Figure 2
Figure 2. The CCL5/CCR5 axis enhances megakaryocyte maturation and proplatelet formation through apoptosis suppression. (A) Recombinant CCL5 (5, 50, or 500 ng/mL) or vehicle control was added to MK cultures on day 4 of maturation. Proplatelet production from MKs was manually quantified after 6 hours as described in Figure 1 and previously.14-16 n = 6; *P < .05, **P < .01, with statistical analysis done by Student t test compared with vehicle control. (B) Maraviroc (10 or 100 nM) was added to indicated cultures and allowed to incubate for 30 minutes. 50 ng/mL CCL5 or vehicle control was then added to MK cultures on day 4 of maturation. Proplatelet production from MKs was manually quantified after 6 hours as described elsewhere.14-16 n = 3; **P < .01, with statistical analysis done by Student t test compared with vehicle control. (C) Representative images of proplatelet production in MKs treated with CCL5 ± maraviroc (MIR). Images were obtained with a Zeiss Axiovert 200 (Carl Zeiss) equipped with a 20× objective, and images obtained and analyzed using Metamorph software (Molecular Devices) and ImageJ. Scale bars represent 20 μm. (D) On day 1 of MK maturation, maraviroc (100 nM) was added to indicated cultures and allowed to incubate for 30 minutes. 50 ng/mL CCL5 or vehicle control was then added to MK cultures. On day 4, MK ploidy was determined using a fluorescence-activated cell sorter (30 000 events per sample) by gating on fluorescence intensity based on DNA binding via propidium iodide (Sigma Aldrich). Statistical analysis was done using 2-way ANOVA with α = 0.05, comparisons made between vehicle control and various treatment groups, as indicated. n = 6; *P < .05, **P < .01, ***P < .005, and ○P < .0001, with data plotted as mean and standard error of the mean and statistical analysis done by 2-way ANOVA with Dunnett’s multiple comparisons test. (E) To interrogate the Akt signaling pathway, maraviroc (100 nM) was added to indicated MK cultures on day 4 of maturation and allowed to incubate for 30 minutes prior to the additional of 50 ng/mL CCL5 or vehicle control. Lysates were generated 15 minutes after the addition of CCL5 and analyzed using the PathScan Akt Signaling Antibody Array (Cell Signaling Technology), which detects phosphorylation levels of 18 proteins in the Akt signaling pathway. The array was performed according to the manufactures instructions, imaged using a G:Box Imaging System (Syngene) and analyzed with ImageJ software. Data were normalized to untreated day 4 MK lysate, and fold changes of 1.5 or greater are shown. (F) Mice were exposed to DSS (5% wt/vol in drinking water) to induce colitis or given untreated drinking water and treated with maraviroc (10mg/kg intraperitoneally, daily) or saline vehicle. After 7 days, mice were euthanized and blood was collected to determine platelet and WBC count (HemaVet, Drew Scientific). Platelet count was correlated with WBC (a marker of inflammation) in each of the four treatment groups using GraphPad Prism 6 software. Pearson’s correlation, P = .018. (G) Proposed model of CCL5/CCR5-induced thrombocytosis. In a state of physiological stress, platelet activation by agonists leads to release of platelet CCL5. CCL5 binds to MK CCR5, causing increased MK maturation and proplatelet formation and a subsequent increase in circulating platelet levels. Thus, a positive feedback loop is established.

The CCL5/CCR5 axis enhances megakaryocyte maturation and proplatelet formation through apoptosis suppression. (A) Recombinant CCL5 (5, 50, or 500 ng/mL) or vehicle control was added to MK cultures on day 4 of maturation. Proplatelet production from MKs was manually quantified after 6 hours as described in Figure 1 and previously.14-16  n = 6; *P < .05, **P < .01, with statistical analysis done by Student t test compared with vehicle control. (B) Maraviroc (10 or 100 nM) was added to indicated cultures and allowed to incubate for 30 minutes. 50 ng/mL CCL5 or vehicle control was then added to MK cultures on day 4 of maturation. Proplatelet production from MKs was manually quantified after 6 hours as described elsewhere.14-16  n = 3; **P < .01, with statistical analysis done by Student t test compared with vehicle control. (C) Representative images of proplatelet production in MKs treated with CCL5 ± maraviroc (MIR). Images were obtained with a Zeiss Axiovert 200 (Carl Zeiss) equipped with a 20× objective, and images obtained and analyzed using Metamorph software (Molecular Devices) and ImageJ. Scale bars represent 20 μm. (D) On day 1 of MK maturation, maraviroc (100 nM) was added to indicated cultures and allowed to incubate for 30 minutes. 50 ng/mL CCL5 or vehicle control was then added to MK cultures. On day 4, MK ploidy was determined using a fluorescence-activated cell sorter (30 000 events per sample) by gating on fluorescence intensity based on DNA binding via propidium iodide (Sigma Aldrich). Statistical analysis was done using 2-way ANOVA with α = 0.05, comparisons made between vehicle control and various treatment groups, as indicated. n = 6; *P < .05, **P < .01, ***P < .005, and ○P < .0001, with data plotted as mean and standard error of the mean and statistical analysis done by 2-way ANOVA with Dunnett’s multiple comparisons test. (E) To interrogate the Akt signaling pathway, maraviroc (100 nM) was added to indicated MK cultures on day 4 of maturation and allowed to incubate for 30 minutes prior to the additional of 50 ng/mL CCL5 or vehicle control. Lysates were generated 15 minutes after the addition of CCL5 and analyzed using the PathScan Akt Signaling Antibody Array (Cell Signaling Technology), which detects phosphorylation levels of 18 proteins in the Akt signaling pathway. The array was performed according to the manufactures instructions, imaged using a G:Box Imaging System (Syngene) and analyzed with ImageJ software. Data were normalized to untreated day 4 MK lysate, and fold changes of 1.5 or greater are shown. (F) Mice were exposed to DSS (5% wt/vol in drinking water) to induce colitis or given untreated drinking water and treated with maraviroc (10mg/kg intraperitoneally, daily) or saline vehicle. After 7 days, mice were euthanized and blood was collected to determine platelet and WBC count (HemaVet, Drew Scientific). Platelet count was correlated with WBC (a marker of inflammation) in each of the four treatment groups using GraphPad Prism 6 software. Pearson’s correlation, P = .018. (G) Proposed model of CCL5/CCR5-induced thrombocytosis. In a state of physiological stress, platelet activation by agonists leads to release of platelet CCL5. CCL5 binds to MK CCR5, causing increased MK maturation and proplatelet formation and a subsequent increase in circulating platelet levels. Thus, a positive feedback loop is established.

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