Qualitative and quantitative histological analysis of the lungs.

<p>Formalin fixed lung tissues were paraffin embedded and sections of 3 µm were cut. These sections were stained with H&E (A) or PAS (B) dyes to determine cellular infiltration and mucus production in lung, respectively. (B) Arrow heads indicate mucus producing goblet cells. (C) Overall lung extension, peri-bronchial and peri-vascular cellular infiltration and interstitial edema were considered as parameters for histological scoring in a double blinded manner. (D) Mucus secretion was assessed by measuring the surface area of mucus-containing goblet cells (Sgc) per total surface of airway epithelial area (Sep) measured. (E) The expression of the MUC5AC gene was quantified using specific primers in a quantitative PCR. Fold increase in the expression of MUC5AC in the positive control (Pos) and experimental (Pos+DT) group are plotted with respect to expression levels observed in the negative control (Neg+DT) group. Expression of GAPDH mRNA was used as internal control for data normalization. Data shown is a representative of three individual experiments using 4–10 mice in each group. Mann Whitney test was used to determine statistical significance. n.s.  =  non-significant.</p

Schematic representation of the experimental set-up.

<p>DEREG-BALB/c mice were sensitized to OVA in presence of alum on day 0 and day 14. Foxp3⁺ Treg depletion was achieved by intra-peritoneal administration of diphtheria toxin (DT) a week post the last sensitization for two consecutive days. Mice were bled through retrobulbar venous plexus one day after the last DT treatment and were analyzed for efficient depletion of Foxp3⁺ Tregs. Negative control (Neg+DT) mice were sham sensitized with PBS in alum and were also treated with DT to rule out any unspecific inflammatory effects. Positive control (Pos) mice were challenged with intra-nasal application of OVA for two consecutive days without any DT treatment. The experimental group of mice (Pos+DT) were challenged with OVA for two consecutive days 24 hr post last DT treatment. Histograms demonstrate the frequency of Foxp3⁺ cells on gated live CD4⁺ T cells, from one representative mouse of each group.</p

Th2 cytokines secretion after selective elimination of Foxp3⁺ Tregs.

<p>Lung draining mediastinal lymph node cells were pooled from each group of mice (n = 4–10) and stimulated <i>ex vivo</i> with OVA. 72 hr post stimulation cell free supernatants were collected and analyzed for Th2 cytokines (IL-4, IL-5 and IL-13) by ELISA using matched antibody pairs. Data shown are a representative of three individual experiments. Histograms represent mean values and error bars represent SD of ELISA replicates performed on four individual stimulations from each group. Mann Whitney test was used to determine statistical significance. *<i>p</i>≤0.05 and n.s. = non-significant.</p

MCMV activates Ly49H⁺ NK cells and expands Ag-specific CD8⁺ T cells from Siglec-H^−/− and wt mice with similar viral clearance in the primary and secondary organs.

<p>Siglec-H^−/− and wt chimeric mice were infected with 5×10⁴ PFU of wt MCMV or mock treated with PBS. (<b>A, B</b>) IFNγ serum concentrations were determined at 36 h p.i. and the MFI of CD69 expression on NK1.1⁺ blood NK cells was quantified by flow cytometry. (<b>C, D</b>) Representative histogram overlays and quantification of KLRG-1 expression on splenic NK1.1⁺ Ly49H⁺ cells at day 8 p.i. (<b>E</b>) Representative FACS plots for H-2D^b M45 tetramer staining and CD62L expression among CD8⁺ splenocytes. (<b>F, G</b>) Quantification of frequencies and absolute numbers of tetramer⁺ CD8⁺ T cells from the tetramer staining shown in (E). (<b>H</b>) Shows frequencies of IFNγ⁺ cells among CD8⁺ T cells after restimulation with the indicated concentrations of H-2D^b M45 peptide. (<b>I</b>) Viral load was measured in the spleen, liver and salivary glands of MCMV infected Siglec-H^−/− and wt mice at day 3, 6 and 8 p.i. Dashed line indicates the limit of detection. Data shown are pooled from 2–3 individual experiments. **<i>p</i><0.01 Students t-test, ns = not significant, nd = not detectable.</p

Characterization of the pDCre x RFP reporter line shows terminal targeting of pDCs.

<p>(<b>A, B</b>) BM and spleen cells from pDCre x RFP mice were stained for Siglec-H, CD11c, MHCII, CD3ε, CD19 and NK1.1 (<b>A</b>) Characterization of the reporter expression (RFP) by different cell types: pDCs were gated as Siglec-H⁺, B cells as Siglec-H⁻ CD19⁺ CD3ε⁻ NK1.1⁻, T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁻, NK-T cells as Siglec-H⁻ CD19⁻ CD3ε⁺ NK1.1⁺, and NK cells as Siglec-H⁻ CD19⁻ CD3ε⁻ CD11c^int NK1.1⁺. FACS plots are representative for two individual experiments. (<b>B</b>) Characterization of the RFP reporter expression by CD11c^hi MHCII^hi cDCs in spleen and CD11c^int CD19⁻ CD3ε⁻ NK1.1⁻ Siglec-H⁻ cells in BM. (<b>C</b>) Quantification of (A, B) showing pooled data from 2 independent experiments using 4–5 mice/group. (<b>D</b>) Phenotypic comparison of pDC markers expressed by RFP⁻ and RFP⁺ pDCs from BM (top panel) and spleen (bottom panel). pDCs were gated as Siglec-H⁺ CD11c^int. Histogram overlays display the isotype controls as dashed line, and the marker expression by RFP⁻ pDCs as grey filled histogram and by RFP⁺ pDCs as bold line. Data shown are from one representative experiment out of two using 4 mice/group. (<b>E</b>) Splenic <i>ex vivo</i> pDCs were purified by FACS sorting from B16-Flt3L treated pDCre x RFP mice and incubated with the indicated MOIs of MCMV <i>in vitro</i>. IFNα/TNFα concentrations were quantified in the supernatants after 24 h incubation by ELISA or cytometric bead assay. Data shown are from one representative experiment out of two using a pool of 3 mice. The differences between RFP⁻ and RFP⁺ pDCs were not significant as calculated by Students t-test. Data displayed in (D, E) are from one out of two individual experiments with similar results. Data are displayed as mean ± SD.</p

IFNα serum levels are elevated in the absence of Siglec-H upon MCMV infection.

<p>Lethally irradiated CD45.1⁺ wt mice were reconstituted with CD45.2⁺ wt or Siglec-H^−/− BM followed by infection with 5×10⁴ PFU of wt MCMV. (<b>A</b>) FACS plots show efficient donor reconstitution in the blood eight weeks after BM transfer (upper panel). Siglec-H and B220 stainings of splenocytes confirm the lack of Siglec-H expression in wt mice reconstituted with Siglec-H^−/− BM (lower panel). (<b>B</b>) The kinetics of serum IFNα levels 1.5, 3 and 6 days p.i. compared between Siglec-H^−/− and wt mice, n = 4–5 mice/group. Data are from one of two individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p

Siglec-H is downregulated upon MCMV infection <i>in vivo</i>.

<p>pDCre x RFP mice were infected with 5×10⁴ PFU MCMV <i>in vivo</i>. (<b>A</b>) Representative FACS plots show CD11c versus Siglec-H of live splenocytes at 36 h p.i. (<b>B</b>) Gating strategy showing exclusion of B-, T-, NK-T, and NK cells by CD19, CD3ε, NK1.1 in a default channel. RFP⁺ pDCs were gated as CD11c^int B220⁺ MHCII^int to exclude MHCII⁻ DC precursors. Histogram overlays show RFP⁺ pDCs from mock (bold line) and MCMV infected (dashed line) pDCre x RFP mice at 36 h p.i. Isotype staining is displayed as grey histogram. Data are representative of 2 independent experiments using 4–5 mice with comparable results.</p

Siglec-H receptor does not play a role in pDC infection.

<p>Flt3-L derived CD11c⁺ B220⁺ pDCs and CD11c⁺ B220⁻ cDCs were sorted from BMDC cultures of wt and Siglec-H^−/− mice. DCs were mock treated with PBS or MCMV-GFP infected at MOI 2. (<b>A</b>) Representative FACS plots show Siglec-H expression and MCMV-GFP expression at 24 h p.i. (<b>B</b>) Quantification of the viral titers per 10⁶ DCs or MEFs at 24 h p.i. (<b>C</b>) Representative histogram plots of CD86 expression on wt and Siglec-H^−/− sorted pDCs (live cell gate) at 24 h p.i. (gray bar = isotype control, gray dashed line = mock treatment, gray solid line = CpG-A treatment, dark bold line = MCMV infection). Data are from one of three individual experiments with similar results. ns = not significant ***<i>p</i><0.001, Students t-test.</p