Generation and Characterization of PTX3-/- Mice

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SUPPLEMENTARY INFORMATION

Generation and characterization of PTX3-/- mice

The targeting vector consisted of a genomic DNA fragment of 8.5 kb encompassing exons 1 through 2 of the mouse ptx3 gene with an IRES (internal ribosomal entry site)-LacZ cassette followed by the PGK-neomycin resistance gene from the pWH9 plasmid (provided by R. Fassler, Lund University, Sweden) integrated in exon 1, 71 bp downstream of the first coding ATG. All methods for culture, selection, and identification of R1 ES cells (kindly provided by A. Nagy, Toronto, Canada) were performed as described 1. Five independently targeted R1 ES cell clones were identified by Southern blot hybridization, using probe A (EcoRI/EcoRV 750 bp fragment) (supplementary figure 1, panel a) after digestion of genomic DNA with EcoRV. No evidence for random integration was detected with the probe B (from the neomycin resistance gene) after digestion with Kpn-1 (supplementary figure 1, panel a). Two of the 5 homologous recombinant Embryonic Stem (ES) cell clones of 235 tested were injected into C57Bl/6 blastocysts to generate germ line chimeras. For genotyping of mice, DNA derived from tail biopsies was amplified by polymerase chain reaction with two primers sets (1: 5’ –AGCAATGCACCTCCCTGCGAT-3’, 5’ –TCCTCGGTGGGATGAAGTCCA-3’; 2: 5’ –CTGCTCTTTACTGAAGGCTC-3’, 5’ –TCCTCGGTGGGATGAAGTCCA-3’) that detected the wild type and targeted allele, respectively. Phenotypic analysis was performed on the two lines derived from independent clones, and results were confirmed in a 129/Sv C57Bl/6 mixed and 129/Sv inbred genetic background. PTX3 +/+ mice were littermates of 129/Sv C57Bl/6 PTX3 -/-, or 129/Sv or C57Bl/6 mice obtained from Charles River, Calco, Italy. PTX3 deficiency at the mRNA and protein level was assessed 4 h after LPS (1 or 300 µg/mouse intravenously or intraperitoneally) or hIL-1 (1 µg/mouse intravenously) treatment 2: LPS or IL-1-treated wild type (PTX3 +/+) mice expressed PTX3 mRNA (supplementary figure 1, panel c) and PTX3 levels rose from undetectable to about 200-400 ng/ml in plasma (supplementary figure 1, panel d). In contrast PTX3-deficient mice showed no expression of the transcript (supplementary figure 1, panel c) and undetectable protein (supplementary figure 1, panel d). Microscopic examination of organs and tissues of young or adult mice (ten months old) did not reveal morphological abnormalities. Complete peripheral blood cell counts and microscopic examination of blood smears of PTX3-deficient mice (n=7) did not show statistically significant differences compared to PTX3 +/+ animals. Heterozygous females and males are normal and fertile and breeding yielded the predicted number of homozygous null mice at a mendelian frequency. However, unexpectedly, when we tried to generate PTX3 deficient mice by breeding homozygous females and males, we observed a severe defect in female fertility.C1q-/- mice were generated as described 3. Procedures involving animals and their care were conformed with institutional guidelines in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international law and policies (EEC Council Directive 86/609, OJ L 358,1,12-12-1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996). All efforts were made to minimize the number of animals used and their suffering.

PTX3 protein purification

PTX3 was expressed in CHO cells and purified under endotoxin-free conditions by immunoaffinity 4. Purified PTX3 was checked for purity by SDS-PAGE and for LPS contamination by Limulusamebocyte lysate assay (Bio-Whittaker) (<124pg LPS/mg protein) and by lack of stimulation of IL-6 production in monocytes. Mock preparation (supernatants of CHO cells transfected with the empty vector and non-PTX3 containing fractions from the purification procedure) had no activity in vitro and in vivo in the assays used in the present study.

Cells

Human monocytes, T cells, monocyte-derived DC (IL-13 and GM-CSF), umbelical vein endothelial cells, fibroblasts and epithelial cells were purified and cultured as described 5-7. Mouse macrophages were obtained by lavage of the lungs or peritoneal cavity of mice injected with thioglycollate 2,8. Mouse DC were generated from CD34+ bone marrow cells or from lungs as described 9,10. The mouse endothelial line 1G11 was cultured as described 11.

Cytokine production and DC activation

For CCL2 production, monocytes were cultured with inactivated A. fumigatus conidia at a ratio of 1:1 or LPS (100 ng/ml) in the presence or absence of 2 µg/ml PTX3 (corresponding to 4.4 x10-8 M) for 24 hours.

DC were cultured in vitro in the presence of A. fumigatus conidia and 0.44 x 10-6 M PTX3 for 24h. IL-12 was measured by ELISA, and MHC-II and CD86 by FACS.

Infections

Invasive Pulmonary Aspergillosis(IPA).The growth and culture conditions of A. fumigatus conidia were as described 12. For intratracheal (i.t.) injection, mice were anesthetized by i.p. injection of 2.5% avertin (Sigma-Aldrich), and a volume of 80 l saline containing 2 x 108 resting conidia (>95% viable, as determined by serial dilution and plating of the inoculum on Sabouraud dextrose agar) were injected under direct vision through the open vocal cords using a 25 gauge metal catheter connected to the outlet of a micropipette. Mice succumbing to fungal challenge were routinely autopsied for histopathological confirmation of IPA. For histology, tissues were excised and immediately fixed in formalin. Sections (3-4 m) of paraffin-embedded tissues were stained with the periodic acid-Schiff or Gomori-Grocott procedures. Cecal ligation and puncture and L. monocitogenes infection were performed as described13,14

Cecal ligation and puncture was performed as described13. Briefly, 8-10 mice per group were anesthetized with 2.5% avertin, the cecum was exteriorized through a laparatomy wound, isolated with a ligation immediately below the ileocecal valve, punctured once with a 25 gauge needle and then returned to the peritoneal cavity. Mice were rehydrated with 1-2 ml of Ringer lactate solution and in some experiments treated with clindamycin (40 mg/Kg) and gentamycin (3.2 mg/Kg) once a day subcutaneously for 4 days (Supplementary figure 2). Mortality was evaluated daily.

L. monocitogenes infection has been performed as described14. Briefly, L. monocitogenes grown overnight in TSB (Oxoid), extensively washed in 0.9% NaCl, counted in a hemocytometer, brought to the described concentrations and injected intraperitoneally in 4-5 mice per group in a final volume of 0.5 ml (Supplementary figure 2). Mortality was evaluated daily for 30 days after infection.

P.aeruginosa (ATCC 10145) pulmonary infection was performed by i.t. injection of 107 bacteria as described for IPA. Mortality was evaluated twice a day.

Phagocytosis and conidiocidal assays.

For phagocytosis, alveolar macrophages (2 x 105cells/200 µl) obtained by plastic adherence from the bronchoalveolar lavage fluid, were incubated at 37° C for 2h with 106 conidia in 6 ml polypropylene tubes (Falcon), in 200 µl of Iscove medium containing 5 µg/ml polymixin B (Sigma) and 50 µg/ml gentamycin but no FCS. Phagocytosis was evaluated on Diff-Quik stained cytospin preparations, as described 12. For the conidiocidal activity, 106macrophages were incubated with 105 conidia for 4 h in 96-well flat-bottomed microtiter plates, as described 15. In selected experiments, 20 µg/ml PTX3 was added. Triton X-100 was then added to the wells, and serial dilutions from each well were made in distilled water. Pour plates (four to six replicate samples) were made by spreading each sample on Sabouraud glucose agar. The number of CFU was determined after 18 h of incubation at 37°C, and the percentage of CFU inhibition (mean ± SE) was determined as follows: percentage of colony-forming inhibition = 100 - ((CFU experimental group/CFU control cultures) x 100). Control cultures consisted of A. fumigatus cells incubated without effector cells.

Legend to supplementary figures

Supplementary figure 1 Targeting of the ptx3 gene

(a). The targeting vector, the ptx3 wild type allele and the homologous recombinant allele are shown. Numbered exons (white blocks), introns (thick line), probes (lettered black blocks), restriction fragments, primers (numbered arrows) are shown. The targeting vector contained a 6 kb SacI fragment, an IRES-lacZ and a PGK-neomycin resistance cassette cloned in the first coding exon (exon I,) and a 2.5 kb SacI/EcoRI fragment.

(b). Southern blot of EcoRV-digested genomic DNA hybridized with probe A (EcoRI/EcoRV fragment), generating a 10 Kb wild type restriction fragment and a 3.5 Kb homologous recombinant restriction fragment.

(c). Northern blot of heart (H), skeletal muscle (SM), liver (L) from PTX3+/+ and -/- mice at 4 h after treatment with 1µg hIL-1 intravenously. 10 µg of total RNA was used in each lane. Ethidium bromide stain of the gel is shown in the lower panel.

(d). PTX3 plasma levels in PTX3+/+ and -/- mice in basal condition and 6-8-16 h after LPS treatment (300 µg/mouse intraperitoneally).

Supplementary figure 2. Normal susceptibility of PTX3 -/- mice to CLP and L. monocytogenes in different experimental conditions.

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