Loss of Bid does not alter the cytokine and chemokine milieu of the joint

<p><b>Copyright information:</b></p><p>Taken from "Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis"</p><p>http://arthritis-research.com/content/9/3/R49</p><p>Arthritis Research & Therapy 2007;9(3):R49-R49.</p><p>Published online 17 May 2007</p><p>PMCID:PMC2206343.</p><p></p> Pro-inflammatory cytokine production in ankle joints following transfer of K/BxN serum. Untreated wild-type (Wt) and Bid-/- mice were euthanized at three, five, or seven days post-serum transfer. Ankles from each mouse (days 3, = 6 (Wt) and n= 8 (Bid-/-); day 5, = 10; day 7, = 12 (Wt) and = 8 (Bid-/-)) were isolated, snap frozen, ground into a fine powder, lysed, and examined for production of tumor necrosis factor (TNF)α and IL-1β using sandwich ELISAs. Chemokine production in ankle joints following transfer of K/BxN serum. Ankles lysates as described above were examined for production of CXC chemokine (KC) and monocyte chemoattractant protein (MCP)-1 using ELISA. Data are shown as μg/μl per joint. Values represent the mean ± standard error, which were compared by Student's -test

Histological scores of ankle sections from wild-type (Wt) and Bid-/- mice

<p><b>Copyright information:</b></p><p>Taken from "Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis"</p><p>http://arthritis-research.com/content/9/3/R49</p><p>Arthritis Research & Therapy 2007;9(3):R49-R49.</p><p>Published online 17 May 2007</p><p>PMCID:PMC2206343.</p><p></p> Bid-/- mice have increased inflammation and joint destruction compared to Wt mice. Ankles isolated from mice (Wt, = 9; Bid-/- = 7) were prepared as described in Figure 2. Ankle sections were evaluated and scored by a pathologist blinded to the study as described in the Materials and methods section. Values represent the mean ± standard error of ankles/time point, which were compared by Student's -test. Increased numbers of lymphocytes and polymorphonuclear (PMNs) cells in inflamed Bid-/- joints. Ankles were prepared as described above. Values represent the mean ± standard error of ankles/time point, which were compared by Student's -test. Arthritic Bid-/- mice have more macrophages in the pannus and in the whole joint. Ankles were examined for F4/80 antigen as described in Materials and methods. The number of positive cells for F4/80 in pannus, synovial lining, and whole joint was determined by a pathologist blinded to the study. Values represent the mean ± standard error of ankles/time point, which were compared by Student's -test

Effect of phosphate on intracellular calcium.

<p>The changes of fluorescence intensity was recorded in endothelial cells treated with 0.5 mM phosphate (A), 1 mM phosphate (B) and PFA +3 mM phosphate (C). Percentage changes in intracellular fluorescent intensity is illustrated in D. N = 5 per condition.</p

Effect of pan-caspase inhibitor z-VAD in preventing apoptosis caused by simulated hypophosphatemia and hyperphosphatemia.

<p>To further define the role of inorganic phosphate on human umbilical vein endothelial cell apoptosis, a pan-caspase inhibitor z-VAD (40 uM) was added to the medium containing various concentrations of phosphate. Incubation with pan-caspase inhibitor z-VAD prevented apoptosis induced by both simulated hyperphosphatemia and hypophosphatemia (Figure 2 A–F). B: hypophosphatemia (0.5 mM); C: hypophosphatemia (0.5 mM) plus z-VAD; D: phosphate (1 mM); E: hyperphosphatemia (3 mM phosphate); F: hyperphosphatemia (3 mM) plus z-VAD. In Figure B–F, data in each quadrant are the percentage of total cells, and shown as mean +/− SEM. p values vs control (1 mM phosphate). N = 6∼9.</p

Effect of inorganic phosphate on endothelial cell proliferation and apoptosis.

<p>We examined the effect of exposure to different concentrations of inorganic phosphate for 24 hours on human umbilical vein endothelial cell proliferation and apoptosis. In Figure 1A, cell number is determined by light microscopy counting of trypan blue-excluding cells, whereas B–D pertain to flow analysis. Incubation in media simulating hypophosphatemia (0.5 mM) or hyperphosphatemia (3 mM phosphate) resulted in a significant reduction of cell numbers (A), decreased cell viability (B) and increased cell dearth (C, statistically significant only seen in hyperphosphatemia) when compared to cells incubated in the medium containing 1 mM phosphate. Similarly, incubation in media simulating hypophosphatemia (0.5 mM, D & E) or hyperphosphatemia (3 mM phosphate, D & G) resulted in a significant increase in apoptotic cells when compared to cells incubated in the medium containing 1 mM phosphate (D & F). In Figure E–G, data in each quadrant are the percentage of total cells, and shown as mean +/− SEM. p values vs control (1 mM phosphate). N = 6∼9.</p

Effect of Akt pathway on endothelial function.

<p>We used PI3K/Akt inhibitor, Ly492002, to explore the role of altered Akt in the endothelial dysfunction induced by simulated hypophosphatemia. Ly492002 had no effect on increased apoptosis induced by low phosphate levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023268#pone-0023268-g005" target="_blank">Figure 5A–E</a>). LY492002 resulted in decreased cell viability in hypophosphatemia. It led to decreased cell viability and increased cell death in physiologic phosphate levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023268#pone-0023268-g005" target="_blank">Figure 5 F & G</a>). On the other hand, Ly492002 was able to block the reduction of eNOS expression induced by simulated hypophospatemia but had no effect on eNOS expression in cells exposed to physiologic phosphate levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023268#pone-0023268-g005" target="_blank">Figure 5H</a>). In Figure A–D, data in each quadrant are the percentage of total cells, and shown as mean +/− SEM. A: 0.5 mM phosphate; B: 1 mM phosphatre; C: 0.5 mM phosphate+Ly492002; D: 1.0 mM phosphate+Ly492002.</p

Effect of inorganic phosphate on eNOS expression and NO production

<p>. We examined the effect of exposure to different concentrations of inorganic phosphate for 24 hours on NO production and eNOS expression. Incubation in media simulating hypophosphatemia (0.5 mM) or hyperphosphatemia (3 mM phosphate) resulted in a significant reduction in eNOS expression (A) and NO production (B). The down-regulation of eNOS following exposure to simulated hyperphosphatemia (3 mM phosphate) was reversed with co-administration of 1 mM PFA, which is a specific inhibitor of phosphate transport across the cell membrane (C). To determine whether abnormal phosphate levels affected the ability of acetylcholine (a physiological activator of eNOS) to stimulate NO production, a set of cells was incubated with 2 µM acetylcholine and the supernatant NO levels before (0 minute) and 5 minute and 15 minute post acetylcholine stimulation was measured. As shown in (D), incubation in medium with 1 mM phosphate resulted in a significant NO production 5 minute post acetylcholine stimulation and remained elevated at 15 minutes. In contrast, incubation in media simulating both hypophosphatemia (0.5 mM) and hyperphosphatemia (3 mM phosphate) completely prevented acetylcholine-induced stimulation of NO generation, pointing to endothelial dysfunction. p values vs control (1 mM phosphate). N = 6∼10.</p

Additional file 1: Table S1. of Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner

Affymetrix QuantiGene 2.0 custom panel 21522 for analysis of macrophage polarization. Figure S1. Genotype validation of splenocyte populations. Figure S2. Genotype validation of BMDMs. Figure S3. TLR9 in vivo activation induces upregulation of serum cytokines and chemokines at similar levels in Casp8 fl/fl and Cre LysM Casp8 fl/fl mice. Figure S4. Caspase-8–deficient splenic myeloid populations are not predisposed to aberrant death. Figure S5. Caspase-8–deficient BMDMs express Fas. Figure S6. Caspase-8–deficient BMDMs undergo caspase-independent cell death in response to apoptotic stimuli. Figure S7. Caspase-8 deficiency in macrophages alters the response to TLR activation in vitro. Figure S8. Caspase-8 deficiency in macrophages alters the genetic profile in response to macrophage polarization in vitro. (PDF 5409 kb

Additional file 2: Table S2. of Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner

ANOVA comparison data from Affymetrix QuantiGene 2.0 custom panel 21522 for analysis of macrophage polarization. (XLS 68 kb