A novel assay of antimycobacterial activity and phagocytosis by human neutrophils

Abstract

Despite abundant evidence that neutrophils arrive early at sites of mycobacterial disease and phagocytose organisms, techniques to assay phagocytosis or killing of mycobacteria by these cells are lacking. Existing assays for measuring the antimycobacterial activity of human leukocytes require cell lysis which introduces new bioactive substances and may be incomplete. They are also time-consuming and carry multiple risks of inaccuracy due to serial dilution and organism clumping. Flow cytometric techniques for measuring phagocytosis of mycobacteria by human cells have failed to adequately address the effects of organism clumping, quenching agents and culture conditions on readouts. Here we present a novel in-tube bioluminescence-based assay of antimycobacterial activity by human neutrophils. The assay yields intuitive results, with improving restriction of mycobacterial bioluminescence as the ratio of cells to organisms increases. We show that lysis of human cells is not required to measure luminescence accurately. We also present a phagocytosis assay in which we have minimised the impact of mycobacterial clumping, investigated the effect of various opsonisation techniques and established the correct usage of trypan blue to identify surface-bound organisms without counting dead cells. The same multiplicity of infection and serum conditions are optimal to demonstrate both internalisation and restriction of mycobacterial growth.

Figure 1

Restriction of mycobacterial luminescence by neutrophils is inversely proportional to multiplicity of infection and requires viable cells. a. Luminescence of BCG-lux (Relative Light Units, RLU) at one hour post-inoculation according to MOI. Column heights represent the mean results from nine separate donors (neutrophils isolated by MicroBeads) performed in triplicate for each MOI; error bars indicate standard deviation (SD). The inoculum was standardised to 200,000 RLU (80,000 CFU) and the number of cells was varied as indicated. The serum control contained no neutrophils. Overall p-value for one-way ANOVA < 0.0001. b. Luminescence of BCG-lux using higher ratio of neutrophils to organisms. Column heights represent the mean results from six different donors (neutrophils isolated by Percoll gradient) performed in triplicate for each donor; error bars indicate SD. Other experimental conditions as in (a). c. Luminescence of M. tb-lux using same MOI as in (b). Column heights represent mean results from four separate donors (neutrophils isolated by MicroBeads) performed in triplicate for each donor; error bars indicate SD. Other experimental conditions as in (a). d. 24 h luminescence readings from same experiments presented in (a). Overall p-value for one-way ANOVA < 0.0001. e. and f. Luminescence of BCG-lux (200,000 RLU/80,000 CFU inoculum) incubated in RPMI-1640 with either 10% serum only (‘Serum’), serum plus viable neutrophils at MOI 0.17:1 (‘Viable cells’) or serum plus neutrophils pre-heat-shocked at 60 °C for 20 min at MOI 0.17:1 (‘Dead cells’); luminescence was measured at 1 h (e) or 24 h (f). Markers represent the mean of triplicate readings per condition (3 donors).

Figure 2

Lysis or washing of cells results in artefactual increases in luminescence only. a. Saponin treatment permeabilises neutrophils. Samples of 400,000 neutrophils in 10% autologous serum were treated with 1 ml 0.1% saponin (red line) or 1 ml PBS (blue line) for 30 min and then incubated with 5 mcl propidium iodide for 20 min before acquisition on the flow cytometer. Figure shows granulocyte gate as set by forward and side scatter and propidium iodide signal (detected in PE-Cy7 channel). b. Saponin appears to increases luminescence in cell samples, but this effect is mediated by PBS alone. 50 mcl BCG-lux (400,000 RLU (130,000 CFU)) was inoculated into either 400 mcl RPMI-1640 plus 50 mcl autologous serum (‘serum’) or 400 mcl neutrophil suspension (MOI = 1 CFU:3 neutrophils) in RPMI-1640 plus 50 mcl autologous serum (‘Cells’). After one hour's incubation samples were allowed to cool and either vortexed and placed immediately in the luminometer (‘immediate’) or had 1 ml 0.1% saponin added, vortexed and incubated for 30 min before measurement in the luminometer (‘saponin’) or had 1 ml PBS added, vortexed and incubated for 30 min before measurement in the luminometer (‘PBS’). Column heights represent mean values from four separate experiments, performed in triplicate at each occasion; error bars represent SD. p-values from paired t-tests. NS = Not significant. c. Ratio of cell luminescence to serum luminescence does not change depending on condition. Ratio of cell:serum luminescence was calculated on samples from (b), also including measurements on serum and cell samples immediately after the addition of PBS or saponin. p > 0.05 across all conditions. Column heights represent means, error bars represent SD. d and e. CFU show similar pattern to RLU readings. RLU (left-hand columns, small checks), and colony forming units (right-hand columns, large checks) in 500 mcl samples containing 10% autologous serum and the number of neutrophils as indicated on the x-axis, initially inoculated with 200,000 RLU (80,000 CFU) BCG-lux. RLU and CFU were measured after 1 h (d) or 24 h (e) incubation at 37 °C. Column heights represent means from two separate experiments, performed in triplicate for each condition; error bars represent SD. * = p < 0.05 versus serum control. Overall p-values for one-way ANOVA: 1 h RLU, 0.019; 1 h CFU, 0.019; 24 h RLU, 0.027; 24 h CFU, 0.010. Above each pair of columns is the mean RLU:CFU ratio for that MOI and time point. f. There is no greater residual luminescence in cell versus serum samples after replacing culture medium. Samples containing 200,000 RLU (80,000 CFU) BCG-lux, 10% autologous serum and either no neutrophils (‘Serum’) or 400,000 neutrophils (‘Cells’) were incubated at 37 °C for one hour. Sample luminescence was either measured directly or culture medium was pipetted away and 500 mcl fresh RPMI-1640 added before measurement (‘Serum pipetted’ and ‘Cells pipetted’). Results represent three separate experiments, performed in duplicate for each condition; p-values derived from paired t-tests.

Figure 3

Gating strategy and interpretation for phagocytosis assay. a. First, doublet signals are excluded by plotting forward scatter area versus forward scatter height. b. Dead cells are excluded on the basis of positivity for eFluor450 Fixable viability dye (signal seen in Pacific Blue channel); note that most dead cells are also positive for trypan blue (signal seen in APC channel). c. Neutrophils are defined as positive for PE-conjugated CD66a, c, e and high side scatter. d. Neutrophils are divided into quadrants on the basis of trypan blue signal (APC) and FITC signal (Alexa Fluor 488). Q1 (FITC positive, trypan blue negative): Internalised organisms only; Q2 (FITC positive, trypan blue positive): Internal and external organisms; Q3 (FITC negative, trypan blue positive): External organisms only; Q4: (FITC negative, trypan blue negative): not associated with organisms.

Figure 4

Assessment of clumping. a. A Forward Scatter versus Side Scatter plot of BCG-lux organisms (with 10% serum in RPMI-1640) demonstrates how organisms can mimic cells; the granulocyte gate was derived from a contemporaneous cell sample from the donor of the serum. b. Confocal microscopy image of a clump of BCG organisms attached to a neutrophil; note that the green fluorescence of individual external organisms has been ‘quenched’ by trypan blue and they now fluoresce red (arrows), while the clump of organisms remains green. Nuclei are stained with Hoechst 33342. c. Samples of BCG-lux at different concentrations in the presence of human serum in RPMI-1640 were processed identically to cell-containing samples and results acquired on the flow cytometer. The number of events in these organism-only samples seen inside a granulocyte gate derived from a contemporaneous cell sample (on the basis of forward and side scatter) are here expressed as a percentage of the number of viable neutrophils in that cell sample; note the logarithmic y-axis and hence results are not shown if they are zero. Each marker represents one donor; lines represent medians. Overall p-value for Kruskal-Wallis test = 0.0028. d. Orthogonal Confocal microscopy images confirm in three dimensions that organisms (here GFP-expressing BCG-lux) are internalised and, as it typical at an MOI of 1 CFU:6 cells, most neutrophils only contain one or two organisms. Nuclei are stained with Hoechst 33342. e. Samples of 8 × 10⁴ CFU FITC-labelled BCG-lux were prepared in 1 ml PBS with 12.5 mcl trypan blue (red line) or without trypan blue (blue line), centrifuged and fixed in 2% paraformaldehyde before acquisition on the flow cytometer. Signal in the Alexa-Fluor 488 (FITC) and APC (trypan blue) channels is represented for both samples; trypan blue treatment results in near-total loss of FITC signal and increase in APC signal.

Figure 5

Determining optimal multiplicity of infection to demonstrate phagocytosis. Percentage of viable neutrophils with internalised organisms or ‘organism associated’ (includes those cells with external organisms only) according to infecting inoculum of pre-opsonised FITC-labelled BCG-lux. The x-axis is a log₁₀ scale, and inocula used for these experiments are indicated. Expressed as MOI (CFU:cells), 1.7 × 10⁴ CFU = 1:30; 8 × 10⁴ CFU = 1:6; 1.7 × 10⁵ CFU = 1:3; 1.7 × 10⁶ RLU = 3:1. Markers represent mean of six experiments, error bars represent standard deviation. Neutrophils isolated by MicroBeads.

Figure 6

Trypan blue stains dead and prolonged fixed cells. a–c. Concordance of eFluor450 Fixable viability dye (fluorescence detected in the Pacific Blue channel) and trypan blue (fluorescence detected in APC channel). Samples of 400,000 neutrophils without organisms were stained with 12.5 mcl trypan blue and 1 mcl eFluor450 Fixable viability dye only; a – freshly isolated cells; b – cells heat-shocked at 60 °C for 20 min; c – sample containing half freshly isolated and half heat-shocked cells. Plots include all singlet events. d. A sample of neutrophils containing FITC-labelled BCG organisms was acquired immediately (blue) and the following day (red); note the shift on the trypan blue axis (APC channel) but little change in the FITC (Alexa-Fluor 488) axis.

Figure 7

Investigation of opsonisation. a. Internalisation and external binding of FITC-labelled BCG-lux by viable MicroBead-isolated neutrophils according to opsonisation. All conditions performed with MOI 1 CFU:3 cells and incubated for 30 min. ‘Pre-opsonised’ – organisms were incubated with autologous serum for 20 min at 37 °C before infection of cells. ‘Non-pre-opsonised’ – organisms and serum were added at the same time. ‘No serum’ – organisms were added without serum. Each marker represents one donor; lines represent means. Overall p-value for one-way ANOVA < 0.0001. b. Plot of FITC signal in viable neutrophils from one donor incubated (1 CFU:3 cells) with labelled organisms for 30 min. Blue line: organisms pre-opsonised for 20 min with autologous serum; red line: organisms pre-opsonised for 20 min with heat inactivated fetal calf serum (contemporaneous sample).