PLCB3 Loss of Function Reduces Pseudomonas aeruginosa-Dependent IL-8 Release in Cystic Fibrosis.

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-ß3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform ß, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.

Transient Receptor Potential Ankyrin 1 Channels Modulate Inflammatory Response in Respiratory Cells from Patients with Cystic Fibrosis.

Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o-) and primary cells from patients with CF were used to: (1) check TRPA1 function modulation, by Fura-2 calcium imaging; (2) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and small interfering RNA silencing; and (3) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to proinflammatory challenges, by quantitative RT-PCR and 27-protein Bioplex assay. TRPA1 channels are expressed in the CF pseudostratified columnar epithelium facing the bronchial lumina exposed to bacteria, where IL-8 is coexpressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the proinflammatory cytokines IL-1ß and TNF-α, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of patients with CF. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells.

Regulation of IL-8 gene expression in gliomas by microRNA miR-93.

BACKGROUND: Different strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas. METHODS: Here we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G. RESULTS: IL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis). CONCLUSION: In conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.

Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans.

Interleukin-12 (IL-12) and IL-23 share a common chain. Yet, their production in response to pathogens is differentially regulated, and their functions are distinct and often antithetic. IL-12 is involved in the induction or amplification of the T-helper (Th) type 1 response, whereas IL-23 has been associated with the generation of the Th17 response and IL-17 production. Mycobacterium tuberculosis and yeast zymosan induce IL-23, but in the absence of other stimuli, no IL-12 is induced in human dendritic cells (DCs). The stimulation of IL-23 by M. tuberculosis was mostly explained by the triggering of Toll-like receptor (TLR2) and the cytoplasmic receptor nucleotide oligomerization domain (NOD)-containing protein 2, whereas zymosan induces IL-23 primarily by stimulating the beta-glucan receptor dectin-1 alone or in combination with TLR2. IL-23, IL-6, transforming growth factor (TGF-beta1), and IL-1beta in supernatants from activated human DCs induce human naive CD4(+) T cells to produce IL-17. These data are consistent with various recent reports that TGF-beta is an inducer of IL-17 production both in human and in mouse cells. However, IL-1 is necessary in combination with some or all of the other cytokines to induce IL-17 production in human T cells. The ability of various stimuli to induce Th17 cells depends not only on their induction of IL-23, IL-6, and TGF-beta production in DCs but also on their ability to activate directly or indirectly the inflammasome and to induce IL-1beta.

The Cross-Talk between Myeloid and Mesenchymal Stem Cells of Human Bone Marrow Represents a Biomarker of Aging That Regulates Immune Response and Bone Reabsorption.

One of the mechanisms that characterizes the aging process of different organs is the accumulation of fat. Different authors have demonstrated that adipose tissue replaces the loss of other cell types, deriving from mesenchymal cells. During aging, there is substitution or trans-differentiation of mesenchymal cells with other cells having the same embryological origin. Newly formed adipocytes were also observed in the trabecular matrix of elderly people's bones, associated with myeloid cells. In this study, we have investigated the relationship between immature myeloid-derived suppressor cells (I-MDSCs) and mesenchymal stem cells (MSCs) in bone marrow (BM) samples harvested from 57 patients subjected to different orthopedic surgeries. Patients aged from 18 to 92 years were considered in order to compare the cellular composition of bone marrow of young and elderly people, considered a biomarker of immunity, inflammation, and bone preservation. The I-MDSC percentage was stable during aging, but in elderly people, it was possible to observe a strong basal immunosuppression of autologous and heterologous T cells' proliferation. We hypothesized that this pattern observed in elders depends on the progressive accumulation in the BM of activating stimuli, including cell-cell contact, or the production of different cytokines and proteins that induce the differentiation of bone marrow mesenchymal stem cells in adipocytes. The collected data provided underline the importance of specific biomarkers of aging that promote a reduction in immune response and incremented inflammatory pathways, leading to bone reabsorption in elderly people.

Prevalence of CYP2C9 polymorphisms in the south of Europe.

CYP2C9 is a major liver enzyme responsible of the metabolism of many clinically important drugs. The presence of CYP2C9 genetic polymorphisms has been associated with marked interindividual variability in its catalytic activity that could result in drug toxicity. Here we present frequencies of the most common CYP2C9 coding variants CYP2C9*2 (C430T) and CYP2C9*3 (A1075C) in representative samples of four regions from Spain (Basque Country, n=358; Catalonia, n=240; Central Spain, n=190 and Galicia, n=288) and one northern Italian region, (Verona, n=164), which range between 0.125 and 0.165 in the case of CYP2C9*2 and between 0.071 and 0.085 for CYP2C9*3. No significant differences between CYP2C9 allele frequencies were found comparing all the sampled populations. A more extensive comparative analysis using allele frequency data of populations widely spread over Europe was performed, showing significant differences in the CYP2C9*2 allele frequencies distribution between some of the regions, being quite homogeneous in the case of CYP2C9*3 variant. The results obtained show that above 40% of our samples carry a mutate allele, which can result in a poor metabolization of low therapeutic index drugs as oral anticoagulants (warfarin, acenocoumarol), oral antidiabetic drugs and some non-steroidal anti-inflammatory drugs. Our study constitutes both a large (n=1240) and robust allele frequency database on CYP2C9 polymorphisms, which represents one of the most numerous CYP2C9*2 and *3 database existing to date.

Interleukin-1 and interferon-γ orchestrate ß-glucan-activated human dendritic cell programming via IκB-ζ modulation.

Recognition of microbial components via innate receptors including the C-type lectin receptor Dectin-1, together with the inflammatory environment, programs dendritic cells (DCs) to orchestrate the magnitude and type of adaptive immune responses. The exposure to ß-glucan, a known Dectin-1 agonist and component of fungi, yeasts, and certain immune support supplements, activates DCs to induce T helper (Th)17 cells that are essential against fungal pathogens and extracellular bacteria but may trigger inflammatory pathology or autoimmune diseases. However, the exact mechanisms of DC programming by ß-glucan have not yet been fully elucidated. Using a gene expression/perturbation approach, we demonstrate that in human DCs ß-glucan transcriptionally activates via an interleukin (IL)-1- and inflammasome-mediated positive feedback late-induced genes that bridge innate and adaptive immunity. We report that in addition to its known ability to directly prime T cells toward the Th17 lineage, IL-1 by promoting the transcriptional cofactor inhibitor of κB-ζ (IκB-ζ) also programs ß-glucan-exposed DCs to express cell adhesion and migration mediators, antimicrobial molecules, and Th17-polarizing factors. Interferon (IFN)-γ interferes with the IL-1/IκB-ζ axis in ß-glucan-activated DCs and promotes T cell-mediated immune responses with increased release of IFN-γ and IL-22, and diminished production of IL-17. Thus, our results identify IL-1 and IFN-γ as regulators of DC programming by ß-glucan. These molecular networks provide new insights into the regulation of the Th17 response as well as new targets for the modulation of immune responses to ß-glucan-containing microorganisms.

Glutathione S-transferase and CYP1A1 gene polymorphisms and non-melanoma skin cancer risk in Italian transplanted patients.

Solid organ transplant recipients are at higher risk of non-melanoma skin cancer (NMSC), especially basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Genetic alterations in the production of detoxifying enzymes such as glutathione S-transferase (GST) and CYP1A1 may enhance this risk. We investigated the frequency of GST genotypes (GSTM1, GSTM3, GSTT1 and GSTP1) and CYP1A1 in 239 transplant recipients: 107 cases with NMSC and 132 controls free from NMSC matched for type of transplanted organ, duration of transplantation, sex and age. Allele GSTP1*A was associated with a higher risk of NMSC [odds ratio (OR) 1.7 (1.1-2.5); P = 0.017]. Homozygosity for allele GSTP1 Val(105) was lower in cases [OR 0.3 (0.1-0.8); P = 0.012], especially in patients with SCC [OR 0.1 (0.0-0.7); P = 0.012]. A higher risk of BCC was found in patients with GSTM1 null/null [null/null versus A + B, OR 3.1 (1.4-6.8); P = 0.003]. Analysis of allelism and interaction between allelic variants showed significant association between combined GSTM1 and CYP1A1 Val(462) genotypes, where individuals homozygous for the risk allele GSTM1 null and carrying also the allele CYP1A1 Val(462), show a higher risk of developing NMSC [OR 4.5 (1.1-21.4); P = 0.03], especially SCC [OR 6.5 (1.4-34.4); P = 0.01]. GSTP1 polymorphisms are associated with both BCC and SCC risk. GSTM1 polymorphisms seem to be involved in BCC risk, while GSTM1 null/null genotype combined with CYP1A1 allele Val(462) are associated with a higher risk for SCC, indicating that allelism and/or interactions between allelic variants at other loci may also influence the risk of NMSC, particularly SCC.