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

Dissociated expression of IL-17, IL-22 and IFN-\u3b3 in naive CD4+ T lymphocytes

IL-17, IL-22 e IFN-\u3b3 sono citochine prodotte soprattutto da cellule T helper (Th) che giocano ruoli differenti ed essenziali nella difesa contro un\u2019ampia variet\ue0 di patogeni, ma sono anche responsabili di danni tessutali che portano a numerose patologie autoimmunitarie. Le cellule Th che producono IL-17 (Th17) e quelle che producono IFN-\u3b3 (Th1) appartengono a sottotipi distinti; tuttavia, si trovano frequentemente sia in vivo che in vitro cellule Th17 che producono IFN-\u3b3, lasciando aperto il dibattito rispetto ai relativi ruoli di Th17 e Th1 in patologie autoimmunitarie. IL-22 \ue8 una citochina che gioca un ruolo fondamentale nella riparazione e nella difesa epiteliale, nella difesa dell\u2019ospite contro infezioni da patogeni extracellulari ed ha un ruolo protettivo nei confronti del danno agli epatociti durante l\u2019epatite acuta. IL-22 \ue8 prodotta principalmente da cellule Th17, ma \ue8 anche stata descritta una popolazione di cellule Th che producono IL-22 in assenza di IL-17 e IFN-\u3b3. Per contribuire a chiarire la relazione tra IL-17, IL-22 e IFN-\u3b3 e per identificare i fattori rilevanti nell\u2019induzione dell\u2019espressione di queste citochine, abbiamo valutato il pattern di espressione di IL-17, IL-22 e IFN-\u3b3 in linfociti T CD4+ naive attivati con anti-CD3 ed anti-CD28. Poich\ue9 il fattore principale che determina la polarizzazione di queste cellule \ue8 rappresentato dalle citochine prodotte dalle cellule presentanti l\u2019antigene attivate da patogeni, abbiamo stimolato linfociti CD4+ T naive in presenza di sopranatanti derivati da cellule dendritiche precondizionate con IFN-\u3b3 e stimolate con zymosan (SNDCzym), che in precedenza abbiamo dimostrato essere in grado di indurre efficientemente l\u2019espressione di IL-17, o con la combinazione delle citochine IL-1\u3b2+IL-6+IL-23, analogamente conosciute per la loro capacit\ue0 di polarizzare una risposta Th17. Inoltre, abbiamo usato il SNDCzym con varie combinazioni di anticorpi neutralizzanti le citochine IL-6, IL-12, IL-23 o con l\u2019antagonista del recettore di IL-1, che inibisce IL-1\u3b1 e IL-1\u3b2, o, in alternativa, con differenti combinazioni di IL-1\u3b2, IL-6 e IL-23 ricombinanti. La comparazione dei risultati ottenuti da questi due sistemi ci ha permesso di chiarire che l\u2019espressione di IL-17, IL-22 e IFN-\u3b3 durante la differenziazione di linfociti T CD4+ naive \ue8 dissociata, e dipendente dalla combinazione di fattori presenti nell\u2019ambiente cellulare. Ancor pi\uf9, ci ha permesso di scoprire che IL-6 e IL-23, comunemente descritte nel topo quali induttori dell\u2019espressione di IL-22, esercitano entrambe ruoli opposti, attivatorio ed inibitorio, sull\u2019espressione di IL-22, in dipendenza del contesto di fattori nel quale agiscono.IL-17, IL-22 and IFN-\u3b3 are cytokines produced mostly by T helper (Th) cells playing different and essential roles in the defence against a wide variety of pathogens, but also responsible for tissue damages leading to several autoimmune diseases. Th cells producing IL-17 (Th17) and Th cells producing IFN-\u3b3 (Th1) belong to distinct subsets; however, Th17 cells producing IFN-\u3b3 are frequently found both in vivo and in vitro, leaving open the debate about the relative roles of Th17 and Th1 in autoimmune diseases. IL-22 is a cytokine playing pivotal roles in the repair and defence of epithelial, in the host defence against infections of extracellular pathogens and a protective role on hepatocytes damage during acute hepatitis. IL-22 is principally produced by Th17 cells, but a population of Th cells producing IL-22 in the absence of IL-17 and of IFN-\u3b3 has been described. To contribute to clarify the relationship among IL-17, IL-22 and IFN-\u3b3 and to identify factors relevant in induction of expression of these cytokines, we evaluated the pattern of expression of IL-17, IL-22 and IFN-\u3b3 in naive CD4+ T lymphocytes activated with anti-CD3 plus anti-CD28. Since the main factor determining the polarization of these cells is represented by cytokines produced by pathogen-activated APCs, we stimulated naive CD4+ T lymphocytes in the presence of a supernatant derived from IFN-\u3b3-primed dendritic cells stimulated with zymosan (SNDCzym), which we previously demonstrated to efficiently induce IL-17 expression, or with a mixture of the cytokines IL-1\u3b2+IL-6+IL-23, analogously known for their capacity to polarize a Th17 response. Moreover, we used the SNDCzym with various combinations of antibodies neutralizing the cytokines IL-6, IL-12, IL-23 or with IL-1 receptor antagonist, inhibiting IL-1\u3b1 and IL-1\u3b2, or with different mixtures of recombinant IL-1\u3b2, IL-6 and IL-23. The comparison of the results obtained from these two systems allowed us to clarify that the expression of IL-17, IL-22 and IFN-\u3b3 during naive CD4+ T lymphocyte differentiation is dissociated, depending on the combination of factors present in the cell environment. More relevantly, it allowed us to uncover that IL-6 and IL-23, commonly described in the mouse as inducers of IL-22 expression, both exert opposed roles, activatory or inhibitory, in IL-22 expression, depending on the context of factors in which they act

Two new highly polymorphic markers in the 3\u2019UTR region of the PLA2G7 gene

We described two new highly polymorphic markers located 31 bp downstream of the last nucleotide of exon 12 in the 3' UTR region of the gene PLA2G7: 1344 +31TG(n) AG(m). Eight and 14 alleles were observed for the AG and TG repeats, respectively. These two markers have the highest heterozygosity until now reported for PLA2G7 gene

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 increasin

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

Regulation of IL-8 gene expression in gliomas by microRNAs

Interleukin-8 (IL-8, or CXCL8) is a major promoter of angiogenesis and invasiveness in human gliomas, where it is expressed and secreted at high levels. Among the different control levels of IL-8 gene expression in gliomagenesis, several have been studied and well characterized, such as hypoxia/anoxia stimulation, response to Fas ligation, death receptor activation, cytosolic Ca2+ transients, TNF-alpha, IL-1, and other cytokines and various cellular stresses. In addition, the expression of the IL-8 gene might be under the control of epigenetic mechanisms(s), such as those regulated by microRNAs. We found that bacterial challenge, which is known to strongly activate IL-8 gene transcription in epithelial cells, is downregulated by miR-93 (Fabbri, 2014). This is of peculiar interest for cancerogenesis since miR-93 has been found involved in the downregulation of expression of VEGF which cooperates, together with IL-8, in glioma angiogenesis. Expression levels of IL-8 and VEGF genes and of miR-93, have been investigated in High Grade and Low Grade Gliomas (HGG and LGG) samples and in U251 human glioma cells. Pre-miR- and anti-miR-93 were transfected in U251 cells to check modulation of candidate target genes (IL-8 and other cytokines relevant to the glioma microenvironment). Both VEGF and IL-8 mRNAs were higly expressed in LGGs (20-200 folds) and HGGs (20-300 folds) in respect to reference RNA from healthy brains. VEGF and IL-8 mRNAs expression correlated directly, whereas MiR-93 correlated inversely with both target genes transcripts in glioma specimens. In silico analyses evidenced consensus sequences for the interaction of miR-93 in the 3'-UTR regions of IL-8 and VEGF genes. Transfection of U251 glioma cells with pre-miR-93 down-modulated both VEGF and IL-8 genes expression, whereas anti-miR-93 resulted in a consistent up-modulation. Our results suggest that microRNAs, including miR-93, might be proposed as relevant post-transcriptional regulators of angiogenesis in human gliomas

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

International audienceThe 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-β3 (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 β, 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 Cystic Fibrosis (CF) patients. The nociceptive Transient Receptor Potential Ankyrin 1 (TRPA1) calcium channels have been recently found involved in non-neurogenic inflammation. Here, we investigated 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 by immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o-) and primary cells from CF patients were utilized to a) check TRPA1 function modulation, by Fura-2 calcium imaging, b) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and siRNA silencing, and c) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to pro-inflammatory challenges, by qRT-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 co-expressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the pro-inflammatory cytokines IL-1\u3b2 and TNF-\u3b1, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of CF patients. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells