Cell proliferation and cytokinesis is impaired in n-cofilin null macrophages.

<p>(A) Cell proliferation was impaired in n-cof^{flx/Δ,Mx1-cre} cultures. Within 120 hours after plating, n-cof^{flx/Δ,Mx1-cre} bm cells did not expand (average of three independent experiments is shown); (B) Cytokinesis defect in n-cof^null cells and accumulation of multinucleated cells (n_cells>150, 2-way ANOVA analysis: Mx1-cre:n-cof^null p<0.0001). ADF is not required for cytokinesis; (C) In n-cof^{flx/Δ,Mx1-cre} cultures DNA synthesis decreased during the first days of bone marrow culture (n_cells>200); (D) TUNEL staining showed slightly increased apoptosis in n-cof^{flx/Δ,Mx1-cre} cultures (n_cells>100).</p

Immunological responses of n-cofilin mutant dendritic cells.

<p>(A) Phagocytosis of FITC-labelled <i>E. coli</i> in n-cof^{flx/Δ,Mx1-cre}, ADF^-/-, ADF^+/-, and control macrophage cultures (n-cof^{wt/wt,Mx1-cre}); (B) Phagocytosis of FITC-zymosan by individual macrophages was scored (n_cells>100). Phagocytosis in ADF^−/− cells was not changed; (C) TNF-α production induced by Zymosan phagocytosis. Macrophages were allowed to ingest zymosan particles and after 16 hours, TNF-α levels were determined in the cell supernatant; (D-E) Impaired T-cell stimulation by n-cofilin mutant dendritic cells. Serial dilutions of OVA_323-339 peptide were presented by n-cof^{fwt/wt,Mx1-cre} or n-cof^{flx/Δ,Mx1-cre} dendritic cells to OT-II T-cells. T cell stimulation was measured by production of IL-2 (D) and IFN-γ (E) after 24 hours; (F-G) T-cell stimulation of ADF^−/− dendritic cells was similar to ADF^+/− controls; (H) MHC class II-induced contact formation is impaired in n-cof^null macrophages. A mix of control macrophages (escaper cells) and n-cof^null macrophages, preincubated with anti-MHC class II antibody were allowed to form contacts with the beads. Control cells form F-actin free contact zones (arrows) with anti-mouse-IgG-coated beads (FITC labeled, green), while n-cof^null macrophages showed a F-actin rich domain (phalloidin, red) at the interface with the bead (arrows).</p

Expression of ADF and n-cofilin in myeloid cells and gene deletion in macrophages.

<p>(A) Western blot analysis of different myeloid cell types. In the middle panel, the filter was probed with n-cofilin and ADF specific antibodies to allow a comparison of relative amounts. Comparable levels of n-cofilin were found in total bone marrow (bm), bone marrow derived macrophages (BMM), peritoneal exudate cells (PEC), dendritic cells (DC) and granulocytes. ADF is down-regulated in BMM and PEC. Actin is shown as loading control; (B) Distribution of ADF and n-cofilin in wildtype BMM. ADF and n-cofilin (green, antibodies) co-localize with F-actin rich domains (red, phalloidin for ADF staining, and anti-actin antibody for n-cofilin staining, see arrows); (C) Time course of n-cofilin deletion in bone marrow derived macrophages after poly(I-C) treatment. Deletion on day 0, 3 and 6 of culture. Upper panel: Southern blot analysis for n-cofilin deletion as indicated by the loss of the flx-allele. Wild type (wt), conditional (<i>flox</i>-cof) and the deleted alleles (Δcof) are indicated. Lower panels: Western blot analysis of the respective cell lysates after n-cofilin deletion. The amount of n-cofilin strongly decreases on day 3 and slightly increases again at day 6, due to cells, which escaped the deletion. Actin and tubulin are shown as loading controls; (D) Upregulation of the macrophage marker CapG during BMM differentiation, ADF and gelsolin expression is not altered upon deletion of n-cofilin; (E, F) Cell shape of n-cof^null and ADF^−/− macrophages. (E) N-cof^null BMM can be identified in n-cof^{flx/Δ,Mx1-cre} cultures, using an anti-n-cofilin antibody (green). N-cof^null BMM had a distinct round shape and showed a high actin signal (red, anti-actin antibody). N-cofilin expressing escaper cells have a regular shape and much lower F-actin content. (F) ADF^−/− macrophages (ADF antibody staining, green) have regular morphology, cell polarity and F-actin content (phalloidin, red).</p

Characterization of n-cofilin null macrophages.

<p>(A) Confocal sections through an adherent n-cof^null macrophage show the unusual cytoskeletal structure and the impaired leading edge formation. F-actin (in red) is shown by phalloidin staining. Note the short extensions emerging from the cell body and the lack of a lamellipodium; (B) Scanning EM of saponin extracted macrophages illustrate a loose network of short filaments in the lamellipodium of control cells, while n-cof^null BMM show densly packed cortical F-actin bundles; (C) Biochemical fractionation showed increased Triton X-100 stable F-actin levels in n-cof^{flx/Δ,Mx1-cre} macrophage cultures (n_culture = 6, p<0.05). ADF^−/− macrophages showed normal F-actin levels; (D) FACS overlay of phalloidin stained macrophages from a day 6 control (grey histogram) and n-cof^{flx/Δ,Mx1-cre} (black line histogram) culture. About 10-fold increase in phalloidin signal intensity was observed in n-cof^null cells, while the escaper cells are represented in the low intensity F-actin peak, similar to the control cells. (E) Cell adhesion on laminin is not altered in n-cofilin mutant macrophage cultures (n-cof^{flx/Δ,Mx1-cre}) at 4 to 24 hours after plating. The average of three independent experiments performed in quadruplicates is shown; (F) Cell spreading was significantly impaired in n-cofilin mutant macrophages (n-cof^null). The spreading area was compared in control (n-cof^{wt/wt,Mx1-cre}), ADF^-/-, ADF^+/− and n-cofilin mutant BMM (n_cells>100, student's test, n-cof^null:control p<0,0001; ADF^−/−:ADF^+/− p = 0,15); (G) N-cof^null BMM fail to polarize. The shape of more than 90% of n-cof^null BMM was apolar, while 75% of control and ADF^+/− cells were either bipolar or multipolar (n_cells>130). ADF^−/− BMM showed a noticeable tendency to increase cell polarization.</p

<i>In vitro</i> and <i>in vivo</i> migration of macrophages lacking n-cofilin.

<p>(A) Random motility of n-cof^null BMM is impaired, ADF^−/− macrophages behave normally. Velocity of center movement over a period of 20 min is shown (n_cells>60, student's test n-cof^null:control p<0,0001); (B) Over a period of 8 hours n-cof^null cells show no significant net translocation, cell protrusions collapse back onto the cell body (see arrows). Migratory tracks are shown by white lines and dots, for two cells each (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036034#pone.0036034.s004" target="_blank">videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036034#pone.0036034.s005" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036034#pone.0036034.s006" target="_blank">S3</a>); (C) <i>In vivo</i> recruitment of n-cof^null cells to sites of inflammation is impaired. Thioglycollate injection (TG) into n-cof^{flx/Δ,Mx1-cre} mice does not lead to recruitment of mutant macrophages (Δcof) to the peritoneum (PEC). N-cofilin depleted cells remain in the bone marrow (bm). Southern blot of the respective mutant cell fractions (n-cof^{flx/Δ,Mx1-cre}) is shown in the left panel, and the corresponding western blot in the right panel. PEC mainly consists of escaper cells as indicated by the equal Δcof/<i>flox</i>-cof signals in Southern blot and the presence of n-cofilin protein. Western blot analysis of control mice (n-cof^{wt/wt,Mx1-cre}), is shown on the right panel to illustrate the normal expression levels of n-cofilin in bm and PEC under these conditions; (D) FACS analysis of bone marrow (bm) and PEC from the experiment in (C). Bone marrow composition after TG injection (TG) and n-cofilin deletion (poly(I−C)+TG) was comparable (left panels). The main portion of bm cells were Mac1/Gr1 positive, few B cells (B220) and T cells (CD3) were detected. By FACS the Mac1 and Gr1 positive cells in bm appear as one population. 3 days after TG treatment, the majority of PECs in controls are positive for the macrophage marker Mac1 (upper right panel). Upon deletion of n-cofilin (poly(I−C)+TG), the PEC population increases in Gr1 (granulocytes) and decreases in Mac1 (macrophages).</p

Protective and Pathogenic Roles of CD8+ T Lymphocytes in Murine <i>Orientia tsutsugamushi</i> Infection

<div><p>T cells are known to contribute to immune protection against scrub typhus, a potentially fatal infection caused by the obligate intracellular bacterium <i>Orientia (O</i>.<i>) tsutsugamushi</i>. However, the contribution of CD8+ T cells to protection and pathogenesis during <i>O</i>. <i>tsutsugamushi</i> infection is still unknown. Using our recently developed BALB/c mouse model that is based on footpad inoculation of the human-pathogenic Karp strain, we show that activated CD8+ T cells infiltrate spleen and lung during the third week of infection. Depletion of CD8+ T cells with monoclonal antibodies resulted in uncontrolled pathogen growth and mortality. Adoptive transfer of CD8+ T cells from infected animals protected naïve BALB/c mice from lethal outcome of intraperitoneal challenge. In C57Bl/6 mice, the pulmonary lymphocyte compartment showed an increased percentage of CD8+ T cells for at least 135 days post <i>O</i>. <i>tsutsugamushi</i> infection. Depletion of CD8+ T cells at 84 days post infection caused reactivation of bacterial growth. In CD8+ T cell-deficient beta 2-microglobulin knockout mice, bacterial replication was uncontrolled, and all mice succumbed to the infection, despite higher serum IFN-γ levels and stronger macrophage responses in liver and lung. Moreover, we show that CD8+ T cells but not NKT cells were required for hepatocyte injury: elevated concentrations of serum alanine aminotransferase and infection-induced subcapsular necrotic liver lesions surrounded by macrophages were found in C57Bl/6 and CD1d-deficient mice, but not in beta 2-microglobulin knockout mice. In the lungs, peribronchial macrophage infiltrations also depended on CD8+ T cells. In summary, our results demonstrate that CD8+ T cells restrict growth of <i>O</i>. <i>tsutsugamushi</i> during acute and persistent infection, and are required to protect from lethal infections in BALB/c and C57BL/6 mice. However, they also elicit specific pathologic tissue lesions in liver and lung.</p></div

Dissemination of <i>Orientia tsutsugamushi</i> and Inflammatory Responses in a Murine Model of Scrub Typhus

<div><p>Central aspects in the pathogenesis of scrub typhus, an infection caused by <i>Orientia (O.) tsutsugamushi</i>, have remained obscure. Its organ and cellular tropism are poorly understood.</p><p>The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of <i>O. tsutsugamushi</i> infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy <i>traD</i> genes, we quantitatively monitored the spread of <i>O. tsutsugamushi</i> Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected <i>O. tsutsugamushi</i> at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.</p><p>Our data show that upon inoculation to the skin, <i>O. tsutsugamushi</i> spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of <i>O. tsutsugamushi</i> infection.</p></div

Decreased macrophage recruitment to pulmonary BALT in β₂m^-/- mice.

<p>β₂m^-/- mice or C57BL/6 controls were footpad-infected with <i>O</i>. <i>tsutsugamushi</i>. 16 or 17 days p.i., mice were sacrificed. <b>A,</b> Consecutive lung sections, stained for IBA1 or iNOS (top, middle) and cryosections, stained for IBA1 (green) and DAPI (blue) (bottom). Arrowheads indicate IBA1+ or iNOS+ cells in BALT regions (immunohistochemistry) or IBA1+ cells at peribronchial lining (immune fluorescence). Scale bars, parenchyma: 100 μm; BALT: 200 μm. Images show representative regions. Graphs show percentages of stain⁺ areas per total tissue area from 4 representative regions of each mouse (n = 3). β₂m^-/- were compared to C57BL/6 controls by two-way ANOVA with Bonferroni’s posttest. ns: not significant; *** p<0.001 <b>B,</b> Immunofluorescence of bronchiolar lining, stained with antibody against the <i>O</i>. <i>tsutsugamushi</i> 56 kD antigen (red) and DAPI (blue) overlayed with DIC. Images were captured with an Olympus confocal microscope. Bottom panel shows region of higher magnification. Arrowhead indicates a bacterium at the luminal side inside an epithelial cell. Scale bars: 100 μm (overview); 20 μm (detail). parench.: parenchyma.</p

C57BL/6 wildtype and CD1d^-/- but not CD8+ T cell deficient β₂m^-/- mice control <i>O</i>. <i>tsutsugamushi</i> infection.

<p><b>A-C,</b> β₂m^-/- mice or C57BL/6 controls were footpad-infected with <i>O</i>. <i>tsutsugamushi</i> without further treatment. <b>A,</b> Survival curve. Data from one of two representative experiments (n = 5). Significance was determined by Mantel-Cox test. <b>B,</b> Bacteremia at indicated time points (left) and <i>O</i>. <i>tsutsugamushi</i> burdens in target organs at days 16 or 17 p.i. (right) are depicted. Shown are mean values ± SD from one of two representative experiments (bacteremia, n = 5) or pooled data from two independent experiments (organ loads, mean ± SD, n = 6, lung: n = 3). β₂m^-/- mice were compared to C57BL/6 controls by student’s t-tests. <b>C,</b> IFN-γ was measured by ELISA in serum from β₂m^-/- mice or C57BL/6 controls at days 16 or 17 p.i. IFN-γ levels were compared by student’s t-test. <b>D,</b> CD1d^-/- mice or C57BL/6 controls were footpad-infected with <i>O</i>. <i>tsutsugamushi</i>. Bacteremia was measured at indicated time points by qPCR. <b>A-D,</b> * p<0.05; ** p<0.01; *** p<0.001.</p

Time course of symptoms and bacterial loads in organs of <i>O. tsutsugamushi</i>-infected mice.

<p>BALB/c mice received an inoculum containing 5×10³<i>sfu</i> of <i>O. tsutsugamushi</i> in the hind footpad. A, The course of infection was monitored by assessing the clinical score. B–D, At 3, 7, 10, 14, 17, 21 and 28 days p.i., mice were sacrificed, and loads of <i>O. tsutsugamushi</i> in DNA extracts of blood or indicated organs were determined by <i>traD</i> qPCR (<i>n</i> = 4). B, Bacteremia. C, Secondary lymphoid organs (peripheral lymph node (LN), spleen). D, Lung, brain, heart and liver. Graphs show combined results of two independent experiments (n = 4; mean ± SD). E, Organ loads on day 14 p.i. Bm, bone marrow (n = 6–10, mean ± SD. Data were pooled from 2–3 independent experiments. ** p<0.01 comparing lung to any other group, as determined by unpaired two-tailed <i>t</i> test).</p