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. 2022 Feb 1;107(2):558-561.
doi: 10.3324/haematol.2021.279864.

Interferon α-induced SAMHD1 regulates human cultured megakaryocyte apoptosis and proplatelet formation

Seema Bhatlekar  1 , Shancy Jacob  1 , Emilia Tugolukova  1 , Bhanu K Manne  2 , Yasuhiro Kosaka  1 , Phillipe Loher  3 , Ryan M O'Connell  4 , Vicente Planelles  5 , Matthew T Rondina  6 , Isidore Rigoutsos  7 , Paul F Bray  8
Affiliations

Interferon α-induced SAMHD1 regulates human cultured megakaryocyte apoptosis and proplatelet formation

Seema Bhatlekar et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Interferona regulates platelet production. (A to E) CD34+ hematopoietic stem cells and progenitor cells were isolated by immunomagnetic separation from human umbilical cord blood. Cells were cultured for 13 days in stem cells expansion media supplemented with thrombopoietin that promotes megakaryocyte (MK) differentiation. 1,000 units/milliliter (U/mL) of human interferon α (INFα) and phosphate-buffered saline (PBS) (used as a negative control) was added at day 9 and further incubated until day 13. All assays mentioned in panels A to E were performed at day 13. (A) MK proplatelet formation (PPF) was counted blinded as to the IFNa treatment. At least 200 cells were counted per culture (n=4). (B) Representative images of day 13 cultured MK treated with PBS or IFNa. Treated MK were plated on fibrinogen on day 12 overnight, and fixed with 4% paraformaldehyde, stained with Alexa Fluor 488 Phalloidin (green) and a nuclear stain, DAPI (blue). Images were taken by a confocal microscope at 40X oil objective lens. (C) Platelet-like particles (PLP) were collected from IFNa or PBS-treated MK cultures and stained with APC labeled anti-CD41a antibody at 37°C for 10 minute and measured by flow cytometry (n=3). PLP were gated based on human peripheral blood platelets. (D) MK were stained with APC-labeled CD41a and PE-labeled CD42a antibodies, and CD41a+ CD42a+ MK (a marker for MK maturation) were assessed by flow cytometry (n=3). (E) Cultured MK treated with IFNa or PBS were stained with APC-labeled CD41a and propidium iodide, and ploidy was assessed by flow cytometry (n=3). The quantification of the ploidy distribution is shown on the y-axis by calculating the percentage of cells with 2n, 4n, 8n and 16n. Apoptotic population were gated out. Statistical significance was determined by two-tailed paired t-test (A to E). Error bars represent mean ± standard error of mean. (F) 25,000 units of murine IFNa or PBS (negative control) were administered intraperitoneally in wild-type mice for consecutive three days (n=5 per group). On day 4, mice blood was harvested by cardiac puncture and platelet count was measured by Hemavet. Statistical significance was determined by two-tailed unpaired t-test with Welch’s correction. Error bars represent mean ± standard error of mean.
Figure 2.
Figure 2.
Interferon α upregulates. dNTP hydrolase enzyme SAMHD1 in cultured megakaryocytes. CD34+ hematopoietic stem cells and progenitor cells were isolated by immunomagnetic separation from human umbilical cord blood. Cells were cultured in megakaryocyte (MK) promoting conditions by supplementation with thrombopoietin for 13 days. 1,000 units per milliliter (U/mL) of interferon α (INFα) and phosphate-buffered saline (PBS) (negative control) was added in 3 independent cultures at day 13 for 8 hours. (Dose and time course studies were performed in Meg-01 to select optimal concentrations and times, data not shown). CD61+ MK were separated by immunomagnetic beads and total RNA was obtained by mirVana kit. RNA samples with RIN score of >8 were used for RNA sequencing. The library preparation was done using TruSeq standard total RNA sample preparation kit with RNA depletion (Illumina Inc.). RNA sequensing was performed using NextSeq500 instrument with read length of 50 basepair single-end read. On average 68.9 million reads were obtained from each sample and genome mapping was on average 94.5% for all samples. (A) Volcano plot shows the relationship between the P-values and the fold change in normalized expression for IFNa or PBS treated cultured human MK. Differentially expressed genes (false discovery rate [FDR] <0.05) on IFNa treatment was plotted. SAMHD1 is shown in red. (B) Real-time polymerase chain reaction analysis validates the upregulation of SAMHD1 in IFNa-treated CD61+ MK compared to PBS control. Actin was used as housekeeping gene. Log fold change of SAMHD1 mRNA levels is plotted for IFNa vs. PBS control (n=3). Statistical significance was determined by one sample t-test. (C) Representative immunoblot shows increase in SAMHD1 protein in IFNa-treated MK compared to PBS control. Actin is used as loading control.
Figure 3.
Figure 3.
Interferon α-induced SAMHD1 expression regulates human megakaryocyte (MK) proplatelet formation and MK apoptosis. (A) Plot of Pearson correlation (R) between platelet count and SAMHD1 mRNA levels in 154 healthy donors. Dotted lines represent 95% confidence intervals. (B to D) CRISPR/Cas9 knock-down of SAMHD1 in CD34+ derived human umbilical cord cells were performed at day 3. Cells were cultured in MK differentiation promoting conditions, and treated with 1,000 U/mL interferon α (INFα) and phosphate-buffered saline (PBS) control. On day 13, MK treated with IFNa or PBS were assayed as mentioned below. (B) Representative immunoblot of SAMHD1 after CRISPR-Cas9 knock-down (denoted as “SAMHD1 cr” throughout the figure) in day 13 human CD34+ derived cultured MK with or without IFNa. Guide RNA not targeting known genes are used as negative control with or without IFNa (“neg cr” throughout the figure). Fold changes of densitometric quantification of SAMHD1 immunoblots, normalized to actin is shown on right (n=5). (C) The percentage of proplatelet formation (PPF) MK, scored blinded, for SAMHD1 cr vs neg cr, with or without IFNa is shown (n=6). (D) The mean fluorescence intensity (MFI) of annexin V binding (a marker of apoptosis-induced phosphatidylserine expression) on IFNa stimulation in SAMHD1 cr vs. neg cr MK, with or without IFNa is plotted (n=5). Statistical significance was determined by paired t-test (B to D). Error bars represent mean ± standard error of mean.
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