In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans

Materials and Reagents

1. Candida albicans strains, SC5314 and ATCC18804
   
2. Mice
   Note: 2x per WT or Dectin-1 KO mice on C57BL/6 background should give sufficient macrophages for a full 24-well plate experiment. Mice were age and sex matched.
   
3. Thioglycollate-elicited macrophages
   Note: This method has been described in detail in Kerrigan et al. (2012).
   
4. RPMI 1640 medium (Life Technologies, catalog number: 11875-093 ) 
5. 70% ethanol
6. Sabouraud Dextrose broth (Oxoid Limited, catalog number: CM0147 )
   
7. Rhodamine Green-X (Life Technologies, Invitrogen^TM, catalog number: R-6113 )
   
8. Difco Thioglycollate Broth (BD Biosciences, catalog number: 225710 )
   
9. Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
   
10. OptEIA TNF kit (BD Biosciences, catalog number: 555268 )
    
11. Zymosan (Life Technologies, Invitrogen^TM, catalog number: Z2849 )
    
12. Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
    
13. Fetal Bovine/Calf Serum (FCS) (Sigma-Aldrich, catalog number: F6178 )
    
14. EDTA (Sigma-Aldrich, catalog number: E9884 )
    

Equipment

1. Titer-Tek Fluoroskan II (Labsystems)
   
2. Shaker
   Note: The shaker temperature was controlled at 30 °C and the speed was between 150 to 200 rpm.
   
3. Centrifuge (Eppendorf, model: 5810R with swing bucket rotor S-4-72 )
   
4. Light microscope
   
5. Haemocytometer
   
6. 24-well plates (BD Biosciences)
   
7. 2 ml Eppendorf tube
   
8. 96-well plates (black in color for fluorescence measurement) (BD Biosciences)
   
9. Rubber back of syringe plunger
   Note: The make or brands for above equipment are not important for reproducibility.
   

Procedure

1. Growth and Labeling of Candida albicans
   1. Start C. albicans cultures by inoculating frozen stocks into 5 ml Sabouraud Dextrose broth and incubate for 16 to 24 h at 30 °C with shaking at 200 rpm.
      
   2. Centrifuge the cells for 5 min at 3,000 rpm and wash three times in 10 ml Dulbecco’s PBS.
      
   3. Count the yeast cells under the microscope using a haemocytometer.
      
   4. Adjust the cell density to at least 3.2 x 10⁶ yeast/ml, or as desirable in PBS and transfer into a 2 ml eppendorf tube.
      
   5. Add Rhodamine Green-X to a concentration of 200 μg/ml.
      
   6. Cover the tube with foil and incubate with gentle agitation at room temperature for 30 to 45 min.
      
   7. Centrifuge the labelled cells at 3,000 rpm for 5 min and resuspend in PBS.
      
   8. Wash the cells about ten times with 2 ml PBS or until free Rhodamine Green-X is removed.
   
   
2. Macrophage binding and pro-inflammatory assays
   Notes:
   1.  Extraction of macrophages is done in a hood within animal facility.
      
   2. The treatment of macrophages with Candida albicans is performed in a hood in a tissue culture BSL2 area.
      
   3. The hood surface is sprayed with 70% ethanol to prevent sample contamination.
   1. Inject mice intraperitoneally with 1 ml of thioglycollate broth (Kerrigan et al., 2012). Gently hold the mouse to allow free space on the site of injection. Use a syringe to inject the thioglycollate broth in the lower quadrant of the abdomen; be careful not to prick vital organs such as the bladder or intestines.
      
   2. After four days, isolate peritoneal exudate cells by lavage with ice-cold 5 ml PBS containing 5 mM EDTA.
      
   3. Centrifuge the peritoneal cells at 1,000 rpm for 10 min and resuspend the pellet in 5 ml RPMI medium containing 10% (volume/volume) heat-inactivated FCS (by heating at 60 °C for 30 min).
      Note: All FCS used in the following steps are heat-inactivated.
      
   4. Count macrophages on a haemocytometer, dilute them to 5.0 x 10⁵ cells/ml and seed them at a density of 2.5 x 10⁵ cells/well in a 24-well plate with RPMI medium containing 10% FCS. Incubate the plate overnight at 37 °C (without any agitation).
      
   5. Aspirate wells and add 0.5 ml fresh RPMI plus 10% FCS to the macrophages that are attached to the wells.
      
   6. Add the Rhodamine Green-X-labelled C. albicans at the MOI (multiplicity of infection) of 5:1 or 10:1 to the macrophages.
      
   7. Fluorescein isothiocyanate–labelled zymosan can be used as a control at MOI of 25:1. Zymosan is made of Beta-glucan particles that are recognized by macrophage receptors and induce pro-inflammatory responses; Dectin-1 is a well-studied receptor for these particles (Brown et al., 2003).
      Notes:
      1. Zymosan is labelled with FITC according to manufacturer’s (Molecular Probe) instructions as described also in detail in Kerrigan et al. (2012).
         
      2. The expected results are that Zymosan will bind macrophage and induce proinflammatory responses in a Dectin-1 dependent manner.
   8. Incubate the plate on ice for 30 min to allow the particles to settle, and then a further 30 min incubation at 37 °C, 5% CO₂.
      
   9. Wash the wells three times with 0.5 ml RPMI plus 10% FCS to remove the unbound particles.
      
   10. Add 500 μl of the RPMI plus 10% FCS to the wells and incubate at 37 °C for 3 h, 5% CO₂.
       
   11. Aliquot 150 μl of the supernatant and store at -80 °C for later analysis of pro-inflammatory cytokine production.
       
   12. Wash the wells and lyse the cells by adding 150 μl 3% Triton X-100 (pH 7.5). Triton X-100 is diluted to 3% (v/v) in distilled water.
       
   13. Detach the cells using rubber back of syringe plunger and add 100 μl into black 96-well plates. Cell scraper can also be used.
       
   14. To quantify the binding of fungal particles to macrophages, measure the fluorescence at the excitation of 490 nm and the emission at 514 nm on a Titer-Tek Fluoroskan II. Expected results are as shown in Figure 4b in Marakalala et al. (2013).
       
   15. For pro-inflammatory responses, use ELISA (OptEIA TNF kit) to measure the TNF concentrations of the supernatant aliquots which had been stored at -80 °C.
       Note: Pro-inflammatory responses to hyphae can be measured similarly, except that macrophages are added directly to the wells that already contain live or heat-killed hyphae. See below for detailed description of hyphal induction.
   
   
3. Pro-inflammatory responses to hyphae
   1. To induce hyphae formation, grow and quantify C. albicans as stated in steps A1-3.
      
   2. Incubate 2.5 x 10⁶ yeast/well (cell density of about 3.2 x 10⁶ yeast/ml or as per required amount) with RPMI medium for 3 h at 37 °C. View the cells under the microscope to confirm hyphae formation.
      
   3. Heat-kill the hyphae by incubating at 65 °C for 2 h, or use live hyphae if desired.
   4. Add 2.5 x 10⁵ of thioglycollate-elicited macrophages (densisty is 5.0 x 10⁵ cells/ml directly the hyphae-containing wells).
   5. Incubate the plates overnight at 37 °C, 5% CO₂.
   6. Take the supernatant samples and store at -80 °C until needed for cytokine analysis.
      

Acknowledgments

This protocol was adapted from previous publications including (Marakalala et al., 2008; Brown et al., 2003; Kerrigan et al., 2012, and Taylor et al., 2007). I also acknowledge the Sydney Brenner Fellowship, National Research Foundation and Medical Research Council of South Africa for funding.

References

1. Brown, G. D., Herre, J., Williams, D. L., Willment, J. A., Marshall, A. S. and Gordon, S. (2003). Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197(9): 1119-1124.
   
2. Kerrigan, A. M., de Sousa Mda, G. and Brown, G. D. (2012). Simple assays for measuring innate interactions with fungi. Methods Mol Biol 845: 303-317.
   
3. Marakalala, M. J., Vautier, S., Potrykus, J., Walker, L. A., Shepardson, K. M., Hopke, A., Mora-Montes, H. M., Kerrigan, A., Netea, M. G., Murray, G. I., Maccallum, D. M., Wheeler, R., Munro, C. A., Gow, N. A., Cramer, R. A., Brown, A. J. and Brown, G. D. (2013). Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1. PLoS Pathog 9(4): e1003315.
   
4. Taylor, P. R., Tsoni, S. V., Willment, J. A., Dennehy, K. M., Rosas, M., Findon, H., Haynes, K., Steele, C., Botto, M., Gordon, S. and Brown, G. D. (2007). Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol 8(1): 31-38.