The suppression of maternal-fetal leukemia inhibitory factor signal relay pathway by maternal immune activation impairs brain development in mice.

Recent studies in rodents suggest that maternal immune activation (MIA) by viral infection is associated with schizophrenia and autism in offspring. Although maternal IL-6 is though t to be a possible mediator relating MIA induced these neuropsychiatric disorders, the mechanism remains to be elucidated. Previously, we reported that the maternal leukemia inhibitory factor (LIF)-placental ACTH-fetal LIF signaling relay pathway (maternal-fetal LIF signal relay) promotes neurogenesis of fetal cerebrum in rats. Here we report that the maternal-fetal LIF signal relay in mice is suppressed by injection of polyriboinosinic-polyribocytidylic acid into dams, which induces MIA at 12.5 days post-coitum. Maternal IL-6 levels and gene expression of placental suppressor of cytokine signaling 3 (Socs3) increased according to the severity of MIA and gene expression of placental Socs3 correlated with maternal IL-6 levels. Furthermore, we show that MIA causes reduction of LIF level in the fetal cerebrospinal fluid, resulting in the decreased neurogenesis in the cerebrum. These findings suggest that maternal IL-6 interferes the maternal-fetal LIF signal relay by inducing SOCS3 in the placenta and leads to decreased neurogenesis

Response of leukemia inhibitory factor (LIF)–adrenocorticotropic hormone (ACTH)–LIF signaling to maternal LIF stimulation in mice.

<p>(A) Chronological change of LIF levels in fetal cerebrospinal fluid (CSF). (B) <i>Pomc</i> expression levels in the placenta. (C) ACTH level in fetal serum. (D) LIF level in fetal CSF. Reactivity to maternal LIF stimulation in placenta and fetus were examined at 3 h after the injection at 12.5 and 13.5 days post-coitum (dpc). Mouse recombinanat LIF (rLIF) was administered to dams at 5 μg/kg body weight. *, <i>p</i> < 0.05 vs. control at 12.5 dpc. **, <i>p</i> < 0.05 versus control at 13.5 dpc. Number of dams = 3 or 4 in each group. <i>Error bars</i>, SEM.</p

Maternal immune activation (MIA) induced changes of placental gp130 related cytokines gene expression and JAK/STAT3 signaling.

<p>(A) Expression of <i>Il-6st</i>. (B) Expression of <i>Il-6r</i>. (C) Expression of <i>Lifr</i>. (D) Expression of suppressor of cytokine signaling 3 (<i>Socs3</i>) at 13.5 days post-coitum (dpc). (E) Analysis of STAT3 phosphorylation at 13.5 dpc. (F) Correlation of suppressor of cytokine signaling 3 (<i>Socs3</i>) mRNA expression and maternal interleukin-6 (IL-6). Placental <i>Socs3</i> expression was upregulated with the polyriboinosinic-polyribocytidylic acid [poly (I:C)] dosage. polyriboinosinic-polyribocytidylic acid [poly (I:C)] at 12.5 dpc, placenta were analysed 3 h after the injection (A, B, C). Expression of <i>Socs3</i> and the activation of JAK2/STAT3 signaling were analysed 24 h after the injection (D and E). Controls, Poly 4 and Poly 20 were blotted in the same membrane (E). Maternal IL-6 in control was not detected and is represented as Zero in the Fig (F). Controls were injected with an equal volume of saline (0.01 ml/g body weight). #, <i>p</i> < 0.01. *, <i>p</i> < 0.05. Number of dams = 3 or 4 in each group, two foetuses were collected as a pooled sample from each litter. <i>Error bars</i>, SEM.</p

Alteration of fetal adrenocorticotropic hormone (ACTH) and leukemia inhibitory factor (LIF) levels by maternal immune activation (MIA).

<p>(A) Concentration of ACTH in fetal serum (FS) and LIF in fetal cerebrospinal fluid (CSF) at 3 h after MIA at 12.5 days post-coitum (dpc). (B) Concentration of ACTH in FS and LIF in fetal CSF at 13.5 dpc. (C) Correlation of LIF level in CSF and ACTH level in FS. MIA was induced by an intraperitoneal (i.p.) injection of 4 or 20 mg/kg polyriboinosinic-polyribocytidylic acid [poly (I:C)] at 12.5 dpc. Controls were injected with an equal volume of saline (0.01 ml/g body weight). Gray bars: ACTH concentration, open circles: LIF concentration. *, <i>p</i> < 0.05 vs. control in FS. #, <i>p</i> < 0.05 vs. control in CSF. Number of dams = 3 or 4 in each group. <i>Error bars</i>, SEM.</p

Changes in interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) concentrations in the maternal serum in response to maternal immune activation (MIA).

<p>(A) Concentration of IL-6 in maternal serum (MS). (B) Concentration of LIF in MS. MIA was induced by an intraperitoneal (i.p.) injection of 1, 4 or 20 mg/kg polyriboinosinic-polyribocytidylic acid [poly (I:C)] at 12.5 days post-coitum (dpc), maternal serum was analysed 3 h after the injection. Controls were injected with an equal volume of saline (0.01 ml/g body weight). Injection of polyriboinosinic-polyribocytidylic acid [poly (I:C)] at 12.5 dpc induced IL-6 in MS in a dose dependent manner. In contrast, the concentration of LIF in MS was increased by the injection of poly (I:C) at 1 and 4 mg/kg; the concentration of LIF in MS was decreased to the control level by the injection of poly (I:C) at 20 mg/kg. Cont: control, Poly 4: poly (I:C) 4 mg/kg, Poly 20: poly (I:C) 20 mg/kg. *, <i>p</i> < 0.05. Number of dams = 3 or 4 in each group. <i>Error bars</i>, SEM.</p

Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

<div><p>In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.</p></div

MCRs differentially regulate EPO-induced phosphorylation of ERK, STAT5 and AKT in erythroblasts during differentiation.

<p><b>(A)</b> Phosphorylation of ERK, STAT and AKT in erythroblasts. Erythroblasts at M0, M3 and M6 were starved for 3 h in HPGM without cofactors and subsequently incubated for 15 min with or without EPO. (<b>B</b>)<b>–</b>(<b>E</b>), Synergistic effects on EPO downstream signaling of ACTH, and the signal inhibition by nAbs. After starvation with HPGM without cofactors for 3 h, the cells were incubated for 15 min with nAbs and reacted with EPO for 15 min. EPO-induced phosphorylation of ERK is not altered by the addition of 0.1 nM ACTH39 to erythroblasts at M0. n = 3; error bars, s.e.m <b>(B)</b>. The phosphorylation of ERK is decreased by nAbs of MC2R and MC5R <b>(C)</b>. ACTH39 enhances EPO-induced phosphorylation of STAT5 in erythroblasts at M3 <b>(D)</b>. Phosphorylation of STAT5 is inhibited by nAbs of MC1R <b>(E)</b>. <b>(F)</b> and <b>(G),</b> AKT phosphorylation by ACTH and, the signal inhibition by nAbs. Treatment with ACTH39 for 15 min without starvation causes phosphorylation of AKT in erythroblasts at M6 <b>(F)</b>. The phosphorylation of AKT is inhibited by MC5R-nAb <b>(G)</b>. EPO, 3 U/ml EPO; ACTH, 0.1 nM ACTH39. n = 3; error bars, s.e.m. IgG, 10 μg/ml normal IgG; MC1R, 10 μg/ml anti-MC1R nAb; MC2R, 5 μg/ml anti-MC2R nAb; MC5R, 10 μg/ml anti-MC5R nAb. n = 3; error bars, s.e.sm.</p

MCR expressions during erythroblast differentiation.

<p>(<b>A</b>) Schematic diagram of an <i>in vitro</i> differentiation protocol for deriving erythroblasts from human HPCs. Human CD34⁺ HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN₂). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; IL-6, interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. (<b>B</b>) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. (<b>C</b>) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.</p