The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene-function relationships. However, relatively little is known about the physiology of neutrophils from wild-type mice. Do they behave like human neutrophils, or are there species-specific differences that need to be considered when extrapolating results from mice to humans? How do we isolate neutrophils from mice? For practical reasons, many studies on mouse neutrophils are done with bone marrow cells. However, human bone marrow neutrophils appear to be heterogeneous and functionally immature. We have isolated and compared neutrophils from mouse bone marrow and from peripheral blood obtained by tail bleeding. Using the same Percoll® density gradient for both preparations, we have obtained morphologically mature neutrophils from bone marrow and blood. Both cell populations responded to formylmethionyl-leucyl-phenylalanine (fMLF) with primary and secondary granule release and superoxide production. Quantitative analysis of our data revealed minor differences between cells from bone marrow and blood. Superoxide production and primary granule release were stimulated at lower fMLF concentrations in blood neutrophils. However, the amplitude and the kinetics of maximal responses were similar. The principal difference was the lifespan of the two cell populations. Bone marrow cells survived significantly longer in culture, which may suggest that they are receiving antiapoptic signals that are absent in the blood. Our data suggest that mice have a large reservoir of functionally competent neutrophils in their bone marrow. This reservoir may be needed to replace circulating neutrophils rapidly during infection.

Trefoil factor (TFF)1 is synthesized and secreted by the normal stomach mucosa and by the gastrointestinal cells of injured tissues. The link between mouse TFF1 inactivation and the fully penetrant antropyloric tumor phenotype prompted the classification of TFF1 as a gastric tumor suppressor gene. Accordingly, altered expression, deletion, and/or mutations of the TFF1 gene are frequently observed in human gastric carcinomas. The present study was undertaken to address the nature of the cellular and molecular mechanisms targeted by TFF1 signalling. TFF1 effects were investigated in IEC18, HCT116, and AGS gastrointestinal cells treated with recombinant human TFF1, and in stably transfected HCT116 cells synthesizing constitutive or doxycycline-induced human TFF1. We observed that TFF1 triggers two types of cellular responses. On one hand, TFF1 lowers cell proliferation by delaying G1-S cell phase transition. This results from a TFF1-mediated increase in the levels of cyclin-dependent kinase inhibitors of both the INK4 and CIP subfamilies, leading to lower E2F transcriptional activity. On the other hand, TFF1 protects cells from chemical-, anchorage-free–, or Bad-induced apoptosis. In this process, TFF1 signalling targets the active form of caspase-9. Together, these results provide the first evidence of a dual antiproliferative and antiapoptotic role for TFF1. Similar paradoxical functions have been reported for tumor suppressor genes involved in cell differentiation, a function consistent with TFF1.

Despite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19‐7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation‐associated apoptosis (+16%). Decreased production (–58%) of S ‐adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (–57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long‐lasting consequences.—Akchiche, N., Bossenmeyer‐Pourié, C., Kerek, R., Martin, N., Pourié, G., Koziel, V., Helle, D., Alberto, J.‐M., Ortiou, S., Camadro, J.‐M., Léger, T., Guéant, J.‐L., Daval, J.‐L. Homocysteinylation of neuronal proteins contributes to folate deficiency‐associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells. FASEB J. 26, 3980–3992 (2012). www.fasebj.org

The methyl donors folate (vitamin B9) and vitamin B12 are centrepieces of the one-carbon metabolism that has a key role in transmethylation reactions, and thus in epigenetic and epigenomic regulations. Low dietary intakes of folate and vitamin B12 are frequent, especially in pregnant women and in the elderly, and deficiency constitutes a risk factor for various diseases, including neurological and developmental disorders. In this respect, both vitamins are essential for normal brain development, and have a role in neuroplasticity and in the maintenance of neuronal integrity. The consequences of a methyl donor deficiency (MDD) were studied both in vivo in rats exposed in utero, and in vitro in hippocampal progenitors (H19-7 cell line). Deficiency was associated with growth retardation at embryonic day 20 (E20) and postnatally with long-term brain defects in selective areas. mRNA and protein levels of the transcription factor Stat3 were found to be decreased in the brains of deprived fetuses and in differentiating progenitors (62 and 48% for total Stat3 protein, respectively), along with a strong reduction in its phosphorylation at both Tyr⁷⁰⁵ and Ser⁷²⁷ residues. Vitamin shortage also affected upstream kinases of Stat3 signaling pathway (phospho-Erk1/2, phospho-Src, phospho-JNK, and phospho-p38) as well as downstream target gene products (Bcl-2 and Bcl-xL), thus promoting apoptosis. Conversely, the expression of the Stat3 regulator miR-124 was upregulated in deficiency conditions (≥65%), and its silencing by using siRNA partly restored Stat3 signaling in hippocampal neurons by increasing specifically the phosphorylation of Erk1/2 and Src kinases. Furthermore, miR-124 siRNA improved the phenotype of deprived cells, with enhanced neurite outgrowth. Taken together, our data suggest that downregulation of Stat3 signaling by miR-124 would be a key factor in the deleterious effects of MDD on brain development.