Proinflammatory Chemokine CXCL10 Research Articles

Abstract 191: NF-?B p65 Methylation by Protein Arginine Methyltransferase 5 (PRMT5) is Necessary for the Transcriptional Induction of CXCL10 by TNF-a

TNF-α initiates the expression of genes involved in the recruitment, adhesion, and transmigration of leukocytes to sites of inflammation. Here, we report that the protein arginine methyltransferase PRMT5 is required for the transcriptional induction of the pro-inflammatory chemokine CXCL10 (IP-10) in endothelial cells. Depletion of PRMT5 by siRNA results in significantly diminished TNF-α-induced CXCL10 mRNA expression, but does not affect expression of other chemokines, such as MCP-1 or IL-8. Chromatin immunoprecipitation experiments of the CXCL10 proximal promoter show the presence of symmetrical dimethylated arginine (sDMA)-containing proteins upon exposure to TNF-α. This methylation is completely lost when PRMT5 is removed from cells by siRNA. Using immunoprecipitation, we show that PRMT5 enhances CXCL10 expression by methylating the RelA (p65) subunit of NF-κB. In summary, we have identified that PRMT5 is a novel regulator of CXCL10 expression. Further, we have discovered that PRMT5 methylates NF-κB, a finding which may further knowledge of the post-translational code governing NF-κB regulation and target specificity.

Sensitivity of PTEN-deficient prostate carcinoma cells to ionizing radiation through inhibition of treatment-induced CXCL8 signaling.

154 Background: We recently demonstrated that loss of PTEN in CaP cell lines increases expression of the pro-inflammatory chemokine CXCL8. Increased expression and signalling of this chemokine is detectable in PIN-foci and increases with disease progression. Our objective was to determine how induction of CXCL8 signalling affects the sensitivity of PTEN-deficient CaP cells to radiotherapy, a major treatment modality used in locally-confined CaP. Methods: Established cell-based models of CaP with differential expression of PTEN were subjected to ionizing radiation (IR) under normal or hypoxic culture conditions. Changes in chemokine expression in CaP cells were analyzed by real-time PCR, immunoblotting, and ELISA. siRNA- or shRNA-directed strategies were used to repress PTEN expression and/or attenuate autocrine CXCL8 signalling by down-regulating CXCR1 and CXCR2 gene expression.The effects of modulating chemokine signaling on cell viability following exposure to clinically-relevant doses of IR were assessed by colony formation assays, cell proliferation analysis, and analysis of apoptosis and/or cellular senescence. Results: Exposure to IR (3 Gy) in normoxic and hypoxic cells increased gene expression of CXCL8 and its receptors CXCR1 and CXCR2, a response potentiated in PTEN depleted cells. Loss of PTEN had different effects on the sensitivity of CaP cell lines; shRNA-mediated loss of PTEN conferred increased radioresistance in DU145 cells but increased the sensitivity of 22Rv1 cells to IR. However, despite these variable responses, siRNA-mediated repression of CXCR1/2 expression increased the sensitivity of all PTEN-depleted cell-based models to IR, under both normal and hypoxic conditions. Interestingly, inhibition of CXCL8 signalling reinforced IR-induced senescence in PTEN-depleted 22Rv1 cells (Tp53 WT) but increased apoptosis in PTEN-depleted DU145 cells (Tp53 mutant). Conclusions: Targeting IR-induced autocrine CXCL8 signalling is a novel therapeutic strategy to enhance the sensitivity of PTEN-deficient CaP cells to IR, taking on added significance in cells harbouring mutations or loss of Tp53 expression.

Anti-Inflammatory Dimethylfumarate: A Potential New Therapy for Asthma?

Asthma is a chronic inflammatory disease of the airways, which results from the deregulated interaction of inflammatory cells and tissue forming cells. Beside the derangement of the epithelial cell layer, the most prominent tissue pathology of the asthmatic lung is the hypertrophy and hyperplasia of the airway smooth muscle cell (ASMC) bundles, which actively contributes to airway inflammation and remodeling. ASMCs of asthma patients secrete proinflammatory chemokines CXCL10, CCL11, and RANTES which attract immune cells into the airways and may thereby initiate inflammation. None of the available asthma drugs cures the disease—only symptoms are controlled. Dimethylfumarate (DMF) is used as an anti-inflammatory drug in psoriasis and showed promising results in phase III clinical studies in multiple sclerosis patients. In regard to asthma therapy, DMF has been anecdotally reported to reduce asthma symptoms in patients with psoriasis and asthma. Here we discuss the potential use of DMF as a novel therapy in asthma on the basis of in vitro studies of its inhibitory effect on ASMC proliferation and cytokine secretion in ASMCs.

Expression and detrimental role of hematopoietic prostaglandin D synthase in spinal cord contusion injury

Prostaglandin D(2) (PGD(2) ) is a potent inflammatory mediator, which is implicated in both the initiation and resolution of inflammation in peripheral non-neural tissues. Its role in the central nervous system has not been fully elucidated. Spinal cord injury (SCI) is associated with an acute inflammatory response, which contributes to secondary tissue damage that worsens functional loss. We show here, with the use of hematopoietic prostaglandin D synthase (HPGDS) deficient mice and a HPGDS selective inhibitor (HQL-79), that PGD(2) plays a detrimental role after SCI. We also show that HPGDS is expressed in macrophages in the injured mouse spinal cord and contributes to the increase in PGD(2) in the contused spinal cord. HPGDS(-/-) mice also show reduced secondary tissue damage and reduced expression of the proinflammatory chemokine CXCL10 as well as an increase in IL-6 and TGFβ-1 expression in the injured spinal cord. This was accompanied by a reduction in the expression of the microglia/macrophage activation marker Mac-2 and an increase in the antioxidant metallothionein III. Importantly, HPGDS deficient mice exhibit significantly better locomotor recovery after spinal cord contusion injury than wild-type (Wt) mice. In addition, systemically administered HPGDS inhibitor (HQL-79) also enhanced locomotor recovery after SCI in Wt mice. These data suggest that PGD(2) generated via HPGDS has detrimental effects after SCI and that blocking the activity of this enzyme can be beneficial.

Expression of CXCL8, CXCR1, and CXCR2 in Neurons and Glial Cells of the Human and Rabbit Retina

Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes. The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging. Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells. Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNgamma, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+ CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFbeta, TGFbetaRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFbeta and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum

IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN-gamma secretion in response to erythrocytes infected with Plasmodium falciparum (Pf), a causative agent of human malaria. In addition, direct sensing of Pf infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are individually dispensable for NK cell activation induced by Pf-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages, and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection.

High-dose hydrocortisone reduces expression of the pro-inflammatory chemokines CXCL8 and CXCL10 in SARS coronavirus-infected intestinal cells

Clinical observations and our high-density oligonucleotide microarray results demonstrated increased expression of proinflammatory chemokines after SARS-CoV infection. Here, we investigated the influence of SARS-CoV infection on CXCL8 (interleukin 8) and CXCL10 (interferon-gamma-inducible protein 10) in human intestinal epithelial (Caco2) cells. RT-PCR and ELISA showed time-dependent up-regulation of both chemokines after SARS-CoV infection. Electric mobility shift assay revealed increased DNA binding activity of the cellular transcription factors activator protein 1 (AP-1) and nuclear factor (B (NF-kappaB) in SARS-CoV infected cells. High hydrocortisone concentrations (> or =50 microg/ml) completely prevented increased DNA binding activity of AP-1 and NF-kappaB and inhibited up-regulation of CXCL8 and CXCL10, but did not reduce chemokine expression to basal levels. Ribavirin that does not inhibit SARS-CoV replication in Vero cells inhibited SARS-CoV replication in Caco2 cells at therapeutical concentrations. Hydrocortisone neither influenced SARS-CoV titres alone nor in combination with ribavirin. Our results show that corticosteroids may be of limited benefit in the suppression of chemokine production by SARS-CoV-infected cells.

Role of type I interferon in the bacillus Calmette-Guérin-induced expression of CXCL10 from human monocytes.

BACKGROUND: The proinflammatory chemokine CXCL10, in addition to its chemotactic properties, is also involved in the stimulation of natural killer and T-cell migration in Mycobacterium tuberculosis infection. In this study, our experiments were designed to determine the role of interferon (IFN)-alphabeta in the production of CXCL10 by human monocytes infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG). METHODS: The concentrations of CXCL10 in culture supernatants of monocytes infected with M. bovis BCG were determined by enzyme-linked immunosorbent assay. CXCL10 mRNA levels were determined by the reverse transcription-polymerase chain reaction method. RESULTS: We have shown the induction of CXCL10 following infection with M. bovis BCG in a dose-dependent and time-dependent manner. Importantly, the secretion of CXCL10 in response to M. bovis was increased by IFN-alpha. These results were further confirmed by the fact that the addition of an anti-IFN-alphabeta neutralizing antibody completely reversed the stimulatory effect, whereas an isotype-matched control antibody had no significant effect on CXCL10 secretion. It is important to note that no significant effect of type I IFN on CXCL8 production in M. bovis-infected monocytes was observed. This was consistent with the finding by the reverse transcription-polymerase chain reaction method that treatment with anti-IFN-alpha/beta antibodies potentially inhibited CXCL10 mRNA levels, whereas no significant effect was observed on CXCL8 mRNA. Moreover, in THP-1 monocytes and THP-1 macrophages, the addition of exogenous IFN-alpha stimulated CXCL10 secretion. CONCLUSIONS: Collectively, these results indicate that the type I IFN may play an important role to modulate the expression of CXCL10 in M. bovis BCG infection. Studies on M. bovis-induced chemokine secretion could provide important insight into the regulation of the immune response against tuberculosis.

Role of type I interferon in the bacillus Calmette‐Guérin‐induced expression of CXCL10 from human monocytes

The proinflammatory chemokine CXCL10, in addition to its chemotactic properties, is also involved in the stimulation of natural killer and T-cell migration in Mycobacterium tuberculosis infection. In this study, our experiments were designed to determine the role of interferon (IFN)-alphabeta in the production of CXCL10 by human monocytes infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG). The concentrations of CXCL10 in culture supernatants of monocytes infected with M. bovis BCG were determined by enzyme-linked immunosorbent assay. CXCL10 mRNA levels were determined by the reverse transcription-polymerase chain reaction method. We have shown the induction of CXCL10 following infection with M. bovis BCG in a dose-dependent and time-dependent manner. Importantly, the secretion of CXCL10 in response to M. bovis was increased by IFN-alpha. These results were further confirmed by the fact that the addition of an anti-IFN-alphabeta neutralizing antibody completely reversed the stimulatory effect, whereas an isotype-matched control antibody had no significant effect on CXCL10 secretion. It is important to note that no significant effect of type I IFN on CXCL8 production in M. bovis-infected monocytes was observed. This was consistent with the finding by the reverse transcription-polymerase chain reaction method that treatment with anti-IFN-alpha/beta antibodies potentially inhibited CXCL10 mRNA levels, whereas no significant effect was observed on CXCL8 mRNA. Moreover, in THP-1 monocytes and THP-1 macrophages, the addition of exogenous IFN-alpha stimulated CXCL10 secretion. Collectively, these results indicate that the type I IFN may play an important role to modulate the expression of CXCL10 in M. bovis BCG infection. Studies on M. bovis-induced chemokine secretion could provide important insight into the regulation of the immune response against tuberculosis.