Effect of Antiretroviral Therapy on HIV-mediated Impairment of the Neutrophil

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1 Online Data Supplement Effect of Antiretroviral Therapy on HIV-mediated Impairment of the Neutrophil Antimycobacterial Response David M Lowe, Nonzwakazi Bangani, Rene Goliath, Beate Kampmann, Katalin A Wilkinson, Robert J Wilkinson, Adrian R Martineau

2 Supplementary Figure Legends Supplementary Figure E1. Flowchart detailing recruitment, exclusions and loss to follow-up in the case-control study. Supplementary Figure E2. Gating strategy for phagocytosis assay. Singlet signals (derived from Forward Scatter (FSC) Area versus FSC Height), were first gated on efluor450 Viability Dye versus Side Scatter (SSC) to exclude dead cells (not shown). Neutrophils were then defined as cells with high side scatter and high CD66ac,e-PE. Within this population, the percentage of cells which had internalised FITC-labelled M.tuberculosis-lux was defined by Alexa Fluor-488 signal (i.e. Q1 + Q2). Supplementary Figure E3. Gating strategy for cell death assay. First, singlet events were defined on the basis of Forward Scatter Area (FSC-A) versus Forward Scatter Height (FSC-H). All singlet events were gated using FSC versus side scatter area (SSC-A) to define a granulocytes gate or using SSC-A versus the neutrophil surface marker CD66a,c,e-PE to define a total cells gate. These populations were then divided on the basis of efluor450 Viability Dye (Pacific Blue channel) versus Annexin V-FITC (Alexa-Fluor 488 channel). Dual negative events were defined as viable, Annexin V-positive / Viability Dye negative events were defined as apoptotic, Viability Dye positive / Annexin V negative were defined as primary necrotic, and dual positive events were defined as late apoptotic/necrotic. E2

3 Supplementary Figure E1 Participants recruited: HIV infected = 23 HIV uninfected = 22 Samples taken: HIV infected = 22 HIV uninfected = 20 Baseline results included*: HIV infected = 20 HIV uninfected = 20 HIV infected participants completing study = 18 Unable to bleed: HIV infected = 1 HIV uninfected = 2 Excluded, active tuberculosis: HIV infected = 2 Lost to follow-up: After 1 month = 1 After 3 months = 1 * One functional result excluded due to laboratory error E3

4 Supplementary Figure E2 E4

5 Supplementary Figure E3 E5

6 Supplementary Methods Patients and Recruitment HIV-infected patients were recruited from the Ubuntu Site B clinic in Khayelitsha, South Africa if they were eligible for (CD4 count <350 x 10 3 /ml) and agreed to commence antiretroviral therapy. Control patients were recruited from the same community, identified at Ubuntu or the Khayelitsha Youth Centre amongst people who had recently tested negative for HIV. Ethics The studies on HIV-infected participants and HIV-uninfected controls were approved by the University of Cape Town Research Ethics Committee (HREC 545/2010) and all participants provided written informed consent. Organisms and Labelling The plasmid construction and electroporation of organisms has been described previously (E1). 1.5 ml vials of M.tuberculosis-lux stored at -80 o C were defrosted and added to 15ml liquid 7H9 (Becton Dickinson)/ADC (Becton Dickinson) growth medium enriched with 0.05% Tween 80 (Sigma) and 1 μl/ml hygromycin B (Roche diagnostics). Organisms were grown to mid-log phase (72 hours) before use in these assays. Luminescence was measured as previously described (E2, E3). For FITC-labelling, 5 ml of mid-log phase organisms were centrifuged at 2000 x g for 5 minutes and resuspended in 1ml carbonate-bicarbonate buffer (ph 9.6) (sodium hydrogen carbonate, Merck) containing 0.05% Tween 80. 5μl of FITC stock (Sigma) at 100 mg/ml concentration was added and the suspension was incubated at 37 o C E6

7 for 15 minutes. The labelled organisms were centrifuged at 4000 x g for 2 minutes and washed three times in 1ml PBS containing 0.05% Tween 80. Cell Depletion and Neutrophil Isolation For comparative depletion experiments, blood was drawn into heparinised tubes, divided into aliquots and incubated with Miltenyi MicroBeads at 2-8 o C. Volumes and incubation times were as follows: anti-cd15: 50μl/ml, 15 minutes; Basic (unconjugated) MicroBeads used for controls: 50μl/ml, minutes. Blood was then diluted 1:1 with RPMI-1640 and pipetted into Miltenyi Biotec LS columns resting in magnets (MidiMACS Separation Unit, Miltenyi Biotec); columns were pre- primed with 3ml MACS buffer (0.5% bovine serum albumin (Sigma) and 2mM EDTA (Sigma)), all as previously described (E2). Depleted blood was collected into Universal containers, while CD15+ cells were recovered from the appropriate column using fresh medium and the supplied syringe driver. The collected CD15+ cells were counted using a Beckman Coulter Ac.T Diff, a Drew Scientific HemaVet HV950 haematology analyser or via microscopy with counting chambers and diluted if necessary with further RPMI-1640 to reach the required final concentration. Isolated Neutrophil Lux Assay This assay has been previously described in detail (E2). Briefly, granulocytes were resuspended to 1 x 10 6 cells/ml in RPMI-1640 before pipetting 400mcl of cell suspension into a 5 ml Falcon flow cytometry tube (Becton Dickinson) together with 50 mcl autologous serum (final concentration 10% serum) followed by 50 mcl organism suspension, appropriately diluted with sterile PBS to reach multiplicity of infection (MOI) approximately 1 CFU:3 neutrophils. Tubes were capped and rolled to ensure that all organisms were mixed E7

8 with the cell suspension and then incubated on their sides on a rocking plate (20 revolutions per minute (rpm)) at 37 o C. After 1 and 24 hours, samples were allowed to cool to room temperature, briefly vortexed, caps were removed and the tubes were placed in a Berthold Sirius single-tube luminometer for measurement. Serum control samples were prepared with 400mcl RPMI-1640, 50mcl serum and 50mcl organism suspension. 7H9 control samples were prepared with 450mcl 7H9 and 50mcl organism suspension. Results were calculated as the ratio of luminescence in neutrophil samples versus autologous serum controls. Whole Blood Lux Assay This has been described in full previously (E3, E4). Briefly, for depletion experiments, approximately 5 x 10 5 cfu M. tuberculosis-lux at mid-log phase in 100mcl PBS was inoculated into triplicate samples of 900mcl cell-depleted or Basic MicroBead-treated blood already diluted 1:1 with RPMI, as described above. Samples were incubated at 37 C on a rocking platform and mycobacterial luminescence was measured on triplicate 100mcl aliquots after harvesting of assay supernatants and water-lysis of red blood cells in samples at 96 hours. Phagocytosis Assay This has been described in detail previously (E2). Briefly, 400mcl of granulocytes in RPMI at a concentration of 1 x 10 6 /ml were aliquotted into sterile 5 ml flow cytometry tubes. 50mcl autologous serum and 50mcl FITC-labelled M.tuberculosis-lux suspension at MOI of 1 RLU:1 granulocyte was added and samples were incubated on their sides on a rocking plate (20 rpm) at 37 o C for 30 minutes. Samples were centrifuged at 300 x g for 5 minutes at 0 o C and supernatants discarded. 1 mcl efluor450 Fixable Viability Dye (ebiosciences) and 0.5 mcl Phycoerythrin (PE)-conjugated anti-cd66a,c,e antibody (BioLegend) was added to the E8

9 pellets and samples incubated at 0 o C for 12 minutes before adding 1 ml ice-cold PBS and 12.5 mcl syringe-filtered Trypan blue. After centrifuging at 300 x g for 5 minutes, supernatants were aspirated and the pellets resuspended in 500 mcl 4% paraformaldehyde for fixation before acquisition on a Becton Dickinson Fortessa flow cytometer. Single stained and unstained samples were used as compensation controls. The percentage of singlet, viable, CD66a,c,e-positive cells containing FITC was calculated as described (E2) to define internalisation (Supplementary Figure E2). Surface Activation Marker Flow Cytometry 200mcl of blood were transferred to 5ml flow cytometry tubes (Becton Dickinson) and fluorochrome labelled antibodies were added (anti-cd66,a,c,e-pe, Clone ASL32, 2mcl [BioLegend]; anti-cd16-percp Cy5.5, Clone 3G8, 4mcl [Becton Dickinson]; anti-cd11b- PECy7, Clone ICRF44, 5mcl [Becton Dickinson]; anti-cd62l-fitc, Clone DREG-56, 15mcl [Becton Dickinson]; efluor450 Fixable Viability Dye, 1mcl [ebiosciences]). Samples were incubated in the dark for 15 minutes before adding 2ml 1x BD FACS Lysing Solution (Becton Dickinson), briefly vortexing and incubating for a further 10 minutes. Samples were then centrifuged at 500g for 5 minutes, washed in PBS and fixed in 500mcl 2% paraformaldehyde (Alfa Aesar) before acquisition on a BD Fortessa flow cytometer. Single stained and unstained controls were always included for compensation. A minimum of 50,000 events was collected for each sample. Analysis and compensation was performed using FlowJo software from Treestar. The gating strategy is described in Supplementary Figure E3. E9

10 Measurement of CD4 Count, HIV Viral Load, Full Blood Count and C-Reactive Protein These were performed by the South African National Health Laboratory Service by flow cytometry, polymerase chain reaction and nephelometry respectively. Cell Death Assay 400mcl granulocytes at a concentration of 1 x 10 6 /ml in RPMI-1640 with 10% autologous serum, infected with mycobacteria (50mcl suspension, same MOI as the restriction assay), were incubated at 37 o C for 24 hours. Samples were then centrifuged at 500 x g for 6 minutes and supernatants removed and stored at -80 o C for measurement of HNP (see below). Pellets were re-suspended in 500mcl Annexin V binding buffer (Becton Dickinson); 5mcl Annexin V-FITC (Becton Dickinson), 0.5mcl CD66a,c,e-PE and 1mcl efluor450 Viability Dye were added to each sample and incubated for 20 minutes. They were then washed in PBS and resuspended in 4% paraformaldehyde for fixation before acquisition on a BD Fortessa flow cytometer. For gating, neutrophil surface marker positive events were gated amongst singlets using CD66a,c,e-PE versus side scatter (SSC). Alternatively, granulocytes were gated using Forward Scatter (FSC) versus SSC. These events were then divided into quadrants on the basis of Annexin V-FITC and Viability Dye positivity (Supplementary Figure E4), similar to assays previously described (E5). Human Neutrophil Peptide 1-3 ELISA (Hycult Biotech) and Quantiferon Gold In-Tube assays (Cellestis / Qiagen) These were performed according to manufacturers instructions. E10

11 Statistics Parametric data (normality assessed using Kolmogorov-Smirnov testing) was analysed using Student s t-tests for 2 groups (paired when appropriate) or one-way ANOVA for 3 or more groups with post-hoc correction. Non-parametric data was analysed using Mann-Whitney U- tests or Wilcoxon signed rank tests for 2 groups and Kruskal-Wallis testing with post-hoc Dunn s correction for 3 or more groups. Proportions were compared with chi-squared tests. Correlations were performed using Pearson methodology. All statistical analyses were performed using SPSS Version 18.0 (SPSS Inc, Chicago, USA) or GraphPad Prism version 4.0. All reported p-values are two-sided; p <0.05 was inferred as significant. E11

12 Supplementary References E1. Snewin VA, Gares MP, Gaora PO, Hasan Z, Brown IN, Young DB. Assessment of immunity to mycobacterial infection with luciferase reporter constructs. Infect Immun 1999;67: E2. Lowe DM, Bangani N, Mehta MR, Lang DM, Rossi AG, Wilkinson KA, Wilkinson RJ, Martineau AR. A novel assay of antimycobacterial activity and phagocytosis by human neutrophils. Tuberculosis 2013;93: E3. Kampmann B, Gaora PO, Snewin VA, Gares MP, Young DB, Levin M. Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria. J Infect Dis 2000;182: E4. Kampmann B, Tena GN, Mzazi S, Eley B, Young DB, Levin M. Novel human in vitro system for evaluating antimycobacterial vaccines. Infect Immun2004;72: E5. Corleis B, Korbel D, Wilson R, Bylund J, Chee R, Schaible UE. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils. Cell Microbiol 2012;14: E12

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