Selected CLRs, their microbial ligands, and the microbes that have altered ability to infect in the absence of the receptor within mice and humans.

<p>A selection of CLRs are listed with identified microbial ligands and phenotypes observed with different pathogens in both mice and humans, where ↑ denotes enhanced susceptibility of the host and ↓ denotes enhanced resistance.</p>*<p>DC-SIGN is a human molecule with related homologues in the mouse; mouse phenotypes described are for SIGNR3 knockouts.</p>**<p>Exact ligand on DV is unknown.</p>***<p>These experiments were performed using blocking antibodies to CLEC5A. Abbreviations: HIV, human immunodeficiency virus; ManLAM, mannosylated lipoarabinomannan; TDM, trehalose-6, 6′-dimycolate.</p

The role of SR-B1 <i>in vivo</i> following low dose infection with Mtb.

<p>Wild type C57/BL6 and SR-B1^−/− mice were infected with 100 CFU <i>Mycobacterium tuberculosis</i> H37Rv by aerosol route and sacrificed after 2 and 4 months. (A) Body weight was monitored throughout the course of the experiment and is presented as % of original body weight (time of infection) with the SEM shown as solid lines for wild type animals and dashed lines for SR-B1^−/− mice. Lungs of infected animals were analysed at 2 and 4 months for histopathology by H&E staining (B). Also shown is a lung section showing the presence of clefts of accumulated cholesterol that were observed in some inflammatory foci in infected SR-B1^−/− mice. Lung sections after 4 months of infection were further anlaysed by morphometric analysis to calculate the sizes of the inflammatory lesions (C). Lung homogenates were analysed at 2 and 4 months for bacterial burden (D), as well as the production of TNF, IFNγ, IL10, IL12p70 and IL6 (E) with the black circles representing wild type and the open circles representing SR-B1^−/− animals. Shown are the data from individual mice and the median value.</p

Characterization of BCG and Mtb binding to SR-B1 transfected cells.

<p>R6F cells stably transfected with the indicated constructs were incubated with either BCG-lux (left panels) or Mtb-lux (right panels), and quantified for binding by measuring luciferase activity. Experiments were performed in triplicate and normalised to cell number by CFSE staining. (A) Quantification of mycobacterial binding to SR-B1 expressing cells (white bars) or vector control cells (black bars) in the presence or absence of serum, as indicated. Shown is the x-fold increase of luciferase activity compared to vector control which was set as 1. (B) Mycobacteria (BCG-lux or Mtb-lux) were pre-incubated in medium with or without 10% serum, prior binding to R6F-SR-B1 cells also in the presence or absence of 10% serum. Shown is % of luciferase activity relative to binding to R6F-SR-B1 cells in the absence of serum, which was set at 100%. (C) Effect of LDL on binding of mycobacteria to R6F cells expressing SR-B1 or SIGNR1, as indicated. Shown is % of luciferase activity relative to R6F-SR-B1 or R6F-SIGNR1 cells, respectively, in the absence of additives. (D) Effect of BAL fluid and serum (as a control) on binding of BCG-lux or Mtb-lux to SR-B1 expressing R6F cells. Shown is % of luciferase activity relative to R6F-SR-B1 in the absence of additives. (E) Effect of anti-SR-B1 antibodies on binding of BCG-lux or Mtb-lux to SR-B1 or SIGNR1 transfected R6F cells. The white bars show % of luciferase activity relative to binding in the absence of antibody (black bars). (F) SR-B1 expressing R6F cells were incubated with mycobacteria in the presence of increasing concentrations of MβCD. Binding of BCG-lux is shown as % of luciferase activity relative to control (no MβCD).</p

The role of SR-B1 <i>in vivo</i> following high dose infection with Mtb.

<p>Wild type C57/BL6 and SR-B1^−/− mice were infected with 1000 CFU <i>Mycobacterium tuberculosis</i> H37Rv by aerosol route and sacrificed after 4 months. (A) Body weight was monitored throughout the course of the experiment and is presented as % of original body weight (time of infection) with the SEM shown as solid lines for wild type animals and dashed lines for SR-B1^−/− mice. Lungs of infected animals were analysed for bacterial burden (B), TNF, IFNγ, IL10, IL12p70 and IL6 (C), histopathology (D) and inflammatory lesion size (E), with the black circles representing wild type and the open circles representing SR-B1^−/− animals. Shown are the data from individual mice and the median value. *, p<0.05.</p

The role of SR-B1 in mycobacterial recognition in primary cells.

<p>(A) Western Blot assessing expression of SR-B1 in different macrophage populations derived from wild type C57/BL6 and SR-B1^−/− mice, as indicated. (B) Alveolar macrophages and BMDmØ isolated from wild type (black bars) and SR-B1^−/− mice (white bars) were tested for BCG-lux binding in the absence of serum. Shown is the relative luciferase activity (R.L.U.), normalised to cell number by CFSE staining. (C) TNF production at 4 hr or 24 hr after binding of BCG-lux to alveolar macrophages or BMDmØ isolated from wild type (black bars) and SR-B1^−/− mice (white bars). Shown is x-fold increase in TNF production relative to control (no BCG binding). Experiments were normalised to cell number by CFSE staining. (D) Survival of BCG-lux in infected BMDmØ isolated from wild type (solid lines) and SR-B1^−/− mice (dashed lines). After infection of macrophages, unbound mycobacteria were removed and samples taken after the indicated time points to assess luciferase activity. Experiments were performed in triplicate, normalised to cell number by CFSE staining and shown as R.L.U. relative to time point 1.</p

Identification of SR-B1 as a receptor for mycobacteria.

<p>NIH3T3 cells stably transfected with empty vector pFBneo (negative control), SR-B1 or SIGNR1 (positive control), respectively, were incubated with BCG-GFP (A) or BCG-lux (B) and assessed for binding by fluorescence microscopy or luciferase activity, respectively. Shown is the x-fold increase of luciferase activity compared to vector control which was set as 1. Experiments were performed in triplicate and normalised to cell number by CFSE staining. (C) Western Blot showing expression of SR-B1 in various untransfected cell lines as indicated. Cellular lysates from rat liver were included as a control, and GAPDH served as loading control. (D) FACS analysis of R6F cells stably transduced with pFB (vector control), SR-B1 or SIGNR1, and stained with anti-SR-B1 or anti-SIGNRI, as indicated. (E) FACS assay showing binding/uptake of DiI-LDL to R6F cells stably expressing pFB (vector control), SR-B1 or SIGNR1. (F) Binding of FITC-labelled zymosan to R6F cells stably expressing pFB (vector control), SR-B1 or SIGNR1, as quantified by fluorometry. Shown is the x-fold increase of fluorescence compared to vector control which was set as 1. Experiments were performed in triplicate and normalised to cell number by CFSE staining.</p

The effect of cholesterol in SR-B1^−/− mice during Mtb infection.

<p>Wild type C57/BL6 and SR-B1^−/− mice were fed either a low cholesterol diet (LC, 0.15% cholesterol) or a high cholesterol diet (HC, 1.25% cholesterol) throughout the experiment, infected with 100 CFU <i>Mycobacterium tuberculosis</i> H37Rv by aerosol route and sacrificed after 4 months. (A) Levels of serum cholesterol following 2 weeks on the various diets, as indicated, prior to infection (black bars representing wild type, and white bars the SR-B1^−/− animals). (B) Average mouse weight throughout the course of the experiment, presented as % of original body weight ±SEM. 4 months after infection the mice were analyzed for serum cholesterol (C), histopathology (D), bacterial burdens (E), and pulmonary TNF, IFNγ, IL10, IL12p70 and IL6 (F) with the black circles representing wild type and the open circles representing SR-B1^−/− animals. Shown are the data from individual mice and the median value. *, p<0.05.</p

Inhibition of dectin-1 and -2-mediated fungal recognition by prior complement opsonization.

<p><b>A)</b> Representative flow cytometric plots of dectin-2 surface expression on NIH3T3 retrovirally transduced with dectin-2 (left panel) and both dectin-2 and FcεRγ chain (right panel). <b>B)</b> Serum-opsonization inhibits the recognition of zymosan by both dectin-1 and dectin-2. NIH3T3 retrovirally transduced as indicated in the legend were incubated with zymosan that had been pre-incubated with serum at increasing concentrations and the interaction was monitored by flow cytometry. Data represents the mean±SEM of triplicates from a representative of 2 independent experiments. Data in (D) was analyzed by two-way ANOVA, with the significant Interaction statistics demonstrating the reduced binding of zymosan to the transduced cells after prior incubation with increasing concentrations of serum.</p

Assessment of dectin-2 function on inflammatory cells. A)

<p>Flow-cytometric plots showing expression of dectin-2 on the surface of inflammatory neutrophils (Ly-6G⁺) and monocyte/MØ (Ly-6G⁻). Data is pre-gated on Ly-6B.2⁺ cells and is representative of results obtained with pooled cells from 3 independent experiments. <b>B)</b> Assessment of the impact of blockade of dectin-2 (D2.11E4) on the recognition (upper graphs) and response to (lower graphs) zymosan of inflammatory neutrophils and monocyte/MØ from dectin-1-deficient mice. All bars show mean±95% confidence intervals of dectin-2-blocked cells relative to isotype control treated cells (100%, dotted line) from 3 independent experiments (raw representative data, which includes the isotype control data, from one of the 3 independent experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045781#pone.0045781.s002" target="_blank">Figure S2</a>). Data were analysed by two-way ANOVA, two assess the differences caused by complement opsonization (‘C’) or the use of different cell lineages (‘L’). Samples in which the 95% confidence intervals do not overlap with the mean isotype control are specific indicated with a # symbol.</p

Characterization of high dose zymosan peritonitis. A)

<p>Representative flow-cytometric density plots showing the recruitment of inflammatory cells and the clearance of fluorescent (FITC)-labelled zymosan particles. The examples shown are taken from 18 hours after the administration of zymosan. Cells were identified as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045781#s2" target="_blank">methods</a>. The panel on the left shows the gating of Ly-6B⁺ cells, which includes (right) Ly-6G^high neutrophils and Ly-6G⁻ monocytes and MØ. Both neutrophils and monocytes/MØ exhibit association with FITC-labeled zymosan at this time point (right panel). <b>B)</b> Graphical representation of the number of neutrophils (left) and all types of monocyte (Mo) and MØ combined (right) in the peritoneal cavity before and after acute zymosan peritonitis. n = 3 129S6/SvEv mice per group and is representative of 2 independent experiments. Data is shown as mean±SEM and cells were isolated from naïve animals (0 hours), and challenged mice 4, 18, 72 and 168 hours after induction of peritonitis. <b>C)</b> Graphical representation of the percentage of neutrophils and Mo/MØ present that are associated with zymosan, scored by flow-cytometry as indicated in (A) above. <b>D)</b> Photomicrograph, which is representative of neutrophils associated with zymosan in a 4 hour inflammatory infiltrate. Those neutrophils that are associated tend to have multiple particles, but only represent a minority of the total neutrophils present at this time (see (C) above).</p