The affinity columns, IgG Sepharose and anti-properdin monoclonal antibody Sepharose, were prepared as described previously (7). The IgG-Sepharose column was prepared from human non-immune IgG (~26 mg IgG/ml of Sepharose) coupled to CNBr-activated Sepharose (GE Healthcare, UK). For preparation of the anti-human properdin column, CNBr-activated Sepharose (GE Healthcare Life Sciences, UK) was used to couple to anti-properdin mouse monoclonal antibody (2 mg/ml). One liter of human plasma (TCS Biosciences) containing 5 mM EDTA was filtered through Whatman filter paper before applying to IgG Sepharose to deplete C1q (which would otherwise have bound to the IgG on the anti-properdin Sepharose). The column was washed with three bed volume of HEPES buffer (10 mM HEPES, 140 mM NaCl, 0.5 mM EDTA, and pH 7.4). Plasma was then applied to the monoclonal anti-properdin column and washed with the same HEPES buffer. Bound properdin was eluted with 3 M MgCl₂ and the peak fractions were dialyzed against HEPES buffer overnight at 4°C. Contaminants were further removed by applying the pooled protein fractions to a HiTrap Q FF-Sepharose (GE Healthcare) ion-exchange column, followed by washing the column with three bed volumes of 50 mM Tris–HCl, pH 7.5, 50 mM NaCl, and 5 mM EDTA. Properdin did not bind to the Q Sepharose column and appeared in the flow-through free from contaminants as demonstrated by SDS-PAGE.

For the size exclusion chromatography analysis, 50 µl of the proteins at the concentrations varying from 0.3 mg/mL to 1.0 mg/mL were applied to a TSKgel G2000SWXL, 5 µm, 7.8 × 300 mm column (Tosoh Bioscience). The column was equilibrated with buffer containing 50 mM sodium phosphate, pH7.0 and 300 mM NaCl at the flow rate 0.3 ml/min using SCL-10Avp HPLC system (Shimadzu). The absorbance was detected at 230 and 280 nm. The Bio-Rad Gel Filtration Standard (Cat # 151-1901) were used for the protein molecular weight calibration of the column.

The recombinant maltose-binding protein (MBP) fusion proteins MBP-TSR4+5, MBP-TSR4, or MBP-TSR5 were expressed in Escherichia coli as described previously (7, 29). The E. coli BL21 bacterial cells (Life Technologies) were grown in 1 L of Luria–Bertani medium with 100 µg/ml ampicillin, shaking at 37°C until an optical density at 600 nm (OD₆₀₀) of between 0.6 and 0.8 was reached. Protein expression was then induced in the bacterial cell culture with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) for 3 h shaking at 37°C. The cells were then pelleted at 4,500 rpm, 4°C for 10 min, lysed using 50 ml lysis buffer [20 mM Tris–HCl, pH 8.0, 0.5 M NaCl, 1 mM EDTA, 0.25% v/v Tween 20, 5% v/v glycerol, 100 µg/ml lysozyme (Sigma-Aldrich), and 0.1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich)], and incubated for 1 h at 4°C on a rotary shaker. The cell lysate was then sonicated using a Soniprep 150 (MSE, London, UK) at 60 Hz for 30 s with an interval of 2 min (12 cycles) and then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was diluted five-fold with buffer A (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.25% v/v Tween 20) and passed through an amylose resin column (25 ml bed) (New England Biolabs) that was equilibrated in buffer A. The affinity column was washed with buffer A without Tween 20 and with 1 M NaCl, 20 mM Tris–HCl, pH 8.0, 1 mM EDTA, followed by buffer B (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). The MBP-TSR4+5 fusion protein was eluted with 100 ml of buffer B containing 10 mM maltose (Sigma-Aldrich) (affinity elution buffer). Trace contaminants were further removed by applying the fusion protein to a DEAE Sepharose column. Thus, the affinity purified fusion protein in affinity elution buffer was applied to the ion-exchange (5 ml bed) column and washed with three column volumes of low salt buffer containing 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, at pH 7.5. After extensive washing with low salt buffer, the fusion protein eluted at 0.2 M NaCl using a NaCl gradient (50 mM to 1 M). The peak elutions were then passed through Pierce™ High Capacity Endotoxin Removal Resin (Qiagen) to remove LPS. Endotoxin levels were determined using the QCL-1000 Limulus amebocyte lysate system (Lonza), and the assay was linear over a range of 0.1–1.0 EU/ml (10 EU = 1 ng of endotoxin). The endotoxin levels were less than 4 pg/µg of the MBP-TSR4+5.

Mycobacterium bovis BCG (Pasteur strain) were grown in liquid culture using Middlebrook 7H9 media (Sigma-Aldrich), supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween-80, and 10% (v/v) albumin dextrose catalase (ADC) (BD BBL, Becton Dickinson). Green fluorescent protein (GFP)-expressing M. bovis BCG (Danish Strain 1331) containing the pGFPHYG2 plasmid was a kind gift from B. Robertson, Imperial College, London, UK. GFP-M. bovis BCG was grown in the above conditions/media but with the addition of 50 µg/ml of hygromycin to maintain the plasmid. Cultures were incubated at 37°C with agitation (~120 rpm) for 7–10 days until the bacteria had reached the exponential growth phase at OD_600nm = 0.60–1.00.

Mycobacterium bovis BCG, harvested and washed in PBS, was adjusted to a concentration of 1.25 × 10⁹ cells/ml in PBS (OD₆₀₀ = 1 equates to approximately 1 × 10⁹ cell/ml). Then 200 µl of bacterial suspension was dispensed into individual microtiter wells of a 96-well plate (Maxisorp™, NUNC). Plates were incubated at 4°C overnight and washed with buffer 1 [10 mM HEPES pH 7.5, 140 mM NaCl, 0.5 mM EDTA, and 100 µg/ml hen ovalbumin (Sigma-Aldrich)]. Wells were blocked for 2 h at 37°C with buffer 1 + 10% (w/v) Marvel Dried Milk powder.

Human properdin (up to 50 µg/ml) or TSR4+5 (up to 30 µg/ml) were added, in two-fold serial dilutions (100 µl/well) in buffer 1 and incubated for 2 h at 37°C. Individual TSR4 and TSR5 proteins, MBP and BSA were used as negative controls. Microtiter wells were washed three times with buffer 1. Mouse anti-properdin monoclonal antibody (1.19 mg/ml) diluted 1/2,500 in buffer 1 (29) was added to the wells containing properdin. Mouse anti-MBP monoclonal antibody (Sigma-Aldrich) was added to wells containing TSR4+5, TSR4 and TSR5, diluted 1/5,000 in buffer 1, and incubated for 1 h at 37°C. For the BSA negative control, mouse anti-BSA monoclonal antibody (Sigma-Aldrich) was used (1/5,000 dilution). Plates were washed an additional three times in buffer 1 and then incubated with goat anti-mouse IgG-horseradish peroxidase conjugate (Sigma-Aldrich), diluted 1/5,000 in buffer 1. The substrate p-nitrophenol phosphate (Sigma-Aldrich) was then added to each well, and the plates read at 405 nm.

Mycobacterium bovis BCG bacteria (approximately 10⁶ cells) were spotted on poly-l-lysine coated microscope slides (Sigma-Aldrich) and incubated at 37°C for cells to adhere. After washing three times with PBS, bacterial cells were then fixed with 4% paraformaldehyde for 5 min. Slides were washed three times with PBS and then incubated at 37°C for 1 h with 0, 1, or 10 μg/ml of TSR4+5, or 10 µg/ml of BSA (negative control) in buffer 1. Slides were washed three times with PBS, and then the primary monoclonal antibody (mouse anti-MBP) added at 1/500 dilution and incubated for 1 h at room temperature. After washing three times with PBS, goat anti-mouse conjugated with AlexaFluor488 (1/500 dilution) was added as the secondary antibody and incubated for 1 h at room temperature. Slides were then washed three times with PBS and mounted with antifade (Citifluor AF3) PBS solution and viewed using a LeicaDM4000 Fluorescence microscope. Images were processed using Image J.¹

THP-1 macrophage cells were cultured in RPMI-1640 (Gibco) (RPMI) containing 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), 100 µg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow in 5% CO₂ at 37°C for approximately 3 days before passaging. Cells were resuspended in RPMI and adjusted to 1 × 10⁶ cells/well (in 1.8 ml) in a 24-well plate. To induce adherence onto the wells, THP-1 cells were treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) into RPMI-1640 without FBS, penicillin or streptomycin and left to settle for at least 30 min before adding 200 µl of bacterial culture (1 × 10⁹ bacteria/ml).

M. bovis BCG bacteria were pelleted at mid-exponential phase, at an OD_600nm = 0.6–1.0 by centrifugation at 1,000 × g for 10 min at 4°C. The mycobacterial pellet was resuspended in the buffer 1. This mycobacterial culture was then separated into different microfuge tubes and treated with varying concentrations of properdin (2 or 20 µg/ml) or MBP-TSR4+5 (1 or 5 µg/ml). Control samples were left untreated, and all were incubated for 2 h at 37°C for binding to occur. The mycobacterial suspension was washed once in growth medium before resuspending in RPMI medium without FBS, penicillin, or streptomycin. 200 µl of the mycobacterial suspension was added to each well of THP-1 cells. Mycobacterial concentration was adjusted to give approximate multiplicity of infection (MOI) ratio of 10:1.

Plates were gently swirled and incubated at 37°C, 5% CO₂ for up to 48 h to allow mycobacterial uptake. THP-1 cells were sampled at 15, 30, and 45 min and 1, 2, and 6 h. Supernatants were collected after 24 and 48 h of incubation for multiplex analysis. Plates were washed three times with PBS to remove extracellular bacteria. THP-1 cells were then lifted by adding 1 ml of 0.25% trypsin to the wells and incubated for 10 min at 37°C, 5% CO₂. THP-1 cells were collected by centrifugation at 1,000 × g for 10 min at 4°C.

To recover and count the ingested mycobacteria, THP-1 cells were lysed by resuspending the cell pellets in 1 ml of sterile water, followed by a series of vortex mixing for 10 min at room temperature. 24-well plates containing 2 ml Middlebrook 7H10 agar with 10% Oleic Acid+ADC (OADC) (BD, BBL, Becton Dickinson) were prepared. Four serial 1/10 dilutions were made, and 10 µl of the concentrated mycobacterial suspension and diluted suspension from each time point was spotted onto the 7H10 agar wells. The 24-well plates were secured with parafilm and wrapped in aluminum foil, inverted and incubated at 37°C for 10–14 days. Wells were photographed, and the colony-forming unit (CFU) count determined. The same procedure was used to quantify the initial input number of bacteria incubated with THP-1 cells.

THP-1 cells were cultured as described above and seeded at 1 × 10⁵ cells per 13 mm coverslip and differentiated with PMA as described above. GFP-expressing M. bovis BCG was incubated with 0, 1, 10 µg/ml of TSR4+5 for 2 h at 37°C in buffer 1. Cells were also incubated with 10 µg/ml of BSA as a negative control. Cells were washed twice in PBS and then resuspended in plain RPMI media. 1 × 10⁶ GFP-M. bovis BCG was added to the THP-1 cells (MOI of 10:1) and incubated for phagocytosis for 2 h at 37°C. THP-1 cells were then washed three times in PBS to remove extracellular bacteria and then fixed in 4% paraformaldehyde for 5 min. After washing three times in PBS, THP-1 cells were incubated with 2 µg/ml of AlexaFluor546-conjugated wheat germ agglutinin (Invitrogen) to reveal the plasma membrane. Cells were then washed three times and mounted using Vectashield antifade with DAPI (Vector Labs) to reveal nucleus. Slides were observed under a Leica DM4000 fluorescence microscope at 40× magnification. Images were processed using Image J (see text footnote 1).

THP-1 cell pellets were collected from each time point as described above. RNA extraction was performed using the GenElute Mammalian Total RNA Purification Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Samples were then treated with DNase I (Sigma-Aldrich) to remove any contaminating DNA according to the manufacturer’s protocol. The amount of RNA was measured using the NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific) at 260 nm, and the ratio of absorbance at 260 and 280 nm was used to assess the purity of the RNA. Complementary DNA (cDNA) was synthesized using High Capacity RNA to cDNA Kit (Applied Biosystems, UK) according to the manufacturer’s protocol. Primer sequences (Table 1) were designed and analyzed for specificity using the nucleotide Basic Local Alignment Search Tool and Primer-BLAST.²

PCR was performed on all cDNA samples to assess the quality of the cDNA. The qPCR assays were performed for the expression of pro- and anti-inflammatory cytokines. The qPCR reaction consisted of 5 µl Power SYBR Green MasterMix (Applied Biosystems), 75 nM of forward and reverse primer, 500 ng template cDNA in a 10 µl final reaction volume. qPCR was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems). The initial steps were 2 min incubation at 50°C followed by 10 min incubation at 95°C, the template was then amplified for 40 cycles under these conditions: 15 s incubation at 95°C and 1 min incubation at 60°C. Samples were normalized using the expression of human 18S rRNA. Data were analyzed using the relative quantification (RQ) Manager Version 1.2.1 (Applied Biosystems). Cycle threshold (Ct) values for each cytokine target gene were calculated, and the relative expression of each cytokine target gene was calculated using the RQ value, using the formula: RQ = 2^−ΔΔCt for each cytokine target gene, and comparing relative expression with that of the 18S rRNA constitutive gene product. Assays were conducted twice in triplicate.

Supernatants were collected from the phagocytosis assay at 24 and 48 h to determine the levels of secreted cytokines (IL-6, IL-10, IL-12p40, IL-12p70, IL-1α, IL-1β, TNF-α, IL-13, IL-15, IL-17A, IL-9, and TNF-β), chemokines (MCP-3, MDC, Eotaxin, Fractalkine, GRO, IL-8, IP-10, MCP-1, and MIP-1α), growth factors (IL-2, EGF, FGF-2, G-CSF, GM-CSF, IL-3, IL-4, IL-5, IL-7, and VEGF), and other related ligands and receptors (IFN-α2, IFN-ϒ, FLT-3L, IL-1RA, and sCD40L). MagPix Milliplex kit (EMD Millipore) was used to measure immune response following the manufacturer’s protocol. 25 µl of assay buffer was added to each well of a 96-well plate, followed by the addition of 25 µl of standard, controls or supernatants of cells treated with M. bovis BCG in the presence or absence of properdin and MBP-TSR4+5. 25 µl of magnetic beads coupled to analytes of interest was added in each well and incubated for 18 h at 4°C. The 96-well plate was washed with the assay buffer, and 25 µl of detection antibodies was incubated with the beads for 1 h at room temperature. 25 µl of streptavidin–phycoerythrin was then added to each well and incubated for 30 min at room temperature with shaking at 750 rpm. Following a washing step, 150 µl of sheath fluid was added to each well, and the plate was read using the Luminex Magpix instrument. Assays were conducted in duplicate.

Analysis of data for statistical significance was conducted using GraphPad Prism 6 for Windows (GraphPad Software, Inc.). Statistical analyses were made using two-way ANOVA for mRNA expression data and a one-way ANOVA for the multiplex data. p Values < 0.05 were considered statistically significant, unless otherwise stated (non-significant).

Human properdin was purified from human plasma. SDS-PAGE, followed by western blotting using antihuman properdin polyclonal antibodies, showed a distinct band at 55 kDa (Figure 1A), which was the expected molecular weight of the glycosylated monomer. The two biologically active modules of properdin TSR4 and TSR5 were expressed together in tandem as previously described, fused to MBP (7), and is also shown on an SDS-PAGE gel, which has a molecular weight of 55 kDa (Figure 1B). Using gel filtration chromatography, we found that human properdin eluted as a mixture of monomer, dimer and trimer; a negligible amount probably formed aggregates. Nearly 60% of MBP-TSR4+5 appeared as a monomer while nearly 40% was found to migrate as a dimer (data not shown).

The binding of properdin to M. bovis BCG was observed to be in a dose-dependent manner; BSA was used as a negative control that showed almost no binding (Figure 2A). TSR4+5 binding was also observed to be in a dose-dependent manner. MBP was used as a negative control (Figure 2B). The two binding curves cannot be compared quantitatively, as different detection antibodies were used. Because the MBP-TSR4+5 recombinant protein and properdin monomer have about the same molecular weight, 5 µg of TSR4+5 corresponds in molar terms to about 5 µg of properdin monomer (Figures 1A,B). The binding of a mixture of the two separately expressed TSR4, and TSR5 is much lower than that of the combined expressed TSR4+5 (Figure 2B). For this comparison, the same detection antibody was used. These results suggest that both TSR4 and TSR5 modules contribute to the interaction with M. bovis BCG, and that TSR4+5 binds with similar characteristics to that of whole properdin on M. bovis BCG surface. These results were further confirmed using microscopy where TSR4+5 specifically bound to M. bovis BCG in a dose-dependent manner (Figure 2C).

Properdin inhibited the uptake of M. bovis BCG by THP-1 cells. At a concentration of 20 µg/ml, uptake of M. bovis BCG was significantly reduced by properdin (Figure 3A). TSR4+5 was also able to substantially inhibit uptake of M. bovis BCG by THP-1 cells (Figure 3B). The effect of properdin and TSR4+5 on the phagocytosis of M. bovis BCG was dose dependent. The input number of M. bovis BCG was about 7.8 × 10⁶ CFU/ml, which was the total number of bacteria added to THP-1 cells. Without properdin or TSR4+5, 5.0 × 10⁶ CFU/ml of M. bovis BCG was phagocytosed which was approximately 66% efficiency of phagocytosis compared with the input number. At the highest concentration tested, properdin showed an inhibition of uptake of approximately 60% compared with M. bovis BCG with no properdin (Figure 3A). For TSR4+5, the effect on M. bovis BCG was slightly lower at approximately 40% inhibition (Figure 3B). These results were also confirmed by microscopy, with TSR4+5 having a suppressive effect on the uptake of GFP-expressing M. bovis BCG by THP-1 cells (Figure 3C). PMA stimulation was used to induce differentiation of THP-1 cells before incubation with M. bovis BCG. PMA has been shown to activate protein kinase C and increase cell adherence and expression of surface markers associated with macrophage differentiation (30). These data show that (i) properdin has an anti-opsonic effect on M. bovis BCG, inhibiting phagocytosis; and (ii) TSR4+5 modules play a major role in this interaction of M. bovis BCG and macrophages. These observations demonstrate, for the first time, a novel, non-complement-related role for properdin in host–pathogen interactions in tuberculosis.

The effect of properdin on the inflammatory response during the phagocytosis of M. bovis BCG was measured. The gene expression of key pro- and anti-inflammatory cytokines in tuberculosis was determined using quantitative real-time PCR. Our data showed that properdin significantly enhanced the upregulation of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 from THP-1 cells challenged by M. bovis BCG (Figure 4A), particularly at the initial stage of uptake (within the first hour of phagocytosis), which decreased gradually toward the later stages of phagocytosis. The increase in TNF-α transcript was particularly striking as TNF-α is well known for activating macrophages for killing of intracellular mycobacteria. In addition, TNF-α is a key mediator in the early stages of granuloma formation. By contrast, the anti-inflammatory cytokines measured from THP-1 cells (IL-10 and TGF-β) were shown to be downregulated in the presence of properdin, when challenged by M. bovis BCG (Figure 4B). IL-12 also appeared to be downregulated (Figure 4A). Properdin, therefore, appears to play an important role in pro-inflammatory cytokine production by macrophages infected by M. bovis BCG, which may have significant implications in shaping the adaptive immune response during M. tuberculosis infection.

Cytokine gene expression by THP-1 cells infected with M. bovis BCG were also studied in the presence of TSR4+5, which revealed that TSR4+5 also has a significant effect on the pro-inflammatory response. TNF-α was upregulated (Figure 5A), while IL-10 was found to be downregulated (Figure 5B), during the first hour of phagocytosis. IL-12 was also shown to be significantly downregulated (Figure 5A). These data mirror the observations for properdin, and hence, validate the importance of TSR4+5 in the binding of properdin to M. bovis BCG and in its modulation of the inflammatory response. These data are also similar to recent published observations of another complement regulatory protein, factor H (19), thus offering potentially novel insights into the involvement of these proteins in host–pathogen interactions in tuberculosis.

The inflammatory response during the phagocytosis of M. bovis BCG by THP-1 cells was further determined by measuring the secretion of cytokines, chemokines, and other growth factors using the Multiplex analysis of supernatants collected at 24 and 48 h post phagocytosis (Figures 6A–D). The secretion of pro-inflammatory cytokines TNF-α, IL-1β, and IL-1α was significantly enhanced by treatment with properdin or TSR4+5 at the 24 h time point (Figure 6A). The enhancement of these pro-inflammatory cytokines can be critical for controlling mycobacterial infection, particularly in the formation of the granuloma. However, by 48 h, there was a decrease in the production of pro-inflammatory cytokines (IL-6, IL-12p40, IL-12p70, IL-1α, IL-1β, TNF-α, IL-13, IL-15, and IL-9) in the presence of properdin- and TSR4+5-treated M. bovis BCG (Figure 6A). Properdin and TSR4+5 also downregulated the anti-inflammatory response such as IL-10 after 24 and 48 h of phagocytosis, although this was less pronounced for IL-12 at 48 h (Figure 6A). These observations again mirror the initial responses observed in cytokine gene expression of during the first few hours of phagocytosis, in the presence of properdin or TSR4+5 (Figures 5A,B). The effect of properdin and TSR4+5 also led to marked downregulation of a number of growth factors MCP-3 (24 h), MDC, Eotaxin, Fractalkine (24 h), GRO (24 h), IP-10, MCP-1, MIP-1, VEGF, G-CSF (48 h), GM-CSF (48 h), and VEGF (24 h) (Figures 6B,C). Additional ligands and receptors (IFN-α2, IFN-γ, FLT-3L, IL-1RA, and sCD40L) did not show any significant changes (Figure 6D).