Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine

It was reported recently that histamine induced Toll-like receptor (TLR)2 and TLR4 expression in endothelial cells and enhanced their sensitivity to Gram-positive and Gram-negative bacteria; and that TLRs were expressed in airway epithelial cells and that several inflammatory mediators modulated their expression. However, little is known of potential influence of histamine on TLRs in pulmonary epithelial cells. In the present study, effects of histamine on expression of TLRs in both human A549 and NCI-H292 cell lines were examined by using real-time quantitative RT-PCR analysis, flow cytometry and immunofluorescent staining. The results revealed that both cell types constitutively expressed mRNAs for TLR1-TLR10. Histamine up-regulated the expression of TLR3 mRNA by 12.3- and 11.6-fold, respectively in both cell types. The time course showed that histamine induced TLR3 mRNA expression was initiated at 30 min, nearly reached peak levels after 2 h and was sustained at least until 12 h. Histamine also induced TLR3 protein expression in A549 and NCI-H292 cells. Histamine and poly (I:C), a specific TLR3 ligand stimulated interleukin (IL)-8 secretion from both cell types. Moreover, histamine enhanced poly (I:C)-induced IL-8 secretion and phosphorylation of NF-kappaB in the two cell types, and histamine H1 receptor antagonists inhibited the action of histamine. In conclusion, histamine selectively up-regulated expression of TLR3, and stimulated IL-8 secretion from the cells. Histamine also enhanced poly (I:C) induced IL-8 secretion and phosphorylation of NF-kappaB. These observations suggest that histamine might play an important role in enhancing the innate immune responses of airway to viral infection.     

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)] induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-kappaB and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-beta mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-gamma inducible protein 10 (IP10), myxovirus resistance gene A and 2',5'-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-beta expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.     

Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture

The goal of this study was to examine the role of E₂ in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E₂ on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human β-defensin-2 (HBD2), tumor necrosis factor (TNF)-α, IL-8, and nuclear factor (NF)-kB. When incubated with E₂ for 24–48 h, we found that E₂ stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus, E₂ increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E₂ had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1β on mRNA expression of TNF-α, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E₂ inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E₂. Further, it suggests that with E₂ regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.     

Effect of oestradiol on PAMP-mediated CCL20/MIP-3 alpha production by mouse uterine epithelial cells in culture

The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3 alpha (CCL20/MIP-3 alpha) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3 alpha with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3 alpha increased rapidly (4 hr) in both apical and basolateral secretions. Time-course studies indicated that responses to PAMPs added to the apical surface persisted for 12-72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3 alpha. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3 alpha. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3 alpha secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3 alpha is produced in response to PAMPs. Since CCL20/MIP-3 alpha is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3 alpha production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.     

Human oviductal epithelial cells express Toll-like receptor 3 and respond to double-stranded RNA: Fallopian tube-specific mucosal immunity against viral infection

BACKGROUND: The aim of this study was to evaluate the site-specific immunoregulatory mechanisms against viral infection in human Fallopian tubes. METHODS: We therefore investigated the effects of double-stranded RNA (dsRNA) on the production of interleukin (IL)-6, IL-8 and granulocyte chemotactic protein-2 (GCP-2) by cultured oviductal epithelial cells (OECs) using enzyme-linked immunosorbent assays. Phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha) protein after dsRNA stimulation and the expression of Toll-like receptor (TLR) 3 in these cells were also evaluated by western blot analysis. RESULTS: Polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that antagonizes TLR3, stimulated the secretion of IL-6, IL-8 and GCP-2 by OECs. Poly I:C-induced production of these cytokines by OECs was inhibited by the pretreatment of these cells with anti-TLR3 antibody. The phosphorylation of IkappaB-alpha protein was detected in OECs after stimulation by poly I:C. The expression of TLR3 was also detected in OECs. CONCLUSION: These results suggest that the epithelial cells of the human Fallopian tube have evolved a unique, site-specific mechanism for recognizing viral infection. TLR3-mediated production of proinflammatory cytokines and chemokines in OECs in response to viral dsRNA may be important for antiviral immunity in the human female reproductive tract.     

Differential responses of murine vaginal and uterine epithelial cells prior to and following herpes simplex virus type 2 (HSV-2) infection

PROBLEM: This study was undertaken to evaluate the susceptibility of upper and lower reproductive tract epithelial cells (ECs) to herpes simplex virus type 2 (HSV-2) infection and examine their cytokine secretion patterns prior to and following infection. METHOD OF STUDY: Primary EC cultures, grown from murine vaginal and uterine tissue, were inoculated with HSV-2. Viral shedding was measured in apical and basolateral compartments. Multi-analyte bead-based immunoassays run on Luminex, were used to analyse cytokine profiles. RESULTS: Both vaginal and uterine ECs became productively infected with HSV-2, ex-vivo. Uterine ECs displayed varying degrees of infection, dependent on transepithelial resistance of the monolayers. Co-culturing stromal cells did not significantly change levels of viral shedding from ECs. Uterine ECs and epithelial-stromal co-cultures constitutively secreted interleukin (IL)-1alpha, IL-6, mouse homologue of human IL-8 (KC) and monocyte chemotactic protein-1 (MCP-1), while vaginal epithelial-stromal co-cultures secreted granulocyte-macrophage colony stimulating factor (GM-CSF) and KC. Following exposure to HSV-2, IL-6 and MCP-1 levels decreased in uterine EC cultures. CONCLUSIONS: This data shows that ECs from the upper and lower reproductive tract have different cytokine secretion profiles and respond differentially to infection. HSV-2 may be able to suppress epithelial cytokine secretion as a strategy to evade host immune system.     

聚肌胞苷酸刺激的上皮细胞对成纤维细胞活化的影响及EMMPRIN的作用

 目的：探讨聚肌胞苷酸［poly(I:C)］作用上皮细胞后对气道成纤维细胞增殖和转分化的影响及细胞外基质金属蛋白酶诱导因子(EMMPRIN)的作用。方法：以poly(I:C)作用于人肺泡上皮细胞,将细胞上清刺激人气道成纤维细胞或种植在胶原凝胶中的成纤维细胞,观察后者增殖、α-平滑肌肌动蛋白(α-SMA)和EMMPRIN表达及凝胶收缩;以EMMPRIN刺激成纤维细胞,检测细胞增殖、α-SMA表达及MMP-2、-9活性水平;应用p38 MAPK和ERK1/2抑制剂,观察对α-SMA表达及EMMPRIN分泌的影响。结果：Poly(I:C)作用的上皮细胞上清诱导成纤维细胞增殖、α-SMA和EMMPRIN表达及凝胶收缩;EMMPRIN剂量依赖性增强细胞增殖、α-SMA表达及MMP-2、-9活性;poly(I:C)作用的上皮细胞上清激活成纤维细胞p38 MAPK和ERK1/2信号通路,两者特异性阻断剂减弱成纤维细胞α-SMA和EMMPRIN表达。结论：Poly(I:C)作用上皮细胞后诱导成纤维细胞增殖、α-SMA表达和凝胶收缩,p38 MAPK和ERK1/2信号通路介导了上述过程,EMMPRIN是其中重要介质。     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

Expression of Toll‐like receptor‐3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2

Anti‐microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti‐microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in‐situ hybridization. Moreover, we examined the regulation of LCN2 in HT‐29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll‐like receptor (TLR)‐3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up‐regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de‐novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT‐29 was enhanced by both interleukin (IL)‐1β and the TLR‐3 ligand poly(I:C), and TLR‐3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.     

TLR3激动剂poly(I:C)对胃癌BGC-823细胞生物学特征的影响

目的:poly(I:C)是双链RNA(dsRNA)的结构类似物,可被模式识别受体TLR3、RLRs识别,具有抗肿瘤作用。本文旨在研究poly(I:C)对胃癌BGC-823细胞生物学特征的影响并进行机制探讨。方法:以胃腺癌细胞系BGC-823细胞为模型,采用半定量RT-PCR检测TLR1-TLR10 mRNA基础表达水平,然后用MTT法、CCK-8法、EdU掺入法检测poly(I:C)对BGC-823细胞的增殖能力的影响;MTT法、CFSE-7-AAD法评价NK细胞对poly(I:C)处理后的BGC-823细胞杀伤能力的影响;实时定量PCR法检测细胞因子mRNA水平的变化;流式细胞术分析BGC-823细胞中TLR3、Cyclin D1表达水平以及NKL细胞中CD107a、Perforin和TNF-α的表达水平。结果:TLR3在BGC-823细胞有明显表达。poly(I:C)可抑制BGC-823细胞的增殖,这一过程的主要分子特征是DNA合成减弱、Cyclin D1的表达水平降低、炎症因子的mRNA表达水平下降;同时NK细胞对于poly(I:C)处理后的BGC-823细胞的杀伤功能增强,NK细胞表达CD107a、Perforin、TNF-α的能力提高。结论:poly(I:C)可直接抑制肿瘤的生长并提高BGC-823细胞对NK细胞杀伤的敏感性,为设计和利用基于TLR的抗肿瘤治疗药物提供有益的启示。     

Tuning the immune response of dendritic cells to surface-assembled poly(I:C) on microspheres through synergistic interactions between phagocytic and TLR3 signaling

The artificial dsRNA polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a potent adjuvant candidate for vaccination, as it strongly drives cell-mediated immunity. However, because of its effects on non-immune bystander cells, poly(I:C) administration may bear danger for the development of autoimmune diseases. Thus poly(I:C) should be applied in the lowest dose possible. We investigated microspheres carrying surface-assembled poly(I:C) as a two-in-one adjuvant formulation to stimulate maturation of monocyte-derived dendritic cells (MoDCs). Negatively charged polystyrene microspheres were equipped with a poly(ethylene glycol) corona through electrostatically driven surface assembly of a library of polycationic poly(l-lysine)-graft-poly(ethylene glycol) copolymers, PLL-g-PEG. Stable surface assembly of poly(I:C) was achieved by incubation of polymer-coated microspheres in an aqueous poly(I:C) solution. Surface-assembled poly(I:C) exhibited a strongly enhanced efficacy to stimulate maturation of MoDCs by up to two orders of magnitude, as compared to free poly(I:C). Multiple phagocytosis events were the key factor to enhance the efficacy. The cytokine secretion pattern of MoDCs after exposure to surface-assembled poly(I:C) differed from that of free poly(I:C), while their ability to stimulate T cell proliferation was similar. Overall, phagocytic signaling plays an important role in defining the resulting immune response to such two-in-one adjuvant formulations.     

Toll-like receptor engagement stimulates anti-snRNP autoreactive B cells for activation

Autoreactive B cells are the source of pathogenic autoantibodies (autoAb) in systemic lupus erythematosus (SLE). Previous studies have demonstrated that anti-small nuclear ribonucleoprotein particles (snRNP) B cells from normal background mice tolerize T cells in the periphery and do not secrete autoAb. In this study, we examined whether these anti-snRNP B cells can be activated for autoAb production by the engagement of Toll-like receptors (TLR). Anti-snRNP B cells proliferated vigorously and secreted abundant anti-snRNP autoAb upon exposure to CpG or polyriboinosinic polyribocytidylic acid [poly (I:C)] in vitro. In addition, the costimulatory molecules CD80 and CD86 were up-regulated. While both anti-snRNP B cells and wild-type B cells produced similar levels of IL-6 and IL-10, anti-snRNP B cells secreted predominately IFN-gamma in response to CpG or poly (I:C) stimulation. Furthermore, we showed that in vivo engagement of TLR stimulated immature anti-snRNP B cells to further differentiate and produce autoAb and form germinal centers. The activated anti-snRNP B cells became expanded and migrated into the T-B cell interface. Moreover, TLR engagement directly or indirectly activated autoreactive B cells via a CD4 T cell-independent manner. These results provide in vitro and in vivo evidence that BCR/TLR co-engagement promotes the activation of anti-snRNP B cells for autoAb production.     

IFN-alpha regulates Toll-like receptor-mediated IL-27 gene expression in human macrophages

IL-27 is a novel member of the IL-12 cytokine family. IL-27 has pro- and anti-inflammatory properties, and it controls the responses of adaptive immunity. It promotes the differentiation of na?ve Th cells and suppresses the effector functions of Th17 cells. Biologically active IL-27 is a heterodimer composed of EBV-induced gene 3 (EBI3) and p28 proteins. We report that TLR-dependent expression of IL-27 in human macrophages is mediated by IFN-alpha. Stimulation of macrophages with agonists for TLR3 {polyinosinic:polycytidylic acid [poly(I:C)]}, TLR4 (LPS), or TLR7/8 (R848) results in concurrent expression of EBI3 and p28. The p28 expression is inhibited with neutralizing anti-IFN-alpha antibodies. Unlike poly(I:C), LPS, and R848, TLR2 agonist (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride does not stimulate macrophages to produce IFN-alpha, and therefore, it is not able to turn on the expression of p28. There is an IFN-stimulated response element (ISRE) in the p28 gene promoter. IFN-alpha enhances the expression of IFN regulatory factor 1 (IRF-1) in macrophages and induces binding of IRF-1 to the p28 ISRE site. The data provide a mechanistic basis for the IFN-alpha-mediated activation of IL-27. The data emphasize a role of IFN-alpha in immune responses, which rely on the recognition of pathogens by TLRs.     

B type CpG-DNA suppresses poly(I:C)-induced BLyS expression and production in human tonsillar fibroblasts

Although B lymphocyte stimulator (BLyS) has potent costimulatory effects on B cells, the details of BLyS-expression in tonsillar fibroblasts remain unexplored. We examined the effect of the Toll-like receptor (TLR) ligands on BLyS-expression in human tonsillar fibroblasts as well as the crosstalk that occurs among different TLR ligands. The expression of BLyS mRNA by tonsillar fibroblasts was strongly induced in the presence of polyinosinic–polycytidylic acid (poly(I:C)) that is a ligand, of TLR3. We also revealed that DNA containing CpG motifs (CpG-DNA), coding for a TLR9 ligand, markedly suppressed the poly(I:C)-induced mRNA expression and protein production of BLyS. B type CpG-DNA decreased the poly(I:C)-induced phosphorylation of inhibitor kappa B alpha (IκBα) and its degradation. Pre-incubation with nuclear factor kappa B (NF-κB) signaling inhibitors reduced the poly(I:C)-induced BLyS-expression. These results indicate that human tonsillar fibroblasts strongly induce BLyS-expression and production that can be inhibited by CpG-DNA and regulated through NF-κB signaling.     

Viruses induce high expression of BAFF by salivary gland epithelial cells through TLR- and type-I IFN-dependent and -independent pathways

B cell activating factor (BAFF) plays a key role in promoting B lymphocyte activation. We investigated whether danger signals induce BAFF secretion by cultured salivary gland epithelial cells (SGEC), which are the target of primary Sjögren's syndrome, a prototypic systemic autoimmune disease. SGEC cultures were established from minor salivary glands obtained from ten patients with pSS or sicca symptoms. BAFF mRNA and protein were measured after stimulation of the different Toll‐like receptors (TLR) by agonists or viruses. The expression of TLR2, –3, and –7 was detected in SGEC. Poly (I:C) (a synthetic TLR3 agonist) and reovirus‐1 (a dsRNA virus) induced high expression of BAFF mRNA (multiplied by a factor of 246 ± 39 (SEM) and 347 ± 66, respectively) and of BAFF protein secretion (58.49 ± 4.34 pg/mL and 69.73 ± 5.67). Inhibition of both the endosomal (by chloroquine) and IFN (by anti‐IFNAR antibody) pathways partly inhibited BAFF expression. Treatment with both dsRNA virus and poly (I:C) induced high levels of BAFF mRNA and protein expression by SGEC, through pathways dependent on and independent of TLR and dependent on and independent of IFN. BAFF induction by target organs of autoimmune diseases after viral infection may be a link between innate immunity and autoimmunity.