Recombinant murine NEU3 expression
Chinese hamster ovary (CHO-K1) cells were cultured in CDM4CHO medium with l-glutamine (Cat# SH30557.02, Hyclone/Cytiva, Marlborough, MA, USA). Cells (1 × 105) were mixed with 2 μg of 100 μg/ml of murine NEU3 expression plasmid (MR223297; Origene, Rockville, MD, USA) in 100 μl PBS (GE Lifesciences, Marlborough, MA, USA) and were transfected by electroporation using a 4D-Nucleofector System (Lonza, Walkersville, MD, USA) following the manufacturer’s protocol. Before use, the plasmid was sequenced for verification as previously described [25, 27,28,29]. The transfected cells were kept at room temperature for 15 min for recovery, after which the CHO-K1 cells were cultured in 25 ml CDM4CHO medium in a humidified incubator at 37 °C with 5% CO2. After 24 h, 400 μg/ml of G418 (345812; Calbiochem EMD, San Diego, CA) was added to select for transfected cells. After 10 days, the cells were collected and lysed, and c-Myc–tagged recombinant murine NEU3 was purified using a Myc-Trap agarose kit (ytak-20; Chromotek, Hauppauge, NY, USA) following the manufacturer’s protocol. Recombinant protein was checked for protein concentration by OD 260/280/320 using a Take3 micro-volume plate with a SynergyMX plate reader (BioTek, Winooski, VT, USA). NEU3 is 51 kDa, and was further purified by centrifugation at 10,000×g for 5 min at 4 °C through an Amicon Ultra 0.5 ml 100 kDa cutoff spin filter (Millipore, Billerica, MA, USA). The NEU3 was analyzed for size and purity by PAGE on, and Coomassie staining of, 4–20% Tris/glycine gels (Bio-Rad, Hercules, CA, USA), as described previously [25, 27,28,29]. The NEU3 was stored in 50 μl of 10% glycerol, 100 mM glycine, and 25 mM Tris–HCl, pH 7.3, at 4 °C.
Recombinant murine inactive NEU3 variant generation
Starting with the murine NEU3 expression plasmid MR223297, a variant mutated to change the tyrosines at positions 179 and 181 to phenylalanines was generated using a QuikChange II Site-Directed Mutagenesis Kit (#200523; Agilent, Santa Clara, CA, USA) and the primer 5′ CATCCCCGCCTTCGCCTTCTATGTCTCACGTTGG 3′, with the underscored sequences representing the point mutation sites. Other workers found that these two mutations eliminate NEU3 sialidase activity . The resulting plasmid was sequenced to confirm the point mutations and absence of other mutations.
Cell isolation and culture
Human peripheral blood was collected from healthy volunteers who gave written consent with specific approval from the Texas A&M University human subjects review board (IRB2009-0671D). All methods were performed in accordance with the relevant guidelines and regulations. Blood collection, isolation of peripheral blood mononuclear cells (PBMC), and cell culture were done as described previously [22, 23, 25, 30]. Murine spleen cells were isolated by forcing diced spleen fragments through a 100 μm cell strainer (BD Biosciences, San Jose, CA, USA) using the plunger of a 1 ml syringe (BD Medical, Franklin Lakes, NJ, USA), as described previously . To determine the activity of native and mutated NEU3 we assayed the ability of NEU3 to induce extracellular accumulation of IL-6 from human PBMC and murine spleen cells [23, 25], using human (BioLegend) and murine (PeproTech, Cranbury, NJ, USA) IL-6 ELISA kits following the manufacturers’ protocols.
Mouse models of pulmonary inflammation and fibrosis.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Texas A&M University Animal Use and Care Committee (IACUC 2020-0272). All procedures were performed under 4% isoflurane in oxygen anesthesia, and all efforts were made to minimize suffering. Animals were housed with a 12-h/12-h light–dark cycle with free access to food and water, and all procedures were performed between 09:00 and noon. To induce inflammation and fibrosis, 7–8 week old 20–25 g male and female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were given an oropharyngeal aspiration of 3 U/kg (equivalent to 0.06 U/20 g mouse) bleomycin (2246–10; BioVision Incorporated, Milpitas, CA, USA) in 50 µl of 0.9% saline or oropharyngeal saline alone, as a control, as previously described [22, 23, 25, 32]. Starting 10 days after saline or bleomycin had been administered, some of the mice received an oropharyngeal aspiration of 15 ng of recombinant (rec) murine NEU3 or mutated NEU3 in 50 µl of 0.9% saline, or saline, every 48 h. An additional cohort of mice received only 15 ng of recombinant murine NEU3, mutated NEU3, or saline, every 48 h over the course of 21 days. Independent sets of animal experiments were performed three times over the course of 6 months. Three male and three female mice that did not receive saline, bleomycin, or NEU3 were defined as naïve mice. All the mice were monitored twice daily to observe any sign of distress. At the indicated time points, mice were euthanized by CO2 inhalation, and bronchoalveolar lavage fluid (BALF) and lung tissue was obtained and analyzed as described previously [22, 23, 25, 32, 33].
Staining of bronchoalveolar lavage fluid (BALF) cells
BALF cells were counted and processed to prepare cell spots as described previously [22, 23, 25, 32]. After air drying for 48 h at room temperature, some of the cell spots were fixed and immunochemistry was performed as described previously [22, 23, 25, 32]. Briefly, slides with cell spots were incubated with antibodies diluted to 5 µg/ml in PBS containing 2% IgG-free BSA (BSA-50, Rockland Immunochemicals, Pottstown, PA, USA). Antibodies included anti-CD3 (NB600-1441, rabbit clone SP7, Novus Biologicals, Centennial, CO, USA) to detect T-cells, anti-CD11b (101202, clone M1/70, BioLegend, San Diego, CA, USA) to detect blood and inflammatory macrophages, anti-CD11c (M100-3, clone 223H7, MBL International, Woburn, MA, USA) to detect alveolar macrophages and dendritic cells, anti-CD45 (147702, clone I3/2.3, BioLegend) for total leukocytes, anti-Ly6g (127602, clone 1A8, BioLegend) to detect neutrophils, with isotype-matched irrelevant rat (BioLegend) and rabbit (Novus Biologicals) antibodies as controls. After washing six times with PBS for 5 min each, the slides were incubated at room temperature for 30 min with 1 µg/ml biotinylated donkey anti-rat (NBP1-75379, Novus) or biotinylated donkey F(ab′)2 anti-rabbit (711-066-152, Jackson Laboratories, West Grove, PA, USA) secondary antibodies. After washing, the biotinylated antibodies were detected with a 1/500 dilution of ExtrAvidin alkaline phosphatase (SA-5100-1, Vector Laboratories, Newark, CA, USA). Staining was developed with the Vector Red Alkaline Phosphatase Kit (SK-5100, Vector), following the manufacturers’ protocols. Slides were then counterstained with Gill’s hematoxylin (Sigma-Aldrich). Using a 40 × objective, at least 150 cells from each stained BALF spot were examined and the percent positive cells was recorded.
After collecting BALF, the lungs from the mice were harvested and inflated with Surgipath frozen section compound (#3801480, Leica, Buffalo Grove, IL, USA), frozen on dry ice, and stored at − 80 °C. 10 μm cryosections of lungs were placed on Superfrost Plus glass slides (VWR) and were air dried for 48 h. Immunohistochemistry was done as previously described [22, 23, 25, 32] and as detailed above using anti-CD3, anti-CD11b, anti-CD11c, anti-CD45, anti-Ly6g, or anti-Mac2 (clone M3/38; BioLegend) antibodies with isotype-matched irrelevant rat and rabbit as controls. Positively stained cells were counted from randomly selected fields, and presented as the number of positive cells per mm2, as described previously [22, 23, 25, 32]. Lung sections were also stained with Sirius red to detect collagen and analyzed as previously described [23, 25, 34, 35]. Briefly, images were converted to RGB stacks using Image J, and then the green channel (which shows the red staining) was analyzed. Intensity of staining was determined using the threshold tool, which was kept the same for analyzing each set of images. The area stained as a percentage of the total area of the lung tissue was then determined using Image J. Other investigators and ourselves have shown that histological analysis of fibrosis and collagen content by sirius red staining, correlates well with collagen content as measured by antibody staining and hyproxyproline and sirius red (Sircol) assays [23, 25, 34,35,36,37,38]. Lung sections were also stained using rabbit polyclonal anti-collagen VI antibodies (NB120-6588, Novus Biologicals), as described previously [34, 39]. Lung sections stained with picrosirius red were assessed histologically for fibrosis, using the Ashcroft system, as described previously [38, 40].
Statistical analysis was performed using Prism Version 7.05 (GraphPad Software, La Jolla, CA, USA). Statistical significance between two groups was determined by t test, or between multiple groups using analysis of variance (ANOVA) with both Bonferroni’s and Sidak’s post-tests, and significance was defined as p < 0.05.