Flow cytometry analysis of liver granuloma during acute schistosomiasis.

<p>BALB/c mice were infected with 100 <i>S. mansoni</i> cercariae, killed 8 weeks later and liver granuloma-associated leukocytes isolated before 4-colour flow cytometry analysis. (A) Gating strategy for analysis of surface expression of CD11b and I-A/I-E on SSC^high and live (7-AAD⁻) liver granuloma-associated leukocytes. Outlined regions define CD11b⁺I-A/I-E⁻ and CD11b⁺I-A/I-E^high, respectively. (B) CD11b⁺I-A/I-E⁻, and CD11b⁺I-A/I-E^high gated populations in A were analyzed for F4/80, Gr-1, Siglec-F, CD204, MMR, Dectin-1, CD68, CD80, CD86 and IL-4Rα expression, respectively. Greyscale histograms show relevant isotype control. (C) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺I-A/I-E⁻ cells as described in A. F4/80 and Gr-1 double staining contour plot is shown. Outlined regions were sorted for cytospin and morphological analysis. (D) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺F4/80⁺ cells as described in A. MHC-II (I-A/I-E) and scavenger receptor-A (CD204) double staining contour plot is shown. Outlined region was sorted for cytospin and morphological analysis. Data are representative of three independent experiments with similar results.</p

IL-4Rα-expressing macrophages do not affect cytokine responses in granulomas.

<p><i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice were infected with 100 <i>S. mansoni</i> cercariae and killed 8 weeks later. Mesenteric lymph node (mLN) cells or liver granuloma-associated leukocytes (Liver) were then isolated and restimulated overnight with 20 µg/ml SEA before analyzed for <i>ex vivo</i> cytokine production capability. Staining of surface CD4 was followed by detection of IL-4, IL-10 and IFN-γ secretion in a cytokine catch assay or detection of intracellular levels of IL-13 after 4h of monensin treatment, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#s2" target="_blank">Methods</a>. Live gate was placed on lymphocytes according to their forward- and side-scatter by flow cytometry. (A) Representative contour plots of IFN-γ, IL-4, IL-10 and IL-13-producing lymphocytes. Quadrant bars were set up on unstimulated cells and numbers represent percent cells in each quadrant. Data represent one out of three independent experiments with similar results. (B) Percentages of IFN-γ, IL-4, IL-10 or IL-13-producing cells were determined in gated CD4⁺ or non-CD4⁺ cell populations from mLN or liver granuloma-associated lymphocytes (Liver) (mean±SEM, <i>n</i> = 4). Data are representative of three independent experiments. *<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.001 compared to control <i>Il4ra^−/lox</i> mice.</p

Localisation of mannose receptor and Ym1-expressing granuloma macrophages in close contact with <i>S. mansoni</i> eggs depends on their IL-4Rα signalling.

<p>Livers were collected at 8 weeks p.i. from <i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice and immunofluorescent stainings performed on cryosections. (A) Representative micrographs of MMR (panels v–viii), Ym1 (panels xiii–xvi), CD204 (panels xxi–xxiv) and iNOS (panels xxix–xxxii) stainings of liver cryosections as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#s2" target="_blank">Methods</a>. Stainings with secondary antibody (MMR, iNOS), streptavidin alone (Ym1) or isotype-control (CD204) is shown for each corresponding staining (MMR = i–iv, Ym1 = ix–xii, CD204 = xvii–xx, iNOS = xxv–xxviii). Note that MMR⁺ and Ym1⁺ cells are restricted to the periphery of <i>LysM^creIl4ra^−/lox</i> granulomas. Outlined regions represent the areas magnified in B. (B) Representative micrographs of liver cryosections stained with scavenger receptor (CD204) for macrophages detection (panels i–viii, green) or iNOS for classically activated macrophages detection (panels ix–xvi, green); and co-stained for MMR (panels i–iv and ix–xii, red) or Ym1 (panels v–viii and xiii–xvi, red). Note the low frequency of CD204⁺ macrophages co-expressing MMR⁺ or Ym1⁺ cells around the parasite eggs (panels ii and vi) but the high levels of iNOS⁺ cells (panels x and xiv) in <i>LysM^creIl4ra^−/lox</i> mice, suggesting these macrophages to be classically activated. White arrows indicate the parasite eggs. Original magnification: 400×. Data represent one of three independent experiments.</p

IL-10 signalling drives mannose receptor and Ym1 expression in macrophages independently of their IL-4Rα expression.

<p>Peritoneal cells were harvested 7 days after injection of 3000 <i>S. mansoni</i> purified eggs in the peritoneum of <i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>or <i>Il4ra^−/−</i> mice. (A) Representative histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages gated on FSC^highSSC^lowCD11b⁺F4/80⁺ cells after exclusion of peritoneal eosinophils (FSC^lowSSC^highCD11b⁺F4/80⁺) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#pntd.0000689-Taylor1" target="_blank">[37]</a>. Black, isotype control; red, <i>Il4ra^−/lox</i>; blue, <i>LysM^creIl4ra^−/lox</i>; green, <i>Il4ra^−/−</i>. Broken lines show threshold for positive signal. Data are representative of two independent experiments of pooled samples (<i>n</i> = 3). (B) Anti-IL-10 receptor treatment. Four mice out of eight in each group received 4µg of anti-IL-10R i.p. at day 0, 4 and 6 post-injection. Overlays of representative monoparametric histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages are shown as described in A. Bracketed line indicates positive signal. Dotted line, isotype control; greyscale, untreated mice; bold line, mice treated with anti-IL-10R. (C) Percent and mean fluorescent intensities of peritoneal macrophages expressing MMR or Ym1 based on flow cytometry analysis in B. Data is representative of two independent experiments with mean±SEM (<i>n</i> = 3). *<i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Mannose receptor and Ym1 expression in granuloma macrophages of <i>LysM^creIl4ra^−/lox</i> mice.

<p><i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice were infected with 100 <i>S. mansoni</i> cercariae, killed 8 weeks later and liver granuloma-associated leukocytes purified. (A) CD11b⁺I-A/I-E^highCD204⁺ granuloma macrophages were sorted to high purity (>98%) and RNA extracted before analysis of expression levels of genes associated with macrophage activation by real time RT-PCR: IL-4Rα (<i>Il4ra</i>), nitric oxide synthase (<i>Nos2</i>), arginase 1 (<i>Arg1</i>), FIZZ1 (<i>Retnla</i>), macrophage mannose receptor (<i>Mrc1</i>), and Ym1 (<i>Chi3l3</i>). Relative expression levels normalized to r12S house-keeping gene are shown as fold-change over <i>Il4ra^−/lox</i> mice values. Data is representative of two independent experiments with analysis of pooled samples (<i>n</i> = 4). Bars show mean±SD of analyses performed in triplicates. (B) 4-colour flow cytometry analysis of granuloma leukocytes gated on CD11b⁺I-A/I-E^highCD204⁺ cells. Representative monoparametric histograms show stainings of surface macrophage mannose receptor (MMR) or intracellular Ym1 expression. Data is representative of four independent experiments of pooled samples (<i>n</i> = 4). Greyscale histograms show relevant isotype control. (C) Mean fluorescent intensities of MMR and Ym1 stainings based on analysis by flow cytometry as presented in B. Data show results of individual mice for 2 independent experiments. (D) 4-colour flow cytometry analysis of granuloma leukocytes gated on CD11b⁺I-A/I-E^high cells. Representative contour plots show MMR/Ym1 double staining and outlined regions show MMR-positive staining. Data is representative of two independent experiments of pooled samples (<i>n</i> = 4). (E) 4-colour flow cytometry analysis of granuloma leukocytes. Analysis gate (R1) was placed on SSC^highCD11b⁺MMR⁺7-AAD⁻ and histograms show isotype IgG2a (upper panel) and anti-IL-4Rα (lower panel) stainings. Data is representative of two independent experiments of pooled samples (<i>n</i> = 4). Black, <i>Il4ra^−/lox</i>; grey, <i>LysM^creIl4ra^−/lox</i>. * <i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Trypanosome infected IgM^−/− mice produce compensatory IgD titers.

<p>Sera were collected from <i>T. brucei</i> AnTat 1.1E infected WT and IgM^−/− BALB/c mice, and were analyzed as serial dilutions (log 10) in a solid phase VSG coated ELISA, using isotype-specific antibodies for detection. Sera were collected throughout the first peak and clearance phase from 5 individual mice. Values are presented as means.</p

The role of IgM in VSG-specific protection.

<p>(A) BALB/c WT (□), IgM^−/− (▴) and µMT (▪) mice were infected with 5000 pleomorphic AnTat 1.1E parasites. (B) BALB/c WT mice were infected with 5000 monomorphic AnTat 1.1 (•) or MITat 1.4 (▒) parasites. (C) AnTat 1.1E infected BALB/c WT (□) mice were super-infected (S.I.) on day 6 or (D) on day 10 with 5000 parasites of a homologous, monomorphic AnTat 1.1 (•) or a non-homologous monomorphic, MITat 1.4 (▒) strain. (E) AnTat 1.1E infected BALB/c µMT (□) and (F) BALB/c IgM^−/− (□) mice were super-infected with AnTat 1.1 (•) or MITat 1.4 (▒) parasites using the same strategy described above in C–D. (G) BALB/c WT (□) and BALB/c ^nu/nu (▾) mice were infected with 5000 AnTat 1.1E parasites and (H) were super-infected AnTat 1.1 (•) or MITat 1.4 (▒) parasites using the same strategy described above in C–D. All primary and super-infections were done by intra-peritoneal inoculation of 5000 parasites. Mortality was recorded using 10 mice per experimental group and the results were compared to mice that only received the primary infection. One out of 3 representative experiments is shown.</p

Quantitative analysis of VSG switching.

<p>Switching of VSG expression was followed by real time RT-PCR, by amplification of peak stage parasite RNA with primers specific for VSG AnTat 1.1E and the housekeeping genes tubulin-zeta (<i>Tubulin</i>). (A) RNA was isolated from WT derived and (B) µMT derived parasites on the first and second peak, respectively occurring on day 6 and 14. Expression of VSG AnTat 1.1E specific RNA on the second peak was calculated and compared to the first peak, after normalization of the first and second peak VSG AnTat 1.1E specific RNA to tubulin-zeta gene expression.. One representative of two experiments are shown. (C) Similarly, total VSG mRNA was amplified from first and second peak µMT derived parasites, using VSG-Uni primers, and results were normalized using <i>Tubulin</i> gene expression. (D) To assess parasite proliferation in µMT mice, the occurrence of dividing ‘long slender’ parasites versus intermediate and non-dividing ‘short stumpy’ parasites was analyzed by light microscopy on day 4 and compared to the chronic infection stage (up to day 22).</p

Trypanosomiasis-associated anemia is present in µMT mice.

<p>(A) C57BL/6 WT (○) and µMT (•) mice were infected with a pleomorphic <i>T. brucei</i> AnTat 1.1E clone by intra-peritoneal inoculation of 5000 parasites. (B) BALB/c WT (□), µMT (▪) and IgM^−/− (▴) mice were infected by intra-peritoneal inoculation of 5000 with the pleomorphic AnTat 1.1E clone. RBC counts were followed by microscopy analysis of tail-cut blood samples. Values are presented as mean±SD of 10 individual mice per group.</p

Cellular composition of liver granulomas depending on macrophage/neutrophil or T cell-specific IL-4Rα deletion.

<p>Cellular composition of liver granulomas depending on macrophage/neutrophil or T cell-specific IL-4Rα deletion.</p