Synergistic Proinflammatory Responses by IL-17A and Toll-Like Receptor 3 in Human Airway Epithelial Cells

Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.     

Toll-Like Receptor Agonists Synergistically Increase Proliferation and Activation of B Cells by Epstein-Barr Virus

Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.Epstein-Barr virus (EBV), a herpesvirus, is a very successful infectious agent: it establishes and maintains latent infection in >95% of human beings worldwide. This success is related to EBV''s varied strategies to maintain itself in its preferred host cell type, the B cell, by establishing different modes of latent infection (46). Some of these modes (latency modes 0, I, and II) are characterized by a resting B-cell phenotype and expression of a very limited set of EBV proteins (from none to four). In contrast, latency III involves the expression of at least 12 EBV latent-cycle gene products (10 proteins and 2 RNAs) (30, 31), which in their combined action profoundly alter the B cell''s appearance and behavior by inducing B-cell activation associated with proliferation, altered receptor expression, and cytokine secretion, as well as causing enhanced antigen presentation (31).In these various features, EBV infection of the latency III type resembles physiological activation of B cells in germinal centers even in its molecular details, because EBV closely mimics or constitutively activates some of the B cell''s main signaling pathways. Exogenous physiological signals leading to B-cell activation have been classified as “signal 1,” the stimulation of the B-cell receptor (BCR) by antigen binding; “signal 2,” the stimulation of CD40 by the CD40 ligand molecule, expressed on activated helper T cells; and “signal 3,” the stimulation of Toll-like receptors (TLRs) by microbial components, such as unmethylated CpG DNA, or their mimics. All three signals together are required for maximal proliferation of naive B cells (47). However, stimulation with TLR ligands alone, for example, CpG DNA, is sufficient to cause transient B-cell activation, including proliferation and induction of immune effector molecules such as CD86, a T-cell-costimulatory molecule (24). Additional immune effectors, the cytokines interleukin-6 (IL-6), IL-10, and IL-12, are induced when CpG stimulation is combined with strong CD40 stimulation (55).For primary infection of B cells, it is well established that EBV''s latent membrane proteins LMP2A (10, 39) and LMP1 (22) mimic signaling by the BCR and CD40, respectively. It is less clear whether and how EBV generates a potential signal 3 in the course of primary B-cell infection. A role of the TLR7 pathway has been proposed, based on the observation that EBV infection of naive B cells elevates the expression of TLR7 and its downstream signaling mediators (40). Additional mechanisms have recently been proposed to explain how EBV might trigger TLRs or other pattern recognition receptors in other cellular systems. For example, the Epstein-Barr virus-encoded small RNAs (EBERs) were described to trigger the retinoic acid-inducible gene I (RIG-I)-encoded protein, a receptor for various viral RNAs, in Burkitt''s lymphoma cells (48, 49). TLR2 signaling in monocytes is activated by binding of EBV particles to the cells (21) or by extracellular provision of EBV dUTPase (2).However, a physiologically relevant signal 3 need not originate in EBV itself. Other microbial agents present at the site of EBV infection might influence EBV infection, B-cell transformation, and virus release. For example, infectious mononucleosis (IM), a frequent consequence of primary EBV infection in adolescents and adults, is usually accompanied by tonsillitis with characteristic massive bacterial colonization (50), a likely source of TLR agonists acting on local EBV-infected B cells. Here we investigate the effects of CpG DNA and other exogenous TLR ligands on EBV-driven B-cell proliferation, clonal outgrowth, and induction of activation-associated cellular receptors and cytokines.     

Interferon Production and Resistance to Viral Infection Induced by Poly I · Poly C in Chick Embryo Cells

Two different growth media, one based on Eagle's minimum essential medium (MEM) and the other on Earle's balanced salt solution–lactalbumin hydrolysate–yeast extract (YLE), were used for growing primary chick embryo cells (CEC), and resistance to viral infection and interferon production induced by polyinosinic-polycytidylic acid (poly I·poly C) were compared. In CEC grown in Eagle's MEM, treatment with poly I·poly C at a concentration as low as 1.0 ng/ml was sufficient to induce a detectable resistance to infection with vesicular stomatitis virus (VSV), while more than 300-fold concentrated poly I·poly C was required to induce a similar resistance when the cells were grown in YLE. The cells grown in YLE did not produce an appreciable amount of interferon, whereas a significantly higher level of interferon was produced by the cells grown in Eagle's MEM. A similar phenomenon was observed in the interferon production of chick embryo cells treated with ultraviolet light (UV)-irradiated Newcastle disease virus (NDV) and in the induction of resistance to vaccinia virus in cells treated with poly I·poly C. It was found that the response of cells, bathing in one growth medium, to poly I·poly C was not affected by replacing it with the other at the same time with the addition of poly I·poly C, and that the response of CEC was strongly dependent upon the medium used for cultivation. These facts suggested that the observed difference in the response of cells to poly I·poly C was not due to a direct interaction between the inducer and medium components but to the physiological state of CEC established during their growth. Which component of YLE was responsible for such a lowered response of cells to poly I·poly C was also examined, and the marked reduction of PDD₅₀ by the replacement of lactalbumin hydrolysate of YLE with amino acids and the increase of PDD₅₀ by addition of lactalbumin hydrolysate to MEM suggested that lactalbumin hydrolysate might play an important role in this phenomenon.     

Toll-Like Receptor Expression and Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/β) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

Toll-Like Receptor Ligands LPS and Poly (I:C) Exacerbate Airway Hyperresponsiveness in a Model of Airway Allergy in Mice,Independently of Inflammation

It is well-established that bacterial and viral infections have an exacerbating effect on allergic asthma, particularly aggravating respiratory symptoms, such as airway hyperresponsiveness (AHR). The mechanism by which these infections alter AHR is unclear, but some studies suggest that Toll-like receptors (TLRs) play a role. In this study, we investigated the impact of TLR3 and TLR4 ligands on AHR and airway inflammation in a model of pre-established allergic inflammation. Female BALB/c mice were sensitised and challenged intranasally (i.n.) with either PBS or ovalbumin (OVA) and subsequently i.n. challenged with poly (I:C) (TLR3) or LPS (TLR4) for four consecutive days. The response to methacholine was measured in vivo; cellular and inflammatory mediators were measured in blood, lung tissue and broncheoalveolar lavage fluid (BALF). OVA challenge resulted in an increase in AHR to methacholine, as well as increased airway eosinophilia and TH2 cytokine production. Subsequent challenge with TLR agonists resulted in a significant increase in AHR, but decreased TLR-specific cellular inflammation and production of immune mediators. Particularly evident was a decline in LPS-induced neutrophilia and neutrophil-associated cytokines following LPS and poly (I:C) treatment. The present data indicates that TLRs may play a pivotal role in AHR in response to microbial infection in allergic lung inflammation. These data also demonstrate that aggravated AHR occurs in the absence of an exacerbation in airway inflammation and that allergic inflammation impedes a subsequent inflammatory response to TLRs. These results may parallel clinical signs of microbial asthma exacerbation, including an extended duration of illness and increased respiratory symptoms.     

白介素17对脂多糖诱导人支气管上皮细胞炎症损伤的影响

目的:探讨白介素17A(interleukin-17A, IL-17A)对脂多糖(lipopolysaccharide, LPS)诱导的人支气管上皮细胞(16-HBE)炎症损伤的影响及其可能机制。方法:体外培养16-HBE细胞系,予LPS、IL-17A进行干预,分为空白对照组、LPS组、IL-17A组、IL-17A+LPS组。采用酶联免疫吸附法(Enzyme linked immunosorbent assay, Elisa)测定细胞培养液上清中IL-4、IFN-γ、IL-6、IL-8等炎症因子的水平,蛋白印迹法(Western Blot, WB)检测细胞中丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPKs)信号通路相关蛋白:细胞外调节蛋白激酶(ERK)、P38蛋白激酶(P38)、c-Jun氨基末端激酶(JNK)的表达及其相应磷酸化蛋白(P-ERK、P-P38、P-JNK)的表达。体外培养16-HBE细胞系,予LPS、IL-17A以及ERK1/2抑制剂(U0126)、p38抑制剂(SB203580)和JNK抑制剂(SP600125),分为空白对照组、LPS+IL-17A组、IL-17A+LPS+UO126组、IL-17A+LPS+SB203580组、IL-17A+LPS+SP600125组。采用酶联免疫吸附法测定细胞培养液上清中IFN-γ、IL-4、IL-6、IL-8等炎症因子水平。结果:与空白对照组比较,LPS、IL-17A组,细胞上清中IL-6、IL-8的表达明显升高(P0.01),IL-4的表达降低(空白组vs LPS组P0.01,空白组vs IL-17A组P0.05),细胞内磷酸化ERK、P38、JNK蛋白的表达明显增加(空白组vs LPS组P0.05,空白组vs IL-17A组P0.01)。IL-17A+LPS组细胞上清中IL-6、IL-8、IL-4水平及细胞内P-ERK、P-P38、P-JNK的表达较LPS、IL-17A组更高(P0.05)。添加ERK、P38和JNK抑制剂后,与LPS+IL-17A组对比,IL-17A+LPS+U0126组、IL-17A+LPS+SB203580组和IL-17A+LPS+SP600125组细胞上清中IL-6、IL-8、IL-4水平下降(P0.05)。结论:IL-17A可能通过上调IL-6、IL-8的表达加重LPS诱导的16-HBE细胞炎症损伤,MAPKs可能是这一过程中的重要信号转导通路。     

Constitutive Production of Macrophage Colony-Stimulating Factor and Interleukin-6 by Human Ovarian Surface Epithelial Cells

Normal and neoplastic epithelial cells produce growth factors that can affect cells from different lineages. Epithelial ovarian cancers produce M-CSF and IL-6. In the present study, production of these cytokines has been measured in the apparently normal epithelial cells from which epithelial ovarian neoplasms are thought to arise. Epithelial cells from the surface of premenopausal human ovaries were established in short-term cultures. The cells bound anti-cytokeratin antibodies and exhibited characteristic epithelial morphology by light and transmission electron microscopy. M-CSF and IL-6 were detected in supernatants from cultures of these cells, using assays specific for each factor. Cytokine levels were comparable to those in supernatants from ovarian and breast cancer cell lines. M-CSF expression could also be demonstrated by immunohistochemical analysis with specific rabbit heteroantiserum. Thus, M-CSF and IL-6 are produced constitutively by normal as well as by neoplastic ovarian epithelium.     

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.     

Production of Interleukin-1 by Primary Cultured Parenchymal Liver Cells (Hepatocytes)

The production of interleukin-1 (IL-1) by cultured parenchymal liver cells was revealed by a biological assay with an IL-1-dependent cell line, Northern blot analysis, and in situ hybridization. Inhibition experiments on the IL-1 activity with anti IL-1α antibody also support the presence of IL-1α in the supernatant of cultured parenchymal liver cells. Based on these results, we discuss the possibility of IL-1 production by parenchymal liver cells in vivo.     

Mechanical-Stretch of C2C12 Myoblasts Inhibits Expression of Toll-Like Receptor 3 (TLR3) and of Autoantigens Associated with Inflammatory Myopathies

Recent studies in patients suffering from inflammatory autoimmune myopathies suggested that moderate exercise training improves or at least stabilizes muscle strength and function without inducing disease flares. However, the precise mechanisms involved in this beneficial effect have not been extensively studied. Here we used a model of in vitro stretched C2C12 myoblasts to investigate whether mechanical stretch could influence myoblast proliferation or the expression of proinflammatory genes. Our results demonstrated that cyclic mechanical stretch stimulated C2C12 cell cycling and early up-regulation of the molecules related to mechanical-stretch pathway in muscle (calmodulin, nNOS, MMP-2, HGF and c-Met). Unexpectedly, mechanical stretch also reduced the expression of TLR3 and of proteins known to represent autoantigens in inflammatory autoimmune myopathies (Mi-2, HRS, DNA-PKcs, U1-70). Interestingly, stimulation or inhibition of calmodulin, NOS, HGF or c-Met molecules in vitro affected the expression of autoantigens and TLR3 proteins confirming their role in the inhibition of autoantigens and TLR3 during mechanical stretch. Overall, this study demonstrates for the first time that mechanical stretch could be beneficial by reducing expression of muscle autoantigens and of pro-inflammatory TLR3 and may provide new insight to understand how resistance training can reduce the symptoms associated with myositis.     

Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.     

Poly(C) synthesis by class I and class II CCA-adding enzymes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.