the pharmacological inhibitors of P2 X7 R,caspase-1 and TLR4 treated to THP-1 macrophages attenuates ECM deposition in LX-2 cells. CONCLUSION Both ECM producing in HSCs and inflammatory cytokines secreting from macrophages were regulated by P2 X7 R, suggesting a therapeutic utility of P2 x7 R blockade in liver fibrosis treatment.     

Miltefosine triggers a strong proinflammatory cytokine response during visceral leishmaniasis: role of TLR4 and TLR9

Visceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is associated with irregular fever, weight loss, hepatosplenomegaly and anemia. The therapeutic arsenal against VL is limited and the recent advent of a novel immunomodulatory drug, Miltefosine has shown promising results for effective treatment of VL but its dependence on Toll like receptors (TLR) has not been explored. In this study, we have shown that the non-cytotoxic dose (5 μM) of Miltefosine could render significant protection corresponding to 88% and 95% reduction in intracellular parasite load at 24 h and 48 h in L. donovani infected THP1 cells. This was accompanied by a strong proinflammatory cytokine response in the form of IFN-γ, IL-12 and TNF-α as evident by enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR). This Miltefosine induced proinflammatory cytokine response in infected THP1 cells was also accompanied by simultaneous 10- and 12-fold increase in TLR4 mRNA and TLR9 mRNA. These changes in cytokine response and TLR expression were also studied in peripheral blood mononuclear cells (PBMC) of VL patients treated with Miltefosine by RT-PCR which showed similar results as in THP1 cells. Thereby, suggesting a probable dependence of Miltefosine on TLR4 and TLR9 in triggering a proinflammatory response.     

Expression of IL-27 p28 by Theiler's virus-infected macrophages depends on TLR3 and TLR7 activation of JNK-MAP-kinases

Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease (DD) due to infection of macrophages, stimulation of macrophage Toll-like receptor (TLR)3 and TLR7 pathways, activation of Mitogen-activated protein kinases (MAPK)s, and production of macrophages cytokines. Because expression of IL-27, a macrophage cytokine composed of p28 and EBI3 subunits, has been implicated in DD, we examined IL-27 subunit mRNA expression during TMEV infection of RAW264.7 cells, a macrophage cell line. TMEV infection of RAW264.7 cells did not affect cell viability, resulted in viral RNA replication, as well as p28 and EBI3 expression. Expression of p28 in TMEV-infected RAW264.7 cells depended on TLR3 and TLR7, as well as JNK but not p38 or ERK MAPKs. Since TMEV causes DD in SJL/J but not B10.S mice we determined the difference in expression of IL-27 subunit mRNA in SJL/J compared to B10.S macrophages. SJL/J macrophages expressed significantly more p28 mRNA after TMEV infection and after stimulation with TLR3 and TLR7 agonists compared with B10.S macrophages. Therefore, macrophages expression of IL-27 p28 mRNA in response to TMEV is due to activation of TLR3, TLR7, and JNK MAPKs pathways.     

A vitamin D3 analog augmented interleukin-8 production by human monocytic cells in response to various microbe-related synthetic ligands,especially NOD2 agonistic muramyldipeptide

Active metabolite vitamin D₃, 1α,25-dihydroxyvitamin D₃, is a pleiotropic factor and exhibits various physiological functions, including immunomodulating activities. In this study, the possible regulation of innate immune responses of human monocytic cells by a vitamin D₃ analog was examined. Human monocytic THP-1 cells were pre-treated with OCT, vitamin D₃ analog, 1α,25-dihydroxy-22-oxavitamin D₃, followed by stimulation with various chemically synthesized Toll-like receptors (TLR) and NOD1 and NOD2 ligands. OCT-treated cells produced more IL-8 than non-treated cells upon stimulation with various chemically-synthesized ligands: TLR2-agonistic lipopeptide (FSL-1), TLR3-agonistic poly I:C, TLR4-agonistic lipid A (E. coli-type LA-15-PP), NOD1-agonistic FK565 and NOD2-agonistic muramyldipeptide (MDP). Among the ligands, MDP was the highest inducer of IL-8 production in OCT-treated THP-1 cells, and IL-8 production increased depending on the treatment time until 72 h. OCT up-regulated the expression of NOD2 in THP-1 cells, and OCT-treated cells exhibited higher activation of p38, JNK and ERK in the MAPK pathway, IκBα in the NF-κB pathway, and TAK1 upstream in response to MDP than non-treated cells. Analysis using siRNA against NOD2 and inhibitors of specific signal molecules indicated that the existence of NOD2 and activation of the above signaling molecules are required for enhanced production of IL-8 in OCT-treated THP-1 cells. These findings suggested that NOD2, NF-κB and MAPK pathways are involved in the activity of OCT to augment the response of human monocytic cells to MDP.     

Hsa_circ_0001204 modulates inflammatory response of macrophages infected by Mycobacterium tuberculosis via TLR4/NF-κB signalling pathway

Circular RNAs (circRNAs) play a vital role in the regulation of Mycobacterium tuberculosis (M.tb) by macrophages. In this project, the potential role of hsa_circ_0001204 in M.tb-infected macrophages is explored. Hsa_circ_0001204 was determined in the patients with tuberculosis (TB) and M.tb-infected macrophages. Its effect on the survival of M.tb and the apoptosis and inflammation of M.tb-infected macrophages was evaluated. Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signalling was detected by western blotting and immunofluorescence. TB patients and M.tb-infected THP-1 cells showed the significant downregulation of hsa_circ_0001204. Upregulating hsa_circ_0001204 reduced M.tb survival and suppressed the apoptosis and inflammatory response of THP-1 cells. The TLR4/NF-κB signalling pathway could be inhibited by hsa_circ_0001204 overexpression, which was activated by M.tb-infection. Hsa_circ_0001204 confers protective effects in M.tb-infected THP-1 cells, at least partly via the inhibition of TLR4/NF-κB signalling pathway.