Human blood specimens
The procurement of human blood and all experimental procedures were approved by the Human Research Ethics Committee of the Women’s and Children’s Health Network (WCHN), Adelaide, South Australia, in accordance with The National Statement on Ethical Conduct in Human Research (2007, updated 2018) (National Health and Medical Research Council Act 1992). Venous blood was collected from healthy adult volunteers by venipuncture with their informed consent, under approval number HREC/15/WCHN/21.
The mouse monoclonal antibody (clone 3C9, for flow cytometry, 0.2 µg; for western blotting, 1:3000) that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, San Francisco, CA). The rabbit recombinant monoclonal anti-CD11b antibody (ab133357, clone EPR1344, 1:1000), and mouse IgG1 isotype control antibody (ab37355) were purchased from Abcam. The mouse monoclonal anti-CD11c antibody (clone N-19, 1:1000) and goat PE-conjugated anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology. The mouse monoclonal anti-GAPDH (clone 71.1, 1:20,000) was obtained from Sigma-Aldrich. The polyclonal HRP-conjugated rabbit anti-mouse (P0260), anti-goat (P0449) and goat anti-rabbit (P0448) immunoglobulin antibodies (1:2000) were obtained from Dako.
Roswell Park Memorial Institute (RPMI) 1640 tissue culture medium, Hank’s buffered saline solution (HBSS), foetal calf serum (FCS), L-glutamine, penicillin and streptomycin were purchased from SAFC Biosciences. Dithiothreitol (DTT), benzamidine, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), 1α,25-dihydroxyvitamin D3 (1,25D) and 25-dihydroxyvitamin D3 (25D) were purchased from Sigma-Aldrich. Stock solutions of 1,25D and 25D were prepared to 10−3 M in 95% ethanol and stored at −80 °C. Pam3CSK4 was purchased from Invivogen, with stock preparation at 1 mg ml−1 in endotoxin-free water and storage at −20 °C. Aprotinin was purchased from Merck.
Cell preparation and culture
Peripheral blood mononuclear cells (PBMC) or cord blood mononuclear cells were prepared by density gradient centrifugation of blood on Ficoll-Paque PLUS (GE Healthcare). The interface layer containing PBMC was harvested and cells were washed in RPMI 1640 medium. Monocytes were purified from the MC following seeding of the latter at 2 × 107 per autologous plasma-coated 6-cm culture dish (TPP) and incubation at 37 °C, 5% CO2/air, in a high humidity incubator for 2 h. Non-adherent cells were removed by three gentle washes resulting in >90% monocytes purity, and each dish replenished with 4 mL of RPMI 1640 supplemented with 2 mmol L−1 L-glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 10% FCS, pH 7.4. Experiments either utilized total PBMC or purified monocytes. Cells were stimulated with either, 1,25D, 25D, Pam3CSK4, or diluent and cultured for the duration specified in the ‘Results’ section. Cells were harvested after either 3 days (for CRIg mRNA analysis) or 5 days (for CRIg protein analysis or phagocytosis assays) culture by gentle scraping with a ‘rubber policeman’.
Quantitative PCR assays
The quantitative PCR (qPCR) assays were performed as previously described8. In brief, total RNA was extracted from harvested cells using TRIzol reagent (Invitrogen). cDNA was prepared using iScript cDNA synthesis kit (Bio-Rad). qPCR analysis was performed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad) with the following conditions: initial denaturation for 5 min at 95 °C followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s using an iQ5 Real-Time Detection System with iQ5 Optical System v2.1 software (Bio-Rad). Data were normalized to the expression of a control gene GAPDH for each experiment. The primer pairs used were for human CRIg (Forward: 5′-ACACTTATGGCCGTCCCAT-3′; Reverse: 5′-TGTACCAGCCACTTCACCAA-3′), CD11b (F: 5′-CCTGGTGTTCTTGGTGCCC-3′ and R: 5′-TCCTTGGTGTGGCACGTACTC-3′) CD11c (F: 5′-CCGATTGTTCCATGCCTCAT-3′; R: 5′-AACCCCAATTGCATAGCGG-3′), CYP27B1 (F: 5′-TGGCCCAGATCCTAACACATTT-3′) (R: 5′-GTCCGGGTCTTGGGTCTAACT-3′)34 and GAPDH (F: 5′-GAGTCAACGGATTTGGTCGT-3′; R: 5′-GACAAGCTTCCCGTTCTCAGCCT-3′).
Staphylococcus aureus bioparticle uptake quantitation by flow cytometry
Briefly, 1 × 106 macrophages in HBSS with 8% human AB serum, were incubated with 80 µg pHrodo™ Red S. aureus Bioparticles™ (Invitrogen), in a final volume of 400 µL in 12 × 75 mm round-bottom tubes18. These were gassed with 5% CO2/air and capped, with incubation at 37 °C for 1 h. Following washing in HBSS, samples were acquired using a BD FACSCanto I flow cytometer with FACSDiva 8.0, with analysis using FlowJo 10.1 software (FlowJo LLC) to determine bioparticle uptake by changes in median fluorescence intensity in the PE channel. For experiments run in the presence of the anti-CRIg blocking antibody, 1 × 106 macrophages were incubated at room temperature with either 10 μg/mL mouse anti-human CRIg monoclonal antibodies (clone 6H8, Santa Cruz Biotechnology; clone 6C9, Genentech) or mouse IgG1 isotype control antibodies for 15 min prior to conducting the phagocytosis assay as above.
Candida albicans particle uptake quantitation by microscopy
This phagocytosis assay was performed essentially as described previously7,8. Briefly, 1 × 105C. albicans yeast particles were added to 5 × 104 macrophages in a final volume of 0.5 ml HBSS. Complement-containing human AB serum was added to a final concentration of 10%. The cells were incubated for 15 min at 37 °C on a rocking platform. Following removal of unphagocytosed yeast particles by differential centrifugation at 175 × g for 5 min, the remaining macrophages in the pellet were cytocentrifuged onto a microscope slide and stained with Giemsa. The particles in phagocytic vacuoles were enumerated, with phagocytosis was scored as both the number of macrophages that had engulfed >4 fungi as well as the number of fungi engulfed per cell.
Assessing cell surface CRIg and CD11b expression
Macrophage surface CRIg expression was determined by flow cytometry7. Briefly, harvested cells were incubated in 12 × 75 mm round-bottom tubes on ice with 100 μg purified human IgG (Kiovig, Baxter) for 15 min. This was followed by addition of 0.2 µg of either anti-human CRIg or mouse IgG1 isotype control antibodies, with further incubation for 20 min. Cells were washed with 2 mL PBS with centrifugation at 500 × g for 5 min. Goat anti-mouse IgG PE secondary antibody was then added, with continued incubation in the dark on ice for 20 min. For experiments assessing CD11b, 0.2 μg of anti-CD11b (PE) were added to concurrent tubes set up in the absence of anti-CRIg primary antibody. Following washing twice more, the cells were acquired (50,000 event minimum) on a BD FACSCanto I with FACSDiva 8.0, and data analysed using FlowJo 10.1.
Western blotting assays
Protein analysis in harvested macrophages was performed using western blot essentially as previously described8. Lysates were generated from macrophages in each culture dish with 100 µL of buffer containing 20 mmol L−1 HEPES, pH 7.4, 0.5% Nonidet P-40 (v/v), 100 mmol L−1 NaCl, 1 mmol L−1 EDTA, 2 mmol L−1 Na3VO4, 2 mmol L−1 DTT, 1 mmol L−1 PMSF and 1 µg mL−1 of each protease inhibitor, benzamidine, leupeptin and pepstatin A. Total protein in the soluble fractions was quantitated using the Qubit™ Protein Assay Kit on a Qubit 3.0 (Invitrogen), prior to the addition of Laemmli buffer. Samples were boiled at 100 °C for 5 min and 60 µg of protein were subjected to 10% SDS-PAGE at 170 V for ~1 h, using the Mini-PROTEAN 3 system (Bio-Rad). The samples were transferred onto nitrocellulose membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). The extent of protein transfer was ascertained using 0.1% Ponceau S membrane staining. After blocking in TBST with 5% skim milk (blocking solution), the membrane was incubated with either mouse anti-human CRIg, rabbit anti-human CD11b, or mouse anti-human CD11c antibodies in blocking solution overnight at 4 °C. The membrane was washed in blocking solution (3 × 5 min) and then incubated with the appropriate secondary HRP-conjugated antibody (anti-mouse, anti-rabbit or anti-goat IgG) in blocking solution for 1 h at room temperature. Immunoreactive material was detected using the Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer), with protein bands visualized on a ChemiDoc™ XRS+ Imager and quantitated using Image Lab™ Software, Version 3.0 (Bio-Rad). For GAPDH determination, stained membranes were subjected to antibody stripping using ReBlot Plus Mild Solution (Millipore) and incubated with mouse anti-human GAPDH antibody, followed by the staining and visualization steps as described above.
Statistics and reproducibility
Graphpad Prism 8.0 (Graphpad Software) was used for statistical analysis. Mean differences were compared using t-tests (for comparisons of two groups) or one-way ANOVA followed by multiple comparison tests (for comparisons of three or more groups). P values <0.05 were considered to be statistically significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.