ERK1 Regulates the Hematopoietic Stem Cell Niches

The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1−/− HSC are impaired, suggesting that the ERK1−/−-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1−/− defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments

Deletion of ERK1 impairs osteoclast formation without any effects on osteoblasts.

<p>WT and ERK1^−/− mouse femur sections were stained with Trichrome Goldner-Masson stain (A) and revealed for TRAP activity (B). Ob.S/BS and Oc.S/BS parameters were calculated from Goldner trichrome and TRAP activity sections, respectively. Data are the means±S.E.M. (n  = 5 per group). Data represent the mean±SEM, the Mann-Whitney test was used to calculate the <i>P</i> value.</p

ERK1^−/− mice have a reduced fraction of bone marrow monocytes.

<p>(A) Representative FACS plot of BM monocytes defined as Gr1⁺CD115⁺ cells. (B) Proportion of monocytes as described above in the total BM in WT (n = 11) and ERK1^−/− (n = 12). Data represent the mean±SEM. The <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1^−/− microenvironment induces a defect in WT HSC activity.

<p>(A) Scheme of transplantation assay. (B) Analysis of WT donor cells engraftment in WT (n = 10) and ERK1^−/− mice (n = 10) primary recipient 24 weeks after transplantation. (C) Analysis of WT donor cells engraftment in WT (n = 9 per group) and ERK1^−/− mice (n = 16) secondary recipient 24 weeks after transplantation. In panels B and C, total donor cells are shown as a percentage of live cells. Individual lineages are shown as a percentage of donor-derived cells. Data represent the mean±SEM, the <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1^−/− microenvironment alters the lodging and the homing efficiencies of BM cells.

<p>Lodging of the bone marrow cells into a non irradiated host were quantified 3 h (A) and 24 h (B) after injection. Homing of the bone marrow cells into a lethally irradiated host were quantified 3 h (C) and 24 h (D) after injection. Results are presented as scatter plots showing the percentage of recovered CFSE⁺ cells in the BM 3 and 24 hours after transplantation. The non parametric Mann-Whiney test was used to calculate the <i>P</i> value. Horizontal bars show the mean values.</p

ERK1 deficiency impairs osteoclastogenesis.

<p>(A) Total number of osteoclasts per well following 5 days of culture under osteoclastogenic condition (n = 3) (B) Representative picture of TRAP-positive multinucleated osteoclasts generated from BMNCs of WT and ERK1^−/− mice. (C) Relative expression of cathepsin K (CTSK), calcitonin receptor (CaR), and receptor activator of nuclear factor <i>kappa</i> B (RANK) in WT and ERK1^−/− osteoclasts. (D) Representative pictures of the bone resorption pits formed by WT and ERK1^−/− derived osteoclasts. (E) Bar graph representing the quantification of the resorptive area per dentin slice for WT and ERK1^−/− osteoclasts (n = 3). Data represent the mean± SEM. The <i>t</i>-test was used to calculate the <i>P</i> value.</p

ERK1 loss alters the bone architecture.

<p>Cortical and trabecular parameters in WT and ERK1^−/− mice (n = 5 for each group). (A) midshaft diaphysis cortical thickness, (B) bone volume/tissue volume (% BV/TV), (C) trabecular thickness (Tb.Th), (D) trabecular number (Tb.N), (E) trabecular separation (Tb.S). The non parametric Mann-Whiney test was used to calculate the <i>P</i> value. Horizontal bars show the mean values.</p

Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs.

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</p

Comparison between the antiviral effect of IFITMs reported in the literature for the different viruses and mediated by the pool of IFITM proteins in target cells, with the negative imprinting of the virion particles infectivity reported in this study.

<p>Given their high identity, the antiviral effects of IFITM2 and 3 are presented together, separately from those of IFITM1. Variations in the magnitude of the antiviral effects reported in the different studies have not been taken into account here, as they are likely influenced by the specific experimental conditions used, so that the effects of IFITMs on viral infectivity are presented as negative, absent (none), or controversial, even when a single conflicting report exists. When data in the literature was not directly comparable to ours (i.e. the same virus was not used), data was compared to its closest relative, marked in <i>italicus</i>. The effects of the expression of IFITMs in target cells against AAV and MeV were measured in this study and are presented in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s008" target="_blank">S6 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.g007" target="_blank">Fig 7C</a>, respectively.</p