Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.     

Apoptosis of airway epithelial cells in response to meconium

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.     

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.     

Modeling an immune response to influenza A virus infection in alveolar epithelial cells

Influenza A viruses (IAV) have been the cause of several influenza pandemics in history and are a significant threat for the next global pandemic. Hospitalized influenza patients often have excess interferon production and a dysregulated immune response to the IAV infection. Obtaining a better understanding of the mechanisms of IAV infection that induce these harmful effects would help drug developers and health professionals create more effective treatments for IAV infection and improve patient outcomes. IAV stimulates viral sensors and receptors expressed by alveolar epithelial cells, like RIG-I and toll-like receptor 3 (TLR3). These two pathways coordinate with one another to induce expression of type III interferons to combat the infection. Presented here is a queuing theory-based model of these pathways that was designed to analyze the timing and amount of interferons produced in response to IAV single stranded RNA and double-stranded RNA detection. The model accurately represents biological data showing the necessary coordination of the RIG-I and TLR3 pathways for effective interferon production. This model can serve as the framework for future studies of IAV infection and identify new targets for potential treatments.     

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3.     

Characterization and expression of Megalobrama amblycephala toll‐like receptor 22 involved in the response to Aeromonas hydrophila

The toll‐like receptors (TLR) tlr22 was identified and characterized for the first time in one of the economically most important freshwater fish species in China, Megalobrama amblycephala. The full‐length cDNA (4039 bp) of M. amblycephala tlr22 contains an open reading frame of 2706 bp, encoding a 901 amino‐acid long polypeptide. The putative polypeptide contains 16 leucine‐rich repeat (LRR) motifs, an LRR C‐terminal, a transmembrane region and a cytoplasmic toll–interleukin‐1 receptor (TIR) domain. Phylogenetic analyses revealed that M. amblycephala Tlr22 shared the closest relationship with a grass carp ortholog. tlr22 was constitutively expressed in nine tissues and during 10 developmental stages studied, albeit with varying expression levels. Along with many pathological changes observed after Aeromonas hydrophila bacterium infection, tlr22 and myd88 mRNA were significantly upregulated in blood, head kidney, spleen and intestine, indicating that tlr22 is involved in the immune response. These results provide an insight into tlr22 regulation mechanisms in the innate immune response to bacterial infection.     

Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells

To test the hypothesis that the excess inflammatory response in cystic fibrosis airway epithelial cells is the result of differential activation of genes in response to a laboratory strain of Pseudomonas aeruginosa (PAO1), a 48-h time course of genes expressed following PAO1 stimulation (10(9) CFU for 1 h) was studied in two pairs of airway epithelial cells: 9/HTEo- and 16HBE14o-, each with a matched normal and CF phenotype pair. cRNA was hybridized to Affymetrix HG-U95Av2 chips and pairwise comparisons against zero time (no PAO1) were calculated for each time point. PAO1 elicited profound changes in both cell pairs: for 9/HTEo-, 144 genes changed significantly in the normal pair, and 116 for the CF pair; for the 16HBE14o- pair, 57 genes changed significantly for the normal pair and 53 for the CF pair. Changes were much greater in the 9/HTEo- than in the 16HBE14o- pair, but basal levels of expression of inflammatory genes are higher in the 16HBE14o- pair, so 16HBE14o- was used mainly to corroborate the results of 9/HTEo-. Clustering analysis indicated that the pattern of gene expression is similar in the CF cells and their normal counterparts. However, there were substantial quantitative differences in gene expression. Thus, the difference between CF and normal resides in the magnitude, not the pattern, of the changes.     

肠道菌群参与宿主免疫应答的作用及机制研究进展

肠道菌群作为动物体内重要的组成部分,能够直接参与机体的免疫调控作用,促进机体免疫系统发育,维持正常免疫功能。同时,免疫系统对肠道菌群又有调控和制约作用。本文主要综述了肠道菌群的组成以及影响肠道菌群变化的因素,系统阐述了肠道菌群与疾病相互作用的机制,总结了肠道菌群在宿主感染与免疫应答中的作用,为开展肠道菌群参与机体免疫应答的机制方面的研究提供新的思路。     

poly(I:C) synergizes with proteasome inhibitors to induce apoptosis in cervical cancer cells

Cervical cancer is one of the most common malignancies in women, with a poor survival rate. Thus, there is a need to define effective combination strategies to improve therapy. In this study, we report that dsRNA poly(I:C) up-regulated the expression of IFNβ and apoptosis-associated genes in cervical cancer cells, activating both intrinsic and extrinsic apoptotic pathways, and eventually inducing cell death. Similarly, proteasome inhibitors also effectively induced cervical cancer cell apoptosis, probably through prevention of p53 degradation, inhibiting NF-κB signal activation and decreasing BCL-2 expression. Importantly, the combination of poly(I:C) with proteasome inhibitors enhanced caspase-8 and caspase-9 activation, and synergistically induced cervical cancer cell apoptosis. Both activated p38 signals and increased ROS levels, and their combination extended these effects. Collectively, we show that the activation of multiple pro-apoptotic pathways by poly(I:C) and proteasome inhibitors underpin a synergistic effect on inducing cervical cancer cell death, suggesting a potential therapeutic combination with clinical relevance.     

Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA

Scavenger receptors and Toll-like receptors (TLRs) cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (ds)RNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.     

Diesel exhaust enhances virus- and poly(I:C)-induced Toll-like receptor 3 expression and signaling in respiratory epithelial cells

Prior exposure of respiratory epithelial cells to an aqueous-trapped solution of diesel exhaust (DE(as)) enhances the susceptibility to influenza infections. Here, we examined the effect of DE(as) on the Toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analyses showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DE(as) and then infected with influenza A or treated with polyriboinosinic acid-polyribocytidylic acid [poly(I:C)], a synthetic form of double-stranded RNA. Exposure to DE(as) before infection with influenza or stimulation with poly(I:C) significantly upregulated the expression of TLR3. Additionally, preexposure to DE(as) significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant-negative mutant form of TNF receptor-associated factor 6 reversed the effects of DE(as) on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3 dependent. Similarly, preexposure to DE(as) significantly increased nuclear levels of interferon regulatory factor 3 and the expression of IFN-beta in response to poly(I:C). Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, was able to abate the effect of DE(as) on poly(I:C)-induced IFN-beta expression. Together, these results indicate that exposure of respiratory epithelial cells to DE(as) could potentially alter the response to viral infections by increasing the expression and function of TLR3.