Additional file 6: of Effect of a rare genetic variant of TM7SF4 gene on osteoclasts of patients with Paget’s disease of bone

Raw data of Western Blot analyses. (DOC 30 kb

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

14 avril 19161916/04/14 (A17,N5562)

Signalling pathways induced by specific proteinase-activated receptor 2 (PAR-2) activation

<p><b>Copyright information:</b></p><p>Taken from "Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study"</p><p>http://arthritis-research.com/content/9/6/R121</p><p>Arthritis Research & Therapy 2007;9(6):R121-R121.</p><p>Published online 21 Nov 2007</p><p>PMCID:PMC2246240.</p><p></p> Representative Western blot of time and dose curves of PAR-2 signalling in osteoarthritic chondrocytes (= 3 or 4). Phosphorylated forms of Erk1/2 (p-Erk1/2) , p38 (p-p38) , and JNK (p-JNK) treated with PAR-2-activating peptide (PAR-2-AP) at 0 (-), 10, 100, and 400 μM in the absence or presence of interleukin 1 beta (IL-1β) (100 pg/mL) for 0 to 60 minutes . values indicate the comparison between the untreated (-) and the PAR-2-AP-treated chondrocytes. For p-Erk1/2, all of the times and concentrations showed a statistically significant increase (< 0.03). For p-p38, statistical significance was reached for each concentration at 5 and 15 minutes (< 0.03). values indicate the comparison between the untreated and the IL-1β-treated chondrocytes in the absence or presence of PAR-2-AP. Erk1/2, extracellular signal-regulated kinase 1/2; JNK, c-jun N-terminal kinase

Production of metalloproteinase (MMP)-1 , MMP-13 , and cyclooxygenase 2 (COX-2) following interleukin 1 beta (IL-1β) and specific proteinase-activated receptor 2 (PAR-2) activation

<p><b>Copyright information:</b></p><p>Taken from "Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study"</p><p>http://arthritis-research.com/content/9/6/R121</p><p>Arthritis Research & Therapy 2007;9(6):R121-R121.</p><p>Published online 21 Nov 2007</p><p>PMCID:PMC2246240.</p><p></p> Immunostaining data of osteoarthritic cartilage untreated (= 12) and treated with IL-1β (= 12), PAR-2-activating peptide (PAR-2-AP) 1 μM (= 3), PAR-2-AP 10 μM (= 4), PAR-2-AP 100 μM (= 4), and PAR-2-AP 400 μM (= 9). values indicate the comparison with the untreated (CTL) specimens

Proteinase-activated receptor 2 (PAR-2) gene expression and protein synthesis

<p><b>Copyright information:</b></p><p>Taken from "Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study"</p><p>http://arthritis-research.com/content/9/6/R121</p><p>Arthritis Research & Therapy 2007;9(6):R121-R121.</p><p>Published online 21 Nov 2007</p><p>PMCID:PMC2246240.</p><p></p> mRNA levels, as determined by real-time quantitative polymerase chain reaction as described in Materials and methods, in normal (= 4) and osteoarthritis (= 6) chondrocytes. PAR-2 immunostaining in normal (= 4) and osteoarthritis (= 4) cartilage. The percentage of positive chondrocytes represents the number of chondrocytes staining positive for PAR-2 of the total number of chondrocytes. Data are expressed as median and range and are presented as box plots, in which the boxes represent the first and third quartiles, the line within the box represents the median, and the lines outside the box represent the spread of values. values indicate the comparison of normal to osteoarthritis cartilage using the Mann-Whitney test. Representative sections showing PAR-2 immunostaining in normal and osteoarthritis cartilage. The arrows refer to positive chondrocytes

Pyrin is not required for the development of EAE induced by adoptive transfer of encephalitogenic T cells.

<p>Monitoring of EAE in C57BL/6 mice expressing (black circles) or lacking pyrin (white squares) after adoptive transfer of lymphocytes isolated from MOG-immunized wild-type mice and reactivated <i>in vitro</i>. All mice were included in the analyses, except for the clinical scoring (bottom graph), which included only mice that had developed clinical signs of EAE at the end of the study (i.e., after 21 days). No significant intergenotype difference was detected according to Wilcoxon tests (<i>P</i>≥0.2). Additional statistics are provided in the table. Sample size: 8 (Pyrin^+/+) or 7 (Pyrin^−/−).</p

PTX increases pyrin expression in myeloid cells via a TLR4-dependent pathway.

<p><b><i>a</i></b>, Quantification of pyrin mRNA by qRT-PCR in peritoneal leukocytes from TLR4-knockout and wild-type mice harvested 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from all the other groups according to Wilcoxon tests (<i>P</i>≤0.014). Sample size: 8 (PTX groups) or 4 (PBS groups). <b><i>b</i></b>, Bivariate analysis showing a positive correlation between the amounts of pyrin and IL-1β mRNAs (Spearman's test, <i>P</i><0.0001, R = 0.98). <b><i>c</i></b>, Quantification of pyrin mRNA by qRT-PCR in peritoneal leukocytes harvested from mice 6 h after injection of PTX or PBS, and sorted using the CD11b, F4/80 and Ly6G markers. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.0081). Sample size: 6–7 per group. <b><i>d</i></b>, Pyrin detection by Western blotting in peritoneal cells from mice killed 6 h after injection of PTX or PBS. β-actin was used as loading control.</p

Both IL-6 and IL-1β are produced in tissues exposed to PTX, but only IL-6 reaches increased levels in the circulation.

<p><b><i>a</i></b>, Quantification of the mRNAs encoding IL-6 and IL-1β by qRT-PCR in different tissues from mice killed 3 or 6 h after intraperitoneal injection of PTX (20 µg/kg) or PBS. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i><0.05). Sample size: 4–10 (PBS groups) or 7–10 (PTX groups). <b><i>b</i></b>, Quantification of IL-6 and IL-1β by ELISA in peritoneal fluid and plasma samples. *Significantly different from the corresponding PBS group according to the Wilcoxon test (<i>P</i>≤0.0003). Sample size: 3–9 (PBS groups) or 4–10 (PTX groups).</p

The Inflammasome Pyrin Contributes to Pertussis Toxin-Induced IL-1β Synthesis, Neutrophil Intravascular Crawling and Autoimmune Encephalomyelitis

<div><p>Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.</p></div

PTX requires pyrin to induce neutrophil intravascular crawling and to maximally promote EAE.

<p><b><i>a</i></b>, Micrographs showing rod-shaped leukocytes stained for Ly6G or CD3 by immunohistochemistry in brain sections from a PTX-treated mouse (C57BL/6 background). Scale bar: 5 µm. <b><i>b</i></b>, Stereological counts of rod-shaped Ly6G⁺ and CD3⁺ leukocytes in the cerebral cortex of C57BL/6 mice deficient (white bars) or not (black bars) in pyrin and killed 24 h after treatment with PTX (2 injections of 20 µg/kg given 2 days apart) or PBS. *Significantly different from the corresponding pyrin-deficient group according to the Wilcoxon test (<i>P</i> = 0.0050). Sample size: 6–7 (PBS groups) or 7–10 (PTX groups). <b><i>c</i></b>, Monitoring of EAE in PTX-treated 2D2 mice expressing (black circles) or lacking pyrin (white squares). All mice were included in the analyses, except for the clinical scoring (right graph), which included only mice that had developed clinical signs of EAE at the end of the study (i.e., after 21 days). *Time at which a significant intergenotype difference was found according to Wilcoxon tests (<i>P</i>≤0.044). The Kaplan-Meier curves (EAE incidence) were significantly different according to the Wilcoxon test (<i>P</i> = 0.013). Additional statistics are provided in the table. Sample size: 12 (2D2 Pyrin^+/+) or 19 (2D2 Pyrin^−/−). <b><i>d</i></b>, Micrographs of spinal cord sections showing comparable infiltration of CD3⁺ T cells in 2D2 mice expressing or not pyrin with an EAE score of 3, but few cells in a mouse that did not develop EAE. These mice were killed 21 days after the first PTX injection. Scale bar: 100 µm.</p