PTX3 and lupus nephritis in B6<i>^lpr</i> mice.

<p>A: Kidney sections were stained with PAS. Images are representative for at least 12 mice in each group, original magnification ×400. B: The composite activity index of lupus nephritis (0–24) was assessed as described in methods. Data represent means ± SEM of 8–12 mice in each group. C: Urinary albumin/creatinine ratios were determined at 6 months as a functional marker of glomerular damage. D: Renal P-selectin and E-selectin mRNA levels were determined by real-time RT-PCR and are expressed as means of the ratio of the specific mRNA versus that of 18S rRNA ± SEM (n = 12 in each group). E: Neutrophils, macrophages or T lymphocytes numbers were quantify, data represent means ± SEM from at least 12 mice in each group. F: C9 complement staining was performed on kidney sections. No significant differences were detected between the two genotypes.</p

PTX3 and B cell subsets in B6<i>^lpr</i> mice.

<p>A: Flow cytometry was used to determine the total number of distinct B cell subsets in spleens of 6 months old B6<i>^{lpr/Ptx3−/−}</i> and B6<i>^lpr</i> wild type mice. The histogram presents means ± SEM of 8–14 mice in each group. B: Spleens from the same mice were stained for IgM to localize plasma cell areas. The images are representative for 6 mice in each group. Original magnification ×100.</p

Lung damage and gene expression in B6<i>^lpr</i> mice.

<p>A: Peribronchial and interstitial inflammation was scored from 0–4 as described in methods. Data represent means ± SEM of 8–12 mice in each group. B: Neutrophils, macrophages or T lymphocytes numbers were quantify, data represent means ± SEM from at least 12 mice in each group, * p<0.05, **p<0.005, ***p<0.0005 versus B6<i>^lpr</i> mice. C: RNA was isolated from lungs of 6 months old B6<i>^{lpr/Ptx3−/−}</i> and B6<i>^lpr</i> wild-type mice for real-time RT-PCR. Data are expressed as means of the ratio of the specific mRNA from at least 5 mice versus that of 18S rRNA ± SEM. * p<0.05 versus B6<i>^lpr</i> mice. D: Lung sections were stained with anti- P-selectin P. Images are representative for at least 12 mice in each group. Original magnification ×100. E: Protein samples were prepared from lungs of B6<i>^lpr</i> or B6<i>^{Ptx3−/−lpr}</i> mice. PTX3 Western blot indicates the quantitiative (20 µg protein load per lane) P-selectin protein expression. The histogram represents the ratio of P-selectin to the respective β-actin expression. Data represent means ± SEM from three independent experiments.</p

Lack of PTX3 specifically affects T cells subsets in B6<i>^lpr</i> mice.

<p>A: Spleen monocytes, neutrophils, CD11b+ and CD11c+ cells were quantified in B6<i>^{lpr/Ptx3−/−}</i> and B6<i>^lpr</i> wild-type mice by flow cytometry also using surface MHCII and CD40 expression as a marker of cell activation as described in methods. Data represent means ± SEM from 10 mice in each group. B: Serum IL-12p40 levels were determined in 6 months old B6<i>^lpr</i> of both genotypes by ELISA and are shown as dot blot. C: Flow cytometry was used to determine the total number of distinct T cell subsets in spleens of 6 months old B6<i>^{lpr/Ptx3−/−}</i> and B6<i>^lpr</i> wild type mice. The graphs in B–C present means ± SEM of 8–14 mice in each group * p<0.05.</p

PTX3 fosters the phagocytic uptake of apoptotic cells.

<p>A: 2×10⁵ pHrodo-labeled apoptotic cells were injected intraperitoneally into B6<i>lprPtx3</i>-deficient and B6<i>lpr</i> wildtype mice and 45 min later peritoneal lavage fluids where prepared for flow cytometry. Figure A displays the percentage of F4/80+ peritoneal macrophages of all cells in lavage fluids which where not different in B6<i>lpr</i> (black bar) from B6<i>lpr Ptx3</i>-deficient mice (white bar). Figure B displays the number of F4/80+ cells also positive for pHrodo, a dye which gets only activated in the acidic environment of phagolysosomes. Data represent means ± SEM from three independent experiments; * p<0.05 versus wild type. Figure C displays representative dot plots from this experiment. C: Representative density plots of F4/80+Phrodo+ cells in B6<i>lprPtx3</i>-deficient and B6<i>lpr</i> wildtype mice.</p

PTX3 and lung injury in B6<i>^lpr</i> mice.

<p>Lung sections were stained either with PAS or with specific antibodies for neutrophils, macrophages or T lymphocytes. Images are representative for at least 12 mice in each group. Original magnification ×100 (PAS) and ×200 (immunostaining).</p

PTX3 expression in mice.

<p>A: RNA was isolated from organs of 6 week old B6<i>^lpr</i> mice for real-time RT-PCR. Data are expressed as means of the ratio of the specific mRNA versus that of 18S rRNA ± SEM. B: Protein samples were prepared from spleens, kidneys, and lungs of B6<i>^lpr</i> mice at 1, 3, and 6 months of age. PTX3 Western blot indicates the quantitiative (20 µg protein load per lane) PTX3 protein expression in each organ over time. The histogram represents the ratio of PTX3 expression to the expression of the β-actin loading control. Data represent means ± SEM from three independent experiments.</p

PTX3 and lymphoproliferation in B6<i>^lpr</i> mice.

<p>A: Body weight increased similarly over time in <i>Ptx3</i>-deficient or wild type B6<i>^lpr</i> mice. Data are means ± SEM from at least 18 mice in each group. B: At 6 months of age <i>Ptx3</i>-deficient B6<i>^lpr</i> mice revealed splenomegaly and hyperplasia of cervical lymph nodes as compared to age-matched B6<i>^lpr</i> control mice. Quantitative data on spleen and lymphnode weights are means ± SEM from at least 18 mice in each group, * p<0.05 versus B6<i>^lpr</i> mice.</p

SAP enhances the neutralizing activity of SP-D and MBL against WT but not SAP^R Mem/71-Bel.

<p>Dilutions of (i) SAP, SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ alone, or (ii) SP-D and MBL were prepared in TBS supplemented with 10 mM Ca²⁺ and 0.60 µg/ml SAP, and incubated at 37°C for 15 mins to allow for interactions between different proteins. After this time, an equivalent volume of WT or SAP^R Mem71-Bel was added and incubated at 37°C for 30 mins and the amount of infectious virus remaining was determined by fluorescent-focus reduction assay. Final concentrations after incubation with virus were SAP alone (0.6 µg/ml), SP-D alone (0.3 µg/ml), MBL alone (2.5 µg/ml), SAP+SP-D (0.6 µg/ml +0.3 µg/ml) and SAP+MBL (0.6 µg/ml +2.5 µg/ml final). Neutralization data for WT (left panel) or SAP^R Mem/71-Bel (right panel) are expressed as the mean (±1 SD) percent of the number of fluorescent foci in each virus only control and are pooled data from two independent experiments, each performed in triplicate. **p<0.01, ***p<0.001, One-way ANOVA, Tukey’s post hoc.</p

Serum Amyloid P Is a Sialylated Glycoprotein Inhibitor of Influenza A Viruses

<div><p>Members of the pentraxin family, including PTX3 and serum amyloid P component (SAP), have been reported to play a role in innate host defence against a range of microbial pathogens, yet little is known regarding their antiviral activities. In this study, we demonstrate that human SAP binds to human influenza A virus (IAV) strains and mediates a range of antiviral activities, including inhibition of IAV-induced hemagglutination (HA), neutralization of virus infectivity and inhibition of the enzymatic activity of the viral neuraminidase (NA). Characterization of the anti-IAV activity of SAP after periodate or bacterial sialidase treatment demonstrated that α(2,6)-linked sialic acid residues on the glycosidic moiety of SAP are critical for recognition by the HA of susceptible IAV strains. Other proteins of the innate immune system, namely human surfactant protein A and porcine surfactant protein D, have been reported to express sialylated glycans which facilitate inhibition of particular IAV strains, yet the specific viral determinants for recognition of these inhibitors have not been defined. Herein, we have selected virus mutants in the presence of human SAP and identified specific residues in the receptor-binding pocket of the viral HA which are critical for recognition and therefore susceptibility to the antiviral activities of SAP. Given the widespread expression of α(2,6)-linked sialic acid in the human respiratory tract, we propose that SAP may act as an effective receptor mimic to limit IAV infection of airway epithelial cells.</p> </div