Background
Serious myelosuppression is one of the fatal diseases manifested as a suppression on all types of hematopoietic cells and remains as one of the most difficult diseases to treat to date. Myelosuppression is either primary including aplastic anemia (AA) and myelodysplastic syndrome (MDS) or secondary to various disease status including chemotherapy and accidental/therapeutic radiation exposure [
1‐
3]. Among the severe complications of myelosuppression, thrombocytopenia is immediately life threatening. Platelet transfusion is currently applied to treat severe thrombocytopenia; however, it has various side effects being common with the transfusion of blood components, and the endogenous generation of platelet is not ameliorated with such therapy [
4,
5]. A more etiological treatment is the administration of recombinant thrombopoietin (TPO) which exerts significant effects in promoting platelet generation; however, it also has some side effects such as induction of autoantibodies due to the cross-reactions with endogenous TPO [
4,
6,
7]. Since the effects of TPO receptor agonists are mild [
6,
8,
9], exploration for new approaches to treat severe myelosuppression has potential translational values.
TPO has been established as a main cytokine to promote the generation of megakaryocytes and platelets by acting on its receptor, c-mpl [
10‐
12]. The most established role of TPO is to promote the generation and differentiation of the hematopoietic stem cells in the bone marrow by acting on the TPO receptors, particularly the megakaryocytic stem/progenitor cells differentiated to megakaryocytes [
13‐
15]. The TPO receptors are also found in the leukocytic and erythrocytic hematopoietic stem cells to be involved in the generation of leukocytes and erythrocytes in adulthood bone marrow [
14,
16,
17].
Hydrogen sulfide (H
2S) is endogenously generated in mammals via the H
2S-generating enzymes cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [
18‐
20]. H
2S was considered as a metabolite without any physiological roles at the very start. The first piece of evidence reflecting a physiological role of H
2S was found in organ bath experiments where an H
2S donor, sodium hydrosulfide (NaHS), caused relaxation of the isolated vessels [
21]. In the following years, H
2S has been found to regulate the function of the cardiovascular system [
22‐
24], the nervous system [
25‐
27], the endocrine system [
28,
29], and the immune system [
30‐
32], among which the cardiovascular role of H
2S is the most well established, including a regulation of vascular tone [
33,
34] and remodeling [
35‐
37], a protection against ischemia/reperfusion injury of the cardiomyocytes and myocardium [
38,
39], regulation of ion channels [
22,
40], and promotion of angiogenesis [
41‐
43]. To date, any potential role of H
2S in hematopoiesis is unknown.
Here, we report a novel protective effect of the gas molecule, H
2S, against fatal myelosuppression being dependent on c-mpl. This is an unexpected finding in our studies investigating the role of endothelial progenitor cells in the proangiogenic effects of H
2S which we found previously [
41,
44,
45]. Though our data showed that the endothelial progenitor cells were not involved in these H
2S effects, these pilot experiments revealed a novel promoting effect of H
2S on hematopoiesis. This prompts us to propose a hypothesis that H
2S may ameliorate myelosuppression.
In the present study, an H2S donor (NaHS) was applied to treat fatal myelosuppression in a mice model exposed to radiation and this is the first effort to examine the role of the gas transmitter H2S on hematopoiesis.
Discussion
This is the first time that the gas transmitter H2S has been found to protect against fatal myelosuppression with improvement in bone marrow morphology, circulating platelet levels, and final survival. The increased survival in H2S-treated mice may be ascribed to the decreased internal bleeding. This idea was supported by less bleeding time and fecal occult blood found in these H2S-treated mice. Moreover, these effects were found at physiologically relevant dosages of H2S since the treatment just recovered the decrease in plasma H2S levels in radiation-exposed mice. Therefore, these findings have potential translational values and may be further explored for new approaches to treat fatal diseases resulting from severe myelosuppression.
In the embryonic period, hematopoiesis mainly occurs in the liver where the hematopoietic stem cells were differentiated into various types of blood cells [
50‐
52]. Here, we show a novel protective role of H
2S in fatal myelosuppression, i.e., H
2S ameliorates internal bleeding and improves survival by promoting generation of megakaryocytes and platelets. Future works are required to further validate this hypothesis using multiple approaches including bone marrow transplantation experiments.
We found here that the hematopoietic effects of H
2S were blunted in the c-mpl
−/− mice exposed to radiation and also in the fetal liver cells isolated from the c-mpl
−/− mice. These data suggest that the TPO receptors are essential for H
2S to promote hematopoiesis. In the adult stage, the TPO receptors are expressed on different maturation stages of megakaryocyte and early hematopoietic stem/progenitor cells in the bone marrow [
53‐
55]. The predominant role of the TPO receptors is to mediate the signals to induce proliferation and differentiation of megakaryocyte progenitor cells to generate megakaryocytes and platelets [
10‐
12]. In addition, since the TPO receptors are also expressed on the hematopoietic stem/progenitor cells, it is also involved in the expansion of erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells [
14,
16,
17]. Although there are also some evidence showing expression of the TPO receptors in the tissues outside of the bone marrow, e.g., in placenta, fetal liver, fetal blood, cord blood, and peripheral blood, bone marrow is the main place of hematopoiesis in adulthood [
51,
52,
56]. In this context, if the hematopoietic effect of H
2S is dependent on the TPO receptors, these H
2S effects are mediated via the TPO receptors located in the hematopoietic stem/progenitor cells in the bone marrow. Indeed, the absence of more direct evidence using bone marrow transplantation models is a limitation of our current study.
The mechanisms about how H
2S would activate the TPO receptors are unknown. How a molecule as small as H
2S targets its “receptors” is one of the most challenging questions in the field. Our previous studies aim to find that the “receptor” of H
2S shows that VEGFR2 serves as a direct target molecule for H
2S to induce angiogenesis. The study further shows that H
2S targets a specific molecular switch, i.e., the Cys1024-Cys1045 motif, to regulate the conformation and function of VEGFR2 by sulfur-sulfur nucleophilic attack [
44]. This suggests a new mechanism beyond the typical docking between a ligand and its receptor. Some theories of atomic biology involving molecular orbitals are involved in this H
2S-induced regulation of VEGFR2. Interestingly, the TPO receptors and VEGFR2 belong to the receptor tyrosine kinase family. Some members of this kinase family, e.g., EGFR, have been recently found to contain an intracellular kinase domain similar to that of the VEGFR2 also with a molecular switch labile to H
2S regulation [
57]. This gives rise to an idea that the TPO receptors might also contain a similar intracellular kinase domain with a molecular switch sensitive to H
2S regulation. Indeed, much future works are required to examine this hypothesis.
In TPO
−/− mice exposed to radiation, H
2S also failed to ameliorate internal hemorrhage and improve survival despite a significant increase in circulating platelet levels. In addition, H
2S promoted megakaryocytes/platelets generation in cultured fetal liver cells isolated from TPO
−/− mice but not in c-mpl
−/− mice. These data suggest an essential role of the TPO receptor but not TPO for H
2S to promote hematopoiesis. The question is why H
2S increased platelet levels in TPO
−/− mice without ameliorating bleeding and subsequent mortality? This discrepancy might be ascribed to a significantly decreased survival in TPO
−/− mice treated with vehicle as compared with that in c-mpl
−/− mice (
P < 0.01, Additional file
1: Figure S1). Myelosuppression might be too severe to be recovered by H
2S in these TPO
−/− mice, whereas the TPO receptors were present in the cells isolated from the TPO
−/− mice and might serve as a target molecule for H
2S to promote hematopoiesis.
In addition, H
2S also increased circulating leukocytes in WT mice exposed to radiation at day 14 though erythrocytes were not increased (Additional file
2: Figure S2). Our present data are not sufficient to clarify whether such an improvement in circulating leukocyte counting contributes to an increase in survival in mice exposed to radiation due to an improvement in the immune system.
Methods
Animals
Wild-type C57BL/6 mice (WT mice) were purchased from the Department of Laboratory Animal Science of Fudan University and acclimatized for at least 2 weeks before experiments. c-mpl is the receptor of TPO; the
c-mpl knockout mice (c-mpl
−/− mice) were provided by The Jackson Laboratory (Bar Harbor, ME, USA) with an 80 % decrease in the number of platelets in comparison to that of wild-type C57BL/6 mice [
58,
59] (the strain name of these c-mpl
−/− mice is C57BL/6J-
Mpl
hlb219
/J with the stock number 005124). Likewise, TPO-deficient mice (TPO
−/− mice) have a >80 % decrease in their platelets [
59,
60]. The embryos of TPO
−/− mice were gifts kindly provided by Dr. Fred de Sauvage (Genentech Inc., USA) and developed into mice through the Embryo Recovery Service of The Jackson Laboratory (these TPO
−/−mice were produced by targeted mutation on the background of C57BL/6J mice; for more details, see de Sauvage [
60]). Both TPO
−/− and c-mpl
−/− mice were hybridized with WT mice and then their offspring were genotyped according to the protocol provided by The Jackson Laboratory. Homozygote animals were used in the experiments while heterozygote mice were maintained for breeding. Only female mice were used in the experiments. Mice were given free access to food and water while housed in a clean environment with appropriate temperature and humidity. At the time of experiments, all mice were 6–8 weeks old weighting 18–21 g. Considering that in the experiment mice will die or suffer severe sickness in the process, we set at least 10 mice per group at the beginning and repeated the experiment if necessary. To ensure the reliability of results, mice were randomly allocated to each group. All procedures were approved by the Institutional Animal Care and Use Committee, Fudan University Shanghai Medical College (20120302-098).
Irradiation-induced myelosuppression
The Cs-137 instrument (Gammacell-40, MDS Nordion, CA) was used as the γ-ray irradiation source in this study. Six- to 8-week-old mice were either exposed to 7.5 Gy (WT mice) or 5.0 Gy (TPO−/− and c-mpl−/− mice) cesium (137Cs)-emitted radiation for total body irradiation (TBI) to build up the myelosuppression model. The TPO−/− and c-mpl−/− mice were more sensitive to radiation exposure, and the mortality was too high to perform subsequent experiments in these mice. We performed a pilot study to explore the intensity of radiation exposure in these mice to cause radiation-induced myelosuppression with a mortality within the range that enables the subsequent experiments to be performed. The results showed 5.0 Gy as the radiation intensity for the TPO−/− and c-mpl−/− mice.
Platelet counts
Platelet counts were measured by automatic blood cell analyzer (XN-1000, Sysmex, Kobe, Japan). About 80–100 μL peripheral blood was obtained from the angular vein of individual mice then pumped into a 1.5-mL Eppendorf tube coated with ethylenediaminetetraacetic acid (EDTA) and mixed immediately. For platelet counting, whole blood was used without any dilution. During all experiments, every single mouse was sampled at 7-day intervals. Frequency and timing of sampling of each individual mouse was identical in all types of mice in this study. In this part, the operator of the automatic blood analyzer was blinded to the sample groups. Results were excluded in cases where there was clotting in the sample.
Bleeding time test
The tails of mice were placed horizontally then amputated about 3 mm from the tip and immersed into saline solution pre-warmed and maintained at 37 °C. The time from severing to cessation of bleeding was recorded as bleeding time, and the longest observation time was 20 min to avoid over bleeding which frequently occurred in the TPO−/− and c-mpl−/− mice. Researchers were blinded to the sample groups.
Fecal occult blood test
A fecal occult blood test kit (Baso, Zhuhai, China) was used to examine occult gastrointestinal bleeding according to protocols provided by the manufacturer. Briefly, 20–30 mg feces were smeared on the window of the test card then dripped with a drop of developer A (pyramidon). After sufficient penetration, a drop of developer B (alcohol and hydrogen peroxide) was dripped and then the results were read by comparing with a color card within 2 min. Researchers were blinded to the sample groups.
Bone marrow megakaryocytes counts
The femur and tibia were collected from some mice sacrificed at day 7 during the experimental period. After fixing in 10 % formaldehyde and dehydrated, the bone samples were embedded into paraffin, sliced, and stained with hematoxylin and eosin. The morphology of the bone marrow was assessed via light microscopy (Leica DMLB, Leica, Germany), and megakaryocytes were counted by an observer blinded to the sample groups. For WT mice, no less than 10 visions were observed in each sample, whereas for TPO−/− and c-mpl−/− mice the megakaryocytes were counted throughout the whole slice of the samples.
Cell culture
Mouse fetal livers were collected at E14, cut to pieces, digested with 0.125 % Tryspin (Life, Thermo fisher scientific, Waltham, MA, USA), and then filtered successively through 200-mesh (pore size, 74 μm) and 400-mesh (pore size, 37 μm) stainless steel screens. The yielded cells were cultured in RPMI1640 (Gibco, Thermo Fisher scientific, Waltham, MA, USA) supplemented with 10 % FBS (HyClone, Logan, Utah, USA) and incubated at 37 °C in 5 % CO2.
Flow cytometry
Cultured fetal liver cells were stained with PE-CD61 (104308, 1:100, Biolegend, San Diego, CA), FITC-CD41 (13903, 1:200, BioLegend, San Diego, CA), or 7-AAD viability staining solution (420403, 1:20, BioLegend, San Diego, CA) according to instructions provided by the manufacturer. Briefly, about one million cells were collected and resuspended in 100 μL phosphate-buffered saline (PBS) and then stained with respective antibodies according to its recommended usage for 30 min at 4 °C. Then the cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then the cells were washed in 1 mL PBS twice before being resuspended in 500 μL PBS. FACS analysis was performed with a flow cytometer (FACSCalibur, BD, USA). More than 10,000 cells were acquired and analyzed by CELLQuest. For each experiment of this part, fetal liver cells isolated from at least three individual mice were used and for at least three repetitions for each group. The operator of the flow cytometer was blinded to the sample groups.
Administration of H2S and TPO
NaHS (Sigma-Aldrich, St. Louis, MO, USA) was applied as a donor of H
2S [
45] in the present study. For the in vivo administration, NaHS and recombinant murine TPO (R&D, NASDAQ, USA) were freshly diluted in sterile saline or PBS for each administration. A single dose of TPO (15 μg kg
−1) was given by intraperitoneal injection after radiation exposure whereas NaHS was injected once a day during the experimental period. For in vitro experiments, recombinant murine TPO (PeproTech, NJ, USA) was freshly diluted with cell culture medium and added to the cultured cells with a single dose of 10 ng mL
−1 given at the first day of the experiments. NaHS was diluted with cell culture medium and added to the cultured cells every 8 h.
Scanning electron microscopy
Specimen were first double fixed with 2.5 % glutaraldehyde and 1 % osmium tetroxide and then gradiently dehydrated with ethanol followed by dehydration in HCP-2 critical point dryer (Haitchi, Tokyo, Japan) with liquid CO2. The dehydrated specimen was coated with gold palladium and then observed in a JSM-6360LV scanning electron microscope (JEOL, Tokyo, Japan).
Confocal microscopy
Cultured fetal liver cells were stained with PE-CD61 (12-0611, 1:40, eBioscience, San Diego, CA), FITC-CD41 (11-0411, 1:200, eBioscience, San Diego, CA), and the DAPI staining solution (C1006, 100 μL, Beyotime, China) and then examined with a confocal laser scanning microscope LSM 710 (Zeiss, Jena, Germany).
Measurement of plasma H2S
Plasma H
2S levels in some mice were measured according to a method previously described [
61]. The concentrations were determined using the standard curve generated with a standard NaHS solution.
Measurement of plasma TPO
Plasma TPO levels in some mice were measured using the Mouse Thrombopoietin Quantiken ELISA Kit (R&D, NASDAQ, USA) according to the manufacturer’s instructions.
Statistical analysis
Results were expressed as the mean ± standard error. Unless specifically stated, the data show normal distribution. For two groups, independent-sample t test was used. For more than two groups, statistical analysis was performed with one-way ANOVA followed by LSD if the variance is homogeneous or Tamhane’s T2 test when the variance is not homogeneous. For the fecal occult blood assay, the data were transformed in rank cases first and then analyzed with one-way ANOVA as above. Comparisons of mortality were performed with Kaplan-Meier survival curves, in which log-rank test was used to evaluate differences in survival. P < 0.05 was considered as statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
H-DL and YC performed the experiments, A-JZ performed the experiments and drafted the manuscript, J-JX performed the pilot experiments, and Y-CZ designed the study and wrote the manuscript. All authors read and approved the final manuscript.