MiR-125a Is a critical modulator for neutrophil development

<div><p>MicroRNAs are universal post-transcriptional regulators in genomes. They have the ability of buffering gene expressional programs, contributing to robustness of biological systems and playing important roles in development, physiology and diseases. Here, we identified a microRNA, miR-125a, as a positive regulator of granulopoiesis. <i>MiR125a</i> knockout mice show reduced infiltration of neutrophils in the lung and alleviated tissue destruction after endotoxin challenge as a consequence of decreased neutrophil numbers. Furthermore, we demonstrated that this significant reduction of neutrophils was due to impaired development of granulocyte precursors to mature neutrophils in an intrinsic manner. We showed that <i>Socs3</i>, a critical repressor for granulopoiesis, was a target of miR-125a. Overall, our study revealed a new microRNA regulating granulocyte development and supported a model in which miR-125a acted as a fine-tuner of granulopoiesis.</p></div

MiR-125a regulates maturation of neutrophils by targeting <i>Socs3 in vivo</i>.

<p>(A) Flow cytometry analysis of GFP⁺ bone marrow neutrophils after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Bar graphs indicated numbers of GFP⁺ neutrophils per femur and tibia. (B) Flow cytometry analysis of GFP⁺ myeloid precursor cell populations after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. Bar graphs indicated numbers of GFP⁺ GMPs (upper) or CMPs (lower) per femur and tibia. (C) Flow cytometry analysis of neutrophils incorporating BrdU in bone marrow GFP⁺ GMPs (upper) or CMPs (lower). Bar graphs indicate the mean fluorescence intencities of BrdU-incorporating GMPs (upper) or CMPs (lower). ns, none significant difference, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

<i>Socs3</i> ia a target of miR-125a.

<p>(A) Protein expression of SOCS3 in bone marrow neutrophils from <i>MiR125a</i>^<i>+/+</i> and <i>MiR125a</i>^<i>-/-</i> mice. Cell lysates were analyzed by immunoblot using SOCS3 antibody. (B) Schematic presentation of a potential miR-125a binding sites in the 3’UTR regions of Socs3. Sequences below indicate the mutant form of this site. (C) Luciferase reporter gene assay performed on 293T cells transfected with plasmids on which the luciferase reporter gene fused to the fragment of wild-type or mutant 3’UTRs of Socs3. Values were normalized to a firefly gene’s activity on the same construct (mean±s.d., n = 3). (D) The mRNA expression of Socs3 in sorted GFP⁺ GMPs which were transduced with retrovirus of Socs3 shRNA or a control(Ctrl) shRNA. (E-G) 1000 GMPs were sorted from <i>MiR125a</i>^<i>-/-</i> bone marrow lin^- cells which were transduced with retrovirus of Socs3 shRNA or a Ctrl shRNA and then cultivated in G-CSF containing methylcellulose media. Photographed CFUs (E), colony numbers (F) and cell number per CFUs (G) were shown. Representative data were from three independent experiments. <i>**P</i><0.01 (Student’s <i>t</i>-test).</p

Impaired G-CSF signaling in <i>MiR125a</i>^-/- neutrophils.

<p>(A) Proliferation of bone marrow neutrophils in respond to various concentrations of G-CSF. 2x10⁴ bone marrow neutrophils were cultured with G-CSF in various concentrations for 24 hours. Cell number were then counted. (B) Flow cytometry analysis of BrdU incorporating bone marrow neutrophils in response to G-CSF. Bar graphs show the average percentage of BrdU-incorporating neutrophils. (C) Apoptosis of bone marrow neutrophils in response to G-CSF for 48 hours. The bar graph shows the percentage of Annexin V + neutrophils. (D) Activation of STAT1, ERK and STAT3 in response to G-CSF. Bone marrow neutrophils were stimulated with 10 ng/ml G-CSF for 15 min, 30 min, 60 min and 120 min. Cell lysates were analyzed by immunoblot using antibodies specific for phosphorylated and total STATs, ERK and GAPDH. Representative data are from three independent experiments. (E) The ratio of phosphorylated STAT1, ERK and STAT3 vs. total STAT1, ERK and STAT3. Image J was used to quantitatively analyze the western blots results in (D). All values were represented as mean±s.d., n = 3 mice of each genotype. Ns, none significant difference, ** <i>P</i><0.01, *<i>P</i> <0.05 (Student’s <i>t</i>-test).</p

Impaired differentiation from granulocyte progenitors to mature neutrophils in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of myeloid precursor cell populations of 8-week-old mice. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. The right panel shows the overall number of precursors per bone marrow sample isolated from femurs and tibiae (mean±s.d.,n = 6 mice of each genotype). (B-C) Colony numbers of bone marrow cells in methylcellulose colony assays. Myeloid precursors were analyzed in complete methylcellulose medium containing SCF, IL-3, IL-6, and EPO (B) or varying concentrations of G-CSF (C). Values were represented as mean±s.d., n = 3 mice of each genotype. (D-G) 1000 GMPs were sorted from <i>MiR125a</i>^<i>+/+</i> or <i>MiR125a</i>^<i>-/-</i> mice and cultivate in G-CSF containing methylcellulose media. Colony numbers (D), photographed CFUs (E), cell number per CFUs (F) and total cell number of 1000 CFUs (g) were shown. Values were represented as mean±s.d., n = 3 mice of each genotype. ns, none significant difference,*<i>P</i><0.05, ***<i>P</i><0.001(Student’s <i>t</i>-test).</p

Lower mortality and neutrophil infiltration in LPS-induced lethal septic shock in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of infiltrating neutrophils from lungs of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice challenged with 25 mg/kg LPS after 24 hours. Single cell suspensions of lung cells were previously gated with CD45. Neutrophils were stained with CD11b-Percp cy5.5 and Ly6G-APC. Bar graph shows the average percentage of infiltrating neutrophils (mean±s.d.,n = 3 mice of each genotype). (B) Hematoxylinand-eosin staining of lung sections from WT and KO mice 24 hours after 25 mg/kg LPS injection. Bar graph is the histopathological severity score of lung sections. Histopathological severity of randomly selected fields from the lung sections were graded as 0 (no findings or normal), 1 (mild), 2 (moderate) or 3 (severe) for each of the four parameters(congestion, edema, hemorrhage and inflammation). Theses results were confirmed by a blinded independent researcher. (C) Serum concentrations of aspartate aminotransferase (ALT), blood urea nitrogen (BUN), creatine kinase (CK) and creatinine (CREA) in <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice 24 h after injection of 25 mg/kg LPS (mean±s.d.,n = 5 mice of each genotype,). (D) Survival of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice (n = 10 each genotype) intraperitoneally challenged with 45 mg/kg LPS. Data are presented as a Kaplan-Meier plot. P<0.05 (log-rank test). (E) TNF-α and IL-6 concentrations in serum of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice 2h after intraperitoneal injection of 45 mg/kg LPS (mean±s.d., n = 5 mice of each genotype). ns, no significant difference (Student’s <i>t</i>-test). (F) Wild-type mice were first depleted of endogenous macrophages by pre-treatment with clodronate liposomes and then were transplanted with 1x10⁷ <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> bone marrow derived macrophages 6 hours before intraperitoneal injection with 45 mg/kg LPS. Survival percentage of these mice are presented as a Kaplan-Meier plot (n = 7 mice of each genotype;p = 0.7114, log-rank test).*<i>P</i><0.05,**<i>P</i><0.01, ***<i>P</i><0.001.</p

Hematological Parameters of <i>MiR125a</i>^<i>-/-</i> mice.

<p>Hematological Parameters of <i>MiR125a</i>^<i>-/-</i> mice.</p

MiR-125a regulates maturation of neutrophils by targeting <i>Socs3 in vivo</i>.

<p>(A) Flow cytometry analysis of GFP⁺ bone marrow neutrophils after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Bar graphs indicated numbers of GFP⁺ neutrophils per femur and tibia. (B) Flow cytometry analysis of GFP⁺ myeloid precursor cell populations after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. Bar graphs indicated numbers of GFP⁺ GMPs (upper) or CMPs (lower) per femur and tibia. (C) Flow cytometry analysis of neutrophils incorporating BrdU in bone marrow GFP⁺ GMPs (upper) or CMPs (lower). Bar graphs indicate the mean fluorescence intencities of BrdU-incorporating GMPs (upper) or CMPs (lower). ns, none significant difference, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

Decreased neutrophils in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of bone marrow (upper panel) and peripheral blood (lower panel). Neutrophils were stained with CD11b-Percp cy5.5 and Ly6G-APC. Bar graphs indicated numbers of neutrophils per femur. Values were represented as mean±s.d., n = 5 mice of each genotype. (B) Flow cytometry analysis of bone marrow neutrophils after bone marrow transplantation for 6 weeks. Bar graphs indicated total numbers of neutrophils. Values were represented as mean±s.d.,n = 5 mice of each genotype. (C) Morphological character of neutrophils in <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice. Peripheral blood (original magnification, x1000) of control and knockout mice were stained with Giemsa. (D) Expression of miR-125a during myeloid development (mean±s.d.,n = 3). HSC, hematopoietic stem cells; CMP, common myeloid progenitors; GMP, granulocyte–monocyte progenitors; MEP, megakaryocyte erythroid progenitors; BM-N, bonemarrow neutrophils. **<i>P</i><0.01, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

Decreased proliferation of immature neutrophils in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of three subpopulations in CD11b⁺Gr-1⁺ neutrophils in bone marrow. Mature neutrophils (mNeu) indicate CD11b^hi Gr-1^hi cells. Immature neutrophils (imNeu) indicate CD11b^lowGr-1^hi cells and promyelocytes/myelocytes (pro/mye) indicate CD11b^intGr-1^int cells. The bar graph shows the average numbers of these subpopulations in <i>MiR125a</i>^<i>+/+</i> and <i>MiR125a</i>^<i>-/-</i> mice. Values were represented as mean±s.d., n = 5 mice of each genotype. (B) Flow cytometry analysis of three populations of CD11b⁺Gr-1⁺ neutrophils incorporating BrdU in bone marrow after in vivo pulsing BrdU for 72 hours. The bar graph indicates the average percentage of intensities of BrdU-incorporating cells (mean±s.d., n = 5). Ns, none significant difference, *<i>P</i><0.05, ***<i>P</i><0.001 (Student’s <i>t</i>-test).</p