Lung anti-pneumococcal immunity is restored in chimeric Mincle KO mice reconstituted with the hematopoietic system of WT mice.

<p>Mincle KO mice received whole body irradiation (8 Gy), followed by transplantation with bone marrow cells (10⁷ cells/mouse i.v.) from either WT mice (WT onto KO, white bars), or Mincle KO mice (KO onto KO, transplantation controls, black bars). Seven weeks later, mice were infected orotracheally with 10⁷ CFU/mouse of type 19F <i>S</i>. <i>pneumoniae</i>. (A-C) Determination of BAL fluid cellular constituents (A, alveolar macrophages; B, alveolar exudate macrophages; C, alveolar recruited neutrophils) under baseline conditions (CL), or in response to infection, as indicated. (D) Analysis of Mincle expression on the cell surface of alveolar recruited neutrophils in bronchoalveolar lavage from <i>S</i>. <i>pneumoniae</i>-infected (24 h) Mincle KO mice reconstituted with WT (solid lines) or Mincle KO bone marrow cells (dashed lines). Grey histogram, isotype-stained negative control. (E,F) Determination of CFU counts in BAL fluids (E) or lung tissue (F), as indicated. (G-K) BAL fluid levels of proinflammatory cytokines TNFα (G), KC (H), IL-1β (I), or anti-inflammatory cytokines IL1ra (J), and IL-10 (K) in WT onto KO mice, or KO onto KO mice, as indicated. The data are shown as mean ± SD of n = 5 mice (A-F) or n = 4 mice (G-K) per time point and treatment group, and are representative of two experiments (Mann-Whitney U test).</p

Characterization of glycolipid Glc-DAG purified from <i>S</i>. <i>pneumoniae</i>.

<p>(A) NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with live <i>S</i>. <i>pneumoniae</i> at MOI 0.2, 2, 20 and 200 for 18 h followed by determination of the percentage of GFP-expressing reporter cells by flow cytometry. (B) FcRγ or Mincle+FcRγ expressing NFAT-GFP reporter cells (4 x 10⁴ cells/well) were incubated with aqueous or C:M fraction of <i>S</i>. <i>pneumoniae</i> lysates for 18 h, followed by determination of GFP-expressing reporter cells by flow cytometry. (C) HPTLC result of HPLC fractionated C:M portion of pneumococcal lysates with putative ligand indicated by an arrow head (copper acetate stain). (D) FcRγ (white bars) or Mincle + FcRγ (black bars) NFAT-GFP reporter cell assay of HPLC fractions obtained from scratched and purified HPTLC bands of <i>S</i>. <i>pneumoniae</i> lysates shown in (C). Experiments were repeated three times with similar results. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006038#ppat.1006038.s002" target="_blank">S2 Fig</a>. (E-H) Effect of TDM (E), or <i>S</i>. <i>pneumoniae</i>-derived Glc-DAG (F), or synthetic Glc-DAG (C14:0/C14:0, (G), or C18:0/C18:0, (H)) to trigger GFP reporter activation in Mincle/FcRγ (black dots), or FcRγ only (white dots) expressing NFAT-GFP reporter cells.</p

Expression of Mincle in the lungs of mice after infection with <i>S</i>. <i>pneumoniae</i>.

<p>(A) WT mice were infected with <i>S</i>. <i>pneumoniae</i> (1 x 10⁷ CFU/mouse) or were mock-infected (PBS), and lungs were harvested at the given time points and Mincle mRNA levels were analyzed. Values are shown as mean ± SD (n = 3 mice per time point and treatment group). CL (control): Mock-infection relative to untreated control, all other values in (A), relative to mock-infection. * p<0.05, relative to mock-infection. (B) Flow sorted alveolar macrophages and neutrophils purified from lung tissue of mock- versus <i>S</i>. <i>pneumoniae</i>-infected WT mice were subjected to Mincle mRNA analysis by real-time RT-PCR. Data are shown as mean ± SD (n = 4–6 mice for mock-infected and n = 5 mice for <i>S</i>. <i>pneumoniae</i>-infected mice). * p<0.05 relative to neutrophils. (C) Mincle mRNA levels in neutrophils collected by bronchoalveolar lavage from the lungs of patients with pneumococcal pneumonia, relative to peripheral blood neutrophils collected from the same patients. Data are shown as mean ± SD (n = 3 patients). * p<0.05 relative to PB neutrophils. (D-J) WT mice were left untreated (D, and CL in H-J), or were infected with <i>S</i>. <i>pneumoniae</i> (10⁶ CFU/mouse). At 0 h, 24 h, 48 h, and 72 h post-infection, expression of Mincle was analyzed on BAL AM (D (0 h), E (24 h), and H), and BAL ExMacs (F (48 h), and I), and BAL neutrophils (G (48 h), and J) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006038#ppat.1006038.s001" target="_blank">S1 Fig</a>). Note that CL in (I,J) represents Mincle expression on resident alveolar macrophages (I) and neutrophils (J) purified from lung tissue of mock-infected WT mice. Horizontal bars: median values (n = 5–8 mice). Experiments were repeated two times with similar results. ** p<0.01, *** p<0.001, relative to mock-infection ((Mann-Whitney U test). ExMacs, exudate macrophages.</p

Impact of Mincle deficiency on lung proinflammatory cytokine release and macrophage necrosis in mice infected with <i>S</i>. <i>pneumoniae</i>.

<p>WT and Mincle KO mice were mock-infected or infected with <i>S</i>. <i>pneumoniae</i> (10⁷ CFU/mouse), and at indicated time points post-infection, BAL fluid TNF-α (A), KC (B), IL-1β (C), IL-10 (D), and IL-1ra (E) cytokine levels were measured. Data are shown as mean ± SD of n = 5–8 mice per time point and treatment group (12 h, n = 4 mice). Data are representative of two independent experiments with similar results. (F) WT and Mincle KO mice were mock-infected or low-dose infected with <i>S</i>. <i>pneumoniae</i> (5 x 10⁵ CFU/mouse). At the indicated time points, the percentage of necrotic macrophages (propidium iodide^pos/annexin V^neg) in BAL fluid was determined by flow cytometry. Values are shown as mean ± SD with n = 3 (0 h values) or 8 mice (12 and 24 h values) per time point and treatment group and are representative of two experiments. *p<0.05, **p<0.01, ***p<0.001 relative to WT mice. (Mann-Whitney U test).</p

Mincle is an essential receptor for Glc-DAG purified from <i>S</i>. <i>pneumoniae</i>.

<p>(A,B) AM were collected from the lungs of untreated WT and Mincle KO mice, or were collected by bronchoalveolar lavage from healthy volunteers (C,D). After adherence purification, murine or human AM were seeded into vehicle (isopropanol), or Glc-DAG coated (5 μg/well), or TDM coated (1 μg/well) 96-well plates (7.5 x 10⁴ cells/well), or were stimulated with LTA (dissolved in sterile water) at 1 μg/ml, as indicated. After 24 and 48 h (A,B), or after 24 h (C-F), murine (A,B, E,F) or human (C,D) TNF-α or IL-1ra release was determined in cell culture supernatants by ELISA. Data are shown as mean ± SD of 4 determinations per time point and treatment condition and the data are representative of two independent experiments. In (C,D), cytokine values are depicted as mean ± SD of quadruplicate determinations per time point and treatment condition collected from human AM of one healthy individual, with similar data obtained from two other healthy individuals. *p<0.05, **p<0.01 compared to vehicle treated AM and +p<0.05, ++p<0.01 compared to AM from WT mice (unpaired t-test).</p