ERK Signaling Is Essential for Macrophage Development

<div><p>Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of <i>Erk1</i>^<i>-/-</i><i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of <i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from <i>Erk1</i>^<i>-/-</i><i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of <i>Erk1</i>^<i>-/-</i><i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} mice was enriched for CD11b⁺ myeloid cells, CD11b^hi Gr-1^hi neutrophils, Lin^- c-Kit⁺ Sca–1⁺ hematopoietic stem cells, and Lin^- c-Kit⁺ CD34⁺ CD16/32⁺ granulocyte-macrophage progenitors. Culture of bone marrow Lin^- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from <i>Erk1</i>^<i>-/-</i><i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.</p></div

ERK signaling is required for some macrophage responses to M-CSF.

<p>(A) Wild-type bone marrow-derived macrophages were treated with 2 ng/ml M-CSF for the indicated times, lysed, and analyzed by Western blotting. Data represent two independent experiments. (B) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO vehicle (all others) and then treated with 2 ng/ml M-CSF for 10 min, lysed, and analyzed by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2 respectively were 33.7, 31.9 and 32.3 for β-actin; 43.7, 36.1 and 6.81 for ERK2; and 31.3, 54.9 and 14.0 for p-ERK2. Data represent two independent experiments. (C) and (D) Bone marrow-derived macrophages were pre-treated for 30 min with U0126 or DMSO and then treated with 2 ng/ml M-CSF for 24 h. RNA expression was measured by qRT-PCR. Each data point represents mean ± standard deviation of triplicate samples. Data are representative of three independent experiments. (E) Bone marrow macrophages were pre-treated with U0126 or DMSO, treated with 2 ng/ml M-CSF for 24 h, collected by detaching the cells using PBS with 10 mM EDTA, stained with 2 μg/ml anti-CD115, and analyzed by flow cytometry. Data represent two independent experiments. Labels: -, untreated; +, treated with M-CSF; B6, C57BL/6J; E1, <i>Erk1</i>^<i>-/-</i>; E2, <i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>}; U, U0126-treated C57BL/6J. **: <i>P</i><0.01, *: <i>P</i><0.05, NS: <i>P</i>>0.05.</p

Complete blood count data.

<p>Data are displayed as means ± standard deviations. NS indicates not significant, <i>P</i>>0.05. Abbreviations: WBC, white blood cells; RBC, red blood cells; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width; MPV, mean platelet volume.</p><p>Complete blood count data.</p

Antibodies used for flow cytometry.

<p>^aExperimental panels refer to: Lineage, bone marrow lineage committed cell populations; FACS, bone marrow myeloid cell sorting by FACS; BMM, cultured bone marrow-derived macrophage immunophenotyping; Differentiation, <i>in vitro</i> myeloid lineage differentiation from Lin^- cells; Progenitors, bone marrow hematopoietic progenitor populations.</p><p>^bLineage cocktail consists of a mixture of FITC-conjugated anti-CD3ε (clone 145-2C11), anti-Gr–1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-B220 (clone RA3-6B2), and anti-TER–199 (clone Ter–119) and was used according to the manufacturer’s instructions.</p><p>Antibodies used for flow cytometry.</p

Macrophage marker expression and responses to TLR2 stimulation are intact in ERK1/2 macrophages.

<p>(A) Bone marrow-derived macrophages grown in LADMAC medium for 7 d were rested overnight in D10F, and then stained for CD11b, CD18, M-CSF receptor and F4/80, and analyzed by flow cytometry. Data are representative of two independent experiments. (B) Bone marrow-derived macrophages from LADMAC-stimulated cultures were rested overnight in D10F, pre-treated for 30 min with U0126 or DMSO vehicle (all others), and then treated with or without 30 nM Pam₃CSK₄ for 15 min, lysed, and analyzed for ERK phosphorylation by Western blotting. Densitometric values (in arbitrary units) for LysM, E1 and ERK1/2, respectively, were 33.12, 30.29 and 36.6 for β-actin; 35.2, 26.5 and 2.94 for ERK2; and 28.88, 34.69 and 6.36 for p-ERK2. Results are representative of two independent experiments. (C) and (D). Bone marrow-derived macrophages were pre-treated with U0126 or DMSO as in (B) and then treated with 30 nM Pam₃CSK₄ for 2 h (C) or 4 h (D). Gene expression was measured by qRT-PCR. These time points were previously determined to demonstrate maximal expression of <i>Il10</i> and <i>Il12b</i>, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140064#pone.0140064.ref021" target="_blank">21</a>]. Results represent two independent experiments and show means ± standard deviations. Labels: -, untreated; +, treated with Pam₃CSK₄; B6, C57BL/6J; E1, <i>Erk1</i>^<i>-/-</i>; E2, <i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>}; U, U0126-treated C57BL/6J. ****: <i>P</i><0.0001, **: <i>P</i><0.01, *: <i>P</i><0.05, NS: <i>P</i>>0.05.</p

Production of monocytes and macrophages by <i>in vitro</i> culture of Lin^- progenitors from ERK1/2 and LysM bone marrow.

<p>(A) Western blot of Lin^- cells from LysM and ERK1/2 bone marrow. (B) Cytospin preparation showing representative cells generated by culture of Lin^- cells for 4 d in 5 ng/ml IL–3, 10 ng/ml IL–6, and 10 ng/ml SCF (similar results were observed with M-CSF in addition to these growth factors). (C) Lin^- cells were grown in IL–3, IL–6, and SCF and monitored daily for acquisition of neutrophil (CD11b⁺ Gr–1⁺) and monocyte (CD11b⁺ Gr–1^-) lineage markers by flow cytometry. (D) Cells were grown and analyzed as in (C) but with the addition of 5 ng/ml M-CSF. (E) and (F) Total yields of cells from cultures of Lin^- cells in myeloid-stimulating cytokines. Data in (A) and (B) are representative of two independent experiments; data in (C)-(F) show the means ± standard deviations of triplicate samples and are representative of two independent experiments.</p

Bone marrow of ERK1/2 mice contains increased numbers of mature myeloid and lymphoid cells with retention of ERK2 expression in monocytes but not granulocytes.

<p>(A) Bone marrow cells from LysM and ERK1/2 mice (six of each) were analyzed by <i>ex vivo</i> flow cytometry to assess lineage-committed cells. (B-I) Cell numbers were calculated from total bone marrow cell counts and the percentage of each population in the gate. (J) Neutrophils (CD11b^hi Gr-1^hi) and monocytes (CD11b⁺ Gr–1^-) from LysM and ERK1/2 bone marrow were collected by FACS and analyzed by Western blotting for expression of ERK and β-actin. Data are expressed as means ± standard deviations. Results are representative of two independent experiments.</p

LysM and ERK1/2 mice express the expected mutations in germline DNA, but macrophages from ERK1/2 mice selectively retain expression of ERK2 protein.

<p>(A) DNA from clipped tails of LysM and ERK1/2 mice (three of each) was amplified to detect the mutant or wild-type alleles for <i>Lyz2</i>, <i>Erk1</i>, and <i>Erk2</i>. (B) Macrophages were derived from bone marrow cultures stimulated for 7 d with LADMAC-conditioned medium containing M-CSF, and expression of ERK1 and ERK2 was detected by Western blotting. Diagonal arrows indicate the position of the ERK2 band, expressed in ERK1/2 macrophages but greatly reduced in <i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>} macrophages. Results are representative of three independent experiments. Labels: B6, C57BL/6J; E1 E2^f/f, <i>Erk1</i>^<i>-/-</i><i>Erk2</i>^{<i>flox/flox</i>}; E1, <i>Erk1</i>^<i>-/-</i>; E2, <i>Erk2</i>^{<i>flox/flox</i>}<i>Lyz2</i>^{<i>Cre/Cre</i>}.</p

ERK1/2 mice have increased numbers of hematopoietic stem cells (LSK cells), common lymphoid progenitors, and granulocyte-macrophage progenitors on <i>ex vivo</i> flow cytometry analysis of progenitor populations.

<p>(A) Bone marrow cells from LysM and ERK1/2 mice (six of each) were counted using a hemacytometer and then analyzed by flow cytometry using the indicated gating strategy and the antibody panel described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140064#pone.0140064.t001" target="_blank">Table 1</a>. (B-F) Cell numbers from each population of immature hematopoietic cells were extrapolated from the total bone marrow cell counts and the percentage of each population following analysis in FlowJo. Data are represented as means ± standard deviations. Results are representative of two independent experiments.</p

SPR analysis of substrate binding to LprG.

<p>(<b>A</b>) Schematic representation of mycobacterial glycolipids and lipoglycans used in this study. (<b>B–F</b>) Substrate binding to LprG was assessed by SPR. LprG was immobilized on a CM5 sensor chip. Sensograms were obtained by injecting increasing concentrations of (<b>B</b>) PIM₂ (<b>C</b>) PIM₆ (<b>D</b>) LM (<b>E</b>) ManLAM and (<b>F</b>) PI-LAM. Binding was measured as response units (RU). Binding curves were calculated with BIA evaluation 3.1 software with subtraction of non-specific binding of the substrates to the sensor chip control cells without immobilized LprG. Results are representative of three independent experiments.</p