Role of retinoic acid or vitamin D analogue in models of human keratinocyte apoptosis Interactions with the IGF system

Available from British Library Document Supply Centre- DSC:DXN060380 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Toll-like receptor-4 drives pro-inflammatory cytokine response & tissue degradation in human bacterial keratitis

Purpose: : Toll-like Receptor-4 (TLR4) is a key component of the innate immune response during bacterial infections. Pathways and downstream effectors relating to TLR signalling in human bacterial keratitis (BK) remain unknown. By activating the TLR4 signalling cascade with bacterial lipoploysaccharide (LPS), we investigated whether TLR4 influenced matrix metalloproteases (MMP-2, MMP-9) and cytokine expression in diseased human primary corneal fibroblast (CF) cells are altered.Methods: : Human primary CF cells from patients with severe corneal ulceration from patients with gram negative bacterial keratitis were grown ex vivo and cultured in conjunction with healthy controls. CF cells were treated with exogenous LPS derived from Pseudomonas Aeruginosa.Results: : TLR4, MMP-2 and MMP-9 were constitutively expressed in both ulcerated and control CF cells. Diseased CF cells showed greater responsiveness to LPS stimulation. TLR4 and MMP-9 expression was dose-dependently increased by LPS. MMP-2 expression was not affected by LPS. Analysis on cytokine expression revealed that IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ and TNF-α expression increased following LPS treatment but only in the diseased cells.Conclusions: : TLR4 activation with LPS increases TLR4, MMP-9 and cytokine expression in CF cells cultured from human BK patients. Over expression of these products may provide a local mechanism to eradicate bacterial infection but also contribute to corneal ulceration and perforation. This is the first description of the effect of TLR4 activation in human bacterial keratitis

The interleukin 13 (IL-13) pathway in human macrophages is modulated by microRNA-155 via direct targeting of interleukin 13 receptor ?1 (IL13R?1)

Macrophages play a central role in the balance and efficiency of the immune response and are at the interface between innate and adaptive immunity. Their phenotype is a delicate equilibrium between the M1 (classical, pro-Th(1)) and M2 (alternative, pro-Th(2)) profiles. This balance is regulated by cytokines such as interleukin 13 (IL-13), a typical pro-M2-Th(2) cytokine that has been related to allergic disease and asthma. IL-13 binds to IL-13 receptor ?1 (IL13R?1), a component of the Type II IL-4 receptor, and exerts its effects by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) through phosphorylation. MicroRNAs are short (?22 nucleotide) inhibitory non-coding RNAs that block the translation or promote the degradation of their specific mRNA targets. By bioinformatics analysis, we found that microRNA-155 (miR-155) is predicted to target IL13R?1. This suggested that miR-155 might be involved in the regulation of the M1/M2 balance in macrophages by modulating IL-13 effects. miR-155 has been implicated in the development of a healthy immune system and function as well as in the inflammatory pro-Th(1)/M1 immune profile. Here we have shown that in human macrophages, miR-155 directly targets IL13R?1 and reduces the levels of IL13R?1 protein, leading to diminished activation of STAT6. Finally we also demonstrate that miR-155 affects the IL-13-dependent regulation of several genes (SOCS1, DC-SIGN, CCL18, CD23, and SERPINE) involved in the establishment of an M2/pro-Th(2) phenotype in macrophages. Our work shows a central role for miR-155 in determining the M2 phenotype in human macrophages

Lipopolysaccharide regulation of toll-like receptor-4 and matrix metalloprotease-9 in human primary corneal fibroblast cells

Purpose: Toll-like Receptor 4 (TLR4) is a key component of the innate immune response related to microbial keratitis (MK). Pathways and downstream effectors relating to TLR signalling remain unknown in human bacterial MK. To this effect, by activating the TLR4 signalling cascade with LPS we investigated whether TLR4, matrix metalloproteases (MMP)-2, MMP-9 and cytokine expression in diseased human primary corneal fibroblast (CF) cells are altered. Methods: Human primary CF cells from patients with severe corneal ulceration were cultured in conjunction with healthy control CF cells and treated with lipopolysaccharides (LPS) derived from Pseudomonas aeruginosa. Results: TLR4, MMP-2 and MMP-9 were constitutively expressed in both ulcerated and control CF cells. Diseased CF cells showed greater responsiveness to LPS stimulation. TLR4 and MMP-9 expression was dose-dependently increased by LPS. MMP-2 expression was not affected by LPS. Analysis on cytokine expression revealed that IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFN? and TNF? expression increased following LPS treatment but only in the diseased cells. Conclusions: TLR4 activation with LPS increases TLR4, MMP-9 and cytokine expression in CF cells cultured from human MK patients. Over expression of these products may provide a local mechanism to eradicate bacterial infection but may also aid corneal ulceration and perforation

A microRNA network dysregulated in asthma controls IL-6 production in bronchial epithelial cells

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-?, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-?, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-? and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying diseas

The cutaneous biochemical redox barrier: A component of the innate immune defenses against sensitization by highly reactive environmental xenobiotics

Contact allergy to environmental xenobiotics is a common and important problem, but it is unclear why some chemicals are potent sensitizers and others weak/nonsensitizers. We explored this by investigating why similar chemicals, 2,4-dinitrochlorobenzene (DNCB) and 2,4-dinitrothiocyanobenzene (DNTB), differ in their ability to induce contact hypersensitivity (CHS). DNCB induced CHS in humans, whereas at similar doses DNTB did not. However, following DNCB sensitization, DNTB elicited CHS in vivo and stimulated DNCB-responsive T cells in vitro, suggesting that differences in response to these compounds lie in the sensitization phase. In contrast to DNCB, DNTB failed to induce emigration of epidermal Langerhans cells in naive individuals. Examination for protein dinitrophenylation in skin revealed that DNCB penetrated into the epidermis, whereas DNTB remained bound to a thiol-rich band within the stratum corneum. DNTB reacted rapidly with reduced glutathione in vitro and was associated with a decrease in the free thiol layer in the stratum corneum, but not in the nucleated epidermis. By contrast, DNCB required GST facilitation to react with gluthathione and, following penetration through the stratum corneum, depleted thiols in the viable epidermis. Chemical depletion of the thiol-rich band or removing it by tape stripping allowed increased penetration of DNTB into the epidermis. Our results suggest that the dissimilar sensitizing potencies of DNCB and DNTB in humans are determined by a previously undescribed outer epidermal biochemical redox barrier, a chemical component of the innate immune defense mechanisms that defend against sensitization by highly reactive environmental chemicals

Individual microRNA modulatory effects on inflammatory genes.

<p>BEAS2B cells were transfected with anti-miRNA oligonucleotides against miR-18a, miR-27a, miR-128 and miR-155 or Anti-miRNA Control. Cells were harvested 24 h post-transfection. Inflammatory genes were analysed by RT-qPCR. No symbol: non-significant. n = 4 * = p<0.05; ** = p<0.01, ratio paired t-test.</p

A microRNA network affects IL-6 secretion in bronchial epithelial cells.

<p>BEAS2B cells were transfected with anti-miRNA oligonucleotides against miR-18a, miR-27a, miR-128 and miR-155 separately or together (Anti-MiX) or Anti-miRNA Control. Cells were harvested 24 h post-transfection. IL-8 (<i>A</i>) and IL-6 (<i>B</i>) secretion was measured by ELISA. n = 4 ns: non-significant. * = p<0.05; ** = p<0.01, ratio paired t-test.</p

A microRNA network modulates IL-6 and IL-8 mRNA expression.

<p>BEAS2B cells were transfected with anti-miRNA oligonucleotides against miR-18a, miR-27a, miR-128 and miR-155 together (Anti-MiX) or Anti-miRNA Control. Cells were harvested 24 h post-transfection. Inflammatory genes were analysed by RT-qPCR. n = 6. No symbol: non-significant. * = p<0.05; ** = p<0.01, ratio paired t-test.</p

MiR-27a and miR-128 directly target the 3′UTR of SMAD2.

<p><i>A</i> Renilla luciferase construct harbouring an SMAD2 3′-UTR fragment containing the predicted binding sites for miR-27a and miR-128 (wild type, WT) or its mutated version for both miRNAs binding (MUT) was co-transfected with either an empty expression vector, a miR-27a over-expressing vector (WT+27a and MUT+27a, respectively) or an miR-128 over-expressing vector (WT+128 and MUT+miR-128, respectively. One of three independent experiments is shown. Where no significance is shown = non-significant, ***p≤0.001. Error bars indicate Standard Error. <i>B</i> Schematic representing the binding region (and mutation site) of miR-27 and miR-128 in SMAD2 3′UTR.</p