Morphological changes in alveolar macrophages (AMs) in macrophage scavenger receptor (MSR) enhancer-promoter dominant negative (DN) MafB transgenic (TG) mice.

<div><p>(A) Transmission electric microscopy revealed that the high electron density area (arrow) in the nucleus was increased in TG mice. Original magnification: ×7000.</p> <p>(B) The high electron density area in each nucleus was quantified (as described in <i>Materials and Methods</i>) and compared. The fraction of high electron density area in the nuclei of TG mice was significantly increased, compared with wild-type (WT) mice.</p> <p>(C) Scanning electron microscopy revealed that the morphology of pseudopods of AMs was different. In WT mice, pseudopod formations were narrow rod-like structures (‘filopodium-like’) while they were flat ruffled structures in the TG mice (‘lamellipodium-like’). Original magnification: ×8000.</p> <p>(D) Formation of actin filament (F-actin) was investigated using fluorescent-labeled phalloidin. In WT mice, alveolar macrophages projected more pseudopods than in TG mice. F-actin was dominantly localized in pseudopods, and fluorescent signal was stronger in the alveolar macrophages of WT mice, compared with TG mice. Top and second panel: original magnification: ×200. Third and bottom panel: original magnification: ×630. WT: wild-type mice, TG: transgenic mice.</p></div

Phagocytosis assay of alveolar macrophages (AMs) in macrophage scavenger receptor (MSR) enhancer-promoter dominant negative (DN) MafB transgenic (TG) mice.

<div><p>(A) (B) The phagocytic capacity of TG and wild-type (WT) AMs for IgG coated phycoerythrin (PE)-labeled polystyrene beads was analyzed using flow cytometry. The representative histogram data of WT (A) and TG (B) were shown.</p> <p>(A) Phagocytic capacity was significantly reduced in AMs of TG mice, compared to those of WT mice.</p> <p>(B) Rho GTPase activity was significantly reduced in TG mice compared with WT mice.</p></div

Repression of MafB activity in macrophages of macrophage scavenger receptor (MSR) enhancer-promoter dominant negative (DN) MafB transgenic (TG) mice.

<div><p>(A) The total amount of MafB plus DN MafB gene expression was assessed by RT-PCR in wild-type (WT) mice and TG mice. A DN MafB primer set that could amplify both DN MafB and endogenous MafB mRNA was used. PCR products were significantly increased in TG mice, compared with WT mice (n = 6 for both).</p> <p>(B and C) Bone marrow derived macrophages (BMDMs) from TG and WT mice were stimulated with 4-HNE to enhance endogenous MafB expression. Subsequently, BMDMs were transfected with control vector or 3× Maf recognition element (MARE) reporter plasmid vector. MafB expression was confirmed with a β-lactamase reporter assay using flow cytometry (n = 6 in each group). Dot plot data of both WT and TG BMDMs were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073963#pone-0073963-g003" target="_blank">Figure 3B and 3C</a>, respectively. The β-lactamase-positive cells had blue fluorescence.</p> <p>(D) The numbers of β-lactamase-positive cells were significantly lower in BMDMs of TG than those of WT.</p></div

Increased apoptosis in macrophages of macrophage scavenger receptor (MSR) enhancer-promoter dominant negative (DN) MafB transgenic (TG) mice.

<p>(A) RT-PCR: Expression of caspase-3 was significantly increased in BAL cells of TG mice compared with WT mice (n = 6 for both). (B) TUNEL staining: TUNEL positive cells were increased in the spleen of TG mice, compared with WT mice. Original magnification: ×100.</p

An increase in alveolar monocyte-like cells in macrophage scavenger receptor (MSR)-dominant negative (DN) MafB transgenic mice.

<p>In BAL cells from MSR-DN MafB transgenic mice, mononuclear cells were frequently observed compared with wild-type (WT) mice (arrow). Their cytoplasmic granularities were less than alveolar macrophages, but they had pseudopods on the cell surface, a typical finding for phagocytes. They were also positive for Mac-3, suggesting that they were monocytic lineage cells, namely alveolar monocyte. Original magnification: ×1000.</p

Verification of the plasmid vector containing human macrophage scavenger receptor (MSR) enhancer-promoter and the vector that enables expression of dominant negative (DN) MafB under the control of the MSR enhancer-promoter.

<p>(A) The reporter plasmid containing the MSR enhancer-promoter (<i>pMSR-EP-Bla</i>) was transfected into the RAW264.7 macrophage cell line, and reporter activity was assessed as described in the <i>Materials and Methods</i> in the online supplement. Positive activity of MSR enhancer-promoter was confirmed by the presence of ‘blue cells’ (as indicated in the photograph). (B) <i>pcMSR-EP-DN-MafB</i> and empty control plasmid were transfected into RAW264.7 cells. DN MafB protein in cell homogenates was evaluated by immunoblotting using a DDDDK-tag antibody. As an internal control, the amount of beta actin was also assessed. A 15 KDa band, estimated size of DN MafB, was only detected in RAW264.7 cells transfected with <i>pcMSR-EP-DN-MafB</i>.</p

Verification of mice expressing dominant negative (DN) MafB only in macrophages under the control of the human macrophage scavenger receptor (MSR) enhancer-promoter.

<div><p>(A) DN MafB mRNA expression was confirmed by RT-PCR using specific primers between the MafB sequence and the DDDDK tag sequence. DN MafB mRNA was positive in alveolar macrophages, peritoneal macrophages and the liver of transgenic (TG) mice, but negative in all of these in wild-type (WT) mice. (B) The lungs of TG mice were double stained with a DDDDK tag antibody (top panel: phycoerythrin, red) and a Mac-3 antibody (center panel: FITC, green). As shown in the bottom panel, DDDDK-tag positive cells and Mac-3 positive cells were merged, indicating that alveolar macrophages were positive for DN MafB. In the lungs of WT mice, DDDDK tag positive cells were not observed (data not shown). Original magnification: ×1000. (C) DDDDK-tag was immunostained in the liver, kidney, and spleen of TG and WT mice. Positive cells for DDDDK were only confirmed in the liver, kidney and spleen of MSR-DN MafB TG mice.</p> <p>Original magnification: ×1000. Counterstain: haematoxylin.</p> <p>(D) RT-PCR. In hematopoietic progenitor cells, neither DN-MafB (top) nor MSR (middle) mRNA expressions were detected (lane 1). After M-CSF treatment for 7 days, both DN-MafB and MSR were expressed in the macrophages derived from hematopoietic progenitor cells (lane2). GAPDH (bottom): internal control.</p></div

Phosphorylations of MAP kinase and nuclear transcriptional factor, NF-κB after TAC operation.

<p>A. Increase in phosphorylations of MAPK and NF-κB after TAC operation in PTX3-KO mice (left panel) and in PTX3-TG mice (right panel). B. Modulation of ERK1/2 and NF-κB p65 phosphorylation in PTX3-KO (left), PTX3-TG (right), compared with WT mice. C. ERK1/2 phosphorylation after recombinant PTX3 stimulation (left panel; cardiac myocytes, right panel; cardiac fibroblasts). *P<0.01 vs. sham-operated mice of the same strain. Results are expressed as mean ± SD (n = 8).</p

Hypertrophic changes after TAC operation.

<p>A. Plasma concentrations of IL-6 at 5 days after TAC or sham operation in PTX3-KO mice (left) and PTX3-TG mice (right). B. IL-6 expression after TAC detected by quantitative PCR in PTX3-KO mice (left) and PTX3-TG mice (right). C. Representative photographs of hearts at 4 weeks after TAC and sham operation in PTX3-KO mice (left) and PTX3-TG mice (right). *<i>P</i><0.01 vs. sham mice of the same strain. Results are expressed as mean ± SD (n = 6–8).</p

Gravimetric and echocardiographic data of PTX3-KO mice after TAC.

<p>WT, Wild type; PTX3-KO, PTX3 systemic knockout mice; TAC, thoracic aortic constriction; BW, body weight; HW, heart weight; LW, lung wet weight; IVS, interventricular septal wall thickness; LVEDD, left ventricular end-diastolic diameter; LVFS, left ventricular fractional shortening. All data are shown as mean ± SD (n = 10 per group).</p>*<p><i>P</i><0.05 and</p>**<p><i>P</i><0.01 vs. sham-operated mice of the same strain;</p>#<p><i>P</i><0.05 and</p>##<p><i>P</i><0.01 vs. TAC-operated WT mice at each time point, respectively.</p