As in our Toll-like Receptor (TLR) signaling screen, stimulated ex vivo thioglycolate-elicited peritoneal macrophages are used to discover components involved in sensing the adjuvant alum, an aluminium salt that adsorbs and precipitates protein antigens in solution thereby improving vaccine immunogenicity by facilitating the slow release of antigen from the vaccine depot formed at the site of inoculation.  Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists release only traces of IL-1β, despite inducement of IL-1β mRNA.  In contrast, cells costimulated with TLR agonists plus alum release large amounts of IL-1β due to caspase-1 activation (1).  Alum is now known to activate caspase-1 via the Nlrp3 inflammasome, leading to secretion of the cytokine IL-1β (2).  Nlrp3 is a NOD-like receptor capable of sensing stimuli of microbial origin as well as endogenous markers of cellular damage (3).

 

The alum macrophage screen is designed to probe for additional components of the alum sensing pathway in macrophages isolated from N-ethyl-N-nitrosourea (ENU)-mutagenized mice.  Because inflammasome activation requires two signals for the production of mature IL-1β, macrophages are first primed with lipopolysaccharide (LPS) and then exposed to aluminium adjuvants.  Appropriate macrophage responses to alum are assayed by examining IL-1β levels. 

4% (w/v) Brewer’s thioglycolate medium powder (BBL Microbiology Systems, Cockeysville, MD) is added to distilled water pre-warmed to 37°C. Solution is autoclaved to sterilize and stored away from light.

 

Hepes-buffered saline solution (Gibco, Invitrogen, Carlsbad, CA )

5% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA)

200 IU/mL penicillin (Gibco)

200 mg/mL streptomycin (Gibco)

 

Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA)

5% (v/v) heat-inactivated fetal bovine serum

200 IU/mL penicillin

200 mg/mL streptomycin

 

2 mg MTT (Sigma)/mL sterile PBS.

 

90% (v/v) isopropanol

0.5% (w/v) SDS

0.04N HCl

Adjust volume with distilled water.

 

1 mg/ml (Alexis, San Diego, CA, cat # ALX-581-007)

 

Nigericin (sigma N7143)

 

IL-1β ELISA (eBioscience, San Diego, CA, cat # 88-7913)

 

0.5 ml Tween 20 (Sigma)

1L PBS

 

Table 1 |  IL-1β ELISA plate layout.

 

 

Thioglycolate solution should be carefully prepared to ensure maximal PEC recovery.  Solvent should be warmed to 37°C prior to solute addition to assist solubility, and subsequently autoclaved at least once, since this increases inflammatory potential (4).  Aging the solution for at least 1 month also enhances activity (5), in which case it should be stored away from light and at room temperature.

 

While stimulated PEC supernatants may be stored indefinitely at -20°C, PECs stored at 4°C remain viable and responsive to stimulation for up to four days.  Prompt completion of the initial assay therefore allows any remaining cells to be used for confirmation of putative mutants.  A minimum of three weeks should be allowed between thioglycolate injections to allow for sufficient PEC recovery.

   1.  Li, H., Nookala, S., and Re, F. (2007) Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release, J Immunol. 178, 5271-5276.

   2.  Eisenbarth, S. C., Colegio, O. R., O'Connor, W., Sutterwala, F. S., and Flavell, R. A. (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants, Nature 453, 1122-1126.

   3.  Mariathasan, S. and Monack, D. M. (2007) Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation, Nat. Rev. Immunol. 7, 31-40.

   4.  Davies, J. Q. and Gordon, S. (2005) Isolation and culture of murine macrophages, Methods Mol. Biol. 290, 91-103.

   5.  Li, Y. M., Baviello, G., Vlassara, H., and Mitsuhashi, T. (1997) Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages, J. Immunol. Methods 201, 183-188.