Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

<div><p>Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.</p> </div

Rat MCL is expressed on monocytes, macrophages and granulocytes. A,

<p>mAb WEN42 is specific for MCL. The specificity of WEN42 was tested in flow cytometry analysis of BWN3G cells stably expressing rat MCL or Mincle. <b>B,</b> Flow cytometry analysis of rat MCL surface expression on different cell types isolated from different organs, as indicated. Histograms display cell subsets gated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057406#s2" target="_blank">materials and methods</a> section. Black line histograms: MCL. Filled grey histograms: isotype control.</p

Immunostaining of rat spleen.

<p>Serial-cut frozen sections were stained with mAbs towards <b>A,</b> rat MCL <b>B,</b> rat CD169 <b>C,</b> rat MHC class II <b>D,</b> human MHC class I (negative control) and visualized with peroxidase-conjugated secondary antibody and DAB substrate. RP: red pulp. PALS: periarteriolar lymphoid sheath. FOLL: follicle. MZ: marginal zone.</p

Phagocytosis of anti-MCL-coated beads. A,

<p>Comparative surface expression of the receptors, numbers represent median fluorescence intensity. <b>B.</b> Imaging flow cytometry analysis of a RMW cell showing a cell interacting with two beads. Channel 1 shows visible light image; bead-fluorescence is shown in channel 10, with DyLight-594 counterstaining in channel 4. Black arrowhead shows an internalized bead (green fluorescence) <b>C</b>. Percentage of cells with internalized beads, or with only surface-bound beads. <b>D.</b> Efficiency of phagocytosis expressed as a ratio of cells phagocytosing beads to cells with only surface-bound beads. 10,000 cells events were collected.</p

RMW is a rat myeloid cell line. A–D

<p>TEM ultrastructure of RMW cells. M: mitochondria. LB: lipid bodies. RER: rough endoplasmic reticulum. MV: multivesicular endosomes. G, Golgi. <b>E</b>, Flow cytometry analysis of RMW surface markers. Solid lines show the indicated surface markers; grey filled histograms correspond to isotype control.</p

Regulation of MCL surface expression under the effect of different stimuli.

<p><b>A,</b> Rats were injected intraperitoneally with zymosan, peritoneal cells were obtained after 24 h and stained with mAbs for flow cytometry analysis. Discontinuous line histogram shows staining in the zymosan treated group, dotted line histogram shows MCL staining in the PBS control group. Histograms display MCL expression on granular cells (including mast cells, eosinophils and neutrophils) and macrophages (MΦ) as indicated. The MCL⁺ fraction of granular cells likely reflects influx of neutrophils from the blood. MFI values are shown, calculated as the median of the fluorescence intensity. <b>B,</b> Resident peritoneal macrophages were cultured overnight with the indicated substances. Solid line histogram shows MCL staining. Grey filled histograms correspond to isotype controls.</p

Infection with purified <i>Piscine orthoreovirus</i> demonstrates a causal relationship with heart and skeletal muscle inflammation in Atlantic salmon

<div><p>Viral diseases pose a significant threat to the productivity in aquaculture. Heart- and skeletal muscle inflammation (HSMI) is an emerging disease in Atlantic salmon (<i>Salmo salar</i>) farming. HSMI is associated with <i>Piscine orthoreovirus</i> (PRV) infection, but PRV is ubiquitous in farmed Atlantic salmon and thus present also in apparently healthy individuals. This has brought speculations if additional etiological factors are required, and experiments focusing on the causal relationship between PRV and HSMI are highly warranted. A major bottleneck in PRV research has been the lack of cell lines that allow propagation of the virus. To bypass this, we propagated PRV in salmon, bled the fish at the peak of the infection, and purified virus particles from blood cells. Electron microscopy, western blot and high-throughput sequencing all verified the purity of the viral particles. Purified PRV particles were inoculated into naïve Atlantic salmon. The purified virus replicated in inoculated fish, spread to naïve cohabitants, and induced histopathological changes consistent with HSMI. PRV specific staining was demonstrated in the pathological lesions. A dose-dependent response was observed; a high dose of virus gave earlier peak of the viral load and development of histopathological changes compared to a lower dose, but no difference in the severity of the disease. The experiment demonstrated that PRV can be purified from blood cells, and that PRV is the etiological agent of HSMI in Atlantic salmon.</p></div

Expression of innate antiviral genes in blood cells in challenge experiment #2.

<p>Relative expression of type I interferon (IFNab) (A) and Mx (B) in controls (grey), the PRV-High (red) and the PRV-Low (green) group (n = 6). Target Ct values are normalized against the expression level of elongation factor (EF)1α, and fold induction relative to the mean level of control fish (0 wpc) is shown. Statistical analysis comparing PRV-High and PRV-Low was performed using Mann-Whitney test at each time point, *p < 0.05, asterisk color (red and green) indicate the significantly higher group.</p

Primers and probe used in this study.

<p>Primers and probe used in this study.</p

Summary cohabitant group.

<p>(A) PRV RNA in blood cells and heart measured by RT-qPCR, shown as individual and mean Ct-values from 2 to 8 weeks post challenge (wpc). (B) Amount of σ1- and μNS -protein measured by flow cytometry, shown as mean fluorescence intensity (MFI) for individual fish and group mean. (C) Histopathological score in the heart (total cardiac score) and red skeletal muscle shown as individual and group mean from 4 to 8 wpc. (D) Light microscopic images at 8 wpc in PRV-Low; (D) Heart lesions in the ventrical including severe epicarditis (arrowhead) and inflammation in the compact (*) and spongy myocardium (**) consistent with HSMI. (E) Mild inflammation in the red skeletal muscle (arrowhead), presence of melanin (*). Scale bar 20 μm. Color coding: PRV-High (red), PRV-Low (green), positive control (black) and negative control (grey). n = 6.</p